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Chilkoti, Ashutosh

Overview:

Ashutosh Chilkoti is the Alan L. Kaganov Professor of Biomedical Engineering and Chair of the Department of Biomedical Engineering at Duke University. 



My research in biomolecular engineering and biointerface science focuses on the development of new molecular tools and technology's that borrow from molecular biology, protein engineering, polymer chemistry and surface science that we then exploit for the development of applications that span the range from bioseparations, plasmonic biosensors, low-cost clinical diagnostics, and drug delivery.

Positions:

Alan L. Kaganov Professor of Biomedical Engineering

Biomedical Engineering
Pratt School of Engineering

Professor of Biomedical Engineering

Biomedical Engineering
Pratt School of Engineering

Professor in the Department of Mechanical Engineering and Materials Science

Pratt School of Engineering
Pratt School of Engineering

Professor in the Department of Chemistry

Chemistry
Trinity College of Arts & Sciences

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Chair of the Department of Biomedical Engineering

Biomedical Engineering
Pratt School of Engineering

Education:

Ph.D. 1991

Ph.D. — University of Washington

News:

Grants:

Magnetically directed single cell transcriptome analysis in HIV latency

Administered By
Medicine, Pulmonary, Allergy, and Critical Care Medicine
AwardedBy
National Institutes of Health
Role
Investigator
Start Date
August 01, 2014
End Date
August 31, 2021

Pharmacological Sciences Training Program

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Participating Faculty Member
Start Date
July 01, 1975
End Date
June 30, 2020

Training in Medical Imaging

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 15, 2003
End Date
August 31, 2019

Self-assembled microporous networks for cell culture

Administered By
Biomedical Engineering
AwardedBy
EMD Millipore
Role
Principal Investigator
Start Date
April 01, 2017
End Date
March 31, 2019

Center for Environmental Implications of Nanotechnology

Administered By
Pratt School of Engineering
AwardedBy
National Science Foundation
Role
Investigator
Start Date
October 01, 2008
End Date
August 31, 2018

MRI: Acquisition of a Triple Quadrupole LC/MS System

Administered By
Chemistry
AwardedBy
National Science Foundation
Role
Major User
Start Date
August 01, 2015
End Date
July 31, 2018

Elastin Fusion Proteins

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2000
End Date
June 30, 2018

Acquisition of a MALDI-TOF Mass Spectrometer System at Duke University

Administered By
Chemistry
AwardedBy
North Carolina Biotechnology Center
Role
Major User
Start Date
March 28, 2017
End Date
March 27, 2018

Non-immunogenic PEG conjugates of Biologics

Administered By
Biomedical Engineering
AwardedBy
North Carolina Biotechnology Center
Role
Principal Investigator
Start Date
September 01, 2016
End Date
February 28, 2018

Assessing the Manufacturability of a "Next-Gen" Non-Immunogenic PEG-like Conjugate

Administered By
Biomedical Engineering
AwardedBy
North Carolina Biotechnology Center
Role
Co Investigator
Start Date
October 01, 2016
End Date
September 30, 2017

One-Pot Synthesis of Multicomponent Protein-Carbohydrate Antigens

Administered By
Biomedical Engineering
AwardedBy
Pfizer, Inc.
Role
Principal Investigator
Start Date
September 12, 2016
End Date
September 11, 2017

Treating Melanoma with a Molecularly Engineered, Long Circulating Immunotoxin

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 15, 2015
End Date
June 30, 2017

Collaborative Research: Supramolecular Materials by Nucleic Acid Block Copolymer Selfassembly

Administered By
Mechanical Engineering and Materials Science
AwardedBy
National Science Foundation
Role
Co-Principal Investigator
Start Date
July 15, 2014
End Date
June 30, 2017

Medical Scientist Training Program

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1997
End Date
June 30, 2017

University Training Program in Biomolecular and Tissue Engineering

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1994
End Date
June 30, 2017

Texas State University PREM: Center on Interfaces in Materials

Administered By
Pratt School of Engineering
AwardedBy
Texas State University
Role
Co Investigator
Start Date
September 01, 2012
End Date
May 31, 2017

OptiDiag: Optimizing diagnosis and management of acute malnutrition

Administered By
Pediatrics, Endocrinology
AwardedBy
Action Contre La Faim
Role
Consultant
Start Date
May 14, 2015
End Date
May 13, 2017

Thermally Triggered Multivalent Targeting of Tumors

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2007
End Date
June 30, 2016

Thermally Targeted Drug Delivery by Elastin Biopolymers

Administered By
Pratt School of Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 2002
End Date
April 30, 2016

Protease Operated Depot for Delivery of GLP-1

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2012
End Date
March 31, 2016

Site-specific antibody-toxin conjugates for cancer therapy

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 01, 2015
End Date
March 02, 2016

Local Radionuclide Delivery to Solid Tumors by Injectible Biopolymer

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
May 17, 2009
End Date
January 31, 2016

Site-Specific Growth of Stealth Polymer from Peptide Therapeutics

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 30, 2011
End Date
August 31, 2015

Surface Initiated Enzymatic Polymerization of DNA Nanostructures for Highly Amplified Sensing

Administered By
Biomedical Engineering
AwardedBy
National Science Foundation
Role
Principal Investigator
Start Date
September 01, 2010
End Date
August 31, 2015

2-D Diffusion Assay on Polymer Brush for POC Cardiac Infarction Diagnosis

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 17, 2012
End Date
June 30, 2015

Live-Animal Micro-CT System

Administered By
Orthopaedics
AwardedBy
National Institutes of Health
Role
Major User
Start Date
May 15, 2012
End Date
May 14, 2014

SAXS System

Administered By
Pratt School of Engineering
AwardedBy
National Science Foundation
Role
Co Investigator
Start Date
August 01, 2012
End Date
July 31, 2013

Inhibition of metastasis-initiating cells by chimeric polypeptide nanoparticles

Administered By
Pratt School of Engineering
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 01, 2010
End Date
December 31, 2012

Delivery of Peptide Therapeutics by Molecular Release Depots

Administered By
Pratt School of Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 30, 2010
End Date
August 31, 2012

Detection of Biomolecules by Nanoscale Plasmon Ruler

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 2009
End Date
July 31, 2012

Genetically Designed Materials for Cartilage Repair

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Investigator
Start Date
September 20, 2003
End Date
July 31, 2012

Small Animal PET / CT Molecular Imaging

Administered By
Radiology, Nuclear Medicine
AwardedBy
National Institutes of Health
Role
Major User
Start Date
April 01, 2011
End Date
March 31, 2012

Plasmonic assay for detection of biomarkers at the single-molecule level

Administered By
Pratt School of Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 2009
End Date
July 31, 2011

Intravital point-scanning confocal microscope

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 06, 2010
End Date
May 05, 2011

Molecular Imaging Using Hyperspectral Darkfield Microscope of Nanoparticles

Administered By
Biomedical Engineering
AwardedBy
National Science Foundation
Role
Co-Principal Investigator
Start Date
April 01, 2007
End Date
March 31, 2011

NIRT: Hierarchical Bionanomanufacturing

Administered By
Pratt School of Engineering
AwardedBy
National Science Foundation
Role
Co-Principal Investigator
Start Date
September 15, 2006
End Date
August 31, 2010

Thermally-Induced Intra-Articular Drug Delivery System

Administered By
Orthopaedics
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
September 01, 2006
End Date
June 30, 2009

Training in Biomolecular and Tissue Engineering

Administered By
Orthopaedics
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 20, 2003
End Date
June 30, 2009

Elastin Fusion Proteins

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2000
End Date
March 31, 2009

Nanophotonics for Select Agent Detection

Administered By
Pratt School of Engineering
AwardedBy
Centers for Disease Control and Prevention
Role
Principal Investigator
Start Date
September 15, 2003
End Date
September 14, 2008

Nanophotonics for Select Agent Detection

Administered By
Pratt School of Engineering
AwardedBy
Centers for Disease Control and Prevention
Role
Principal Investigator
Start Date
September 15, 2003
End Date
September 14, 2008

Nanophotonics for Select Agent Detection

Administered By
Pratt School of Engineering
AwardedBy
Centers for Disease Control and Prevention
Role
Principal Investigator
Start Date
September 15, 2003
End Date
September 14, 2008

NER: Integrated Magnetic and Chemical Assembly Techniques for Building Nanoparticle Arrays

Administered By
Mechanical Engineering and Materials Science
AwardedBy
National Science Foundation
Role
Co-Principal Investigator
Start Date
July 15, 2006
End Date
June 30, 2008

pH Sensitive Elastic-Like-Peptides for Tumor Targeting

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2006
End Date
June 30, 2008

Biointerface Science GRC

Administered By
Pratt School of Engineering
AwardedBy
Army Research Office
Role
Principal Investigator
Start Date
October 01, 2006
End Date
September 30, 2007

Thermally Targeted Drug Delivery by Elastin Biopolymers

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 2002
End Date
July 31, 2007

NIRT

Administered By
Pratt School of Engineering
AwardedBy
National Science Foundation
Role
Co-Principal Investigator
Start Date
September 01, 2002
End Date
August 31, 2006

Upgrade of a Shared Instrumentation Resource in the PSOE: The Laser Scanning Confocal Microscope

Administered By
Biomedical Engineering
AwardedBy
Lord Foundation of North Carolina
Role
Principal Investigator
Start Date
May 31, 2002
End Date
June 30, 2005

Elastin Fusion Proteins

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2000
End Date
March 31, 2005

The AVS2004 Biomaterials Interfaces Symposium

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 24, 2004
End Date
February 28, 2005

Imaging XPS

Administered By
Pratt School of Engineering
AwardedBy
National Science Foundation
Role
Principal Investigator
Start Date
August 01, 2002
End Date
July 31, 2004

Elastin Bioelastomers for Microactuation

Administered By
Biomedical Engineering
AwardedBy
Office of Naval Research
Role
Principal Investigator
Start Date
January 01, 2000
End Date
December 31, 2002

CAREER: Smart Protein-Polymer Conjugates for Biotechnology

Administered By
Biomedical Engineering
AwardedBy
National Science Foundation
Role
Principal Investigator
Start Date
October 01, 1998
End Date
April 30, 2002

(98-0048) CAREER: Smart Protein - Polymer Conjugates for Biotechnology

Administered By
Biomedical Engineering
AwardedBy
National Science Foundation
Role
Principal Investigator
Start Date
May 15, 1998
End Date
April 30, 2002

Engineered Protein-Polymer Conjugates for Biotechnology

Administered By
Biomedical Engineering
AwardedBy
National Science Foundation
Role
Principal Investigator
Start Date
May 15, 1998
End Date
April 30, 2002

Elastin Nanobiosensors

Administered By
Biomedical Engineering
AwardedBy
National Science Foundation
Role
Principal Investigator
Start Date
March 01, 2000
End Date
February 28, 2002

(97-0915) Extrinsic Modulation of Protein-Ligand Recognition

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 1998
End Date
October 31, 2000

Extrinsic Modulation of Protein/Ligand Recognition

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 1998
End Date
October 31, 2000

An Engineering Outreach Program for High School

Administered By
Biomedical Engineering
AwardedBy
Lord Foundation of North Carolina
Role
Principal Investigator
Start Date
May 01, 1998
End Date
June 30, 1999

Laboratories in Biomedical and Cellular Engineering

Administered By
Biomedical Engineering
AwardedBy
Lord Foundation of North Carolina
Role
Principal Investigator
Start Date
May 01, 1998
End Date
June 30, 1999

(98-0050) Isothermal Titration Calorimetry for Biological Research

Administered By
Biomedical Engineering
AwardedBy
Division of Undergraduate Education
Role
Principal Investigator
Start Date
April 01, 1998
End Date
March 31, 1999

(97-0056) Combined Atomic Force and Total Internal Reflection Fluorescence Microscopy for Biological Research

Administered By
Biomedical Engineering
AwardedBy
National Science Foundation
Role
Principal Investigator
Start Date
February 15, 1997
End Date
January 31, 1999

(97-0929) Matching Funds for Combined Atomic Force & Optical...

Administered By
Biomedical Engineering
AwardedBy
Lord Foundation of North Carolina
Role
Principal Investigator
Start Date
June 01, 1997
End Date
May 31, 1998
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Awards:

Distinguished Alumnus Award . Indian Institute of Technology Delhi, India .

Type
International
Awarded By
Indian Institute of Technology Delhi, India
Date
January 01, 2015

Fellow. National Academy of Inventors.

Type
National
Awarded By
National Academy of Inventors
Date
January 01, 2014

Pritzker Distinguished Lecture Award. Biomedical Engineering Society.

Type
National
Awarded By
Biomedical Engineering Society
Date
January 01, 2013

Clemson Award for Contributions to the Literature. Society for Biomaterials.

Type
National
Awarded By
Society for Biomaterials
Date
January 01, 2011

Clemson Award for Contributions to the Literature. Society for Biomaterials.

Type
National
Awarded By
Society for Biomaterials
Date
January 01, 2011

Humboldt Senior Researcher Award. Alexander Von Humboldt Foundation.

Type
International
Awarded By
Alexander Von Humboldt Foundation
Date
January 01, 2010

Stansell Family Distinguished Research Award. Duke University, Pratt School of Engineering.

Type
School
Awarded By
Duke University Pratt School of Engineering
Date
January 01, 2008

Fellows. American Institute for Medical and Biological Engineering.

Type
National
Awarded By
American Institute for Medical and Biological Engineering
Date
January 01, 1996

Publications:

Cargo self-assembly rescues affinity of cell-penetrating peptides to lipid membranes

© 2017 The Author(s).Although cationic cell-penetrating peptides (CPPs) are able to bind to cell membranes, thus promoting cell internalization by active pathways, attachment of cargo molecules to CPPs invariably reduces their cellular uptake. We show here that CPP binding to lipid bilayers, a simple model of the cell membrane, can be recovered by designing cargo molecules that self-assemble into spherical micelles and increase the local interfacial density of CPP on the surface of the cargo. Experiments performed on model giant unilamellar vesicles under a confocal laser scanning microscope show that a family of thermally responsive elastin-like polypeptides that exhibit temperature-triggered micellization can promote temperature triggered attachment of the micelles to membranes, thus rescuing by self-assembly the cargo-induced loss of the CPP affinity to bio-membranes.

Authors
Weinberger, A; Walter, V; MacEwan, SR; Schmatko, T; Muller, P; Schroder, AP; Chilkoti, A; Marques, CM
MLA Citation
Weinberger, A, Walter, V, MacEwan, SR, Schmatko, T, Muller, P, Schroder, AP, Chilkoti, A, and Marques, CM. "Cargo self-assembly rescues affinity of cell-penetrating peptides to lipid membranes." Scientific Reports 7 (March 6, 2017).
Source
scopus
Published In
Scientific Reports
Volume
7
Publish Date
2017
DOI
10.1038/srep43963

Strong, Tough, Stretchable, and Self-Adhesive Hydrogels from Intrinsically Unstructured Proteins.

Strong, tough, stretchable, and self-adhesive hydrogels are designed with intrinsically unstructured proteins. The extraordinary mechanical properties exhibited by these materials are enabled by an integration of toughening mechanisms that maintain high elasticity and dissipate mechanical energy within the protein networks.

Authors
Gonzalez, MA; Simon, JR; Ghoorchian, A; Scholl, Z; Lin, S; Rubinstein, M; Marszalek, P; Chilkoti, A; López, GP; Zhao, X
MLA Citation
Gonzalez, MA, Simon, JR, Ghoorchian, A, Scholl, Z, Lin, S, Rubinstein, M, Marszalek, P, Chilkoti, A, López, GP, and Zhao, X. "Strong, Tough, Stretchable, and Self-Adhesive Hydrogels from Intrinsically Unstructured Proteins." Advanced materials (Deerfield Beach, Fla.) 29.10 (March 2017).
PMID
28060425
Source
epmc
Published In
Advanced Materials
Volume
29
Issue
10
Publish Date
2017
DOI
10.1002/adma.201604743

Site-Specific and Stoichiometric Stealth Polymer Conjugates of Therapeutic Peptides and Proteins.

As potent and selective therapeutic agents, peptides and proteins are an important class of drugs, but they typically have suboptimal pharmacokinetic profiles. One approach to solve this problem is their conjugation with "stealth" polymers. Conventional methods for conjugation of this class of polymers to peptides and proteins are typically carried out by reactions that have poor yield and provide limited control over the site of conjugation and the stoichiometry of the conjugate. To address these limitations, new chemical and biological approaches have been developed that provide new molecular tools in the bioconjugation toolbox to create stealth polymer conjugates of peptides and proteins with exquisite control over their properties. This review article highlights these recent advances in the synthesis of therapeutic peptide- and protein-stealth polymer conjugates.

Authors
Ozer, I; Chilkoti, A
MLA Citation
Ozer, I, and Chilkoti, A. "Site-Specific and Stoichiometric Stealth Polymer Conjugates of Therapeutic Peptides and Proteins." Bioconjugate chemistry 28.3 (March 2017): 713-723.
PMID
27998056
Source
epmc
Published In
Bioconjugate Chemistry
Volume
28
Issue
3
Publish Date
2017
Start Page
713
End Page
723
DOI
10.1021/acs.bioconjchem.6b00652

Characterisation of hydration and nanophase separation during the temperature response in hydrophobic/hydrophilic elastin-like polypeptide (ELP) diblock copolymers.

To understand the complex nanoscale dehydration process during the lower critical solution temperature (LCST) based inverse phase transition of a class of thermoresponsive biopolymers, diblock elastin-like polypeptides (ELPs) were investigated by spin probing continuous wave electron paramagnetic resonance (CW EPR) spectroscopy. The diblock copolymers composed of a hydrophobic block and a hydrophilic block showed different mechanisms of a temperature-driven phase transition. While the phase transition temperature is a function of the hydrophobic mass fraction of the diblock ELPs, the hydrophilic block length determines the molecular structure of the polymer aggregates formed above the transition temperature. When the weight ratio of hydrophilic block length to hydrophobic block length is greater than or equal to 0.3, the polymer aggregates consist of a hydrophobic core and a hydrophilic corona. The interface of these two regions become permeable at temperatures above the transition temperature. In case of smaller ratios, the aggregating hydrophobic parts of the polymer enclose the hydrated hydrophilic blocks, that are too small to form a hydrophilic corona, leading to bigger and less dense aggregates of higher polarity.

Authors
Widder, K; MacEwan, SR; Garanger, E; Núñez, V; Lecommandoux, S; Chilkoti, A; Hinderberger, D
MLA Citation
Widder, K, MacEwan, SR, Garanger, E, Núñez, V, Lecommandoux, S, Chilkoti, A, and Hinderberger, D. "Characterisation of hydration and nanophase separation during the temperature response in hydrophobic/hydrophilic elastin-like polypeptide (ELP) diblock copolymers." Soft matter 13.9 (March 2017): 1816-1822.
PMID
28169384
Source
epmc
Published In
Soft Matter
Volume
13
Issue
9
Publish Date
2017
Start Page
1816
End Page
1822
DOI
10.1039/c6sm02427k

Encapsulating a Hydrophilic Chemotherapeutic into Rod-Like Nanoparticles of a Genetically Encoded Asymmetric Triblock Polypeptide Improves Its Efficacy

Authors
Bhattacharyya, J; Weitzhandler, I; Ho, SB; McDaniel, JR; Li, X; Tang, L; Liu, J; Dewhirst, M; Chilkoti, A
MLA Citation
Bhattacharyya, J, Weitzhandler, I, Ho, SB, McDaniel, JR, Li, X, Tang, L, Liu, J, Dewhirst, M, and Chilkoti, A. "Encapsulating a Hydrophilic Chemotherapeutic into Rod-Like Nanoparticles of a Genetically Encoded Asymmetric Triblock Polypeptide Improves Its Efficacy." Advanced Functional Materials 27.12 (March 2017): 1605421-1605421.
Source
crossref
Published In
Advanced Functional Materials
Volume
27
Issue
12
Publish Date
2017
Start Page
1605421
End Page
1605421
DOI
10.1002/adfm.201605421

Quantitative Mapping of the Spatial Distribution of Nanoparticles in Endo-Lysosomes by Local pH.

Understanding the intracellular distribution and trafficking of nanoparticle drug carriers is necessary to elucidate their mechanisms of drug delivery and is helpful in the rational design of novel nanoparticle drug delivery systems. The traditional immunofluorescence method to study intracellular distribution of nanoparticles using organelle-specific antibodies is laborious and subject to artifacts. As an alternative, we developed a new method that exploits ratiometric fluorescence imaging of a pH-sensitive Lysosensor dye to visualize and quantify the spatial distribution of nanoparticles in the endosomes and lysosomes of live cells. Using this method, we compared the endolysosomal distribution of cell-penetrating peptide (CPP)-functionalized micelles to unfunctionalized micelles and found that CPP-functionalized micelles exhibited faster endosome-to-lysosome trafficking than unfunctionalized micelles. Ratiometric fluorescence imaging of pH-sensitive Lysosensor dye allows rapid quantitative mapping of nanoparticle distribution in endolysosomes in live cells while minimizing artifacts caused by extensive sample manipulation typical of alternative approaches. This new method can thus serve as an alternative to traditional immunofluorescence approaches to study the intracellular distribution and trafficking of nanoparticles within endosomes and lysosomes.

Authors
Wang, J; MacEwan, SR; Chilkoti, A
MLA Citation
Wang, J, MacEwan, SR, and Chilkoti, A. "Quantitative Mapping of the Spatial Distribution of Nanoparticles in Endo-Lysosomes by Local pH." Nano letters 17.2 (February 2017): 1226-1232.
PMID
28033711
Source
epmc
Published In
Nano Letters
Volume
17
Issue
2
Publish Date
2017
Start Page
1226
End Page
1232
DOI
10.1021/acs.nanolett.6b05041

Poly(oligo(ethylene glycol) methyl ether methacrylate) Brushes on High-κ Metal Oxide Dielectric Surfaces for Bioelectrical Environments.

Advances in electronics and life sciences have generated interest in "lab-on-a-chip" systems utilizing complementary metal oxide semiconductor (CMOS) circuitry for low-power, portable, and cost-effective biosensing platforms. Here, we present a simple and reliable approach for coating "high-κ" metal oxide dielectric materials with "non-fouling" (protein- and cell-resistant) poly(oligo(ethylene glycol) methyl ether methacrylate (POEGMA) polymer brushes as biointerfacial coatings to improve their relevance for biosensing applications utilizing advanced electronic components. By using a surface-initiated "grafting from" strategy, POEGMA films were reliably grown on each material, as confirmed by ellipsometric measurements and X-ray photoelectron spectroscopy (XPS) analysis. The electrical behavior of these POEGMA films was also studied to determine the potential impact on surrounding electronic devices, yielding information on relative permittivity and breakdown field for POEGMA in both dry and hydrated states. We show that the incorporation of POEGMA coatings significantly reduced levels of nonspecific protein adsorption compared to uncoated high-κ dielectric oxide surfaces as shown by protein resistance assays. These attributes, combined with the robust dielectric properties of POEGMA brushes on high-κ surfaces open the way to incorporate this protein and cell resistant polymer interface into CMOS devices for biomolecular detection in a complex liquid milieu.

Authors
Joh, DY; McGuire, F; Abedini-Nassab, R; Andrews, JB; Achar, RK; Zimmers, Z; Mozhdehi, D; Blair, R; Albarghouthi, F; Oles, W; Richter, J; Fontes, CM; Hucknall, AM; Yellen, BB; Franklin, AD; Chilkoti, A
MLA Citation
Joh, DY, McGuire, F, Abedini-Nassab, R, Andrews, JB, Achar, RK, Zimmers, Z, Mozhdehi, D, Blair, R, Albarghouthi, F, Oles, W, Richter, J, Fontes, CM, Hucknall, AM, Yellen, BB, Franklin, AD, and Chilkoti, A. "Poly(oligo(ethylene glycol) methyl ether methacrylate) Brushes on High-κ Metal Oxide Dielectric Surfaces for Bioelectrical Environments." ACS applied materials & interfaces 9.6 (February 2017): 5522-5529.
PMID
28117566
Source
epmc
Published In
ACS Applied Materials and Interfaces
Volume
9
Issue
6
Publish Date
2017
Start Page
5522
End Page
5529
DOI
10.1021/acsami.6b15836

Phase Behavior and Self-Assembly of Perfectly Sequence-Defined and Monodisperse Multiblock Copolypeptides.

This paper investigates how the properties of multiblock copolypeptides can be tuned by their block architecture, defined by the size and distribution of blocks along the polymer chain. These parameters were explored by the precise, genetically encoded synthesis of recombinant elastin-like polypeptides (ELPs). A family of ELPs was synthesized in which the composition and length were conserved while the block length and distribution were varied, thus creating 11 ELPs with unique block architectures. To our knowledge, these polymers are unprecedented in their intricately and precisely varied architectures. ELPs exhibit lower critical solution temperature (LCST) behavior and micellar self-assembly, both of which impart easily measured physicochemical properties to the copolymers, providing insight into polymer hydrophobicity and self-assembly into higher order structures, as a function of solution temperature. Even subtle variation in block architecture changed the LCST phase behavior and morphology of these ELPs, measured by their temperature-triggered phase transition and nanoscale self-assembly. Size and morphology of polypeptide micelles could be tuned solely by controlling the block architecture, thus demonstrating that when sequence can be precisely controlled, nanoscale self-assembly of polypeptides can be modulated by block architecture.

Authors
MacEwan, SR; Weitzhandler, I; Hoffmann, I; Genzer, J; Gradzielski, M; Chilkoti, A
MLA Citation
MacEwan, SR, Weitzhandler, I, Hoffmann, I, Genzer, J, Gradzielski, M, and Chilkoti, A. "Phase Behavior and Self-Assembly of Perfectly Sequence-Defined and Monodisperse Multiblock Copolypeptides." Biomacromolecules 18.2 (February 2017): 599-609.
PMID
28094978
Source
epmc
Published In
Biomacromolecules
Volume
18
Issue
2
Publish Date
2017
Start Page
599
End Page
609
DOI
10.1021/acs.biomac.6b01759

Cell-Based Biohybrid Drug Delivery Systems: The Best of the Synthetic and Natural Worlds.

The goal of drug delivery is to deliver therapeutics to the site of disease while reducing unwanted side effects. In recent years, a diverse variety of synthetic nano and microparticles have been developed as drug delivery systems. The success of these systems for drug delivery lies in their ability to overcome biological barriers such as the blood-brain barrier, to evade immune clearance and avoid nonspecific biodistribution. This Review provides an overview of recent advances in the design of biohybrid drug delivery systems, which combine cells with synthetic systems to overcome some of these biological hurdles. Examples include eukaryotic cells, such as stem cells, red blood cells, immune cells, platelets, and cancer cells that are used to carry drug-loaded synthetic particles. Synthetic particles can also be cloaked with naturally derived cell membranes and thereby evade immune clearance, exhibit prolonged systemic circulation, and target specific tissues by capitalizing on the interaction/homing tendency of certain cells and their membrane components to particular tissues. Different designs of cell-based biohybrid systems and their applications, as well as their promise and limitations, are discussed herein.

Authors
Banskota, S; Yousefpour, P; Chilkoti, A
MLA Citation
Banskota, S, Yousefpour, P, and Chilkoti, A. "Cell-Based Biohybrid Drug Delivery Systems: The Best of the Synthetic and Natural Worlds." Macromolecular bioscience 17.1 (January 2017).
PMID
27925398
Source
epmc
Published In
Macromolecular Bioscience
Volume
17
Issue
1
Publish Date
2017
DOI
10.1002/mabi.201600361

Silver nanoparticle toxicity is related to coating materials and disruption of sodium concentration regulation.

Silver nanoparticles (AgNPs) have been increasingly commercialized and their release into the environment is imminent. Toxicity of AgNP has been studied with a wide spectrum of organisms, yet the mechanism of toxicity remains largely unknown. This study systematically compared toxicity of 10 AgNPs of different particle diameters and coatings to Japanese medaka (Oryzias latipes) larvae to understand how characteristics of AgNP relate to toxicity. Dissolution of AgNPs was largely dependent on particle size, but their aggregation behavior and toxicity were more dependent on coating materials. 96 h lethal concentration 50% (LC50) values correlated with AgNP aggregate size rather than size of individual nanoparticles. Of the AgNPs studied, the dissolved Ag concentration in the test suspensions did not account for all of the observed toxicity, indicating the role of NP-specific characteristics in resultant toxicity. Exposure to AgNP led to decrease of sodium concentration in the tissue and increased expression of Na(+)/K(+ )ATPase. Gene expression patterns also suggested that toxicity was related to disruption of sodium regulation and not to oxidative stress.

Authors
Kwok, KWH; Dong, W; Marinakos, SM; Liu, J; Chilkoti, A; Wiesner, MR; Chernick, M; Hinton, DE
MLA Citation
Kwok, KWH, Dong, W, Marinakos, SM, Liu, J, Chilkoti, A, Wiesner, MR, Chernick, M, and Hinton, DE. "Silver nanoparticle toxicity is related to coating materials and disruption of sodium concentration regulation." Nanotoxicology 10.9 (November 2016): 1306-1317.
Website
http://hdl.handle.net/10161/13016
PMID
27345576
Source
epmc
Published In
Nanotoxicology (Informa)
Volume
10
Issue
9
Publish Date
2016
Start Page
1306
End Page
1317
DOI
10.1080/17435390.2016.1206150

Magnetophoretic transistors in a tri-axial magnetic field.

The ability to direct and sort individual biological and non-biological particles into spatially addressable locations is fundamentally important to the emerging field of single cell biology. Towards this goal, we demonstrate a new class of magnetophoretic transistors, which can switch single magnetically labeled cells and magnetic beads between different paths in a microfluidic chamber. Compared with prior work on magnetophoretic transistors driven by a two-dimensional in-plane rotating field, the addition of a vertical magnetic field bias provides significant advantages in preventing the formation of particle clumps and in better replicating the operating principles of circuits in general. However, the three-dimensional driving field requires a complete redesign of the magnetic track geometry and switching electrodes. We have solved this problem by developing several types of transistor geometries which can switch particles between two different tracks by either presenting a local energy barrier or by repelling magnetic objects away from a given track, hereby denoted as "barrier" and "repulsion" transistors, respectively. For both types of transistors, we observe complete switching of magnetic objects with currents of ∼40 mA, which is consistent over a range of particle sizes (8-15 μm). The switching efficiency was also tested at various magnetic field strengths (50-90 Oe) and driving frequencies (0.1-0.6 Hz); however, we again found that the device performance only weakly depended on these parameters. These findings support the use of these novel transistor geometries to form circuit architectures in which cells can be placed in defined locations and retrieved on demand.

Authors
Abedini-Nassab, R; Joh, DY; Albarghouthi, F; Chilkoti, A; Murdoch, DM; Yellen, BB
MLA Citation
Abedini-Nassab, R, Joh, DY, Albarghouthi, F, Chilkoti, A, Murdoch, DM, and Yellen, BB. "Magnetophoretic transistors in a tri-axial magnetic field." Lab on a chip 16.21 (October 3, 2016): 4181-4188.
PMID
27714014
Source
epmc
Published In
Lab on a Chip
Volume
16
Issue
21
Publish Date
2016
Start Page
4181
End Page
4188
DOI
10.1039/c6lc00878j

Controlled release of biologics for the treatment of type 2 diabetes.

Type 2 diabetes is a rapidly growing disease that poses a significant burden to the United States healthcare system. Despite the many available treatments for the disease, close to half of diagnosed type 2 diabetes cases are not properly managed, largely due to inadequate patient adherence to prescribed treatment regimens. Methods for improving delivery - and thereby easing administration - of type 2 drugs have the potential to greatly improve patient health. This review focuses on two peptide drugs - insulin and glucagon-like peptide 1 (GLP-1) - for treatment of type 2 diabetes. Peptide drugs offer the benefits of high potency and specificity but pose a significant delivery challenge due to their inherent instability and short half-life. The development of insulin and GLP-1 analogs highlights the broad spectrum of drug delivery strategies that have been used to solve these problems. Numerous structural modifications and formulations have been introduced to optimize absorption, residence time, stability, route of delivery and frequency of administration. Continual improvements in delivery methods for insulin and GLP-1 receptor agonists are paving the way towards better patient compliance and improved disease management, and thereby enhanced patient quality of life.

Authors
Gilroy, CA; Luginbuhl, KM; Chilkoti, A
MLA Citation
Gilroy, CA, Luginbuhl, KM, and Chilkoti, A. "Controlled release of biologics for the treatment of type 2 diabetes." Journal of controlled release : official journal of the Controlled Release Society 240 (October 2016): 151-164.
PMID
26655062
Source
epmc
Published In
Journal of Controlled Release
Volume
240
Publish Date
2016
Start Page
151
End Page
164
DOI
10.1016/j.jconrel.2015.12.002

Crystallization kinetics of binary colloidal monolayers.

Experiments and simulations are used to study the kinetics of crystal growth in a mixture of magnetic and nonmagnetic particles suspended in ferrofluid. The growth process is quantified using both a bond order parameter and a mean domain size parameter. The largest single crystals obtained in experiments consist of approximately 1000 particles and form if the area fraction is held between 65-70% and the field strength is kept in the range of 8.5-10.5 Oe. Simulations indicate that much larger single crystals containing as many as 5000 particles can be obtained under impurity-free conditions within a few hours. If our simulations are modified to include impurity concentrations as small as 1-2%, then the results agree quantitatively with the experiments. These findings provide an important step toward developing strategies for growing single crystals that are large enough to enable follow-on investigations across many subdisciplines in condensed matter physics.

Authors
Pham, AT; Seto, R; Schönke, J; Joh, DY; Chilkoti, A; Fried, E; Yellen, BB
MLA Citation
Pham, AT, Seto, R, Schönke, J, Joh, DY, Chilkoti, A, Fried, E, and Yellen, BB. "Crystallization kinetics of binary colloidal monolayers." Soft matter 12.37 (October 2016): 7735-7746.
PMID
27477956
Source
epmc
Published In
Soft Matter
Volume
12
Issue
37
Publish Date
2016
Start Page
7735
End Page
7746
DOI
10.1039/c6sm01072e

Developing Precisely Defined Drug-Loaded Nanoparticles by Ring-Opening Polymerization of a Paclitaxel Prodrug.

Nanoparticles with high paclitaxel (PTX) loading and low systemic toxicity are prepared in scalable and versatile manner via one-step ring-opening polymerization of a prodrug monomer consisting of PTX that is appended to a cyclic carbonate through a hydrolysable ester linker. Initiating this monomer from a hydrophilic macroinitiator results in an amphiphilic diblock copolymer that spontaneously self-assembles into well-defined nanoparticles with tunable size.

Authors
Liu, J; Pang, Y; Bhattacharyya, J; Liu, W; Weitzhandler, I; Li, X; Chilkoti, A
MLA Citation
Liu, J, Pang, Y, Bhattacharyya, J, Liu, W, Weitzhandler, I, Li, X, and Chilkoti, A. "Developing Precisely Defined Drug-Loaded Nanoparticles by Ring-Opening Polymerization of a Paclitaxel Prodrug." Advanced healthcare materials 5.15 (August 2016): 1868-1873.
PMID
27111757
Source
epmc
Published In
Advanced healthcare materials
Volume
5
Issue
15
Publish Date
2016
Start Page
1868
End Page
1873
DOI
10.1002/adhm.201600230

A Modular Method for the High-Yield Synthesis of Site-Specific Protein-Polymer Therapeutics.

A versatile method is described to engineer precisely defined protein/peptide-polymer therapeutics by a modular approach that consists of three steps: 1) fusion of a protein/peptide of interest with an elastin-like polypeptide that enables facile purification and high yields; 2) installation of a clickable group at the C terminus of the recombinant protein/peptide with almost complete conversion by enzyme-mediated ligation; and 3) attachment of a polymer by a click reaction with near-quantitative conversion. We demonstrate that this modular approach is applicable to various protein/peptide drugs and used it to conjugate them to structurally diverse water-soluble polymers that prolong the plasma circulation duration of these proteins. The protein/peptide-polymer conjugates exhibited significantly improved pharmacokinetics and therapeutic effects over the native protein/peptide upon administration to mice. The studies reported here provide a facile method for the synthesis of protein/peptide-polymer conjugates for therapeutic use and other applications.

Authors
Pang, Y; Liu, J; Qi, Y; Li, X; Chilkoti, A
MLA Citation
Pang, Y, Liu, J, Qi, Y, Li, X, and Chilkoti, A. "A Modular Method for the High-Yield Synthesis of Site-Specific Protein-Polymer Therapeutics." Angewandte Chemie (International ed. in English) 55.35 (August 2016): 10296-10300.
PMID
27439953
Source
epmc
Published In
Angewandte Chemie International Edition
Volume
55
Issue
35
Publish Date
2016
Start Page
10296
End Page
10300
DOI
10.1002/anie.201604661

Creating cellular patterns using genetically engineered, gold- and cell-binding polypeptides.

Patterning cells on material surfaces is an important tool for the study of fundamental cell biology, tissue engineering, and cell-based bioassays. Here, the authors report a simple approach to pattern cells on gold patterned silicon substrates with high precision, fidelity, and stability. Cell patterning is achieved by exploiting adsorbed biopolymer orientation to either enhance (gold regions) or impede (silicon oxide regions) cell adhesion at particular locations on the patterned surface. Genetic incorporation of gold binding domains enables C-terminal chemisorption of polypeptides onto gold regions with enhanced accessibility of N-terminal cell binding domains. In contrast, the orientation of polypeptides adsorbed on the silicon oxide regions limit the accessibility of the cell binding domains. The dissimilar accessibility of cell binding domains on the gold and silicon oxide regions directs the cell adhesion in a spatially controlled manner in serum-free medium, leading to the formation of well-defined cellular patterns. The cells are confined within the polypeptide-modified gold regions and are viable for eight weeks, suggesting that bioactive polypeptide modified surfaces are suitable for long-term maintenance of patterned cells. This study demonstrates an innovative surface-engineering approach for cell patterning by exploiting distinct ligand accessibility on heterogeneous surfaces.

Authors
Li, L; Mo, C-K; Chilkoti, A; Lopez, GP; Carroll, NJ
MLA Citation
Li, L, Mo, C-K, Chilkoti, A, Lopez, GP, and Carroll, NJ. "Creating cellular patterns using genetically engineered, gold- and cell-binding polypeptides." Biointerphases 11.2 (June 27, 2016): 021009-.
PMID
27233531
Source
epmc
Published In
Biointerphases: an open access journal for the biomaterials interface community
Volume
11
Issue
2
Publish Date
2016
Start Page
021009
DOI
10.1116/1.4952452

Magnetophoretic Conductors and Diodes in a 3D Magnetic Field.

We demonstrate magnetophoretic conductor tracks that can transport single magnetized beads and magnetically labeled single cells in a 3-dimensional time-varying magnetic field. The vertical field bias, in addition to the in-plane rotating field, has the advantage of reducing the attraction between particles, which inhibits the formation of particle clusters. However, the inclusion of a vertical field requires the re-design of magnetic track geometries which can transport magnetized objects across the substrate. Following insights from magnetic bubble technology, we found that successful magnetic conductor geometries defined in soft magnetic materials must be composed of alternating sections of positive and negative curvature. In addition to the previously studied magnetic tracks taken from the magnetic bubble literature, a drop-shape pattern was found to be even more adept at transporting small magnetic beads and single cells. Symmetric patterns are shown to achieve bi-directional conduction, whereas asymmetric patterns achieve unidirectional conduction. These designs represent the electrical circuit corollaries of the conductor and diode, respectively. Finally, we demonstrate biological applications in transporting single cells and in the size based separation of magnetic particles.

Authors
Abedini-Nassab, R; Joh, DY; Van Heest, M; Baker, C; Chilkoti, A; Murdoch, DM; Yellen, BB
MLA Citation
Abedini-Nassab, R, Joh, DY, Van Heest, M, Baker, C, Chilkoti, A, Murdoch, DM, and Yellen, BB. "Magnetophoretic Conductors and Diodes in a 3D Magnetic Field." Advanced functional materials 26.22 (June 2016): 4026-4034.
PMID
27418922
Source
epmc
Published In
Advanced Functional Materials
Volume
26
Issue
22
Publish Date
2016
Start Page
4026
End Page
4034

Combinatorial codon scrambling enables scalable gene synthesis and amplification of repetitive proteins.

Most genes are synthesized using seamless assembly methods that rely on the polymerase chain reaction (PCR). However, PCR of genes encoding repetitive proteins either fails or generates nonspecific products. Motivated by the need to efficiently generate new protein polymers through high-throughput gene synthesis, here we report a codon-scrambling algorithm that enables the PCR-based gene synthesis of repetitive proteins by exploiting the codon redundancy of amino acids and finding the least-repetitive synonymous gene sequence. We also show that the codon-scrambling problem is analogous to the well-known travelling salesman problem, and obtain an exact solution to it by using De Bruijn graphs and a modern mixed integer linear programme solver. As experimental proof of the utility of this approach, we use it to optimize the synthetic genes for 19 repetitive proteins, and show that the gene fragments are amenable to PCR-based gene assembly and recombinant expression.

Authors
Tang, NC; Chilkoti, A
MLA Citation
Tang, NC, and Chilkoti, A. "Combinatorial codon scrambling enables scalable gene synthesis and amplification of repetitive proteins." Nature materials 15.4 (April 2016): 419-424.
PMID
26726995
Source
epmc
Published In
Nature Materials
Volume
15
Issue
4
Publish Date
2016
Start Page
419
End Page
424
DOI
10.1038/nmat4521

Injectable polypeptide micelles that form radiation crosslinked hydrogels in situ for intratumoral radiotherapy.

Intratumoral radiation therapy - 'brachytherapy' - is a highly effective treatment for solid tumors, particularly prostate cancer. Current titanium seed implants, however, are permanent and are limited in clinical application to indolent malignancies of low- to intermediate-risk. Attempts to develop polymeric alternatives, however, have been plagued by poor retention and off-target toxicity due to degradation. Herein, we report on a new approach whereby thermally sensitive micelles composed of an elastin-like polypeptide (ELP) are labeled with the radionuclide (131)I to form an in situ hydrogel that is stabilized by two independent mechanisms: first, body heat triggers the radioactive ELP micelles to rapidly phase transition into an insoluble, viscous coacervate in under 2 min; second, the high energy β-emissions of (131)I further stabilize the depot by introducing crosslinks within the ELP depot over 24h. These injectable brachytherapy hydrogels were used to treat two aggressive orthotopic tumor models in athymic nude mice: a human PC-3 M-luc-C6 prostate tumor and a human BxPc3-luc2 pancreatic tumor model. The ELP depots retained greater than 52% and 70% of their radioactivity through 60 days in the prostate and pancreatic tumors with no appreciable radioactive accumulation (≤ 0.1% ID) in off-target tissues after 72h. The (131)I-ELP depots achieved >95% tumor regression in the prostate tumors (n=8); with a median survival of more than 60 days compared to 12 days for control mice. For the pancreatic tumors, ELP brachytherapy (n=6) induced significant growth inhibition (p=0.001, ANOVA) and enhanced median survival to 27 days over controls.

Authors
Schaal, JL; Li, X; Mastria, E; Bhattacharyya, J; Zalutsky, MR; Chilkoti, A; Liu, W
MLA Citation
Schaal, JL, Li, X, Mastria, E, Bhattacharyya, J, Zalutsky, MR, Chilkoti, A, and Liu, W. "Injectable polypeptide micelles that form radiation crosslinked hydrogels in situ for intratumoral radiotherapy." Journal of controlled release : official journal of the Controlled Release Society 228 (April 2016): 58-66.
PMID
26928529
Source
epmc
Published In
Journal of Controlled Release
Volume
228
Publish Date
2016
Start Page
58
End Page
66
DOI
10.1016/j.jconrel.2016.02.040

Maleimide-Functionalized Poly(2-Oxazoline)s and Their Conjugation to Elastin-Like Polypeptides.

The design of drug delivery systems capable of efficiently delivering poorly soluble drugs to target sites still remains a major challenge. Such materials require several different functionalities; typically, these materials should be biodegradable and nontoxic, nonimmunogenic, responsive to their environment, and soluble in aqueous solution while retaining the ability to solubilize hydrophobic drugs. Here, a polypeptide-polymer hybrid of elastin-like polypeptides (ELPs) and poly(2-oxazoline)s (POx) is reported. This paper describes the chemical synthesis, physical characteristics, and drug loading potential of these novel hybrid macromolecules. A novel method is introduced for terminal functionalization of POx with protected maleimide moieties. Following recovery of the maleimide group via a retro Diels-Alder reaction, the consecutive Michael addition of thiol-functionalized ELPs yields the desired protein-polymer conjugate. These conjugates form nanoparticles in aqueous solution capable of solubilizing the anti-cancer drug paclitaxel with up to 8 wt% loading.

Authors
Nawroth, JF; McDaniel, JR; Chilkoti, A; Jordan, R; Luxenhofer, R
MLA Citation
Nawroth, JF, McDaniel, JR, Chilkoti, A, Jordan, R, and Luxenhofer, R. "Maleimide-Functionalized Poly(2-Oxazoline)s and Their Conjugation to Elastin-Like Polypeptides." Macromolecular bioscience 16.3 (March 2016): 322-333.
PMID
26756582
Source
epmc
Published In
Macromolecular Bioscience
Volume
16
Issue
3
Publish Date
2016
Start Page
322
End Page
333
DOI
10.1002/mabi.201500376

Spatiotemporally photoradiation-controlled intratumoral depot for combination of brachytherapy and photodynamic therapy for solid tumor.

In an attempt to spatiotemporally control both tumor retention and the coverage of anticancer agents, we developed a photoradiation-controlled intratumoral depot (PRCITD) driven by convection enhanced delivery (CED). This intratumoral depot consists of recombinant elastin-like polypeptide (ELP) containing periodic cysteine residues and is conjugated with a photosensitizer, chlorin-e6 (Ce6) at the N-terminus of the ELP. We hypothesized that this cysteine-containing ELP (cELP) can be readily crosslinked through disulfide bonds upon exposure to oxidative agents, specifically the singlet oxygen produced during photodynamic stimulation. Upon intratumoral injection, CED drives the distribution of the soluble polypeptide freely throughout the tumor interstitium. Formation and retention of the depot was monitored using fluorescence molecular tomography imaging. When imaging shows that the polypeptide has distributed throughout the entire tumor, 660-nm light is applied externally at the tumor site. This photo-radiation wavelength excites Ce6 and generates reactive oxygen species (ROS) in the presence of oxygen. The ROS induce in situ disulfide crosslinking of the cysteine thiols, stabilizing the ELP biopolymer into a stable therapeutic depot. Our results demonstrate that this ELP design effectively forms a hydrogel both in vitro and in vivo. These depots exhibit high stability in subcutaneous tumor xenografts in nude mice and significantly improved intratumoral retention compared to controls without crosslinking, as seen by fluorescent imaging and iodine-125 radiotracer studies. The photodynamic therapy provided by the PRCITD was found to cause significant tumor inhibition in a Ce6 dose dependent manner. Additionally, the combination of PDT and intratumoral radionuclide therapy co-delivered by PRCITD provided a greater antitumor effect than either monotherapy alone. These results suggest that the PRCITD could provide a stable platform for delivering synergistic, anti-cancer drug depots.

Authors
Mukerji, R; Schaal, J; Li, X; Bhattacharyya, J; Asai, D; Zalutsky, MR; Chilkoti, A; Liu, W
MLA Citation
Mukerji, R, Schaal, J, Li, X, Bhattacharyya, J, Asai, D, Zalutsky, MR, Chilkoti, A, and Liu, W. "Spatiotemporally photoradiation-controlled intratumoral depot for combination of brachytherapy and photodynamic therapy for solid tumor." Biomaterials 79 (February 2016): 79-87.
PMID
26702586
Source
epmc
Published In
Biomaterials
Volume
79
Publish Date
2016
Start Page
79
End Page
87
DOI
10.1016/j.biomaterials.2015.11.064

Site-Specific Zwitterionic Polymer Conjugates of a Protein Have Long Plasma Circulation.

Many proteins suffer from suboptimal pharmacokinetics (PK) that limit their utility as drugs. The efficient synthesis of polymer conjugates of protein drugs with tunable PK to optimize their in vivo efficacy is hence critical. We report here the first study of the in vivo behavior of a site-specific conjugate of a zwitterionic polymer and a protein. To synthesize the conjugate, we first installed an initiator for atom-transfer radical polymerization (ATRP) at the N terminus of myoglobin (Mb-N-Br). Subsequently, in situ ATRP was carried out in aqueous buffer to grow an amine-functionalized polymer from Mb-N-Br. The cationic polymer was further derivatized to two zwitterionic polymers by treating the amine groups of the cationic polymer with iodoacetic acid to obtain poly(carboxybetaine methacrylate) with a one-carbon spacer (PCBMA; C1 ), and sequentially with 3-iodopropionic acid and iodoacetic acid to obtain PCBMA(mix) with a mixture of C1 and C2 spacers. The Mb-N-PCBMA polymer conjugates had a longer in vivo plasma half-life than a PEG-like comb polymer conjugate of similar molecular weights (MW). The structure of the zwitterion plays a role in controlling the in vivo behavior of the conjugate, as the PCBMA conjugate with a C1 spacer had significantly longer plasma circulation than the conjugate with a mixture of C1 and C2 spacers.

Authors
Bhattacharjee, S; Liu, W; Wang, W-H; Weitzhandler, I; Li, X; Qi, Y; Liu, J; Pang, Y; Hunt, DF; Chilkoti, A
MLA Citation
Bhattacharjee, S, Liu, W, Wang, W-H, Weitzhandler, I, Li, X, Qi, Y, Liu, J, Pang, Y, Hunt, DF, and Chilkoti, A. "Site-Specific Zwitterionic Polymer Conjugates of a Protein Have Long Plasma Circulation." Chembiochem : a European journal of chemical biology 16.17 (November 2015): 2451-2455.
PMID
26481301
Source
epmc
Published In
Chembiochem
Volume
16
Issue
17
Publish Date
2015
Start Page
2451
End Page
2455
DOI
10.1002/cbic.201500439

Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers.

Proteins and synthetic polymers that undergo aqueous phase transitions mediate self-assembly in nature and in man-made material systems. Yet little is known about how the phase behaviour of a protein is encoded in its amino acid sequence. Here, by synthesizing intrinsically disordered, repeat proteins to test motifs that we hypothesized would encode phase behaviour, we show that the proteins can be designed to exhibit tunable lower or upper critical solution temperature (LCST and UCST, respectively) transitions in physiological solutions. We also show that mutation of key residues at the repeat level abolishes phase behaviour or encodes an orthogonal transition. Furthermore, we provide heuristics to identify, at the proteome level, proteins that might exhibit phase behaviour and to design novel protein polymers consisting of biologically active peptide repeats that exhibit LCST or UCST transitions. These findings set the foundation for the prediction and encoding of phase behaviour at the sequence level.

Authors
Quiroz, FG; Chilkoti, A
MLA Citation
Quiroz, FG, and Chilkoti, A. "Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers." Nature materials 14.11 (November 2015): 1164-1171.
PMID
26390327
Source
epmc
Published In
Nature Materials
Volume
14
Issue
11
Publish Date
2015
Start Page
1164
End Page
1171
DOI
10.1038/nmat4418

Prediction of solvent-induced morphological changes of polyelectrolyte diblock copolymer micelles.

Self-assembly processes of polyelectrolyte block copolymers are ubiquitous in industrial and biological processes; understanding their physical properties can also provide insights into the design of polyelectrolyte materials with novel and tailored properties. Here, we report systematic analysis on how the ionic strength of the solvent and the length of the polyelectrolyte block affect the self-assembly and morphology of the polyelectrolyte block copolymer materials by constructing a salt-dependent morphological phase diagram using an implicit solvent ionic strength (ISIS) method for dissipative particle dynamics (DPD) simulations. This diagram permits the determination of the conditions for the morphological transition into a specific shape, namely vesicles or lamellar aggregates, wormlike/cylindrical micelles, and spherical micelles. The scaling behavior for the size of spherical micelles is predicted, in terms of radius of gyration (R(g,m)) and thickness of corona (Hcorona), as a function of solvent ionic strength (c(s)) and polyelectrolyte length (NA), which are R(g,m) ∼ c(s)(-0.06)N(A)(0.54) and Hcorona ∼ c(s)(-0.11)N(A)(0.75). The simulation results were corroborated through AFM and static light scattering measurements on the example of the self-assembly of monodisperse, single-stranded DNA block-copolynucleotides (polyT50-b-F-dUTP). Overall, we were able to predict the salt-responsive morphology of polyelectrolyte materials in aqueous solution and show that a spherical-cylindrical-lamellar change in morphology can be obtained through an increase in solvent ionic strength or a decrease of polyelectrolyte length.

Authors
Li, NK; Fuss, WH; Tang, L; Gu, R; Chilkoti, A; Zauscher, S; Yingling, YG
MLA Citation
Li, NK, Fuss, WH, Tang, L, Gu, R, Chilkoti, A, Zauscher, S, and Yingling, YG. "Prediction of solvent-induced morphological changes of polyelectrolyte diblock copolymer micelles." Soft matter 11.42 (November 2015): 8236-8245.
PMID
26315065
Source
epmc
Published In
Soft Matter
Volume
11
Issue
42
Publish Date
2015
Start Page
8236
End Page
8245
DOI
10.1039/c5sm01742d

Characterizing the Switching Thresholds of Magnetophoretic Transistors.

The switching thresholds of magnetophoretic transistors for sorting cells in microfluidic environments are characterized. The transistor operating conditions require short 20-30 mA pulses of electrical current. By demonstrating both attractive and repulsive transistor modes, a single transistor architecture is used to implement the full write cycle for importing and exporting single cells in specified array sites.

Authors
Abedini-Nassab, R; Joh, DY; Van Heest, MA; Yi, JS; Baker, C; Taherifard, Z; Margolis, DM; Garcia, JV; Chilkoti, A; Murdoch, DM; Yellen, BB
MLA Citation
Abedini-Nassab, R, Joh, DY, Van Heest, MA, Yi, JS, Baker, C, Taherifard, Z, Margolis, DM, Garcia, JV, Chilkoti, A, Murdoch, DM, and Yellen, BB. "Characterizing the Switching Thresholds of Magnetophoretic Transistors." Advanced materials (Deerfield Beach, Fla.) 27.40 (October 2015): 6176-6180.
Website
http://hdl.handle.net/10161/10827
PMID
26349853
Source
epmc
Published In
Advanced Materials
Volume
27
Issue
40
Publish Date
2015
Start Page
6176
End Page
6180
DOI
10.1002/adma.201502352

Protein-polymer conjugation-moving beyond PEGylation.

In this review, we summarize-from a materials science perspective-the current state of the field of polymer conjugates of peptide and protein drugs, with a focus on polymers that have been developed as alternatives to the current gold standard, poly(ethylene glycol) (PEG). PEGylation, or the covalent conjugation of PEG to biological therapeutics to improve their therapeutic efficacy by increasing their circulation half-lives and stability, has been the gold standard in the pharmaceutical industry for several decades. After years of research and development, the limitations of PEG, specifically its non-degradability and immunogenicity have become increasingly apparent. While PEG is still currently the best polymer available with the longest clinical track record, extensive research is underway to develop alternative materials in an effort to address these limitations of PEG. Many of these alternative materials have shown promise, though most of them are still in an early stage of development and their in vivo distribution, mechanism of degradation, route of elimination and immunogenicity have not been investigated to a similar extent as for PEG. Thus, further in-depth in vivo testing is essential to validate whether any of the alternative materials discussed in this review qualify as a replacement for PEG.

Authors
Qi, Y; Chilkoti, A
MLA Citation
Qi, Y, and Chilkoti, A. "Protein-polymer conjugation-moving beyond PEGylation." Current opinion in chemical biology 28 (October 2015): 181-193. (Review)
PMID
26356631
Source
epmc
Published In
Current Opinion in Chemical Biology
Volume
28
Publish Date
2015
Start Page
181
End Page
193
DOI
10.1016/j.cbpa.2015.08.009

Structural Evolution of a Stimulus-Responsive Diblock Polypeptide Micelle by Temperature Tunable Compaction of its Core

Authors
Garanger, E; MacEwan, SR; Sandre, O; Brûlet, A; Bataille, L; Chilkoti, A; Lecommandoux, S
MLA Citation
Garanger, E, MacEwan, SR, Sandre, O, Brûlet, A, Bataille, L, Chilkoti, A, and Lecommandoux, S. "Structural Evolution of a Stimulus-Responsive Diblock Polypeptide Micelle by Temperature Tunable Compaction of its Core." Macromolecules 48.18 (September 22, 2015): 6617-6627.
Source
crossref
Published In
Macromolecules
Volume
48
Issue
18
Publish Date
2015
Start Page
6617
End Page
6627
DOI
10.1021/acs.macromol.5b01371

Elastin-like polypeptides as models of intrinsically disordered proteins.

Elastin-like polypeptides (ELPs) are a class of stimuli-responsive biopolymers inspired by the intrinsically disordered domains of tropoelastin that are composed of repeats of the VPGXG pentapeptide motif, where X is a "guest residue". They undergo a reversible, thermally triggered lower critical solution temperature (LCST) phase transition, which has been utilized for a variety of applications including protein purification, affinity capture, immunoassays, and drug delivery. ELPs have been extensively studied as protein polymers and as biomaterials, but their relationship to other disordered proteins has heretofore not been established. The biophysical properties of ELPs that lend them their unique material behavior are similar to the properties of many intrinsically disordered proteins (IDP). Their low sequence complexity, phase behavior, and elastic properties make them an interesting "minimal" artificial IDP, and the study of ELPs can hence provide insights into the behavior of other more complex IDPs. Motivated by this emerging realization of the similarities between ELPs and IDPs, this review discusses the biophysical properties of ELPs, their biomedical utility, and their relationship to other disordered polypeptide sequences.

Authors
Roberts, S; Dzuricky, M; Chilkoti, A
MLA Citation
Roberts, S, Dzuricky, M, and Chilkoti, A. "Elastin-like polypeptides as models of intrinsically disordered proteins." FEBS letters 589.19 Pt A (September 2015): 2477-2486. (Review)
PMID
26325592
Source
epmc
Published In
FEBS Letters
Volume
589
Issue
19 Pt A
Publish Date
2015
Start Page
2477
End Page
2486
DOI
10.1016/j.febslet.2015.08.029

A paclitaxel-loaded recombinant polypeptide nanoparticle outperforms Abraxane in multiple murine cancer models.

Packaging clinically relevant hydrophobic drugs into a self-assembled nanoparticle can improve their aqueous solubility, plasma half-life, tumour-specific uptake and therapeutic potential. To this end, here we conjugated paclitaxel (PTX) to recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into ∼60 nm near-monodisperse nanoparticles that increased the systemic exposure of PTX by sevenfold compared with free drug and twofold compared with the Food and Drug Administration-approved taxane nanoformulation (Abraxane). The tumour uptake of the CP-PTX nanoparticle was fivefold greater than free drug and twofold greater than Abraxane. In a murine cancer model of human triple-negative breast cancer and prostate cancer, CP-PTX induced near-complete tumour regression after a single dose in both tumour models, whereas at the same dose, no mice treated with Abraxane survived for >80 days (breast) and 60 days (prostate), respectively. These results show that a molecularly engineered nanoparticle with precisely engineered design features outperforms Abraxane, the current gold standard for PTX delivery.

Authors
Bhattacharyya, J; Bellucci, JJ; Weitzhandler, I; McDaniel, JR; Spasojevic, I; Li, X; Lin, C-C; Chi, J-TA; Chilkoti, A
MLA Citation
Bhattacharyya, J, Bellucci, JJ, Weitzhandler, I, McDaniel, JR, Spasojevic, I, Li, X, Lin, C-C, Chi, J-TA, and Chilkoti, A. "A paclitaxel-loaded recombinant polypeptide nanoparticle outperforms Abraxane in multiple murine cancer models." Nature communications 6 (August 4, 2015): 7939-.
PMID
26239362
Source
epmc
Published In
Nature Communications
Volume
6
Publish Date
2015
Start Page
7939
DOI
10.1038/ncomms8939

Bio-inspired synthesis of hybrid silica nanoparticles templated from elastin-like polypeptide micelles.

The programmed self-assembly of block copolymers into higher order nanoscale structures offers many attractive attributes for the development of new nanomaterials for numerous applications including drug delivery and biosensing. The incorporation of biomimetic silaffin peptides in these block copolymers enables the formation of hybrid organic-inorganic materials, which can potentially enhance the utility and stability of self-assembled nanostructures. We demonstrate the design, synthesis and characterization of amphiphilic elastin-like polypeptide (ELP) diblock copolymers that undergo temperature-triggered self-assembly into well-defined spherical micelles. Genetically encoded incorporation of the silaffin R5 peptide at the hydrophilic terminus of the diblock ELP leads to presentation of the silaffin R5 peptide on the coronae of the micelles, which results in localized condensation of silica and the formation of near-monodisperse, discrete, sub-100 nm diameter hybrid ELP-silica particles. This synthesis method, can be carried out under mild reaction conditions suitable for bioactive materials, and will serve as the basis for the development and application of functional nanomaterials. Beyond silicification, the general strategies described herein may also be adapted for the synthesis of other biohybrid nanomaterials as well.

