Co-reporter:Jing Wang, Michael Dzuricky, and Ashutosh Chilkoti
Nano Letters October 11, 2017 Volume 17(Issue 10) pp:5995-5995
Publication Date(Web):August 30, 2017
DOI:10.1021/acs.nanolett.7b02225
Many promising targeting ligands are hydrophobic peptides, and these ligands often show limited accessibility to receptors, resulting in suboptimal targeting. A systematic study to elucidate the rules for the design of linkers that optimize their presentation on nanoparticles has not been carried out to date. In this study, we recombinantly synthesized an elastin-like polypeptide diblock copolymer (ELPBC) that self-assembles into monodisperse micelles. AHNP and EC1, two hydrophobic ErbB2-targeted peptide ligands, were incorporated at the C-terminus of the ELPBC with an intervening peptide linker. We tested more than 20 designs of peptide linkers, where the linker could be precisely engineered at the gene level to systematically investigate the molecular parameters—sequence, length, and charge—of the peptide linker that optimally assist ligands in targeting the ErbB2 receptor on cancer cells. We found that peptide linkers with a minimal length of 12 hydrophilic amino acids and an overall cationic charge—and that impart a zeta potential of the micelle that is close to neutral—were necessary to enhance the uptake of peptide-modified ELPBC micelles by cancer cells that overexpress the ErbB2 receptor. This work advances our understanding of the optimal presentation of hydrophobic ligands by nanoparticles and suggests design rules for peptide linkers for targeted delivery by polymer micelles, an emerging class of nanoparticle carriers for drugs and imaging agents.Keywords: affinity targeting; elastin-like polypeptide; ErbB2; hydrophobic ligand; Linker optimization; nanoparticle;
Co-reporter:Daniel Y. Joh;Felicia McGuire;Roozbeh Abedini-Nassab;Rohan K. Achar;Joseph B. Andrews;Zackary Zimmers;Darush Mozhdehi;Rebecca Blair;Faris Albarghouthi;Cassio M. Fontes;Jacob Richter;William Oles;Angus M. Hucknall;Aaron D. Franklin;Benjamin B. Yellen
ACS Applied Materials & Interfaces February 15, 2017 Volume 9(Issue 6) pp:5522-5529
Publication Date(Web):January 24, 2017
DOI:10.1021/acsami.6b15836
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.Keywords: biointerface; breakdown; capacitance−voltage; metal oxide dielectric; nonfouling; permittivity; POEGMA; polymer brush;
Co-reporter:Sarah R. MacEwanIsaac Weitzhandler, Ingo Hoffmann, Jan Genzer, Michael GradzielskiAshutosh Chilkoti
Biomacromolecules 2017 Volume 18(Issue 2) pp:
Publication Date(Web):January 17, 2017
DOI:10.1021/acs.biomac.6b01759
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.
Co-reporter:Jing WangSarah R. MacEwan, Ashutosh Chilkoti
Nano Letters 2017 Volume 17(Issue 2) pp:
Publication Date(Web):December 30, 2016
DOI:10.1021/acs.nanolett.6b05041
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.Keywords: cell-penetrating peptide; Fluorescence imaging; lysosome; nanoparticle;
Co-reporter:Sarah R. MacEwan;Dr. Ashutosh Chilkoti
Angewandte Chemie International Edition 2017 Volume 56(Issue 24) pp:6712-6733
Publication Date(Web):2017/06/06
DOI:10.1002/anie.201610819
AbstractThe molecular complexity and heterogeneity of cancer has led to a persistent, and as yet unsolved, challenge to develop cures for this disease. The pharmaceutical industry focuses the bulk of its efforts on the development of new drugs, but an alternative approach is to improve the delivery of existing drugs with drug carriers that can manipulate when, where, and how a drug exerts its therapeutic effect. For the treatment of solid tumors, systemically delivered drug carriers face significant challenges that are imposed by the pathophysiological barriers that lie between their site of administration and their site of therapeutic action in the tumor. Furthermore, drug carriers face additional challenges in their translation from preclinical validation to clinical approval and adoption. Addressing this diverse network of challenges requires a systems engineering approach for the rational design of optimized carriers that have a realistic prospect for translation from the laboratory to the patient.
