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Our group recently developed a family of side-chain amino acid-functionalized poly(S-alkyl-l-homocysteines), Xaa-CH (Xaa = generic amino acid), which possess the ability to form environmentally responsive coacervates in water. In an effort to further study how the molecular structure affects polypeptide coacervate formation, we prepared side-chain amino acid-functionalized poly(S-alkyl-rac-cysteines), Xaa-rac-C, via post-polymerization modification of poly(dehydroalanine), ADH. The use of the ADH platform allowed straightforward synthesis of a diverse range of side-chain amino acid-functionalized polypeptides via direct reaction of unprotected l-amino acid 2-mercaptoethylamides with ADH. Despite their differences in the main-chain structure, we found that Xaa-rac-C can form coacervates with properties similar to those seen with Xaa-CH. These results suggest that the incorporation of side-chain amino acids onto polypeptides may be a way to generally favor coacervation. The incorporation of l-methionine in Met-rac-C allowed the preparation of coacervates with improved stability against high ionic strength media. Further, the presence of additional thioether groups in Met-rac-C resulted in an increased solubility change upon oxidation allowing facile reversible redox switching of coacervate formation in aqueous media.

ConspectusThere is considerable recent interest in the synthesis and development of peptide-based materials as mimics of natural biological assemblies that utilize proteins and peptides to form organized structures and develop beneficial properties. Due to their potential compatibility with living organisms, synthetic peptide materials are also being developed for applications such as cell grafting, therapeutic delivery, and implantable diagnostic devices. One desirable feature for such applications is the ability to design materials that can respond to stimuli by changes in their structure or properties under biologically relevant conditions. Peptide and protein assemblies can respond to stimuli, such as changes in temperature, solution pH, ions present in media, or interactions with other biomacromolecules. An exciting area of emerging research is focused on how biology uses the chemistry of sulfur-containing amino acids as a means to regulate biological processes. These concepts have been utilized and expanded in recent years to enable the development of peptide materials with readily switchable properties.The incorporation of sulfur atoms in polypeptides, peptides, and proteins provides unique sites that can be used to alter the physical and biological properties of these materials. Sulfur-containing amino acid residues, most often cysteine and methionine, are able to undergo a variety of selective chemical and enzyme-mediated reactions, which can be broadly characterized as redox or alkylation processes. These reactions often proceed under physiologically relevant conditions, can be reversible, and are significant in that they can alter residue polarity as well as conformations of peptide chains. These sulfur-based reactions are able to switch molecular and macromolecular properties of peptides and proteins in living systems and recently have been applied to synthetic peptide materials. Naturally occurring "sulfur switches" can be reversible or irreversible and are often triggered by enzymatic activity. Sulfur switches in peptide materials can also be triggered in vitro using oxidation/reduction and alkylation as well as photochemical reactions. The application of sulfur switches to peptide materials has greatly expanded the scope of these switches due to the ability to readily incorporate a wide variety of noncanonical sulfur-containing synthetic amino acids.Sulfur switches have been shown to provide considerable potential to reversibly alter peptide material properties under mild physiologically relevant conditions. An important molecular feature of sulfur-containing amino acid residues was found to be the location of sulfur atoms in the side chains. The variation of sulfur atom positions from the backbone by single bond lengths was found to significantly affect polypeptide chain conformations upon oxidation-reduction or alkylation/dealkylation reactions. With the successful adaptation of sulfur switches to peptide materials, future studies can explore how these switches affect how these materials interact with biological systems. This Account provides an overview of the different types of sulfur switch reactions found in biology and their properties and the elaboration of these switches in synthetic systems with a focus on recent developments and applications of reversible sulfur switches in peptide materials.