Authors
Han, W; MacEwan, SR; Chilkoti, A; López, GP
MLA Citation
Han, W, MacEwan, SR, Chilkoti, A, and López, GP. "Bio-inspired synthesis of hybrid silica nanoparticles templated from elastin-like polypeptide micelles." Nanoscale 7.28 (July 2015): 12038-12044.
PMID
26114664
Source
epmc
Published In
Nanoscale
Volume
7
Issue
28
Publish Date
2015
Start Page
12038
End Page
12044
DOI
10.1039/c5nr01407g

Elastin-like Polypeptide Diblock Copolymers Self-Assemble into Weak Micelles.

The self-assembly of synthetic diblock copolymers has been extensively studied experimentally and theoretically. In contrast, self-assembly of polypeptide diblock copolymers has so far been mostly studied experimentally. We discovered that the theory developed for synthetic diblock copolymer does not fully explain the self-assembly of elastin-like polypeptide diblock copolymers, leading us to generalize the theory to make it applicable for these polypeptides. We demonstrated that elastin-like polypeptide diblocks self-assemble into weak micelles with dense cores and almost unstretched coronas, a state not previously observed for synthetic diblock copolymers. Weak micelles form if the surface tension at the core-corona interface is low compared to that expected of a micelle with a dense core. The predictions of the theory of weak micelles for the critical micelle temperature, hydrodynamic radius, and aggregation number of elastin-like polypeptide diblocks are in reasonable agreement with the experimentally measured values. The unique and unprecedented control of amphiphilicity in these recombinant peptide polymers reveals a new micellar state that has not been previously observed in synthetic diblock copolymer systems.

Authors
Hassouneh, W; Zhulina, EB; Chilkoti, A; Rubinstein, M
MLA Citation
Hassouneh, W, Zhulina, EB, Chilkoti, A, and Rubinstein, M. "Elastin-like Polypeptide Diblock Copolymers Self-Assemble into Weak Micelles." Macromolecules 48.12 (June 11, 2015): 4183-4195.
PMID
27065492
Source
epmc
Published In
Macromolecules
Volume
48
Issue
12
Publish Date
2015
Start Page
4183
End Page
4195

Bioinspired reversibly cross-linked hydrogels comprising polypeptide micelles exhibit enhanced mechanical properties

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.Noncovalently cross-linked networks are attractive hydrogel platforms because of their facile fabrication, dynamic behavior, and biocompatibility. The majority of noncovalently cross-linked hydrogels, however, exhibits poor mechanical properties, which significantly limit their utility in load bearing applications. To address this limitation, hydrogels are presented composed of micelles created from genetically engineered, amphiphilic, elastin-like polypeptides that contain a relatively large hydrophobic block and a hydrophilic terminus that can be cross-linked through metal ion coordination. To create the hydrogels, heat is firstly used to trigger the self-assembly of the polypeptides into monodisperse micelles that display transition metal coordination motifs on their coronae, and subsequently cross-link the micelles by adding zinc ions. These hydrogels exhibit hierarchical structure, are stable over a large temperature range, and exhibit tunable stiffness, self-healing, and fatigue resistance. Gels with polypeptide concentration of 10%, w/v, and higher show storage moduli of ≈1 MPa from frequency sweep tests and exhibit self-healing within minutes. These reversibly cross-linked, hierarchical hydrogels with enhanced mechanical properties have potential utility in a variety of biomedical applications.

Authors
Ghoorchian, A; Simon, JR; Bharti, B; Han, W; Zhao, X; Chilkoti, A; Lõpez, GP
MLA Citation
Ghoorchian, A, Simon, JR, Bharti, B, Han, W, Zhao, X, Chilkoti, A, and Lõpez, GP. "Bioinspired reversibly cross-linked hydrogels comprising polypeptide micelles exhibit enhanced mechanical properties." Advanced Functional Materials 25.21 (June 1, 2015): 3122-3130.
Source
scopus
Published In
Advanced Functional Materials
Volume
25
Issue
21
Publish Date
2015
Start Page
3122
End Page
3130
DOI
10.1002/adfm.201500699

Doxorubicin-conjugated polypeptide nanoparticles inhibit metastasis in two murine models of carcinoma.

Drug delivery vehicles are often assessed for their ability to control primary tumor growth, but the outcome of cancer treatment depends on controlling or inhibiting metastasis. Therefore, we studied the efficacy of our genetically encoded polypeptide nanoparticle for doxorubicin delivery (CP-Dox) in the syngeneic metastatic murine models 4T1 and Lewis lung carcinoma. We found that our nanoparticle formulation increased the half-life, maximum tolerated dose, and tumor accumulation of doxorubicin. When drug treatment was combined with primary tumor resection, greater than 60% of the mice were cured in both the 4T1 and Lewis lung carcinoma models compared to 20% treated with free drug. Mechanistic studies suggest that metastasis inhibition and survival increase were achieved by preventing the dissemination of viable tumor cells from the primary tumor.

Authors
Mastria, EM; Chen, M; McDaniel, JR; Li, X; Hyun, J; Dewhirst, MW; Chilkoti, A
MLA Citation
Mastria, EM, Chen, M, McDaniel, JR, Li, X, Hyun, J, Dewhirst, MW, and Chilkoti, A. "Doxorubicin-conjugated polypeptide nanoparticles inhibit metastasis in two murine models of carcinoma." Journal of controlled release : official journal of the Controlled Release Society 208 (June 2015): 52-58.
PMID
25637704
Source
epmc
Published In
Journal of Controlled Release
Volume
208
Publish Date
2015
Start Page
52
End Page
58
DOI
10.1016/j.jconrel.2015.01.033

Bioinspired Reversibly Cross-linked Hydrogels Comprising Polypeptide Micelles Exhibit Enhanced Mechanical Properties

Authors
Ghoorchian, A; Simon, JR; Bharti, B; Han, W; Zhao, X; Chilkoti, A; López, GP
MLA Citation
Ghoorchian, A, Simon, JR, Bharti, B, Han, W, Zhao, X, Chilkoti, A, and López, GP. "Bioinspired Reversibly Cross-linked Hydrogels Comprising Polypeptide Micelles Exhibit Enhanced Mechanical Properties." Advanced Functional Materials 25.21 (June 2015): 3122-3130.
Source
crossref
Published In
Advanced Functional Materials
Volume
25
Issue
21
Publish Date
2015
Start Page
3122
End Page
3130
DOI
10.1002/adfm.201500699

Sustained intra-articular delivery of IL-1RA from a thermally-responsive elastin-like polypeptide as a therapy for post-traumatic arthritis.

Post-traumatic arthritis (PTA) is a rapidly progressive form of arthritis that develops due to joint injury, including articular fracture. Current treatments are limited to surgical restoration and stabilization of the joint; however, evidence suggests that PTA progression is mediated by the upregulation of pro-inflammatory cytokines, such as interleukin-1 (IL-1) or tumor necrosis factor-α (TNF-α). Although these cytokines provide potential therapeutic targets for PTA, intra-articular injections of anti-cytokine therapies have proven difficult due to rapid clearance from the joint space. In this study, we examined the ability of a cross-linked elastin-like polypeptide (xELP) drug depot to provide sustained intra-articular delivery of IL-1 and TNF-α inhibitors as a beneficial therapy. Mice sustained a closed intra-articular tibial plateau fracture; treatment groups received a single intra-articular injection of drug encapsulated in xELP. Arthritic changes were assessed 4 and 8 weeks after fracture. Inhibition of IL-1 significantly reduced the severity of cartilage degeneration and synovitis. Inhibition of TNF-α alone or with IL-1 led to deleterious effects in bone morphology, articular cartilage degeneration, and synovitis. These findings suggest that IL-1 plays a critical role in the pathogenesis of PTA following articular fracture, and sustained intra-articular cytokine inhibition may provide a therapeutic approach for reducing or preventing joint degeneration following trauma.

Authors
Kimmerling, KA; Furman, BD; Mangiapani, DS; Moverman, MA; Sinclair, SM; Huebner, JL; Chilkoti, A; Kraus, VB; Setton, LA; Guilak, F; Olson, SA
MLA Citation
Kimmerling, KA, Furman, BD, Mangiapani, DS, Moverman, MA, Sinclair, SM, Huebner, JL, Chilkoti, A, Kraus, VB, Setton, LA, Guilak, F, and Olson, SA. "Sustained intra-articular delivery of IL-1RA from a thermally-responsive elastin-like polypeptide as a therapy for post-traumatic arthritis." European cells & materials 29 (January 31, 2015): 124-139.
PMID
25636786
Source
epmc
Published In
European cells & materials
Volume
29
Publish Date
2015
Start Page
124
End Page
139

A noncanonical function of sortase enables site-specific conjugation of small molecules to lysine residues in proteins.

We provide the first demonstration that isopeptide ligation, a noncanonical activity of the enzyme sortase A, can be used to modify recombinant proteins. This reaction was used in vitro to conjugate small molecules to a peptide, an engineered targeting protein, and a full-length monoclonal antibody with an exquisite level of control over the site of conjugation. Attachment to the protein substrate occurred exclusively through isopeptide bonds at a lysine ε-amino group within a specific amino acid sequence. This reaction allows more than one molecule to be site-specifically conjugated to a protein at internal sites, thereby overcoming significant limitations of the canonical native peptide ligation reaction catalyzed by sortase A. Our method provides a unique chemical ligation procedure that is orthogonal to existing methods, supplying a new method to site-specifically modify lysine residues that will be a valuable addition to the protein conjugation toolbox.

Authors
Bellucci, JJ; Bhattacharyya, J; Chilkoti, A
MLA Citation
Bellucci, JJ, Bhattacharyya, J, and Chilkoti, A. "A noncanonical function of sortase enables site-specific conjugation of small molecules to lysine residues in proteins." Angewandte Chemie (International ed. in English) 54.2 (January 2015): 441-445.
PMID
25363491
Source
epmc
Published In
Angewandte Chemie International Edition
Volume
54
Issue
2
Publish Date
2015
Start Page
441
End Page
445
DOI
10.1002/anie.201408126

Ring-opening polymerization of prodrugs: a versatile approach to prepare well-defined drug-loaded nanoparticles.

The synthesis of polymer-drug conjugates from prodrug monomers consisting of a cyclic polymerizable group that is appended to a drug through a cleavable linker is achieved by organocatalyzed ring-opening polymerization. The monomers polymerize into well-defined polymer prodrugs that are designed to self-assemble into nanoparticles and release the drug in response to a physiologically relevant stimulus. This method is compatible with structurally diverse drugs and allows different drugs to be copolymerized with quantitative conversion of the monomers. The drug loading can be controlled by adjusting the monomer(s)/initiator feed ratio and drug release can be encoded into the polymer by the choice of linker. Initiating these monomers from a poly(ethylene glycol) macroinitiator results in amphiphilic diblock copolymers that spontaneously self-assemble into micelles with a long plasma circulation, which is useful for systemic therapy.

Authors
Liu, J; Liu, W; Weitzhandler, I; Bhattacharyya, J; Li, X; Wang, J; Qi, Y; Bhattacharjee, S; Chilkoti, A
MLA Citation
Liu, J, Liu, W, Weitzhandler, I, Bhattacharyya, J, Li, X, Wang, J, Qi, Y, Bhattacharjee, S, and Chilkoti, A. "Ring-opening polymerization of prodrugs: a versatile approach to prepare well-defined drug-loaded nanoparticles." Angewandte Chemie (International ed. in English) 54.3 (January 2015): 1002-1006.
PMID
25427831
Source
epmc
Published In
Angewandte Chemie International Edition
Volume
54
Issue
3
Publish Date
2015
Start Page
1002
End Page
1006
DOI
10.1002/anie.201409293

Noncanonical self-assembly of highly asymmetric genetically encoded polypeptide amphiphiles into cylindrical micelles.

Elastin-like polypeptides (ELPs) are a class of biopolymers consisting of the pentameric repeat (VPGαG)n based on the sequence of mammalian tropoelastin that display a thermally induced soluble-to-insoluble phase transition in aqueous solution. We have discovered a remarkably simple approach to driving the spontaneous self-assembly of high molecular weight ELPs into nanostructures by genetically fusing a short 1.5 kDa (XGy)z assembly domain to one end of the ELP. Classical theories of self-assembly based on the geometric mass balance of hydrophilic and hydrophobic block copolymers suggest that these highly asymmetric polypeptides should form spherical micelles. Surprisingly, when sufficiently hydrophobic amino acids (X) are presented in a periodic sequence such as (FGG)8 or (YG)8, these highly asymmetric polypeptides self-assemble into cylindrical micelles whose length can be tuned by the sequence of the morphogenic tag. These nanostructures were characterized by light scattering, tunable resistive pulse sensing, fluorescence spectrophotometry, and thermal turbidimetry, as well as by cryogenic transmission electron microscopy (cryo-TEM) and small-angle neutron scattering (SANS). These short assembly domains provide a facile strategy to control the size, shape, and stability of stimuli responsive polypeptide nanostructures.

Authors
McDaniel, JR; Weitzhandler, I; Prevost, S; Vargo, KB; Appavou, M-S; Hammer, DA; Gradzielski, M; Chilkoti, A
MLA Citation
McDaniel, JR, Weitzhandler, I, Prevost, S, Vargo, KB, Appavou, M-S, Hammer, DA, Gradzielski, M, and Chilkoti, A. "Noncanonical self-assembly of highly asymmetric genetically encoded polypeptide amphiphiles into cylindrical micelles." Nano letters 14.11 (November 2014): 6590-6598.
PMID
25268037
Source
epmc
Published In
Nano Letters
Volume
14
Issue
11
Publish Date
2014
Start Page
6590
End Page
6598
DOI
10.1021/nl503221p

Abstract 4481: Genetically encoded chimeric polypeptide nanoparticles for gemcitabine delivery to solid tumors

Authors
Bhattacharyya, J; Chilkoti, A
MLA Citation
Bhattacharyya, J, and Chilkoti, A. "Abstract 4481: Genetically encoded chimeric polypeptide nanoparticles for gemcitabine delivery to solid tumors." October 1, 2014.
Source
crossref
Published In
Cancer Research
Volume
74
Issue
19 Supplement
Publish Date
2014
Start Page
4481
End Page
4481
DOI
10.1158/1538-7445.AM2014-4481

Abstract 4583: In situ photocontrolled intratumoral depot for combined photodynamic therapy and brachytherapy for solid tumor

Authors
Liu, W; Mukerji, R; Li, X; Schaal, J; Bhattacharyya, J; Zalutsky, M; Chilkoti, A
MLA Citation
Liu, W, Mukerji, R, Li, X, Schaal, J, Bhattacharyya, J, Zalutsky, M, and Chilkoti, A. "Abstract 4583: In situ photocontrolled intratumoral depot for combined photodynamic therapy and brachytherapy for solid tumor." October 1, 2014.
Source
crossref
Published In
Cancer Research
Volume
74
Issue
19 Supplement
Publish Date
2014
Start Page
4583
End Page
4583
DOI
10.1158/1538-7445.AM2014-4583

Molecular description of the LCST behavior of an elastin-like polypeptide.

Elastin-like polypeptides (ELPs) with the repeat sequence of VPGVG are widely used as a model system for investigation of lower critical solution temperature (LCST) transition behavior. In this paper, the effect of temperature on the structure, dynamics and association of (VPGVG)18 in aqueous solution is investigated using atomistic molecular dynamics simulations. Our simulations show that as the temperature increases the ELP backbones undergo gradual conformational changes, which are attributed to the formation of more ordered secondary structures such as β-strands. In addition, increasing temperature changes the hydrophobicity of the ELP by exposure of hydrophobic valine-side chains to the solvent and hiding of proline residues. Based on our simulations, we conclude that the transition behavior of (VPGVG)18 can be attributed to a combination of thermal disruption of the water network that surrounds the polypeptide, reduction of solvent accessible surface area of the polypeptide, and increase in its hydrophobicity. Simulations of the association of two (VPGVG)18 molecules demonstrated that the observed gradual changes in the structural properties of the single polypeptide chain are enough to cause the aggregation of polypeptides above the LCST. These results lead us to propose that the LCST phase behavior of poly(VPGVG) is a collective phenomenon that originates from the correlated gradual changes in single polypeptide structure and the abrupt change in properties of hydration water around the peptide and is a result of a competition between peptide-peptide and peptide-water interactions. This is a computational study of an important intrinsically disordered peptide system that provides an atomic-level description of structural features and interactions that are relevant in the LCST phase behavior.

Authors
Li, NK; García Quiroz, F; Hall, CK; Chilkoti, A; Yingling, YG
MLA Citation
Li, NK, García Quiroz, F, Hall, CK, Chilkoti, A, and Yingling, YG. "Molecular description of the LCST behavior of an elastin-like polypeptide." Biomacromolecules 15.10 (October 2014): 3522-3530.
PMID
25142785
Source
epmc
Published In
Biomacromolecules
Volume
15
Issue
10
Publish Date
2014
Start Page
3522
End Page
3530
DOI
10.1021/bm500658w

Nanoparticle-Film Plasmon Ruler Interrogated with Transmission Visible Spectroscopy.

The widespread use of plasmonic nanorulers (PNRs) in sensing platforms has been plagued by technical challenges associated with the development of methods to fabricate precisely controlled nanostructures with high yield and characterize them with high throughput. We have previously shown that creating PNRs in a nanoparticle-film (NP-film) format enables the fabrication of an extremely large population of uniform PNRs with 100% yield using a self-assembly approach, which facilitates high-throughput PNR characterization using ensemble spectroscopic measurements and eliminates the need for expensive microscopy systems required by many other PNR platforms. We expand upon this prior work herein, showing that the NP-film PNR can be made compatible with aqueous sensing studies by adapting it for use in a transmission localized surface plasmon resonance spectroscopy format, where the coupled NP-film resonance responsible for the PNR signal is directly probed using an extinction measurement from a standard spectrophotometer. We designed slide holders that fit inside standard spectrophotometer cuvettes and position NP-film samples so that the coupled NP-film resonance can be detected in a collinear optical configuration. Once the NP-film PNR samples are cuvette-compatible, it is straightforward to calibrate the PNR in aqueous solution and use it to characterize dynamic, angstrom-scale distance changes resulting from pH-induced swelling of polyelectrolyte (PE) spacer layers as thin as 1 PE layer and also of a self-assembled monolayer of an amine-terminated alkanethiol. This development is an important step toward making PNR sensors more user-friendly and encouraging their widespread use in various sensing schemes.

Authors
Hill, RT; Kozek, KM; Hucknall, A; Smith, DR; Chilkoti, A
MLA Citation
Hill, RT, Kozek, KM, Hucknall, A, Smith, DR, and Chilkoti, A. "Nanoparticle-Film Plasmon Ruler Interrogated with Transmission Visible Spectroscopy." ACS photonics 1.10 (October 2014): 974-984.
PMID
25541618
Source
epmc
Published In
ACS Photonics
Volume
1
Issue
10
Publish Date
2014
Start Page
974
End Page
984
DOI
10.1021/ph500190q

Applications of elastin-like polypeptides in drug delivery

Elastin-like polypeptides (ELPs) are biopolymers inspired by human elastin. Their lower critical solution temperature phase transition behavior and biocompatibility make them useful materials for stimulus-responsive applications in biological environments. Due to their genetically encoded design and recombinant synthesis, the sequence and size of ELPs can be exactly defined. These design parameters control the structure and function of the ELP with a precision that is unmatched by synthetic polymers. Due to these attributes, ELPs have been used extensively for drug delivery in a variety of different embodiments - as soluble macromolecular carriers, self-assembled nanoparticles, cross-linked microparticles, or thermally coacervated depots. These ELP systems have been used to deliver biologic therapeutics, radionuclides, and small molecule drugs to a variety of anatomical sites for the treatment of diseases including cancer, type 2 diabetes, osteoarthritis, and neuroinflammation. © 2014 Elsevier B.V.

Authors
Macewan, SR; Chilkoti, A
MLA Citation
Macewan, SR, and Chilkoti, A. "Applications of elastin-like polypeptides in drug delivery." Journal of Controlled Release 190 (September 28, 2014): 314-330.
Source
scopus
Published In
Journal of Controlled Release
Volume
190
Publish Date
2014
Start Page
314
End Page
330
DOI
10.1016/j.jconrel.2014.06.028

Co-opting biology to deliver drugs.

The goal of drug delivery is to improve the safety and therapeutic efficacy of drugs. This review focuses on delivery platforms that are either derived from endogenous pathways, long-circulating biomolecules and cells or that piggyback onto long-circulating biomolecules and cells. The first class of such platforms is protein-based delivery systems--albumin, transferrin, and fusion to the Fc domain of antibodies--that have a long-circulation half-life and are designed to transport different molecules. The second class is lipid-based delivery systems-lipoproteins and exosomes-that are naturally occurring circulating lipid particles. The third class is cell-based delivery systems--erythrocytes, macrophages, and platelets--that have evolved, for reasons central to their function, to exhibit a long life-time in the body. The last class is small molecule-based delivery systems that include folic acid. This article reviews the biology of these systems, their application in drug delivery, and the promises and limitations of these endogenous systems for drug delivery.

Authors
Yousefpour, P; Chilkoti, A
MLA Citation
Yousefpour, P, and Chilkoti, A. "Co-opting biology to deliver drugs." Biotechnology and bioengineering 111.9 (September 2014): 1699-1716. (Review)
PMID
24916780
Source
epmc
Published In
Biotechnology & Bioengineering
Volume
111
Issue
9
Publish Date
2014
Start Page
1699
End Page
1716
DOI
10.1002/bit.25307

Applications of elastin-like polypeptides in drug delivery.

Elastin-like polypeptides (ELPs) are biopolymers inspired by human elastin. Their lower critical solution temperature phase transition behavior and biocompatibility make them useful materials for stimulus-responsive applications in biological environments. Due to their genetically encoded design and recombinant synthesis, the sequence and size of ELPs can be exactly defined. These design parameters control the structure and function of the ELP with a precision that is unmatched by synthetic polymers. Due to these attributes, ELPs have been used extensively for drug delivery in a variety of different embodiments-as soluble macromolecular carriers, self-assembled nanoparticles, cross-linked microparticles, or thermally coacervated depots. These ELP systems have been used to deliver biologic therapeutics, radionuclides, and small molecule drugs to a variety of anatomical sites for the treatment of diseases including cancer, type 2 diabetes, osteoarthritis, and neuroinflammation.

Authors
MacEwan, SR; Chilkoti, A
MLA Citation
MacEwan, SR, and Chilkoti, A. "Applications of elastin-like polypeptides in drug delivery." Journal of controlled release : official journal of the Controlled Release Society 190 (September 2014): 314-330. (Review)
PMID
24979207
Source
epmc
Published In
Journal of Controlled Release
Volume
190
Publish Date
2014
Start Page
314
End Page
330
DOI
10.1016/j.jconrel.2014.06.028

Surface initiated polymerization and its applications in medicine

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "Surface initiated polymerization and its applications in medicine." August 10, 2014.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
248
Publish Date
2014

Non-chromatographic purification of recombinant elastin-like polypeptides and their fusions with peptides and proteins from Escherichia coli.

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Authors
MacEwan, SR; Hassouneh, W; Chilkoti, A
MLA Citation
MacEwan, SR, Hassouneh, W, and Chilkoti, A. "Non-chromatographic purification of recombinant elastin-like polypeptides and their fusions with peptides and proteins from Escherichia coli." Journal of visualized experiments : JoVE 88 (June 9, 2014).
PMID
24961229
Source
epmc
Published In
Journal of Visualized Experiments
Issue
88
Publish Date
2014
DOI
10.3791/51583

Simple assay for proteases based on aggregation of stimulus-responsive polypeptides.

Unregulated changes in protease activity are linked to many diseases including cancer. Fast, accurate, and low-cost assays for detection of these changes are being explored for early diagnosis and monitoring of these diseases and can also be used as platforms for the discovery of new drugs. We report a new methodology for the simple detection and quantification of protease activity in buffer and human serum. The assay is based on recombinant diblock polypeptides that undergo temperature- or salt-triggered micellization in water. The coronae of the micelles are linked to the water-insoluble cores by a peptide substrate that is cleaved in the presence of the target protease. Protease cleavage of the diblock polypeptide triggers the aggregation of the core-forming segment, leading to a change in solution optical density, which can be used to detect the presence of, and to quantify the concentration of, protease. We used matrix metalloproteinase-1 (MMP-1) as a model protease and found peptide aggregation time to be proportional to enzyme concentration over a range from endogenous MMP-1 level in human serum (∼3 ng/mL) to 100 ng/mL (0.15-5 nM) in 40% human serum and 1-100 ng/mL in buffer. The assay does not require any intermediate steps or sophisticated data analysis, and the modular design of the assay system is amenable to straightforward adaptation for the detection of a wide range of proteases.

Authors
Ghoorchian, A; Chilkoti, A; López, GP
MLA Citation
Ghoorchian, A, Chilkoti, A, and López, GP. "Simple assay for proteases based on aggregation of stimulus-responsive polypeptides." Analytical chemistry 86.12 (June 2014): 6103-6110.
PMID
24832919
Source
epmc
Published In
Analytical Chemistry
Volume
86
Issue
12
Publish Date
2014
Start Page
6103
End Page
6110
DOI
10.1021/ac5012574

Enzymatic polymerization of high molecular weight DNA amphiphiles that self-assemble into star-like micelles

High molecular weight ssDNA amphiphiles are synthesized by enzymatic polymerization. These highly asymmetric diblock DNA copolymers self-assemble into "hairy", star-like micelles, shown in the AFM image and the DPD snapshot. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Authors
Tang, L; Tjong, V; Li, N; Yingling, YG; Chilkoti, A; Zauscher, S
MLA Citation
Tang, L, Tjong, V, Li, N, Yingling, YG, Chilkoti, A, and Zauscher, S. "Enzymatic polymerization of high molecular weight DNA amphiphiles that self-assemble into star-like micelles." Advanced Materials 26.19 (May 21, 2014): 3050-3054.
Source
scopus
Published In
Advanced Materials
Volume
26
Issue
19
Publish Date
2014
Start Page
3050
End Page
3054
DOI
10.1002/adma.201306049

Enzymatic polymerization of high molecular weight DNA amphiphiles that self-assemble into star-like micelles.

High molecular weight ssDNA amphiphiles are synthesized by enzymatic polymerization. These highly asymmetric diblock DNA copolymers self-assemble into "hairy", star-like micelles, shown in the AFM image and the DPD snapshot.

Authors
Tang, L; Tjong, V; Li, N; Yingling, YG; Chilkoti, A; Zauscher, S
MLA Citation
Tang, L, Tjong, V, Li, N, Yingling, YG, Chilkoti, A, and Zauscher, S. "Enzymatic polymerization of high molecular weight DNA amphiphiles that self-assemble into star-like micelles." Advanced materials (Deerfield Beach, Fla.) 26.19 (May 2014): 3050-3054.
PMID
24497034
Source
epmc
Published In
Advanced Materials
Volume
26
Issue
19
Publish Date
2014
Start Page
3050
End Page
3054
DOI
10.1002/adma.201306049

Rational design of "heat seeking" drug loaded polypeptide nanoparticles that thermally target solid tumors.

This paper demonstrates the first example of targeting a solid tumor that is externally heated to 42 °C by "heat seeking" drug-loaded polypeptide nanoparticles. These nanoparticles consist of a thermally responsive elastin-like polypeptide (ELP) conjugated to multiple copies of a hydrophobic cancer drug. To rationally design drug-loaded nanoparticles that exhibit thermal responsiveness in the narrow temperature range between 37 and 42 °C, an analytical model was developed that relates ELP composition and chain length to the nanoparticle phase transition temperature. Suitable candidates were designed based on the predictions of the model and tested in vivo by intravital confocal fluorescence microscopy of solid tumors, which revealed that the nanoparticles aggregate in the vasculature of tumors heated to 42 °C and that the aggregation is reversible as the temperature reverts to 37 °C. Biodistribution studies showed that the most effective strategy to target the nanoparticles to tumors is to thermally cycle the tumors between 37 and 42 °C. These nanoparticles set the stage for the targeted delivery of a range of cancer chemotherapeutics by externally applied mild hyperthermia of solid tumors.

Authors
McDaniel, JR; MacEwan, SR; Li, X; Radford, DC; Landon, CD; Dewhirst, M; Chilkoti, A
MLA Citation
McDaniel, JR, MacEwan, SR, Li, X, Radford, DC, Landon, CD, Dewhirst, M, and Chilkoti, A. "Rational design of "heat seeking" drug loaded polypeptide nanoparticles that thermally target solid tumors." Nano letters 14.5 (May 2014): 2890-2895.
PMID
24738626
Source
epmc
Published In
Nano Letters
Volume
14
Issue
5
Publish Date
2014
Start Page
2890
End Page
2895
DOI
10.1021/nl5009376

Genetically encoded polypeptide nanoparticles for delivery of drugs

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "Genetically encoded polypeptide nanoparticles for delivery of drugs." March 16, 2014.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
247
Publish Date
2014

"Smart" DNA interfaces.

This review focuses on surface-grafted DNA, and its use as a molecular building block that exploits its unique properties as a directional (poly)anion that exhibits molecular recognition. The selected examples highlight innovative applications of DNA at surfaces and interfaces ranging from molecular diagnostics and sequencing to biosensing.

Authors
Tjong, V; Tang, L; Zauscher, S; Chilkoti, A
MLA Citation
Tjong, V, Tang, L, Zauscher, S, and Chilkoti, A. ""Smart" DNA interfaces." Chem Soc Rev 43.5 (March 7, 2014): 1612-1626. (Review)
PMID
24352168
Source
pubmed
Published In
Chemical Society Reviews
Volume
43
Issue
5
Publish Date
2014
Start Page
1612
End Page
1626
DOI
10.1039/c3cs60331h

Growing polymers from peptides and proteins: A biomedical perspective

This review covers the development of the in situ growth of polymers from biomolecules, also termed the "grafting from" conjugation methodology. We trace the evolution of this field of research, with an emphasis on recently developed methods to synthesize polymer conjugates of peptides and proteins with control over the site of conjugation, the stoichiometry of the conjugate, and the chain length and polydispersity of the polymer. We highlight the functional properties and potential biomedical applications of the peptide/protein-polymer conjugates, including conjugates with more advanced architecture and self-assembly behaviour. © 2014 The Royal Society of Chemistry.

Authors
Qi, Y; Chilkoti, A
MLA Citation
Qi, Y, and Chilkoti, A. "Growing polymers from peptides and proteins: A biomedical perspective." Polymer Chemistry 5.2 (January 21, 2014): 266-276.
Source
scopus
Published In
Polymer Chemistry
Volume
5
Issue
2
Publish Date
2014
Start Page
266
End Page
276
DOI
10.1039/c3py01089a

Non-chromatographic purification of recombinant elastin-like polypeptides and their fusions with peptides and proteins from Escherichia coli.

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Authors
MacEwan, SR; Hassouneh, W; Chilkoti, A
MLA Citation
MacEwan, SR, Hassouneh, W, and Chilkoti, A. "Non-chromatographic purification of recombinant elastin-like polypeptides and their fusions with peptides and proteins from Escherichia coli." Journal of visualized experiments : JoVE 88 (January 1, 2014).
Source
scopus
Published In
Journal of Visualized Experiments
Issue
88
Publish Date
2014

The language of protein polymers

© 2014 American Chemical Society.Proteins are heteropolymers of one or more amino acid residues arranged in a molecularly defined fashion. The precise control of amino acid sequence in protein biosynthesis programs the folding of these heteropolymers into diverse three-dimensional structures. The language of proteins, however, as seen in nature, encompasses limitless amino acid "phrases" (heteropolymers) written in peptide "words" (amino acid motifs) that span the entire structural spectrum from tightly folded to unstructured. Because protein sequences do not always have an obvious syntactic unit (word), herein we focus on protein polymers that repeat one or more syntactic motifs-units with a characteristic fold, biological activity or physical property (e.g., elasticity, phase behavior). We review the biosynthesis and sequence-controlled behavior of protein polymers that altogether span the gap between folded proteins and unstructured polymers. Learning to speak the language of protein polymers promises to merge the science of protein design and the materials science of synthetic polymers. Paradoxically, while protein structure is largely foreign to polymer chemists, the study and synthesis of unstructured, polymer-like proteins has been-till recently-similarly foreign to structural biologists. Interesting possibilities in materials science emerge from acquiring the capacity to read, write and speak the language of protein polymers.

Authors
Quiroz, FG; Chilkoti, A
MLA Citation
Quiroz, FG, and Chilkoti, A. "The language of protein polymers." January 1, 2014.
Source
scopus
Published In
ACS Symposium Series
Volume
1170
Publish Date
2014
Start Page
15
End Page
33
DOI
10.1021/bk-2014-1170.ch002

Genetically encoded “smart” peptide polymers for biomedicine

Authors
Mastria, E; Chilkoti, A
MLA Citation
Mastria, E, and Chilkoti, A. "Genetically encoded “smart” peptide polymers for biomedicine." MRS Bulletin 39.01 (January 2014): 35-43.
Source
crossref
Published In
MRS bulletin / Materials Research Society
Volume
39
Issue
01
Publish Date
2014
Start Page
35
End Page
43
DOI
10.1557/mrs.2013.313

Controlled apoptosis by a thermally toggled nanoscale amplifier of cellular uptake.

Internalization into cancer cells is a significant challenge in the delivery of many anticancer therapeutics. Drug carriers can address this challenge by facilitating cellular uptake of cytotoxic cargo in the tumor, while preventing cellular uptake in healthy tissues. Here we describe an extrinsically controlled drug carrier, a nanopeptifier, that amplifies cellular uptake by modulating the activity of cell-penetrating peptides with thermally toggled self-assembly of a genetically encoded polypeptide nanoparticle. When appended with a proapoptotic peptide, the nanopeptifier creates a cytotoxic switch, inducing apoptosis only in its self-assembled state. The nanopeptifier provides a new approach to tune the cellular uptake and activity of anticancer therapeutics by an extrinsic thermal trigger.

Authors
MacEwan, SR; Chilkoti, A
MLA Citation
MacEwan, SR, and Chilkoti, A. "Controlled apoptosis by a thermally toggled nanoscale amplifier of cellular uptake." Nano letters 14.4 (January 2014): 2058-2064.
PMID
24611762
Source
epmc
Published In
Nano Letters
Volume
14
Issue
4
Publish Date
2014
Start Page
2058
End Page
2064
DOI
10.1021/nl5002313

Co-opting biology to deliver drugs

The goal of drug delivery is to improve the safety and therapeutic efficacy of drugs. This review focuses on delivery platforms that are either derived from endogenous pathways, long-circulating biomolecules and cells or that piggyback onto long-circulating biomolecules and cells. The first class of such platforms is protein-based delivery systems-albumin, transferrin, and fusion to the Fc domain of antibodies-that have a long-circulation half-life and are designed to transport different molecules. The second class is lipid-based delivery systems-lipoproteins and exosomes-that are naturally occurring circulating lipid particles. The third class is cell-based delivery systems-erythrocytes, macrophages, and platelets-that have evolved, for reasons central to their function, to exhibit a long life-time in the body. The last class is small molecule-based delivery systems that include folic acid. This article reviews the biology of these systems, their application in drug delivery, and the promises and limitations of these endogenous systems for drug delivery. Biotechnol. Bioeng. 2014;111: 1699-1716. © 2014 Wiley Periodicals, Inc.

Authors
Yousefpour, P; Chilkoti, A
MLA Citation
Yousefpour, P, and Chilkoti, A. "Co-opting biology to deliver drugs." Biotechnology and Bioengineering 111.9 (2014): 1699-1716.
Source
scival
Published In
Biotechnology & Bioengineering
Volume
111
Issue
9
Publish Date
2014
Start Page
1699
End Page
1716
DOI
10.1002/bit.25307

Sortase-mediated initiator attachment for in situ grafting of a PEG-based polymer brush from the C terminus of green fluorescent protein.

Authors
Qi, Y; Amiram, M; Gao, W; Chilkoti, A
MLA Citation
Qi, Y, Amiram, M, Gao, W, and Chilkoti, A. "Sortase-mediated initiator attachment for in situ grafting of a PEG-based polymer brush from the C terminus of green fluorescent protein." J Control Release 172.1 (November 28, 2013): e121-.
PMID
24242415
Source
pubmed
Published In
Journal of Controlled Release
Volume
172
Issue
1
Publish Date
2013
Start Page
e121
DOI
10.1016/j.jconrel.2013.08.288

A depot-forming glucagon-like peptide-1 fusion protein reduces blood glucose for five days with a single injection.

Peptide drugs are an exciting class of pharmaceuticals for the treatment of a variety of diseases; however, their short half-life dictates multiple and frequent injections causing undesirable side effects. Herein, we describe a novel peptide delivery system that seeks to combine the attractive features of prolonged circulation time with a prolonged release formulation. This system consists of glucagon-like peptide-1, a type-2 diabetes drug fused to a thermally responsive, elastin-like-polypeptide (ELP) that undergoes a soluble-insoluble phase transition between room temperature and body temperature, thereby forming an injectable depot. We synthesized a set of GLP-1-ELP fusions and verified their proteolytic stability and potency in vitro. Significantly, a single injection of depot forming GLP-1-ELP fusions reduced blood glucose levels in mice for up to 5 days, 120 times longer than an injection of the native peptide. These findings demonstrate the unique advantages of using ELPs to release peptide-ELP fusions from a depot combined with enhanced systemic circulation to create a tunable peptide delivery system.

Authors
Amiram, M; Luginbuhl, KM; Li, X; Feinglos, MN; Chilkoti, A
MLA Citation
Amiram, M, Luginbuhl, KM, Li, X, Feinglos, MN, and Chilkoti, A. "A depot-forming glucagon-like peptide-1 fusion protein reduces blood glucose for five days with a single injection." J Control Release 172.1 (November 28, 2013): 144-151.
PMID
23928357
Source
pubmed
Published In
Journal of Controlled Release
Volume
172
Issue
1
Publish Date
2013
Start Page
144
End Page
151
DOI
10.1016/j.jconrel.2013.07.021

A genetically engineered thermally responsive sustained release curcumin depot to treat neuroinflammation.

Radiculopathy, a painful neuroinflammation that can accompany intervertebral disc herniation, is associated with locally increased levels of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). Systemic administration of TNF antagonists for radiculopathy in the clinic has shown mixed results, and there is growing interest in the local delivery of anti-inflammatory drugs to treat this pathology as well as similar inflammatory events of peripheral nerve injury. Curcumin, a known antagonist of TNFα in multiple cell types and tissues, was chemically modified and conjugated to a thermally responsive elastin-like polypeptide (ELP) to create an injectable depot for sustained, local delivery of curcumin to treat neuroinflammation. ELPs are biopolymers capable of thermally-triggered in situ depot formation that have been successfully employed as drug carriers and biomaterials in several applications. ELP-curcumin conjugates were shown to display high drug loading, rapidly release curcumin in vitro via degradable carbamate bonds, and retain in vitro bioactivity against TNFα-induced cytotoxicity and monocyte activation with IC50 only two-fold higher than curcumin. When injected proximal to the sciatic nerve in mice via intramuscular (i.m.) injection, ELP-curcumin conjugates underwent a thermally triggered soluble-insoluble phase transition, leading to in situ formation of a depot that released curcumin over 4days post-injection and decreased plasma AUC 7-fold.

Authors
Sinclair, SM; Bhattacharyya, J; McDaniel, JR; Gooden, DM; Gopalaswamy, R; Chilkoti, A; Setton, LA
MLA Citation
Sinclair, SM, Bhattacharyya, J, McDaniel, JR, Gooden, DM, Gopalaswamy, R, Chilkoti, A, and Setton, LA. "A genetically engineered thermally responsive sustained release curcumin depot to treat neuroinflammation." J Control Release 171.1 (October 10, 2013): 38-47.
Website
http://hdl.handle.net/10161/7787
PMID
23830979
Source
pubmed
Published In
Journal of Controlled Release
Volume
171
Issue
1
Publish Date
2013
Start Page
38
End Page
47
DOI
10.1016/j.jconrel.2013.06.032

Genetically engineered polypeptide conjugates and fusions for drug delivery

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "Genetically engineered polypeptide conjugates and fusions for drug delivery." September 8, 2013.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
246
Publish Date
2013

Enzyme catalyzed polymerization of DNA amphiphiles that self-assemble into star-like micelles

Authors
Tang, L; Tjong, V; Gu, R; Chilkoti, A; Zauscher, S
MLA Citation
Tang, L, Tjong, V, Gu, R, Chilkoti, A, and Zauscher, S. "Enzyme catalyzed polymerization of DNA amphiphiles that self-assemble into star-like micelles." September 8, 2013.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
246
Publish Date
2013

Syntax of "smart" peptide polymers governs their function

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "Syntax of "smart" peptide polymers governs their function." September 8, 2013.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
246
Publish Date
2013

Elastin-based amphiphilic copolymers as precision building blocks for controlled self-assembly

Authors
Garanger, E; Mac Ewan, S; Brulet, A; Bataille, L; Sandre, O; Chilkoti, A; Lecommandoux, S
MLA Citation
Garanger, E, Mac Ewan, S, Brulet, A, Bataille, L, Sandre, O, Chilkoti, A, and Lecommandoux, S. "Elastin-based amphiphilic copolymers as precision building blocks for controlled self-assembly." September 8, 2013.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
246
Publish Date
2013

Actively targeting solid tumours with thermoresponsive drug delivery systems that respond to mild hyperthermia.

A diverse range of drug delivery vehicles have been developed to specifically target chemotherapeutics to solid tumours while avoiding systemic dose-limiting toxicity. Many of these active targeting strategies display limited efficacy because they rely on subtle differences in expression patterns between pathogenic tissue and healthy tissue. In contrast, drug delivery systems that exploit thermoresponsive behaviour allow a clinician to spatially and temporally control the accumulation and/or release of the toxic agents within tumour tissue by simply applying mild hyperthermia (defined as 39-43 °C) to the desired site. Although thermally sensitive materials comprise a significant portion of the literature on novel drug delivery systems, only a few systems have been methodically tuned to respond within this narrowly defined physiological temperature range in an in vivo environment. This review discusses the materials and strategies developed to control the primary tumour through the combined application of hyperthermia and chemotherapy.

Authors
McDaniel, JR; Dewhirst, MW; Chilkoti, A
MLA Citation
McDaniel, JR, Dewhirst, MW, and Chilkoti, A. "Actively targeting solid tumours with thermoresponsive drug delivery systems that respond to mild hyperthermia." Int J Hyperthermia 29.6 (September 2013): 501-510. (Review)
PMID
23924317
Source
pubmed
Published In
International Journal of Hyperthermia (Informa)
Volume
29
Issue
6
Publish Date
2013
Start Page
501
End Page
510
DOI
10.3109/02656736.2013.819999

Spectroscopic study of a DNA brush synthesized in situ by surface initiated enzymatic polymerization.

We used a combination of synchrotron-based X-ray photoelectron spectroscopy (XPS) and angle-resolved near-edge X-ray absorption fine structure (NEXAFS) spectroscopy to study the chemical integrity, purity, and possible internal alignment of single-strand (ss) adenine deoxynucleotide (poly(A)) DNA brushes. The brushes were synthesized by surface-initiated enzymatic polymerization (SIEP) on a 25-mer of adenine self-assembled monolayer (SAM) on gold (A25-SH), wherein the terminal 3'-OH of the A25-SH serve as the initiation sites for SIEP of poly(A). XPS and NEXAFS spectra of poly(A) brushes were found to be almost identical to those of A25-SH initiator, with no unambiguous traces of contamination. Apart from the well-defined chemical integrity and contamination-free character, the brushes were found to have a high degree of orientational order, with an upright orientation of individual strands, despite their large thickness up to ~55 nm, that corresponds to a chain length of at least several hundred nucleotides for individual ssDNA molecules. The orientational order exhibited by these poly(A) DNA brushes, mediated presumably by base stacking, was found to be independent of the brush thickness as long as the packing density was high enough. The well-defined character and orientational ordering of the ssDNA brushes make them a potentially promising system for different applications.

Authors
Khan, MN; Tjong, V; Chilkoti, A; Zharnikov, M
MLA Citation
Khan, MN, Tjong, V, Chilkoti, A, and Zharnikov, M. "Spectroscopic study of a DNA brush synthesized in situ by surface initiated enzymatic polymerization." J Phys Chem B 117.34 (August 29, 2013): 9929-9938.
PMID
23899324
Source
pubmed
Published In
The Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces and Biophysical
Volume
117
Issue
34
Publish Date
2013
Start Page
9929
End Page
9938
DOI
10.1021/jp404774x

A unified model for de novo design of elastin-like polypeptides with tunable inverse transition temperatures.

Elastin-like polypeptides (ELPs) are stimulus-responsive peptide polymers that exhibit inverse temperature phase transition behavior, causing an ELP to aggregate above its inverse transition temperature (T(t)). Although this property has been exploited in a variety of biotechnological applications, de novo design of ELPs that display a specific T(t) is not trivial because the T(t) of an ELP is a complex function of several variables, including its sequence, chain length, polypeptide concentration, and the type and concentration of cosolutes in solution. This paper provides a quantitative model that predicts the T(t) of a family of ELPs (Val-Pro-Gly-Xaa-Gly, where Xaa = Ala and/or Val) from their composition, chain length, and concentration in phosphate buffered saline. This model will enable de novo prediction of the amino acid sequence and chain length of ELPs that will display a predetermined T(t) in physiological buffer within a specified concentration regime, thereby greatly facilitating the design of new ELPs for applications in medicine and biotechnology.

Authors
McDaniel, JR; Radford, DC; Chilkoti, A
MLA Citation
McDaniel, JR, Radford, DC, and Chilkoti, A. "A unified model for de novo design of elastin-like polypeptides with tunable inverse transition temperatures." Biomacromolecules 14.8 (August 12, 2013): 2866-2872.
PMID
23808597
Source
pubmed
Published In
Biomacromolecules
Volume
14
Issue
8
Publish Date
2013
Start Page
2866
End Page
2872
DOI
10.1021/bm4007166

Sortase-catalyzed initiator attachment enables high yield growth of a stealth polymer from the C terminus of a protein.

Conventional methods for synthesizing protein/peptide-polymer conjugates, as a means to improve the pharmacological properties of therapeutic biomolecules, typically have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of site- specificity, which results in heterogeneous products with significantly compromised bioactivity. To address these limitations, the use of sortase A from Staphylococcus aureus is demonstrated to site-specifically attach an initiator solely at the C-terminus of green fluorescent protein (GFP), followed by in situ growth of a stealth polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeds with high specificity and near-complete (≈95%) product conversion. Subsequent in situ ATRP in aqueous buffer produces 1:1 stoichiometric conjugates with >90% yield, low dispersity, and no denaturation of the protein. This approach introduces a simple and useful method for high yield synthesis of protein/peptide-polymer conjugates.

Authors
Qi, Y; Amiram, M; Gao, W; McCafferty, DG; Chilkoti, A
MLA Citation
Qi, Y, Amiram, M, Gao, W, McCafferty, DG, and Chilkoti, A. "Sortase-catalyzed initiator attachment enables high yield growth of a stealth polymer from the C terminus of a protein." Macromol Rapid Commun 34.15 (August 2013): 1256-1260.
PMID
23836349
Source
pubmed
Published In
Macromolecular Rapid Communications
Volume
34
Issue
15
Publish Date
2013
Start Page
1256
End Page
1260
DOI
10.1002/marc.201300460

Hydration layer coupling and cooperativity in phase behavior of stimulus responsive peptide polymers.

It is shown that hydrophilic (backbone) and hydrophobic (side chain) hydration layers of elastin-like polypeptides (ELPs), a class of stimulus responsive peptide polymers that exhibit lower critical solution temperature (LCST) phase transition behavior, can exist in a coupled and decoupled state. The decoupled hydration state consists of hydrophobic and hydrophilic hydration layers that respond independently to temperature, while the coupled hydration state is characterized by a common, cooperative dehydration of both hydration layers. It is further shown that the primary sequence of an ELP can be tuned to exhibit either of the hydration layer coupling modes. Charged side chains lead to decoupling, while strongly hydrophobic side chains trigger stronger interaction between hydrophilic and hydrophobic hydration, leading to coupling of both layers. Further, for aprotic residues this coupling is fostered by decreasing bulkiness of hydrophobic side chains due to larger hydration numbers and water molecules mediating coupling between side chain and backbone hydration shells. For coupled hydration shells, the LCST phase transition characterized by spin probing continuous wave electron paramagnetic resonance spectroscopy is reminiscent of a first-order process even on nanoscopic length scales. In contrast, analogous synthetic polymers exhibit nanoscale phase transitions over a broad temperature range, indicating that their nanoscale phase behavior is not of first order. Hence, our results indicate that ELPs are the first identified class of polymers that exhibit a first-order inverse phase transition on nanoscopic length scales. These results may also provide insights into the role of hydration layers in governing the structure-function relationship of intrinsically disordered proteins.

Authors
Kurzbach, D; Hassouneh, W; McDaniel, JR; Jaumann, EA; Chilkoti, A; Hinderberger, D
MLA Citation
Kurzbach, D, Hassouneh, W, McDaniel, JR, Jaumann, EA, Chilkoti, A, and Hinderberger, D. "Hydration layer coupling and cooperativity in phase behavior of stimulus responsive peptide polymers." J Am Chem Soc 135.30 (July 31, 2013): 11299-11308.
PMID
23822733
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
135
Issue
30
Publish Date
2013
Start Page
11299
End Page
11308
DOI
10.1021/ja4047872

Calcium binding peptide motifs from calmodulin confer divalent ion selectivity to elastin-like polypeptides.

Calcium-sensitive elastin-like polypeptides (CELPs) were synthesized by periodically interspersing a calcium-binding peptide sequence from calmodulin within an elastin-like polypeptide (ELP) with the goal of creating thermal and calcium responsive peptide polymers. The CELPs exhibit high sensitivity to calcium compared to monovalent cations but do not exhibit the exquisite selectivity for calcium over other divalent cations, such as magnesium, that is displayed by calmodulin. The CELPs were further used as a building block for the synthesis of calcium-sensitive nanoparticles by fusing a hydrophilic, noncalcium-sensitive ELP block with a CELP block that becomes more hydrophobic upon calcium binding. We show that addition of calcium at concentrations between 50 and 500 mM imparts sufficient amphiphilicity to the diblock polypeptide between 33 and 46 °C to trigger its self-assembly into monodisperse spherical micelles with a hydrodynamic radius of ∼50 nm.

Authors
Hassouneh, W; Nunalee, ML; Shelton, MC; Chilkoti, A
MLA Citation
Hassouneh, W, Nunalee, ML, Shelton, MC, and Chilkoti, A. "Calcium binding peptide motifs from calmodulin confer divalent ion selectivity to elastin-like polypeptides." Biomacromolecules 14.7 (July 8, 2013): 2347-2353.
PMID
23705904
Source
pubmed
Published In
Biomacromolecules
Volume
14
Issue
7
Publish Date
2013
Start Page
2347
End Page
2353
DOI
10.1021/bm400464s

Interactions of elastin-like polypeptides with model membranes on GUVs

Authors
Weinberger, A; Macewan, S; Schmatko, T; Schroder, A; Chilkoti, A; Marques, CM
MLA Citation
Weinberger, A, Macewan, S, Schmatko, T, Schroder, A, Chilkoti, A, and Marques, CM. "Interactions of elastin-like polypeptides with model membranes on GUVs." July 2013.
Source
wos-lite
Published In
European Biophysics Journal
Volume
42
Publish Date
2013
Start Page
S158
End Page
S158

Predicting transition temperatures of elastin-like polypeptide fusion proteins.

Elastin-like polypeptides (ELPs) are thermally sensitive peptide polymers that undergo thermally triggered phase separation and this behavior is imparted to soluble proteins when they are fused to an ELP. The transition temperature of the ELP fusion protein is observed to be different than that of a free ELP, indicating that the surface properties of the fused protein modulate the thermal behavior of ELPs. Understanding this effect is important for the rational design of applications that exploit the phase transition behavior of ELP fusion proteins. We had previously developed a biophysical model that explained the effect of hydrophobic proteins on depressing the transition temperature of ELP fusion proteins relative to free ELP. Here, we extend the model to elucidate the effect of hydrophilic proteins on the thermal behavior of ELP fusion proteins. A linear correlation was found between overall residue composition of accessible protein surface weighted by a characteristic transition temperature for each residue and the difference in transition temperatures between the ELP protein fusion and the corresponding free ELP. In breaking down the contribution of residues to polar, nonpolar, and charged, the model revealed that charged residues are the most important parameter in altering the transition temperature of an ELP fusion relative to the free ELP.

Authors
Christensen, T; Hassouneh, W; Trabbic-Carlson, K; Chilkoti, A
MLA Citation
Christensen, T, Hassouneh, W, Trabbic-Carlson, K, and Chilkoti, A. "Predicting transition temperatures of elastin-like polypeptide fusion proteins." Biomacromolecules 14.5 (May 13, 2013): 1514-1519.
PMID
23565607
Source
pubmed
Published In
Biomacromolecules
Volume
14
Issue
5
Publish Date
2013
Start Page
1514
End Page
1519
DOI
10.1021/bm400167h

Abstract 5615: Evaluation of the antitumoral efficacy and toxicity of injectable, polymeric radionuclide seeds in a noninvasive preclinical prostate tumor model.

Authors
Schaal, JL; Liu, W; Li, X; Zalutsky, M; Chilkoti, A
MLA Citation
Schaal, JL, Liu, W, Li, X, Zalutsky, M, and Chilkoti, A. "Abstract 5615: Evaluation of the antitumoral efficacy and toxicity of injectable, polymeric radionuclide seeds in a noninvasive preclinical prostate tumor model." April 15, 2013.
Source
crossref
Published In
Cancer Research
Volume
73
Issue
8 Supplement
Publish Date
2013
Start Page
5615
End Page
5615
DOI
10.1158/1538-7445.AM2013-5615

Abstract 4509: Genetically encoded chimeric polypeptide nanoparticles for paclitaxel delivery to triple negative breast cancer.

Authors
Bhattacharyya, J; McDaniel, J; Chilkoti, A
MLA Citation
Bhattacharyya, J, McDaniel, J, and Chilkoti, A. "Abstract 4509: Genetically encoded chimeric polypeptide nanoparticles for paclitaxel delivery to triple negative breast cancer." April 15, 2013.
Source
crossref
Published In
Cancer Research
Volume
73
Issue
8 Supplement
Publish Date
2013
Start Page
4509
End Page
4509
DOI
10.1158/1538-7445.AM2013-4509

Abstract 4341: A novel injectable polymer liquid that self-assembles into radioactive “seeds” for brachytherapy.

Authors
Liu, W; McDaniel, JR; Li, X; Schaal, J; Bhattacharyya, J; Zalutsky, MR; Chilkoti, A
MLA Citation
Liu, W, McDaniel, JR, Li, X, Schaal, J, Bhattacharyya, J, Zalutsky, MR, and Chilkoti, A. "Abstract 4341: A novel injectable polymer liquid that self-assembles into radioactive “seeds” for brachytherapy." April 15, 2013.
Source
crossref
Published In
Cancer Research
Volume
73
Issue
8 Supplement
Publish Date
2013
Start Page
4341
End Page
4341
DOI
10.1158/1538-7445.AM2013-4341

Delivery of paclitaxel with elastin-like polypeptides

Authors
Caudill, C; Bhattacharyya, J; Chilkoti, A
MLA Citation
Caudill, C, Bhattacharyya, J, and Chilkoti, A. "Delivery of paclitaxel with elastin-like polypeptides." April 7, 2013.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
245
Publish Date
2013

PHARMACOKINETICS OF A THERMALLY RESPONSIVE CURCUMIN CONJUGATE FOR LOCAL ANTAGONISM OF NEUROINFLAMMATION IN DISC HERNIATION

Authors
Sinclair, SM; Bhattacharyya, J; Little, D; Bowles, RD; Gooden, DM; Toone, EJ; Chilkoti, A; Setton, LA
MLA Citation
Sinclair, SM, Bhattacharyya, J, Little, D, Bowles, RD, Gooden, DM, Toone, EJ, Chilkoti, A, and Setton, LA. "PHARMACOKINETICS OF A THERMALLY RESPONSIVE CURCUMIN CONJUGATE FOR LOCAL ANTAGONISM OF NEUROINFLAMMATION IN DISC HERNIATION." April 2013.
Source
wos-lite
Published In
Osteoarthritis and Cartilage
Volume
21
Publish Date
2013
Start Page
S292
End Page
S293

Three-in-one chromatography-free purification, tag removal, and site-specific modification of recombinant fusion proteins using sortase A and elastin-like polypeptides.

Authors
Bellucci, JJ; Amiram, M; Bhattacharyya, J; McCafferty, D; Chilkoti, A
MLA Citation
Bellucci, JJ, Amiram, M, Bhattacharyya, J, McCafferty, D, and Chilkoti, A. "Three-in-one chromatography-free purification, tag removal, and site-specific modification of recombinant fusion proteins using sortase A and elastin-like polypeptides." Angew Chem Int Ed Engl 52.13 (March 25, 2013): 3703-3708.
PMID
23424160
Source
pubmed
Published In
Angewandte Chemie International Edition
Volume
52
Issue
13
Publish Date
2013
Start Page
3703
End Page
3708
DOI
10.1002/anie.201208292

Injectable protease-operated depots of glucagon-like peptide-1 provide extended and tunable glucose control.

Peptide drugs are an exciting class of pharmaceuticals increasingly used for the treatment of a variety of diseases; however, their main drawback is a short half-life, which dictates multiple and frequent injections and an undesirable "peak-and-valley" pharmacokinetic profile, which can cause undesirable side-effects. Synthetic prolonged release formulations can provide extended release of biologically active native peptide, but their synthetic nature can be an obstacle to production and utilization. Motivated by these limitations, we have developed a new and entirely genetically encoded peptide delivery system--Protease Operated Depots (PODs)--to provide sustained and tunable release of a peptide drug from an injectable s.c. depot. We demonstrate proof-of-concept of PODs, by fusion of protease cleavable oligomers of glucagon-like peptide-1, a type-2 diabetes drug, and a thermally responsive, depot-forming elastin-like-polypeptide that undergoes a thermally triggered inverse phase transition below body temperature, thereby forming an injectable depot. We constructed synthetic genes for glucagon-like peptide-1 PODs and demonstrated their high-yield expression in Escherichia coli and facile purification by a nonchromatographic scheme we had previously developed. Remarkably, a single injection of glucagon-like peptide-1 PODs was able to reduce blood glucose levels in mice for up to 5 d, 120 times longer than an injection of the native peptide drug. These findings demonstrate that PODs provide the first genetically encoded alternative to synthetic peptide encapsulation schemes for sustained delivery of peptide therapeutics.

Authors
Amiram, M; Luginbuhl, KM; Li, X; Feinglos, MN; Chilkoti, A
MLA Citation
Amiram, M, Luginbuhl, KM, Li, X, Feinglos, MN, and Chilkoti, A. "Injectable protease-operated depots of glucagon-like peptide-1 provide extended and tunable glucose control." Proc Natl Acad Sci U S A 110.8 (February 19, 2013): 2792-2797.
PMID
23359691
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
110
Issue
8
Publish Date
2013
Start Page
2792
End Page
2797
DOI
10.1073/pnas.1214518110

Self-assembly of thermally responsive nanoparticles of a genetically encoded peptide polymer by drug conjugation.

Authors
McDaniel, JR; Bhattacharyya, J; Vargo, KB; Hassouneh, W; Hammer, DA; Chilkoti, A
MLA Citation
McDaniel, JR, Bhattacharyya, J, Vargo, KB, Hassouneh, W, Hammer, DA, and Chilkoti, A. "Self-assembly of thermally responsive nanoparticles of a genetically encoded peptide polymer by drug conjugation." Angew Chem Int Ed Engl 52.6 (February 4, 2013): 1683-1687.
PMID
23280697
Source
pubmed
Published In
Angewandte Chemie International Edition
Volume
52
Issue
6
Publish Date
2013
Start Page
1683
End Page
1687
DOI
10.1002/anie.201200899

Direct fluorescence detection of RNA on microarrays by surface-initiated enzymatic polymerization.

We report the first demonstration of surface-initiated enzymatic polymerization (SIEP) for the direct detection of RNA in a fluorescence microarray format. This new method incorporates multiple fluorophores into an RNA strand using the two-step sequential and complementary reactions catalyzed by yeast poly(A) polymerase (PaP) to incorporate deoxyadenosine triphosphate (dATP) at the 3'-OH of an RNA molecule, followed by terminal deoxynucleotidyl transferase (TdT) to catalyze the sequential addition of a mixture of natural and fluorescent deoxynucleotides (dNTPs) at the 3'-OH of an RNA-DNA hybrid. We found that the 3'-end of RNA can be efficiently converted into DNA (∼50% conversion) by polymerization of dATP using yeast PaP, and the short DNA strand appended to the end of the RNA by PaP acts as the initiator for the TdT-catalyzed polymerization of longer DNA strands from a mixture of natural and fluorescent dNTPs that contain up to ∼45 Cy3 fluorophores per 1 kb DNA. We obtained an ∼2 pM limit of detection (LOD) and a 3 log-linear dynamic range for hybridization of a short 21 base-long RNA target to an immobilized peptide nucleic acid probe, while fragmented mRNA targets from three different full length mRNA transcripts yielded a ∼10 pM LOD with a similar dynamic range in a microarray format.