Co-reporter:Sarah R. MacEwan;Dr. Ashutosh Chilkoti
Angewandte Chemie 2017 Volume 129(Issue 24) pp:6814-6837
Publication Date(Web):2017/06/06
DOI:10.1002/ange.201610819
AbstractDie molekulare Komplexität und Heterogenität von Krebs ist ein ungelöstes Problem bei der Entwicklung von Tumortherapeutika. Zwar konzentriert die pharmazeutische Industrie den Großteil ihrer Forschungen auf die Entwicklung neuer Wirkstoffe, ein alternativer Ansatz besteht aber darin, die Verabreichung vorhandener Wirkstoffe mithilfe von Wirkstoff-Trägern zu verbessern. Für die Behandlung solider Tumoren stehen systemisch verabreichte Wirkstoff-Träger vor erheblichen Hürden, die auf die pathophysiologischen Barrieren zurückzuführen sind, die zwischen der Verabreichungsstelle und dem Ort ihrer therapeutischen Wirkung im Tumor liegen. Darüber hinaus bestehen noch zusätzliche Hürden bezüglich der Translation der Wirkstoffe von der präklinischen Untersuchung zu ihrer klinischen Zulassung und Übernahme in die klinische Praxis. Die Überwindung dieser vielfältigen Hürden macht einen systemtechnischen Ansatz erforderlich, um eine bessere rationale Entwicklung optimierter Träger zu ermöglichen, die eine realistische Aussicht auf die Translation vom Labor zum Patienten haben.
Co-reporter:Jing Wang, Jayanta Bhattacharyya, Eric Mastria, Ashutosh Chilkoti
Journal of Controlled Release 2017 Volume 260(Volume 260) pp:
Publication Date(Web):28 August 2017
DOI:10.1016/j.jconrel.2017.05.032
Nanoscale carriers with an acid-labile linker between the carrier and drug are commonly used for drug delivery. However, their efficacy is potentially limited by inefficient linker cleavage, and lysosomal entrapment of drugs. To address these critical issues, we developed a new imaging method that spatially overlays the location of a nanoparticle and the released drug from the nanoparticle, on a map of the local intracellular pH that delineates individual endosomes and lysosomes, and the therapeutic intracellular target of the drug—the nucleus. We used this method to quantitatively map the intracellular fate of micelles of a recombinant polypeptide conjugated with doxorubicin via an acid-labile hydrazone linker as a function of local pH and time within live cells. We found that hydrolysis of the acid-labile linker is incomplete because the pH range of 4–7 in the endosomes and lysosomes does not provide complete cleavage of the drug from the nanoparticle, but that once cleaved, the drug escapes the acidic endo-lysosomal compartment into the cytosol and traffics to its therapeutic destination—the nucleus. This study also demonstrated that unlike free drug, which enters the cytosol directly through the cell membrane and then traffics into the nucleus, the nanoparticle-loaded drug almost exclusively traffics into endosomes and lysosomes upon intracellular uptake, and only reaches the nucleus after acid-triggered drug release in the endo-lysosomes. This methodology provides a better and more quantitative understanding of the intracellular behavior of drug-loaded nanoparticles, and provides insights for the design of the next-generation of nanoscale drug delivery systems.Download high-res image (120KB)Download full-size image
Co-reporter:Jayanta Bhattacharyya;Xiu-Rong Ren;Robert A. Mook, Jr.;Jiangbo Wang;Ivan Spasojevic;Richard T. Premont;Xinghai Li;Wei Chen
Nanoscale (2009-Present) 2017 vol. 9(Issue 34) pp:12709-12717
Publication Date(Web):2017/08/31
DOI:10.1039/C7NR01973D
Abnormal Wnt activity is a major mechanism responsible for many diseases, including cancer. Previously, we reported that the anthelmintic drug Niclosamide (NIC) inhibits Wnt/β-catenin signaling and suppresses colon cancer cell growth. Although the pharmacokinetic properties of NIC are appropriate for use as an anthelmintic agent, its low solubility, low bioavailability and low systemic exposure limit its usefulness in treating systemic diseases. To overcome these limitations, we conjugated NIC to recombinant chimeric polypeptides (CPs), and the CP-NIC conjugate spontaneously self-assembled into sub-100 nm near-monodisperse nanoparticles. CP-NIC nanoparticles delivered intravenously act as a pro-drug of NIC to dramatically increase exposure of NIC compared to dosing with free NIC. CP-NIC improved anti-tumor activity compared to NIC in a xenograft model of human colon cancer. Because NIC has multiple biological activities, CP-NIC could be used for treatment of multiple diseases, including cancer, bacterial and viral infection, type II diabetes, NASH and NAFLD.
Co-reporter:Dr. Kelli M. Luginbuhl;Dr. Davoud Mozhdehi;Michael Dzuricky;Parisa Yousefpour;Fred C. Huang;Nicholas R. Mayne;Kristen L. Buehne; Dr. Ashutosh Chilkoti
Angewandte Chemie International Edition 2017 Volume 56(Issue 45) pp:13979-13984
Publication Date(Web):2017/11/06
DOI:10.1002/anie.201704625
AbstractInspired by biohybrid molecules that are synthesized in Nature through post-translational modification (PTM), we have exploited a eukaryotic PTM to recombinantly synthesize lipid–polypeptide hybrid materials. By co-expressing yeast N-myristoyltransferase with an elastin-like polypeptide (ELP) fused to a short recognition sequence in E. coli, we show robust and high-yield modification of the ELP with myristic acid. The ELP's reversible phase behavior is retained upon myristoylation and can be tuned to span a 30–60 °C. Myristoylated ELPs provide a versatile platform for genetically pre-programming self-assembly into micelles of varied size and shape. Their lipid cores can be loaded with hydrophobic small molecules by passive diffusion. Encapsulated doxorubicin and paclitaxel exhibit cytotoxic effects on 4T1 and PC3-luc cells, respectively, with potencies similar to chemically conjugated counterparts, and longer plasma circulation than free drug upon intravenous injection in mice.
Co-reporter:Jeffrey L. Schaal, Xinghai Li, Eric Mastria, Jayanta Bhattacharyya, Michael R. Zalutsky, Ashutosh Chilkoti, Wenge Liu
Journal of Controlled Release 2016 Volume 228() pp:58-66
Publication Date(Web):28 April 2016
DOI:10.1016/j.jconrel.2016.02.040
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 131I 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 131I further stabilize the depot by introducing crosslinks within the ELP depot over 24 h. 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 72 h. The 131I-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.
Co-reporter:Jinyao Liu;Yan Pang;Jayanta Bhattacharyya;Wenge Liu;Isaac Weitzhler;Xinghai Li
Advanced Healthcare Materials 2016 Volume 5( Issue 15) pp:1868-1873
Publication Date(Web):
DOI:10.1002/adhm.201600230
Co-reporter:Dr. Yan Pang;Dr. Jinyao Liu;Yizhi Qi;Dr. Xinghai Li ; Ashutosh Chilkoti
Angewandte Chemie International Edition 2016 Volume 55( Issue 35) pp:10296-10300
Publication Date(Web):
DOI:10.1002/anie.201604661
Abstract
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.
Co-reporter:Dr. Yan Pang;Dr. Jinyao Liu;Yizhi Qi;Dr. Xinghai Li ; Ashutosh Chilkoti
Angewandte Chemie 2016 Volume 128( Issue 35) pp:10452-10456
Publication Date(Web):
DOI:10.1002/ange.201604661
Abstract
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.
Co-reporter:Yizhi Qi, Ashutosh Chilkoti
Current Opinion in Chemical Biology 2015 Volume 28() pp:181-193
Publication Date(Web):October 2015
DOI:10.1016/j.cbpa.2015.08.009
•Limitations of conjugating poly(ethylene glycol) to proteins and peptides have become increasingly apparent.•Various polymers have shown promise as PEG alternatives.•Extensive future in vivo testing is essential to validate their suitability to replace PEG.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.
Co-reporter:Eric M. Mastria, Mingnan Chen, Jonathan R. McDaniel, Xinghai Li, Jinho Hyun, Mark W. Dewhirst, Ashutosh Chilkoti
Journal of Controlled Release 2015 Volume 208() pp:52-58
Publication Date(Web):28 June 2015
DOI:10.1016/j.jconrel.2015.01.033
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.Doxorubicin-conjugated polypeptide nanoparticles (CP-Dox) inhibit metastasis by preventing the escape of viable cancer cells from the primary tumor to a greater extent than freely dissolved doxorubicin (free Dox).
Co-reporter:Dr. Jinyao Liu;Dr. Wenge Liu;Isaac Weitzhler;Dr. Jayanta Bhattacharyya;Dr. Xinghai Li;Dr. Jing Wang;Yizhi Qi;Dr. Somnath Bhattacharjee ;Dr. Ashutosh Chilkoti
Angewandte Chemie International Edition 2015 Volume 54( Issue 3) pp:1002-1006
Publication Date(Web):
DOI:10.1002/anie.201409293
Abstract
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.
Co-reporter:Joseph J. Bellucci;Dr. Jayanta Bhattacharyya ; Ashutosh Chilkoti
Angewandte Chemie 2015 Volume 127( Issue 2) pp:451-455
Publication Date(Web):
DOI:10.1002/ange.201408126
Abstract
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.
Co-reporter:Dr. Jinyao Liu;Dr. Wenge Liu;Isaac Weitzhler;Dr. Jayanta Bhattacharyya;Dr. Xinghai Li;Dr. Jing Wang;Yizhi Qi;Dr. Somnath Bhattacharjee ;Dr. Ashutosh Chilkoti
Angewandte Chemie 2015 Volume 127( Issue 3) pp:1016-1020
Publication Date(Web):
DOI:10.1002/ange.201409293
Abstract
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.