Authors
Tjong, V; Yu, H; Hucknall, A; Chilkoti, A
MLA Citation
Tjong, V, Yu, H, Hucknall, A, and Chilkoti, A. "Direct fluorescence detection of RNA on microarrays by surface-initiated enzymatic polymerization." Anal Chem 85.1 (January 2, 2013): 426-433.
PMID
23194025
Source
pubmed
Published In
Analytical Chemistry
Volume
85
Issue
1
Publish Date
2013
Start Page
426
End Page
433
DOI
10.1021/ac303132j

Surface Patterning

Authors
Hill, RT; Chilkoti, A
MLA Citation
Hill, RT, and Chilkoti, A. "Surface Patterning." Biomaterials Science: An Introduction to Materials: Third Edition. January 1, 2013. 276-301.
Source
scopus
Publish Date
2013
Start Page
276
End Page
301
DOI
10.1016/B978-0-08-087780-8.00028-0

Encapsulation of Stimuli-Responsive Fusion Proteins in Silica: Thermally Responsive Metal Ion-Sensitive Hybrid Membranes

Authors
Li, L; Im, O; Chilkoti, A; López, GP
MLA Citation
Li, L, Im, O, Chilkoti, A, and López, GP. "Encapsulation of Stimuli-Responsive Fusion Proteins in Silica: Thermally Responsive Metal Ion-Sensitive Hybrid Membranes." MRS Proceedings 1498 (January 2013).
Source
crossref
Published In
MRS Online Proceedings Library
Volume
1498
Publish Date
2013
DOI
10.1557/opl.2013.332

Harnessing the power of cell-penetrating peptides: activatable carriers for targeting systemic delivery of cancer therapeutics and imaging agents.

Targeted delivery of cancer therapeutics and imaging agents aims to enhance the accumulation of these molecules in a solid tumor while avoiding uptake in healthy tissues. Tumor-specific accumulation has been pursued with passive targeting by the enhanced permeability and retention effect, as well as with active targeting strategies. Active targeting is achieved by functionalization of carriers to allow specific interactions between the carrier and the tumor environment. Functionalization of carriers with ligands that specifically interact with overexpressed receptors on cancer cells represents a classic approach to active tumor targeting. Cell-penetrating peptides (CPPs) provide a non-specific and receptor-independent mechanism to enhance cellular uptake that offers an exciting alternative to traditional active targeting approaches. While the non-specificity of CPP-mediated internalization has the intriguing potential to make this approach applicable to a wide range of tumor types, their promiscuity is, however, a significant barrier to their clinical utility for systemically administered applications. Many approaches have been investigated to selectively turn on the function of systemically delivered CPP-functionalized carriers specifically in tumors to achieve targeted delivery of cancer therapeutics and imaging agents.

Authors
MacEwan, SR; Chilkoti, A
MLA Citation
MacEwan, SR, and Chilkoti, A. "Harnessing the power of cell-penetrating peptides: activatable carriers for targeting systemic delivery of cancer therapeutics and imaging agents." Wiley Interdiscip Rev Nanomed Nanobiotechnol 5.1 (January 2013): 31-48. (Review)
PMID
22977001
Source
pubmed
Published In
Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology
Volume
5
Issue
1
Publish Date
2013
Start Page
31
End Page
48
DOI
10.1002/wnan.1197

Effect of detergents on the thermal behavior of elastin-like polypeptides.

Elastin-like polypeptide (ELP) fusions have been designed to allow large-scale, nonchromatographic purification of many soluble proteins by using the inverse transition cycling (ITC) method; however, the sensitivity of the aqueous lower critical solubility phase transition temperature (T(t)) of ELPs to the addition of cosolutes, including detergents, may be a potential hindrance in purification of proteins with surface hydrophobicity in such a manner. To identify detergents that are known to solubilize such proteins (e.g., membrane proteins) and that have little effect on the T(t) of the ELP, we screened a number of detergents with respect to their effects on the T(t) and secondary structures of a model ELP (denoted here as ELP180). We found that mild detergents (e.g., n-dodecyl-β-D-maltoside, Triton-X100, and 3-[(3-cholamidopropyl) dimethylamino]-1-propanesulfonate) do not alter the phase transition behavior or structure (as probed by circular dichroism) of ELP180. This result is in contrast to previous studies that showed a strong effect of other detergents (e.g., sodium dodecylsulfate) on the T(t) of ELPs. Our results clearly indicate that mild detergents do not preclude ITC-based separation of ELPs, and thus that ELP fusions may prove to be useful in the purification of detergent-solubilized recombinant hydrophobic proteins, including membrane proteins, which are otherwise notoriously difficult to extract and purify by conventional separation methods (e.g., chromatography).

Authors
Thapa, A; Han, W; Simons, RH; Chilkoti, A; Chi, EY; López, GP
MLA Citation
Thapa, A, Han, W, Simons, RH, Chilkoti, A, Chi, EY, and López, GP. "Effect of detergents on the thermal behavior of elastin-like polypeptides." Biopolymers 99.1 (January 2013): 55-62.
PMID
23097230
Source
pubmed
Published In
Biopolymers
Volume
99
Issue
1
Publish Date
2013
Start Page
55
End Page
62
DOI
10.1002/bip.22137

Cylindrical polymer brushes with elastin-like polypeptide side chains

Monodisperse high molar mass elastin-like polypeptide macromonomers comprising 20 pentasequences (M = 8332 g/mol) were radically polymerized to high degrees of polymerization Pw = 590. Polymerization was conducted in water well above the lower phase transition temperature, i.e., in the phase separated regime. The resulting polymers adopt a cylindrical shape as demonstrated by AFM pictures of solutions spin-cast on mica. The directional persistence of the cylindrical brushes was determined by static light scattering to Kuhn statistical segments lengths lk = 120 nm at 5 mM aqueous NaCl solution which decreased to lk = 54 nm at 0.65 M NaCl. Upon polymerization the phase transition temperature drops significantly and the transition interval becomes sharper. The change of the hydrodynamic radius of the cylindrical brushes was monitored by dynamic light scattering as a function of temperature and revealed a continuous decrease from 20 to 36 C, above of which aggregates of several hundred nm in size start to form prior to phase separation. © 2013 American Chemical Society.

Authors
Weller, D; McDaniel, JR; Fischer, K; Chilkoti, A; Schmidt, M
MLA Citation
Weller, D, McDaniel, JR, Fischer, K, Chilkoti, A, and Schmidt, M. "Cylindrical polymer brushes with elastin-like polypeptide side chains." Macromolecules 46.12 (2013): 4966-4971.
Source
scival
Published In
Macromolecules
Volume
46
Issue
12
Publish Date
2013
Start Page
4966
End Page
4971
DOI
10.1021/ma400917t

Sortase-Catalyzed Initiator Attachment Enables High Yield Growth of a Stealth Polymer from the C Terminus of a Protein

Conventional methods for synthesizing protein/peptide-polymer conjugates, as a means to improve the pharmacological properties of therapeutic biomolecules, typically have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of site- specificity, which results in heterogeneous products with significantly compromised bioactivity. To address these limitations, the use of sortase A from Staphylococcus aureus is demonstrated to site-specifically attach an initiator solely at the C-terminus of green fluorescent protein (GFP), followed by in situ growth of a stealth polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeds with high specificity and near-complete (≈95%) product conversion. Subsequent in situ ATRP in aqueous buffer produces 1:1 stoichiometric conjugates with >90% yield, low dispersity, and no denaturation of the protein. This approach introduces a simple and useful method for high yield synthesis of protein/peptide-polymer conjugates. A general method is developed for the high-yield synthesis of C-terminally site-specific and one-to-one stoichiometric protein/peptide-polymer conjugates. Sortase A from Staphylococcus aureus is used to site-specifically attach an initiator solely at the C-terminus of green fluorescent protein, followed by in situ growth of a stealth polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) by atom transfer radical polymerization. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Authors
Qi, Y; Amiram, M; Gao, W; McCafferty, DG; Chilkoti, A
MLA Citation
Qi, Y, Amiram, M, Gao, W, McCafferty, DG, and Chilkoti, A. "Sortase-Catalyzed Initiator Attachment Enables High Yield Growth of a Stealth Polymer from the C Terminus of a Protein." Macromolecular Rapid Communications 34.15 (2013): 1256-1260.
Source
scival
Published In
Macromolecular Rapid Communications
Volume
34
Issue
15
Publish Date
2013
Start Page
1256
End Page
1260
DOI
10.1002/marc.201300460

Amplified fluorescence detection of DNA and RNA hybridization by surface initiated enzymatic polymerization (SIEP)

Detection of nucleic acid on-chip has been of great interest due to its potential as research tools and for diagnostic applications. We present a new on-chip, isothermal signal amplification technique for the direct detection of unmodified DNA and RNA on microarrays using terminal deoxy nucleotidyl transferase (TdT), a template-independent DNA polymerase that catalyzes the sequential addition of deoxynucleotides (dNTPs) at the 3'-OH group of a DNA primer and yeast poly(A) polymerase (PaP), an RNA polymerase that catalyzes polyadenylation at the 3'-OH an RNA primer. We utilized their ability to catalyze the formation of long polynucleotide and TdT's ability to incorporate unnatural nucleotide substrates such as fluorescent nucleotides (F-dNTP) into a long polymer chain of single stranded DNA (ssDNA) from the 3'-OH of a target DNA or RNA that is tethered on the surface through hybridization. We obtained dose reponse curves of hybridized DNA and RNA with LOD in -1-10 pM.

Authors
Tjong, V; Yu, H; Hucknall, A; Chilkoti, A
MLA Citation
Tjong, V, Yu, H, Hucknall, A, and Chilkoti, A. "Amplified fluorescence detection of DNA and RNA hybridization by surface initiated enzymatic polymerization (SIEP)." Technical Proceedings of the 2013 NSTI Nanotechnology Conference and Expo, NSTI-Nanotech 2013 3 (2013): 32-36.
Source
scival
Published In
Technical Proceedings of the 2013 NSTI Nanotechnology Conference and Expo, NSTI-Nanotech 2013
Volume
3
Publish Date
2013
Start Page
32
End Page
36

Plasmonic waveguide modes of film-coupled metallic nanocubes.

A metallic nanoparticle positioned over a metal film offers great advantages as a highly controllable system relevant for probing field-enhancement and other plasmonic effects. Because the size and shape of the gap between the nanoparticle and film can be controlled to subnanometer precision using relatively simple, bottom-up fabrication approaches, the film-coupled nanoparticle geometry has recently been applied to enhancing optical fields, accessing the quantum regime of plasmonics, and the design of surfaces with controlled reflectance. In the present work, we examine the plasmon modes associated with a silver nanocube positioned above a silver or gold film, separated by an organic, dielectric spacer layer. The film-coupled nanocube is of particular interest due to the formation of waveguide cavity-like modes between the nanocube and film. These modes impart distinctive scattering characteristics to the system that can be used in the creation of controlled reflectance surfaces and other applications. We perform both experimental spectroscopy and numerical simulations of individual nanocubes positioned over a metal film, finding excellent agreement between experiment and simulation. The waveguide mode description serves as a starting point to explain the optical properties observed.

Authors
Lassiter, JB; McGuire, F; Mock, JJ; Ciracì, C; Hill, RT; Wiley, BJ; Chilkoti, A; Smith, DR
MLA Citation
Lassiter, JB, McGuire, F, Mock, JJ, Ciracì, C, Hill, RT, Wiley, BJ, Chilkoti, A, and Smith, DR. "Plasmonic waveguide modes of film-coupled metallic nanocubes." Nano Lett 13.12 (2013): 5866-5872.
PMID
24199752
Source
pubmed
Published In
Nano Letters
Volume
13
Issue
12
Publish Date
2013
Start Page
5866
End Page
5872
DOI
10.1021/nl402660s

Controlled-reflectance surfaces with film-coupled colloidal nanoantennas.

Efficient and tunable absorption is essential for a variety of applications, such as designing controlled-emissivity surfaces for thermophotovoltaic devices, tailoring an infrared spectrum for controlled thermal dissipation and producing detector elements for imaging. Metamaterials based on metallic elements are particularly efficient as absorbing media, because both the electrical and the magnetic properties of a metamaterial can be tuned by structured design. So far, metamaterial absorbers in the infrared or visible range have been fabricated using lithographically patterned metallic structures, making them inherently difficult to produce over large areas and hence reducing their applicability. Here we demonstrate a simple method to create a metamaterial absorber by randomly adsorbing chemically synthesized silver nanocubes onto a nanoscale-thick polymer spacer layer on a gold film, making no effort to control the spatial arrangement of the cubes on the film. We show that the film-coupled nanocubes provide a reflectance spectrum that can be tailored by varying the geometry (the size of the cubes and/or the thickness of the spacer). Each nanocube is the optical analogue of a grounded patch antenna, with a nearly identical local field structure that is modified by the plasmonic response of the metal's dielectric function, and with an anomalously large absorption efficiency that can be partly attributed to an interferometric effect. The absorptivity of large surface areas can be controlled using this method, at scales out of reach of lithographic approaches (such as electron-beam lithography) that are otherwise required to manipulate matter on the nanoscale.

Authors
Moreau, A; Ciracì, C; Mock, JJ; Hill, RT; Wang, Q; Wiley, BJ; Chilkoti, A; Smith, DR
MLA Citation
Moreau, A, Ciracì, C, Mock, JJ, Hill, RT, Wang, Q, Wiley, BJ, Chilkoti, A, and Smith, DR. "Controlled-reflectance surfaces with film-coupled colloidal nanoantennas." Nature 492.7427 (December 6, 2012): 86-89.
PMID
23222613
Source
pubmed
Published In
Nature
Volume
492
Issue
7427
Publish Date
2012
Start Page
86
End Page
89
DOI
10.1038/nature11615

Brachytherapy using injectable seeds that are self-assembled from genetically encoded polypeptides in situ.

Brachytherapy is a common clinical technique involving implantation of sealed radioactive "seeds" within a tumor to selectively irradiate the tumor mass while minimizing systemic toxicity. To mitigate the disadvantages associated with complex surgical implantation and subsequent device removal procedures, we have developed an alternative approach using a genetically encoded peptide polymer solution composed of a thermally responsive elastin-like polypeptide (ELP) radiolabeled with (131)I that self-assembles into radionuclide seeds upon intratumoral injection. The formation of these nontoxic and biodegradable polymer seeds led to prolonged intratumoral retention (~85% ID/tumor 7 days postinjection) of the radionuclide, elicited a tumor growth delay in 100% of the tumors in two human xenografts (FaDu and PC-3), and cured more than 67% of tumor-bearing animals after a single administration of labeled ELP. These results suggest that in situ self-assembly of biodegradable and injectable radionuclide-containing polypeptide seeds could be a promising therapeutic alternative to conventional brachytherapy.

Authors
Liu, W; McDaniel, J; Li, X; Asai, D; Quiroz, FG; Schaal, J; Park, JS; Zalutsky, M; Chilkoti, A
MLA Citation
Liu, W, McDaniel, J, Li, X, Asai, D, Quiroz, FG, Schaal, J, Park, JS, Zalutsky, M, and Chilkoti, A. "Brachytherapy using injectable seeds that are self-assembled from genetically encoded polypeptides in situ." Cancer Res 72.22 (November 15, 2012): 5956-5965.
PMID
23155121
Source
pubmed
Published In
Cancer Research
Volume
72
Issue
22
Publish Date
2012
Start Page
5956
End Page
5965
DOI
10.1158/0008-5472.CAN-12-2127

Overcoming limitations in nanoparticle drug delivery: triggered, intravascular release to improve drug penetration into tumors.

Traditionally, the goal of nanoparticle-based chemotherapy has been to decrease normal tissue toxicity by improving drug specificity to tumors. The enhanced permeability and retention effect can permit passive accumulation into tumor interstitium. However, suboptimal delivery is achieved with most nanoparticles because of heterogeneities of vascular permeability, which limits nanoparticle penetration. Furthermore, slow drug release limits bioavailability. We developed a fast drug-releasing liposome triggered by local heat that has already shown substantial antitumor efficacy and is in human trials. Here, we show that thermally sensitive liposomes (Dox-TSL) release doxorubicin inside the tumor vasculature. Real-time confocal imaging of doxorubicin delivery to murine tumors in window chambers and histologic analysis of flank tumors illustrates that intravascular drug release increases free drug in the interstitial space. This increases both the time that tumor cells are exposed to maximum drug levels and the drug penetration distance, compared with free drug or traditional pegylated liposomes. These improvements in drug bioavailability establish a new paradigm in drug delivery: rapidly triggered drug release in the tumor bloodstream.

Authors
Manzoor, AA; Lindner, LH; Landon, CD; Park, J-Y; Simnick, AJ; Dreher, MR; Das, S; Hanna, G; Park, W; Chilkoti, A; Koning, GA; ten Hagen, TLM; Needham, D; Dewhirst, MW
MLA Citation
Manzoor, AA, Lindner, LH, Landon, CD, Park, J-Y, Simnick, AJ, Dreher, MR, Das, S, Hanna, G, Park, W, Chilkoti, A, Koning, GA, ten Hagen, TLM, Needham, D, and Dewhirst, MW. "Overcoming limitations in nanoparticle drug delivery: triggered, intravascular release to improve drug penetration into tumors." Cancer Res 72.21 (November 1, 2012): 5566-5575.
PMID
22952218
Source
pubmed
Published In
Cancer Research
Volume
72
Issue
21
Publish Date
2012
Start Page
5566
End Page
5575
DOI
10.1158/0008-5472.CAN-12-1683

Fabrication of ssDNA/oligo(ethylene glycol) monolayers and complex nanostructures by an irradiation-promoted exchange reaction.

Authors
Khan, MN; Tjong, V; Chilkoti, A; Zharnikov, M
MLA Citation
Khan, MN, Tjong, V, Chilkoti, A, and Zharnikov, M. "Fabrication of ssDNA/oligo(ethylene glycol) monolayers and complex nanostructures by an irradiation-promoted exchange reaction." Angew Chem Int Ed Engl 51.41 (October 8, 2012): 10303-10306.
PMID
22987725
Source
pubmed
Published In
Angewandte Chemie International Edition
Volume
51
Issue
41
Publish Date
2012
Start Page
10303
End Page
10306
DOI
10.1002/anie.201204245

Plasmon ruler with angstrom length resolution.

We demonstrate a plasmon nanoruler using a coupled film nanoparticle (film-NP) format that is well-suited for investigating the sensitivity extremes of plasmonic coupling. Because it is relatively straightforward to functionalize bulk surface plasmon supporting films, such as gold, we are able to precisely control plasmonic gap dimensions by creating ultrathin molecular spacer layers on the gold films, on top of which we immobilize plasmon resonant nanoparticles (NPs). Each immobilized NP becomes coupled to the underlying film and functions as a plasmon nanoruler, exhibiting a distance-dependent resonance red shift in its peak plasmon wavelength as it approaches the film. Due to the uniformity of response from the film-NPs to separation distance, we are able to use extinction and scattering measurements from ensembles of film-NPs to characterize the coupling effect over a series of very short separation distances-ranging from 5 to 20 Å-and combine these measurements with similar data from larger separation distances extending out to 27 nm. We find that the film-NP plasmon nanoruler is extremely sensitive at very short film-NP separation distances, yielding spectral shifts as large as 5 nm for every 1 Å change in separation distance. The film-NP coupling at extremely small spacings is so uniform and reliable that we are able to usefully probe gap dimensions where the classical Drude model of the conducting electrons in the metals is no longer descriptive; for gap sizes smaller than a few nanometers, either quantum or semiclassical models of the carrier response must be employed to predict the observed wavelength shifts. We find that, despite the limitations, large field enhancements and extreme sensitivity persist down to even the smallest gap sizes.

Authors
Hill, RT; Mock, JJ; Hucknall, A; Wolter, SD; Jokerst, NM; Smith, DR; Chilkoti, A
MLA Citation
Hill, RT, Mock, JJ, Hucknall, A, Wolter, SD, Jokerst, NM, Smith, DR, and Chilkoti, A. "Plasmon ruler with angstrom length resolution." ACS nano 6.10 (October 2012): 9237-9246.
PMID
22966857
Source
epmc
Published In
ACS Nano
Volume
6
Issue
10
Publish Date
2012
Start Page
9237
End Page
9246
DOI
10.1021/nn3035809

Uptake of silver nanoparticles and toxicity to early life stages of Japanese medaka (Oryzias latipes): effect of coating materials.

Silver nanoparticles (AgNPs) with antimicrobial properties are perhaps the most deployed engineered nanomaterials in consumer products. Almost all AgNPs are coated with organic materials to enhance their dispersion in water. Contributions of coatings to the toxicity of NPs have received little attention. Studies using AgNPs with one of three different coating materials (citrate (Cit), gum arabic (GA), and polyvinylpyrrolidone (PVP)) showed significantly different toxicity. GA AgNP proved to be the most toxic, while PVP and Cit AgNP exhibited similar and lower toxicity. However, all AgNPs were about three to ten times less toxic than AgNO(3) when their toxicities were compared on a mass-concentration basis. Evidence for NP-specific toxicity was observed with longer time for initiation of toxicity and increased incidence of resultant spinal flexure of medaka exposed to AgNPs, compared to AgNO(3). Hyperspectral imaging of 6 μm paraffin sections of fish exposed to AgNPs revealed AgNPs and their aggregates in tissues of fish. Gill distribution was ubiquitous, while small amounts were found in other organs, including the liver and brain. AgNPs were observed regularly in the gut lumen, but rarely in mural elements and mesentery. These results suggest that while ingestion was common, gills were the principal sites of AgNP uptake. In conclusion, AgNPs is a source of toxic Ag ions, while itself contribute partially to its toxicity to fish, and which interact with skin surface and were taken up via the gills.

Authors
Kwok, KWH; Auffan, M; Badireddy, AR; Nelson, CM; Wiesner, MR; Chilkoti, A; Liu, J; Marinakos, SM; Hinton, DE
MLA Citation
Kwok, KWH, Auffan, M, Badireddy, AR, Nelson, CM, Wiesner, MR, Chilkoti, A, Liu, J, Marinakos, SM, and Hinton, DE. "Uptake of silver nanoparticles and toxicity to early life stages of Japanese medaka (Oryzias latipes): effect of coating materials." Aquat Toxicol 120-121 (September 15, 2012): 59-66.
PMID
22634717
Source
pubmed
Published In
Aquatic Toxicology
Volume
120-121
Publish Date
2012
Start Page
59
End Page
66
DOI
10.1016/j.aquatox.2012.04.012

Biomolecular toolbox for the in situ synthesis of protein-polymer conjugates

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "Biomolecular toolbox for the in situ synthesis of protein-polymer conjugates." August 19, 2012.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
244
Publish Date
2012

Rheological properties of cysteine-containing elastin-like polypeptide solutions and hydrogels.

The rheological properties of cysteine-containing elastin-like polypeptide (Cys-ELP) solutions and Cys-ELP hydrogels are reported. The Cys-ELP solutions exhibit a surprisingly high apparent viscosity at low shear rate. The high viscosity is attributed to the formation of an interfacial cross-linked "skin" at the sample surface, rather than the bulk of the Cys-ELP solution. At higher shear rate, the interfacial cross-linked film breaks, and its influence on the viscosity of the Cys-ELP solution can be ignored. Cys-ELP hydrogels are formed by mixing Cys-ELP and hydrogen peroxide (H(2)O(2)). At fixed concentration of Cys-ELP, the gelation time can be tuned by the concentration of H(2)O(2). Cys-ELP hydrogels have the typical characteristics of covalent cross-linked networks, as the storage moduli are larger than the loss moduli and are independent of frequency in dynamic oscillatory frequency sweep experiments. The plateau moduli obtained from linear frequency sweep experiments are much lower than those estimated from the number of thiol groups along the Cys-ELP chain, indicating that only a small fraction of thiols form elastically active cross-links. From the small value of the fraction of elastically active cross-links, the Cys-ELP hydrogel is concluded to be an inhomogenous network. Under steady shear, a 2.5 wt % Cys-ELP hydrogel shear thickens at shear rates lower than that necessary for fracture.

Authors
Xu, D; Asai, D; Chilkoti, A; Craig, SL
MLA Citation
Xu, D, Asai, D, Chilkoti, A, and Craig, SL. "Rheological properties of cysteine-containing elastin-like polypeptide solutions and hydrogels." Biomacromolecules 13.8 (August 13, 2012): 2315-2321.
PMID
22789001
Source
pubmed
Published In
Biomacromolecules
Volume
13
Issue
8
Publish Date
2012
Start Page
2315
End Page
2321
DOI
10.1021/bm300760s

Probing the ultimate limits of plasmonic enhancement.

Metals support surface plasmons at optical wavelengths and have the ability to localize light to subwavelength regions. The field enhancements that occur in these regions set the ultimate limitations on a wide range of nonlinear and quantum optical phenomena. We found that the dominant limiting factor is not the resistive loss of the metal, but rather the intrinsic nonlocality of its dielectric response. A semiclassical model of the electronic response of a metal places strict bounds on the ultimate field enhancement. To demonstrate the accuracy of this model, we studied optical scattering from gold nanoparticles spaced a few angstroms from a gold film. The bounds derived from the models and experiments impose limitations on all nanophotonic systems.

Authors
Ciracì, C; Hill, RT; Mock, JJ; Urzhumov, Y; Fernández-Domínguez, AI; Maier, SA; Pendry, JB; Chilkoti, A; Smith, DR
MLA Citation
Ciracì, C, Hill, RT, Mock, JJ, Urzhumov, Y, Fernández-Domínguez, AI, Maier, SA, Pendry, JB, Chilkoti, A, and Smith, DR. "Probing the ultimate limits of plasmonic enhancement." Science (New York, N.Y.) 337.6098 (August 2012): 1072-1074.
Website
http://hdl.handle.net/10161/7576
PMID
22936772
Source
epmc
Published In
Science
Volume
337
Issue
6098
Publish Date
2012
Start Page
1072
End Page
1074
DOI
10.1126/science.1224823

Chemistry. Nanomaterials for drug delivery.

Authors
Hubbell, JA; Chilkoti, A
MLA Citation
Hubbell, JA, and Chilkoti, A. "Chemistry. Nanomaterials for drug delivery." Science 337.6092 (July 20, 2012): 303-305.
PMID
22822138
Source
pubmed
Published In
Science
Volume
337
Issue
6092
Publish Date
2012
Start Page
303
End Page
305
DOI
10.1126/science.1219657

Protein polymer hydrogels by in situ, rapid and reversible self-gelation.

Protein-based biomaterials are an important class of materials for applications in biotechnology and medicine. The exquisite control of their composition, stereochemistry, and chain length offers unique opportunities to engineer biofunctionality, biocompatibility, and biodegradability into these materials. Here, we report the synthesis of a thermally responsive peptide polymer-based hydrogel composed of a recombinant elastin-like polypeptide (ELP) that rapidly forms a reversibly cross-linked hydrogel by the formation of intermolecular disulfide cross-links. To do so, we designed and synthesized ELPs that incorporate periodic cysteine residues (cELPs), and show that cELPs are thermally responsive protein polymers that display rapid gelation under physiologically relevant, mild oxidative conditions. Gelation of cELPs, at concentrations as low as 2.5 wt%, occurs in ≈ 2.5 min upon addition a low concentration of hydrogen peroxide (0.3 wt%). We show the utility of these hydrogels for the sustained release of a model protein in vitro, and demonstrate the ability of this injectable biomaterial to pervade tumors to maximize tumor coverage and retention time upon intratumoral injection. cELPs represent a new class of injectable reversibly cross-linked hydrogels with properties intermediate between ELP coacervates and chemically cross-linked ELP hydrogels that will find useful applications in drug delivery and tissue engineering.

Authors
Asai, D; Xu, D; Liu, W; Garcia Quiroz, F; Callahan, DJ; Zalutsky, MR; Craig, SL; Chilkoti, A
MLA Citation
Asai, D, Xu, D, Liu, W, Garcia Quiroz, F, Callahan, DJ, Zalutsky, MR, Craig, SL, and Chilkoti, A. "Protein polymer hydrogels by in situ, rapid and reversible self-gelation." Biomaterials 33.21 (July 2012): 5451-5458.
PMID
22538198
Source
pubmed
Published In
Biomaterials
Volume
33
Issue
21
Publish Date
2012
Start Page
5451
End Page
5458
DOI
10.1016/j.biomaterials.2012.03.083

Digital switching of local arginine density in a genetically encoded self-assembled polypeptide nanoparticle controls cellular uptake.

Cell-penetrating peptides (CPPs) are a class of molecules that enable efficient internalization of a wide variety of cargo in diverse cell types, making them desirable for delivery of anticancer drugs to solid tumors. For CPPs to be useful, it is important to be able to turn their function on in response to an external trigger that can be spatially localized in vivo. Here we describe an approach to turning on CPP function by modulation of the local density of arginine (Arg) residues by temperature-triggered micelle assembly of diblock copolymer elastin-like polypeptides (ELP(BC)s). A greater than 8-fold increase in cellular uptake occurs when Arg residues are presented on the corona of ELP(BC) micelles, as compared to the same ELP(BC) at a temperature in which it is a soluble unimer. This approach is the first to demonstrate digital 'off-on' control of CPP activity by an extrinsic thermal trigger in a clinically relevant temperature range by modulation of the interfacial density of Arg residues on the exterior of a nanoparticle.

Authors
Macewan, SR; Chilkoti, A
MLA Citation
Macewan, SR, and Chilkoti, A. "Digital switching of local arginine density in a genetically encoded self-assembled polypeptide nanoparticle controls cellular uptake." Nano Lett 12.6 (June 13, 2012): 3322-3328.
PMID
22625178
Source
pubmed
Published In
Nano Letters
Volume
12
Issue
6
Publish Date
2012
Start Page
3322
End Page
3328
DOI
10.1021/nl301529p

Unexpected multivalent display of proteins by temperature triggered self-assembly of elastin-like polypeptide block copolymers.

We report herein the unexpected temperature triggered self-assembly of proteins fused to thermally responsive elastin-like polypeptides (ELPs) into spherical micelles. A set of six ELP block copolymers (ELP(BC)) differing in hydrophilic and hydrophobic block lengths were genetically fused to two single domain proteins, thioredoxin (Trx) and a fibronectin type III domain (Fn3) that binds the α(v)β(3) integrin. The self-assembly of these protein-ELP(BC) fusions as a function of temperature was investigated by UV spectroscopy, light scattering, and cryo-TEM. Self-assembly of the ELP(BC) was unexpectedly retained upon fusion to the two proteins, resulting in the formation of spherical micelles with a hydrodynamic radius that ranged from 24 to 37 nm, depending on the protein and ELP(BC). Cryo-TEM images confirmed the formation of spherical particles with a size that was consistent with that measured by light scattering. The bioactivity of Fn3 was retained when presented by the ELP(BC) micelles, as indicated by the enhanced uptake of the Fn3-decorated ELP(BC) micelles in comparison to the unimer by cells that overexpress the α(v)β(3) integrin. The fusion of single domain proteins to ELP(BC)s may provide a ubiquitous platform for the multivalent presentation of proteins.

Authors
Hassouneh, W; Fischer, K; MacEwan, SR; Branscheid, R; Fu, CL; Liu, R; Schmidt, M; Chilkoti, A
MLA Citation
Hassouneh, W, Fischer, K, MacEwan, SR, Branscheid, R, Fu, CL, Liu, R, Schmidt, M, and Chilkoti, A. "Unexpected multivalent display of proteins by temperature triggered self-assembly of elastin-like polypeptide block copolymers." Biomacromolecules 13.5 (May 14, 2012): 1598-1605.
PMID
22515311
Source
pubmed
Published In
Biomacromolecules
Volume
13
Issue
5
Publish Date
2012
Start Page
1598
End Page
1605
DOI
10.1021/bm300321n

Doxorubicin-conjugated chimeric polypeptide nanoparticles that respond to mild hyperthermia.

This paper reports the design, physicochemical characterization and in vitro cytotoxicity of a thermally responsive chimeric polypeptide (CP), derived from an elastin-like polypeptide (ELP). The CP self-assembles into ~40 nm diameter nanoparticles upon conjugation of multiple copies of doxorubicin (Dox), and displays a nanoparticle-to-aggregate phase transition between 39 and 42 °C in media, a temperature range suitable for mild hyperthermia of solid tumors. The CP-Dox nanoparticle is stable upon dilution to low micromolar concentrations, and is cytotoxic at both 37 and 42 °C. A thermally responsive nanoparticle formulation of Dox may prove to be broadly useful in hyperthermia targeted chemotherapy of a variety of solid tumors.

Authors
McDaniel, JR; Macewan, SR; Dewhirst, M; Chilkoti, A
MLA Citation
McDaniel, JR, Macewan, SR, Dewhirst, M, and Chilkoti, A. "Doxorubicin-conjugated chimeric polypeptide nanoparticles that respond to mild hyperthermia." J Control Release 159.3 (May 10, 2012): 362-367.
PMID
22421424
Source
pubmed
Published In
Journal of Controlled Release
Volume
159
Issue
3
Publish Date
2012
Start Page
362
End Page
367
DOI
10.1016/j.jconrel.2012.02.030

Probing dynamically tunable localized surface plasmon resonances of film-coupled nanoparticles by evanescent wave excitation.

The localized surface plasmon resonance (LSPR) spectrum associated with a gold nanoparticle (NP) coupled to a gold film exhibits extreme sensitivity to the nanogap region where the fields are tightly localized. The LSPR of an ensemble of film-coupled NPs can be observed using an illumination scheme similar to that used to excite the surface plasmon resonance (SPR) of a thin metallic film; however, in the present system, the light is used to probe the highly sensitive distance-dependent LSPR of the gaps between NPs and film rather than the delocalized SPR of the film. We show that the SPR and LSPR spectral contributions can be readily distinguished, and we compare the sensitivities of both modes to displacements in the average gap between a collection of NPs and the gold film. The distance by which the NPs are suspended in solution above the gold film is fixed via a thin molecular spacer layer and can be further modulated by subjecting the NPs to a quasistatic electric field. The observed LSPR spectral shifts triggered by the applied voltage can be correlated with angstrom scale displacements of the NPs, suggesting the potential for chip-scale or flow-cell plasmonic nanoruler devices with extreme sensitivity.

Authors
Mock, JJ; Hill, RT; Tsai, Y-J; Chilkoti, A; Smith, DR
MLA Citation
Mock, JJ, Hill, RT, Tsai, Y-J, Chilkoti, A, and Smith, DR. "Probing dynamically tunable localized surface plasmon resonances of film-coupled nanoparticles by evanescent wave excitation." Nano Lett 12.4 (April 11, 2012): 1757-1764.
PMID
22429053
Source
pubmed
Published In
Nano Letters
Volume
12
Issue
4
Publish Date
2012
Start Page
1757
End Page
1764
DOI
10.1021/nl204596h

Triple stimulus-responsive polypeptide nanoparticles that enhance intratumoral spatial distribution.

To address the limited tumor penetration of nanoparticle drug delivery vehicles, we report the first pH-responsive polypeptide micelle that dissociates at the low extracellular pH of solid tumors. This histidine-rich elastin-like polypeptide block copolymer self-assembles at 37 °C into spherical micelles that are stabilized by Zn(2+) and are disrupted as the pH drops from 7.4 to 6.4. These pH-sensitive micelles demonstrate better in vivo penetration and distribution in tumors than a pH-insensitive control.

Authors
Callahan, DJ; Liu, W; Li, X; Dreher, MR; Hassouneh, W; Kim, M; Marszalek, P; Chilkoti, A
MLA Citation
Callahan, DJ, Liu, W, Li, X, Dreher, MR, Hassouneh, W, Kim, M, Marszalek, P, and Chilkoti, A. "Triple stimulus-responsive polypeptide nanoparticles that enhance intratumoral spatial distribution." Nano Lett 12.4 (April 11, 2012): 2165-2170.
PMID
22417133
Source
pubmed
Published In
Nano Letters
Volume
12
Issue
4
Publish Date
2012
Start Page
2165
End Page
2170
DOI
10.1021/nl300630c

Field-induced nanolithography for the patterning of non-fouling polymer brushes

Authors
Ferris, R; Hucknall, A; Kwon, BS; Chilkoti, A; Zauscher, S
MLA Citation
Ferris, R, Hucknall, A, Kwon, BS, Chilkoti, A, and Zauscher, S. "Field-induced nanolithography for the patterning of non-fouling polymer brushes." March 25, 2012.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
243
Publish Date
2012

Genetically encoded stimulus responsive elastin-like polypeptides: Applications in drug delivery

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "Genetically encoded stimulus responsive elastin-like polypeptides: Applications in drug delivery." March 25, 2012.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
243
Publish Date
2012

Conjugates of polypeptides and synthetic polymers with oligonucleotides: Stars, micelles and multiblockcopolymers

Authors
Fluegel, S; Kiefer, T; Heimann, N; Buehler, J; Fischer, K; McDaniel, J; Chilkoti, A; Schmidt, M
MLA Citation
Fluegel, S, Kiefer, T, Heimann, N, Buehler, J, Fischer, K, McDaniel, J, Chilkoti, A, and Schmidt, M. "Conjugates of polypeptides and synthetic polymers with oligonucleotides: Stars, micelles and multiblockcopolymers." March 25, 2012.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
243
Publish Date
2012

Stimulus-Responsive Polymers as Intelligent Coatings for Biosensors: Architectures, Response Mechanisms, and Applications

Authors
Tjong, V; Zhang, J; Chilkoti, A; Zauscher, S
MLA Citation
Tjong, V, Zhang, J, Chilkoti, A, and Zauscher, S. "Stimulus-Responsive Polymers as Intelligent Coatings for Biosensors: Architectures, Response Mechanisms, and Applications." (February 3, 2012): 1-30. (Chapter)
Source
scopus
Publish Date
2012
Start Page
1
End Page
30
DOI
10.1002/9781118181249.ch1

Protein switches

Authors
Christensen, T; Hassouneh, W; Callahan, DJ; Chilkoti, A
MLA Citation
Christensen, T, Hassouneh, W, Callahan, DJ, and Chilkoti, A. "Protein switches." Comprehensive Biophysics 3 (2012): 238-266.
Source
scival
Published In
Comprehensive Biophysics
Volume
3
Publish Date
2012
Start Page
238
End Page
266
DOI
10.1016/B978-0-12-374920-8.00316-7

Fusions of elastin-like polypeptides to pharmaceutical proteins.

Elastin-like polypeptides (ELPs) are a class of stimulus-responsive biopolymers whose physicochemical properties and biocompatibility are particularly suitable for in vivo applications, such as drug delivery and tissue engineering. The lower critical solution temperature (LCST) behavior of ELPs allows them to be utilized as soluble macromolecules below their LCST, or as self-assembled nanoscale particles such as micelles, micron-scale coacervates, or viscous gels above their LCST, depending on the ELP architecture. As each ELP sequence is specified at its genetic level, functionalization of an ELP with peptides and proteins is simple to accomplish by the fusion of a gene encoding an ELP with that of the peptide or protein of interest. Protein ELP fusions, where the appended protein serves a therapeutic or targeting function, are suitable for applications in which the ELP can improve the systemic pharmacokinetics and biodistribution of the protein, or can be used as an injectable depot for sustained, local protein delivery. Here we describe considerations in the design of therapeutic protein ELP fusions and provide details of their gene design, expression, and purification.

Authors
Hassouneh, W; MacEwan, SR; Chilkoti, A
MLA Citation
Hassouneh, W, MacEwan, SR, and Chilkoti, A. "Fusions of elastin-like polypeptides to pharmaceutical proteins." Methods Enzymol 502 (2012): 215-237.
PMID
22208987
Source
pubmed
Published In
Methods in Enzymology
Volume
502
Publish Date
2012
Start Page
215
End Page
237
DOI
10.1016/B978-0-12-416039-2.00024-0

Raman antenna formed by molecule/plasmonic nanostructure hybrid system

A nano-antenna composed of a particle and a polarizable surface provides control of the spatial distribution and high enhancement of Raman scattering. This structure may serve as a stable platform for single molecule detection. © 2010 Optical Society of America.

Authors
Chen, SY; Mock, JJ; Hill, RT; Chilkoti, A; Smith, DR; Lazarides, AA
MLA Citation
Chen, SY, Mock, JJ, Hill, RT, Chilkoti, A, Smith, DR, and Lazarides, AA. "Raman antenna formed by molecule/plasmonic nanostructure hybrid system." Optics InfoBase Conference Papers (December 1, 2011).
Source
scopus
Published In
Optics InfoBase Conference Papers
Publish Date
2011

Field-induced nanolithography for patterning of non-fouling polymer brush surfaces.

Authors
Ferris, R; Hucknall, A; Kwon, BS; Chen, T; Chilkoti, A; Zauscher, S
MLA Citation
Ferris, R, Hucknall, A, Kwon, BS, Chen, T, Chilkoti, A, and Zauscher, S. "Field-induced nanolithography for patterning of non-fouling polymer brush surfaces." Small 7.21 (November 4, 2011): 3032-3037.
PMID
21901825
Source
pubmed
Published In
Small
Volume
7
Issue
21
Publish Date
2011
Start Page
3032
End Page
3037
DOI
10.1002/smll.201100923

Hyperspectral molecular imaging of multiple receptors using immunolabeled plasmonic nanoparticles.

This work presents simultaneous imaging and detection of three different cell receptors using three types of plasmonic nanoparticles (NPs). The size, shape, and composition-dependent scattering profiles of these NPs allow for a system of multiple distinct molecular markers using a single optical source. With this goal in mind, tags consisting of anti-epidermal growth factor receptor gold nanorods, anti-insulin-like growth factor 1-R silver nanospheres, and human epidermal growth factor receptor 2Ab gold nanospheres were developed to monitor the expression of receptors commonly overexpressed by cancer cells. These labels were chosen because they scatter strongly in distinct spectral windows. A hyperspectral darkfield microspectroscopy system was developed to record the scattering spectra of cells labeled with these molecular tags. Simultaneous monitoring of multiple tags may lead to applications such as profiling of cell line immunophenotype and investigation of receptor signaling pathways. Single, dual, and triple tag experiments were performed to analyze NP tag specificity as well as their interactions. Distinct resonance peaks were observed in these studies, showing the ability to characterize cell lines using conjugated NPs. However, interpreting shifts in these peaks due to changes in a cellular dielectric environment may be complicated by plasmon coupling between NPs bound to proximal receptors and other coupling mechanisms due to the receptors themselves.

Authors
Seekell, K; Crow, MJ; Marinakos, S; Ostrander, J; Chilkoti, A; Wax, A
MLA Citation
Seekell, K, Crow, MJ, Marinakos, S, Ostrander, J, Chilkoti, A, and Wax, A. "Hyperspectral molecular imaging of multiple receptors using immunolabeled plasmonic nanoparticles." J Biomed Opt 16.11 (November 2011): 116003-.
PMID
22112108
Source
pubmed
Published In
Journal of Biomedical Optics
Volume
16
Issue
11
Publish Date
2011
Start Page
116003
DOI
10.1117/1.3646529

In vivo tumor targeting by a NGR-decorated micelle of a recombinant diblock copolypeptide.

Antivascular targeting is a promising strategy for tumor therapy. This strategy has the potential to overcome many of the transport barriers associated with targeting tumor cells in solid tumors, because the tumor vasculature is directly accessible to targeting vehicles in systemic circulation. We report a novel nanoscale delivery system consisting of multivalent polymer micelles to target receptors that are preferentially upregulated in the tumor vasculature and perivascular cells, specifically CD13. To this end we utilized amphiphilic block copolymers, composed of a genetically engineered elastin-like polypeptide (ELP) that self-assemble into monodisperse spherical micelles. These polymer micelles were functionalized by incorporating the NGR tripeptide ligand, which targets the CD13 receptor, on their corona. We examined the self-assembly and in vivo tumor targeting by these NGR-functionalized nanoparticles and show that multivalent presentation of NGR by micelle self-assembly selectively targets the tumor vasculature by targeting CD13. Furthermore, we show greater vascular retention and extravascular accumulation of nanoparticles in tumor tissue compared to normal tissue, although the enhancement is modest. These results suggest that enhanced delivery to solid tumors can be achieved by targeting upregulated receptors in the tumor vasculature with multivalent ligand-presenting nanoparticles, but additional work is required to optimize such systems for multivalent targeting.

Authors
Simnick, AJ; Amiram, M; Liu, W; Hanna, G; Dewhirst, MW; Kontos, CD; Chilkoti, A
MLA Citation
Simnick, AJ, Amiram, M, Liu, W, Hanna, G, Dewhirst, MW, Kontos, CD, and Chilkoti, A. "In vivo tumor targeting by a NGR-decorated micelle of a recombinant diblock copolypeptide." J Control Release 155.2 (October 30, 2011): 144-151.
PMID
21763734
Source
pubmed
Published In
Journal of Controlled Release
Volume
155
Issue
2
Publish Date
2011
Start Page
144
End Page
151
DOI
10.1016/j.jconrel.2011.06.044

Dual-order snapshot spectral imaging of plasmonic nanoparticles.

The development of truly scalable, multiplexed optical microarrays requires a detection platform capable of simultaneous detection of multiple signals in real-time. We present a technique we term dual-order snapshot spectroscopic imaging (DOSSI) and demonstrate that it can be effectively used to collect spectrally resolved images of a full field of view of sparsely located spots in real time. Resonant peaks of plasmonic gold nanoparticles were tracked as a function of their surrounding refractive index. Measurement uncertainty analysis indicated that the spectral resolution of DOSSI in the described configuration is approximately 0.95 nm. Further, real-time measurements by DOSSI allowed discrimination between optically identical nanoparticles that were functionalized with two homologous small molecule ligands that bound to the same protein, albeit with different affinity, based purely on their different molecular interaction kinetics-a feat not possible with slower raster-type hyperspectral imaging systems, or other dark-field optical detection systems that solely rely on end point measurements. Kinetic measurements of plasmon bands by DOSSI can be performed with a relatively simple optical system, thereby opening up the possibility of developing low-cost detectors for arrayed plasmonic diagnostics.

Authors
Nusz, GJ; Marinakos, SM; Rangarajan, S; Chilkoti, A
MLA Citation
Nusz, GJ, Marinakos, SM, Rangarajan, S, and Chilkoti, A. "Dual-order snapshot spectral imaging of plasmonic nanoparticles." Appl Opt 50.21 (July 20, 2011): 4198-4206.
PMID
21772408
Source
pubmed
Published In
Applied Optics
Volume
50
Issue
21
Publish Date
2011
Start Page
4198
End Page
4206

Amplified on-chip fluorescence detection of DNA hybridization by surface-initiated enzymatic polymerization.

We describe the incorporation of multiple fluorophores into a single stranded DNA (ssDNA) chain using terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase that catalyzes the sequential addition of deoxynucleotides (dNTPs) at the 3'-OH group of an oligonucleotide primer; we term this methodology surface initiated enzymatic polymerization (SIEP) of DNA. We found that long (>1 Kb) ssDNA homopolymer can be grown by SIEP, and that the length of the ssDNA product is determined by the monomer to oligonucleotide initiator ratio. We observed efficient initiation (≥50%) and narrow polydispersity of the extended product when fluorescently labeled nucleotides are incorporated. TdT's ability to incorporate fluorescent dNTPs into a ssDNA chain was characterized by examining the effect of the molar ratios of fluorescent dNTP to natural dNTP on the degree of fluorophore incorporation and the length of the polymerized DNA strand. These experiments allowed us to optimize the polymerization conditions to incorporate up to ~50 fluorescent Cy3-labeled dNTPs per kilobase into a ssDNA chain. With the goal of using TdT as an on-chip labeling method, we also quantified TdT mediated signal amplification on the surface by immobilizing ssDNA oligonucleotide initiators on a glass surface followed by SIEP of DNA. The incorporation of multiple fluorophores into the extended DNA chain by SIEP translated to a ~45 fold signal amplification compared to the incorporation of a single fluorophore. SIEP was then employed to detect hybridization of DNA, by the posthybridization, on-chip polymerization of fluorescently labeled ssDNA that was grown from the 3'-OH of target strands that hybridized to DNA probes that were printed on a surface. A dose-response curve for detection of DNA hybridization by SIEP was generated, with a ~1 pM limit of detection and a linear dynamic range of 2 logs.

Authors
Tjong, V; Yu, H; Hucknall, A; Rangarajan, S; Chilkoti, A
MLA Citation
Tjong, V, Yu, H, Hucknall, A, Rangarajan, S, and Chilkoti, A. "Amplified on-chip fluorescence detection of DNA hybridization by surface-initiated enzymatic polymerization." Anal Chem 83.13 (July 1, 2011): 5153-5159.
PMID
21604676
Source
pubmed
Published In
Analytical Chemistry
Volume
83
Issue
13
Publish Date
2011
Start Page
5153
End Page
5159
DOI
10.1021/ac200946t

Self-assembly of monodisperse oligonucleotide-elastin block copolymers into stars and compound micelles.

Authors
Fluegel, S; Buehler, J; Fischer, K; McDaniel, JR; Chilkoti, A; Schmidt, M
MLA Citation
Fluegel, S, Buehler, J, Fischer, K, McDaniel, JR, Chilkoti, A, and Schmidt, M. "Self-assembly of monodisperse oligonucleotide-elastin block copolymers into stars and compound micelles." Chemistry 17.20 (May 9, 2011): 5503-5506.
PMID
21469235
Source
pubmed
Published In
Chemistry - A European Journal
Volume
17
Issue
20
Publish Date
2011
Start Page
5503
End Page
5506
DOI
10.1002/chem.201100436

Micro- and nanostructured poly[oligo(ethylene glycol)methacrylate] brushes grown from photopatterned halogen initiators by atom transfer radical polymerization.

Photolithographic techniques have been used to fabricate polymer brush micro- and nanostructures. On exposure to UV light with a wavelength of 244 nm, halogens were selectively removed from films of chloromethylphenyltrichlorosilane and 3-(2-bromoisobutyramido)propyl-triethoxysilane on silicon dioxide. Patterning was achieved at the micrometer scale, by using a mask in conjunction with the incident laser beam, and at the nanometer scale, by utilizing interferometric lithography (IL). Friction force microscopy images of patterned surfaces exhibited frictional contrast due to removal of the halogen but no topographical contrast. In both cases the halogenated surface was used as an initiator for surface atom-transfer radical polymerization. Patterning of the surface by UV lithography enabled the definition of patterns of initiator from which micro- and nanostructured poly[oligo(ethylene glycol)methacrylate] bottle brushes were grown. Micropatterned brushes formed on both surfaces exhibited excellent resistance to protein adsorption, enabling the formation of protein patterns. Using IL, brush structures were formed that covered macroscopic areas (approximately 0.5 cm²) but exhibited a full width at half maximum height as small as 78 nm, with a period of 225 nm. Spatially selective photolytic removal of halogens that are immobilized on a surface thus appears to be a simple, rapid, and versatile method for the formation of micro- and nanostructured polymer brushes and for the control of protein adsorption.

Authors
Ahmad, SA; Leggett, GJ; Hucknall, A; Chilkoti, A
MLA Citation
Ahmad, SA, Leggett, GJ, Hucknall, A, and Chilkoti, A. "Micro- and nanostructured poly[oligo(ethylene glycol)methacrylate] brushes grown from photopatterned halogen initiators by atom transfer radical polymerization." Biointerphases 6.1 (March 2011): 8-15.
PMID
21428690
Source
pubmed
Published In
Biointerphases: an open access journal for the biomaterials interface community
Volume
6
Issue
1
Publish Date
2011
Start Page
8
End Page
15
DOI
10.1116/1.3553579

Spatially addressable chemoselective C-terminal ligation of an intein fusion protein from a complex mixture to a hydrazine-terminated surface.

Protein immobilization on surfaces is useful in many areas of research, including biological characterization, antibody purification, and clinical diagnostics. A critical limitation in the development of protein microarrays and heterogeneous protein-based assays is the enormous amount of work and associated costs in the purification of proteins prior to their immobilization onto a surface. Methods to address this problem would simplify the development of interfacial diagnostics that use a protein as the recognition element. Herein, we describe an approach for the facile, site-specific immobilization of proteins on a surface without any preprocessing or sample purification steps that ligates an intein fusion protein at its C-terminus by reaction with a hydrazine group presented by a surface. Furthermore, we demonstrate that this methodology can directly immobilize a protein directly from cell lysate onto a protein-resistant surface. This methodology is also compatible with soft lithography and inkjet printing so that one or more proteins can be patterned on a surface without the need for purification.

Authors
Yang, P; Marinakos, SM; Chilkoti, A
MLA Citation
Yang, P, Marinakos, SM, and Chilkoti, A. "Spatially addressable chemoselective C-terminal ligation of an intein fusion protein from a complex mixture to a hydrazine-terminated surface." Langmuir 27.4 (February 15, 2011): 1463-1471.
PMID
21142101
Source
pubmed
Published In
Langmuir
Volume
27
Issue
4
Publish Date
2011
Start Page
1463
End Page
1471
DOI
10.1021/la104186n

A highly parallel method for synthesizing DNA repeats enables the discovery of 'smart' protein polymers.

Robust high-throughput synthesis methods are needed to expand the repertoire of repetitive protein-polymers for different applications. To address this need, we developed a new method, overlap extension rolling circle amplification (OERCA), for the highly parallel synthesis of genes encoding repetitive protein-polymers. OERCA involves a single PCR-type reaction for the rolling circle amplification of a circular DNA template and simultaneous overlap extension by thermal cycling. We characterized the variables that control OERCA and demonstrated its superiority over existing methods, its robustness, high-throughput and versatility by synthesizing variants of elastin-like polypeptides (ELPs) and protease-responsive polymers of glucagon-like peptide-1 analogues. Despite the GC-rich, highly repetitive sequences of ELPs, we synthesized remarkably large genes without recursive ligation. OERCA also enabled us to discover 'smart' biopolymers that exhibit fully reversible thermally responsive behaviour. This powerful strategy generates libraries of repetitive genes over a wide and tunable range of molecular weights in a 'one-pot' parallel format.

Authors
Amiram, M; Quiroz, FG; Callahan, DJ; Chilkoti, A
MLA Citation
Amiram, M, Quiroz, FG, Callahan, DJ, and Chilkoti, A. "A highly parallel method for synthesizing DNA repeats enables the discovery of 'smart' protein polymers." Nat Mater 10.2 (February 2011): 141-148.
PMID
21258353
Source
pubmed
Published In
Nature Materials
Volume
10
Issue
2
Publish Date
2011
Start Page
141
End Page
148
DOI
10.1038/nmat2942

NANOSCALE SELF-ASSEMBLY FOR DELIVERY OF THERAPEUTICS AND IMAGING AGENTS.

Self-assemblies are complex structures spontaneously organized from simpler subcomponents, primarily through noncovalent interactions. These structures are being exploited as delivery vehicles of therapeutic and imaging agents. They have two unique advantages in comparison to other vehicles: 1) they are able to assume complex structures that are difficult to attain by chemical synthesis, and 2) the dissociation of self-assembled structures can be triggered by external stimuli, which can serve as a mechanism of payload release. In this review, we discuss two naturally occurring (proteins and viral capsids) and five engineered self-assemblies (vesicles, micelles, proteins, hydrogels, and inclusion complexes) that are under development for delivery of drugs and imaging agents. For each class of self-assembled supramolecular structures, we examine its structural and physicochemical properties, and potential applications within the context of assembly, loading, and payload release.

Authors
Chen, M; McDaniel, JR; Mackay, JA; Chilkoti, A
MLA Citation
Chen, M, McDaniel, JR, Mackay, JA, and Chilkoti, A. "NANOSCALE SELF-ASSEMBLY FOR DELIVERY OF THERAPEUTICS AND IMAGING AGENTS." Technol Innov 13.1 (January 1, 2011): 5-25.
PMID
24077873
Source
pubmed
Published In
Technology and Innovation
Volume
13
Issue
1
Publish Date
2011
Start Page
5
End Page
25
DOI
10.3727/194982411X13003853539948

Plasmonic flow cytometry by immunolabeled nanorods.

Fluorescence-based flow cytometry measures multiple cellular characteristics, including levels of receptor expression, by assessing the fluorescence intensity from a population of cells whose cell surface receptors are bound by a fluorescently labeled antibody or ligand for that receptor. Functionalized noble metal nanoparticles provide a complementary method of receptor labeling based on plasmonics for population analysis by flow cytometry. The potential benefits of using plasmonic nanoparticles to label cell surface receptors in flow cytometry include scattering intensity from a single particle that is equivalent to fluorescence intensity of 10⁵ fluorescein molecules, biocompatibility and low cytotoxicity, and nonquenching optical properties. The large spectral tunability of nanorods also provides convenient access to plasmonic markers with peak surface plasmon resonances ranging from 600 to 2,200 nm, unlike gold nanosphere markers that are limited to visible wavelengths. Gold nanorod-based plasmonic flow cytometry is demonstrated herein by comparing the scattering of cells bound to anti-epidermal growth factor receptor (EGFR)-conjugated nanorods to the emission of cells bound to anti-EGFR-conjugated fluorescent labels. EGFR-expressing cells exhibited a statistically significant six-fold increase in scattering when labeled with anti-EGFR-conjugated nanorods compared with labeling with IgG1-conjugated nanorods. Large scattering intensities were observed despite using a 1,000-fold lower concentration of nanorod-conjugated antibody relative to the fluorescently labeled antibody.

Authors
Crow, MJ; Marinakos, SM; Cook, JM; Chilkoti, A; Wax, A
MLA Citation
Crow, MJ, Marinakos, SM, Cook, JM, Chilkoti, A, and Wax, A. "Plasmonic flow cytometry by immunolabeled nanorods." Cytometry A 79.1 (January 2011): 57-65.
PMID
21182183
Source
pubmed
Published In
Cytometry
Volume
79
Issue
1
Publish Date
2011
Start Page
57
End Page
65
DOI
10.1002/cyto.a.20994

The use of micropatterning to control smooth muscle myosin heavy chain expression and limit the response to transforming growth factor β1 in vascular smooth muscle cells.

In the healthy artery, contractile vascular smooth muscle cells (VSMCs) have an elongated shape and are highly aligned but transition to a synthetic phenotype in culture, while additionally becoming well spread and randomly organized. Thus, controlling VSMC phenotype is a challenge in tissue engineering. In this study, we investigated the effects of micropatterning on contractile protein expression in VSMCs at low and high passage and in the presence of transforming growth factor beta 1 (TGFβ1). Micropatterning led to significantly decreased cell area, increased elongation, and increased alignment compared to non-patterned VSMCs independent of passage number. In the presence of serum, micropatterning led to increased smooth muscle myosin heavy chain (SM-MHC) and α-actin expression in low passage VSMCs, but had no effect on high passage VSMCs. Micropatterning was as effective as TGFβ1 in up-regulating SM-MHC at low passage; however, micropatterning limited VSMC response to TGFβ1 at both low and high passage. Investigation of TGFβ receptor 1 revealed higher expression in non-patterned VSMCs compared to patterned at high passage. Our studies demonstrate that micropatterning is an important regulator of SM-MHC expression in contractile VSMCs and that it may provide a mechanism for phenotype stabilization in the presence of growth factors.

Authors
Williams, C; Brown, XQ; Bartolak-Suki, E; Ma, H; Chilkoti, A; Wong, JY
MLA Citation
Williams, C, Brown, XQ, Bartolak-Suki, E, Ma, H, Chilkoti, A, and Wong, JY. "The use of micropatterning to control smooth muscle myosin heavy chain expression and limit the response to transforming growth factor β1 in vascular smooth muscle cells." Biomaterials 32.2 (January 2011): 410-418.
PMID
20858564
Source
pubmed
Published In
Biomaterials
Volume
32
Issue
2
Publish Date
2011
Start Page
410
End Page
418
DOI
10.1016/j.biomaterials.2010.08.105

In situ growth of a thermoresponsive polymer from a genetically engineered elastin-like polypeptide

We report the in situ growth of a thermoresponsive dumbbell-like polymer conjugate of a genetically engineered triblock elastin-like polypeptide (tELP). Atom transfer radical polymerization (ATRP) was used to directly grow a PEG-like polymer selectively from the first and third blocks of the tELP to form a poly(oligo(ethylene glycol) methyl ether methacrylate) (poly(OEGMA)) brush conjugate with quantitative yield. We found that in situ growth of poly(OEGMA) from tELP significantly changed the inverse phase transition and rheological behaviors of tELP. Dynamic light scattering (DLS) and turbidity measurements as a function of temperature showed that the inverse phase transition behavior of the conjugate was not determined by the tELP but by poly(OEGMA). Oscillatory rheological measurements indicated that the conjugate started to form a physical hydrogel at a temperature of 55 °C. In vitro enzymatic degradation studies showed that the conjugate could be degraded by collagenase. These results suggest that this class of conjugates may be potentially useful as an injectable, thermoresponsive drug carrier for local drug delivery and as a scaffold for tissue engineering. © 2011 The Royal Society of Chemistry.

Authors
Gao, W; Xu, D; Lim, DW; Craig, SL; Chilkoti, A
MLA Citation
Gao, W, Xu, D, Lim, DW, Craig, SL, and Chilkoti, A. "In situ growth of a thermoresponsive polymer from a genetically engineered elastin-like polypeptide." Polymer Chemistry 2.7 (2011): 1561-1566.
Source
scival
Published In
Polymer Chemistry
Volume
2
Issue
7
Publish Date
2011
Start Page
1561
End Page
1566
DOI
10.1039/c1py00074h

Micro- and nanostructured poly[oligo(ethylene glycol)methacrylate] brushes grown from photopatterned halogen initiators by atom transfer radical polymerization.