Co-reporter:Elisabeth Garanger, Sarah R. MacEwan, Olivier Sandre, Annie Brûlet, Laure Bataille, Ashutosh Chilkoti, and Sébastien Lecommandoux
Macromolecules 2015 Volume 48(Issue 18) pp:6617-6627
Publication Date(Web):September 9, 2015
DOI:10.1021/acs.macromol.5b01371
With a perfectly defined primary structure, both in terms of monomer sequence and chain length, recombinant polypeptides obtained by protein engineering techniques allow the investigation of structure–property relationships at a level of detail that is difficult to achieve with traditional synthetic polymers because of the precision with which their sequence can be defined. In the present work, we have studied the behavior and temperature-triggered self-assembly of a series of diblock recombinant elastin-like polypeptides (ELPs) with the goal of elucidating the mechanism of their self-assembly into micelles. Aqueous solutions of diblock ELPs were studied below and above their critical micellar temperature (CMT) by multiangle light scattering and small-angle neutron scattering techniques. Below the CMT, the radius of gyration of soluble ELP chains follows a power law as a function of molecular weight with an exponent value close to 0.5 that is characteristic of Gaussian coil conformations. As the temperature reaches the CMT, attractive interactions between the more hydrophobic block of diblock ELP chains leads to the self-assembly of monodisperse spherical micelles at thermodynamic equilibrium. Above the CMT, micelles expel water molecules from their core whose densification is evidenced by the monotonic increase in the light and neutron scattering intensities as a function of temperature. The behaviors of these different diblock ELPs in solution and as self-assembled nanoparticles above the CMT following universal experimental scaling laws make them analogous to synthetic amphiphilic diblock copolymers (star-like vs crew-cut micelle models). These studies also shed light on the important role of water in the thermal behavior of these thermally responsive self-assembling diblock polypeptides and suggest a new design parameter—thermally triggered desolvation and densification of the core of micelles—that can be fine-tuned at the sequence level to control the density of self-assembled polymer nanoparticles.
Co-reporter:Joseph J. Bellucci;Dr. Jayanta Bhattacharyya ; Ashutosh Chilkoti
Angewandte Chemie International Edition 2015 Volume 54( Issue 2) pp:441-445
Publication Date(Web):
DOI:10.1002/anie.201408126
Abstract
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.
Co-reporter:Dr. Somnath Bhattacharjee;Dr. Wenge Liu;Dr. Wei-Han Wang;Isaac Weitzhler;Dr. Xinghai Li;Yizhi Qi;Dr. Jinyao Liu;Dr. Yan Pang;Dr. Donald F. Hunt;Dr. Ashutosh Chilkoti
ChemBioChem 2015 Volume 16( Issue 17) pp:2451-2455
Publication Date(Web):
DOI:10.1002/cbic.201500439
Abstract
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.
Co-reporter:Wafa Hassouneh, Ekaterina B. Zhulina, Ashutosh Chilkoti, and Michael Rubinstein
Macromolecules 2015 Volume 48(Issue 12) pp:4183-4195
Publication Date(Web):June 11, 2015
DOI:10.1021/acs.macromol.5b00431
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.
Co-reporter:Vinalia Tjong, Lei Tang, Stefan Zauscher and Ashutosh Chilkoti
Chemical Society Reviews 2014 vol. 43(Issue 5) pp:1612-1626
Publication Date(Web):18 Dec 2013
DOI:10.1039/C3CS60331H
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.
Co-reporter:Lei Tang;Vinalia Tjong;Nan Li;Yaroslava G. Yingling;Stefan Zauscher
Advanced Materials 2014 Volume 26( Issue 19) pp:3050-3054
Publication Date(Web):
DOI:10.1002/adma.201306049
Co-reporter:Sarah R. MacEwan and Ashutosh Chilkoti
Nano Letters 2014 Volume 14(Issue 4) pp:2058-2064
Publication Date(Web):March 10, 2014
DOI:10.1021/nl5002313
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.
Co-reporter:Jonathan R. McDaniel, Isaac Weitzhandler, Sylvain Prevost, Kevin B. Vargo, Marie-Sousai Appavou, Daniel A. Hammer, Michael Gradzielski, and Ashutosh Chilkoti
Nano Letters 2014 Volume 14(Issue 11) pp:6590-6598
Publication Date(Web):September 30, 2014
DOI:10.1021/nl503221p
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.
Co-reporter:Jonathan R. McDaniel, Sarah R. MacEwan, Xinghai Li, D. Christopher Radford, Chelsea D. Landon, Mark Dewhirst, and Ashutosh Chilkoti
Nano Letters 2014 Volume 14(Issue 5) pp:2890-2895
Publication Date(Web):April 16, 2014
DOI:10.1021/nl5009376
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.
Co-reporter:Yizhi Qi and Ashutosh Chilkoti
Polymer Chemistry 2014 vol. 5(Issue 2) pp:266-276
Publication Date(Web):11 Oct 2013
DOI:10.1039/C3PY01089A
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.
Co-reporter:Ali Ghoorchian, Ashutosh Chilkoti, and Gabriel P. López
Analytical Chemistry 2014 Volume 86(Issue 12) pp:6103
Publication Date(Web):May 16, 2014
DOI:10.1021/ac5012574
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.
Co-reporter:Vinalia Tjong, Hua Yu, Angus Hucknall, and Ashutosh Chilkoti
Analytical Chemistry 2013 Volume 85(Issue 1) pp:426
Publication Date(Web):November 29, 2012
DOI:10.1021/ac303132j
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.
Co-reporter:Yizhi Qi;Miriam Amiram;Weiping Gao;Dewey G. McCafferty
Macromolecular Rapid Communications 2013 Volume 34( Issue 15) pp:1256-1260
Publication Date(Web):
DOI:10.1002/marc.201300460
Co-reporter:Trine Christensen, Wafa Hassouneh, Kimberley Trabbic-Carlson, and Ashutosh Chilkoti
Biomacromolecules 2013 Volume 14(Issue 5) pp:
Publication Date(Web):March 25, 2013
DOI:10.1021/bm400167h
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.
Co-reporter:Wafa Hassouneh, Michelle L. Nunalee, M. Coleman Shelton, and Ashutosh Chilkoti
Biomacromolecules 2013 Volume 14(Issue 7) pp:
Publication Date(Web):May 24, 2013
DOI:10.1021/bm400464s
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.
Co-reporter:Jonathan R. McDaniel;Dr. Jayanta Bhattacharyya;Kevin B. Vargo;Wafa Hassouneh; Daniel A. Hammer; Ashutosh Chilkoti
Angewandte Chemie International Edition 2013 Volume 52( Issue 6) pp:1683-1687
Publication Date(Web):
DOI:10.1002/anie.201200899
Co-reporter:Jonathan R. McDaniel, D. Christopher Radford, and Ashutosh Chilkoti
Biomacromolecules 2013 Volume 14(Issue 8) pp:
Publication Date(Web):July 1, 2013
DOI:10.1021/bm4007166
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 (Tt). Although this property has been exploited in a variety of biotechnological applications, de novo design of ELPs that display a specific Tt is not trivial because the Tt 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 Tt 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 Tt in physiological buffer within a specified concentration regime, thereby greatly facilitating the design of new ELPs for applications in medicine and biotechnology.
Co-reporter:Jonathan R. McDaniel;Dr. Jayanta Bhattacharyya;Kevin B. Vargo;Wafa Hassouneh; Daniel A. Hammer; Ashutosh Chilkoti
Angewandte Chemie 2013 Volume 125( Issue 6) pp:1727-1731
Publication Date(Web):
DOI:10.1002/ange.201200899
Co-reporter:Joseph J. Bellucci;Dr. Miriam Amiram;Dr. Jayanta Bhattacharyya; Dewey McCafferty; Ashutosh Chilkoti
Angewandte Chemie 2013 Volume 125( Issue 13) pp:3791-3796
Publication Date(Web):
DOI:10.1002/ange.201208292
Co-reporter:Joseph J. Bellucci;Dr. Miriam Amiram;Dr. Jayanta Bhattacharyya; Dewey McCafferty; Ashutosh Chilkoti
Angewandte Chemie International Edition 2013 Volume 52( Issue 13) pp:3703-3708
Publication Date(Web):
DOI:10.1002/anie.201208292
Co-reporter:Miriam Amiram;Xinghai Li;Mark N. Feinglos;Kelli M. Luginbuhl
PNAS 2013 Volume 110 (Issue 8 ) pp:2792-2797
Publication Date(Web):2013-02-19
DOI:10.1073/pnas.1214518110
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.
Co-reporter:Daniel J. Callahan, Wenge Liu, Xinghai Li, Matthew R. Dreher, Wafa Hassouneh, Minkyu Kim, Piotr Marszalek, and Ashutosh Chilkoti
Nano Letters 2012 Volume 12(Issue 4) pp:2165-2170
Publication Date(Web):March 14, 2012
DOI:10.1021/nl300630c
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 Zn2+ 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.