Photolithographic techniques have been used to fabricate polymer brush micro- and nanostructures. On exposure to UV light with a wavelength of 244 nm, halogens were selectively removed from films of chloromethylphenyltrichlorosilane and 3-(2-bromoisobutyramido)propyl-triethoxysilane on silicon dioxide. Patterning was achieved at the micrometer scale, by using a mask in conjunction with the incident laser beam, and at the nanometer scale, by utilizing interferometric lithography (IL). Friction force microscopy images of patterned surfaces exhibited frictional contrast due to removal of the halogen but no topographical contrast. In both cases the halogenated surface was used as an initiator for surface atom-transfer radical polymerization. Patterning of the surface by UV lithography enabled the definition of patterns of initiator from which micro- and nanostructured poly[oligo(ethylene glycol)methacrylate] bottle brushes were grown. Micropatterned brushes formed on both surfaces exhibited excellent resistance to protein adsorption, enabling the formation of protein patterns. Using IL, brush structures were formed that covered macroscopic areas (approximately 0.5 cm2) but exhibited a full width at half maximum height as small as 78 nm, with a period of 225 nm. Spatially selective photolytic removal of halogens that are immobilized on a surface thus appears to be a simple, rapid, and versatile method for the formation of micro- and nanostructured polymer brushes and for the control of protein adsorption.

Authors
Ahmad, SA; Leggett, GJ; Hucknall, A; Chilkoti, A
MLA Citation
Ahmad, SA, Leggett, GJ, Hucknall, A, and Chilkoti, A. "Micro- and nanostructured poly[oligo(ethylene glycol)methacrylate] brushes grown from photopatterned halogen initiators by atom transfer radical polymerization." Biointerphases 6.1 (2011): 8-15.
Source
scival
Published In
Biointerphases: an open access journal for the biomaterials interface community
Volume
6
Issue
1
Publish Date
2011
Start Page
8
End Page
15
DOI
10.1116/1.3553579

Raman antenna formed by molecule/plasmonic nanostructure hybrid system

A nano-antenna composed of a particle and a polarizable surface provides control of the spatial distribution and high enhancement of Raman scattering. This structure may serve as a stable platform for single molecule detection. © 2011 OSA.

Authors
Chen, S-Y; Mock, JJ; Hill, RT; Chilkoti, A; Smith, DR; Lazarides, AA
MLA Citation
Chen, S-Y, Mock, JJ, Hill, RT, Chilkoti, A, Smith, DR, and Lazarides, AA. "Raman antenna formed by molecule/plasmonic nanostructure hybrid system." 2011 Conference on Lasers and Electro-Optics: Laser Science to Photonic Applications, CLEO 2011 (2011).
Source
scival
Published In
2011 Conference on Lasers and Electro-Optics: Laser Science to Photonic Applications, CLEO 2011
Publish Date
2011

Hyperspectral molecular imaging of multiple receptors using immunolabeled plasmonic nanoparticles

This work presents simultaneous imaging and detection of three types of cell receptors using three types of plasmonic nanoparticles. The size, shape, and composition-dependent scattering profiles of these particles allow for a system of multiple distinct molecular markers using a single optical source. With this goal in mind, a system of tags consisting of anti-EGFR gold nanorods, anti-IGF1R silver nanospheres, and anti-HER-2 gold nanospheres was developed for monitoring the expression of three commonly overexpressed receptors in cancer cells. These labels were chosen because they each scatter strongly in a distinct spectral window. A hyperspectral dark-field microscope was developed to record the scattering spectra of cells labeled with these molecular tags. The ability to monitor multiple tags simultaneously may lead to applications such as profiling the immunophenotype of cell lines and gaining better knowledge of receptor signaling pathways. Single, dual, and triple tag experiments were performed to analyze the specificity of the nanoparticle tags as well as their effect on one another. While distinct resonance peaks in these studies show the ability to characterize cell lines using conjugated nanoparticles, shifts in these peaks also indicate changes in the cellular dielectric environment which may not be distinct from plasmon coupling between nanoparticles bound to proximal receptors. © 2011 SPIE.

Authors
Crow, MJ; Seekell, K; Marinakos, S; Ostrander, J; Chilkoti, A; Wax, AP
MLA Citation
Crow, MJ, Seekell, K, Marinakos, S, Ostrander, J, Chilkoti, A, and Wax, AP. "Hyperspectral molecular imaging of multiple receptors using immunolabeled plasmonic nanoparticles." Progress in Biomedical Optics and Imaging - Proceedings of SPIE 7911 (2011).
Source
scival
Published In
Proceedings of SPIE
Volume
7911
Publish Date
2011
DOI
10.1117/12.874093

Applications of elastin-like polypeptides in tissue engineering.

Elastin-like polypeptides (ELPs) have found utility in tissue engineering applications, not only because they are biocompatible, biodegradable, and non-immunogenic, but also because their amino acid sequence and molecular weight can be precisely controlled at the genetic or synthetic level, affording exquisite control over final protein functionality. This review presents a basic overview of ELP properties and modifications that are relevant to tissue engineering, as well as a discussion of the application of ELPs to cartilage, intervertebral disc, vascular graft, liver, ocular, and cell sheet engineering.

Authors
Nettles, DL; Chilkoti, A; Setton, LA
MLA Citation
Nettles, DL, Chilkoti, A, and Setton, LA. "Applications of elastin-like polypeptides in tissue engineering." Adv Drug Deliv Rev 62.15 (December 30, 2010): 1479-1485. (Review)
PMID
20385185
Source
pubmed
Published In
Advanced Drug Delivery Reviews
Volume
62
Issue
15
Publish Date
2010
Start Page
1479
End Page
1485
DOI
10.1016/j.addr.2010.04.002

Drug delivery to solid tumors by elastin-like polypeptides.

Thermally responsive elastin-like polypeptides (ELPs) are a promising class of recombinant biopolymers for the delivery of drugs and imaging agents to solid tumors via systemic or local administration. This article reviews four applications of ELPs to drug delivery, with each delivery mechanism designed to best exploit the relationship between the characteristic transition temperature (T(t)) of the ELP and body temperature (T(b)). First, when T(t)≫T(b), small hydrophobic drugs can be conjugated to the C-terminus of the ELP to impart the amphiphilicity needed to mediate the self-assembly of nanoparticles. These systemically delivered ELP-drug nanoparticles preferentially localize to the tumor site via the EPR effect, resulting in reduced toxicity and enhanced treatment efficacy. The remaining three approaches take direct advantage of the thermal responsiveness of ELPs. In the second strategy, where T(b)

Authors
McDaniel, JR; Callahan, DJ; Chilkoti, A
MLA Citation
McDaniel, JR, Callahan, DJ, and Chilkoti, A. "Drug delivery to solid tumors by elastin-like polypeptides." Adv Drug Deliv Rev 62.15 (December 30, 2010): 1456-1467. (Review)
PMID
20546809
Source
pubmed
Published In
Advanced Drug Delivery Reviews
Volume
62
Issue
15
Publish Date
2010
Start Page
1456
End Page
1467
DOI
10.1016/j.addr.2010.05.004

Gold nanoparticles on polarizable surfaces as Raman scattering antennas.

Surface plasmons supported by metal nanoparticles are perturbed by coupling to a surface that is polarizable. Coupling results in enhancement of near fields and may increase the scattering efficiency of radiative modes. In this study, we investigate the Rayleigh and Raman scattering properties of gold nanoparticles functionalized with cyanine deposited on silicon and quartz wafers and on gold thin films. Dark-field scattering images display red shifting of the gold nanoparticle plasmon resonance and doughnut-shaped scattering patterns when particles are deposited on silicon or on a gold film. The imaged radiation patterns and individual particle spectra reveal that the polarizable substrates control both the orientation and brightness of the radiative modes. Comparison with simulation indicates that, in a particle-surface system with a fixed junction width, plasmon band shifts are controlled quantitatively by the permittivity of the wafer or the film. Surface-enhanced resonance Raman scattering (SERRS) spectra and images are collected from cyanine on particles on gold films. SERRS images of the particles on gold films are doughnut-shaped as are their Rayleigh images, indicating that the SERRS is controlled by the polarization of plasmons in the antenna nanostructures. Near-field enhancement and radiative efficiency of the antenna are sufficient to enable Raman scattering cyanines to function as gap field probes. Through collective interpretation of individual particle Rayleigh spectra and spectral simulations, the geometric basis for small observed variations in the wavelength and intensity of plasmon resonant scattering from individual antenna on the three surfaces is explained.

Authors
Chen, S-Y; Mock, JJ; Hill, RT; Chilkoti, A; Smith, DR; Lazarides, AA
MLA Citation
Chen, S-Y, Mock, JJ, Hill, RT, Chilkoti, A, Smith, DR, and Lazarides, AA. "Gold nanoparticles on polarizable surfaces as Raman scattering antennas." ACS Nano 4.11 (November 23, 2010): 6535-6546.
Website
http://hdl.handle.net/10161/4100
PMID
21038892
Source
pubmed
Published In
ACS Nano
Volume
4
Issue
11
Publish Date
2010
Start Page
6535
End Page
6546
DOI
10.1021/nn101644s

Quantitative model of the phase behavior of recombinant pH-responsive elastin-like polypeptides.

Quantitative models are required to engineer biomaterials with environmentally responsive properties. With this goal in mind, we developed a model that describes the pH-dependent phase behavior of a class of stimulus responsive elastin-like polypeptides (ELPs) that undergo reversible phase separation in response to their solution environment. Under isothermal conditions, charged ELPs can undergo phase separation when their charge is neutralized. Optimization of this behavior has been challenging because the pH at which they phase separate, pHt, depends on their composition, molecular weight, concentration, and temperature. To address this problem, we developed a quantitative model to describe the phase behavior of charged ELPs that uses the Henderson-Hasselbalch relationship to describe the effect of side-chain ionization on the phase-transition temperature of an ELP. The model was validated with pH-responsive ELPs that contained either acidic (Glu) or basic (His) residues. The phase separation of both ELPs fit this model across a range of pH. These results have important implications for applications of pH-responsive ELPs because they provide a quantitative model for the rational design of pH-responsive polypeptides whose transition can be triggered at a specified pH.

Authors
Mackay, JA; Callahan, DJ; Fitzgerald, KN; Chilkoti, A
MLA Citation
Mackay, JA, Callahan, DJ, Fitzgerald, KN, and Chilkoti, A. "Quantitative model of the phase behavior of recombinant pH-responsive elastin-like polypeptides." Biomacromolecules 11.11 (November 8, 2010): 2873-2879.
Website
http://hdl.handle.net/10161/4020
PMID
20925333
Source
pubmed
Published In
Biomacromolecules
Volume
11
Issue
11
Publish Date
2010
Start Page
2873
End Page
2879
DOI
10.1021/bm100571j

Chain stiffness of elastin-like polypeptides.

Authors
Fluegel, S; Fischer, K; McDaniel, JR; Chilkoti, A; Schmidt, M
MLA Citation
Fluegel, S, Fischer, K, McDaniel, JR, Chilkoti, A, and Schmidt, M. "Chain stiffness of elastin-like polypeptides." Biomacromolecules 11.11 (November 8, 2010): 3216-3218.
Website
http://hdl.handle.net/10161/4021
PMID
20961120
Source
pubmed
Published In
Biomacromolecules
Volume
11
Issue
11
Publish Date
2010
Start Page
3216
End Page
3218
DOI
10.1021/bm100965y

Intracellular uptake and associated toxicity of silver nanoparticles in Caenorhabditis elegans.

Silver nanoparticles (AgNPs) are frequently used as antimicrobials. While the mechanism(s) by which AgNPs are toxic are unclear, their increasing use raises the concern that release into the environment could lead to environmental toxicity. We characterized the physicochemical behavior, uptake, toxicity (growth inhibition), and mechanism of toxicity of three AgNPs with different sizes and polyvinylpyrrolidone (PVP) or citrate coatings to the nematode Caenorhabditis elegans. We used wild-type (N2) C. elegans and strains expected to be sensitive to oxidative stress (nth-1, sod-2 and mev-1), genotoxins (xpa-1 and nth-1), and metals (mtl-2). Using traditional and novel analytical methods, we observed significant aggregation and extra-organismal dissolution of silver, organismal uptake and, in one case, transgenerational transfer of AgNPs. We also observed growth inhibition by all tested AgNPs at concentrations in the low mg/L levels. A metallothionein-deficient (mtl-2) strain was the only mutant tested that exhibited consistently greater AgNP sensitivity than wild-type. Although all tested AgNPs were internalized (passed cell membranes) in C. elegans, at least part of the toxicity observed was mediated by ionic silver. Finally, we describe a modified growth assay that permits differentiation between direct growth-inhibitory effects and indirect inhibition mediated by toxicity to the food source.

Authors
Meyer, JN; Lord, CA; Yang, XY; Turner, EA; Badireddy, AR; Marinakos, SM; Chilkoti, A; Wiesner, MR; Auffan, M
MLA Citation
Meyer, JN, Lord, CA, Yang, XY, Turner, EA, Badireddy, AR, Marinakos, SM, Chilkoti, A, Wiesner, MR, and Auffan, M. "Intracellular uptake and associated toxicity of silver nanoparticles in Caenorhabditis elegans." Aquat Toxicol 100.2 (October 15, 2010): 140-150.
PMID
20708279
Source
pubmed
Published In
Aquatic Toxicology
Volume
100
Issue
2
Publish Date
2010
Start Page
140
End Page
150
DOI
10.1016/j.aquatox.2010.07.016

Leveraging nanoscale plasmonic modes to achieve reproducible enhancement of light.

The strongly enhanced and localized optical fields that occur within the gaps between metallic nanostructures can be leveraged for a wide range of functionality in nanophotonic and optical metamaterial applications. Here, we introduce a means of precise control over these nanoscale gaps through the application of a molecular spacer layer that is self-assembled onto a gold film, upon which gold nanoparticles (NPs) are deposited electrostatically. Simulations using a three-dimensional finite element model and measurements from single NPs confirm that the gaps formed by this process, between the NP and the gold film, are highly reproducible transducers of surface-enhanced resonant Raman scattering. With a spacer layer of roughly 1.6 nm, all NPs exhibit a strong Raman signal that decays rapidly as the spacer layer is increased.

Authors
Hill, RT; Mock, JJ; Urzhumov, Y; Sebba, DS; Oldenburg, SJ; Chen, S-Y; Lazarides, AA; Chilkoti, A; Smith, DR
MLA Citation
Hill, RT, Mock, JJ, Urzhumov, Y, Sebba, DS, Oldenburg, SJ, Chen, S-Y, Lazarides, AA, Chilkoti, A, and Smith, DR. "Leveraging nanoscale plasmonic modes to achieve reproducible enhancement of light." Nano Lett 10.10 (October 13, 2010): 4150-4154.
Website
http://hdl.handle.net/10161/4095
PMID
20804206
Source
pubmed
Published In
Nano Letters
Volume
10
Issue
10
Publish Date
2010
Start Page
4150
End Page
4154
DOI
10.1021/nl102443p

In situ growth of a PEG-like polymer from the C terminus of an intein fusion protein improves pharmacokinetics and tumor accumulation.

This paper reports a general in situ method to grow a polymer conjugate solely from the C terminus of a recombinant protein. GFP was fused at its C terminus with an intein; cleavage of the intein provided a unique thioester moiety at the C terminus of GFP that was used to install an atom transfer radical polymerization (ATRP) initiator. Subsequent in situ ATRP of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) yielded a site-specific (C-terminal) and stoichiometric conjugate with high yield and good retention of protein activity. A GFP-C-poly(OEGMA) conjugate (hydrodynamic radius (R(h)): 21 nm) showed a 15-fold increase in its blood exposure compared to the protein (R(h): 3.0 nm) after intravenous administration to mice. This conjugate also showed a 50-fold increase in tumor accumulation, 24 h after intravenous administration to tumor-bearing mice, compared to the unmodified protein. This approach for in situ C-terminal polymer modification of a recombinant protein is applicable to a large subset of recombinant protein and peptide drugs and provides a general methodology for improvement of their pharmacological profiles.

Authors
Gao, W; Liu, W; Christensen, T; Zalutsky, MR; Chilkoti, A
MLA Citation
Gao, W, Liu, W, Christensen, T, Zalutsky, MR, and Chilkoti, A. "In situ growth of a PEG-like polymer from the C terminus of an intein fusion protein improves pharmacokinetics and tumor accumulation." Proc Natl Acad Sci U S A 107.38 (September 21, 2010): 16432-16437.
PMID
20810920
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
107
Issue
38
Publish Date
2010
Start Page
16432
End Page
16437
DOI
10.1073/pnas.1006044107

Elastin-like polypeptides as a purification tag for recombinant proteins.

This unit presents a recombinant protein purification method that employs an elastin-like polypeptide (ELP) as a purification tag. ELPs undergo a sharp and reversible phase transition when heated above their lower critical solution temperature. ELPs retain this behavior when they are fused to a protein, and thereby provide a simple method to isolate a recombinant ELP fusion protein from cell contaminants by cycling the solution through the insoluble and soluble phase of the ELP fusion protein using a procedure that is termed Inverse Transition Cycling. This method does not require the use of chromatography, so it is cost-effective, easy to scale up, and easy to multiplex.

Authors
Hassouneh, W; Christensen, T; Chilkoti, A
MLA Citation
Hassouneh, W, Christensen, T, and Chilkoti, A. "Elastin-like polypeptides as a purification tag for recombinant proteins." Curr Protoc Protein Sci Chapter 6 (August 2010): Unit-6.11.
PMID
20814933
Source
pubmed
Published In
Current Protocols in Protein Science
Volume
Chapter 6
Publish Date
2010
Start Page
Unit
End Page
6.11
DOI
10.1002/0471140864.ps0611s61

Stimulus-responsive macromolecules and nanoparticles for cancer drug delivery.

Nanoparticles and macromolecular carriers have been widely used to increase the efficacy of chemotherapeutics, largely through passive accumulation provided by the enhanced permeability and retention effect. Stimulus-responsive peptide and polymer vehicles can further enhance the efficacy of antitumor therapeutics compared with the administration of free drug by three mechanisms: increasing the overall accumulation within solid tumors; providing a homogeneous spatial distribution in tumor tissues; and increasing the intracellular localization of anticancer therapeutics. This article highlights recent developments in 'smart' - stimulus-responsive - peptide, polymer and lipid drug carriers designed to enhance the localization and efficacy of therapeutic payloads as compared with free drug.

Authors
MacEwan, SR; Callahan, DJ; Chilkoti, A
MLA Citation
MacEwan, SR, Callahan, DJ, and Chilkoti, A. "Stimulus-responsive macromolecules and nanoparticles for cancer drug delivery." Nanomedicine (Lond) 5.5 (July 2010): 793-806. (Review)
PMID
20662649
Source
pubmed
Published In
Nanomedicine (London, England)
Volume
5
Issue
5
Publish Date
2010
Start Page
793
End Page
806
DOI
10.2217/nnm.10.50

Protein patterning by UV-induced photodegradation of poly(oligo(ethylene glycol) methacrylate) brushes.

The UV photodegradation of protein-resistant poly(oligo(ethylene glycol) methacrylate) (POEGMA) bottle-brush films, grown on silicon oxide by surface-initiated atom radical transfer polymerization, was studied using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Exposure to light with a wavelength of 244 nm caused a loss of polyether units from the brush structure and the creation of aldehyde groups that could be derivatized with amines. An increase was measured in the coefficient of friction of the photodegraded polymer brush compared to the native brush, attributed to the creation of a heterogeneous surface film, leading to increased energy dissipation through film deformation and the creation of new polar functional groups at the surface. Exposure of the films through a photomask yielded sharp, well-defined patterns. Analysis of topographical images showed that physical removal of material occurred during exposure, at a rate of 1.35 nm J(-1) cm(2). Using fluorescence microscopy, the adsorption of labeled proteins onto the exposed surfaces was studied. It was found that protein strongly adsorbed to exposed areas, while the masked regions retained their protein resistance. Exposure of the film to UV light from a scanning near-field optical microscope yielded submicrometer-scale patterns. These data indicate that a simple, rapid, one-step photoconversion of the poly(OEGMA) brush occurs that transforms it from a highly protein-resistant material to one that adsorbs protein and can covalently bind amine-containing molecules and that this photoconversion can be spatially addressed with high spatial resolution.

Authors
Alang Ahmad, S; Hucknall, A; Chilkoti, A; Leggett, GJ
MLA Citation
Alang Ahmad, S, Hucknall, A, Chilkoti, A, and Leggett, GJ. "Protein patterning by UV-induced photodegradation of poly(oligo(ethylene glycol) methacrylate) brushes." Langmuir 26.12 (June 15, 2010): 9937-9942.
Website
http://hdl.handle.net/10161/4084
PMID
20356046
Source
pubmed
Published In
Langmuir
Volume
26
Issue
12
Publish Date
2010
Start Page
9937
End Page
9942
DOI
10.1021/la100438d

A versatile diffractive maskless lithography for single-shot and serial microfabrication.

We demonstrate a diffractive maskless lithographic system that is capable of rapidly performing both serial and single-shot micropatterning. Utilizing the diffractive properties of phase holograms displayed on a spatial light modulator, arbitrary intensity distributions were produced to form two and three dimensional micropatterns/structures in a variety of substrates. A straightforward graphical user interface was implemented to allow users to load templates and change patterning modes within the span of a few minutes. A minimum resolution of approximately 700 nm is demonstrated for both patterning modes, which compares favorably to the 232 nm resolution limit predicted by the Rayleigh criterion. The presented method is rapid and adaptable, allowing for the parallel fabrication of microstructures in photoresist as well as the fabrication of protein microstructures that retain functional activity.

Authors
Jenness, NJ; Hill, RT; Hucknall, A; Chilkoti, A; Clark, RL
MLA Citation
Jenness, NJ, Hill, RT, Hucknall, A, Chilkoti, A, and Clark, RL. "A versatile diffractive maskless lithography for single-shot and serial microfabrication." Opt Express 18.11 (May 24, 2010): 11754-11762.
Website
http://hdl.handle.net/10161/4242
PMID
20589036
Source
pubmed
Published In
Optics express
Volume
18
Issue
11
Publish Date
2010
Start Page
11754
End Page
11762

Injectable intratumoral depot of thermally responsive polypeptide-radionuclide conjugates delays tumor progression in a mouse model.

This study evaluated a biodegradable drug delivery system for local cancer radiotherapy consisting of a thermally sensitive elastin-like polypeptide (ELP) conjugated to a therapeutic radionuclide. Two ELPs (49 kDa) were synthesized using genetic engineering to test the hypothesis that injectable biopolymeric depots can retain radionuclides locally and reduce the growth of tumors. A thermally sensitive polypeptide, ELP(1), was designed to spontaneously undergo a soluble-insoluble phase transition (forming viscous microparticles) between room temperature and body temperature upon intratumoral injection, while ELP(2) was designed to remain soluble upon injection and to serve as a negative control for the effect of aggregate assembly. After intratumoral administration of radionuclide conjugates of ELPs into implanted tumor xenografts in nude mice, their retention within the tumor, spatio-temporal distribution, and therapeutic effect were quantified. The residence time of the radionuclide-ELP(1) in the tumor was significantly longer than the thermally insensitive ELP(2) conjugate. In addition, the thermal transition of ELP(1) significantly protected the conjugated radionuclide from dehalogenation, whereas the conjugated radionuclide on ELP(2) was quickly eliminated from the tumor and cleaved from the biopolymer. These attributes of the thermally sensitive ELP(1) depot improved the antitumor efficacy of iodine-131 compared to the soluble ELP(2) control. This novel injectable and biodegradable depot has the potential to control advanced-stage cancers by reducing the bulk of inoperable tumors, enabling surgical removal of de-bulked tumors, and preserving healthy tissues.

Authors
Liu, W; MacKay, JA; Dreher, MR; Chen, M; McDaniel, JR; Simnick, AJ; Callahan, DJ; Zalutsky, MR; Chilkoti, A
MLA Citation
Liu, W, MacKay, JA, Dreher, MR, Chen, M, McDaniel, JR, Simnick, AJ, Callahan, DJ, Zalutsky, MR, and Chilkoti, A. "Injectable intratumoral depot of thermally responsive polypeptide-radionuclide conjugates delays tumor progression in a mouse model." J Control Release 144.1 (May 21, 2010): 2-9.
PMID
20117157
Source
pubmed
Published In
Journal of Controlled Release
Volume
144
Issue
1
Publish Date
2010
Start Page
2
End Page
9
DOI
10.1016/j.jconrel.2010.01.032

Morphing low-affinity ligands into high-avidity nanoparticles by thermally triggered self-assembly of a genetically encoded polymer.

Multivalency is the increase in avidity resulting from the simultaneous interaction of multiple ligands with multiple receptors. This phenomenon, seen in antibody-antigen and virus-cell membrane interactions, is useful in designing bioinspired materials for targeted delivery of drugs or imaging agents. While increased avidity offered by multivalent targeting is attractive, it can also promote nonspecific receptor interaction in nontarget tissues, reducing the effectiveness of multivalent targeting. Here, we present a thermal targeting strategy--dynamic affinity modulation (DAM)--using elastin-like polypeptide diblock copolymers (ELP(BC)s) that self-assemble from a low-affinity to high-avidity state by a tunable thermal "switch", thereby restricting activity to the desired site of action. We used an in vitro cell binding assay to investigate the effect of the thermally triggered self-assembly of these ELP(BC)s on their receptor-mediated binding and cellular uptake. The data presented herein show that (1) ligand presentation does not disrupt ELP(BC) self-assembly; (2) both multivalent ligand presentation and upregulated receptor expression are needed for receptor-mediated interaction; (3) increased size of the hydrophobic segment of the block copolymer promotes multivalent interaction with membrane receptors, potentially due to changes in the nanoscale architecture of the micelle; and (4) nanoscale presentation of the ligand is important, as presentation of the ligand by micrometer-sized aggregates of an ELP showed a low level of binding/uptake by receptor-positive cells compared to its presentation on the corona of a micelle. These data validate the concept of thermally triggered DAM and provide rational design parameters for future applications of this technology for targeted drug delivery.

Authors
Simnick, AJ; Valencia, CA; Liu, R; Chilkoti, A
MLA Citation
Simnick, AJ, Valencia, CA, Liu, R, and Chilkoti, A. "Morphing low-affinity ligands into high-avidity nanoparticles by thermally triggered self-assembly of a genetically encoded polymer." ACS Nano 4.4 (April 27, 2010): 2217-2227.
Website
http://hdl.handle.net/10161/4103
PMID
20334355
Source
pubmed
Published In
ACS Nano
Volume
4
Issue
4
Publish Date
2010
Start Page
2217
End Page
2227
DOI
10.1021/nn901732h

Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes.

This paper reports a new strategy, recursive directional ligation by plasmid reconstruction (PRe-RDL), to rapidly clone highly repetitive polypeptides of any sequence and specified length over a large range of molecular weights. In a single cycle of PRe-RDL, two halves of a parent plasmid, each containing a copy of an oligomer, are ligated together, thereby dimerizing the oligomer and reconstituting a functional plasmid. This process is carried out recursively to assemble an oligomeric gene with the desired number of repeats. PRe-RDL has several unique features that stem from the use of type IIs restriction endonucleases: first, PRe-RDL is a seamless cloning method that leaves no extraneous nucleotides at the ligation junction. Because it uses type IIs endonucleases to ligate the two halves of the plasmid, PRe-RDL also addresses the major limitation of RDL in that it abolishes any restriction on the gene sequence that can be oligomerized. The reconstitution of a functional plasmid only upon successful ligation in PRe-RDL also addresses two other limitations of RDL: the significant background from self-ligation of the vector observed in RDL, and the decreased efficiency of ligation due to nonproductive circularization of the insert. PRe-RDL can also be used to assemble genes that encode different sequences in a predetermined order to encode block copolymers or append leader and trailer peptide sequences to the oligomerized gene.

Authors
McDaniel, JR; Mackay, JA; Quiroz, FG; Chilkoti, A
MLA Citation
McDaniel, JR, Mackay, JA, Quiroz, FG, and Chilkoti, A. "Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes." Biomacromolecules 11.4 (April 12, 2010): 944-952.
Website
http://hdl.handle.net/10161/4022
PMID
20184309
Source
pubmed
Published In
Biomacromolecules
Volume
11
Issue
4
Publish Date
2010
Start Page
944
End Page
952
DOI
10.1021/bm901387t

Neural network analysis identifies scaffold properties necessary for in vitro chondrogenesis in elastin-like polypeptide biopolymer scaffolds.

The successful design of biomaterial scaffolds for articular cartilage tissue engineering requires an understanding of the impact of combinations of material formulation parameters on diverse and competing functional outcomes of biomaterial performance. This study sought to explore the use of a type of unsupervised artificial network, a self-organizing map, to identify relationships between scaffold formulation parameters (crosslink density, molecular weight, and concentration) and 11 such outcomes (including mechanical properties, matrix accumulation, metabolite usage and production, and histological appearance) for scaffolds formed from crosslinked elastin-like polypeptide (ELP) hydrogels. The artificial neural network recognized patterns in functional outcomes and provided a set of relationships between ELP formulation parameters and measured outcomes. Mapping resulted in the best mean separation amongst neurons for mechanical properties and pointed to crosslink density as the strongest predictor of most outcomes, followed by ELP concentration. The map also grouped formulations together that simultaneously resulted in the highest values for matrix production, greatest changes in metabolite consumption or production, and highest histological scores, indicating that the network was able to recognize patterns amongst diverse measurement outcomes. These results demonstrated the utility of artificial neural network tools for recognizing relationships in systems with competing parameters, toward the goal of optimizing and accelerating the design of biomaterial scaffolds for articular cartilage tissue engineering.

Authors
Nettles, DL; Haider, MA; Chilkoti, A; Setton, LA
MLA Citation
Nettles, DL, Haider, MA, Chilkoti, A, and Setton, LA. "Neural network analysis identifies scaffold properties necessary for in vitro chondrogenesis in elastin-like polypeptide biopolymer scaffolds." Tissue Eng Part A 16.1 (January 2010): 11-20.
Website
http://hdl.handle.net/10161/3372
PMID
19754250
Source
pubmed
Published In
Tissue Engineering, Part A
Volume
16
Issue
1
Publish Date
2010
Start Page
11
End Page
20
DOI
10.1089/ten.tea.2009.0134

Elastin-like polypeptides: biomedical applications of tunable biopolymers.

Artificial repetitive polypeptides have grown in popularity as a bioinspired alternative to synthetic polymers. The genetically encoded synthesis, monodispersity, potential lack of toxicity, and biocompatibility are attractive features of these biopolymers for biological applications. Elastin-like polypeptides (ELPs) are one such class of biopolymers that are of particular interest because of their "smart"-stimuli responsive-properties. Herein, we discuss the genetically encoded design and recombinant synthesis of ELPs that enable precise control of their physicochemical properties and which have led to a wide range of biomedical applications of these biopolymers in the last decade.

Authors
MacEwan, SR; Chilkoti, A
MLA Citation
MacEwan, SR, and Chilkoti, A. "Elastin-like polypeptides: biomedical applications of tunable biopolymers." Biopolymers 94.1 (2010): 60-77. (Review)
PMID
20091871
Source
pubmed
Published In
Biopolymers
Volume
94
Issue
1
Publish Date
2010
Start Page
60
End Page
77
DOI
10.1002/bip.21327

Self-assembling chimeric polypeptide-doxorubicin conjugate nanoparticles that abolish tumours after a single injection.

New strategies to self-assemble biocompatible materials into nanoscale, drug-loaded packages with improved therapeutic efficacy are needed for nanomedicine. To address this need, we developed artificial recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into sub-100-nm-sized, near-monodisperse nanoparticles on conjugation of diverse hydrophobic molecules, including chemotherapeutics. These CPs consist of a biodegradable polypeptide that is attached to a short Cys-rich segment. Covalent modification of the Cys residues with a structurally diverse set of hydrophobic small molecules, including chemotherapeutics, leads to spontaneous formation of nanoparticles over a range of CP compositions and molecular weights. When used to deliver chemotherapeutics to a murine cancer model, CP nanoparticles have a fourfold higher maximum tolerated dose than free drug, and induce nearly complete tumour regression after a single dose. This simple strategy can promote co-assembly of drugs, imaging agents and targeting moieties into multifunctional nanomedicines.

Authors
MacKay, JA; Chen, M; McDaniel, JR; Liu, W; Simnick, AJ; Chilkoti, A
MLA Citation
MacKay, JA, Chen, M, McDaniel, JR, Liu, W, Simnick, AJ, and Chilkoti, A. "Self-assembling chimeric polypeptide-doxorubicin conjugate nanoparticles that abolish tumours after a single injection." Nat Mater 8.12 (December 2009): 993-999.
PMID
19898461
Source
pubmed
Published In
Nature Materials
Volume
8
Issue
12
Publish Date
2009
Start Page
993
End Page
999
DOI
10.1038/nmat2569

Self-assembling chimeric polypeptide-doxorubicin conjugate nanoparticles that abolish tumours after a single injection

Authors
MacKay, JA; Chen, M; McDaniel, JR; Liu, W; Simnick, AJ; Chilkoti, A
MLA Citation
MacKay, JA, Chen, M, McDaniel, JR, Liu, W, Simnick, AJ, and Chilkoti, A. "Self-assembling chimeric polypeptide-doxorubicin conjugate nanoparticles that abolish tumours after a single injection." NATURE MATERIALS 8.12 (December 2009): 993-999.
Source
wos-lite
Published In
Nature Materials
Volume
8
Issue
12
Publish Date
2009
Start Page
993
End Page
999
DOI
10.1038/NMAT2569

Hydrogen bonding of beta-turn structure is stabilized in D(2)O.

The lower critical solution temperature (LCST) of elastin-like polypeptides (ELPs) was investigated as a function of ELP chain length and guest residue chemistry. These measurements were made in both D(2)O and H(2)O. Differences in the LCST values with heavy and light water were correlated with secondary structure formation of the polypeptide chains. Such structural information was obtained by circular dichroism and infrared measurements. Additional thermodynamic data were obtained by differential scanning calorimetry. It was found that there is a greater change in the LCST value between H(2)O and D(2)O for those polypeptides which form the greatest amount of beta-turn/beta-aggregate structure. Moreover, these same molecules were the least hydrophobic ELPs. Therefore, hydrogen bonding rather than hydrophobicity was the key factor in the stabilization of the collapsed state of ELPs in D(2)O compared with H(2)O.

Authors
Cho, Y; Sagle, LB; Iimura, S; Zhang, Y; Kherb, J; Chilkoti, A; Scholtz, JM; Cremer, PS
MLA Citation
Cho, Y, Sagle, LB, Iimura, S, Zhang, Y, Kherb, J, Chilkoti, A, Scholtz, JM, and Cremer, PS. "Hydrogen bonding of beta-turn structure is stabilized in D(2)O." J Am Chem Soc 131.42 (October 28, 2009): 15188-15193.
PMID
19919159
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
131
Issue
42
Publish Date
2009
Start Page
15188
End Page
15193
DOI
10.1021/ja9040785

In situ growth of a stoichiometric PEG-like conjugate at a protein's N-terminus with significantly improved pharmacokinetics.

The challenge in the synthesis of protein-polymer conjugates for biological applications is to synthesize a stoichiometric (typically 1:1) conjugate of the protein with a monodisperse polymer, with good retention of protein activity, significantly improved pharmacokinetics and increased bioavailability, and hence improved in vivo efficacy. Here we demonstrate, using myoglobin as an example, a general route to grow a PEG-like polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) [poly(OEGMA)], with low polydispersity and high yield, solely from the N-terminus of the protein by in situ atom transfer radical polymerization (ATRP) under aqueous conditions, to yield a site-specific (N-terminal) and stoichiometric conjugate (1:1). Notably, the myoglobin-poly(OEGMA) conjugate [hydrodynamic radius (R(h)): 13 nm] showed a 41-fold increase in its blood exposure compared to the protein (R(h): 1.7 nm) after IV administration to mice, thereby demonstrating that comb polymers that present short oligo(ethylene glycol) side chains are a class of PEG-like polymers that can significantly improve the pharmacological properties of proteins. We believe that this approach to the synthesis of N-terminal protein conjugates of poly(OEGMA) may be applicable to a large subset of protein and peptide drugs, and thereby provide a general methodology for improvement of their pharmacological profiles.

Authors
Gao, W; Liu, W; Mackay, JA; Zalutsky, MR; Toone, EJ; Chilkoti, A
MLA Citation
Gao, W, Liu, W, Mackay, JA, Zalutsky, MR, Toone, EJ, and Chilkoti, A. "In situ growth of a stoichiometric PEG-like conjugate at a protein's N-terminus with significantly improved pharmacokinetics." Proc Natl Acad Sci U S A 106.36 (September 8, 2009): 15231-15236.
PMID
19706892
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
106
Issue
36
Publish Date
2009
Start Page
15231
End Page
15236
DOI
10.1073/pnas.0904378106

Early metabolite levels predict long-term matrix accumulation for chondrocytes in elastin-like polypeptide biopolymer scaffolds.

The development of cartilage tissue engineering scaffolds could greatly benefit from methods to evaluate the interactions of cells with scaffolds that are rapid, are nondestructive, and can be carried out at early culture times. Motivated by this rationale, the objective of the current study was to evaluate whether the concentration of metabolites in scaffold-cell cultures at early culture times could predict matrix synthesis in the same samples at longer culture times. Metabolite and matrix synthesis were measured for 16 different formulations of cell-laden elastin-like polypeptide hydrogels. Metabolites were measured at days 4 and 7 of culture, while matrix accumulation was evaluated at day 28. Four of the 16 formulations resulted in molar ratios of lactate:glucose near 2, indicating anaerobic metabolism of glucose, which resulted in collagen:glycosaminoglycan accumulation ratios near those of native tissue. Lactate and pyruvate concentrations were found to significantly correlate with both sulfated glycosaminoglycan and hydroxyproline accumulation, with better fits for the latter. Lactate was found to be the strongest predictor of both matrix components, suggesting that measuring this metabolite at very early culture times may be useful for evaluating the status of tissue engineering constructs in a rapid and nondestructive manner.

Authors
Nettles, DL; Chilkoti, A; Setton, LA
MLA Citation
Nettles, DL, Chilkoti, A, and Setton, LA. "Early metabolite levels predict long-term matrix accumulation for chondrocytes in elastin-like polypeptide biopolymer scaffolds." Tissue Eng Part A 15.8 (August 2009): 2113-2121.
PMID
19193139
Source
pubmed
Published In
Tissue Engineering, Part A
Volume
15
Issue
8
Publish Date
2009
Start Page
2113
End Page
2121
DOI
10.1089/ten.tea.2008.0448

Fusion order controls expression level and activity of elastin-like polypeptide fusion proteins.

We have previously developed a method to purify recombinant proteins, termed inverse transition cycling (ITC) that eliminates the need for column chromatography. ITC exploits the inverse solubility phase transition of an elastin-like polypeptide (ELP) that is fused to a protein of interest. In ITC, a recombinant ELP fusion protein is cycled through its phase transition, resulting in separation of the ELP fusion protein from other Escherichia coli contaminants. Herein, we examine the role of the position of the ELP in the fusion protein on the expression levels and yields of purified protein for four recombinant ELP fusion proteins. Placing the ELP at the C-terminus of the target protein (protein-ELP) results in a higher expression level for the four ELP fusion proteins, which also translates to a greater yield of purified protein. The position of the fusion protein also has a significant impact on its specific activity, as ELP-protein constructs have a lower specific activity than protein-ELP constructs for three out of the four proteins. Our results show no difference in mRNA levels between protein-ELP and ELP-protein fusion constructs. Instead, we suggest two possible explanations for these results: first, the translational efficiency of mRNA may differ between the fusion protein in the two orientations and second, the lower level of protein expression and lower specific activity is consistent with a scenario that placement of the ELP at the N-terminus of the fusion protein increases the fraction of misfolded, and less active conformers, which are also preferentially degraded compared to fusion proteins in which the ELP is present at the C-terminal end of the protein.

Authors
Christensen, T; Amiram, M; Dagher, S; Trabbic-Carlson, K; Shamji, MF; Setton, LA; Chilkoti, A
MLA Citation
Christensen, T, Amiram, M, Dagher, S, Trabbic-Carlson, K, Shamji, MF, Setton, LA, and Chilkoti, A. "Fusion order controls expression level and activity of elastin-like polypeptide fusion proteins." Protein Sci 18.7 (July 2009): 1377-1387.
PMID
19533768
Source
pubmed
Published In
Protein Science
Volume
18
Issue
7
Publish Date
2009
Start Page
1377
End Page
1387
DOI
10.1002/pro.157

Versatile synthesis and micropatterning of nonfouling polymer brushes on the wafer scale.

In this article, the authors describe new approaches to synthesize and pattern surfaces with poly[oligo(ethylene glycol) methyl methacrylate] (POEGMA) polymer brushes synthesized by surface-initiated atom transfer radical polymerization. These patterned coatings confer "nonfouling" properties protein and cell resistance-to the surface in a biological milieu. The versatile routes for the synthesis of POEGMA demonstrated here offer clear advantages over other techniques previously used in terms of their simplicity, reliability, and ability to pattern large-area substrates. They also demonstrate that POEGMA polymer brushes can be patterned directly by photolithography, plasma ashing, and reactive ion etching to create patterns at the micro- and nanoscale over large areas with high throughput and repeatability, while preserving the protein and cell resistance of the POEGMA brush.

Authors
Hucknall, A; Simnick, AJ; Hill, RT; Chilkoti, A; Garcia, A; Johannes, MS; Clark, RL; Zauscher, S; Ratner, BD
MLA Citation
Hucknall, A, Simnick, AJ, Hill, RT, Chilkoti, A, Garcia, A, Johannes, MS, Clark, RL, Zauscher, S, and Ratner, BD. "Versatile synthesis and micropatterning of nonfouling polymer brushes on the wafer scale." Biointerphases 4.2 (June 2009): FA50-FA57.
PMID
20408717
Source
pubmed
Published In
Biointerphases: an open access journal for the biomaterials interface community
Volume
4
Issue
2
Publish Date
2009
Start Page
FA50
End Page
FA57
DOI
10.1116/1.3151968

Biological applications of polymer brushes. Preface.

Authors
Zauscher, S; Chilkoti, A
MLA Citation
Zauscher, S, and Chilkoti, A. "Biological applications of polymer brushes. Preface." Biointerphases 4.2 (June 2009): FA1-FA2.
PMID
20408712
Source
pubmed
Published In
Biointerphases: an open access journal for the biomaterials interface community
Volume
4
Issue
2
Publish Date
2009
Start Page
FA1
End Page
FA2
DOI
10.1116/1.3149787

Rational selection of gold nanorod geometry for label-free plasmonic biosensors.

We present the development of an analytical model that can be used for the rational design of a biosensor based on shifts in the local surface plasmon resonance (LSPR) of individual gold nanoparticles. The model relates the peak wavelength of light scattered by an individual plasmonic nanoparticle to the number of bound analyte molecules and provides an analytical formulation that predicts relevant figures-of-merit of the sensor such as the molecular detection limit (MDL) and dynamic range as a function of nanoparticle geometry and detection system parameters. The model calculates LSPR shifts for individual molecules bound by a nanorod, so that the MDL is defined as the smallest number of bound molecules that is measurable by the system, and the dynamic range is defined as the maximum number of molecules that can be detected by a single nanorod. This model is useful because it will allow a priori design of an LSPR sensor with figures-of-merit that can be optimized for the target analyte. This model was used to design an LSPR sensor based on biotin-functionalized gold nanorods that offers the lowest MDL for this class of sensors. The model predicts a MDL of 18 streptavidin molecules for this sensor, which is in good agreement with experiments and estimates. Further, we discuss how the model can be utilized to guide the development of future generations of LSPR biosensors.

Authors
Nusz, GJ; Curry, AC; Marinakos, SM; Wax, A; Chilkoti, A
MLA Citation
Nusz, GJ, Curry, AC, Marinakos, SM, Wax, A, and Chilkoti, A. "Rational selection of gold nanorod geometry for label-free plasmonic biosensors." ACS Nano 3.4 (April 28, 2009): 795-806.
PMID
19296619
Source
pubmed
Published In
ACS Nano
Volume
3
Issue
4
Publish Date
2009
Start Page
795
End Page
806
DOI
10.1021/nn8006465

Precision engineered control of biomolecules at interfaces by polymer brushes

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "Precision engineered control of biomolecules at interfaces by polymer brushes." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 237 (March 22, 2009).
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
237
Publish Date
2009

Fabrication of elastin-like polypeptide nanoparticles for drug delivery by electrospraying.

The development of environmentally responsive drug carriers requires new methods for assembling stimuli-responsive nanoparticulates. This communication describes a novel application of electrospray to construct bioresponsive peptide-based particulates, which can encapsulate drugs. These particles are composed from genetically engineered elastin-like polypeptides (ELPs), a biodegradable, biocompatible, and bioresponsive polymer. To generate nanoparticles (300-400 nm in diameter), ELPs and drugs are codissolved in organic solvent, accelerated across a voltage gradient, dried by evaporation during transit, and collected from a target surface. These findings indicate that particle diameter, polydispersity, and morphology are strong functions of the solvent concentration, spraying voltage, and polymer molecular weight. Surprisingly, the loading of drug at 20 w/w% did not influence particle morphology; furthermore, drug release from these particles correlated with the pH-dependent solubility of the parent ELPs. These studies suggest that electrospray is an efficient and flexible method for generating stimuli-responsive drug particles.

Authors
Wu, Y; MacKay, JA; McDaniel, JR; Chilkoti, A; Clark, RL
MLA Citation
Wu, Y, MacKay, JA, McDaniel, JR, Chilkoti, A, and Clark, RL. "Fabrication of elastin-like polypeptide nanoparticles for drug delivery by electrospraying." Biomacromolecules 10.1 (January 12, 2009): 19-24.
PMID
19072041
Source
pubmed
Published In
Biomacromolecules
Volume
10
Issue
1
Publish Date
2009
Start Page
19
End Page
24
DOI
10.1021/bm801033f

Designing interfaces for optical biosensors

I will describe two interfaces for optical biosensors: (1) a label-free biosensor that exploits the local surface plasmon resonance of noble metal nanostructures; and (2) a polymer brush interface on glass that abolishes non-specific adsorption leading to a femtomolar limit-of-detection of protein analytes in whole blood. © 2009 Optical Society of America.

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "Designing interfaces for optical biosensors." Optics InfoBase Conference Papers (January 1, 2009).
Source
scopus
Published In
Optics InfoBase Conference Papers
Publish Date
2009

Simple fabrication of antibody microarrays on nonfouling polymer brushes with femtomolar sensitivity for protein analytes in serum and blood

The fabrication of antibody (Ab) arrays on Poly(oligo(ethylene glycol) methacrylate) POEGMA brushes with several significant features has been reported. Ellipsometry in air of POEGMA brushes grown on oxidized silicon wafers under identical conditions indicated a polymer brush thickness of (105±2) nm. The non covalent adsorption of antibodies provides a simple and robust procedure for the stable immobilization of the capture antibody, which avoids the need for chemical activation and deactivation of the surface. Antibody arrays spotted on the POEGMA brushes require only minimal washing steps, and do not require blocking steps during interrogation, which simplifies the assay and reduces the time required to perform the assay. The femtomolar Limit-Of-Detection (LOD) in serum and blood and the wide dynamic range of antibody arrays spotted on POEGMA brushes suggest that these microarrays will be useful for the quantification of low-abundance protein biomarkers directly from complex mixtures with minimal sample pre-processing, with application in proteomics and clinical diagnostics.

Authors
Hucknall, A; Kim, D-H; Rangarajan, S; Hill, RT; Reichert, WM; Chilkoti, A
MLA Citation
Hucknall, A, Kim, D-H, Rangarajan, S, Hill, RT, Reichert, WM, and Chilkoti, A. "Simple fabrication of antibody microarrays on nonfouling polymer brushes with femtomolar sensitivity for protein analytes in serum and blood." Advanced Materials 21.19 (2009): 1968-1971.
Source
scival
Published In
Advanced Materials
Volume
21
Issue
19
Publish Date
2009
Start Page
1968
End Page
1971
DOI
10.1002/adma.200803125

In pursuit of zero: Polymer brushes that resist the adsorption of proteins

Protein resistant or "non-fouling" surfaces are of great interest for a variety of biomedical and biotechnology applications. This article briefly reviews the development of protein resistant surfaces, followed by recent research on a new methodology to fabricate non-fouling surfaces by surface-initiated polymerization. We show that polymer brushes synthesized by surfaceinitiated polymerization that present short oligo(ethylene glycol) side chains are exceptionally resistant to protein adsorption and cell adhesion. The importance of the protein and cell resistance conferred by these polymer brushes is illustrated by their use as substrates for the fabrication of antibody microarrays that exhibit femtomolar limits of detection in complex fluids such as serum and blood with relaxed requirements for intermediate wash steps. This example highlights the important point that the reduction in background noise afforded by protein-resistant surfaces can greatly simplify the development of ultrasensitive heterogeneous, surface-based clinical and proteomic assays with increased sensitivity and utility. © 2009 WILEY-VCH Verlag GmbH & Co. KGaA,.

Authors
Hucknall, A; Rangarajan, S; Chilkoti, A
MLA Citation
Hucknall, A, Rangarajan, S, and Chilkoti, A. "In pursuit of zero: Polymer brushes that resist the adsorption of proteins." Advanced Materials 21.23 (2009): 2441-2446.
Source
scival
Published In
Advanced Materials
Volume
21
Issue
23
Publish Date
2009
Start Page
2441
End Page
2446
DOI
10.1002/adma.200900383

Simultaneous molecular imaging of EGFR and HER2 using hyperspectral darkfield microscopy and immunotargeted nanoparticles

Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER2) contribute to the regulation of cell proliferation, and when jointly over-expressed are associated with several types of cancer. The ability to monitor both receptors simultaneously results in a more accurate indicator of degree of cancerous activity than either receptor alone. Plasmonic nanoparticles (NPs) show promise as a potential EGFR and HER2 biomarker over alternatives such as fluorophores and quantum dots, which are limited by their cytotoxicity and photobleaching. To observe immunolabeled NPs bound to receptor-expressing cells, our past experiments were conducted using a novel optical darkfield microspectroscopy system. We implemented an epi-illumination darkfield broadband light train, which allows for darkfield analysis of live cells in culture with enhanced NP contrast. Under this setup, molecularly specific binding of NPs immunolabeled with anti-EGFR was confirmed. We have since adapted our darkfield setup, which previously only obtained spectral information from a line imaging spectrometer, to incorporate hyperspectral imaging capabilities, allowing widefield data acquisition within seconds. The new system has been validated through observation of shifts in the peak wavelength of scattering by gold NPs on silanated cover glasses using several immersion media. Peak resonant scattering wavelengths match well with that predicted by Mie theory. We will further demonstrate the potential of the system with simultaneous molecular imaging of multiple receptors in vitro using labeled EGFR+/HER2+ SK-BR-3 human breast cancer cells with anti-EGFR immunolabeled gold nanospheres and anti-HER2 immunolabeled gold nanorods, with each scattering in different spectral windows. Additional trials will be performed to demonstrate molecularly specific binding using EGFR+/HER2- MDA-MB-468 and HER2+/EGFR- MDA-MB-453 breast cancer cells. © 2009 SPIE.

Authors
Crow, M; Marinakos, S; Chilkoti, A; Wax, A
MLA Citation
Crow, M, Marinakos, S, Chilkoti, A, and Wax, A. "Simultaneous molecular imaging of EGFR and HER2 using hyperspectral darkfield microscopy and immunotargeted nanoparticles." Progress in Biomedical Optics and Imaging - Proceedings of SPIE 7192 (2009).
Source
scival
Published In
Proceedings of SPIE
Volume
7192
Publish Date
2009
DOI
10.1117/12.808968

Allosteric actuation of inverse phase transition of a stimulus-responsive fusion polypeptide by ligand binding.

We report herein a biopolymer actuator with a modular design that allosterically transduces ligand binding into an aqueous demixing phase transition. The biopolymer actuator consists of two modular domains: a ligand binding protein domain, calmodulin (CaM), that is fused to a transducer domain, a stimulus-responsive elastin-like polypeptide (ELP) that exhibits a reversible lower critical solution temperature (LCST) phase transition. We demonstrate that binding of calcium to CaM spontaneously triggers the phase transition of the attached ELP, leading to formation of meso-microscale particles depending on the chain length of the ELP. This behavior is reversible as chelation of the bound calcium results in dissolution of the assembled particles, is selective for calcium as opposed to magnesium, and is abolished by the binding of a peptide ligand that is specific to calcium-bound CaM. These results are, to our knowledge, the first demonstration of biomolecular recognition-triggered, allosteric regulation of the LCST phase transition of a polymer and are significant because they expand the available triggers of the LCST transition of stimulus-responsive polymers to biochemical ligand binding. The ability to allosterically trigger the LCST transition of ELPs by biomolecular recognition will be useful for developing "smart" polymer actuators that capitalize upon the myriad ligand-protein pairs that are available from biology and for application in the design of selective pull-down assays in proteomics, drug delivery, and nanoscale biomolecular devices.

Authors
Kim, B; Chilkoti, A
MLA Citation
Kim, B, and Chilkoti, A. "Allosteric actuation of inverse phase transition of a stimulus-responsive fusion polypeptide by ligand binding." J Am Chem Soc 130.52 (December 31, 2008): 17867-17873.
PMID
19055326
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
130
Issue
52
Publish Date
2008
Start Page
17867
End Page
17873
DOI
10.1021/ja8059057

Effects of Hofmeister anions on the phase transition temperature of elastin-like polypeptides.

The modulation of the lower critical solution temperature (LCST) of two elastin-like polypeptides (ELPs) was investigated in the presence of 11 sodium salts that span the Hofmeister series for anions. It was found that the hydrophobic collapse/aggregation of these ELPs generally followed the series. Specifically, kosmotropic anions decreased the LCST by polarizing interfacial water molecules involved in hydrating amide groups on the ELPs. On the other hand, chaotropic anions lowered the LCST through a surface tension effect. Additionally, chaotropic anions showed salting-in properties at low salt concentrations that were related to the saturation binding of anions with the biopolymers. These overall mechanistic effects were similar to those previously found for the hydrophobic collapse and aggregation of poly(N-isopropylacrylamide), PNIPAM. There is, however, a crucial difference between PNIPAM and ELPs. Namely, PNIPAM undergoes a two-step collapse process as a function of temperature in the presence of sufficient concentrations of kosmotropic salts. By contrast, ELPs undergo collapse in a single step in all cases studied herein. This suggests that the removal of water molecules from around the amide moieties triggers the removal of hydrophobic hydration waters in a highly coupled process. There are also some key differences between the LCST behavior of the two ELPs. Specifically, the more hydrophilic ELP V5A2G(3)-120 construct displays collapse/aggregation behavior that is consistent with a higher concentration of anions partitioning to polymer/aqueous interface as compared to the more hydrophobic ELP V(5)-120. It was also found that larger anions could bind with ELP V5A2G(3)-120 more readily in comparison with ELP V(5)-120. These latter results were interpreted in terms of relative binding site accessibility of the anion for the ELP.

Authors
Cho, Y; Zhang, Y; Christensen, T; Sagle, LB; Chilkoti, A; Cremer, PS
MLA Citation
Cho, Y, Zhang, Y, Christensen, T, Sagle, LB, Chilkoti, A, and Cremer, PS. "Effects of Hofmeister anions on the phase transition temperature of elastin-like polypeptides." J Phys Chem B 112.44 (November 6, 2008): 13765-13771.
PMID
18842018
Source
pubmed
Published In
The Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces and Biophysical
Volume
112
Issue
44
Publish Date
2008
Start Page
13765
End Page
13771
DOI
10.1021/jp8062977

Temperature sensitive peptides: engineering hyperthermia-directed therapeutics.

PURPOSE: Recent progress suggests that short peptide motifs can be engineered into biopolymers with specific temperature dependent behavior. This review discusses peptide motifs capable of thermo-responsive behavior, and broadly summarizes design approaches that exploit these peptides as drug carriers. This review focuses on one class of thermally responsive peptide-based biopolymers, elastin-like polypeptides in greater detail. ANALYSIS: Four peptide motifs are presented based on leucine zippers, human collagen, human elastin, and silkworm silk that are potential building blocks for thermally responsive biopolymers. When these short motifs (<7 amino acids) are repeated many times, they generate biopolymers with higher order structure and complex temperature triggered behaviors. These structures are thermodynamically modulated, making them intrinsically temperature sensitive. These four motifs can be categorized by the directionality and reversibility of association. Elastin-like polypeptides (ELPs) are one promising motif that reversibly associates during heating. ELPs aggregate sharply above an inverse phase transition temperature, which depends on polymer hydrophobicity, molecular weight, and concentration. ELPs can be modified with chemotherapeutics, are biodegradable, are biocompatible, have low immunogenicity, and have terminal pharmacokinetic half-lives >8 h. ELP block copolymers can reversibly form micelles in response to hyperthermia, and this behavior can modulate the binding avidity of peptide ligands. When high molecular weight ELPs are systemically administered to mice they accumulate in tumors; furthermore, hyperthermia can initiate the ELP phase transition and double the concentration of peptide in the tumor. CONCLUSIONS: Temperature sensitive peptides are a powerful engineering platform that will enable new strategies for hyperthermia-directed drug delivery.

Authors
Mackay, JA; Chilkoti, A
MLA Citation
Mackay, JA, and Chilkoti, A. "Temperature sensitive peptides: engineering hyperthermia-directed therapeutics." Int J Hyperthermia 24.6 (September 2008): 483-495. (Review)
PMID
18608590
Source
pubmed
Published In
International Journal of Hyperthermia (Informa)
Volume
24
Issue
6
Publish Date
2008
Start Page
483
End Page
495
DOI
10.1080/02656730802149570

Surface plasmon optical study of the interfacial phase transition of elastinlike polypeptide grafted on gold.

The conformational changes in elastinlike polypeptides (ELPs) grafted to a solid/solution interface via different architectures were studied using surface plasmon resonance spectroscopy and surface plasmon field-enhanced fluorescence spectroscopy (SPFS). SPFS provides a simple and convenient optical method to study the influence of the grafting method and the graft density on the conformational changes in ELPs at the solid-solution interface as a function of environmental variables. A typical response of the ELP, consistent with its stimuli responsiveness, was a gradual collapse upon increasing the ionic strength; this effect was inversely correlated with the surface graft density of the ELP.

Authors
Xu, F; Joon, HM; Trabbic-Carlson, K; Chilkoti, A; Knoll, W
MLA Citation
Xu, F, Joon, HM, Trabbic-Carlson, K, Chilkoti, A, and Knoll, W. "Surface plasmon optical study of the interfacial phase transition of elastinlike polypeptide grafted on gold." Biointerphases 3.3 (September 2008): 66-74.
PMID
20408702
Source
pubmed
Published In
Biointerphases: an open access journal for the biomaterials interface community
Volume
3
Issue
3
Publish Date
2008
Start Page
66
End Page
74
DOI
10.1116/1.2965133

Hydration and conformational mechanics of single, end-tethered elastin-like polypeptides.

We investigated the effect of temperature, ionic strength, solvent polarity, and type of guest residue on the force-extension behavior of single, end-tethered elastin-like polypeptides (ELPs), using single molecule force spectroscopy (SMFS). ELPs are stimulus-responsive polypeptides that contain repeats of the five amino acids Val-Pro-Gly-Xaa-Gly (VPGXG), where Xaa is a guest residue that can be any amino acid with the exception of proline. We fitted the force-extension data with a freely jointed chain (FJC) model which allowed us to resolve small differences in the effective Kuhn segment length distributions that largely arise from differences in the hydrophobic hydration behavior of ELP. Our results agree qualitatively with predictions from recent molecular dynamics simulations and demonstrate that hydrophobic hydration modulates the molecular elasticity for ELPs. Furthermore, our results show that SMFS, when combined with our approach for data analysis, can be used to study the subtleties of polypeptide-water interactions and thus provides a basis for the study of hydrophobic hydration in intrinsically unstructured biomacromolecules.

Authors
Valiaev, A; Lim, DW; Schmidler, S; Clark, RL; Chilkoti, A; Zauscher, S
MLA Citation
Valiaev, A, Lim, DW, Schmidler, S, Clark, RL, Chilkoti, A, and Zauscher, S. "Hydration and conformational mechanics of single, end-tethered elastin-like polypeptides." J Am Chem Soc 130.33 (August 20, 2008): 10939-10946.
PMID
18646848
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
130
Issue
33
Publish Date
2008
Start Page
10939
End Page
10946
DOI
10.1021/ja800502h

PMSE 446-Architecture of polymer-drug conjugate controls in vivo fate and efficacy

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "PMSE 446-Architecture of polymer-drug conjugate controls in vivo fate and efficacy." August 17, 2008.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
236
Publish Date
2008

Synthesis and characterization of a thermally-responsive tumor necrosis factor antagonist.