Co-reporter:Sarah R. MacEwan and Ashutosh Chilkoti
Nano Letters 2012 Volume 12(Issue 6) pp:3322-3328
Publication Date(Web):May 24, 2012
DOI:10.1021/nl301529p
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 (ELPBCs). A greater than 8-fold increase in cellular uptake occurs when Arg residues are presented on the corona of ELPBC micelles, as compared to the same ELPBC 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.
Co-reporter:Daisuke Asai, Donghua Xu, Wenge Liu, Felipe Garcia Quiroz, Daniel J. Callahan, Michael R. Zalutsky, Stephen L. Craig, Ashutosh Chilkoti
Biomaterials 2012 33(21) pp: 5451-5458
Publication Date(Web):
DOI:10.1016/j.biomaterials.2012.03.083
Co-reporter:Wafa Hassouneh, Karl Fischer, Sarah R. MacEwan, Robert Branscheid, Chuan Lawrence Fu, Rihe Liu, Manfred Schmidt, and Ashutosh Chilkoti
Biomacromolecules 2012 Volume 13(Issue 5) pp:
Publication Date(Web):April 19, 2012
DOI:10.1021/bm300321n
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 (ELPBC) 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 αvβ3 integrin. The self-assembly of these protein–ELPBC fusions as a function of temperature was investigated by UV spectroscopy, light scattering, and cryo-TEM. Self-assembly of the ELPBC 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 ELPBC. 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 ELPBC micelles, as indicated by the enhanced uptake of the Fn3-decorated ELPBC micelles in comparison to the unimer by cells that overexpress the αvβ3 integrin. The fusion of single domain proteins to ELPBCs may provide a ubiquitous platform for the multivalent presentation of proteins.
Co-reporter:Donghua Xu, Daisuke Asai, Ashutosh Chilkoti, and Stephen L. Craig
Biomacromolecules 2012 Volume 13(Issue 8) pp:
Publication Date(Web):July 12, 2012
DOI:10.1021/bm300760s
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 (H2O2). At fixed concentration of Cys-ELP, the gelation time can be tuned by the concentration of H2O2. 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.
Co-reporter:Weiping Gao, Donghua Xu, Dong Woo Lim, Stephen L. Craig and Ashutosh Chilkoti
Polymer Chemistry 2011 vol. 2(Issue 7) pp:1561-1566
Publication Date(Web):17 May 2011
DOI:10.1039/C1PY00074H
We report the in situgrowth 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 situgrowth 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.
Co-reporter:Vinalia Tjong, Hua Yu, Angus Hucknall, Srinath Rangarajan, and Ashutosh Chilkoti
Analytical Chemistry 2011 Volume 83(Issue 13) pp:5153
Publication Date(Web):May 23, 2011
DOI:10.1021/ac200946t
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.
Co-reporter:Peng Yang, Stella M. Marinakos, and Ashutosh Chilkoti
Langmuir 2011 Volume 27(Issue 4) pp:1463-1471
Publication Date(Web):December 13, 2010
DOI:10.1021/la104186n
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.
Co-reporter:Weiping Gao;Wenge Liu;Michael R. Zalutsky;Trine Christensen
PNAS 2010 Volume 107 (Issue 38 ) pp:16432-16437
Publication Date(Web):2010-09-21
DOI:10.1073/pnas.1006044107
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 (Rh): 21 nm) showed a 15-fold increase in its blood exposure compared to the protein (Rh: 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.
Co-reporter:Shahrul Alang Ahmad, Angus Hucknall, Ashutosh Chilkoti and Graham J. Leggett
Langmuir 2010 Volume 26(Issue 12) pp:9937-9942
Publication Date(Web):March 31, 2010
DOI:10.1021/la100438d
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 cm2. 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.
Co-reporter:Andrew J. Simnick, C. Alexander Valencia, Rihe Liu and Ashutosh Chilkoti
ACS Nano 2010 Volume 4(Issue 4) pp:2217
Publication Date(Web):March 24, 2010
DOI:10.1021/nn901732h
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 (ELPBCs) 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 ELPBCs on their receptor-mediated binding and cellular uptake. The data presented herein show that (1) ligand presentation does not disrupt ELPBC 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.Keywords: block copolymer; ligand−receptor; multivalency; polypeptide; self-assembly
Co-reporter:Jonathan R. McDaniel, J. Andrew MacKay, Felipe García Quiroz and Ashutosh Chilkoti
Biomacromolecules 2010 Volume 11(Issue 4) pp:
Publication Date(Web):February 25, 2010
DOI:10.1021/bm901387t
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.