Numerous antagonists of tumor necrosis factor alpha (TNFalpha) have been developed to attenuate inflammation and accompanying pain in many disease processes. Soluble TNF receptor type II (sTNFRII) is one such antagonist that sequesters TNFalpha away from target receptors and attenuates its activity. Systemic delivery of soluble TNF receptors or other antagonists may have deleterious side effects associated with immune suppression, so that strategies for locally targeted drug delivery are of interest. Elastin-like polypeptides (ELPs) are biopolymers capable of in situ drug depot formation through thermally-driven supramolecular complexes at physiological temperatures. A recombinant fusion protein between ELP and sTNFRII was designed and evaluated for retention of bivalent functionality. Thermal sensitivity was observed by formation of supramolecular submicron-sized particles at 32 degrees C, with gradual resolubilization from the depot observed at physiological temperatures. In vitro refolding of the sTNFRII domain was required and the purified product exhibited an equilibrium dissociation constant for interacting with TNFalpha that was seven-fold higher than free sTNFRII. Furthermore, anti-TNF activity was observed in inhibiting TNFalpha-mediated cytotoxicity in the murine L929 fibrosarcoma assay. Potential advantages of this ELP-sTNFRII fusion protein as an anti-TNFa drug depot include facility of injection, in situ depot formation, low endotoxin content, and functionality against TNFalpha.

Authors
Shamji, MF; Chen, J; Friedman, AH; Richardson, WJ; Chilkoti, A; Setton, LA
MLA Citation
Shamji, MF, Chen, J, Friedman, AH, Richardson, WJ, Chilkoti, A, and Setton, LA. "Synthesis and characterization of a thermally-responsive tumor necrosis factor antagonist." J Control Release 129.3 (August 7, 2008): 179-186.
PMID
18547669
Source
pubmed
Published In
Journal of Controlled Release
Volume
129
Issue
3
Publish Date
2008
Start Page
179
End Page
186
DOI
10.1016/j.jconrel.2008.04.021

Distance-dependent plasmon resonant coupling between a gold nanoparticle and gold film.

We present an experimental analysis of the plasmonic scattering properties of gold nanoparticles controllably placed nanometers away from a gold metal film. We show that the spectral response of this system results from the interplay between the localized plasmon resonance of the nanoparticle and the surface plasmon polaritons of the gold film, as previously predicted by theoretical studies. In addition, we report that the metal film induces a polarization to the single nanoparticle light scattering, resulting in a doughnut-shaped point spread function when imaged in the far-field. Both the spectral response and the polarization effects are highly sensitive to the nanoparticle-film separation distance. Such a system shows promise in potential biometrology and diagnostic devices.

Authors
Mock, JJ; Hill, RT; Degiron, A; Zauscher, S; Chilkoti, A; Smith, DR
MLA Citation
Mock, JJ, Hill, RT, Degiron, A, Zauscher, S, Chilkoti, A, and Smith, DR. "Distance-dependent plasmon resonant coupling between a gold nanoparticle and gold film." Nano Lett 8.8 (August 2008): 2245-2252.
PMID
18590340
Source
pubmed
Published In
Nano Letters
Volume
8
Issue
8
Publish Date
2008
Start Page
2245
End Page
2252
DOI
10.1021/nl080872f

In situ crosslinking elastin-like polypeptide gels for application to articular cartilage repair in a goat osteochondral defect model.

The objective of this study was to evaluate an injectable, in situ crosslinkable elastin-like polypeptide (ELP) gel for application to cartilage matrix repair in critically sized defects in goat knees. One cylindrical, osteochondral defect in each of seven animals was filled with an aqueous solution of ELP and a biocompatible, chemical crosslinker, while the contralateral defect remained unfilled and served as an internal control. Joints were sacrificed at 3 (n = 3) or 6 (n = 4) months for MRI, histological, and gross evaluation of features of biomaterial performance, including integration, cellular infiltration, surrounding matrix quality, and new matrix in the defect. At 3 months, ELP-filled defects scored significantly higher for integration by histological and gross grading compared to unfilled defects. ELP did not impede cell infiltration but appeared to be partly degraded. At 6 months, new matrix in unfilled defects outpaced that in ELP-filled defects and scored significantly better for MRI evidence of adverse changes, as well as integration and proteoglycan-containing matrix via gross and histological grading. The ELP-crosslinker solution was easily delivered and formed stable, well-integrated gels that supported cell infiltration and matrix synthesis; however, rapid degradation suggests that ELP formulation modifications should be optimized for longer-term benefits in cartilage repair applications.

Authors
Nettles, DL; Kitaoka, K; Hanson, NA; Flahiff, CM; Mata, BA; Hsu, EW; Chilkoti, A; Setton, LA
MLA Citation
Nettles, DL, Kitaoka, K, Hanson, NA, Flahiff, CM, Mata, BA, Hsu, EW, Chilkoti, A, and Setton, LA. "In situ crosslinking elastin-like polypeptide gels for application to articular cartilage repair in a goat osteochondral defect model." Tissue Eng Part A 14.7 (July 2008): 1133-1140.
PMID
18433311
Source
pubmed
Published In
Tissue Engineering, Part A
Volume
14
Issue
7
Publish Date
2008
Start Page
1133
End Page
1140
DOI
10.1089/ten.tea.2007.0245

PMSE 206-Architecture of polymer-drug conjugate controls in vivo fate and efficacy

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "PMSE 206-Architecture of polymer-drug conjugate controls in vivo fate and efficacy." April 6, 2008.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
235
Publish Date
2008

POLY 351-Thermally responsive elastin biopolymers for drug delivery

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "POLY 351-Thermally responsive elastin biopolymers for drug delivery." April 6, 2008.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
235
Publish Date
2008

An injectable and in situ-gelling biopolymer for sustained drug release following perineural administration.

STUDY DESIGN: This study evaluated whether the aggregation behavior of a thermally responsive elastin-like polypeptide (ELP) prolongs protein residence time at the dorsal root ganglion (DRG). This work involves development of a sustained-release drug delivery vehicle to provide high and sustained levels of biologic therapeutics to the dorsal root ganglion while minimizing systemic exposure. OBJECTIVE: To study the potential of the ELP biopolymer to sustain release and lower systemic exposure of bioactive peptides following perineural administration. SUMMARY OF BACKGROUND DATA: Anticytokine treatment for lumbar radiculopathy may offer clinical improvement, but exposes patients to systemic toxicities of immunosuppression. ELPs are environmentally responsive polypeptides that undergo a phase transition on heating to form an insoluble aggregate. Drug conjugates with ELP exhibit both temperature-sensitivity and in vitro bioactivity. Monomer resolubilization yields solution-phase molecules, and this reversible aggregation behavior may create a perineural drug depot to sustain drug delivery to an inflamed nerve. METHODS: This experiment involved 48 rats in which radiolabeled ELPs (aggregating or soluble) were injected overlying the L5 dorsal root ganglion. Animals were killed at 6 different time points, and radioactivity associated with the injected segment, serum, and other tissues was evaluated. RESULTS: The aggregating ELP demonstrated a 7-fold longer perineural half-life compared with the soluble ELP. This supports the hypothesis that the aggregating ELP forms a depot from which slow resolubilization and clearance provides sustained, local protein release. Furthermore, serum radioactivity reached a lower peak for the aggregating group, demonstrating slower absorption of the aggregating protein into the systemic circulation. CONCLUSION: These results suggest that ELP aggregation confer the benefit of perineural compartment longevity for bioactive therapeutics delivered fused with this carrier. This may sustain release of potent immunomodulator therapeutics to treat local neuroinflammation. Desirable features include delivery of high local doses and protection against systemic exposure and associated toxicity.

Authors
Shamji, MF; Whitlatch, L; Friedman, AH; Richardson, WJ; Chilkoti, A; Setton, LA
MLA Citation
Shamji, MF, Whitlatch, L, Friedman, AH, Richardson, WJ, Chilkoti, A, and Setton, LA. "An injectable and in situ-gelling biopolymer for sustained drug release following perineural administration." Spine (Phila Pa 1976) 33.7 (April 1, 2008): 748-754.
PMID
18379401
Source
pubmed
Published In
Spine
Volume
33
Issue
7
Publish Date
2008
Start Page
748
End Page
754
DOI
10.1097/BRS.0b013e3181695773

Label-free plasmonic detection of biomolecular binding by a single gold nanorod.

We report the use of individual gold nanorods as plasmonic transducers to detect the binding of streptavidin to individual biotin-conjugated nanorods in real time on a surface. Label-free detection at the single-nanorod level was performed by tracking the wavelength shift of the nanorod-localized surface plasmon resonant scattering spectrum using a dark-field microspectroscopy system. The lowest streptavidin concentration that was experimentally measured was 1 nM, which is a factor of 1000-fold lower than the previously reported detection limit for streptavidin binding by biotinylated single plasmonic nanostructures. We believe that the current optical setup is able to reliably measure wavelength shifts as small as 0.3 nm. Binding of streptavidin at 1 nM concentration induces a mean resonant wavelength shift of 0.59 nm suggesting that we are currently operating at close to the limit of detection of the system.

Authors
Nusz, GJ; Marinakos, SM; Curry, AC; Dahlin, A; Höök, F; Wax, A; Chilkoti, A
MLA Citation
Nusz, GJ, Marinakos, SM, Curry, AC, Dahlin, A, Höök, F, Wax, A, and Chilkoti, A. "Label-free plasmonic detection of biomolecular binding by a single gold nanorod." Anal Chem 80.4 (February 15, 2008): 984-989.
PMID
18197636
Source
pubmed
Published In
Analytical Chemistry
Volume
80
Issue
4
Publish Date
2008
Start Page
984
End Page
989
DOI
10.1021/ac7017348

Temperature triggered self-assembly of polypeptides into multivalent spherical micelles.

We report herein thermally responsive elastin-like polypeptides (ELPs) in a linear AB diblock architecture with an N-terminal peptide ligand that self-assemble into spherical micelles when heated slightly above body temperature. A series of 10 ELP block copolymers (ELP(BC)'s ) with different molecular weights and hydrophilic-to-hydrophobic block ratios were genetically synthesized by recursive directional ligation. The self-assembly of these polymers from unimers into micelles was investigated by light scattering, fluorescence spectroscopy, and cryo-TEM. These ELP(BC)'s undergo two phase transitions as a function of solution temperature: a unimer-to-spherical micelle transition at an intermediate temperature and a micelle-to-bulk aggregate transition at a higher temperature when the hydrophilic-to-hydrophobic block ratio is between 1:2 and 2:1. The critical micelle temperature is controlled by the length of the hydrophobic block, and the size of the micelle is controlled by both the total ELP(BC) length and hydrophilic-to-hydrophobic block ratio. These polypeptide micelles display a critical micelle concentration in the range 4-8 microM demonstrating the high stability of these structures. These studies have also identified a subset of ELP(BC)'s bearing terminal peptide ligands that are capable of forming multivalent spherical micelles that present multiple copies of the ligand on their corona in the clinically relevant temperature range 37-42 degrees C and target cancer cells. These ELP(BC)'s may be useful for drug targeting by thermally triggered multivalency. More broadly, the design rules uncovered by this study should be applicable to the design of other thermally reversible nanoparticles for diverse applications in medicine and biology.

Authors
Dreher, MR; Simnick, AJ; Fischer, K; Smith, RJ; Patel, A; Schmidt, M; Chilkoti, A
MLA Citation
Dreher, MR, Simnick, AJ, Fischer, K, Smith, RJ, Patel, A, Schmidt, M, and Chilkoti, A. "Temperature triggered self-assembly of polypeptides into multivalent spherical micelles." J Am Chem Soc 130.2 (January 16, 2008): 687-694.
PMID
18085778
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
130
Issue
2
Publish Date
2008
Start Page
687
End Page
694
DOI
10.1021/ja0764862

Peptide-based Biopolymers in Biomedicine and Biotechnology.

Peptides are emerging as a new class of biomaterials due to their unique chemical, physical, and biological properties. The development of peptide-based biomaterials is driven by the convergence of protein engineering and macromolecular self-assembly. This review covers the basic principles, applications, and prospects of peptide-based biomaterials. We focus on both chemically synthesized and genetically encoded peptides, including poly-amino acids, elastin-like polypeptides, silk-like polymers and other biopolymers based on repetitive peptide motifs. Applications of these engineered biomolecules in protein purification, controlled drug delivery, tissue engineering, and biosurface engineering are discussed.

Authors
Chow, D; Nunalee, ML; Lim, DW; Simnick, AJ; Chilkoti, A
MLA Citation
Chow, D, Nunalee, ML, Lim, DW, Simnick, AJ, and Chilkoti, A. "Peptide-based Biopolymers in Biomedicine and Biotechnology." Mater Sci Eng R Rep 62.4 (January 2008): 125-155.
PMID
19122836
Source
pubmed
Published In
Materials Science and Engineering: R: Reports
Volume
62
Issue
4
Publish Date
2008
Start Page
125
End Page
155
DOI
10.1016/j.mser.2008.04.004

In situ cross-linking of elastin-like polypeptide block copolymers for tissue repair.

Rapid cross-linking of elastin-like polypeptides (ELPs) with hydroxymethylphosphines (HMPs) in aqueous solution is attractive for minimally invasive in vivo implantation of biomaterials and tissue engineering scaffolds. In order to examine the independent effect of the location and number of reactive sites on the chemical cross-linking kinetics of ELPs and the mechanical properties of the resulting hydrogels, we have designed ELP block copolymers comprised of cross-linkable, hydrophobic ELP blocks with periodic Lys residues (A block) and aliphatic, hydrophilic ELP blocks with no cross-linking sites (B block); three different block architectures, A, ABA, and BABA were synthesized in this study. All ELP block copolymers were rapidly cross-linked with HMPs within several minutes under physiological conditions. The inclusion of the un-cross-linked hydrophilic block, its length relative to the cross-linkable hydrophobic block, and the block copolymer architecture all had a significant effect on swelling ratios of the cross-linked hydrogels, their microstructure, and mechanical properties. Fibroblasts embedded in the ELP hydrogels survived the cross-linking process and remained viable for at least 3 days in vitro when the gels were formed from an equimolar ratio of HMPs and Lys residues of ELPs. DNA quantification of the embedded cells indicated that the cell viability within triblock ELP hydrogels was statistically greater than that in the monoblock gels at day 3. These results suggest that the mechanical properties of ELP hydrogels and the microenvironment that they present to cells can be tuned by the design of the block copolymer architecture.

Authors
Lim, DW; Nettles, DL; Setton, LA; Chilkoti, A
MLA Citation
Lim, DW, Nettles, DL, Setton, LA, and Chilkoti, A. "In situ cross-linking of elastin-like polypeptide block copolymers for tissue repair." Biomacromolecules 9.1 (January 2008): 222-230.
PMID
18163573
Source
pubmed
Published In
Biomacromolecules
Volume
9
Issue
1
Publish Date
2008
Start Page
222
End Page
230
DOI
10.1021/bm7007982

Patterning materials with biomolecules

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "Patterning materials with biomolecules." 8th World Biomaterials Congress 2008 4 (2008): 2249-2250.
Source
scival
Published In
8th World Biomaterials Congress 2008
Volume
4
Publish Date
2008
Start Page
2249
End Page
2250

Fabrication of fiber-optic sensors based on plasmon resonances of metallic nanostructures

This paper describes the fabrication of portable and robust fiber optic sensors - mainly chemical and biological sensors - on standard communication grade optical fibers. Metallic nanostructures such as nanoparticles, nanopillars, nanorods, and nanoholes in optically thick metallic films were employed to precisely control the enhancement and absorption of light so as to form sensitive and specific optical sensors based on optical fibers. The fiber-optic sensors employ localized plasmon resonances (LSPRs) of metallic nanostructures formed on the fiber end-face as well as surface plasmon resonances associated with nanoholes in optically thick metallic films as a means of transducing the input optical signal. These metallic nanostructures were formed on the cleaved end-face of multimode and single mode optical fibers and were employed in both reflection and transmission modes. Metallic nanoparticles, nanorods, and nanopillars were formed on the tip or surface of the optical fiber by either employing chemical means or by first depositing a 10-200 nm layer of metallic film on the tip of the fiber and then employing focused ion beam (FIB) milling to pattern out the metallic nanoparticles and nanopillars from the film. Ordered arrays of nanoholes were formed in optically thick (150-230 nm) metallic films by employing FIB milling. Gold (Au) nanostructures are chemically stable and result in plasmon resonance related dips in the transmission spectrum in the visible spectral region. Hence, gold was selected as the material of choice for the fabrication of the plasmon resonance sensors. © The Electrochemical Society.

Authors
Dhawan, A; Gerhold, M; Marinakos, S; Leonard, D; Wang, H-N; Chilkoti, A; Russell, P; Vo-Dinh, T
MLA Citation
Dhawan, A, Gerhold, M, Marinakos, S, Leonard, D, Wang, H-N, Chilkoti, A, Russell, P, and Vo-Dinh, T. "Fabrication of fiber-optic sensors based on plasmon resonances of metallic nanostructures." ECS Transactions 11.14 (2008): 41-55.
Source
scival
Published In
ECS Transactions
Volume
11
Issue
14
Publish Date
2008
Start Page
41
End Page
55
DOI
10.1149/1.2902314

Photothermal optical coherence tomography for molecular contrast imaging

Photothermal optical coherence tomography with laser-heated gold nanorods as the photothermal source is proposed as a novel molecular imaging technique. A description of the technique and validation experiments are reported. © Optical Society of America.

Authors
Skala, MC; Marinakos, S; Chilkoti, A; Izatt, JA
MLA Citation
Skala, MC, Marinakos, S, Chilkoti, A, and Izatt, JA. "Photothermal optical coherence tomography for molecular contrast imaging." Biomedical Optics, BIOMED 2008 (2008): BWF5-.
Source
scival
Published In
Biomedical Optics, BIOMED 2008
Publish Date
2008
Start Page
BWF5

Surface-initiated enzymatic polymerization of DNA.

We describe a technique to synthesize DNA homopolymers on a surface using surface-initiated enzymatic polymerization (SIEP) with terminal deoxynucleotidyl transferase (TdTase), an enzyme that repetitively adds mononucleotides to the 3'-end of oligonucleotides. The thickness of the synthesized DNA layer was found to depend on the deoxymononucleotide monomer, in the order of dATP > dTTP > dGTP approximately dCTP. In addition, the composition and the surface density of oligonucleotide initiators were also important in controlling the extent of DNA polymerization. The extension of single-stranded DNA chains by SIEP was further verified by their binding to antibodies specific to oligonucleotides. TdTase-mediated SIEP can also be used to grow spatially defined three-dimensional DNA structures by soft lithography and is a new tool for bioinspired fabrication at the micro- and nanoscale.

Authors
Chow, DC; Chilkoti, A
MLA Citation
Chow, DC, and Chilkoti, A. "Surface-initiated enzymatic polymerization of DNA." Langmuir 23.23 (November 6, 2007): 11712-11717.
PMID
17929953
Source
pubmed
Published In
Langmuir
Volume
23
Issue
23
Publish Date
2007
Start Page
11712
End Page
11717
DOI
10.1021/la701630g

Development and characterization of a fusion protein between thermally responsive elastin-like polypeptide and interleukin-1 receptor antagonist: sustained release of a local antiinflammatory therapeutic.

OBJECTIVE: Interleukin-1 receptor antagonist (IL-1Ra) has been evaluated for the intraarticular treatment of osteoarthritis. Such administration of proteins may have limited utility because of their rapid clearance and short half-life in the joint. The fusion of a drug to elastin-like polypeptides (ELPs) promotes the formation of aggregating particles that form a "drug depot" at physiologic temperatures, a phenomenon intended to prolong the presence of the drug. The purpose of this study was to develop an injectable drug depot composed of IL-1Ra and ELP domains and to evaluate the properties and bioactivity of the recombinant ELP-IL-1Ra fusion protein. METHODS: Fusion proteins between IL-1Ra and 2 distinct sequences and molecular weights of ELP were overexpressed in Escherichia coli. Environmental sensitivity was demonstrated by turbidity and dynamic light scattering as a function of temperature. IL-1Ra domain activity was evaluated by surface plasmon resonance, and in vitro antagonism of IL-1-mediated lymphocyte and thymocyte proliferation, as well as IL-1-induced tumor necrosis factor alpha (TNFalpha) expression and matrix metalloproteinase 3 (MMP-3) and ADAMTS-4 messenger RNA expression in human intervertebral disc fibrochondrocytes. IL-1Ra immunoreactivity was assessed before and after proteolytic degradation of the ELP partner. RESULTS: Both fusion proteins underwent supramolecular aggregation at subphysiologic temperatures and slowly resolubilized at 37 degrees C. Interaction with IL-1 receptor was slower in association but equivalent in dissociation as compared with the commercial antagonist. Anti-IL-1 activity was demonstrated by inhibition of lymphocyte and thymocyte proliferation and by decreased TNFalpha expression and ADAMTS-4 and MMP-3 transcription by fibrochondrocytes. ELP domain proteolysis liberated a peptide of comparable size and immunoreactivity as the commercial IL-1Ra. This peptide was more bioactive against lymphocyte proliferation, nearly equivalent to the commercial antagonist. CONCLUSION: The ELP-IL-1Ra fusion protein proved to retain the characteristic ELP inverse phase-transitioning behavior as well as the bioactivity of the IL-1Ra domain. This technology represents a novel drug carrier designed to prolong the presence of bioactive peptides following intraarticular delivery.

Authors
Shamji, MF; Betre, H; Kraus, VB; Chen, J; Chilkoti, A; Pichika, R; Masuda, K; Setton, LA
MLA Citation
Shamji, MF, Betre, H, Kraus, VB, Chen, J, Chilkoti, A, Pichika, R, Masuda, K, and Setton, LA. "Development and characterization of a fusion protein between thermally responsive elastin-like polypeptide and interleukin-1 receptor antagonist: sustained release of a local antiinflammatory therapeutic." Arthritis Rheum 56.11 (November 2007): 3650-3661.
PMID
17968946
Source
pubmed
Published In
Arthritis and Rheumatism
Volume
56
Issue
11
Publish Date
2007
Start Page
3650
End Page
3661
DOI
10.1002/art.22952

Elastin biopolymers for drug delivery

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "Elastin biopolymers for drug delivery." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 234 (August 19, 2007).
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
234
Publish Date
2007

POLY 627-Fabrication of bioconjugated and hybrid polymeric nanostructures by field-induced scanning probe lithography

Authors
Garcia, A; Hucknall, A; Johannes, M; Clark, R; Chilkoti, A; Zauscher, S
MLA Citation
Garcia, A, Hucknall, A, Johannes, M, Clark, R, Chilkoti, A, and Zauscher, S. "POLY 627-Fabrication of bioconjugated and hybrid polymeric nanostructures by field-induced scanning probe lithography." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 234 (August 19, 2007).
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
234
Publish Date
2007

The effect of covalently immobilized rhIL-1ra-ELP fusion protein on the inflammatory profile of LPS-stimulated human monocytes.

The objective of this research was to investigate whether immobilized anti-inflammatory cytokines will signal changes in the inflammatory profile of cultured monocytes. A fusion protein of recombinant human IL-1 receptor antagonist and elastin-like peptide (IL-1ra-ELP) was expressed in Escherichia coli. THP-1 human monocytes were cultured on either carboxyl-terminated self-assembled monolayers (SAMs), or SAMs with either covalently immobilized or soluble IL-1ra-ELP. LPS-stimulated monocytes exposed to either soluble or immobilized IL-1ra-ELP were prevented from cell differentiation, showed attenuated expression of pro-inflammatory cytokines, and had increased production of anti-inflammatory and pro-wound healing cytokines. These results suggest that immobilized anti-inflammatory cytokines have the potential to be immunomodulatory biomaterials.

Authors
Kim, D-H; Smith, JT; Chilkoti, A; Reichert, WM
MLA Citation
Kim, D-H, Smith, JT, Chilkoti, A, and Reichert, WM. "The effect of covalently immobilized rhIL-1ra-ELP fusion protein on the inflammatory profile of LPS-stimulated human monocytes." Biomaterials 28.23 (August 2007): 3369-3377.
PMID
17482260
Source
pubmed
Published In
Biomaterials
Volume
28
Issue
23
Publish Date
2007
Start Page
3369
End Page
3377
DOI
10.1016/j.biomaterials.2007.04.010

Multifunctional thermally transitioning oligopeptides prepared by ring-opening metathesis polymerization.

Authors
Roberts, SK; Chilkoti, A; Setton, LA
MLA Citation
Roberts, SK, Chilkoti, A, and Setton, LA. "Multifunctional thermally transitioning oligopeptides prepared by ring-opening metathesis polymerization." Biomacromolecules 8.8 (August 2007): 2618-2621.
PMID
17625908
Source
pubmed
Published In
Biomacromolecules
Volume
8
Issue
8
Publish Date
2007
Start Page
2618
End Page
2621
DOI
10.1021/bm0702713

Plasmonic detection of a model analyte in serum by a gold nanorod sensor.

We describe the fabrication of a label-free, chip-based biosensor based on the localized surface plasmon resonance (LSPR) of gold nanorods. Gold nanorods were chemisorbed onto a mercaptosilane-modified glass substrate, followed by conjugation of biotin to the nanorods. Streptavidin binding to biotin was monitored by the wavelength shift of the LSPR peak in the UV-vis extinction spectrum of the immobilized gold nanorods due to the change in local refractive index at the gold nanorod surface induced by streptavidin binding. The limit of detection of the sensor is 0.005 microg/mL (94 pM) in PBS and 1 microg/mL (19 nM) in serum, and the dynamic range spans 94 pM to 0.19 microM. The advantages of the nanorod-based sensor over an LSPR sensor that we had previously fabricated from gold nanospheres (Nath, N.; Chilkoti, A. Anal. Chem. 2002, 74, 504-509; J. Fluoresc. 2004, 14, 377-389; Anal. Chem. 2004, 76, 5370-5378) are the significantly lower detection limit and the internal self-reference that the signal of the nanorod sensor provides based on the measurement of peak wavelength shift.

Authors
Marinakos, SM; Chen, S; Chilkoti, A
MLA Citation
Marinakos, SM, Chen, S, and Chilkoti, A. "Plasmonic detection of a model analyte in serum by a gold nanorod sensor." Anal Chem 79.14 (July 15, 2007): 5278-5283.
PMID
17567106
Source
pubmed
Published In
Analytical Chemistry
Volume
79
Issue
14
Publish Date
2007
Start Page
5278
End Page
5283
DOI
10.1021/ac0706527

Toward a systems engineering approach to cancer drug delivery.

Authors
Dreher, MR; Chilkoti, A
MLA Citation
Dreher, MR, and Chilkoti, A. "Toward a systems engineering approach to cancer drug delivery." J Natl Cancer Inst 99.13 (July 4, 2007): 983-985.
PMID
17596569
Source
pubmed
Published In
Journal of the National Cancer Institute
Volume
99
Issue
13
Publish Date
2007
Start Page
983
End Page
985
DOI
10.1093/jnci/djm042

Force-induced prolyl cis-trans isomerization in elastin-like polypeptides.

Elastin-like polypeptides (ELPs) are stimulus-responsive polymers that contain repeats of five amino acids, Val-Pro-Gly-Xaa-Gly (VPGXG), where Xaa is a guest residue that can be any amino acid with the exception of proline. While studying the conformational mechanics of ELPs over a range of solvent conditions by single-molecule force spectroscopy, we noticed that some force-extension curves showed temperature-independent, extensional transitions that could not be fitted with a freely jointed chain or worm-like chain model. Here we show that the observed molecular elongation results from the force-induced peptidyl-prolyl cis-trans isomerization in prolines, which are repeated every fifth residue in the main chain of ELPs. Control experiments with poly(L-proline) demonstrate the similarity of the conformational transition between poly(L-proline) and ELPs. In contrast, the force-extension behavior of poly(L-lysine) showed no deviation in the relevant force range. Force-extension curves in hysteresis experiments showed an elongational difference between extension and relaxation pathways that suggests that the cis conformational state of the prolines could be exhausted on the time scale of the experiment. We present further computational evidence for this mechanism by Monte Carlo simulation of the force-extension behavior using an elastically coupled, two-state model. We believe ours is the first demonstration of force-induced prolyl cis-trans isomerization in proline-containing polypeptides. Our results suggest that single-molecule force spectroscopy could provide an alternate means to assay this important conformational transition in polypeptides.

Authors
Valiaev, A; Lim, DW; Oas, TG; Chilkoti, A; Zauscher, S
MLA Citation
Valiaev, A, Lim, DW, Oas, TG, Chilkoti, A, and Zauscher, S. "Force-induced prolyl cis-trans isomerization in elastin-like polypeptides." J Am Chem Soc 129.20 (May 23, 2007): 6491-6497.
PMID
17469821
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
129
Issue
20
Publish Date
2007
Start Page
6491
End Page
6497
DOI
10.1021/ja070147r

Thermal cycling enhances the accumulation of a temperature-sensitive biopolymer in solid tumors.

The delivery of anticancer therapeutics to solid tumors remains a critical problem in the treatment of cancer. This study reports a new methodology to target a temperature-responsive macromolecular drug carrier, an elastin-like polypeptide (ELP) to solid tumors. Using a dorsal skin fold window chamber model and intravital laser scanning confocal microscopy, we show that the ELP forms micron-sized aggregates that adhere to the tumor vasculature only when tumors are heated to 41.5 degrees C. Upon return to normothermia, the vascular particles dissolve into the plasma, increasing the vascular concentration, which drives more ELPs across the tumor blood vessel and significantly increases its extravascular accumulation. These observations suggested that thermal cycling of tumors would increase the exposure of tumor cells to ELP drug carriers. We investigated this hypothesis in this study by thermally cycling an implanted tumor in nude mice from body temperature to 41.5 degrees C thrice within 1.5 h, and showed the repeated formation of adherent microparticles of ELP in the heated tumor vasculature in each thermal cycle. These results suggest that thermal cycling of tumors can be repeated multiple times to further increase the accumulation of a thermally responsive polymeric drug carrier in solid tumors over a single heat-cool cycle. More broadly, this study shows a new approach--tumor thermal cycling--to exploit stimuli-responsive polymers in vivo to target the tumor vasculature or extravascular compartment with high specificity.

Authors
Dreher, MR; Liu, W; Michelich, CR; Dewhirst, MW; Chilkoti, A
MLA Citation
Dreher, MR, Liu, W, Michelich, CR, Dewhirst, MW, and Chilkoti, A. "Thermal cycling enhances the accumulation of a temperature-sensitive biopolymer in solid tumors." Cancer Res 67.9 (May 1, 2007): 4418-4424.
PMID
17483356
Source
pubmed
Published In
Cancer Research
Volume
67
Issue
9
Publish Date
2007
Start Page
4418
End Page
4424
DOI
10.1158/0008-5472.CAN-06-4444

Improved non-chromatographic purification of a recombinant protein by cationic elastin-like polypeptides.

This paper reports an improvement in the purification of thioredoxin (Trx) expressed from E. coli by inverse transition cycling (ITC) using cationic elastin-like polypeptides (ELPs). Two ELP libraries having 2% and 5% lysine residues and molecular weights ranging from 4 to 61.1 kDa showed greater salt sensitivity in their inverse transition behavior than purely aliphatic ELPs. Expression yield of Trx-ELP fusions was an unpredictable function of guest residue composition, but reducing the molecular weight of the ELP tag generally increased Trx yield. A cationic 4.3 kDa ELP is the shortest ELP used to purify any protein by ITC to date. A 15.9 kDa ELP with a guest residue composition of K:V:F of 1:7:1 was found to be the optimal cationic tag to purify Trx, as it provided 50% greater Trx yield and only required one-fifth the added NaCl for purification of Trx as compared to previously used aliphatic ELP tags.

Authors
Lim, DW; Trabbic-Carlson, K; Mackay, JA; Chilkoti, A
MLA Citation
Lim, DW, Trabbic-Carlson, K, Mackay, JA, and Chilkoti, A. "Improved non-chromatographic purification of a recombinant protein by cationic elastin-like polypeptides." Biomacromolecules 8.5 (May 2007): 1417-1424.
PMID
17407348
Source
pubmed
Published In
Biomacromolecules
Volume
8
Issue
5
Publish Date
2007
Start Page
1417
End Page
1424
DOI
10.1021/bm060849t

Rapid cross-linking of elastin-like polypeptides with (hydroxymethyl)phosphines in aqueous solution.

In situ gelation of injectable polypeptide-based materials is attractive for minimally invasive in vivo implantation of biomaterials and tissue engineering scaffolds. We demonstrate that chemically cross-linked elastin-like polypeptide (ELP) hydrogels can be rapidly formed in aqueous solution by reacting lysine-containing ELPs with an organophosphorous cross-linker, beta-[tris(hydroxymethyl)phosphino]propionic acid (THPP) under physiological conditions. The mechanical properties of the cross-linked ELP hydrogels were largely modulated by the molar concentration of lysine residues in the ELP and the pH at which the cross-linking reaction was carried out. Fibroblasts embedded in ELP hydrogels survived the cross-linking process and were viable after in vitro culture for 3 days. DNA quantification of ELP hydrogels with encapsulated fibroblasts indicated that there was no significant difference in DNA content between day 0 and day 3 when ELP hydrogels were formed with an equimolar ratio of THPP and lysine residues of the ELPs. These results suggest that THPP cross-linking may be a biocompatible strategy for the in situ formation of cross-linked hydrogels.

Authors
Lim, DW; Nettles, DL; Setton, LA; Chilkoti, A
MLA Citation
Lim, DW, Nettles, DL, Setton, LA, and Chilkoti, A. "Rapid cross-linking of elastin-like polypeptides with (hydroxymethyl)phosphines in aqueous solution." Biomacromolecules 8.5 (May 2007): 1463-1470.
PMID
17411091
Source
pubmed
Published In
Biomacromolecules
Volume
8
Issue
5
Publish Date
2007
Start Page
1463
End Page
1470
DOI
10.1021/bm061059m

Analysis of total uncertainty in spectral peak measurements for plasmonic nanoparticle-based biosensors.

One goal of recent research on plasmonic nanoparticle-based sensors is maximizing nanoparticle sensitivity or shift of resonance peak wavelength per refractive index change. Equally important is a measurement system's peak location uncertainty or shift resolution. We provide systematic analyses and discuss optimization of factors that determine peak location uncertainty, reporting values as low as 0.3 nm for the presented scheme. This type of analysis is important, in part, because it provides a means of evaluating detection thresholds for biosensor applications such as analyte binding. We estimate thresholds of 310 streptavidin molecules for the presented scheme and 20 molecules with system improvements.

Authors
Curry, A; Nusz, G; Chilkoti, A; Wax, A
MLA Citation
Curry, A, Nusz, G, Chilkoti, A, and Wax, A. "Analysis of total uncertainty in spectral peak measurements for plasmonic nanoparticle-based biosensors." Appl Opt 46.10 (April 1, 2007): 1931-1939.
PMID
17356640
Source
pubmed
Published In
Applied Optics
Volume
46
Issue
10
Publish Date
2007
Start Page
1931
End Page
1939

Fabrication of gold nanowires by electric-field-induced scanning probe lithography and in situ chemical development.

Authors
Lee, W-K; Chen, S; Chilkoti, A; Zauscher, S
MLA Citation
Lee, W-K, Chen, S, Chilkoti, A, and Zauscher, S. "Fabrication of gold nanowires by electric-field-induced scanning probe lithography and in situ chemical development." Small 3.2 (February 2007): 249-254.
PMID
17199247
Source
pubmed
Published In
Small
Volume
3
Issue
2
Publish Date
2007
Start Page
249
End Page
254
DOI
10.1002/smll.200600396

Microcantilever sensing and actuation with end-grafted stimulus-responsive elastin-like polypeptides.

Stimulus-responsive elastin-like polypeptides (ELPs) grafted onto surfaces are of significant technical interest because they can be exploited for force generation, in sensing applications, or as molecular switches with tunable properties. Changes in the conformational state of grafted ELPs, induced by a phase transition or changes in osmotic pressure, lead to significant changes in the surface stress in the ELP graft layer and translate into detectable changes in microcantilever deflection. In this study, we investigate the conformational mechanics of ELPs in response to changes in solution pH and ionic strength using atomic force microscopy (AFM) microcantilever deflection and quartz crystal microbalance (QCM) measurements. We show that the use of genetically encoded, surface-grafted ELPs is exciting for cantilever actuation and sensing because commonly available microfabricated cantilever springs offer a simple and nonintrusive way to detect changes in solvent type, temperature, and pH, promising great potential for sensing applications in microfluidic devices.

Authors
Valiaev, A; Abu-Lail, NI; Lim, DW; Chilkoti, A; Zauscher, S
MLA Citation
Valiaev, A, Abu-Lail, NI, Lim, DW, Chilkoti, A, and Zauscher, S. "Microcantilever sensing and actuation with end-grafted stimulus-responsive elastin-like polypeptides." Langmuir 23.1 (January 2, 2007): 339-344.
PMID
17190524
Source
pubmed
Published In
Langmuir
Volume
23
Issue
1
Publish Date
2007
Start Page
339
End Page
344
DOI
10.1021/la0616698

Purification of recombinant proteins from Escherichia coli at low expression levels by inverse transition cycling.

Authors
Christensen, T; Trabbic-Carlson, K; Liu, W; Chilkoti, A
MLA Citation
Christensen, T, Trabbic-Carlson, K, Liu, W, and Chilkoti, A. "Purification of recombinant proteins from Escherichia coli at low expression levels by inverse transition cycling." Anal Biochem 360.1 (January 1, 2007): 166-168.
PMID
17084802
Source
pubmed
Published In
Analytical Biochemistry
Volume
360
Issue
1
Publish Date
2007
Start Page
166
End Page
168
DOI
10.1016/j.ab.2006.09.020

Biomedical and biotechnological applications of Elastin-like polypeptides

Elastin-like polypeptides (ELPs) are a unique class of genetically encoded biopolymers with tunable thermosensitivity and biocompatibility. This review provides an introduction to ELPs and an overview of their design and synthesis. It also discusses applications of these polypeptides for drug delivery, tissue engineering, protein purification, and biosensing.

Authors
Simnick, AJ; Lim, DW; Chow, D; Chilkoti, A
MLA Citation
Simnick, AJ, Lim, DW, Chow, D, and Chilkoti, A. "Biomedical and biotechnological applications of Elastin-like polypeptides." Polymer Reviews 47.1 (2007): 121-154.
Source
scival
Published In
Polymer Reviews
Volume
47
Issue
1
Publish Date
2007
Start Page
121
End Page
154
DOI
10.1080/15583720601109594

Single molecule nanomechanics of HIV-1 envelope glycoproteins and elastin-like polypeptides

Authors
Lam, Y; Valiaev, A; Lim, D-W; Chilkoti, A; Alam, SM; Zauscher, S
MLA Citation
Lam, Y, Valiaev, A, Lim, D-W, Chilkoti, A, Alam, SM, and Zauscher, S. "Single molecule nanomechanics of HIV-1 envelope glycoproteins and elastin-like polypeptides." Proceedings of the SEM Annual Conference and Exposition on Experimental and Applied Mechanics 2007 1 (2007): 624-625.
Source
scival
Published In
Proceedings of the SEM Annual Conference and Exposition on Experimental and Applied Mechanics 2007
Volume
1
Publish Date
2007
Start Page
624
End Page
625

Stimulus responsive elastin biopolymers: Applications in medicine and biotechnology.

Elastin-like polypeptides (ELPs) are artificial polypeptides, derived from Val-Pro-Gly-Xaa-Gly (VPGXG) pentapeptide repeats found in human tropoelastin, that reversibly coacervate above a critical temperature. Genetically encodable ELPs are monodisperse, stimuli responsive, and biocompatible, properties that make them attractive for drug delivery and tissue engineering. The potential of ELPs to self-assemble into nanostructures in response to environmental triggers is another interesting feature of these polypeptides that promises to lead to a host of new applications.

Authors
Chilkoti, A; Christensen, T; MacKay, JA
MLA Citation
Chilkoti, A, Christensen, T, and MacKay, JA. "Stimulus responsive elastin biopolymers: Applications in medicine and biotechnology." Curr Opin Chem Biol 10.6 (December 2006): 652-657. (Review)
PMID
17055770
Source
pubmed
Published In
Current Opinion in Chemical Biology
Volume
10
Issue
6
Publish Date
2006
Start Page
652
End Page
657
DOI
10.1016/j.cbpa.2006.10.010

Tumor accumulation, degradation and pharmacokinetics of elastin-like polypeptides in nude mice.

ELPs are genetically engineered, thermally responsive polypeptides that preferentially accumulate in solid tumors subjected to focused, mild hyperthermia. In this paper, we report the biodegradation, pharmacokinetics, tumor localization, and tumor spatial distribution of (14)C-labeled ELPs that were radiolabeled during their biosynthesis in Escheriehia coli. The in vitro degradation rate of a thermally responsive (14)C-labeled ELP1 ([(14)C] ELP1) with a molecular weight of 59.4 kDa, upon exposure to murine serum, was 2.49 wt.%/day. The apparent in vivo degradation rate of ELP1 after intravenous injection of nude mice was 2.46 wt.%/day and its terminal half-life was 8.7 h. The tumor accumulation and spatial distribution of intravenously administered ELP1 and a control ELP that was designed to remain soluble in heated tumors (ELP2) were examined in both heated (41.5 degrees C) and unheated tumors. ELP1 accumulated at a significantly higher concentration in heated tumors than ELP1 in unheated tumors and ELP2 in heated tumors. Quantitative autoradiography of tumor sections provided similar tumor accumulation results as the whole tumor analysis but, in addition, showed that ELP1 had a more homogeneous distribution in heated tumors and a greater concentration in the tumor center than either control treatment.

Authors
Liu, W; Dreher, MR; Furgeson, DY; Peixoto, KV; Yuan, H; Zalutsky, MR; Chilkoti, A
MLA Citation
Liu, W, Dreher, MR, Furgeson, DY, Peixoto, KV, Yuan, H, Zalutsky, MR, and Chilkoti, A. "Tumor accumulation, degradation and pharmacokinetics of elastin-like polypeptides in nude mice." J Control Release 116.2 (November 28, 2006): 170-178.
PMID
16919353
Source
pubmed
Published In
Journal of Controlled Release
Volume
116
Issue
2
Publish Date
2006
Start Page
170
End Page
178
DOI
10.1016/j.jconrel.2006.06.026

Tumor accumulation, degradation and pharmacokinetics of elastin-like polypeptides in nude mice

ELPs are genetically engineered, thermally responsive polypeptides that preferentially accumulate in solid tumors subjected to focused, mild hyperthermia. In this paper, we report the biodegradation, pharmacokinetics, tumor localization, and tumor spatial distribution of C-14-labeled ELPs that were radiolabeled during their biosynthesis in Escheriehia coli. The in vitro degradation rate of a thermally responsive C-14-labeled ELP1 ([C-14] ELP1) with a molecular weight of 59.4 kDa, upon exposure to murine serum, was 2.49 wt.\%/day. The apparent in vivo degradation rate of ELP1 after intravenous injection of nude mice was 2.46 wt.\%/day and its terminal half-life was 8.7 h. The tumor accumulation and spatial distribution of intravenously administered ELP1 and a control ELP that was designed to remain soluble in heated tumors (ELP2) were examined in both heated (41.5 degrees C) and unheated tumors. ELP1 accumulated at a significantly higher concentration in heated tumors than ELP1 in unheated tumors and ELP2 in heated tumors. Quantitative autoradiography of tumor sections provided similar tumor accumulation results as the whole tumor analysis but, in addition, showed that ELP1 had a more homogeneous distribution in heated tumors and a greater concentration in the tumor center than either control treatment. (c) 2006 Elsevier B.V. All rights reserved.

Authors
Liu, WE; Dreher, MR; Furgeson, DY; Peixoto, KV; Yuan, H; Zalutsky, MR; Chilkoti, A
MLA Citation
Liu, WE, Dreher, MR, Furgeson, DY, Peixoto, KV, Yuan, H, Zalutsky, MR, and Chilkoti, A. "Tumor accumulation, degradation and pharmacokinetics of elastin-like polypeptides in nude mice." Journal Of Controlled Release 116.2 (November 2006): 170-178. (Academic Article)
Source
manual
Published In
Journal of Controlled Release
Volume
116
Issue
2
Publish Date
2006
Start Page
170
End Page
178

Purification of an elastin-like fusion protein by microfiltration.

This article describes a simple and potentially scalable microfiltration method for purification of recombinant proteins. This method is based on the fact that when an elastin-like polypeptide (ELP) is fused to a target protein, the inverse phase transition behavior of the ELP tag is imparted to the fusion protein. Triggering the phase transition of a solution of the ELP fusion protein by an increase in temperature, or isothermally by an increase in salt concentration, results in the formation of micron-sized aggregates of the ELP fusion protein. In this article, it is shown that these aggregates are efficiently retained by a microfiltration membrane, while contaminating E. coli proteins passed through the membrane upon washing. Upon reversing the phase transition by flow of Milli-Q water, soluble, pure, and functionally active protein is eluted from the membrane. Proof-of principle of this approach was demonstrated by purifying a fusion of thioredoxin with ELP (Trx-ELP) with greater than 95% recovery of protein and with greater than 95% purity (as estimated from SDS-PAGE gels). The simplicity of this method is demonstrated for laboratory scale purification by purifying Trx-ELP from cell lysate using a syringe and a disposable microfiltration cartridge. The potential scalability of this purification as an automated, continuous industrial-scale process is also demonstrated using a continuous stirred cell equipped with a microfiltration membrane.

Authors
Ge, X; Trabbic-Carlson, K; Chilkoti, A; Filipe, CDM
MLA Citation
Ge, X, Trabbic-Carlson, K, Chilkoti, A, and Filipe, CDM. "Purification of an elastin-like fusion protein by microfiltration." Biotechnol Bioeng 95.3 (October 20, 2006): 424-432.
PMID
16767781
Source
pubmed
Published In
Biotechnology & Bioengineering
Volume
95
Issue
3
Publish Date
2006
Start Page
424
End Page
432
DOI
10.1002/bit.21046

A thermally responsive biopolymer for intra-articular drug delivery.

Intra-articular drug delivery is the preferred standard for targeting pharmacologic treatment directly to joints to reduce undesirable side effects associated with systemic drug delivery. In this study, a biologically based drug delivery vehicle was designed for intra-articular drug delivery using elastin-like polypeptides (ELPs), a biopolymer composed of repeating pentapeptides that undergo a phase transition to form aggregates above their transition temperature. The ELP drug delivery vehicle was designed to aggregate upon intra-articular injection at 37 degrees C, and form a drug 'depot' that could slowly disaggregate and be cleared from the joint space over time. We evaluated the in vivo biodistribution and joint half-life of radiolabeled ELPs, with and without the ability to aggregate, at physiological temperatures encountered after intra-articular injection in a rat knee. Biodistribution studies revealed that the aggregating ELP had a 25-fold longer half-life in the injected joint than a similar molecular weight protein that remained soluble and did not aggregate. These results suggest that the intra-articular joint delivery of ELP-based fusion proteins may be a viable strategy for the prolonged release of disease-modifying protein drugs for osteoarthritis and other arthritides.

Authors
Betre, H; Liu, W; Zalutsky, MR; Chilkoti, A; Kraus, VB; Setton, LA
MLA Citation
Betre, H, Liu, W, Zalutsky, MR, Chilkoti, A, Kraus, VB, and Setton, LA. "A thermally responsive biopolymer for intra-articular drug delivery." J Control Release 115.2 (October 10, 2006): 175-182.
PMID
16959360
Source
pubmed
Published In
Journal of Controlled Release
Volume
115
Issue
2
Publish Date
2006
Start Page
175
End Page
182
DOI
10.1016/j.jconrel.2006.07.022

Tracking the in vivo fate of recombinant polypeptides by isotopic labeling.

We report a method to incorporate a stable isotope (13C) and a radioactive isotope (14C) into a recombinant polypeptide during Escherichia coli culture in M9 minimal medium supplemented with universally labeled 13C- or 14C-labeled glucose. We chose a thermally responsive elastin-like polypeptide (ELP) as a model polypeptide for this study because of its utility in various biotechnology applications such as drug delivery and tissue engineering. High cell densities were obtained by step-wise adaptation of E. coli to M9 medium in addition to supplementing the medium with trace elements that facilitated growth of E. coli. Furthermore, an optimal concentration of isopropyl-beta-d-thiogalactopyranoside was determined for induction of ELP expression to achieve high yield (mg/L culture) of the ELP. The incorporation of carbon isotopes was stoichiometrically related to the ratio of labeled glucose to unlabeled glucose in the culture medium. The isotope-labeled variants retained the physicochemical properties of the unlabeled ELP, specifically its temperature dependent aggregation behavior. As an example of the utility of this method, the in vitro stability of 14C-labeled ELP in PBS and mouse serum was conveniently quantified by SDS-PAGE and autoradiography. In addition, the in vivo stability of the 14C-labeled ELP in plasma was determined along with its plasma pharmacokinetics.

Authors
Liu, W; Dreher, MR; Chow, DC; Zalutsky, MR; Chilkoti, A
MLA Citation
Liu, W, Dreher, MR, Chow, DC, Zalutsky, MR, and Chilkoti, A. "Tracking the in vivo fate of recombinant polypeptides by isotopic labeling." J Control Release 114.2 (August 28, 2006): 184-192.
PMID
16904221
Source
pubmed
Published In
Journal of Controlled Release
Volume
114
Issue
2
Publish Date
2006
Start Page
184
End Page
192
DOI
10.1016/j.jconrel.2006.06.001

Tracking the in vivo fate of recombinant polypeptides by isotopic labeling

Authors
Liu, W; Dreher, MR; Chow, DC; Zalutsky, MR; Chilkoti, A
MLA Citation
Liu, W, Dreher, MR, Chow, DC, Zalutsky, MR, and Chilkoti, A. "Tracking the in vivo fate of recombinant polypeptides by isotopic labeling." August 28, 2006.
Source
wos-lite
Published In
Journal of Controlled Release
Volume
114
Issue
2
Publish Date
2006
Start Page
184
End Page
192
DOI
10.1016/j.jcornel.2006.06.001

Aqueous two-phase system formation kinetics for elastin-like polypeptides of varying chain length.

The kinetics of aqueous two-phase system (ATPS) formation for elastin-like polypeptides (ELP) with defined chemical composition and chain length was investigated by dark field microscopy in an on-chip format with a linear temperature gradient. Scattering intensities from peptide solutions in the presence and absence of sodium dodecyl sulfate (SDS) were recorded as a function of temperature and time, simultaneously. It was found that the formation of the ATPS for three ELPs of different molecular weights (36 075, 59 422, and 129 856 Da) in the absence of SDS followed a coalescence mechanism, and the rate constant and activation energy were independent of chain length. With the introduction of SDS into the ELP solutions, the rate constants were attenuated more strongly with increasing chain length. Moreover, the coalescence process in the presence of SDS showed non-Arrhenius kinetics as a function of temperature. For the two shorter ELPs, ATPS formation occurred via coalescence at all SDS concentrations and temperatures investigated. On the other hand, the coalescence process was greatly suppressed for the longest ELP at elevated temperatures and higher SDS concentrations. Under these circumstances, ATPS formation was forced to proceed via a mixed Ostwald ripening and coalescence mechanism.

Authors
Zhang, Y; Trabbic-Carlson, K; Albertorio, F; Chilkoti, A; Cremer, PS
MLA Citation
Zhang, Y, Trabbic-Carlson, K, Albertorio, F, Chilkoti, A, and Cremer, PS. "Aqueous two-phase system formation kinetics for elastin-like polypeptides of varying chain length." Biomacromolecules 7.7 (July 2006): 2192-2199.
PMID
16827587
Source
pubmed
Published In
Biomacromolecules
Volume
7
Issue
7
Publish Date
2006
Start Page
2192
End Page
2199
DOI
10.1021/bm060254y

Ultra-high expression of a thermally responsive recombinant fusion protein in E. coli.

Elastin-like polypeptides (ELPs) are recombinant peptide-based biopolymers that contain repetitive sequences enriched in glycine, valine, proline, and alanine. Because of the unusually large fraction of these amino acids in ELPs as compared to other cellular proteins, we hypothesized that intracellular pools of these amino acids can be selectively depleted and limit protein yields during expression. In this study, we examined how culture conditions and individual medium components affect protein yields by monitoring cell growth and protein expression kinetics of E. coli expressing an ELP tagged with a green fluorescent protein (GFP). By determining the underlying principles of superior fusion protein yields generated by the hyperexpression protocol, we further improved protein yields through the addition of glycerol and certain amino acids such as proline and alanine and found that amino acid concentrations and the type of basal medium used strongly influenced this beneficial effect. Surprisingly, amino acids other than those that are abundant in ELPs, for example, asparagine, aspartic acid, glutamine, and glutamic acid, also enhanced protein yields even in a nutrient-rich medium. Compared to commonly used Luria-Bertani medium, the protein yield was improved by 36-fold to the remarkable level of 1.6 g/L in shaker flask cultures with a modified medium and optimized culture conditions, which also led to a 8-fold reduction in the cost of the fusion protein. To our knowledge, this is the highest yield of an ELP-fusion protein purified from E. coli cultured in shaker flasks. This study also suggests a useful strategy to improve the yields of other ELP fusion proteins and repetitive polypeptides.

Authors
Chow, DC; Dreher, MR; Trabbic-Carlson, K; Chilkoti, A
MLA Citation
Chow, DC, Dreher, MR, Trabbic-Carlson, K, and Chilkoti, A. "Ultra-high expression of a thermally responsive recombinant fusion protein in E. coli." Biotechnol Prog 22.3 (May 2006): 638-646.
PMID
16739944
Source
pubmed
Published In
Biotechnology Progress
Volume
22
Issue
3
Publish Date
2006
Start Page
638
End Page
646
DOI
10.1021/bp0503742

Analysis of long range correlations due to coherent light scattering from in-vitro cell arrays using angle-resolved low coherence interferometry.

Angle-resolved low coherence interferometry (a/LCI) enables depth-resolved measurements of scattered light that can be used to recover subsurface structural information, such as the size of cell nuclei. Measurements of nuclear morphology, however, can be complicated by coherent scattering between adjacent cell nuclei. Previous studies have eliminated this component by applying a window filter to Fourier transformed angular data, based on the justification that the coherent scattering must necessarily occur over length scales greater than the cell size. To fully study this effect, results of experiments designed to test the validity of this approach are now presented. The a/LCI technique is used to examine light scattered by regular cell arrays, created using stamped adhesive micropatterned substrates. By varying the array spacing, it is demonstrated that cell-to-cell correlations have a predictable effect on light scattering distributions. These results are compared to image analysis of fluorescence micrographs of the cell array samples. The a/LCI results show that the impact of coherent scattering on nuclear morphology measurements can be eliminated through data filtering.

Authors
Pyhtila, JW; Ma, H; Simnick, AJ; Chilkoti, A; Wax, A
MLA Citation
Pyhtila, JW, Ma, H, Simnick, AJ, Chilkoti, A, and Wax, A. "Analysis of long range correlations due to coherent light scattering from in-vitro cell arrays using angle-resolved low coherence interferometry." J Biomed Opt 11.3 (May 2006): 34022-.
PMID
16822071
Source
pubmed
Published In
Journal of Biomedical Optics
Volume
11
Issue
3
Publish Date
2006
Start Page
34022
DOI
10.1117/1.2209561

Protein-resistant polymer coatings on silicon oxide by surface-initiated atom transfer radical polymerization.

The modification of silicon oxide with poly(ethylene glycol) to effectively eliminate protein adsorption has proven to be technically challenging. In this paper, we demonstrate that surface-initiated atom transfer radical polymerization (SI-ATRP) of oligo(ethylene glycol) methyl methacrylate (OEGMA) successfully produces polymer coatings on silicon oxide that have excellent protein resistance in a biological milieu. The level of serum adsorption on these coatings is below the detection limit of ellipsometry. We also demonstrate a new soft lithography method via which SI-ATRP is integrated with microcontact printing to create micropatterns of poly(OEGMA) on glass that can spatially direct the adsorption of proteins on the bare regions of the substrate. This ensemble of methods will be useful in screening biological interactions where nonspecific binding must be suppressed to discern low probability binding events from a complex mixture and to pattern anchorage-dependent cells on glass and silicon oxide.

Authors
Ma, H; Li, D; Sheng, X; Zhao, B; Chilkoti, A
MLA Citation
Ma, H, Li, D, Sheng, X, Zhao, B, and Chilkoti, A. "Protein-resistant polymer coatings on silicon oxide by surface-initiated atom transfer radical polymerization." Langmuir 22.8 (April 11, 2006): 3751-3756.
PMID
16584252
Source
pubmed
Published In
Langmuir
Volume
22
Issue
8
Publish Date
2006
Start Page
3751
End Page
3756
DOI
10.1021/la052796r

Bionanofabrication with polymers and enzymes

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "Bionanofabrication with polymers and enzymes." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 231 (March 26, 2006).
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
231
Publish Date
2006

Fabrication of bioconjugated polymeric nanostructures and metal nanowires by electric field-induced scanning probe lithography

Authors
Lee, W-K; Chen, S; Chilkoti, A; Zauscher, S
MLA Citation
Lee, W-K, Chen, S, Chilkoti, A, and Zauscher, S. "Fabrication of bioconjugated polymeric nanostructures and metal nanowires by electric field-induced scanning probe lithography." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 231 (March 26, 2006).
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
231
Publish Date
2006

Micro-cantilevers decorated with tethered stimulus-responsive polymer brushes and polypeptides for actuation and sensing

Authors
Abu-Lail, N; Kaholek, M; Valiaev, A; Lim, D-W; Chilkoti, A; LaMattina, B; Clark, R; Zauscher, S
MLA Citation
Abu-Lail, N, Kaholek, M, Valiaev, A, Lim, D-W, Chilkoti, A, LaMattina, B, Clark, R, and Zauscher, S. "Micro-cantilevers decorated with tethered stimulus-responsive polymer brushes and polypeptides for actuation and sensing." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 231 (March 26, 2006).
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
231
Publish Date
2006

Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers.

BACKGROUND: Delivery of anticancer therapeutic agents to solid tumors is problematic. Macromolecular drug carriers are an attractive alternative drug delivery method because they appear to target tumors and have limited toxicity in normal tissues. We investigated how molecular weight influences the accumulation of a model macromolecular drug carrier, dextran covalently linked to a fluorophore, in tumors. METHODS: We used dextrans with molecular weights from 3.3 kDa to 2 MDa. Vascular permeability, accumulation, and three-dimensional penetration of these dextrans were simultaneously measured in solid tumors via a dorsal skin fold window chamber, intravital laser-scanning confocal microscopy, and custom image analysis. RESULTS: Increasing the molecular weight of dextran statistically significantly reduced its vascular permeability by approximately two orders of magnitude (i.e., from 154 x 10(-7) cm/s, 95% confidence interval [CI] = 134 to 174 x 10(-7) cm/s, for 3.3-kDa dextran to 1.7 x 10(-7) cm/s, 95% CI = 0.7 to 2.6 x 10(-7) cm/s for 2-MDa dextran; P < .001, two-sided Kruskal-Wallis test) but increased its plasma half-life, which provided ample time for extravasation (i.e., to enter tumor tissue from the vasculature). Tumor accumulation was maximal for dextrans with molecular weights between 40 and 70 kDa. Dextrans of 3.3 and 10 kDa penetrated deeply (greater than 35 microm) and homogeneously into tumor tissue from the vessel wall. After a 30-minute period, a high concentration was observed only approximately 15 microm from the vessel wall for 40- to 70-kDa dextrans and only 5 microm for 2-MDa dextrans. CONCLUSIONS: Increasing the molecular weight of dextran statistically significantly reduced its tumor vascular permeability. Dextrans of 40 and 70 kDa had the highest accumulation in solid tumors but were largely concentrated near the vascular surface.

Authors
Dreher, MR; Liu, W; Michelich, CR; Dewhirst, MW; Yuan, F; Chilkoti, A
MLA Citation
Dreher, MR, Liu, W, Michelich, CR, Dewhirst, MW, Yuan, F, and Chilkoti, A. "Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers." J Natl Cancer Inst 98.5 (March 1, 2006): 335-344.
PMID
16507830
Source
pubmed
Published In
Journal of the National Cancer Institute
Volume
98
Issue
5
Publish Date
2006
Start Page
335
End Page
344
DOI
10.1093/jnci/djj070

Monitoring kinetics of surface initiated atom transfer radical polymerization by quartz crystal microbalance with dissipation.

This article reports that the kinetics of surface-initiated atom transfer radical polymerization can be quantified by the quartz crystal microbalance with dissipation (QCM-D) technique. The kinetics of in situ growth of poly(oligoethylene glycol methylmethacrylate) monitored on a gold-coated QCM-D sensor chip revealed that changes in the experimentally observed frequency (DeltaF) and dissipation (DeltaD) as a function of polymerization time were a function of the initiator density, and that the experimental response could be predicted from a continuum model.

Authors
Ma, H; Textor, M; Clark, RL; Chilkoti, A
MLA Citation
Ma, H, Textor, M, Clark, RL, and Chilkoti, A. "Monitoring kinetics of surface initiated atom transfer radical polymerization by quartz crystal microbalance with dissipation." Biointerphases 1.1 (March 2006): 35-.
PMID
20408613
Source
pubmed
Published In
Biointerphases: an open access journal for the biomaterials interface community
Volume
1
Issue
1
Publish Date
2006
Start Page
35
DOI
10.1116/1.2190697

Epitope tagging for tracking elastin-like polypeptides.