Co-reporter:J. Andrew MacKay, Daniel J. Callahan, Kelly N. FitzGerald, and Ashutosh Chilkoti
Biomacromolecules 2010 Volume 11(Issue 11) pp:
Publication Date(Web):October 6, 2010
DOI:10.1021/bm100571j
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.
Co-reporter:Angus Hucknall;Dong-Hwan Kim;Srinath Rangarajan;Ryan T. Hill;William M. Reichert
Advanced Materials 2009 Volume 21( Issue 19) pp:1968-1971
Publication Date(Web):
DOI:10.1002/adma.200803125
Co-reporter:Angus Hucknall;Srinath Rangarajan
Advanced Materials 2009 Volume 21( Issue 23) pp:2441-2446
Publication Date(Web):
DOI:10.1002/adma.200900383
Abstract
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 surface-initiated 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.
Co-reporter:Greg J. Nusz, Adam C. Curry, Stella M. Marinakos, Adam Wax and Ashutosh Chilkoti
ACS Nano 2009 Volume 3(Issue 4) pp:795
Publication Date(Web):March 18, 2009
DOI:10.1021/nn8006465
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.Keywords: biosensor; gold nanorods; label-free; limit-of-detection; nanoparticle; plasmonics; surface plasmon resonance
Co-reporter:Yiquan Wu, J. Andrew MacKay, Jonathan R. McDaniel, Ashutosh Chilkoti and Robert L. Clark
Biomacromolecules 2009 Volume 10(Issue 1) pp:
Publication Date(Web):December 10, 2008
DOI:10.1021/bm801033f
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.
Co-reporter:Weiping Gao;Wenge Liu;J. Andrew Mackay;Michael R. Zalutsky;Eric J. Toone
PNAS 2009 Volume 106 (Issue 36 ) pp:15231-15236
Publication Date(Web):2009-09-08
DOI:10.1073/pnas.0904378106
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 (Rh): 13 nm] showed a 41-fold increase in its blood exposure compared to the protein (Rh: 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.
Co-reporter:Dong Woo Lim, Dana L. Nettles, Lori A. Setton and Ashutosh Chilkoti
Biomacromolecules 2008 Volume 9(Issue 1) pp:
Publication Date(Web):December 29, 2007
DOI:10.1021/bm7007982
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.
Co-reporter:Trine Christensen, Kimberly Trabbic-Carlson, Wenge Liu, Ashutosh Chilkoti
Analytical Biochemistry 2007 Volume 360(Issue 1) pp:166-168
Publication Date(Web):1 January 2007
DOI:10.1016/j.ab.2006.09.020
Co-reporter:H. Ma;M. Wells;T. P. Beebe Jr.;A. Chilkoti
Advanced Functional Materials 2006 Volume 16(Issue 5) pp:
Publication Date(Web):16 FEB 2006
DOI:10.1002/adfm.200500426
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 “bottlebrushes” 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.
Co-reporter:Wenge Liu, Matthew R. Dreher, Dominic C. Chow, Michael R. Zalutsky, Ashutosh Chilkoti
Journal of Controlled Release 2006 Volume 114(Issue 2) pp:184-192
Publication Date(Web):28 August 2006
DOI:10.1016/j.jconrel.2006.06.001
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-β-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.
Co-reporter:Wenge Liu, Matthew R. Dreher, Darin Y. Furgeson, Katia V. Peixoto, Hong Yuan, Michael R. Zalutsky, Ashutosh Chilkoti
Journal of Controlled Release 2006 Volume 116(Issue 2) pp:170-178
Publication Date(Web):28 November 2006
DOI:10.1016/j.jconrel.2006.06.026
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 14C-labeled ELPs that were radiolabeled during their biosynthesis in Escheriehia coli. The in vitro degradation rate of a thermally responsive 14C-labeled ELP1 ([14C] 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 °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.
Co-reporter:H. Ma;J. Hyun;Z. Zhang;T. P. Beebe Jr.;A. Chilkoti
Advanced Functional Materials 2005 Volume 15(Issue 4) pp:
Publication Date(Web):23 MAR 2005
DOI:10.1002/adfm.200400088
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.