Elastin-like polypeptides (ELPs) are a class of biocompatible, non-immunogenic and crosslinkable biomaterials that offer promise for use as an injectable scaffold for cartilage repair. In this study, an oligohistidine (His(6)) epitope tag was incorporated at the N-terminus of an ELP using recombinant DNA techniques to permit tracking without compromising on material biocompatibility. His(6)-tagged ELPs were successfully detected by Western blot analysis and quantified by ELISAs following digestion with trypsin. The mass of His(6) tagged ELP fragments freed from a crosslinked ELP hydrogel after digestion with trypsin correlated highly with hydrogel weight loss, providing evidence of the tag's capability to enable tracking of enzymatic degradation of the ELP hydrogel. The His(6) tag also facilitated recognition of crosslinked ELPs from background staining of articular cartilage. These results suggest that the His(6) epitope tag has the potential to track ELP scaffold loss independently of newly formed tissue mass for evaluating matrix remodeling in vivo.

Authors
Ong, SR; Trabbic-Carlson, KA; Nettles, DL; Lim, DW; Chilkoti, A; Setton, LA
MLA Citation
Ong, SR, Trabbic-Carlson, KA, Nettles, DL, Lim, DW, Chilkoti, A, and Setton, LA. "Epitope tagging for tracking elastin-like polypeptides." Biomaterials 27.9 (March 2006): 1930-1935.
PMID
16278015
Source
pubmed
Published In
Biomaterials
Volume
27
Issue
9
Publish Date
2006
Start Page
1930
End Page
1935
DOI
10.1016/j.biomaterials.2005.10.018

Patterning cells in highly deformable microstructures: effect of plastic deformation of substrate on cellular phenotype and gene expression.

We describe the fabrication of deformable microstructures by low-pressure-soft-microembossing (muSEmb) that provides in vitro experimental "test-beds" to investigate the interplay of mechanical and chemical stimuli on cell behavior in a highly controlled environment. Soft microembossing exploits the softness and plasticity of parafilm to fabricate microstructures by pressing a silicon master or an elastomeric poly(dimethylsiloxane) stamp into the parafilm. We demonstrate that a protein-resistant comb polymer can be printed into the raised features of the embossed microstructures, which imparts protein, and hence cell resistance to those regions of the microstructures. These two features of our fabrication methodology-microembossing followed by spatially selective transfer of a nonfouling polymer-forms the core of our strategy to pattern cells within the parafilm microstructures, such that the cells are confined within bottoms of the microstructures. Cell culture experiments demonstrated the preferential cell attachment of NIH 3T3 fibroblasts to the fibronectin (FN) micropatterns by immunofluorescence microscopy. The actin cytoskeleton realigned along the axis of applied mechanical stress, and stretched cells showed altered gene expression of cytoskeletal and matrix proteins in response to mechanical deformation. The use of parafilm as a substrate and muSEmb as a fabrication method provides a simple and widely accessible methodology to investigate cellular behavior under well-defined conditions of plastic deformation and surface ligand density.

Authors
Hyun, J; Chen, J; Setton, LA; Chilkoti, A
MLA Citation
Hyun, J, Chen, J, Setton, LA, and Chilkoti, A. "Patterning cells in highly deformable microstructures: effect of plastic deformation of substrate on cellular phenotype and gene expression." Biomaterials 27.8 (March 2006): 1444-1451.
PMID
16154191
Source
pubmed
Published In
Biomaterials
Volume
27
Issue
8
Publish Date
2006
Start Page
1444
End Page
1451
DOI
10.1016/j.biomaterials.2005.08.018

Structural optimization of a "smart" doxorubicin-polypeptide conjugate for thermally targeted delivery to solid tumors.

A thermoresponsive, genetically engineered, elastin-like polypeptide (ELP) containing a C-terminal cysteine residue was synthesized and purified by inverse transition cycling (ITC) and conjugated to doxorubicin (Dox) molecules through four different pH-sensitive, maleimide-activated, hydrazone linkers. The efficiency of Dox activation, conjugation ratios to ELP and biophysical characterization-hydrodynamic radius (Rh) and the temperature transition kinetics-of the ELP-Dox conjugates and pH-mediated release of Dox were quantified in this study. Conjugation ratios of the maleimide-activated Dox to the thiol group of a unique cysteine in the ELP were close to unity. The Rh of the conjugate increased as the linker length between the ELP backbone and Dox was increased. The linker structure and length had little effect on the Tt of the ELP-Dox conjugates, as all conjugates exhibited Tt's that were similar to the native ELP. However, the ELP-Dox conjugates with longer linkers exhibited slower transition kinetics compared to the ELP-Dox conjugates with shorter linkers. The highest release of the ELP-Dox conjugate by cleavage of the hydrazone bond at pH 4 was nearly 80% over 72 h and was exhibited by the conjugate with the shortest linker.

Authors
Furgeson, DY; Dreher, MR; Chilkoti, A
MLA Citation
Furgeson, DY, Dreher, MR, and Chilkoti, A. "Structural optimization of a "smart" doxorubicin-polypeptide conjugate for thermally targeted delivery to solid tumors." J Control Release 110.2 (January 10, 2006): 362-369.
PMID
16303202
Source
pubmed
Published In
Journal of Controlled Release
Volume
110
Issue
2
Publish Date
2006
Start Page
362
End Page
369
DOI
10.1016/j.jconrel.2005.10.006

Measurement system for the high-throughput characterization of metal nanoparticles for biosensors

We present a system for the rapid characterization of nanoparticles for biosensors, providing concurrent atomic force microscopy and scattering spectra of individual nanoparticles and simultaneous spectra from various nanostructures created by electron beam lithography © 2005 Optical Society of America.

Authors
Curry, A; Nusz, G; Chilkoti, A; Wax, A
MLA Citation
Curry, A, Nusz, G, Chilkoti, A, and Wax, A. "Measurement system for the high-throughput characterization of metal nanoparticles for biosensors." Optics InfoBase Conference Papers (January 1, 2006).
Source
scopus
Published In
Optics InfoBase Conference Papers
Publish Date
2006

Chondrocytic differentiation of human adipose-derived adult stem cells in elastin-like polypeptide.

Human adipose derived adult stem (hADAS) cells have the ability to differentiate into a chondrogenic phenotype in three-dimensional culture and media containing dexamethasone and TGF-beta. The current study examined the potential of a genetically engineered elastin-like polypeptide (ELP) to promote the chondrocytic differentiation of hADAS cells without exogenous chondrogenic supplements. hADAS cells were cultured in ELP hydrogels in either chondrogenic or standard medium at 5% O2 for up to 2 weeks. By day 14, constructs cultured in either medium exhibited significant increases in sulfated glycosaminoglycan (up to 100%) and collagen contents (up to 420%). Immunolabeling confirmed that the matrix formed consisted mainly of type II and not type I collagen. The composition of the constructs cultured in either medium did not differ significantly. To assess the effect of oxygen tension on the differentiation of the above constructs, samples were cultured in standard medium at either 5% or 20% O2 for 7 days and their gene expression profile was evaluated using real time RT-PCR. In both cases, the hADAS-ELP constructs upregulated SOX9 and type II collagen gene expression, while type I collagen was downregulated. However, constructs cultured in 20% O2 highly upregulated type X collagen, which was not detected in the 5% O2 cultures. The study suggests that ELP can promote chondrogenesis for hADAS cells in the absence of exogenous TGF-beta1 and dexamethasone, especially under low oxygen tension conditions.

Authors
Betre, H; Ong, SR; Guilak, F; Chilkoti, A; Fermor, B; Setton, LA
MLA Citation
Betre, H, Ong, SR, Guilak, F, Chilkoti, A, Fermor, B, and Setton, LA. "Chondrocytic differentiation of human adipose-derived adult stem cells in elastin-like polypeptide." Biomaterials 27.1 (January 2006): 91-99.
PMID
16023192
Source
pubmed
Published In
Biomaterials
Volume
27
Issue
1
Publish Date
2006
Start Page
91
End Page
99
DOI
10.1016/j.biomaterials.2005.05.071

Surface-initiated atom transfer radical polymerization of oligo(ethylene glycol) methyl methacrylate from a mixed self-assembled monolayer on gold

This paper describes the in-situ synthesis of an oligo(ethylene glycol)-functionalized polymer brush in which the oligo(ethylene glycol) chains are presented as side-chains from a methacrylate backbone that is anchored to the surface. These polymer "bottle-brushes" have been synthesized by surface-initiated atom transfer radical polymerization (SI-ATRP) of oligo(ethylene glycol) methyl methacrylate (OEGMA) from a mixed self-assembled monolayer (SAM) of an ATRP initiator-functionalized alkanethiol and a diluent, methyl-terminated thiol. The systematic control of the ATRP initiator surface density afforded by the mixed SAM on gold and the polymerization time enables the polymer chain length and surface density to be independently controlled. Surface plasmon resonance (SPR) spectroscopy of fibronectin (Fn) adsorption on poly(OEGMA) grown from the surface of the mixed SAMs on gold shows that above a threshold solution molar ratio of the ATRP-initiator thiol to methyl-terminated thiol of 0.2, and a dry film thickness of -4 nm, Fn adsorption on the surface-initiated poly(OEGMA) coatings was below the detection limit of SPR. The relatively low surface density of the ATRP initiator required to confer protein resistance to the surface suggests that SI-ATRP may be a viable strategy to create protein resistant polymer brushes on real-world materials. © 2006 WILEY-VCH Verlag GmbH &Co. KGaA.

Authors
Ma, H; Wells, M; Jr, TPB; Chilkoti, A
MLA Citation
Ma, H, Wells, M, Jr, TPB, and Chilkoti, A. "Surface-initiated atom transfer radical polymerization of oligo(ethylene glycol) methyl methacrylate from a mixed self-assembled monolayer on gold." Advanced Functional Materials 16.5 (2006): 640-648.
Source
scival
Published In
Advanced Functional Materials
Volume
16
Issue
5
Publish Date
2006
Start Page
640
End Page
648
DOI
10.1002/adfm.200500426

Pretreatment of amphiphilic comb polymer surfaces dramatically affects protein adsorption.

New applications in regenerative biotechnology require the ability to understand and control protein-surface interactions on micrometer and submicrometer length scales. Evidence presented here shows that micropatterned amphiphilic comb polymer films exhibit a pretreatment-dependent behavior with respect to protein adsorption for the proteins fibronectin, laminin, and for serum. A micropatterned surface, consisting of protein-reactive regions, separated by comb polymer, was created and tested for protein adsorption using the surface-sensitive imaging tool TOF-SIMS. Immersion of micropatterned surfaces in solutions of fibronectin or laminin resulted in uniform protein coverage on both the comb polymer and protein-reactive regions. However, preimmersion of similarly patterned surfaces in water for 2 h prior to protein incubation was found to dramatically improve the protein-resistant properties of the comb polymer regions. These results are consistent with poly(ethylene glycol) (PEG) side chain reorientation and/or hydration and poly(methyl methacrylate) (PMMA) backbone segregation away from the interface region.

Authors
Zhang, Z; Ma, H; Hausner, DB; Chilkoti, A; Beebe, TP
MLA Citation
Zhang, Z, Ma, H, Hausner, DB, Chilkoti, A, and Beebe, TP. "Pretreatment of amphiphilic comb polymer surfaces dramatically affects protein adsorption." Biomacromolecules 6.6 (November 2005): 3388-3396.
PMID
16283770
Source
pubmed
Published In
Biomacromolecules
Volume
6
Issue
6
Publish Date
2005
Start Page
3388
End Page
3396
DOI
10.1021/bm050446d

Synthesis and in vitro evaluation of enzymatically cross-linked elastin-like polypeptide gels for cartilaginous tissue repair.

Genetically engineered elastin-like polypeptide (ELP) hydrogels offer unique promise as scaffolds for cartilage tissue engineering because of the potential to promote chondrogenesis and to control mechanical properties. In this study, we designed and synthesized ELPs capable of undergoing enzyme-initiated gelation via tissue transglutaminase, with the ultimate goal of creating an injectable, in situ cross-linking scaffold to promote functional cartilage repair. Addition of the enzyme promoted ELP gel formation and chondrocyte encapsulation in a biocompatible process, which resulted in cartilage matrix synthesis in vitro and the potential to contribute to cartilage mechanical function in vivo. A significant increase in the accumulation of sulfated glycosaminoglycans was observed, and histological sections revealed the accumulation of a cartilaginous matrix rich in type II collagen and lacking in type I collagen, indicative of hyaline cartilage formation. These results provide evidence of chondrocytic phenotype maintenance for cells in the ELP hydrogels in vitro. In addition, the dynamic shear moduli of ELP hydrogels seeded with chondrocytes increased from 0.28 to 1.7 kPa during a 4-week culture period. This increase in the mechanical integrity of cross-linked ELP hydrogels suggests restructuring of the ELP matrix by deposition of functional cartilage extracellular matrix components.

Authors
McHale, MK; Setton, LA; Chilkoti, A
MLA Citation
McHale, MK, Setton, LA, and Chilkoti, A. "Synthesis and in vitro evaluation of enzymatically cross-linked elastin-like polypeptide gels for cartilaginous tissue repair." Tissue Eng 11.11-12 (November 2005): 1768-1779.
PMID
16411822
Source
pubmed
Published In
Tissue Engineering
Volume
11
Issue
11-12
Publish Date
2005
Start Page
1768
End Page
1779
DOI
10.1089/ten.2005.11.1768

Enzymatic fabrication of DNA nanostructures: extension of a self-assembled oligonucleotide monolayer on gold arrays.

Nucleic acid nanostructures are useful as templates for bionanofabrication of composite molecular nanostructures in materials science, molecular electronics, and biosensing. Here, we demonstrate that terminal deoxynucleotidyl transferase, which repetitively adds mononucleotides to the 3' end of a short DNA initiator, can be used to rapidly fabricate DNA nanostructures up to 121 nm high with lateral dimensions from 0.1 to 4 mum in 2 h. These programmable scaffolds can potentially be employed to build more complex nanostructures consisting of natural or unnatural nucleotides with selective docking sites along the single-stranded DNA.

Authors
Chow, DC; Lee, W-K; Zauscher, S; Chilkoti, A
MLA Citation
Chow, DC, Lee, W-K, Zauscher, S, and Chilkoti, A. "Enzymatic fabrication of DNA nanostructures: extension of a self-assembled oligonucleotide monolayer on gold arrays." J Am Chem Soc 127.41 (October 19, 2005): 14122-14123.
PMID
16218572
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
127
Issue
41
Publish Date
2005
Start Page
14122
End Page
14123
DOI
10.1021/ja052491z

Stimulus-responsive elastin-like polypetides as coatings for microcantilevers: Applications for sensing and actuation

Authors
Valiaev, A; Abu-Lail, NI; Lim, DW; Chilkoti, A; Zauscher, S
MLA Citation
Valiaev, A, Abu-Lail, NI, Lim, DW, Chilkoti, A, and Zauscher, S. "Stimulus-responsive elastin-like polypetides as coatings for microcantilevers: Applications for sensing and actuation." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 230 (August 28, 2005): U4317-U4318.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
230
Publish Date
2005
Start Page
U4317
End Page
U4318

Passive and active surfaces to control protein and cell interactions

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "Passive and active surfaces to control protein and cell interactions." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 230 (August 28, 2005): U4150-U4151.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
230
Publish Date
2005
Start Page
U4150
End Page
U4151

Rapid fabrication of DNA nanostructures by enzymatic reactions

Authors
Chow, DC; Chilkoti, A; Lee, WK; Zauscher, S
MLA Citation
Chow, DC, Chilkoti, A, Lee, WK, and Zauscher, S. "Rapid fabrication of DNA nanostructures by enzymatic reactions." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 230 (August 28, 2005): U1069-U1069.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
230
Publish Date
2005
Start Page
U1069
End Page
U1069

Self-cleavable stimulus responsive tags for protein purification without chromatography.

A simple method to purify recombinant proteins is described by fusing a target protein with an intein and an elastin-like polypeptide that only requires NaCl, dithiothreitol, and a syringe filter to isolate the target protein from Escherichia coli lysate. This tripartite fusion system enables rapid isolation of the target protein without the need for affinity chromatography for purification or proteases for cleavage of the target protein from the fusion. The elastin-like polypeptide tag imparts reversible phase transition behavior to the tripartite fusion so that the fusion protein can be selectively aggregated in cell lysate by the addition of NaCl. The aggregates are isolated by microfiltration and resolubilized by reversal of the phase transition in low ionic strength buffer. After resolubilizing the fusion protein, the intein is activated to cleave the target protein from the elastin-intein tag, and the target protein is then isolated from the elastin-intein fusion by an additional phase transition cycle.

Authors
Ge, X; Yang, DSC; Trabbic-Carlson, K; Kim, B; Chilkoti, A; Filipe, CDM
MLA Citation
Ge, X, Yang, DSC, Trabbic-Carlson, K, Kim, B, Chilkoti, A, and Filipe, CDM. "Self-cleavable stimulus responsive tags for protein purification without chromatography." J Am Chem Soc 127.32 (August 17, 2005): 11228-11229.
PMID
16089436
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
127
Issue
32
Publish Date
2005
Start Page
11228
End Page
11229
DOI
10.1021/ja0531125

Substrate effect on refractive index dependence of plasmon resonance for individual silver nanoparticles observed using darkfield microspectroscopy.

We use optical darkfield micro-spectroscopy to characterize the plasmon resonance of individual silver nanoparticles in the presence of a substrate. The optical system permits multiple individual nanoparticles to be identified visually for simultaneous spectroscopic study. For silver particles bound to a silanated glass substrate, we observe changes in the Plasmon resonance due to induced variations in the local refractive index. The shifts in the plasmon resonance are investigated using a simple analytical theory in which the contributions from the substrate and environment are weighted with distance from the nanoparticle. The theory is compared with experimental results to determine a weighting factor which facilitates modeling of environmental refractive index changes using standard Mie code. Use of the optical system for characterizing nanoparticles attached to substrates for biosensing applications is discussed.

Authors
Curry, A; Nusz, G; Chilkoti, A; Wax, A
MLA Citation
Curry, A, Nusz, G, Chilkoti, A, and Wax, A. "Substrate effect on refractive index dependence of plasmon resonance for individual silver nanoparticles observed using darkfield microspectroscopy." Opt Express 13.7 (April 4, 2005): 2668-2677.
PMID
19495158
Source
pubmed
Published In
Optics express
Volume
13
Issue
7
Publish Date
2005
Start Page
2668
End Page
2677

Nano- to micro-scale structures of polymers that repel and attract proteins.

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "Nano- to micro-scale structures of polymers that repel and attract proteins." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 229 (March 13, 2005): U1158-U1158.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
229
Publish Date
2005
Start Page
U1158
End Page
U1158

Biointerface Science

Authors
Chilkoti, A; Hubbell, JA
MLA Citation
Chilkoti, A, and Hubbell, JA. "Biointerface Science." MRS Bulletin 30.03 (March 2005): 175-179.
Source
crossref
Published In
MRS bulletin / Materials Research Society
Volume
30
Issue
03
Publish Date
2005
Start Page
175
End Page
179
DOI
10.1557/mrs2005.48

Measurement system for the high-throughput characterization of metal nanoparticles for biosensors

We present a system for the rapid characterization of nanoparticles for biosensors, providing concurrent atomic force microscopy and scattering spectra of individual nanoparticles and simultaneous spectra from various nanostructures created by electron beam lithography © 2005 Optical Society of America.

Authors
Curry, A; Nusz, G; Chilkoti, A; Wax, A
MLA Citation
Curry, A, Nusz, G, Chilkoti, A, and Wax, A. "Measurement system for the high-throughput characterization of metal nanoparticles for biosensors." Optics InfoBase Conference Papers (January 1, 2005).
Source
scopus
Published In
Optics InfoBase Conference Papers
Publish Date
2005

Force-induced proline cis-trans isomerization in elastin-like polypeptides

Authors
Valiaev, A; Lim, DW; Oas, T; Clark, RL; Chilkoti, A; Zauscher, S
MLA Citation
Valiaev, A, Lim, DW, Oas, T, Clark, RL, Chilkoti, A, and Zauscher, S. "Force-induced proline cis-trans isomerization in elastin-like polypeptides." BIOPHYSICAL JOURNAL 88.1 (January 2005): 168A-168A.
Source
wos-lite
Published In
Biophysical Journal
Volume
88
Issue
1
Publish Date
2005
Start Page
168A
End Page
168A

Nanofabrication with biomolecules

The advances made in biotechnology and nanotechnology for building complex bionanostructures on surfaces with nanometer precision are discussed. The use of biomolecules for nanoscale has become an effective solution for difficult positioning problems in the fabrication of nanostructures. To fabricate efficiently with biomolecules and make use of their unique properties, a radical shift away from the high energy, high vacuum processing paradigm of conventional micro and nanofabrication is needed. An exciting opportunities for bionanofabrication have also emerged in the field of synthetic biology and recombinant protein engineering.

Authors
Chow, DC; Johannes, MS; Lee, W-K; Clark, RL; Zauscher, S; Chilkoti, A
MLA Citation
Chow, DC, Johannes, MS, Lee, W-K, Clark, RL, Zauscher, S, and Chilkoti, A. "Nanofabrication with biomolecules." Materials Today 8.12 SUPPL. 1 (2005): 30-39.
Source
scival
Published In
Materials Today
Volume
8
Issue
12 SUPPL. 1
Publish Date
2005
Start Page
30
End Page
39
DOI
10.1016/S1369-7021(05)71287-6

Fabrication of biofunctionalized quasi-three-dimensional microstructures of a nonfouling comb polymer using soft lithography

This paper describes a simple set of patterning methods that are applicable to diverse substrates and allow the routine and rapid fabrication of protein patterns embedded within a background that consists of quasi-three-dimensional microstructures of a cell-resistant polymer. The ensemble of methods reported here utilizes three components to create topographically nonfouling polymeric structures that present cell-adhesive protein patterns in the regions between the microstructures: the first component is an amphiphilic comb polymer that is comprised of a methyl methacrylate backbone and pendant oligo(ethylene glycol) moieties along the side chain, physically deposited films of which are protein- and cell-resistant. The second component of the fabrication methodology involves the use of different variants of soft lithography, such as microcontact printing to create nonfouling topographical features of the comb polymer that demarcate cell-adhesive regions of the third component: a cell-adhesive extracellular protein or peptide. The ensemble of methods reported in this paper was used to fabricate quasi-three-dimensional patterns that present topographical and biochemical cues on a variety of substrates, and was shown to successfully maintain cellular patterns for up to two months in serum-containing medium. We believe that this, and other such methods under development that allow independent and systematic control of chemistry, topography and substrate compliance will provide versatile "test-beds" for fundamental studies in cell biology as well as allow the discovery of rational design principles for the development of biomaterials and tissue-engineering scaffolds. © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Authors
Ma, H; Hyun, J; Zhang, Z; Jr, TPB; Chilkoti, A
MLA Citation
Ma, H, Hyun, J, Zhang, Z, Jr, TPB, and Chilkoti, A. "Fabrication of biofunctionalized quasi-three-dimensional microstructures of a nonfouling comb polymer using soft lithography." Advanced Functional Materials 15.4 (2005): 529-540.
Source
scival
Published In
Advanced Functional Materials
Volume
15
Issue
4
Publish Date
2005
Start Page
529
End Page
540
DOI
10.1002/adfm.200400088

Targeted drug delivery to solid tumors by thermally responsive polypeptides

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "Targeted drug delivery to solid tumors by thermally responsive polypeptides." European Cells and Materials 10.SUPPL.5 (2005): 11--.
Source
scival
Published In
European cells & materials
Volume
10
Issue
SUPPL.5
Publish Date
2005
Start Page
11-

Polypeptide-solvent interactions measured by single molecule force spectroscopy

Stimulus-responsive biomolecules have attracted a large research interest because of their potential application in various areas such as drug delivery, actuators and sensing devices at the nanoscale. Using single-molecule force spectroscopy (SMFS) we studied elastin-like polypeptides (ELPs). These stimulus-responsive polypeptides undergo an inverse temperature transition, accompanied by a large conformational change, when the solvent quality is changed by increasing the temperature or by addition of salt. Understanding the relationship between peptide sequence and mechanisms of force generation can provide a route to engineer ELPs with desirable mechano-chemical properties. Here we studied the effect of solvent quality and type of guest residue on the mechanical properties of ELPs on the single-molecule level. We used a novel statistical approach to estimate polymer elasticity parameters from model fits to the data. With this approach we were able to resolve small changes in the Kuhn segment length distributions associated with different molecular architectures. We then show that these mechanical differences likely arise from differences in the hydrophobic hydration of sidegoups, in line with recent predictions from molecular dynamics simulations. © 2006 Materials Research Society.

Authors
Valiaev, A; Dong, WL; Chilkoti, A; Schmidler, S; Zauscher, S
MLA Citation
Valiaev, A, Dong, WL, Chilkoti, A, Schmidler, S, and Zauscher, S. "Polypeptide-solvent interactions measured by single molecule force spectroscopy." Materials Research Society Symposium Proceedings 898 (2005): 92-97.
Source
scival
Published In
Materials Research Society Symposium - Proceedings
Volume
898
Publish Date
2005
Start Page
92
End Page
97

Measurement system for the high-throughput characterization of metal nanoparticles for biosensors

We present a system for the rapid characterization of nanoparticles for biosensors, providing concurrent atomic force microscopy and scattering spectra of individual nanoparticles and simultaneous spectra from various nanostructures created by electron beam lithography. © 2005 Optical Society of America.

Authors
Curry, A; Nusz, G; Chilkoti, A; Wax, A
MLA Citation
Curry, A, Nusz, G, Chilkoti, A, and Wax, A. "Measurement system for the high-throughput characterization of metal nanoparticles for biosensors." 2005 Conference on Lasers and Electro-Optics, CLEO 3 (2005): 2160-2162.
Source
scival
Published In
2005 Conference on Lasers and Electro-Optics, CLEO
Volume
3
Publish Date
2005
Start Page
2160
End Page
2162

Predictive modeling of polypeptide hydrogel mechanical properties for cartilage repair using artificial neural networks

Authors
Haider, MA; Nettles, DL; Trabbic-Carlson, K; Chilkoti, A; Setton, LA
MLA Citation
Haider, MA, Nettles, DL, Trabbic-Carlson, K, Chilkoti, A, and Setton, LA. "Predictive modeling of polypeptide hydrogel mechanical properties for cartilage repair using artificial neural networks." Proceedings of the 2005 Summer Bioengineering Conference 2005 (2005): 633-634.
Source
scival
Published In
Proceedings of the 2005 Summer Bioengineering Conference
Volume
2005
Publish Date
2005
Start Page
633
End Page
634

Novel technology for cost-effective expression and purification of recombinant proteins and peptides

Authors
Dagher, S; Rose, D; Chilkoti, A
MLA Citation
Dagher, S, Rose, D, and Chilkoti, A. "Novel technology for cost-effective expression and purification of recombinant proteins and peptides." 2005.
Source
wos-lite
Published In
Biopolymers
Volume
80
Issue
4
Publish Date
2005
Start Page
530
End Page
531

Novel technology for cost-effective expression and purification of recombinant proteins and peptides

Authors
Dagher, S; Rose, D; Chilkoti, A
MLA Citation
Dagher, S, Rose, D, and Chilkoti, A. "Novel technology for cost-effective expression and purification of recombinant proteins and peptides." 2005.
Source
wos-lite
Published In
Biopolymers
Volume
80
Issue
4
Publish Date
2005
Start Page
538
End Page
539

Materials Research Society Symposium Proceedings: Preface

Authors
Wong, JY; Plant, AL; Schmidt, CE; Shea, L; Coury, AJ; Chen, CS; Barron, AE; Klok, HA; Deming, TJ; Saltzman, WM; Chilkoti, A; Luo, D; Uhrich, K
MLA Citation
Wong, JY, Plant, AL, Schmidt, CE, Shea, L, Coury, AJ, Chen, CS, Barron, AE, Klok, HA, Deming, TJ, Saltzman, WM, Chilkoti, A, Luo, D, and Uhrich, K. "Materials Research Society Symposium Proceedings: Preface." December 1, 2004.
Source
scopus
Published In
Materials Research Society Symposium - Proceedings
Volume
EXS
Issue
1
Publish Date
2004

Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion.

Thermally responsive elastin like polypeptides (ELPs) can be used to purify proteins from Escherichia coli culture when proteins are expressed as a fusion with an ELP. Nonchromatographic purification of ELP fusion proteins, termed inverse transition cycling (ITC), exploits the reversible soluble-insoluble phase transition behavior imparted by the ELP tag. Here, we quantitatively compare the expression and purification of ELP and oligohistidine fusions of chloramphenicol acetyltransferase (CAT), blue fluorescent protein (BFP), thioredoxin (Trx), and calmodulin (CalM) from both a 4-h culture with chemical induction of the plasmid-borne fusion protein gene and a 24-h culture without chemical induction. The total protein content and functional activity were quantified at each ITC purification step. For CAT, BFP, and Trx, the 24-h noninduction culture of ELP fusion proteins results in a sevenfold increase in the yield of each fusion protein compared to that obtained by the 4-h-induced culture, and the calculated target protein yield is similar to that of their equivalent oligohistidine fusion. For these proteins, ITC purification of fusion proteins also results in approximately 75% recovery of active fusion protein, similar to affinity chromatography. Compared to chromatographic purification, however, ITC is inexpensive, requires no specialized equipment or reagents, and because ITC is a batch purification process, it is easily scaled up to accommodate larger culture volumes or scaled down and multiplexed for high-throughput, microscale purification; thus, potentially impacting both high-throughput protein expression and purification for proteomics and large scale, cost-effective industrial bioprocessing of pharmaceutically relevant proteins.

Authors
Trabbic-Carlson, K; Liu, L; Kim, B; Chilkoti, A
MLA Citation
Trabbic-Carlson, K, Liu, L, Kim, B, and Chilkoti, A. "Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion." Protein Sci 13.12 (December 2004): 3274-3284.
PMID
15557268
Source
pubmed
Published In
Protein Science
Volume
13
Issue
12
Publish Date
2004
Start Page
3274
End Page
3284
DOI
10.1110/ps.04931604

Nitroxide conjugate of a thermally responsive elastin-like polypeptide for noninvasive thermometry.

Hyperthermia, as an adjuvant with radiation and chemotherapy, has shown promise in the treatment of cancer. The relevant biological effects of a hyperthermia treatment are both time and temperature-dependent, creating a need for accurate thermometry. We present a novel noninvasive thermometry modality that combines a temperature responsive biopolymer, the elastin-like polypeptide (ELP), and nitroxide to produce an ELP-nitroxide conjugate. When examined with electron paramagnetic resonance (EPR) spectroscopy, the ELP-nitroxide conjugate has temperature-dependent spectral line widths whose predictive accuracy is approximately 0.3 degrees C (80 microM). We believe that the temperature-dependent changes observed in the EPR spectrum are due to the combined effect of temperature, viscosity and effective radius on the rotational correlation time of the ELP-nitroxide conjugate.

Authors
Dreher, MR; Elas, M; Ichikawa, K; Barth, ED; Chilkoti, A; Rosen, GM; Halpern, HJ; Dewhirst, M
MLA Citation
Dreher, MR, Elas, M, Ichikawa, K, Barth, ED, Chilkoti, A, Rosen, GM, Halpern, HJ, and Dewhirst, M. "Nitroxide conjugate of a thermally responsive elastin-like polypeptide for noninvasive thermometry." Med Phys 31.10 (October 2004): 2755-2762.
PMID
15543780
Source
pubmed
Published In
Medical physics
Volume
31
Issue
10
Publish Date
2004
Start Page
2755
End Page
2762
DOI
10.1118/1.1782677

Label-free biosensing by surface plasmon resonance of nanoparticles on glass: optimization of nanoparticle size.

The unique optical properties of noble metal nanoparticles have been used to design a label-free biosensor in a chip format. In this paper, we demonstrate that the size of gold nanoparticles significantly affects the sensitivity of the biosensor. Gold nanoparticles with diameters in the range of 12-48 nm were synthesized in solution and sensor chips were fabricated by chemisorption of these nanoparticles on amine-functionalized glass. Sensors fabricated from 39-nm-diameter gold nanoparticles exhibited maximum sensitivity to the change of the bulk refractive index and the largest "analytical volume", defined as the region around the nanoparticle within which a change in refractive index causes a change in the optical properties of the immobilized nanoparticles. The detection limit for streptavidin-biotin binding of a sensor fabricated from 39-nm-diameter nanoparticles was 20-fold better than a previously reported sensor fabricated from 13-nm-diameter gold nanoparticles. We also discuss several other factors that could improve the performance of the next generation of these immobilized metal nanoparticle sensors.

Authors
Nath, N; Chilkoti, A
MLA Citation
Nath, N, and Chilkoti, A. "Label-free biosensing by surface plasmon resonance of nanoparticles on glass: optimization of nanoparticle size." Anal Chem 76.18 (September 15, 2004): 5370-5378.
PMID
15362894
Source
pubmed
Published In
Analytical Chemistry
Volume
76
Issue
18
Publish Date
2004
Start Page
5370
End Page
5378
DOI
10.1021/ac049741z

Mechanics of stimulus-responsive elastin-like polypeptides studied by force spectroscopy.

Authors
Valiaev, A; Lim, DW; Clark, R; Chilkoti, A; Zauscher, S
MLA Citation
Valiaev, A, Lim, DW, Clark, R, Chilkoti, A, and Zauscher, S. "Mechanics of stimulus-responsive elastin-like polypeptides studied by force spectroscopy." August 22, 2004.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
228
Publish Date
2004
Start Page
U485
End Page
U485

Label free colorimetric biosensing using nanoparticles.

In this review article, we discuss a class of biosensors that exploit the change in the colorimetric properties of noble metal nanoparticles in response to biomolecular binding at their surface. Several sensor fabrication techniques as well as sensor configurations are discussed with an emphasis on their strengths and limitations. We conclude by presenting the future prospects and challenges for the successful transition of this technology from the laboratory to a commercial product.

Authors
Nath, N; Chilkoti, A
MLA Citation
Nath, N, and Chilkoti, A. "Label free colorimetric biosensing using nanoparticles." J Fluoresc 14.4 (July 2004): 377-389. (Review)
PMID
15617380
Source
pubmed
Published In
Journal of Fluorescence
Volume
14
Issue
4
Publish Date
2004
Start Page
377
End Page
389

Capture and release of proteins on the nanoscale by stimuli-responsive elastin-like polypeptide "switches".

This article describes the fabrication and characterization of stimulus-responsive elastin-like polypeptide (ELP) nanostructures grafted onto omega-substituted thiolates that were patterned onto gold surfaces by dip-pen nanolithography (DPN). In response to external stimuli such as changes in temperature or ionic strength, ELPs undergo a switchable and reversible, hydrophilic-hydrophobic phase transition at a lower critical solution temperature (LCST). We exploited this phase transition behavior to reversibly immobilize a thioredoxin-ELP (Trx-ELP) fusion protein onto the ELP nanopattern above the LCST. Subsequent binding of an anti-thioredoxin monoclonal antibody (anti-Trx) to the surface-captured thioredoxin showed the presentation of the immobilized protein in a sterically accessible orientation in the nanoarray. We also showed that the resulting Trx-ELP/anti-Trx complex formed above the LCST could be reversibly dissociated below the LCST. These results demonstrate the intriguing potential of ELP nanostructures as generic, reversible, biomolecular switches for on-chip capture and release of a small number (order 100-200) of protein molecules in integrated, nanoscale bioanalytical devices. We also investigated the molecular mechanism underlying this switch by measuring the height changes that accompany the binding and desorption steps and by adhesion force spectroscopy using atomic force microscopy.

Authors
Hyun, J; Lee, W-K; Nath, N; Chilkoti, A; Zauscher, S
MLA Citation
Hyun, J, Lee, W-K, Nath, N, Chilkoti, A, and Zauscher, S. "Capture and release of proteins on the nanoscale by stimuli-responsive elastin-like polypeptide "switches"." J Am Chem Soc 126.23 (June 16, 2004): 7330-7335.
PMID
15186170
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
126
Issue
23
Publish Date
2004
Start Page
7330
End Page
7335
DOI
10.1021/ja049721e

Quantification of the effects of chain length and concentration on the thermal behavior of elastin-like polypeptides.

At a specific temperature, elastin-like polypeptides (ELPs) undergo a sharp solubility transition that can be exploited in a variety of applications in biotechnology and medicine. The temperature of the transition varies with ELP sequence, molecular weight, and concentration. We present a single equation of three parameters that quantitatively predicts the transition temperature as a function of ELP length and concentration for an ELP of a fixed composition. This model should be useful both for the design of new ELP sequences that exhibit a desired transition temperature and for the selection of variables to trigger the phase transition of an ELP for a given application.

Authors
Meyer, DE; Chilkoti, A
MLA Citation
Meyer, DE, and Chilkoti, A. "Quantification of the effects of chain length and concentration on the thermal behavior of elastin-like polypeptides." Biomacromolecules 5.3 (May 2004): 846-851.
PMID
15132671
Source
pubmed
Published In
Biomacromolecules
Volume
5
Issue
3
Publish Date
2004
Start Page
846
End Page
851
DOI
10.1021/bm034215n

Enzymatic nanolithography of a self-assembled oligonucleotide monolayer on gold.

This paper describes a simple strategy to biochemically manipulate a surface at the nanoscale by enzyme dip-pen nanolithography using an endonuclease (DNase I) that is directly patterned on a self-assembled monolayer presenting a terminal oligonucleotide. Physisorbed nanopatterns of DNase I carried out nanoscale enzymology at the surface creating oligonucleotide patterns with the fidelity of the patterned enzyme because of the affinity of the enzyme for the immobilized, oligonucleotide substrate.

Authors
Hyun, J; Kim, J; Craig, SL; Chilkoti, A
MLA Citation
Hyun, J, Kim, J, Craig, SL, and Chilkoti, A. "Enzymatic nanolithography of a self-assembled oligonucleotide monolayer on gold." J Am Chem Soc 126.15 (April 21, 2004): 4770-4771.
PMID
15080668
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
126
Issue
15
Publish Date
2004
Start Page
4770
End Page
4771
DOI
10.1021/ja049956q

Fabrication of "smart" biomolecular and polymeric nanostructures using molecular recognition, surface-initiated nanopolymerization and scanning probe lithography.

Authors
Kaholek, M; Hyun, J; Lee, WK; Chilkoti, A; Zauscher, S
MLA Citation
Kaholek, M, Hyun, J, Lee, WK, Chilkoti, A, and Zauscher, S. "Fabrication of "smart" biomolecular and polymeric nanostructures using molecular recognition, surface-initiated nanopolymerization and scanning probe lithography." March 28, 2004.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
227
Publish Date
2004
Start Page
U861
End Page
U861

Effect of protein fusion on the transition temperature of an environmentally responsive elastin-like polypeptide: a role for surface hydrophobicity?

The limited throughput, scalability and high cost of protein purification by chromatography provide motivation for the development of non-chromatographic protein purification technologies that are cheaper and easier to implement in a high-throughput format for proteomics applications and to scale up for industrial bioprocessing. We have shown that genetic fusion of a recombinant protein to an elastin-like polypeptide (ELP) imparts the environmentally sensitive solubility property of the ELP to the fusion protein, and thereby allows selective separation of the fusion protein from Escherichia coli lysate by aggregation above a critical temperature (T(t)). Further development of ELP fusion proteins as widely applicable purification tools necessitates a quantitative understanding of how fused proteins perturb the ELP T(t) such that purification conditions (T(t)) may be predicted a priori for new recombinant proteins. We report here the effect that fusing six different proteins has on the T(t) of an ELP. A negative correlation between T(t) and the fraction hydrophobic surface area on the fused proteins was observed, which was determined from computer modeling of the available three-dimensional structure. The thermally triggered aggregation behavior of ELP-coated, functionalized gold colloids as well as ligand binding to the tendamistat-ELP fusion protein support the hypothesis that hydrophobic surfaces in molecular proximity to ELPs depress the ELP T(t) by a mechanism analogous to hydrophobic residue substitution in the ELP repeat, Val-Pro-Gly-Xaa-Gly.

Authors
Trabbic-Carlson, K; Meyer, DE; Liu, L; Piervincenzi, R; Nath, N; LaBean, T; Chilkoti, A
MLA Citation
Trabbic-Carlson, K, Meyer, DE, Liu, L, Piervincenzi, R, Nath, N, LaBean, T, and Chilkoti, A. "Effect of protein fusion on the transition temperature of an environmentally responsive elastin-like polypeptide: a role for surface hydrophobicity?." Protein Eng Des Sel 17.1 (January 2004): 57-66.
PMID
14985538
Source
pubmed
Published In
Protein Engineering Design and Selection
Volume
17
Issue
1
Publish Date
2004
Start Page
57
End Page
66
DOI
10.1093/protein/gzh006

Effect of genetic circular permutation near the active site on the activity and stability of an enzyme inhibitor.

We report here the effect of circular permutation on the structure and function of a model protein tendamistat, a 74 amino acid competitive inhibitor of porcine pancreatic alpha-amylase. The activity and stability of wild type and two permuted tendamistat variants were characterized by measurement of alpha-amylase kinetic and thermodynamic binding parameters and their thermodynamics of unfolding. Our results show that large variations in structure and function can occur upon circularly permuting tendamistat near its active site that are not obvious, a priori, from the structure of the native protein and we propose a structural thermodynamic explanation of the experimental observations.

Authors
Piervincenzi, RT; Chilkoti, A
MLA Citation
Piervincenzi, RT, and Chilkoti, A. "Effect of genetic circular permutation near the active site on the activity and stability of an enzyme inhibitor." Biomol Eng 21.1 (January 2004): 33-42.
PMID
14715318
Source
pubmed
Published In
Biomolecular Engineering
Volume
21
Issue
1
Publish Date
2004
Start Page
33
End Page
42

Surface engineering strategies for control of protein and cell interactions

Surface engineering is an important tool in understanding the molecular mechanism of protein adsorption and cell-surface interactions with the aim of rational design of surfaces for different biological applications. This article first briefly summarizes some of the commonly used approaches to control biological interactions at the solid-water interface. Next, two different approaches are presented from the authors' research to engineer surfaces that address some of the problems and limitations of current approaches to modulate protein adsorption at surfaces. The first, "static" strategy involves modifying a surface with an amphiphilic comb polymer that presents short oligoethylene glycol side-chains. Two different fabrication methods to synthesize these non-fouling coatings are described including spin-coating and surface-initiated polymerization. The second, "active" strategy to control protein adsorption involves grafting a stimuli-responsive biopolymer, derived from an oligomeric sequence found in mammalian elastin to a surface. This polypeptide exhibits a lower critical solution temperature (LCST) transition in aqueous solution. Surfaces grafted with these polypeptides exhibit a hydrophilic-hydrophobic transition in response to increased temperature or salt concentration. The change in the interfacial properties can be exploited to create "active" polymer grafts that enable reversible, triggered binding of proteins onto surfaces in response to an external stimulus. © 2004 Elseiver B.V. All rights reserved.

Authors
Nath, N; Hyun, J; Ma, H; Chilkoti, A
MLA Citation
Nath, N, Hyun, J, Ma, H, and Chilkoti, A. "Surface engineering strategies for control of protein and cell interactions." Surface Science 570.1-2 (2004): 98-110.
Source
scival
Published In
Surface Science
Volume
570
Issue
1-2
Publish Date
2004
Start Page
98
End Page
110
DOI
10.1016/j.susc.2004.06.182

"Non-Fouling" Oligo(ethylene glycol)-Functionalized Polymer Brushes Synthesized by Surface-Initiated Atom Transfer Radical Polymerization

The synthesis of (EG) n-functionalized polymer brushes of tunable thicknesses in the 5-50 nm range by surface initiated polymerization was discussed. It was shown that these polymer brushes exhibited no detectable adsorption of proteins, and were cell resistant for up to a month under typical cell culture conditions. It was also shown that the synthesis method was compatible with a range of patterning techniques from the nano- to microscale, which enabled the patterning of cells in a biologically relevant milieu over extended period of time. These non-fouling surfaces and topographical structures were found to be useful as model quasi-three-dimensional systems to investigate the response of cells to chemical and topographical cues.

Authors
Ma, H; Hyun, J; Stiller, P; Chilkoti, A
MLA Citation
Ma, H, Hyun, J, Stiller, P, and Chilkoti, A. ""Non-Fouling" Oligo(ethylene glycol)-Functionalized Polymer Brushes Synthesized by Surface-Initiated Atom Transfer Radical Polymerization." Advanced Materials 16.4 (2004): 338-341.
Source
scival
Published In
Advanced Materials
Volume
16
Issue
4
Publish Date
2004
Start Page
338
End Page
341

Materials Research Society Symposium Proceedings: Preface

Authors
Wong, JY; Plant, AL; Schmidt, CE; Shea, L; Coury, AJ; Chen, CS; Barron, AE; Klok, HA; Deming, TJ; Saltzman, WM; Chilkoti, A; Luo, D; Uhrich, K
MLA Citation
Wong, JY, Plant, AL, Schmidt, CE, Shea, L, Coury, AJ, Chen, CS, Barron, AE, Klok, HA, Deming, TJ, Saltzman, WM, Chilkoti, A, Luo, D, and Uhrich, K. "Materials Research Society Symposium Proceedings: Preface." Materials Research Society Symposium Proceedings EXS.1 (2004): xvii-.
Source
scival
Published In
Materials Research Society Symposium - Proceedings
Volume
EXS
Issue
1
Publish Date
2004
Start Page
xvii

3-D reconstruction of tumor vascular networks

Authors
Prohaska, S; Dreher, M; Dewhirst, MW; Chilkoti, A; Pries, AR; Westerhoff, M; Cornelissen, AJM
MLA Citation
Prohaska, S, Dreher, M, Dewhirst, MW, Chilkoti, A, Pries, AR, Westerhoff, M, and Cornelissen, AJM. "3-D reconstruction of tumor vascular networks." 2004.
Source
wos-lite
Published In
Journal of vascular research
Volume
41
Issue
5
Publish Date
2004
Start Page
463
End Page
463

Fabrication of "smart" protein nanostructures using molecular recognition and dip-pen nano-lithography.

Authors
Zauscher, S; Chilkoti, A; Hyun, J; Lee, WK
MLA Citation
Zauscher, S, Chilkoti, A, Hyun, J, and Lee, WK. "Fabrication of "smart" protein nanostructures using molecular recognition and dip-pen nano-lithography." September 2003.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
226
Publish Date
2003
Start Page
U485
End Page
U485

Non-covalent, environmentally modulated molecular recognition between ELP biopolymers: A convenient route to reversible protein arrays.

Authors
Nath, N; Chilkoti, A; Zauscher, S; Hyun, JH; Lee, WK
MLA Citation
Nath, N, Chilkoti, A, Zauscher, S, Hyun, JH, and Lee, WK. "Non-covalent, environmentally modulated molecular recognition between ELP biopolymers: A convenient route to reversible protein arrays." September 2003.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
226
Publish Date
2003
Start Page
U410
End Page
U410

Evaluation of an elastin-like polypeptide-doxorubicin conjugate for cancer therapy.

Thermally responsive elastin-like polypeptides (ELPs) were synthesized by recombinant DNA techniques and conjugated to doxorubicin through an acid-labile hydrazone bond to enable release of the drug in the acidic environment of lysosomes. The thermal properties, intracellular localization and cytotoxicity of the conjugate were investigated in this study. The conjugation procedure resulted in a mixed population of free ELP and ELP-doxorubicin (ELP-dox) conjugates that exhibit a broader transition than the parent ELP. A simple centrifugation procedure was developed to purify the ELP-dox conjugate from other reactants and resulted in a sharper thermal transition, similar to the parent ELP. The ELP was endocytosed by squamous cell carcinoma cells (FaDu) and trafficked into lysosomes, as observed by the colocalization of the ELP with a lysosome-specific dye through confocal fluorescence microscopy. Interestingly, both the ELP-dox conjugate and free drug exhibited near equivalent in vitro cytotoxicity, although their subcellular localization was significantly different. The free drug was largely concentrated in the nucleus, while the conjugate was dispersed throughout the cytoplasm with limited nuclear accumulation. These differences are significant because they suggest a different mechanism of cytotoxicity for the conjugate as compared with the free drug.

Authors
Dreher, MR; Raucher, D; Balu, N; Michael Colvin, O; Ludeman, SM; Chilkoti, A
MLA Citation
Dreher, MR, Raucher, D, Balu, N, Michael Colvin, O, Ludeman, SM, and Chilkoti, A. "Evaluation of an elastin-like polypeptide-doxorubicin conjugate for cancer therapy." J Control Release 91.1-2 (August 28, 2003): 31-43.
PMID
12932635
Source
pubmed
Published In
Journal of Controlled Release
Volume
91
Issue
1-2
Publish Date
2003
Start Page
31
End Page
43

Swelling and mechanical behaviors of chemically cross-linked hydrogels of elastin-like polypeptides.

Genetically engineered elastin-like polypeptides consisting of Val-Pro-Gly-X-Gly repeats, where X was chosen to be Lys every 7 or 17 pentapeptides (otherwise X was Val), were synthesized and expressed in E. coli, purified, and chemically cross-linked using tris-succinimidyl aminotriacetate to produce hydrogels. Swelling experiments indicate hydrogel mass decreases by 80-90% gradually over an approximate 50 degrees C temperature range. Gels ranged in stiffness from 0.24 to 3.7 kPa at 7 degrees C and from 1.6 to 15 kPa at 37 degrees C depending on protein concentration, lysine content, and molecular weight. Changes in gel stiffness and loss angle with cross-linking formulation suggest a low-temperature gel structure that is nearly completely elastic, where force is transmitted almost exclusively through fully extended polypeptide chains and chemical cross-links, and a high-temperature gel structure, where ELP chains are contracted and force is transmitted through chemical cross-links as well as frictional contact between polypeptide chains.

Authors
Trabbic-Carlson, K; Setton, LA; Chilkoti, A
MLA Citation
Trabbic-Carlson, K, Setton, LA, and Chilkoti, A. "Swelling and mechanical behaviors of chemically cross-linked hydrogels of elastin-like polypeptides." Biomacromolecules 4.3 (May 2003): 572-580.
PMID
12741772
Source
pubmed
Published In
Biomacromolecules
Volume
4
Issue
3
Publish Date
2003
Start Page
572
End Page
580
DOI
10.1021/bm025671z

Nanoparticle biosensors

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "Nanoparticle biosensors." March 2003.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
225
Publish Date
2003
Start Page
U964
End Page
U964

Passive and active nonfouling polymer grafts.

Authors
Chilkoti, A; Ma, HW; Hyun, J; Nath, N
MLA Citation
Chilkoti, A, Ma, HW, Hyun, J, and Nath, N. "Passive and active nonfouling polymer grafts." March 2003.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
225
Publish Date
2003
Start Page
U654
End Page
U655

Fabrication of surface confined, stimulus-responsive polymer nanostructures using dip-pen nanolithography.

Authors
Zauscher, S; Chilkoti, A; Ahn, SJ; Hyun, J; Lee, WK
MLA Citation
Zauscher, S, Chilkoti, A, Ahn, SJ, Hyun, J, and Lee, WK. "Fabrication of surface confined, stimulus-responsive polymer nanostructures using dip-pen nanolithography." March 2003.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
225
Publish Date
2003
Start Page
U655
End Page
U655

Fabrication of a reversible protein array directly from cell lysate using a stimuli-responsive polypeptide.

We report a new method to reversibly bind proteins to a surface in a functionally active orientation directly from cell lysate by exploiting a thermodynamically reversible hydrophilic-hydrophobic lower critical solution temperature (LCST) transition exhibited by a recombinant, stimuli-responsive elastin-like polypeptide (ELP). An ELP is covalently micropatterned on a glass surface against an inert BSA background. The ELP-patterned surface is incubated with the soluble fraction of E. coli lysate containing an expressed ELP fusion protein, which is appended with the same ELP as on the surface. The LCST transition of the grafted ELP and the ELP fusion protein is simultaneously triggered by an external stimulus. The LCST transition results in capture of the ELP fusion protein from solution onto the immobilized ELP by hydrophobic interactions between the grafted ELP and the ELP fusion protein. The captured ELP fusion protein is oriented such that the fusion partner is accessible to binding of its target from solution. We also demonstrate that TRAP is reversible; the bound protein-ligand complex is released from the surface by reversing the LCST transition. The triggered control of interfacial properties provided by an immobilized stimuli-responsive polypeptide at the solid-water interface is an enabling technology that allows reversible and functional presentation of ELP fusion proteins on a surface directly from cell lysate without the necessity of intermediate purification steps and subsequent recovery of the protein-ligand complex for downstream analysis by other analytical techniques. TRAP has application in lab-on-a-chip bioanalytical devices as well as in the fabrication of peptide and protein arrays.

Authors
Nath, N; Chilkoti, A
MLA Citation
Nath, N, and Chilkoti, A. "Fabrication of a reversible protein array directly from cell lysate using a stimuli-responsive polypeptide." Anal Chem 75.4 (February 15, 2003): 709-715.
PMID
12622356
Source
pubmed
Published In
Analytical Chemistry
Volume
75
Issue
4
Publish Date
2003
Start Page
709
End Page
715

Effect of streptavidin affinity mutants on the integrin-independent adhesion of biotinylated endothelial cells.

We have previously shown that the high-affinity streptavidin (SA)-biotin interaction enhanced the initial integrin-mediated adhesion of biotinylated endothelial cells to SA-coated surface by serving as an extrinsic bond to stabilize and enhance the intrinsic fibronectin-integrin binding between the cell and surface. However, the SA-biotin interaction produced considerable detachment by cohesive failure of the membrane. In this study, we examined the hypothesis that reducing the SA-biotin bond affinity could reduce cohesive failure without reducing overall cell detachment. Two mutants of SA, W120F and W120A in which the tryptophan residue at position 120 of the SA molecule was substituted by phenylalanine and alanine, respectively, were characterized and tested in cell adhesion experiments. The binding affinity (K(A)) of SA to adsorbed biotin-labeled bovine serum albumin (b-BSA) ranged from 5.2+/-0.1 x 10(10)M(-1) for wild-type to 3.3+/-0.2 x 10(9)M(-1) for W120F and 4.1+/-1.0 x 10(6)M(-1) for W120A. One hour after cell attachment, the critical shear stress was 26.8+/-2.9 dyn/cm(2) for WT, 26.6+/-3.0 dyn/cm(2) for W120F, and 15.4+/-3.0 dyn/cm(2) for W120A. The focal contact areas of adherent cells were greater for the WT and W120F than the lower affinity mutant, W120A. When shear flow was applied to detach adherent cells, adhesive failure (ligand bond breakage) was favored over cohesive failure (membrane rupture), as the SA binding affinity decreased. Thus, cell adhesion augmented by SA-biotin linkages is dependent on the affinity constants of the SA-biotin bonds, but the reduction in cohesive failure was offset by a reduced strength of adhesion.

Authors
Chan, BP; Chilkoti, A; Reichert, WM; Truskey, GA
MLA Citation
Chan, BP, Chilkoti, A, Reichert, WM, and Truskey, GA. "Effect of streptavidin affinity mutants on the integrin-independent adhesion of biotinylated endothelial cells." Biomaterials 24.4 (February 2003): 559-570.
PMID
12437950
Source
pubmed
Published In
Biomaterials
Volume
24
Issue
4
Publish Date
2003
Start Page
559
End Page
570

Universal route to cell micropatterning using an amphiphilic comb polymer

Micropatterning of localized chemical or biochemical domains has the potential to become a powerful tool to control the behavior of anchorage-dependent cells. However, several problems, such as the limited types of substrates that can be successfully patterned with cells and the lack of reliable, long-term retention of cellular patterns under physiological conditions need to be solved for cellular patterning to become widely applicable for cell-based biosensors, biomaterials, and high-throughput drug screening assay. This paper reports on a simple and generic method to micropattern surfaces with an amphiphilic comb polymer presenting short oligoethylene glycol side chains that enables long-term, spatially resolved attachment and growth of mammalian cells in a biologically relevant milieu on a variety of substrates.

Authors
Hyun, J; Ma, H; Zhang, Z; Jr, TPB; Chilkoti, A
MLA Citation
Hyun, J, Ma, H, Zhang, Z, Jr, TPB, and Chilkoti, A. "Universal route to cell micropatterning using an amphiphilic comb polymer." Advanced Materials 15.7-8 (2003): 576-579.
Source
scival
Published In
Advanced Materials
Volume
15
Issue
7-8
Publish Date
2003
Start Page
576
End Page
579

Conformational Mechanics of Stimulus-Responsive Polypeptides

Stimulus-responsive polymers and polypeptides (SRPs) experience a significant entropic response when exposed to an environmental stimulus, such as a change in temperature. This phase transition directly affects polymer conformation and can potentially be harnessed for force generation in actuation devices on nano- and micro-scales. While interfacial applications of SRPs have been prototypically demonstrated, a systematic investigation of the phase transition behavior at the solid-liquid interface and on the single-molecule level is lacking. In this paper we present results from force-spectroscopy measurements probing the force-extension and conformational behavior of one SRP, elastin-like polypeptides (ELF), below and above their transition temperature. The results indicate that there is no significant difference in the force extension behavior at intermediate and large extensions, but the behavior is dramatically different at small extensions. Results also demonstrated that above the phase transition temperature large, unspecific adhesion forces often gave way to constant force steps upon extension, indicating a collapsed, potentially entangled, hydrophobic state of the ELP. The extension behavior below the phase transition temperature, however, closely followed that of a random polymer coil, without any significant unspecific adhesion forces. The excellent fit of a simple extended freely jointed chain model to the data at intermediate and large extensions suggests that the ELP is in a random conformational state without significant secondary structure. Forces associated with a phase transition therefore arise likely from entropic conformational changes associated with a hydrophobic collapse.

Authors
Valiaev, A; Clark, RL; Chilkoti, A; Zauscher, S
MLA Citation
Valiaev, A, Clark, RL, Chilkoti, A, and Zauscher, S. "Conformational Mechanics of Stimulus-Responsive Polypeptides." Proceedings of SPIE - The International Society for Optical Engineering 5053 (2003): 31-40.
Source
scival
Published In
Proceedings of SPIE - The International Society for Optical Engineering
Volume
5053
Publish Date
2003
Start Page
31
End Page
40
DOI
10.1117/12.484700

Dynamic addressing of a surface pattern by a stimuli-responsive fusion protein

An overview is given of a dynamic protein patterning methodology, thermodynamically reversible addressing of proteins (TRAP), in which a recombinant, stimuli responsive polypeptide is used to dynamically address a fusion protein onto a micropatterned surface template by modulating the interaction between a chemically patterned surface and the stimuli-responsive polypeptide. Immobilization of the fusion protein in spatially defined regions of the micropatterned surface is triggered either by an increase in temperature or ionic strength and the immobilized fusion partner is oriented towards solution and is recognized by an antibody to the fusion protein. TRAP is reversible, because the protein-antibody complex can be resolubilized from the surface by reversing the environmental trigger.

Authors
Frey, W; Meyer, DE; Chilkoti, A
MLA Citation
Frey, W, Meyer, DE, and Chilkoti, A. "Dynamic addressing of a surface pattern by a stimuli-responsive fusion protein." Advanced Materials 15.3 (2003): 248-251.
Source
scival
Published In
Advanced Materials
Volume
15
Issue
3
Publish Date
2003
Start Page
248
End Page
251
DOI
10.1002/adma.200390058

Thermodynamically reversible addressing of a stimuli responsive fusion protein onto a patterned surface template

The sensitivity of an elastin-like polypeptide (ELP) to environmental stimuli is used to reversibly immobilize a fusion partner, thioredoxin (TRX), onto a hydrophobia surface. An ELP, fused to TRX at its C-terminus, adsorbs onto a hydrophobic self-assembled monolayer (SAM) on gold above its inverse phase transition temperature (Tc) and is resolubilized from the surface when the solution temperature is lowered below Tc. We show that the adsorbed fusion partner TRX is recognized by an antibody specific to TRX and that the complex is also resolubilized from the surface below Tc, Adsorption of TRX-ELP is inhibited by hydrophilic surfaces that are defect-free. In situ ellipsometry shows that the ELP-anchored TRX adsorbs above Tc up to a well-defined layer thickness that is greater than a monolayer and that the adsorption and desorption cycle can be repeated. These results are confirmed by atomic force microscopy and by surface plasmon resonance spectroscopy, The preferential adsorption of TRX-ELP on hydrophobic surfaces is used to create a pattern of adsorbed protein on a surface composed of a pattern of hydrophobic and hydrophilic SAMs on gold. We term this method to reversibly address an ELP fusion protein to chemically distinct regions of a patterned surface by an external stimulus "thermodynamically reversible addressing of proteins" (TRAP).