Co-reporter:H. Ma;J. Hyun;P. Stiller;A. Chilkoti
Advanced Materials 2004 Volume 16(Issue 4) pp:
Publication Date(Web):26 FEB 2004
DOI:10.1002/adma.200305830
Co-reporter:J. Hyun;H. Ma;Z. Zhang;T.P. Beebe Jr.;A. Chilkoti
Advanced Materials 2003 Volume 15(Issue 7‐8) pp:
Publication Date(Web):9 APR 2003
DOI:10.1002/adma.200304496
Co-reporter:W. Frey;D.E. Meyer;A. Chilkoti
Advanced Materials 2003 Volume 15(Issue 3) pp:
Publication Date(Web):13 FEB 2003
DOI:10.1002/adma.200390058
Co-reporter:Matthew R. Dreher, Drazen Raucher, Narayanan Balu, O. Michael Colvin, Susan M. Ludeman, Ashutosh Chilkoti
Journal of Controlled Release 2003 Volume 91(1–2) pp:31-43
Publication Date(Web):28 August 2003
DOI:10.1016/S0168-3659(03)00216-5
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.
Co-reporter:N. Nath;A. Chilkoti
Advanced Materials 2002 Volume 14(Issue 17) pp:
Publication Date(Web):29 AUG 2002
DOI:10.1002/1521-4095(20020903)14:17<1243::AID-ADMA1243>3.0.CO;2-M
Surfaces modified with stimuli-responsive polymers (SRPs) dynamically alter their physico-chemical properties in response to changes in their environmental conditions. The triggered control of interfacial properties provided by immobilized SRPs at the solid–water interface has application in the design of biomaterials, regenerable biosensors, and microfluidic bioanalytical devices. In this article, we briefly summarize recent research in this area, followed by two recent examples of research from our laboratory on stimuli-responsive surfaces. First, we present a new assay to quantify the phase transition behavior of SRPs at the solid–water interface. This assay, which is based on the distance-dependent colorimetric properties of gold nanoparticles, provides a technically simple and convenient method to determine the effect of different variables on the lower critical solution temperature (LCST) behavior of SRPs at the solid–water interface. Second, we show that stimuli-responsive surfaces can be created by the immobilization of an elastin-like polypeptide (ELP), a thermally responsive biopolymer, on a glass surface. We exploit the phase transition of the ELP at a surface to reversibly address an ELP fusion protein to a surface. This method, which we term thermodynamically reversible addressing of proteins (TRAP), enables the reversible, spatio-temporal modulation of protein binding at the solid-liquid interface, and will enable the realization of new bioanalytical applications.
Co-reporter:D.E. Meyer, B.C. Shin, G.A. Kong, M.W. Dewhirst, A. Chilkoti
Journal of Controlled Release 2001 Volume 74(1–3) pp:213-224
Publication Date(Web):6 July 2001
DOI:10.1016/S0168-3659(01)00319-4
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°C, the accumulation of a thermally responsive ELP with a LCST of 40°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.
Co-reporter:W. Frey;C. K. Woods;A. Chilkoti
Advanced Materials 2000 Volume 12(Issue 20) pp:
Publication Date(Web):23 OCT 2000
DOI:10.1002/1521-4095(200010)12:20<1515::AID-ADMA1515>3.0.CO;2-J
Co-reporter:Jonathan R. McDaniel, Daniel J. Callahan, Ashutosh Chilkoti
Advanced Drug Delivery Reviews (30 December 2010) Volume 62(Issue 15) pp:1456-1467
Publication Date(Web):30 December 2010
DOI:10.1016/j.addr.2010.05.004
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 (Tt) of the ELP and body temperature (Tb). First, when Tt ≫ Tb, 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 Tb < Tt < 42 °C, an ELP–drug conjugate can be injected in conjunction with external application of mild hyperthermia to the tumor to induce ELP coacervation and an increase in concentration within the tumor vasculature. The third approach utilizes hydrophilic–hydrophobic ELP block copolymers that have been designed to assemble into nanoparticles in response to hyperthermai due to the independent thermal transition of the hydrophobic block, thus resulting in multivalent ligand display of a ligand for spatially enhanced vascular targeting. In the final strategy, ELPs with Tt < Tb are conjugated with radiotherapeutics, injtect intioa tumor where they undergo coacervation to form an injectable drug depot for intratumoral delivery. These injectable coacervate ELP-radionuclide depots display a long residence in the tumor and result in inhibition of tumor growth.
Co-reporter:Dominic Chow, Michelle L. Nunalee, Dong Woo Lim, Andrew J. Simnick, Ashutosh Chilkoti
Materials Science and Engineering: R: Reports (1 September 2008) Volume 62(Issue 4) pp:
Publication Date(Web):1 September 2008
DOI:10.1016/j.mser.2008.04.004
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.
Co-reporter:Vinalia Tjong, Lei Tang, Stefan Zauscher and Ashutosh Chilkoti
Chemical Society Reviews 2014 - vol. 43(Issue 5) pp:NaN1626-1626
Publication Date(Web):2013/12/18
DOI:10.1039/C3CS60331H
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.
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