Authors
Frey, W; Meyer, DE; Chilkoti, A
MLA Citation
Frey, W, Meyer, DE, and Chilkoti, A. "Thermodynamically reversible addressing of a stimuli responsive fusion protein onto a patterned surface template." Langmuir 19.5 (2003): 1641-1653.
Source
scival
Published In
Langmuir
Volume
19
Issue
5
Publish Date
2003
Start Page
1641
End Page
1653
DOI
10.1021/la026359d

Surface engineering strategies for control of protein and cell interactions

Authors
Ma, H; Hyun, J; Nath, N; Chilkoti, A
MLA Citation
Ma, H, Hyun, J, Nath, N, and Chilkoti, A. "Surface engineering strategies for control of protein and cell interactions." European Cells and Materials 6.SUPPL. 1 (2003): 16-17.
Source
scival
Published In
European cells & materials
Volume
6
Issue
SUPPL. 1
Publish Date
2003
Start Page
16
End Page
17

X-ray photoelectron spectroscopy as a tool for bio-surface analysis

Authors
Blomfield, CJ; Roberts, AJ; Hutton, SJ; Ma, H; Hyun, J; Chilkoti, A
MLA Citation
Blomfield, CJ, Roberts, AJ, Hutton, SJ, Ma, H, Hyun, J, and Chilkoti, A. "X-ray photoelectron spectroscopy as a tool for bio-surface analysis." European Cells and Materials 6.SUPPL. 1 (2003): 60--.
Source
scival
Published In
European cells & materials
Volume
6
Issue
SUPPL. 1
Publish Date
2003
Start Page
60-

Design of thermally responsive, recombinant polypeptide carriers for targeted drug delivery.

In this article, we review recombinant DNA methods for the design and synthesis of amino acid-based biopolymers, and briefly summarize an approach, recursive directional ligation (RDL), that we have employed to synthesize oligomeric genes for such biopolymers. We then describe our ongoing research in the use of RDL to synthesize recombinant polypeptide carriers for the targeted delivery of radionuclides, chemotherapeutics and biomolecular therapeutics to tumors. The targeted delivery system uses a thermally responsive, elastin-like polypeptide (ELP) as the drug carrier to enhance the localization of ELP-drug conjugates within a solid tumor that is heated by regional hyperthermia. In the context of this drug delivery application, we discuss the design of ELPs and their recombinant synthesis, which enables the molecular weight and the thermal properties of the polypeptide to be precisely controlled. Finally, our results pertaining to the in vivo targeting of tumors with ELPs are briefly summarized.

Authors
Chilkoti, A; Dreher, MR; Meyer, DE
MLA Citation
Chilkoti, A, Dreher, MR, and Meyer, DE. "Design of thermally responsive, recombinant polypeptide carriers for targeted drug delivery." Adv Drug Deliv Rev 54.8 (October 18, 2002): 1093-1111. (Review)
PMID
12384309
Source
pubmed
Published In
Advanced Drug Delivery Reviews
Volume
54
Issue
8
Publish Date
2002
Start Page
1093
End Page
1111

Targeted drug delivery by thermally responsive polymers.

This review article summarizes recent results on the development of macromolecular carriers for thermal targeting of therapeutics to solid tumors. This approach employs thermally responsive polymers in conjunction with targeted heating of the tumor. The two thermally responsive polymers that are discussed in this article, poly(N-isopropylacrylamide-co-acrylamide) (poly(NIPAAm)) and an artificial elastin-like polypeptide (ELP), were designed to exhibit a soluble-insoluble lower critical solution transition in response to increased temperature slightly above 37 degrees C. In vivo fluorescent videomicroscopy and radiolabel distribution studies of ELP delivery to human tumors implanted in nude mice demonstrated that hyperthermic targeting of the thermally responsive ELP for 1 h provides a approximately two-fold increase in tumor localization compared to the same polypeptide without hyperthermia. Similar results were also obtained for poly(NIPAAm) though the extent of accumulation was somewhat lesser than observed for the ELP. The endocytotic uptake of a thermally responsive ELP was also observed to be significantly enhanced by the thermally triggered phase transition of the polypeptide in cell culture for three different tumor cell lines. Preliminary cytotoxicity studies of an ELP-doxorubicin conjugate indicate that the ELP-doxorubicin conjugate has near equivalent cytotoxicity as free doxorubicin in a cell culture assay.

Authors
Chilkoti, A; Dreher, MR; Meyer, DE; Raucher, D
MLA Citation
Chilkoti, A, Dreher, MR, Meyer, DE, and Raucher, D. "Targeted drug delivery by thermally responsive polymers." Adv Drug Deliv Rev 54.5 (September 13, 2002): 613-630. (Review)
PMID
12204595
Source
pubmed
Published In
Advanced Drug Delivery Reviews
Volume
54
Issue
5
Publish Date
2002
Start Page
613
End Page
630

Characterization of a genetically engineered elastin-like polypeptide for cartilaginous tissue repair.

Elastin-like polypeptides (ELPs) are artificial polypeptides with unique properties that make them attractive as a biomaterial for tissue-engineered cartilage repair. ELPs are composed of a pentapeptide repeat, Val-Pro-Gly-Xaa-Gly (Xaa is any amino acid except Pro), that undergo an inverse temperature phase transition. They are soluble in aqueous solution below their transition temperature (T(t)) but aggregate when the solution temperature is raised above their T(t). This study investigates the rheological behavior of an un-cross-linked ELP, below and above its T(t), and also examines the ability of ELP to promote chondrogenesis in vitro. A thermally responsive ELP with a T(t) of 35 degrees C was synthesized using recombinant DNA techniques. The complex shear modulus of the ELP increased by 3 orders of magnitude as it underwent its inverse temperature phase transition, forming a coacervate, or gel-like, ELP phase. Values for the complex shear moduli of the un-cross-linked ELP coacervate are comparable to those reported previously for collagen, hyaluronan, and cross-linked synthetic hydrogels. Cell culture studies show that chondrocytes cultured in ELP coacervate maintain a rounded morphology and their chondrocytic phenotype, characterized by the synthesis of a significant amount of extracellular matrix composed of sulfated glycosaminoglycans and collagen. These results suggest that ELPs demonstrate great potential for use as in situ forming scaffolds for cartilaginous tissue repair.

Authors
Betre, H; Setton, LA; Meyer, DE; Chilkoti, A
MLA Citation
Betre, H, Setton, LA, Meyer, DE, and Chilkoti, A. "Characterization of a genetically engineered elastin-like polypeptide for cartilaginous tissue repair." Biomacromolecules 3.5 (September 2002): 910-916.
PMID
12217035
Source
pubmed
Published In
Biomacromolecules
Volume
3
Issue
5
Publish Date
2002
Start Page
910
End Page
916

A colorimetric gold nanoparticle sensor to interrogate biomolecular interactions in real-time on a surface.

Authors
Chilkoti, A; Nath, N
MLA Citation
Chilkoti, A, and Nath, N. "A colorimetric gold nanoparticle sensor to interrogate biomolecular interactions in real-time on a surface." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 224 (August 18, 2002): U151-U151.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
224
Publish Date
2002
Start Page
U151
End Page
U151

Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system.

We report a new strategy for the synthesis of genes encoding repetitive, protein-based polymers of specified sequence, chain length, and architecture. In this stepwise approach, which we term "recursive directional ligation" (RDL), short gene segments are seamlessly combined in tandem using recombinant DNA techniques. The resulting larger genes can then be recursively combined until a gene of a desired length is obtained. This approach is modular and can be used to combine genes encoding different polypeptide sequences. We used this method to synthesize three different libraries of elastin-like polypeptides (ELPs); each library encodes a unique ELP sequence with systematically varied molecular weights. We also combined two of these sequences to produce a block copolymer. Because the thermal properties of ELPs depend on their sequence and chain length, the synthesis of these polypeptides provides an example of the importance of precise control over these parameters that is afforded by RDL.

Authors
Meyer, DE; Chilkoti, A
MLA Citation
Meyer, DE, and Chilkoti, A. "Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system." Biomacromolecules 3.2 (March 2002): 357-367.
PMID
11888323
Source
pubmed
Published In
Biomacromolecules
Volume
3
Issue
2
Publish Date
2002
Start Page
357
End Page
367

A colorimetric gold nanoparticle sensor to interrogate biomolecular interactions in real time on a surface.

This paper presents a new label-free optical method to study biomolecular interactions in real time at the surface of an optically transparent substrate. The method relies on the change in the absorbance spectrum of a self-assembled monolayer of colloidal gold on glass, as a function of biomolecular binding to the surface of the immobilized colloids. Using this approach, we demonstrate proof of principle of a label-free optical biosensor to quantify biomolecular interactions in real time on a surface in a commercially available UV-visible spectrophotometer and of a colorimetric end-point assay using an optical scanner. The spectrophotometric sensor shows concentration-dependent binding and a detection limit of 16 nM for streptavidin. The sensor is easy to fabricate, is reproducible in its performance, has minimal technological requirements, namely, the availability of an UV-visible spectrophotometer or an optical scanner, and will enable high-throughput screening of biomolecular interactions in real time in an array-based format.

Authors
Nath, N; Chilkoti, A
MLA Citation
Nath, N, and Chilkoti, A. "A colorimetric gold nanoparticle sensor to interrogate biomolecular interactions in real time on a surface." Anal Chem 74.3 (February 1, 2002): 504-509.
PMID
11838667
Source
pubmed
Published In
Analytical Chemistry
Volume
74
Issue
3
Publish Date
2002
Start Page
504
End Page
509

Creating "smart" surfaces using stimuli responsive polymers

"Smart" surfaces that modulate their physico-chemical properties in response to environmental triggers can be fabricated by functionalization of a surface with stimuli-responsive polymers (SRPs). This article summarizes different approaches to the fabrication of "smart" surfaces and their applications. Recent results that demonstrate reversible micropatterning of proteins using a smart biopolymer as well as a simple, colorimetric assay to characterize the phase transition behavior of SRPs at the solid-water interface are highlighted.

Authors
Nath, N; Chilkoti, A
MLA Citation
Nath, N, and Chilkoti, A. "Creating "smart" surfaces using stimuli responsive polymers." Advanced Materials 14.17 (2002): 1243-1247. (Academic Article)
Source
manual
Published In
Advanced Materials
Volume
14
Issue
17
Publish Date
2002
Start Page
1243
End Page
1247
DOI
10.1002/1521-4095(20020903)14:173.0.CO;2-M

Micropatterns of a cell-adhesive peptide on an amphiphilic comb polymer film

We report in this paper a generic method to modify the surfaces of common polymeric biomaterials that enables spatially resolved attachment and growth of mammalian cells in a biologically relevant milieu. We demonstrate that an amphiphilic comb polymer presenting short oligoethylene glycol side chains can be coated onto a number of different polymeric biomaterials, namely polystyrene, poly(methyl methacrylate), and poly(ethylene terephthalate) from a methanol/water mixture. The comb polymer film is stable in water and presents reactive COOH groups at the oligoethylene glycol chain ends, thereby permitting the surface of the comb polymer to be patterned with a cell adhesive, arg-gly-asp peptide. The micropatterned surfaces spatially confine the attachment and growth of fibroblasts for ∼24 h in 10% serum to the patterned regions.

Authors
Hyun, J; Ma, H; Banerjee, P; Cole, J; Gonsalves, K; Chilkoti, A
MLA Citation
Hyun, J, Ma, H, Banerjee, P, Cole, J, Gonsalves, K, and Chilkoti, A. "Micropatterns of a cell-adhesive peptide on an amphiphilic comb polymer film." Langmuir 18.8 (2002): 2975-2979.
Source
scival
Published In
Langmuir
Volume
18
Issue
8
Publish Date
2002
Start Page
2975
End Page
2979
DOI
10.1021/la015712x

Molecular recognition-mediated fabrication of protein nanostructures by dip-pen lithography

We describe the molecular recognition-mediated, stepwise fabrication of patterned protein nanostructures with feature sizes on the order of 200 nm. First, a self-assembled monolayer (SAM) of 16-mercaplohexadecanoic acid (MHA) is patterned onto gold by dip-pen nanolithography (DPN), and the unpatterned regions are passivated with a protein-resistant oligoethylene glycol-terminated alkanethiol SAM. Next, an amine-terminated biotin derivative is covalently conjugated with the chemically activated MHA SAM nanopattern. The surface is then incubated with streptavidin to form streptavidin nanostructures, mediated by molecular recognition between biotin and streptavidin. Finally, protein nanopatterns are fabricated by molecular recognition-mediated immobilization of biotinylated protein from solution. Our fabrication methodology is generically applicable because of the ubiquity of biotin-tagged molecules

Authors
Hyun, J; Ahn, SJ; Lee, WK; Chilkoti, A; Zauscher, S
MLA Citation
Hyun, J, Ahn, SJ, Lee, WK, Chilkoti, A, and Zauscher, S. "Molecular recognition-mediated fabrication of protein nanostructures by dip-pen lithography." Nano Lett. (USA) 2.11 (2002): 1203-1207. (Academic Article)
Source
manual
Published In
Nano Lett. (USA)
Volume
2
Issue
11
Publish Date
2002
Start Page
1203
End Page
1207
DOI
10.1021/n10257364

An immobilized gold nanoparticle sensor for label free optical detection of biomolecular interactions

We present a new label free optical technique to study biomolecular interactions in real time on a surface. This method monitors changes in the absorbance spectrum of a monolayer of gold on glass as a function of biomolecular binding. Gold nanoparticles with a diameter of 13 nm were chemisorbed onto an amine-terminated glass surface. The absorbance spectrum of the monolayer exhibited both a red shift as well as an increase in the absorbance at peak wavelength as a function of bulk solution refractive index. The increase in absorbance at 550 nm was employed to study the kinetics of fibrinogen adsorption on the immobilized monolayer. The results obtained with the absorbance sensor were compared with those obtained using conventional SPR. This sensor is attractive because of its simplicity: gold nanoparticles are easily prepared with high reproducibility, they can be easily immobilized on glass, and their absorbance spectrum can be easily measured using widely available UV-vis spectrophotometers. Furthermore, this technique should be easily amenable to the design of chips in an array format for application in high-throughput immunoassays and proteomics.

Authors
Nath, N; Chilkoti, A
MLA Citation
Nath, N, and Chilkoti, A. "An immobilized gold nanoparticle sensor for label free optical detection of biomolecular interactions." Proceedings of SPIE - The International Society for Optical Engineering 4626 (2002): 441-448.
Source
scival
Published In
Proceedings of SPIE - The International Society for Optical Engineering
Volume
4626
Publish Date
2002
Start Page
441
End Page
448
DOI
10.1117/12.472110

Hybrid bioinorganic smart membranes that incorporate protein-based molecular switches

Polypeptide- and protein-based components have the potential to greatly enhance the functional character of hybrid organic/inorganic materials by imparting recognition, actuation, or transduction properties to these materials. In this study, we demonstrate a simple method of fabricating hybrid silica/polypeptide membranes that exhibit molecular-level control of permeability in response to an external stimulus, namely, heat. The biomolecular switches employed in this study are elastin-like polypeptides (ELPs), which were genetically engineered to allow for control of molecular size and thermal response. ELPs were chosen for incorporation into silica membranes because they exhibit a lower critical solution temperature (LCST) transition in aqueous solution. We demonstrate here that the LCST behavior of ELPs can be retained when they are entrapped in hydrated silica gels and that the LCST transition of these molecules can be used to toggle the permeability of hybrid membranes. Two different ELPs with different LCSTs and different molecular weights (60 and 13 kDa) were examined. They were incorporated into silica membranes using sol-gel synthesis conditions that resulted in the random encapsulation of the proteins in the silica matrixes such that significant porosity is present only upon hydrophobic collapse of the ELPs. Measurement of the permeation of solutions of poly(ethylene glycols) (PEGs) of various molecular weights through centrifugal and ultrafiltration membranes demonstrated that the ELPs in the hybrid membranes act as molecular switches. Below the LCSTs of the ELPs, the hybrid membranes are impermeable to all of the PEG solutions investigated, regardless of the molecular weight of the PEG; above the LCSTs, they are permeable only to those PEGs investigated with molecular weights less than 5000 Da. PEGs investigated of higher molecular weight did not permeate through the hybrid membranes. Thus, the hybrid ELP-containing membranes act as selective molecular weight cutoff filters whose permeability can be switched on and off.

Authors
Rao, GVR; Balamurugan, S; Meyer, DE; Chilkoti, A; López, GP
MLA Citation
Rao, GVR, Balamurugan, S, Meyer, DE, Chilkoti, A, and López, GP. "Hybrid bioinorganic smart membranes that incorporate protein-based molecular switches." Langmuir 18.5 (2002): 1819-1824.
Source
scival
Published In
Langmuir
Volume
18
Issue
5
Publish Date
2002
Start Page
1819
End Page
1824
DOI
10.1021/la011188i

Creating "smart" surfaces using stimuli responsive polymers

"Smart" surfaces that modulate their physico-chemical properties in response to environmental triggers can be fabricated by functionalization of a surface with stimuli-responsive polymers (SRPs). This article summarizes different approaches to the fabrication of "smart" surfaces and their applications. Recent results that demonstrate reversible micropatterning of proteins using a smart biopolymer as well as a simple, colorimetric assay to characterize the phase transition behavior of SRPs at the solid-water interface are highlighted.

Authors
Nath, N; Chilkoti, A
MLA Citation
Nath, N, and Chilkoti, A. "Creating "smart" surfaces using stimuli responsive polymers." Advanced Materials 14.17 (2002): 1243-1247+1181.
Source
scival
Published In
Advanced Materials
Volume
14
Issue
17
Publish Date
2002
Start Page
1243
End Page
1247+1181
DOI
10.1002/1521-4095(20020903)14:17<1243::AID-ADMA1243>3.0.CO;2-M

A colorimetric gold nanoparticle biosensor: Effect of particle size on sensitivity

We have previously developed a label-free optical method to study biomolecular interactions in real time at the surface of an optically transparent substrate. The method relies on the change in the absorbance spectrum of a self-assembled monolayer of gold nanoparticles on glass as a function of biomolecular binding at the surface of the immobilized nanoparticles. Here, we report upon optimization of this sensor by examining the effect of particle size on the analytical sensitivity of the sensor. Increasing the diameter of the gold nanoparticles from 13 nm to 47 nm is shown to increase the sensitivity of the sensor by a factor of three for the model streptavidin-biotin receptor-ligand pair.

Authors
Nath, N; Chilkoti, A
MLA Citation
Nath, N, and Chilkoti, A. "A colorimetric gold nanoparticle biosensor: Effect of particle size on sensitivity." Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings 1 (2002): 574-575.
Source
scival
Published In
Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings
Volume
1
Publish Date
2002
Start Page
574
End Page
575

Elastin-like polypeptide-doxorubicin conjugates for cancer therapy

Thermally responsive elastin-like polypeptides (ELP) were synthesized by recombinant DNA techniques and conjugated to doxorubicin through an acid labile bond. The thermal properties, release of doxorubicin, uptake of ELP, cytotoxicity and cellular localization of ELP-doxorubicin conjugates were investigated in this study.

Authors
Dreher, M; Raucher, D; Balu, N; Colvin, M; Ludeman, S; Chilkoti, A
MLA Citation
Dreher, M, Raucher, D, Balu, N, Colvin, M, Ludeman, S, and Chilkoti, A. "Elastin-like polypeptide-doxorubicin conjugates for cancer therapy." Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings 1 (2002): 524-525.
Source
scival
Published In
Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings
Volume
1
Publish Date
2002
Start Page
524
End Page
525

Molecular Recognition-Mediated Fabrication of Protein Nanostructures by Dip-Pen Lithography

We describe the molecular recognition-mediated, stepwise fabrication of patterned protein nanostructures with feature sizes on the order of 200 nm. First, a self-assembled monolayer (SAM) of 16-mercaptohexadecanoic acid (MHA) is patterned onto gold by dip-pen nanolithography (DPN), and the unpatterned regions are passivated with a protein-resistant oligoethylene glycol-terminated alkanethiol SAM. Next, an amine-terminated biotin derivative is covalently conjugated with the chemically activated MHA SAM nanopattern. The surface is then incubated with streptavidin to form streptavidin nanostructures, mediated by molecular recognition between biotin and streptavidin. Finally, protein nanopatterns are fabricated by molecular recognition-mediated immobilization of biotinylated protein from solution. Our fabrication methodology is generically applicable because of the ubiquity of biotin-tagged molecules.

Authors
Hyun, J; Ahn, SJ; Lee, WK; Chilkoti, A; Zauscher, S
MLA Citation
Hyun, J, Ahn, SJ, Lee, WK, Chilkoti, A, and Zauscher, S. "Molecular Recognition-Mediated Fabrication of Protein Nanostructures by Dip-Pen Lithography." Nano Letters 2.11 (2002): 1203-1207.
Source
scival
Published In
Nano Letters
Volume
2
Issue
11
Publish Date
2002
Start Page
1203
End Page
1207
DOI
10.1021/nl0257364

A two-step chondrocyte recovery system based on thermally sensitive elastin-like polypeptide scaffolds for cartilage tissue engineering

A "two step" tissue engineering strategy was developed to promote rapid matrix accumulation in cartilage constructs in vitro. Chondrocytes expanded in monolayer were encapsulated and cultured in a genetically engineered, thermally sensitive elastin-like polypeptide (ELP) for ten days. The resulting cell-matrix pellets were recovered from the ELP and cultured on inserts for up to four weeks, where nutrient diffusion was not impeded by the presence of the scaffold. Approximately two-milligram (dry weight) tissue was generated that resembles native articular cartilage in histological appearance and biochemical composition. These results suggest that rapid and large cartilage construct formation is possible in vitro, following a period of early incubation and recovery from the thermally responsive ELP.

Authors
Betre, H; Chilkoti, A; Setton, LA
MLA Citation
Betre, H, Chilkoti, A, and Setton, LA. "A two-step chondrocyte recovery system based on thermally sensitive elastin-like polypeptide scaffolds for cartilage tissue engineering." Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings 1 (2002): 829-830.
Source
scival
Published In
Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings
Volume
1
Publish Date
2002
Start Page
829
End Page
830

Enhanced uptake of a thermally responsive polypeptide by tumor cells in response to its hyperthermia-mediated phase transition.

Elastin-like polypeptides (ELPs) composed of a VPGXG repeat undergo a reversible phase transition in aqueous solution. They are hydrophilic and soluble in aqueous solution below their transition temperature (T(t)), but they become hydrophobic and aggregate when the temperature is raised above their T(t). In this study, we examine the quantitative uptake of a fluorescence-labeled, thermally responsive ELP as a function of ELP concentration between 5 and 15 microM in solution in response to hyperthermia by three cultured cancer cell lines. Flow cytometry of fluorescein-ELP conjugates showed that hyperthermia enhanced the cellular uptake of the thermally responsive ELP in human ovarian carcinoma cells (SKOV-3) and in HeLa cells at a concentration of 10 microM or higher, but not at a concentration of 5 microM, as compared with the uptake of a thermally inactive ELP control. In FaDu cells, hyperthermia stimulated uptake of the thermally responsive ELP at all solution concentrations of ELP between 5 and 15 microM. In particular, a >2-fold greater uptake of thermally responsive ELP compared with the thermally inactive control ELP was observed for FaDu cells at a solution concentration of 15 microM in heated cells. Confocal fluorescence microscopy of tumor cells incubated with a rhodamine conjugate of the thermally responsive ELP showed that the cytoplasm was uniformly stained below the T(t). Above the T(t), fluorescent particles were observed in the cytoplasm, suggesting that these particles are aggregates of the thermally responsive polypeptide resulting from the ELP phase transition. These studies demonstrate that the endocytotic uptake of a thermally responsive ELP is significantly enhanced by the thermally triggered phase transition of the polypeptide.

Authors
Raucher, D; Chilkoti, A
MLA Citation
Raucher, D, and Chilkoti, A. "Enhanced uptake of a thermally responsive polypeptide by tumor cells in response to its hyperthermia-mediated phase transition." Cancer Res 61.19 (October 1, 2001): 7163-7170.
PMID
11585750
Source
pubmed
Published In
Cancer Research
Volume
61
Issue
19
Publish Date
2001
Start Page
7163
End Page
7170

Interfacial phase transition of an environmentally responsive elastin biopolymer adsorbed on functionalized gold nanoparticles studied by colloidal surface plasmon resonance.

The change in optical properties of colloidal gold upon aggregation has been used to develop an experimentally convenient colorimetric method to study the interfacial phase transition of an elastin-like polypeptide (ELP), a thermally responsive biopolymer. Gold nanoparticles, functionalized with a self-assembled monolayer (SAM) of mercaptoundecanoic acid onto which an ELP was adsorbed, exhibit a characteristic red color due to the surface plasmon resonance (SPR) of individual colloids. Raising the solution temperature from 10 degrees C to 40 degrees C thermally triggered the hydrophilic-to-hydrophobic phase transition of the adsorbed ELP resulting in formation of large aggregates due to interparticle hydrophobic interaction. Formation of large aggregates caused a change in color of the colloidal suspension from red to violet due to coupling of surface plasmons in aggregated colloids. The surface phase transition of the ELP was reversible, as seen from the reversible change in color upon cooling the suspension to 10 degrees C. The formation of colloidal aggregates due to the interfacial phase transition of adsorbed ELP was independently verified by dynamic light scattering of ELP-modified gold colloids as a function of temperature. Colloidal SPR provides a simple and convenient colorimetric method to study the influence of the solution environment, interfacial properties, and grafting method on the transition properties of ELPs and other environmentally responsive polymers at the solid-water interface.

Authors
Nath, N; Chilkoti, A
MLA Citation
Nath, N, and Chilkoti, A. "Interfacial phase transition of an environmentally responsive elastin biopolymer adsorbed on functionalized gold nanoparticles studied by colloidal surface plasmon resonance." J Am Chem Soc 123.34 (August 29, 2001): 8197-8202.
PMID
11516269
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
123
Issue
34
Publish Date
2001
Start Page
8197
End Page
8202

Biomedical applications of genetically encoded elastin biopolymers.

Authors
Chilkoti, A
MLA Citation
Chilkoti, A. "Biomedical applications of genetically encoded elastin biopolymers." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 222 (August 2001): U421-U421.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
222
Publish Date
2001
Start Page
U421
End Page
U421

Micropatterning biological molecules on a polymer surface using elastomeric microwells.

Authors
Hyun, J; Chilkoti, A
MLA Citation
Hyun, J, and Chilkoti, A. "Micropatterning biological molecules on a polymer surface using elastomeric microwells." J Am Chem Soc 123.28 (July 18, 2001): 6943-6944.
PMID
11448208
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
123
Issue
28
Publish Date
2001
Start Page
6943
End Page
6944

Drug targeting using thermally responsive polymers and local hyperthermia.

We report a new thermal targeting method in which a thermally responsive drug carrier selectively accumulates in a solid tumor that is maintained above physiological temperature by externally applied, focused hyperthermia. We synthesized two thermally responsive polymers that were designed to exhibit a lower critical solution temperature (LCST) transition slightly above physiological temperature: (1) a genetically engineered elastin-like polypeptide (ELP) and (2) a copolymer of N-isopropylacrylamide (NIPAAm) and acrylamide (AAm). The delivery of systemically injected polymer-rhodamine conjugates to solid tumors was investigated by in vivo fluorescence video microscopy of ovarian tumors implanted in dorsal skin fold window chambers in nude mice, with and without local hyperthermia. When tumors were heated to 42 degrees C, the accumulation of a thermally responsive ELP with a LCST of 40 degrees C was approximately twofold greater than the concentration of the same polymer in tumors that were not heated. Similar results were also obtained for a thermally responsive poly(NIPAAM-co-AAm), though the enhanced accumulation of this carrier in heated tumors was lower than that observed for the thermally responsive ELP. These results suggest that enhanced delivery of drugs to solid tumors can be achieved by conjugation to thermally responsive polymers combined with local heating of tumors.

Authors
Meyer, DE; Shin, BC; Kong, GA; Dewhirst, MW; Chilkoti, A
MLA Citation
Meyer, DE, Shin, BC, Kong, GA, Dewhirst, MW, and Chilkoti, A. "Drug targeting using thermally responsive polymers and local hyperthermia." J Control Release 74.1-3 (July 6, 2001): 213-224.
PMID
11489497
Source
pubmed
Published In
Journal of Controlled Release
Volume
74
Issue
1-3
Publish Date
2001
Start Page
213
End Page
224

Protein purification by fusion with an environmentally responsive elastin-like polypeptide: effect of polypeptide length on the purification of thioredoxin.

Elastin-like polypeptides (ELPs) undergo a reversible, soluble-to-insoluble phase transition in aqueous solution upon heating through a characteristic transition temperature (T(t)). Incorporating a terminal ELP expression tag into the gene of a protein of interest allows ELP fusion proteins to be purified from cell lysate by cycles of environmentally triggered aggregation, separation from solution by centrifugation, and resolubilization in buffer. In this study, we examine the effect of ELP length on the expression and purification of a thioredoxin-ELP fusion protein and show that reducing the size of the ELP tag from 36 to 9 kDa increases the expression yield of thioredoxin by 4-fold, to a level comparable to that of free thioredoxin expressed without an ELP tag, while still allowing efficient purification. However, truncation of the ELP tag also results in a more complex transition behavior than is observed with larger tags. For both the 36 kDa and the 9 kDa ELP tag fused to thioredoxin, dynamic light scattering showed that large aggregates with hydrodynamic radii of approximately 2 microm form as the temperature is raised to above the T(t). These aggregates persist at all temperatures above the T(t) for the thioredoxin fusion with the 36 kDa ELP tag. With the 9 kDa tag, however, smaller particles with hydrodynamic radii of approximately 12 nm begin to form at the expense of the larger, micron-size aggregates as the temperature is further raised above the T(t). Because only large aggregates can be effectively retrieved by centrifugation, efficient purification of fusion proteins with short ELP tags requires selection of solution conditions that favor the formation of the micron-size aggregates. Despite this additional complexity, our results show that the ELP tag can be successfully truncated to enhance the yield of a target protein without compromising its purification.

Authors
Meyer, DE; Trabbic-Carlson, K; Chilkoti, A
MLA Citation
Meyer, DE, Trabbic-Carlson, K, and Chilkoti, A. "Protein purification by fusion with an environmentally responsive elastin-like polypeptide: effect of polypeptide length on the purification of thioredoxin." Biotechnol Prog 17.4 (July 2001): 720-728.
PMID
11485434
Source
pubmed
Published In
Biotechnology Progress
Volume
17
Issue
4
Publish Date
2001
Start Page
720
End Page
728
DOI
10.1021/bp010049o

Phase transition of environmentally responsive elastin biopolymers adsorbed on gold nanoparticles studied by colloidal surface plasmon resonance.

Authors
Chilkoti, A; Nath, N
MLA Citation
Chilkoti, A, and Nath, N. "Phase transition of environmentally responsive elastin biopolymers adsorbed on gold nanoparticles studied by colloidal surface plasmon resonance." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 221 (April 1, 2001): U366-U366.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
221
Publish Date
2001
Start Page
U366
End Page
U366

Targeting a genetically engineered elastin-like polypeptide to solid tumors by local hyperthermia.

Elastin-like polypeptides (ELPs) are biopolymers of the pentapeptide repeat Val-Pro-Gly-Xaa-Gly that undergo an inverse temperature phase transition. They are soluble in aqueous solutions below their transition temperature (T1) but hydrophobically collapse and aggregate at temperatures greater than T1. We hypothesized that ELPs conjugated to drugs would enable thermally targeted drug delivery to solid tumors if their T1 were between body temperature and the temperature in a locally heated region. To test this hypothesis, we synthesized a thermally responsive ELP with a T1 of 41 degrees C and a thermally unresponsive control ELP in Escherichia coli using recombinant DNA techniques. In vivo fluorescence videomicroscopy and radiolabel distribution studies of ELP delivery to human tumors (SKOV-3 ovarian carcinoma and D-54MG glioma) implanted in nude mice demonstrated that hyperthermic targeting of the thermally responsive ELP for 1 h provides a approximately 2-fold increase in tumor localization compared to the same polypeptide without hyperthermia. We observed aggregates of the thermally responsive ELP by fluorescence videomicroscopy within the heated tumor microvasculature but not in control experiments, which demonstrates that the phase transition of the thermally responsive ELP carrier can be engineered to occur in vivo at a specified temperature. By exploiting the phase transition-induced aggregation of these polypeptides, this method provides a new way to thermally target polymer-drug conjugates to solid tumors.

Authors
Meyer, DE; Kong, GA; Dewhirst, MW; Zalutsky, MR; Chilkoti, A
MLA Citation
Meyer, DE, Kong, GA, Dewhirst, MW, Zalutsky, MR, and Chilkoti, A. "Targeting a genetically engineered elastin-like polypeptide to solid tumors by local hyperthermia." Cancer Res 61.4 (February 15, 2001): 1548-1554.
PMID
11245464
Source
pubmed
Published In
Cancer Research
Volume
61
Issue
4
Publish Date
2001
Start Page
1548
End Page
1554

Enhanced TOF-SIMS imaging of a micropatterned protein by stable isotope protein labeling.

Patterning of biomolecules on surfaces is an increasingly important technological goal. Because the fabrication of biomolecule arrays often involves stepwise, spatially resolved derivatization of surfaces, spectroscopic imaging of these arrays is important in their fabrication and optimization. Although imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a powerful method for spatially resolved surface analysis, TOF-SIMS images of micropatterned proteins on organic substrates can be difficult to acquire, because of the lack of high intensity, protein-specific molecular ions that are essential for imaging under static conditions. In contrast, low-mass ions are of suitable intensity for imaging, but can originate from different chemical species on the surface. A potential solution to this problem is to utilize stable isotope labeled proteins, an approach that has heretofore not been explored in TOF-SIMS imaging of micropatterned proteins and peptides. To investigate the feasibility of stable isotope enhanced TOF-SIMS imaging of proteins, we synthesized 15N-labeled streptavidin by labeling of the protein during expression from a recombinant gene. The spatial distribution of streptavidin bound to biotin micropatterns, fabricated on a polymer and on a self-assembled monolayer on gold, was imaged by TOF-SIMS. Imaging of high-intensity, low-m/z secondary ions (e.g., C15N-) unique to streptavidin enabled unambiguous spatial mapping of the micropatterned protein with a lateral resolution of a few micrometers. TOF-SIMS imaging of micropatterned 15N-labeled streptavidin also illustrated the exquisite sensitivity of TOF-SIMS to low fractional coverage of protein (5 A effective thickness) in the background regions of the protein micropattern.

Authors
Belu, AM; Yang, Z; Aslami, R; Chilkoti, A
MLA Citation
Belu, AM, Yang, Z, Aslami, R, and Chilkoti, A. "Enhanced TOF-SIMS imaging of a micropatterned protein by stable isotope protein labeling." Anal Chem 73.2 (January 15, 2001): 143-150.
PMID
11199958
Source
pubmed
Published In
Analytical Chemistry
Volume
73
Issue
2
Publish Date
2001
Start Page
143
End Page
150

Microstamping on an activated polymer surface: Patterning biotin and streptavidin onto common polymeric biomaterials

Microstamping on an activated polymer surface (MAPS) is a methodology that enables biomolecules to be patterned on polymers with micrometer spatial resolution. MAPS combines homogeneous surface derivatization of a polymer to introduce a reactive functional group followed by reactive microcontact printing (μCP) of a biological ligand of interest, linked to an appropriate reactive group. We demonstrate here that polyethylene, polystyrene, poly(methyl methacrylate), and poly(ethylene terephthalate) films can be successfully modified to introduce COOH groups on their surfaces, which can be subsequently patterned by reactive μCP of amine-terminated biotin after derivatization of the COOH groups with pentafluorophenol. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry (TOF-SIMS) confirmed the chemistry of MAPS at each stage of the derivatization of the polymer surfaces and reactive μCP of biotin. Micropatterned biotin surfaces fabricated by MAPS were patterned with streptavidin by exploiting molecular recognition between biotin and streptavidin. The formation of streptavidin patterns was examined by fluorescence microscopy of Alexa488-labeled streptavidin and by TOF-SIMS imaging of 15N-labeled recombinant streptavidin, bound to biotin patterns. The contrast in the streptavidin micropatterns was optimized by examining the effect of blocking agents and streptavidin incubation time. Maximum contrast was obtained for binding of 0.1 μM streptavidin from a buffer containing 0.02% (v/v) Tween 20 detergent for an incubation time of 1 min.

Authors
Hyun, J; Zhu, Y; Liebmann-Vinson, A; Jr, TPB; Chilkoti, A
MLA Citation
Hyun, J, Zhu, Y, Liebmann-Vinson, A, Jr, TPB, and Chilkoti, A. "Microstamping on an activated polymer surface: Patterning biotin and streptavidin onto common polymeric biomaterials." Langmuir 17.20 (2001): 6358-6367.
Source
scival
Published In
Langmuir
Volume
17
Issue
20
Publish Date
2001
Start Page
6358
End Page
6367
DOI
10.1021/la010695x

Surface-initiated free radical polymerization of polystyrene micropatterns on a self-assembled monolayer on gold

We describe the in situ synthesis of nanometer thick films of polystyrene (PS) on a self-assembled monolayer (SAM) on gold by surface-initiated free radical polymerization and further demonstrate that three-dimensional polymer structures with micrometer lateral resolution and nanometer vertical resolution can be fabricated by combining microcontact printing (μCP) with surface-initiated polymerization (SIP). We implemented SIP onto a COOH-terminated SAM on gold using a sequential approach to couple an amine-terminated free radical initiator to the terminal COOH groups presented by the SAM, followed by free radical polymerization of styrene. Each step of SIP was characterized by X-ray photoelectron spectroscopy and ellipsometry, and the PS film and micropattern were also characterized by atomic force microscopy. We typically Obtained PS films with a thickness of 10-20 nm for a polymerization time of 12-24 h and with a root-mean-square roughness of ≤0.5 nm. PS micropatterns were also successfully fabricated by reactive μCP of the initiator onto a COOH-terminated SAM, followed by SIP of styrene. We also demonstrate two potential applications of SIP in biomaterials research: (1) label-free, real-time monitoring of protein adsorption on polymers by surface plasmon resonance (SPR) on nanometer thick homogeneous polymer films fabricated on a SAM on gold and (2) patterning cells on micropatterned polymers fabricated by combining μCP and SIP.

Authors
Hyun, J; Chilkoti, A
MLA Citation
Hyun, J, and Chilkoti, A. "Surface-initiated free radical polymerization of polystyrene micropatterns on a self-assembled monolayer on gold." Macromolecules 34.16 (2001): 5644-5652.
Source
scival
Published In
Macromolecules
Volume
34
Issue
16
Publish Date
2001
Start Page
5644
End Page
5652
DOI
10.1021/ma002125u

Really smart bioconjugates of smart polymers and receptor proteins

Over the past 18 years we have been deeply involved with the synthesis and applications of stimuli-responsive polymer systems, especially polymer-biomolecule conjugates. This article summarizes our work with one of these conjugate systems, specifically polymer-protein conjugates. We include conjugates prepared by random polymer conjugation to lysine amino groups, and also those prepared by site-specific conjugation of the polymer to specific amino acid sites that are genetically engineered into the known amino acid sequence of the protein. We describe the preparation and properties of thermally sensitive random conjugates to enzymes and several affinity recognition proteins. We have also prepared site-specific conjugates to streptavidin with temperature-sensitive polymers, pH-sensitive polymers, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed in this article. (C) 2000 John Wiley and Sons, Inc.

Authors
Hoffman, AS; Stayton, PS; Bulmus, V; Chen, G; Chen, J; Cheung, C; Chilkoti, A; Ding, Z; Dong, L; Fong, R; Lackey, CA; Long, CJ; Miura, M; Morris, JE; Murthy, N; Nabeshima, Y; Park, TG; Press, OW; Shimoboji, T; Shoemaker, S; Yang, HJ; Monji, N; Nowinski, RC; Cole, CA; Priest, JH; Harris, JM; Nakamae, K; Nishino, T; Miyata, T
MLA Citation
Hoffman, AS, Stayton, PS, Bulmus, V, Chen, G, Chen, J, Cheung, C, Chilkoti, A, Ding, Z, Dong, L, Fong, R, Lackey, CA, Long, CJ, Miura, M, Morris, JE, Murthy, N, Nabeshima, Y, Park, TG, Press, OW, Shimoboji, T, Shoemaker, S, Yang, HJ, Monji, N, Nowinski, RC, Cole, CA, Priest, JH, Harris, JM, Nakamae, K, Nishino, T, and Miyata, T. "Really smart bioconjugates of smart polymers and receptor proteins." December 1, 2000.
Source
scopus
Published In
Journal of Biomedical Materials Research
Volume
52
Issue
4
Publish Date
2000
Start Page
577
End Page
586
DOI
10.1002/1097-4636(20001215)52:4<577::AID-JBM1>3.0.CO;2-5

Microstamping of biological ligands on activated polymer surfaces.

Authors
Chilkoti, A; Yang, ZP; Belu, AM
MLA Citation
Chilkoti, A, Yang, ZP, and Belu, AM. "Microstamping of biological ligands on activated polymer surfaces." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 219 (March 26, 2000): U573-U573.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
219
Publish Date
2000
Start Page
U573
End Page
U573

Genetically engineered biopolymer conjugate for thermal targeting of anticancer therapeutics.

Authors
Chilkoti, A; Meyer, DE; Kong, GH; Dewhirst, MW; Foulon, C; Zalutsky, MR
MLA Citation
Chilkoti, A, Meyer, DE, Kong, GH, Dewhirst, MW, Foulon, C, and Zalutsky, MR. "Genetically engineered biopolymer conjugate for thermal targeting of anticancer therapeutics." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 219 (March 26, 2000): U453-U453.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
219
Publish Date
2000
Start Page
U453
End Page
U453

Molecular engineering of proteins and polymers for targeting and intracellular delivery of therapeutics.

There are many protein and DNA based therapeutics under development in the biotechnology and pharmaceutical industries. Key delivery challenges remain before many of these biomolecular therapeutics reach the clinic. Two important barriers are the effective targeting of drugs to specific tissues and cells and the subsequent intracellular delivery to appropriate cellular compartments. In this review, we summarize protein engineering work aimed at improving the stability and refolding efficiency of antibody fragments used in targeting, and at constructing new streptavidin variants which may offer improved performance in pre-targeting delivery strategies. In addition, we review recent work with pH-responsive polymers that mimic the membrane disruptive properties of viruses and toxins. These polymers could serve as alternatives to fusogenic peptides in gene therapy formulations and to enhance the intracellular delivery of protein therapeutics that function in the cytoplasm.

Authors
Stayton, PS; Hoffman, AS; Murthy, N; Lackey, C; Cheung, C; Tan, P; Klumb, LA; Chilkoti, A; Wilbur, FS; Press, OW
MLA Citation
Stayton, PS, Hoffman, AS, Murthy, N, Lackey, C, Cheung, C, Tan, P, Klumb, LA, Chilkoti, A, Wilbur, FS, and Press, OW. "Molecular engineering of proteins and polymers for targeting and intracellular delivery of therapeutics." J Control Release 65.1-2 (March 1, 2000): 203-220.
PMID
10699281
Source
pubmed
Published In
Journal of Controlled Release
Volume
65
Issue
1-2
Publish Date
2000
Start Page
203
End Page
220

Microstamping of a Biological Ligand onto an Activated Polymer Surface

Authors
Yang, Z; Chilkoti, A
MLA Citation
Yang, Z, and Chilkoti, A. "Microstamping of a Biological Ligand onto an Activated Polymer Surface." Advanced Materials 12.6 (March 2000): 413-417.
Source
crossref
Published In
Advanced Materials
Volume
12
Issue
6
Publish Date
2000
Start Page
413
End Page
417
DOI
10.1002/(SICI)1521-4095(200003)12:6<413::AID-ADMA413>3.3.CO;2-R

Engineered fusion protein of an elastin-like polypeptide and autofluorescent proteins: a genetically encodable nanobiosensor

An overview is given on the synthesis and characterization of a protein nanobiosensor produced by gene-level fusion of an elastin-like polypeptide (ELP) with green fluorescent protein (GFP) and its blue mutant (BFP). The sensor is generally encodable, and can thus be expressed within living cells as an in vivo molecular sensor.

Authors
Meyer, D; Chilkoti, A
MLA Citation
Meyer, D, and Chilkoti, A. "Engineered fusion protein of an elastin-like polypeptide and autofluorescent proteins: a genetically encodable nanobiosensor." Annals of Biomedical Engineering 28.SUPPL 1 (2000): 95-. (Academic Article)
Source
manual
Published In
Annals of Biomedical Engineering
Volume
28
Issue
SUPPL 1
Publish Date
2000
Start Page
95

Molecular imaging of a micropatterned biological ligand on an activated polymer surface

We report here molecular characterization of a new method derived from reactive microcontact printing - microstamping on an activated polymer surface (MAPS) - which enables biological ligands and proteins to be patterned on a polymer surface with a spatial resolution of at least 5 μm and good reproducibility. MAPS is a multistep procedure: first, the surface of a polymer is modified, in one or more steps, to introduce a reactive group of interest. In a subsequent step, an elastomeric stamp, inked with a biological ligand containing a complementary terminal reactive group, is brought into contact with the activated surface of the polymer. This results in spatially resolved transfer and coupling of the biological ligand to the reactive surface of the polymer. We used MAPS to pattern biotin on carboxylic acid derivatized poly(ethylene terephthalate) (PET), and subsequently with streptavidin, mediated by the high affinity streptavidin-biotin interaction. X-ray photoelectron spectroscopy of biotin-derivatized PET showed that approximately one in five PET repeat units in the top 50-100 angstroms were functionalized with biotin. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) suggested an increased concentration of PET oligomers in the top 10 angstroms due to chain scission during modification and clearly identified the derivatization of PET with biotin. TOF-SIMS imaging mapped biotin and streptavidin to the stamped regions. TOF-SIMS also imaged the spatial distribution of residual reagents from the multistep derivatization in MAPS, such as pentafluorophenol, Tween 20 surfactant, as well as poly(dimethylsiloxane) (PDMS), which was transferred from the elastomeric PDMS stamp to the surface during MAPS.

Authors
Yang, Z; Belu, AM; Liebmann-Vinson, A; Sugg, H; Chilkoti, A
MLA Citation
Yang, Z, Belu, AM, Liebmann-Vinson, A, Sugg, H, and Chilkoti, A. "Molecular imaging of a micropatterned biological ligand on an activated polymer surface." Langmuir 16.19 (2000): 7482-7492.
Source
scival
Published In
Langmuir
Volume
16
Issue
19
Publish Date
2000
Start Page
7482
End Page
7492
DOI
10.1021/la0000623

Microstamping of biological ligands on activated polymer surfaces

A new methodology, microstamping onto an activated polymer surface (MAPS), is presented. The methodology enables biological ligands and proteins to be patterned onto the surface of polymers. Proof of principle is demonstrated by micropatterning amine-terminated biotin and streptavidin onto different polymeric biomaterials, including PET, PS, PE, and PMMA.

Authors
Chilkoti, A; Hyun, J; Belu, A; Yang, Z
MLA Citation
Chilkoti, A, Hyun, J, Belu, A, and Yang, Z. "Microstamping of biological ligands on activated polymer surfaces." Annals of Biomedical Engineering 28.SUPPL 1 (2000): 92-. (Academic Article)
Source
manual
Published In
Annals of Biomedical Engineering
Volume
28
Issue
SUPPL 1
Publish Date
2000
Start Page
92

Microstamping of a biological ligand onto an activated polymer surface

A method for patterning biological ligands and proteins were patterned onto polymer surfaces is described. Called microstamping onto an activated polymer surface (MAPS), the method is based on a reaction between a microcontact-printed biological ligand and the surface of a polymer that has been derivatized to react specifically with the ligand. Using biotin ligands and polyethylene terephthalate, the method was demonstrated to yield a spatial resolution of at least 5 µm, high contrast and good reproducibility.

Authors
Yang, Z; Chilkoti, A
MLA Citation
Yang, Z, and Chilkoti, A. "Microstamping of a biological ligand onto an activated polymer surface." Advanced Materials 12.6 (2000): 413-417. (Academic Article)
Source
manual
Published In
Advanced Materials
Volume
12
Issue
6
Publish Date
2000
Start Page
413
End Page
417
DOI
10.1002/(SICI)1521-4095(200003)12:63.0.CO;2

Really smart bioconjugates of smart polymers and receptor proteins

Over the past 18 years we have been deeply involved with the synthesis and applications of stimuliresponsive polymer systems, especially polymer-biomolecule conjugates. This article summarizes our work with one of these conjugate systems, specifically polymer-protein conjugates. We include conjugates prepared by random polymer conjugation to lysine amino groups, and also those prepared by site-specific conjugation of the polymer to specific amino acid sites that are genetically engineered into the known amino acid sequence of the protein. We describe the preparation and properties of thermally sensitive random conjugates to enzymes and several affinity recognition proteins. We have also prepared site-specific conjugates to streptavidin with temperature-sensitive polymers, pH-sensitive polymers, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed in this article.

Authors
Hoffman, AS; Stayton, PS; Bulmus, V; Chen, G; Chen, J; Cheung, C; Chilkoti, A; Ding, Z; Dong, L; Fong, R; Lackey, CA; Long, CJ; Miura, M; Morris, JE; Murthy, N; al, E
MLA Citation
Hoffman, AS, Stayton, PS, Bulmus, V, Chen, G, Chen, J, Cheung, C, Chilkoti, A, Ding, Z, Dong, L, Fong, R, Lackey, CA, Long, CJ, Miura, M, Morris, JE, Murthy, N, and al, E. "Really smart bioconjugates of smart polymers and receptor proteins." Journal of Biomedical Materials Research 52.4 (2000): 577-586. (Academic Article)
Source
manual
Published In
Journal of Biomedical Materials Research
Volume
52
Issue
4
Publish Date
2000
Start Page
577
End Page
586
DOI
10.1002/1097-4636(20001215)52:43.0.CO;2-5

Ultraflat nanosphere lithography: functionalized nanostructures for biomedical applications

A new method, so-called ultraflat nanosphere lithography (UNSL), was designed to create nanopatterned surfaces of minimal topography and defined size for different materials capable of self-assembly chemistry. The feasibility of the method was demonstrated using pairs of materials M1 and M2.

Authors
Frey, W; Chilkoti, A
MLA Citation
Frey, W, and Chilkoti, A. "Ultraflat nanosphere lithography: functionalized nanostructures for biomedical applications." Annals of Biomedical Engineering 28.SUPPL 1 (2000): 94-. (Academic Article)
Source
manual
Published In
Annals of Biomedical Engineering
Volume
28
Issue
SUPPL 1
Publish Date
2000
Start Page
94

Ultraflat nanosphere lithography: a new method to fabricate flat nanostructures

Ultraflat nanopatterned surfaces can be created by combining the techniques of nanosphere lithography and ultraflat template stripping, as highlighted here. In this new method-ultraflat nanosphere lithography (UNSL)-nanostructures of one material are embedded in a matrix of a second material. The feasibility of UNSL is demonstrated for several pairs of materials

Authors
Frey, W; Woods, CK; Chilkoti, A
MLA Citation
Frey, W, Woods, CK, and Chilkoti, A. "Ultraflat nanosphere lithography: a new method to fabricate flat nanostructures." Adv. Mater. (Germany) 12.20 (2000): 1515-1519. (Academic Article)
Source
manual
Published In
Adv. Mater. (Germany)
Volume
12
Issue
20
Publish Date
2000
Start Page
1515
End Page
1519
DOI
10.1002/1521-4095(200010)12:203.0.CO;2-J

Light-activated affinity micropatterning of proteins on self-assembled monolayers on gold

We describe a method to pattern proteins onto a photolabile “caged” biotin-derivatized self-assembled monolayer (SAM) on gold, which we term light-activated affinity micropatterning of proteins (LAMP). LAMP is a multistep patterning process with considerable flexibility in its implementation. First, a reactive SAM on gold is formed from a mixture of 11-mercaptoundecanol and 16-mercaptohexadecanoic acid. Next, the carboxylic acid end groups in the SAM are coupled to methyl α-nitropiperonyloxycarbonyl biotin succinimidyl ester (caged biotin ester) through a diamine linker. The caged biotin is then deprotected in regions irradiated by masked UV light, and subsequent incubation with streptavidin results in selective binding of streptavidin to the irradiated regions. Micropatterning of various proteins has been demonstrated with a spatial resolution of ~6 μm by confocal microscopic imaging of fluorophore-labeled proteins, and a contrast ratio of ~4:1 was determined by direct ellipsometric imaging of streptavidin. Immobilization of biotinylated antibodies on the streptavidin pattern indicates that LAMP can enable spatially resolved micropatterning of different biomolecules by repeated cycles of spatially defined photodeprotection of biotin, streptavidin incubation, followed by immobilization of the biotinylated moiety of interest

Authors
Yang, Z; Frey, W; Oliver, T; Chilkoti, A
MLA Citation
Yang, Z, Frey, W, Oliver, T, and Chilkoti, A. "Light-activated affinity micropatterning of proteins on self-assembled monolayers on gold." Langmuir (USA) 16.4 (2000): 1751-1758. (Academic Article)
Source
manual
Published In
Langmuir (USA)
Volume
16
Issue
4
Publish Date
2000
Start Page
1751
End Page
1758
DOI
10.1021/la998079

Genetically engineered polypeptide carrier for thermal targeting of anticancer therapeutics

The thermally-responsive carrier, an elastin-like polypeptide (ELP), is an oligomer of a Val-Pro-Gly-Xaa-Gly pentapeptide repeat. Below a specific, inverse phase transition temperature (Tt), ELPs are highly soluble in aqueous solution. However, upon raising the Tt, they undergo a reversible transition resulting in desolvation and aggregation. Control of the amino acid sequence and the molecular weight of the carrier by genetically encodable synthesis enables precise molecular design of ELPs with an inverse transition of -40 °C. ELP was studied whether it will remain soluble systematically but will accumulate at sites of externally-induced local hyperthermia where the temperature is raised above Tt.

Authors
Meyer, DE; Kong, GA; Dewhirst, MW; Foulon, C; Zalutsky, MR; Chilkoti, A
MLA Citation
Meyer, DE, Kong, GA, Dewhirst, MW, Foulon, C, Zalutsky, MR, and Chilkoti, A. "Genetically engineered polypeptide carrier for thermal targeting of anticancer therapeutics." American Chemical Society, Polymer Preprints, Division of Polymer Chemistry 41.1 (2000): 1020-1021.
Source
scival
Published In
American Chemical Society, Polymer Preprints, Division of Polymer Chemistry
Volume
41
Issue
1
Publish Date
2000
Start Page
1020
End Page
1021

Microstamping of biological ligands on activated polymer surfaces

A new methodology, microstamping onto an activated polymer surface (MAPS), is presented. The methodology enables biological ligands and proteins to be patterned onto the surface of polymers. Proof of principle is demonstrated by micropatterning amine-terminated biotin and streptavidin onto different polymeric biomaterials, including PET, PS, PE, and PMMA.

Authors
Chilkoti, A; Hyun, J; Belu, A; Yang, Z
MLA Citation
Chilkoti, A, Hyun, J, Belu, A, and Yang, Z. "Microstamping of biological ligands on activated polymer surfaces." Annals of Biomedical Engineering 28.SUPPL. 1 (2000): S-92.
Source
scival
Published In
Annals of Biomedical Engineering
Volume
28
Issue
SUPPL. 1
Publish Date
2000
Start Page
S
End Page
92

Ultraflat nanosphere lithography: functionalized nanostructures for biomedical applications

A new method, so-called ultraflat nanosphere lithography (UNSL), was designed to create nanopatterned surfaces of minimal topography and defined size for different materials capable of self-assembly chemistry. The feasibility of the method was demonstrated using pairs of materials M1 and M2.

Authors
Frey, W; Chilkoti, A
MLA Citation
Frey, W, and Chilkoti, A. "Ultraflat nanosphere lithography: functionalized nanostructures for biomedical applications." Annals of Biomedical Engineering 28.SUPPL. 1 (2000): S-94.
Source
scival
Published In
Annals of Biomedical Engineering
Volume
28
Issue
SUPPL. 1
Publish Date
2000
Start Page
S
End Page
94

Light-activated affinity micropatterning of proteins on self-assembled monolayers on gold

We describe a method to pattern proteins onto a photolabile 'caged' biotin-derivatized self-assembled monolayer (SAM) on gold, which we term light-activated affinity micropatterning of proteins (LAMP). LAMP is a multistep patterning process with considerable flexibility in its implementation. First, a reactive SAM on gold is formed from a mixture of 11-mercaptoundecanol and 16-mercaptohexadecanoic acid. Next, the carboxylic acid end groups in the SAM are coupled to methyl α-nitropiperonyloxycarbonyl biotin succinimidyl ester (caged biotin ester) through a diamine linker. The caged biotin is then deprotected in regions irradiated by masked UV light, and subsequent incubation with streptavidin results in selective binding of streptavidin to the irradiated regions. Micropatterning of various proteins has been demonstrated with a spatial resolution of approx. 6 μm by confocal microscopic imaging of fluorophore-labeled proteins, and a contrast ratio of approx. 4:1 was determined by direct ellipsometric imaging of streptavidin. Immobilization of biotinylated antibodies on the streptavidin pattern indicates that LAMP can enable spatially resolved micropatterning of different biomolecules by repeated cycles of spatially defined photodeprotection of biotin, streptavidin incubation, followed by immobilization of the biotinylated moiety of interest.

Authors
Yang, Z; Frey, W; Oliver, T; Chilkoti, A
MLA Citation
Yang, Z, Frey, W, Oliver, T, and Chilkoti, A. "Light-activated affinity micropatterning of proteins on self-assembled monolayers on gold." Langmuir 16.4 (2000): 1751-1758.
Source
scival
Published In
Langmuir
Volume
16
Issue
4
Publish Date
2000
Start Page
1751
End Page
1758
DOI
10.1021/la9908079

Engineered fusion protein of an elastin-like polypeptide and autofluorescent proteins: a genetically encodable nanobiosensor

An overview is given on the synthesis and characterization of a protein nanobiosensor produced by gene-level fusion of an elastin-like polypeptide (ELP) with green fluorescent protein (GFP) and its blue mutant (BFP). The sensor is generally encodable, and can thus be expressed within living cells as an in vivo molecular sensor.

Authors
Meyer, D; Chilkoti, A
MLA Citation
Meyer, D, and Chilkoti, A. "Engineered fusion protein of an elastin-like polypeptide and autofluorescent proteins: a genetically encodable nanobiosensor." Annals of Biomedical Engineering 28.SUPPL. 1 (2000): S-95.
Source
scival
Published In
Annals of Biomedical Engineering
Volume
28
Issue
SUPPL. 1
Publish Date
2000
Start Page
S
End Page
95

Elastin-like polypeptides as thermally targeted drug carriers

Here, we present the tissue distribution of radiolabeled ELP carriers that were injected into athymic mice with implanted tumors (human glioma D54MG). We observed a two-fold increase in ELP accumulation when the tumor was heated versus unheated animals. Stringent controls show that the observed enhancement is largely caused by the phase transition of the ELP carrier. Ongoing optimization studies seek to determine the effect of ELP MW, concentration, and administration protocol on targeting efficiency.

Authors
Meyer, D; Chilkoti, A
MLA Citation
Meyer, D, and Chilkoti, A. "Elastin-like polypeptides as thermally targeted drug carriers." Annals of Biomedical Engineering 28.SUPPL. 1 (2000): S-20.
Source
scival
Published In
Annals of Biomedical Engineering
Volume
28
Issue
SUPPL. 1
Publish Date
2000
Start Page
S
End Page
20

Microstamping of a biological ligand onto an activated polymer surface

A method for patterning biological ligands and proteins were patterned onto polymer surfaces is described. Called microstamping onto an activated polymer surface (MAPS), the method is based on a reaction between a microcontact-printed biological ligand and the surface of a polymer that has been derivatized to react specifically with the ligand. Using biotin ligands and polyethylene terephthalate, the method was demonstrated to yield a spatial resolution of at least 5 μm, high contrast and good reproducibility.

Authors
Yang, Z; Chilkoti, A
MLA Citation
Yang, Z, and Chilkoti, A. "Microstamping of a biological ligand onto an activated polymer surface." Advanced Materials 12.6 (2000): 413-417.
Source
scival
Published In
Advanced Materials
Volume
12
Issue
6
Publish Date
2000
Start Page
413
End Page
417
DOI
10.1002/(SICI)1521-4095(200003)12:6<413::AID-ADMA413>3.0.CO;2-#

Really smart bioconjugates of smart polymers and receptor proteins

Over the past 18 years we have been deeply involved with the synthesis and applications of stimuli-responsive polymer systems, especially polymer-biomolecule conjugates. This article summarizes our work with one of these conjugate systems, specifically polymer-protein conjugates. We include conjugates prepared by random polymer conjugation to lysine amino groups, and also those prepared by site-specific conjugation of the polymer to specific amino acid sites that are genetically engineered into the known amino acid sequence of the protein. We describe the preparation and properties of thermally sensitive random conjugates to enzymes and several affinity recognition proteins. We have also prepared site-specific conjugates to streptavidin with temperature-sensitive polymers, pH-sensitive polymers, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed in this article. (C) 2000 John Wiley and Sons, Inc.

Authors
Hoffman, AS; Stayton, PS; Bulmus, V; Chen, G; Chen, J; Cheung, C; Chilkoti, A; Ding, Z; Dong, L; Fong, R; Lackey, CA; Long, CJ; Miura, M; Morris, JE; Murthy, N; Nabeshima, Y; Park, TG; Press, OW; Shimoboji, T; Shoemaker, S; Yang, HJ; Monji, N; Nowinski, RC; Cole, CA; Priest, JH; Harris, JM; Nakamae, K; Nishino, T; Miyata, T
MLA Citation
Hoffman, AS, Stayton, PS, Bulmus, V, Chen, G, Chen, J, Cheung, C, Chilkoti, A, Ding, Z, Dong, L, Fong, R, Lackey, CA, Long, CJ, Miura, M, Morris, JE, Murthy, N, Nabeshima, Y, Park, TG, Press, OW, Shimoboji, T, Shoemaker, S, Yang, HJ, Monji, N, Nowinski, RC, Cole, CA, Priest, JH, Harris, JM, Nakamae, K, Nishino, T, and Miyata, T. "Really smart bioconjugates of smart polymers and receptor proteins." Journal of Biomedical Materials Research 52.4 (2000): 577-586.
PMID
11033539
Source
scival
Published In
Journal of Biomedical Materials Research
Volume
52
Issue
4
Publish Date
2000
Start Page
577
End Page
586
DOI
10.1002/1097-4636(20001215)52:4<577::AID-JBM1>3.0.CO;2-5

Ultraflat nanosphere lithography: A new method to fabricate flat nanostructures

The ultraflat nanosphere lithography (UNSL) was demonstrated by developing the 150 nanometer features of gold, silver and aluminum. The metals were embedded in a matrix with topographical variations between patterned features and matrix. UNSL combined nanosphere lithography and ultraflat template stripping for the creation of ultraflat nanopatterned structures. The nanospheres were lifted off the surface by rinsing and sonication in a mixture of water and methanol. The results established UNSL as a useful technique for the fabrication of chemically distinct nanoarrays with minimal topographic variation.

Authors
Frey, W; Woods, CK; Chilkoti, A
MLA Citation
Frey, W, Woods, CK, and Chilkoti, A. "Ultraflat nanosphere lithography: A new method to fabricate flat nanostructures." Advanced Materials 12.20 (2000): 1515-1519.
Source
scival
Published In
Advanced Materials
Volume
12
Issue
20
Publish Date
2000
Start Page
1515
End Page
1519
DOI
10.1002/1521-4095(200010)12:20<1515::AID-ADMA1515>3.0.CO;2-J

Streptavidin-biotin binding energetics.

The high affinity energetics in the streptavidin-biotin system provide an excellent model system for studying how proteins balance enthalpic and entropic components to generate an impressive overall free energy for ligand binding. We review here concerted site-directed mutagenesis, biophysical, and computational studies of aromatic and hydrogen bonding interaction energetics between streptavidin and biotin. These results also have provided insight into how streptavidin builds a large activation barrier to dissociation by managing the enthalpic and entropic activation components. Finally, we review recent studies of the biotin dissociation pathway that address the fundamental question of how ligands exit protein binding pockets.

Authors
Stayton, PS; Freitag, S; Klumb, LA; Chilkoti, A; Chu, V; Penzotti, JE; To, R; Hyre, D; Le Trong, I; Lybrand, TP; Stenkamp, RE
MLA Citation
Stayton, PS, Freitag, S, Klumb, LA, Chilkoti, A, Chu, V, Penzotti, JE, To, R, Hyre, D, Le Trong, I, Lybrand, TP, and Stenkamp, RE. "Streptavidin-biotin binding energetics." Biomol Eng 16.1-4 (December 31, 1999): 39-44. (Review)
PMID
10796983
Source
pubmed
Published In
Biomolecular Engineering
Volume
16
Issue
1-4
Publish Date
1999
Start Page
39
End Page
44

X-ray crystallographic studies of streptavidin mutants binding to biotin.

On the basis of high resolution crystallographic studies of streptavidin and its biotin complex, three principal binding motifs have been identified that contribute to the tight binding. A flexible binding loop can undergo a conformational change from an open to a closed form when biotin is bound. Additional studies described here of unbound wild-type streptavidin have provided structural views of the open conformation. Several tryptophan residues packing around the bound biotin constitute the second binding motif, one dominated by hydrophobic interactions. Mutation of these residues to alanine or phenylalanine have variable effects on the thermodynamics and kinetics of binding, but they generate only small changes in the molecular structure. Hydrogen bonding interactions also contribute significantly to the binding energetics of biotin, and the D128A mutation which breaks a hydrogen bond between the protein and a ureido NH group results in a significant structural alteration that could mimic an intermediate on the dissociation pathway. In this review, we summarize the structural aspects of biotin recognition that have been gained from crystallographic analyses of wild-type and site-directed streptavidin mutants.

Authors
Freitag, S; Le Trong, I; Klumb, LA; Chu, V; Chilkoti, A; Stayton, PS; Stenkamp, RE
MLA Citation
Freitag, S, Le Trong, I, Klumb, LA, Chu, V, Chilkoti, A, Stayton, PS, and Stenkamp, RE. "X-ray crystallographic studies of streptavidin mutants binding to biotin." Biomol Eng 16.1-4 (December 31, 1999): 13-19.
PMID
10796980
Source
pubmed
Published In
Biomolecular Engineering
Volume
16
Issue
1-4
Publish Date
1999
Start Page
13
End Page
19

Direct force measurements of the streptavidin-biotin interaction.

The interaction between streptavidin and its ligand, biotin, were studied by direct force measurements. The complimentary approaches of surface force apparatus (SFA) and atomic force microscopy (AFM) were used to elucidate both long-range and short-range adhesive interactions of the streptavidin biotin interaction. The high spatial resolution of the SFA provided a detailed profile of the intersurface forces of apposing surfaces functionalized with streptavidin and biotin. Measurements obtained by the SFA corresponded to long and intermediate-range forces that are important in determining ligand receptor association. AFM was used to measure the unbinding force of individual streptavidin biotin complexes. These measurements revealed the short-range interactions (i.e. hydrophobic and hydrogen bonding forces) that stabilize the intermolecular bond.

Authors
Wong, J; Chilkoti, A; Moy, VT
MLA Citation
Wong, J, Chilkoti, A, and Moy, VT. "Direct force measurements of the streptavidin-biotin interaction." Biomol Eng 16.1-4 (December 31, 1999): 45-55.
PMID
10796984
Source
pubmed
Published In
Biomolecular Engineering
Volume
16
Issue
1-4
Publish Date
1999
Start Page
45
End Page
55

Purification of recombinant proteins by fusion with thermally-responsive polypeptides.

Elastin-like polypeptides (ELPs) undergo a reversible, inverse phase transition. Below their transition temperature (Tt), ELPs are soluble in water, but when the temperature is raised above Tt, phase transition occurs, leading to aggregation of the polypeptide. We demonstrate a method for purification of soluble fusion proteins incorporating an ELP tag. Advantages of this method, termed "inverse transition cycling," include technical simplicity, low cost, ease of scale-up, and capacity for multiplexing. More broadly, the ability to environmentally modulate the physicochemical properties of recombinant proteins by fusion with ELPs will allow diverse applications in bioseparation, immunoassays, biocatalysis, and drug delivery.

Authors
Meyer, DE; Chilkoti, A
MLA Citation
Meyer, DE, and Chilkoti, A. "Purification of recombinant proteins by fusion with thermally-responsive polypeptides." Nat Biotechnol 17.11 (November 1999): 1112-1115.
PMID
10545920
Source
pubmed
Published In
Nature Biotechnology
Volume
17
Issue
11
Publish Date
1999
Start Page
1112
End Page
1115
DOI
10.1038/15100

Polypeptide fusion tag for thermal purification of recombinant proteins.

Authors
Meyer, DE; Chilkoti, A
MLA Citation
Meyer, DE, and Chilkoti, A. "Polypeptide fusion tag for thermal purification of recombinant proteins." ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 217 (March 21, 1999): U177-U177.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
217
Publish Date
1999
Start Page
U177
End Page
U177

Controlling the orientation of immobilized proteins by genetic circular permutation

Controlling the orientation of immobilized proteins is critical to their surface activity. However, most currently used protein immobilization strategies result in a wide range of surface orientations. Because termini are ubiquitous to proteins, we have investigated their use as unique sites for immobilization to create homogeneously oriented protein surfaces with optimized activity. We demonstrate proof-of-concept, for a model protein tendamistat, by immobilizing it on a surface specifically by its N-terminal amine by: expression as a C-terminal fusion to thioredoxin; blocking accessible lysine residues; liberation of the N-terminal amine by proteolytic cleavage of the fusion protein; and immobilization to a carboxylic acid-functionalized surface by tendamistat's N-terminal amine. Protein immobilization by native N or C-terminus, however, of limited utility because the orientation specified by the location of the native termini may be inappropriate for a target application. This limitation can be circumvented by using genetic circular permutation to reposition the termini on the protein scaffold. In studies to date, we have shown that controlling the location of the N-terminus of tendamistat by circular permutation, and consequently the site of immobilization, has a dramatic effect on its surface activity. We are currently extending this approach to incorporate genetically-encoded C-terminal peptide tags in permuted proteins, and complementarily, by exploiting enzyme-assisted reverse proteolysis to immobilize permuted proteins by their engineered C-termini.

Authors
Piervincenzi, RT; Chilkoti, A
MLA Citation
Piervincenzi, RT, and Chilkoti, A. "Controlling the orientation of immobilized proteins by genetic circular permutation." Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings 1 (1999): 93--.
Source
scival
Published In
Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings
Volume
1
Publish Date
1999
Start Page
93-

Light-activated affinity micropatterning of proteins

Protein micropatterning has attracted considerable interest because of its prospective application in the fabrication of biosensors and tissue engineering substrates. Motivated by these potential applications, we have developed a method to micropattern proteins onto self-assembled monolayers (SAMs) on gold, which we term light-activated affinity micropatterning of proteins (LAMP). LAMP is a multi-step patterning process: first, a gold substrate is modified with a mixture of 11-mercapto-undecanol and 16-mercaptohexadecanoic acid to provide a non-fouling, reactive SAM template on gold. Next, the carboxylic acid terminal groups in the binary SAM are coupled to methylnitropiperonyloxy-carbonyl biotin, (`caged' biotin) through a diamine linker, resulting in a mixed MeNPOC-biotinyl/OH-terminated monolayer. Activation of the caged biotin by spatially-defined UV illumination at 350-360 nm reconstitutes biotin in the illuminated region, allowing streptavidin or anti-biotin antibody to be localized in the illuminated regions. We have investigated each fabrication step in LAMP by a variety of surface analytical techniques, including contact angle goniometry, ellipsometry, surface plasmon resonance, and X-ray photoelectron spectroscopy to optimize ligand density and pattern contrast. LAMP can be further extended to spatially-resolved micropatterning of multiple biomolecules by repeated cycles of spatially-defined activation, streptavidin incubation, followed by binding of the biotinylated moiety of interest.

Authors
Yang, ZP; Chilkoti, A
MLA Citation
Yang, ZP, and Chilkoti, A. "Light-activated affinity micropatterning of proteins." Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings 2 (1999): 738--.
Source
scival
Published In
Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings
Volume
2
Publish Date
1999
Start Page
738-

Genetically-engineered polypeptide carrier for thermal targeting of therapeutics

We report the synthesis of a environmentally-responsive polypeptide carrier for thermally-targeted delivery of radionuclide therapeutics. We hypothesized that systemically-injected radionuclide-polypeptide conjugates are likely to be cleared from circulation under physiological conditions, while externally-induced local hyperthermia at tumor sites will result in radionuclide accumulation because of carrier aggregation. We report preliminary in vivo results which support this hypothesis.

Authors
Meyer, DE; Kong, G; Dewhirst, M; Chilkoti, A
MLA Citation
Meyer, DE, Kong, G, Dewhirst, M, and Chilkoti, A. "Genetically-engineered polypeptide carrier for thermal targeting of therapeutics." Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings 2 (1999): 1289--.
Source
scival
Published In
Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings
Volume
2
Publish Date
1999
Start Page
1289-

Structural studies of binding site tryptophan mutants in the high-affinity streptavidin-biotin complex.

Previous thermodynamic and computational studies have pointed to the important energetic role of aromatic contacts in generating the exceptional binding free energy of streptavidin-biotin association. We report here the crystallographic characterization of single site tryptophan mutants in investigating structural consequences of alterations in these aromatic contacts. Four tryptophan residues, Trp79, Trp92, Trp108 and Trp120, play an important role in the hydrophobic binding contributions, which along with a hydrogen bonding network and a flexible binding loop give rise to tight ligand binding (Ka approximately 10(13) M-1). The crystal structures of ligand-free and biotin-bound mutants, W79F, W108F, W120F and W120A, in the resolution range from 1.9 to 2.3 A were determined. Nine data sets for these four different mutants were collected, and structural models were refined to R-values ranging from 0.15 to 0.20. The major question addressed here is how these mutations influence the streptavidin binding site and in particular how they affect the binding mode of biotin in the complex. The overall folding of streptavidin was not significantly altered in any of the tryptophan mutants. With one exception, only minor deviations in the unbound structures were observed. In one crystal form of unbound W79F, there is a coupled shift in the side-chains of Phe29 and Tyr43 toward the mutation site, although in a different crystal form these shifts are not observed. In the bound structures, the orientation of biotin in the binding pocket was not significantly altered in the mutant complex. Compared with the wild-type streptavidin-biotin complex, there were no additional crystallographic water molecules observed for any of the mutants in the binding pocket. These structural studies thus suggest that the thermodynamic alterations can be attributed to the local alterations in binding residue composition, rather than a rearrangement of binding site architectures.

Authors
Freitag, S; Le Trong, I; Chilkoti, A; Klumb, LA; Stayton, PS; Stenkamp, RE
MLA Citation
Freitag, S, Le Trong, I, Chilkoti, A, Klumb, LA, Stayton, PS, and Stenkamp, RE. "Structural studies of binding site tryptophan mutants in the high-affinity streptavidin-biotin complex." J Mol Biol 279.1 (May 29, 1998): 211-221.
PMID
9636711
Source
pubmed
Published In
Journal of Molecular Biology
Volume
279
Issue
1
Publish Date
1998
Start Page
211
End Page
221
DOI
10.1006/jmbi.1998.1735

Conjugates of Stimuli-Responsive Polymers and Biomolecules: Random and Site-Specific Conjugates of Temperature-Sensitive Polymers and Proteins

Intelligent polymers exhibit sharp, reversible phase changes in response to small changes in environmental conditions. For example, a small temperature change can cause a sharp precipitation or gelation of a smart polymer solution. Conjugation of these unusual polymers to biomolecules such as enzymes, ligands, lipids, and drugs can lead to many new and exciting applications in medicine and biotechnology. (1-4) This presentation reviews the principles, methodolgies and applications of these "smart" polymer-biomolecule systems, with special focus on temperature-sensitive polymer-protein conjugates.

Authors
Hoffman, AS; Stayton, PS; Shimoboji, T; Chen, G; Ding, Z; Chilkoti, A; Long, C; Miura, M; Chen, J; Park, T; Monji, N; Cole, C-A; Harris, JM; Nakamae, K
MLA Citation
Hoffman, AS, Stayton, PS, Shimoboji, T, Chen, G, Ding, Z, Chilkoti, A, Long, C, Miura, M, Chen, J, Park, T, Monji, N, Cole, C-A, Harris, JM, and Nakamae, K. "Conjugates of Stimuli-Responsive Polymers and Biomolecules: Random and Site-Specific Conjugates of Temperature-Sensitive Polymers and Proteins." Macromolecular Symposia 118 (1997): 553-563.
Source
scival
Published In
Macromolecular Symposia
Volume
118
Publish Date
1997
Start Page
553
End Page
563

Two-dimensional protein crystallization via metal-ion coordination by naturally occurring surface histidines.

A powerful and potentially general approach to the targeting and crystallization of proteins on lipid interfaces through coordination of surface histidine residues to lipid-chelated divalent metal ions is presented. This approach, which should be applicable to the crystallization of a wide range of naturally occurring or engineered proteins, is illustrated here by the crystallization of streptavidin on a monolayer of an iminodiacetate-Cu(II) lipid spread at the air-water interface. This method allows control of the protein orientation at interfaces, which is significant for the facile production of highly ordered protein arrays and for electron density mapping in structural analysis of two-dimensional crystals. Binding of native streptavidin to the iminodiacetate-Cu lipids occurs via His-87, located on the protein surface near the biotin binding pocket. The two-dimensional streptavidin crystals show a previously undescribed microscopic shape that differs from that of crystals formed beneath biotinylated lipids.

Authors
Frey, W; Schief, WR; Pack, DW; Chen, CT; Chilkoti, A; Stayton, P; Vogel, V; Arnold, FH
MLA Citation
Frey, W, Schief, WR, Pack, DW, Chen, CT, Chilkoti, A, Stayton, P, Vogel, V, and Arnold, FH. "Two-dimensional protein crystallization via metal-ion coordination by naturally occurring surface histidines." Proc Natl Acad Sci U S A 93.10 (May 14, 1996): 4937-4941.
PMID
8643507
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
93
Issue
10
Publish Date
1996
Start Page
4937
End Page
4941

Site-specific conjugation of a temperature-sensitive polymer to a genetically-engineered protein

A novel method for sensitive environmental control of the molecular recognition process between a protein receptor and its ligand is presented. The method is based on the conjugation of a smart, temperature-sensitive polymer, poly(N-isopropylacrylamide), to a specific genetically-engineered site, thiol. It was shown that the site-specific conjugation of a temperature-sensitive polymer to a genetically engineered protein provides a very sensitive temperature control of the ligand-protein recognition process. Such molecular switches could have widespread applications in medicine and biotechnology.

Authors
Shimoboji, T; Stayton, PS; Long, C; Chilkoti, A; Chen, G; Harris, JM; Hoffman, AS
MLA Citation
Shimoboji, T, Stayton, PS, Long, C, Chilkoti, A, Chen, G, Harris, JM, and Hoffman, AS. "Site-specific conjugation of a temperature-sensitive polymer to a genetically-engineered protein." Transactions of the Annual Meeting of the Society for Biomaterials in conjunction with the International Biomaterials Symposium 1 (1996): 897--.
Source
scival
Published In
Transactions of the Annual Meeting of the Society for Biomaterials in conjunction with the International Biomaterials Symposium
Volume
1
Publish Date
1996
Start Page
897-

Control of protein-ligand recognition using a stimuli-responsive polymer.

Stimuli-responsive polymers exhibit reversible phase changes in response to changes in environmental factors such as pH or temperature. Conjugating such polymers to antibodies and proteins provides molecular systems for applications such as affinity separations, immunoassays and enzyme recovery and recycling. Here we show that conjugating a temperature-sensitive polymer to a genetically engineered site on a protein allows the protein's ligand binding affinity to be controlled. We synthesized a mutant of the protein streptavidin to enable site-specific conjugation of the responsive polymer near the protein's binding site. Normal binding of biotin to the modified protein occurs below 32 degrees C, whereas above this temperature the polymer collapses and blocks binding. The collapse of the polymer and thus the enabling and disabling of binding, is reversible. Such environmentally triggered control of binding may find many applications in biotechnology and biomedicine, such as the control of enzyme reaction rates and of biosensor activity, and the controlled release of drugs.

Authors
Stayton, PS; Shimoboji, T; Long, C; Chilkoti, A; Chen, G; Harris, JM; Hoffman, AS
MLA Citation
Stayton, PS, Shimoboji, T, Long, C, Chilkoti, A, Chen, G, Harris, JM, and Hoffman, AS. "Control of protein-ligand recognition using a stimuli-responsive polymer." Nature 378.6556 (November 30, 1995): 472-474.
PMID
7477401
Source
pubmed
Published In
Nature
Volume
378
Issue
6556
Publish Date
1995
Start Page
472
End Page
474
DOI
10.1038/378472a0

The relationship between ligand-binding thermodynamics and protein-ligand interaction forces measured by atomic force microscopy.

The interaction forces between biotin and a set of streptavidin site-directed mutants with altered biotin-binding equilibrium and activation thermodynamics have been measured by atomic force microscopy. The AFM technique readily discriminates differences in interaction force between the site-directed (Trp to Phe or Ala) mutants. The interaction force is poorly correlated with both the equilibrium free energy of biotin binding and the activation free energy barrier to dissociation of the biotin-streptavidin complex. The interaction force is generally well correlated with the equilibrium biotin-binding enthalpy as well as the enthalpic activation barrier, but in the one mutant where these two parameters are altered in opposite directions, the interaction force is clearly correlated with the activation enthalpy of dissociation. These results suggest that the AFM force measurements directly probe the enthalpic activation barrier to ligand dissociation.

Authors
Chilkoti, A; Boland, T; Ratner, BD; Stayton, PS
MLA Citation
Chilkoti, A, Boland, T, Ratner, BD, and Stayton, PS. "The relationship between ligand-binding thermodynamics and protein-ligand interaction forces measured by atomic force microscopy." Biophys J 69.5 (November 1995): 2125-2130.
PMID
8580356
Source
pubmed
Published In
Biophysical Journal
Volume
69
Issue
5
Publish Date
1995
Start Page
2125
End Page
2130
DOI
10.1016/S0006-3495(95)80083-4

Engineered chimeric streptavidin tetramers as novel tools for bioseparations and drug delivery.

We report the construction of chimeric streptavidin tetramers that are composed of subunits of both wild-type (WT) streptavidin and genetically-engineered streptavidin variants designed for enhanced bioseparation and drug delivery performance. Subunit mixing is accomplished by guanidine thiocyanateinduced denaturation of an equimolar mixture of WT streptavidin and the respective site-directed mutant, followed by renaturation and reassociation of mixed tetramers. In the first example, we demonstrate the mixing of WT subunits with an Asn49Cys (N49C) mutant. The WT/n49C tetramers can be used for site-specific and stoichiometric attachment of therapeutics/imaging agents or targeting proteins through the genetically-engineered thiol while retaining unhindered access to biotin-binding at the WT subunits. Second, we demonstrate that the His127Cys mutation (H127C) results in a streptavidin mutant that forms a disulfide-linked dimer under non-reducing conditions. Mixing of H127C and WT streptavidin subunits results in chimeric tetramers where both the stoichiometry (WT:H127C::1:1) and subunit architecture is controlled by the unique disulfide bridge engineered into H127C. In the third example, WT subunits were mixed with the subunits of a site-directed mutant, Trp120Ala (W120A), which displays a biotin dissociation constant that is enhanced by more than 10(4) compared to WT streptavidin. The W120 biotin-binding affinity is sufficiently high (Ka approximately equal to 10(7) M-1) to immobilize the mutant on a biotinagarose affinity chromatography column, but the engineered off-rate allows for facile elution with excess biotin at physiological pH, whereas WT streptavidin is irreversibly immobilized on the column. We demonstrate that the purified WT/W120A chimeric tetramers combine the advantages of both subunits, allowing for irreversible immobilization of biotinylated targets at the WT subunit, while retaining the reversible separation capabilities of the W120A subunits via biotin-agarose affinity chromatography.

Authors
Chilkoti, A; Schwartz, BL; Smith, RD; Long, CJ; Stayton, PS
MLA Citation
Chilkoti, A, Schwartz, BL, Smith, RD, Long, CJ, and Stayton, PS. "Engineered chimeric streptavidin tetramers as novel tools for bioseparations and drug delivery." Biotechnology (N Y) 13.11 (November 1995): 1198-1204.
PMID
9636292
Source
pubmed
Published In
Biotechnology
Volume
13
Issue
11
Publish Date
1995
Start Page
1198
End Page
1204

Investigating the relationship between surface chemistry and endothelial cell growth: partial least-squares regression of the static secondary ion mass spectra of oxygen-containing plasma-deposited films.

The relationship between endothelial cell growth and surface properties of plasma-deposited films (PDFs) was investigated using partial least-squares regression (PLS). PDFs of oxygen-containing precursors were prepared under various conditions, and bovine arterial endothelial cells (BAECs) were grown on these substrates. Secondary ion mass spectrometry (SIMS) in the static mode was used to characterize the surface chemistry of these substrates. The growth of BAECs on the PDFs was correlated to the positive and negative static SIMS spectra of the PDFs by PLS. A good correlation between the SIMS spectra of PDFs and endothelial cell growth was obtained. Qualitative information was also extracted from the multivariate model, giving some information as to the most important variables influencing BAEC growth.

Authors
Chilkoti, A; Schmierer, AE; Pérez-Luna, VH; Ratner, BD
MLA Citation
Chilkoti, A, Schmierer, AE, Pérez-Luna, VH, and Ratner, BD. "Investigating the relationship between surface chemistry and endothelial cell growth: partial least-squares regression of the static secondary ion mass spectra of oxygen-containing plasma-deposited films." Anal Chem 67.17 (September 1, 1995): 2883-2891.
PMID
8779415
Source
pubmed
Published In
Analytical Chemistry
Volume
67
Issue
17
Publish Date
1995
Start Page
2883
End Page
2891

Site-directed mutagenesis studies of the high-affinity streptavidin-biotin complex: contributions of tryptophan residues 79, 108, and 120.

We report the functional characterization of site-directed biotin binding-site mutants of recombinant core streptavidin. The mutagenesis studies were aimed at characterizing the contributions of Trp residues known to contact biotin that have been postulated to control the exceptional binding affinity observed in this system. The functional properties of single site-directed mutants replacing Trp residues with Phe or Ala at positions 79, 108, and 120 were investigated by quantitating the EC50 binding parameters of these mutants to biotin and 2-iminobiotin in an ELISA format. The biotin EC50 for all mutants was the same as wild-type streptavidin, demonstrating that their delta Ka values relative to wild type were < 10(6). The conservative W79F and W108F mutants displayed only a 2- to 3-fold increase in EC50 for 2-iminobiotin, corresponding to an estimated delta Ka < 10, while the W120F mutant displayed a much greater alteration in 2-iminobiotin EC50, corresponding to an estimated delta Ka of 10(2). These delta Ka values are likely to reflect similar changes for biotin. The 2-iminobiotin EC50 values for the Ala mutants fell outside the accessible concentration range of the ELISA assay, demonstrating that these mutations lowered the Ka by a factor of 10(4) to 10(6). Direct estimation of biotin Ka values for W79A, W120A, and W120F in an ultrafiltration binding assay yielded Ka values of 4.3 x 10(7) M-1, 8.6 x 10(6) M-1, and > 5 x 10(9) M-1, respectively, in excellent agreement with the ELISA estimates of delta Ka with 2-iminobiotin as a reporter ligand. The results of these preliminary functional studies suggest that these aromatic side chains contribute significantly to the streptavidin-biotin binding free energy.

Authors
Chilkoti, A; Tan, PH; Stayton, PS
MLA Citation
Chilkoti, A, Tan, PH, and Stayton, PS. "Site-directed mutagenesis studies of the high-affinity streptavidin-biotin complex: contributions of tryptophan residues 79, 108, and 120." Proc Natl Acad Sci U S A 92.5 (February 28, 1995): 1754-1758.
PMID
7878054
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
92
Issue
5
Publish Date
1995
Start Page
1754
End Page
1758

Molecular origins of the slow streptavidin-biotin dissociation kinetics

The association of streptavidin and avidin with biotin is among the strongest known noncovalent protein-ligand interactions (Ka ≈ 2.5 × 1013 M-1) and is controlled by an exceptionally slow off-rate. We have used this model system to elucidate the role of aromatic tryptophan side-chain binding contacts in the dissociation reaction coordinate and relatedly to the construction of the activation barrier and to the structure of the transition state. The significantly lower dissociation t1/2 of conservative Trp to Phe site-directed mutants, 35 h (wild-type) > 5.5 h (W79F) > 2 h (W 108F) > 0.5 h (W 120F), reveals the importance of these Trp contacts in regulating the dissociation rate and also highlights the position dependence of the Trp contributions, most notably the optimized "capping" interaction of Trp 120 with biotin. We have also conducted a transition state analysis of the temperature-dependent dissociation kinetics, which along with the independent estimation of the equilibrium biotin-binding free energies and enthalpies has provided thermodynamic profiles defining the enthalpic, entropic, and free energy barriers to dissociation for the mutants relative to wild-type streptavidin. The increased biotin off-rate for W79F, which contacts the valeric acid moiety of biotin, and for W120F, which partially caps the bicyclic ring system, is caused largely by free energy destabilization of the ligand-bound ground state relative to wild-type streptavidin. This free energy destabilization is controlled by a 2.4 kcal mol-1 entropic destabilization of the ligand-bound W79F ground state relative to wild-type at 298 K, and a 5.1 kcal mol-1 enthalpic destabilization of the ligand-bound W120F ground state relative to wild-type. W79F displays an increased equilibrium binding enthalpy relative to wild-type, and thus streptavidin sacrifices potential binding enthalpy to minimize the entropic costs of biotin immobilization. This energetic role correlates well with the structural role of Trp 79, where the side chain contacts the valeric acid tail of biotin, the most conformationally flexible portion of the ligand and thus the most entropically costly to immobilize. The 17-fold increase in off-rate for W108F, on the other hand, which contacts the bicyclic ring of biotin from a buried position within the biotin-binding site, is largely due to the stabilization of the transition state, and is driven by a large 6.5 kcal mol-1 increase in the favorable activation entropy relative to wild-type streptavidin at 298 K. These results are consistent with a snapshot of the transition state where biotin and/or the protein have moved such that Trp 79 and Trp 120 no longer maintain strong contact with biotin, while the contact with Trp 108 remains energetically significant.

Authors
Chilkoti, A; Stayton, PS
MLA Citation
Chilkoti, A, and Stayton, PS. "Molecular origins of the slow streptavidin-biotin dissociation kinetics." Journal of the American Chemical Society 117.43 (1995): 10622-10628.
Source
scival
Published In
Journal of the American Chemical Society
Volume
117
Issue
43
Publish Date
1995
Start Page
10622
End Page
10628

Dissociation of tetrameric ions of noncovalent streptavidin complexes formed by electrospray ionization

The noncovalent tetrameric association of the protein streptavidin formed by electrospray ionization (ESI) mass spectrometry has been observed intact and dissociated in the gas phase. An extended mass-to-charge ratio range quadrupole mass spectrometer was employed to examine the effects of harsher conditions in the ESI atmosphere-vacuum interface region on the streptavidin tetramer. Thermally induced dissociation caused the mass spectra to exhibit a series of complementary monomer and trimer ions that correspond to decomposition of the tetrameric species. Similar results were obtained with tandem mass spectrometric experiments on a Fourier transform ion cyclotron resonance mass spectrometer by application of sustained off-resonance irradiation (SORI) on a selected tetrameric charge state. The technique of single-frequency quadrupole excitation was used to accomplish selected-ion accumulation of the 14 + charge state of the tetramer during ion injection. Subsequent low energy SORI combined with broadband quadrupole cooling produced the 7 + monomer and 7 + trimer species, as well as the 6 + monomer and 8 + trimer complementary ions. The observed asymmetric breakup of the tetramer is qualitatively explained by using physical models. © 1995 American Society for Mass Spectrometry.

Authors
Schwartz, BL; Bruce, JE; Anderson, GA; Hofstadler, SA; Rockwood, AL; Smith, RD; Chilkoti, A; Stayton, PS
MLA Citation
Schwartz, BL, Bruce, JE, Anderson, GA, Hofstadler, SA, Rockwood, AL, Smith, RD, Chilkoti, A, and Stayton, PS. "Dissociation of tetrameric ions of noncovalent streptavidin complexes formed by electrospray ionization." Journal of the American Society for Mass Spectrometry 6.6 (1995): 459-465.
PMID
24214298
Source
scival
Published In
Journal of The American Society for Mass Spectrometry
Volume
6
Issue
6
Publish Date
1995
Start Page
459
End Page
465
DOI
10.1016/1044-0305(95)00191-F

Investigation of non-covalent ligand binding to the intact streptavidin tetramer by electrospray ionization mass spectrometry

The relative non-covalent binding behavior of two small molecule ligands to the tetrameric protein streptavidin was investigated by electrospray ionization mass spectrometry (ESI-MS). An extended m/z range quadrupole mass spectrometer was employed to observe the intact multimeric form of the protein and to probe the relative stabilities of the tetrameric protein- ligand complexes in the gas phase. Various protein-ligand molar ratio concentrations and incubation times were studied for the ligands biotin (K(d) ~ 10-15 M) and iminobiotin (K(d) ~ 10-7 M), in combination with the adjustment of variables in the ESI atmosphere-vacuum interface region. Positive-ion ESI-MS of a 1 : 7 molar ratio streptavidin-biotin sample produced peaks corresponding to the intact tetrameric complex (16 + to 14 + charge states) with four ligands binding to the active form of the protein. Only the expected specific non-covalent complexes of four ligand molecules binding to the protein tetramer were detected, consistent with known solution behavior and without the appearance of any random aggregation. However, under identical interface conditions, complete iminobiotin binding to the protein was not observed, even when higher ligand concentrations, longer complexation times and gentler interface conditions were employed. The thermally induced dissociation (TID) of the non-covalent complexes was studied by applying more severe conditions in the ESI interface region. In addition, observation of an intact streptavidin-biotinylated oligonucleotide non-covalent complex (17- to 14- tetrameric charge states) by negative-ion ESI-MS at m/z 4500-5000 is presented. The results show that the complexes observed in the mass spectrometer are representative of those that exist in solution, under the ESI conditions employed, and that the ability to observe such complexes is at least qualitatively related to stability in solution.

Authors
Schwartz, BL; Gale, DC; Smith, RD; Chilkoti, A; Stayton, PS
MLA Citation
Schwartz, BL, Gale, DC, Smith, RD, Chilkoti, A, and Stayton, PS. "Investigation of non-covalent ligand binding to the intact streptavidin tetramer by electrospray ionization mass spectrometry." Journal of Mass Spectrometry 30.8 (1995): 1095-1102.
Source
scival
Published In
Journal of Mass Spectrometry
Volume
30
Issue
8
Publish Date
1995
Start Page
1095
End Page
1102
DOI
10.1002/jms.1190300806

Site-specific conjugation of a temperature-sensitive polymer to a genetically-engineered protein.

A genetically-engineered mutant of cytochrome b5, incorporating a unique cysteine residue, was conjugated to maleimide-terminated oligo(N-isopropylacrylamide). The conjugation of the protein by reaction of the cysteine residue, precisely positioned by site-directed mutagenesis techniques, with an activated oligomer containing only one reactive end group in the oligomer chain permits the site-specific and stoichiometric conjugation of the oligomer with the protein. The protein-oligomer conjugate was shown to exhibit lower critical solution temperature (LCST) behavior, similar to the free oligomer. Furthermore, the LCST behavior of the protein-oligomer conjugate is reversible and allows selective precipitation of the conjugate above its LCST.

Authors
Chilkoti, A; Chen, G; Stayton, PS; Hoffman, AS
MLA Citation
Chilkoti, A, Chen, G, Stayton, PS, and Hoffman, AS. "Site-specific conjugation of a temperature-sensitive polymer to a genetically-engineered protein." Bioconjug Chem 5.6 (November 1994): 504-507.
PMID
7873655
Source
pubmed
Published In
Bioconjugate Chemistry
Volume
5
Issue
6
Publish Date
1994
Start Page
504
End Page
507

Static secondary ion mass spectrometric investigation of the surface chemistry of organic plasma-deposited films created from oxygen-containing precursors. 3. Multivariate statistical modeling.

Partial least squares (PLS) multivariate statistical models were developed to predict the surface composition and chemistry of a set of model homopolymers based on their static SIMS fragmentation patterns. In the calibration or model-building step, the positive and negative ion static SIMS spectra of different classes of model homopolymers were related to specific chemical attributes of the polymers. The models were then used to examine the surface chemistry of oxygen-containing plasma-deposited films prepared from a variety of precursors. PLS models were developed to predict the surface oxygen concentration and H/C ratios. The results obtained from the PLS models were compared with experimental results.

Authors
Chilkoti, A; Ratner, BD; Briggs, D
MLA Citation
Chilkoti, A, Ratner, BD, and Briggs, D. "Static secondary ion mass spectrometric investigation of the surface chemistry of organic plasma-deposited films created from oxygen-containing precursors. 3. Multivariate statistical modeling." Anal Chem 65.13 (July 1, 1993): 1736-1745.
PMID
8368525
Source
pubmed
Published In
Analytical Chemistry
Volume
65
Issue
13
Publish Date
1993
Start Page
1736
End Page
1745

Analysis of biomedical polymer surfaces: polyurethanes and plasma-deposited thin films.

The surface characterization of biomaterials is important for understanding the biological reactivity of surfaces and for monitoring surface reproducibility and contamination. Electron spectroscopy for chemical analysis (ESCA), secondary ion mass spectrometry (SIMS), contact-angle methods, vibrational spectroscopic methods, and scanning probe microscopies are briefly reviewed. Examples are presented using these methods to characterize RF plasma-deposited surfaces based upon acetone and oxygen for cell culture and Biomer¿ surfaces.

Authors
Ratner, BD; Tyler, BJ; Chilkoti, A
MLA Citation
Ratner, BD, Tyler, BJ, and Chilkoti, A. "Analysis of biomedical polymer surfaces: polyurethanes and plasma-deposited thin films." Clin Mater 13.1-4 (1993): 71-84. (Review)
PMID
10146244
Source
pubmed
Published In
Clinical Materials
Volume
13
Issue
1-4
Publish Date
1993
Start Page
71
End Page
84

Analysis of polymer surfaces by SIMS. 16. investigation of surface cross-linking in polymer gels of 2-hydroxyethyl methacrylate

This study is part of an ongoing effort to systematically delineate the analytical potential of static SIMS for organic surface analysis. Consistent with this overall goal, this study aims to answer if surface cross-linking in a polymeric system be identified by static SIMS.

Authors
Chilkoti, A; Lopez, GP; Ratner, BD
MLA Citation
Chilkoti, A, Lopez, GP, and Ratner, BD. "Analysis of polymer surfaces by SIMS. 16. investigation of surface cross-linking in polymer gels of 2-hydroxyethyl methacrylate." Macromolecules 26.18 (1993): 4825-4832.
Source
scival
Published In
Macromolecules
Volume
26
Issue
18
Publish Date
1993
Start Page
4825
End Page
4832

Engineered proteins for biomaterials

A molecular adaptor for interfacing environmentally sensitive, soluble polymers and antibody molecules has been developed. The gene coding for the minimally sized, 55 amino acid IgG binding domain from protein G has been constructed by total gene synthesis. This domain is thermally stable, exhibits a highly reversible folding and unfolding equilibrium, and recognizes IgG and Fab molecules with high affinity. These properties make the protein G domain a potentially useful adaptor for non-covalent immobilization of antibodies to soluble polymers and hydrogels. Engineered single-chain Fv antibody fragments have also been constructed and a method for expanding the usefulness of the protein G adaptor to these molecules is proposed. The engineered antibodies also provide a model system for developing general immobilization strategies aimed at maximizing binding affinities and therapeutic responses. The overall goal is to develop optimized engineering designs for functionally optimized antibody-material hybrids.

Authors
Stayton, PS; Chilkoti, A; Long, CJ; Pettit, DK; Tan, PHS; Chen, G; Hoffman, AS
MLA Citation
Stayton, PS, Chilkoti, A, Long, CJ, Pettit, DK, Tan, PHS, Chen, G, and Hoffman, AS. "Engineered proteins for biomaterials." Materials Research Society Symposium Proceedings 292 (1993): 77-82.
Source
scival
Published In
Materials Research Society Symposium Proceedings
Volume
292
Publish Date
1993
Start Page
77
End Page
82

X-ray photoelectron spectroscopy of iodine-doped nonconjugated polymers

The effects of iodine doping on poly(cis-butadiene) and the cis and trans isomers of polyisoprene were examined by core-level X-ray photoelectron spectroscopy (XPS). The XPS results for iodine-doped poly(cis-butadiene) are similar to that for polyisoprene, suggesting that both polymers are doped by a similar mechanism. Core-level XPS of the iodine-doped polymers suggests that the direction of charge transfer is from the backbone carbon to the iodine species. The binding energies of the iodine species in the I3d spectra of the doped polymers indicate the presence of molecular iodine and I3- (and/or I5-) species and the absence of a significant concentration of cationic iodonium-π complex. In conjunction with conductivity measurements, XPS analysis of cryogenically cooled (-80°C) iodine-doped poly(as-isoprene) and poly(transisoprene) provides direct spectroscopic evidence that the conductivity of the doped polymers is due to excess iodine, present in the form of polyiodide chains. © 1993 American Chemical Society.

Authors
Chilkoti, A; Ratner, BD
MLA Citation
Chilkoti, A, and Ratner, BD. "X-ray photoelectron spectroscopy of iodine-doped nonconjugated polymers." Chemistry of Materials 5.6 (1993): 786-792.
Source
scival
Published In
Chemistry of Materials
Volume
5
Issue
6
Publish Date
1993
Start Page
786
End Page
792

A tandem SIMS study of poly(vinyl methyl ether)

Authors
Leggett, GJ; Chilkoti, A; Ratner, BD; Vickerman, JC
MLA Citation
Leggett, GJ, Chilkoti, A, Ratner, BD, and Vickerman, JC. "A tandem SIMS study of poly(vinyl methyl ether)." Surface and Interface Analysis 18.3 (March 1992): 210-216.
Source
crossref
Published In
Surface and Interface Analysis
Volume
18
Issue
3
Publish Date
1992
Start Page
210
End Page
216
DOI
10.1002/sia.740180306

Analysis of polymer surfaces by SIMS: part 15. oxygen-functualized aliphatic homopolymers

The positive and negative ion spectra from simple oxygen-containing aliphatic homopolymers (i.e. ethers, ketones, carboxylates and backbone esters) are reported. Interpretation of the static SIMS fragmentation patterns of these polymers is shown to be related to the structure of the polymer, in particular its heterofunctionality. A systematic investigation of the atomic secondary anions that are ubiquitous to all organic polymers reveals that their relative yields are not devoid of chemical information, but appear to be related to polymer structure.

Authors
Chilkoti, A; Ratner, BD; Briggs, D
MLA Citation
Chilkoti, A, Ratner, BD, and Briggs, D. "Analysis of polymer surfaces by SIMS: part 15. oxygen-functualized aliphatic homopolymers." Surface and Interface Analysis 18.8 (1992): 604-618.
Source
scival
Published In
Surface and Interface Analysis
Volume
18
Issue
8
Publish Date
1992
Start Page
604
End Page
618

Static secondary ion mass spectrometry of organic plasma-deposited films created from stable isotope-labeled precursors. II. Mixtures of acetone and oxygen

A series of plasma-deposited films (PDFs), created by blending controlled ratios of acetone vapor and oxygen in the feed to the plasma reactor, were analyzed by static secondary ion mass spectrometry (SIMS). Examination of the quadrupole-based static SIMS fragmentation patterns of acetone-O2 PDFs created from 13C-labeled acetone generally allowed the hydrocarbon secondary ions to be distinguished from oxygen-containing secondary ions. These results were compared with those obtained via high-mass resolution time-of-flight (TOF)-SIMS. The identified secondary ions were then assigned structural attributes, based on comparison of the static SIMS spectra of the acetone-O2 PDFs with those of conventional hydrocarbon and oxygen-containing polymers. A preliminary investigation to unravel the mechanism of oxygen incorporation in the acetone-O2 PDFs from the two plasma feed components was undertaken through the analysis of PDFs created from 1,2,3 13C-acetone(16O) vapor and 18O2 gas.

Authors
Chilkoti, A; Ratner, BD; Briggs, D; Reich, F
MLA Citation
Chilkoti, A, Ratner, BD, Briggs, D, and Reich, F. "Static secondary ion mass spectrometry of organic plasma-deposited films created from stable isotope-labeled precursors. II. Mixtures of acetone and oxygen." Journal of Polymer Science, Part A: Polymer Chemistry 30.7 (1992): 1261-1278.
Source
scival
Published In
Journal of Polymer Science (In Two Sections)
Volume
30
Issue
7
Publish Date
1992
Start Page
1261
End Page
1278
DOI
10.1002/pola.1992.080300704

Contemporary methods for characterizing complex biomaterial surfaces

There are many methods that we can bring to bear for the characterization of polymeric surfaces. Standard methods include contact angle techniques, attenuated total reflectance infrared (IR) and electron spectroscopy for chemical analysis (ESCA). However, new and modified methods can be used that can greatly enhance the information content available from these traditional methods. In this paper, the application of newer methods to two complex systems, polyetherurethanes and plasma deposited thin films, will be discussed. In particular, external reflection IR, derivatization ESCA, static secondary ion mass spectrometry (SIMS), derivatization SIMS, isotope SIMS, time-of-flight (TOF) SIMS, atomic force microscopy, synchrotron x-ray fluorescence and synchrotron electron yield will be described.

Authors
Ratner, BD; Chilkoti, A; Castner, DG
MLA Citation
Ratner, BD, Chilkoti, A, and Castner, DG. "Contemporary methods for characterizing complex biomaterial surfaces." Clinical Materials 11.1-4 (1992): 25-36.
Source
scival
Published In
Clinical Materials
Volume
11
Issue
1-4
Publish Date
1992
Start Page
25
End Page
36

Substrate temperature effects on film chemistry in plasma deposition of organics. III. Analysis by static secondary ion mass spectrometry

Statie-secondary ion mass spectrometry (SIMS) was used to examine the effect of reducing the substrate temperature during the radio frequency plasma deposition of organic films. Studies of two polymerizable plasma precursors (2-hydroxyethyl methacrylate and acrylic acid) and one nonpolymerizable precursor (acetone) deposited without substrate cooling and with liquid nitrogen cooling are presented. Acetone deposited with methanol/dry ice cooling was also investigated. Spectra of polymerizable precursors were analyzed by comparison to spectra for the corresponding conventionally-polymerized polymer films [i.e., poly(hydroxyethyl methacrylate) and poly(acrylic acid)]. Acetone spectra were interpreted by reference to SIMS analysis of plasma-deposited films prepared from isotopically-labelled acetone and to reference homopolymers. Comparison of the SIMS spectra of films deposited at different substrate temperatures indicates that a reduction in substrate temperature generally results in higher intensity of peaks characteristic of oxygenated ion structures. SIMS also suggests that the reduction of substrate temperature results in less polymer unsaturation and fewer structures which form by hydrogen redistribution during the deposition process. These results support the hypothesis that deposition at low substrate temperatures leads to an increase in the proportion of precursor incorporated into the film without substantial fragmentation. Corroborative results from high resolution x-ray photoelectron spectroscopy (XPS) and assays for precursor functional groups by chemical derivatization reactions in conjunction with XPS are also presented.

Authors
Lopez, GP; Chilkoti, A; Briggs, D; Ratner, BD
MLA Citation
Lopez, GP, Chilkoti, A, Briggs, D, and Ratner, BD. "Substrate temperature effects on film chemistry in plasma deposition of organics. III. Analysis by static secondary ion mass spectrometry." Journal of Polymer Science, Part A: Polymer Chemistry 30.11 (1992): 2427-2441.
Source
scival
Published In
Journal of Polymer Science, Part A: Polymer Chemistry
Volume
30
Issue
11
Publish Date
1992
Start Page
2427
End Page
2441
DOI
10.1002/pola.1992.080301117

Endothelial cell growth on oxygen-containing films deposited by radio-frequency plasmas: the role of surface carbonyl groups.

Polystyrene substrates were modified by radio-frequency plasma deposition from mixtures of various organic vapors (acetone, methane, methanol, and formic acid) and oxygen. The resulting surfaces exhibited a wide range of surface oxygen concentrations, as measured by electron spectroscopy for chemical analysis (ESCA). The surface hydroxyl, carboxyl, and carbonyl groups were derivatized with trifluoroacetic anhydride, trifluoroethanol, or hydrazine, respectively, and their concentrations subsequently determined by ESCA. The growth of bovine aortic endothelial cells was found to increase with the surface carbonyl concentration but did not appear to correlate with the hydroxyl or carboxyl concentrations.

Authors
Ertel, SI; Chilkoti, A; Horbett, TA; Ratner, BD
MLA Citation
Ertel, SI, Chilkoti, A, Horbett, TA, and Ratner, BD. "Endothelial cell growth on oxygen-containing films deposited by radio-frequency plasmas: the role of surface carbonyl groups." Journal of biomaterials science. Polymer edition 3.2 (December 1, 1991): 163-183.
Source
scopus
Published In
Journal of Biomaterials Science, Polymer Edition
Volume
3
Issue
2
Publish Date
1991
Start Page
163
End Page
183

Static secondary-ion mass spectrometric investigation of the surface structure of organic plasma-deposited films prepared from stable-isotope-labeled precursors. 1. Carbonyl precursors.

Stable-isotope-labeled carbonyl precursors (acetaldehyde, acetone, and 2-butanone) were used to create plasma-deposited films (PDFs), which were then examined by positive- and negative-ion static SIMS. This allowed hydrocarbon (HC) fragments to be distinguished from oxygen-containing fragments in the static SIMS spectra of these PDFs. Both the positive- and negative-ion static SIMS fragmentation patterns of conventional HC and oxygen-containing polymers were qualitatively examined in order to assign structural units on the PDF surface that could account for the sallent features in the static SIMS fragmentation patterns of these PDFs.

Authors
Chilkoti, A; Ratner, BD; Briggs, D
MLA Citation
Chilkoti, A, Ratner, BD, and Briggs, D. "Static secondary-ion mass spectrometric investigation of the surface structure of organic plasma-deposited films prepared from stable-isotope-labeled precursors. 1. Carbonyl precursors." Anal Chem 63.15 (August 1, 1991): 1612-1620.
PMID
1952085
Source
pubmed
Published In
Analytical Chemistry
Volume
63
Issue
15
Publish Date
1991
Start Page
1612
End Page
1620

STATIC SECONDARY ION MASS-SPECTROMETRY AND X-RAY PHOTOELECTRON-SPECTROSCOPY OF DEUTERIUM-SUBSTITUTED AND METHYL-SUBSTITUTED POLYSTYRENE

Authors
CHILKOTI, A; CASTNER, DG; RATNER, BD
MLA Citation
CHILKOTI, A, CASTNER, DG, and RATNER, BD. "STATIC SECONDARY ION MASS-SPECTROMETRY AND X-RAY PHOTOELECTRON-SPECTROSCOPY OF DEUTERIUM-SUBSTITUTED AND METHYL-SUBSTITUTED POLYSTYRENE." APPLIED SPECTROSCOPY 45.2 (February 1991): 209-217.
Source
wos-lite
Published In
Applied spectroscopy
Volume
45
Issue
2
Publish Date
1991
Start Page
209
End Page
217
DOI
10.1366/0003702914337588

Direct emission of molecular fragments during the sputtering of poly(4-hydroxystyrene) and determination of ion structures using tandem secondary ion mass spectrometry

Poly(4-hydroxystyrene) (P4HS) has been studied using a tandem secondary ion mass spectrometer under static conditions. The secondary ion mass spectrometry (SIMS) spectrum of P4HS exhibits few ions which are not observed in the SIMS spectrum of polystyrene. The fragmentation of these oxygen-containing ions has been investigated in detail and the relevant neutral losses have been deduced. These ions are found to have structures based upon either the tropyllium ion or the styrene ion. The absence of other oxygen-containing ions is explained in terms of the ready loss of oxygen-containing neutral fragments from P4HS compared with the loss of hydrocarbon fragments. The oxygen-containing tropyllium and styrene ions are, in contrast, readily formed, and in these cases the energy barrier to formation of the oxygen-containing ion is less than the energy barrier to expulsion of oxygen-containing neutral species. © 1991.

Authors
Leggett, GJ; Chilkoti, A; Castner, DG; Ratner, BD; Vickerman, JC
MLA Citation
Leggett, GJ, Chilkoti, A, Castner, DG, Ratner, BD, and Vickerman, JC. "Direct emission of molecular fragments during the sputtering of poly(4-hydroxystyrene) and determination of ion structures using tandem secondary ion mass spectrometry." International Journal of Mass Spectrometry and Ion Processes 108.1 (1991): 29-39.
Source
scival
Published In
International Journal of Mass Spectrometry and Ion Processes
Volume
108
Issue
1
Publish Date
1991
Start Page
29
End Page
39
DOI
10.1016/0168-1176(91)87004-K

X-ray photoelectron spectroscopic investigation of the selectivity of hydroxyl derivatization reactions

The selectivity of trifluoroacetic anhydride (TFAA), used for the vapor-phase derivatization of hydroxyl groups in multifunctional oxygen-containing polymeric surfaces, has been investigated by XPS studies on model epoxide systems. The reactivity of TFAA towards epoxides observed here is discussed in the context of the large amount of published literature implying high selectivity of this reaction towards hydroxyl groups. Acetyl chloride (AcCl) and heptafluorobutyryl chloride (HFBuCl), used to derivatize hydroxyl groups in the literature, were also examined, with regard to cross-reaction with epoxides. Both AcCl and HFBuCl react with epoxides, forming chloroacetates and perfluorinated chloroacetates, respectively. Moreover, HFBuCl is shown to react with double bonds forming perfluorinated chloroacetates, while AcCl is unreactive towards them. The reactivity of HFBuCl towards epoxides and unsaturation compromises its use as a derivatization reagent for hydroxyl groups. However, the unique reaction products formed by exposure of hydroxyls and epoxides to AcCl vapor may allow hydroxyl species to be distinguished from epoxides in multifunctional carbon-oxygen polymeric surfaces.

Authors
Chilkoti, A; Ratner, BD
MLA Citation
Chilkoti, A, and Ratner, BD. "X-ray photoelectron spectroscopic investigation of the selectivity of hydroxyl derivatization reactions." Surface and Interface Analysis 17.8 (1991): 567-574.
Source
scival
Published In
Surface and Interface Analysis
Volume
17
Issue
8
Publish Date
1991
Start Page
567
End Page
574

Plasma-deposited polymeric films prepared from carbonyl-containing volatile precursors: XPS chemical derivatization and static SIMS surface characterization

The surface chemistry of plasma-deposited films (PDF) created from carbonyl precursors was investigated by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and vapor-phase derivatization reactions in conjunction with XPS (derivatization XPS). The selectivity of trifluorpacetic anhydride (TFAA) toward hydroxyl groups and hydrazine toward carbonyl groups was ascertained by reacting them with various polymers with oxygen-containing functional groups likely to be present in the carbon-oxygen PDF of interest. Investigation of the surface chemistry of ultrathin PDF from acetaldehyde, acetone, and 2-butanone demonstrated that under the invariant experimental conditions utilized, the effect of varying the C/O ratio of the precursor had little effect on PDF surface chemistry. The low concentration of carbonyl groups detected suggests extensive fragmentation of the precursor during plasma deposition. The creation of a graded series of oxygen-containing PDF was also explored by blending controlled ratios of O2 gas in the acetone feed to the plasma reactor. Derivatization XPS indicated that the distribution of hydroxyl, carbonyl, and carboxyl functional groups varied across the range of oxygen incorporation in the acetone-O2 PDF. The surface chemistry of the acetone-O2 PDF series and a commercial tissue culture polystyrene surface (Falcon) was compared by XPS, derivatization XPS, and static secondary ion mass spectrometry. © 1991 American Chemical Society.

Authors
Chilkoti, A; Ratner, BD; Briggs, D
MLA Citation
Chilkoti, A, Ratner, BD, and Briggs, D. "Plasma-deposited polymeric films prepared from carbonyl-containing volatile precursors: XPS chemical derivatization and static SIMS surface characterization." Chemistry of Materials 3.1 (1991): 51-61.
Source
scival
Published In
Chemistry of Materials
Volume
3
Issue
1
Publish Date
1991
Start Page
51
End Page
61

Structural elucidation of oxygen-containing plasma deposited films by static secondary ion mass spectrometry

This study focusses on the use of static secondary ion mass spectrometry (SIMS) to unravel the surface chemistry of oxygen-containing organic PDF of interest in cell growth applications. The aims of this study are (1) to elucidate the identity of secondary ions emitted from the PDF surface (2) relate these ions to structural features on the PDF surface (3) develop a quantitative model that is capable of predicting aspects of the surface structure of these PDF. This has been achieved by (1) analysis of stable-isotope labeled PDF to identify the PDF secondary ions, followed by (2) comparison of PDF spectra with appropriate model polymers to relate them to known structural features and (3) the use of multivariate statistical techniques to predict both the concentration of functional groups of interest and the various types of structural units that could be present on the PDF surface.

Authors
Chilkoti, A; Ratner, BD; Briggs, D
MLA Citation
Chilkoti, A, Ratner, BD, and Briggs, D. "Structural elucidation of oxygen-containing plasma deposited films by static secondary ion mass spectrometry." Transactions of the Annual Meeting of the Society for Biomaterials in conjunction with the International Biomaterials Symposium 14 (1991): 180--.
Source
scival
Published In
Transactions of the Annual Meeting of the Society for Biomaterials in conjunction with the International Biomaterials Symposium
Volume
14
Publish Date
1991
Start Page
180-

Endothelial cell growth on oxygen-containing films deposited by radio-frequency plasmas: the role of surface carbonyl groups.

Polystyrene substrates were modified by radio-frequency plasma deposition from mixtures of various organic vapors (acetone, methane, methanol, and formic acid) and oxygen. The resulting surfaces exhibited a wide range of surface oxygen concentrations, as measured by electron spectroscopy for chemical analysis (ESCA). The surface hydroxyl, carboxyl, and carbonyl groups were derivatized with trifluoroacetic anhydride, trifluoroethanol, or hydrazine, respectively, and their concentrations subsequently determined by ESCA. The growth of bovine aortic endothelial cells was found to increase with the surface carbonyl concentration but did not appear to correlate with the hydroxyl or carboxyl concentrations.

Authors
Ertel, SI; Chilkoti, A; Horbett, TA; Ratner, BD
MLA Citation
Ertel, SI, Chilkoti, A, Horbett, TA, and Ratner, BD. "Endothelial cell growth on oxygen-containing films deposited by radio-frequency plasmas: the role of surface carbonyl groups." J Biomater Sci Polym Ed 3.2 (1991): 163-183.
PMID
1768637
Source
pubmed
Published In
Journal of Biomaterials Science, Polymer Edition
Volume
3
Issue
2
Publish Date
1991
Start Page
163
End Page
183

Surface characterization of a poly(styrene/ p ‐hydroxystyrene) copolymer series using x‐ray photoelectron spectroscopy, static secondary ion mass spectrometry, and chemical derivatization techniques

Authors
Chilkoti, A; Castner, DG; Ratner, BD; Briggs, D
MLA Citation
Chilkoti, A, Castner, DG, Ratner, BD, and Briggs, D. "Surface characterization of a poly(styrene/ p ‐hydroxystyrene) copolymer series using x‐ray photoelectron spectroscopy, static secondary ion mass spectrometry, and chemical derivatization techniques." Journal of Vacuum Science & Technology A: Vacuum, Surfaces, and Films 8.3 (May 1990): 2274-2282.
Source
crossref
Published In
Journal of vacuum science & technology. A, Vacuum, surfaces, and films : an official journal of the American Vacuum Society
Volume
8
Issue
3
Publish Date
1990
Start Page
2274
End Page
2282
DOI
10.1116/1.576750

Static SIMS investigation of the surface structure of plasma deposited films prepared from stable isotope-labeled carbonyl precursors

The plasma deposition of organics is an emerging technology, permitting the formation of ultrathin films of technological interest on a variety of substrates. However, the thin film nature of plasma deposited films (PDF) and their complex chemistry, i.e., multifunctional, cross-linked etc., has hindered their characterization. The advent of static secondary ion mass spectrometry (SIMS) as a surface characterization technique provides for the direct interrogation of polymer structure. We have attempted to elucidate aspects of the surface structure of a set of oxygen-functionalized organic PDF's through the use of static SIMS. The static SIMS analysis of PDF's created from carbonyl-functionalized precursors and their site-specific isotopically labeled analogs revealed that the PDF's were primarily HC in nature. Additionally, a mixture of aliphatic and aromatic fragments were observed in their positive-ion static SIMS spectra, which may be related to unsaturation and crosslinking effects.

Authors
Chilkoti, A; Ratner, BD; Briggs, D
MLA Citation
Chilkoti, A, Ratner, BD, and Briggs, D. "Static SIMS investigation of the surface structure of plasma deposited films prepared from stable isotope-labeled carbonyl precursors." Polymeric Materials Science and Engineering, Proceedings of the ACS Division of Polymeric Materials Science and Engineering 62 (1990): 135-139.
Source
scival
Published In
Polymeric Materials Science and Engineering, Proceedings of the ACS Division of Polymeric Materials Science and Engineering
Volume
62
Publish Date
1990
Start Page
135
End Page
139

Effect of monomer type on the surface chemistry of rf-plasma deposited surfaces prepared for cell culture

The characterization techniques used reveal that the differences between plasma deposited films from the three monomers under identical reactor conditions are minor, which suggests that the monomer molecules undergo extensive fragmentation in the plasma; this would help explain the similar nature of the three plasma deposited surfaces. Future investigations will focus on cell growth studies and on more detailed characterization of these surfaces using static secondary ion mass spectrometry (SSIMS).

Authors
Chilkoti, A; Ratner, BD
MLA Citation
Chilkoti, A, and Ratner, BD. "Effect of monomer type on the surface chemistry of rf-plasma deposited surfaces prepared for cell culture." Polymeric Materials Science and Engineering, Proceedings of the ACS Division of Polymeric Materials Science and Engineering 59 (1988): 258-262.
Source
scival
Published In
Polymeric Materials Science and Engineering, Proceedings of the ACS Division of Polymeric Materials Science and Engineering
Volume
59
Publish Date
1988
Start Page
258
End Page
262
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