We report development and preparation of synthetic polypeptide based, coacervate core polyelectrolyte complex micelles, PCMs, in aqueous media, which were characterized and evaluated for the encapsulation and in vitro release of a model single-stranded RNA, polyadenylic acid, poly(A). Cationic, α-helical polypeptides pegylated at their N-termini, PEG113-b-5bn and PEG113-b-5cn, were designed to form coacervate core PCMs upon mixing with multivalent anions in aqueous media. Sodium tripolyphosphate (TPP) and poly(A) were used as model multivalent anions that allowed optimization of polypeptide composition and chain length for formation of stable, nanoscale PCMs. PEG113-b-5c27 was selected for preparation of PCMs that were characterized under different environmental conditions using dynamic light scattering, atomic force microscopy and cryoelectron microscopy. The PCMs were found to efficiently encapsulate poly(A), were stable at physiologically relevant pH and solution ionic strength, and were able to release poly(A) in the presence of excess polyvalent anions. These PCMs were found to be a promising model system for further development of polypeptide based therapeutic delivery vehicles.

We report the preparation of a new water-soluble, nonionic homopolypeptide poly(l-homoserine) as well as poly(l-homoserine) block copolymers with controllable segment lengths. The conformational preferences of poly(l-homoserine) were also determined in both the solid state and in solution. Poly(l-homoserine) is soluble in water and adopts a disordered conformation that makes it a promising addition to the small class of nonionic, water-soluble homopolypeptides with potential for development for applications in biology. Toward this goal, a poly(l-homoserine) containing a block copolypeptide was prepared and found to assemble into micro- and nanoscale vesicles in water.

Via the design of a new, soluble poly(S-alkyl-l-cysteine) precursor, a route was developed for the successful preparation of long-chain poly(dehydroalanine), ADH, as well as the incorporation of dehydroalanine residues and ADH segments into copolypeptides. Based on experimental and computational data, ADH was found to adopt a previously unobserved "hybrid coil" structure, which combines the elements of 25-helical and 310-helical conformations. Analysis of the spectroscopic properties of ADH revealed that it possesses a strong inherent blue fluorescence, which may be amenable for use in imaging applications. ADH also contains reactive electrophilic groups that allowed its efficient modification to functionalized polypeptides after reactions under mild conditions with thiol and amine nucleophiles. The combined structural, spectroscopic, and reactivity properties of ADH make it a unique reactive and fluorescent polypeptide component for utilization in self-assembled biomaterials.

We report the development of new side-chain amino acid-functionalized α-helical homopolypeptides that reversibly form coacervate phases in aqueous media. The designed multifunctional nature of the side-chains was found to provide a means to actively control coacervation via mild, biomimetic redox chemistry as well as allow response to physiologically relevant environmental changes in pH, temperature, and counterions. These homopolypeptides were found to possess properties that mimic many of those observed in natural coacervate forming intrinsically disordered proteins. Despite ordered α-helical conformations that are thought to disfavor coacervation, molecular dynamics simulations of a polypeptide model revealed a high degree of side-chain conformational disorder and hydration around the ordered backbone, which may explain the ability of these polypeptides to form coacervates. Overall, the modular design, uniform nature, and ordered chain conformations of these polypeptides were found to provide a well-defined platform for deconvolution of molecular elements that influence biopolymer coacervation and tuning of coacervate properties for downstream applications.

Selective lectin binding and sorting was achieved using thermosensitive glycoconjugates derived from recombinant elastin-like polypeptides (ELPs) in simple centrifugation-precipitation assays. A recombinant ELP, (VPGXG)40, containing periodically spaced methionine residues was used to enable chemoselective postsynthetic modification via thioether alkylation using alkyne functional epoxide derivatives. The resulting sulfonium groups were selectively demethylated to give alkyne functionalized homocysteine residues, which were then reacted with azido-functionalized monosaccharides to obtain ELP glycoconjugates with periodic saccharide functionality. These modifications were also found to allow modulation of ELP temperature dependent water solubility. The multivalent ELP glycoconjugates were evaluated for specific recognition, binding and separation of the lectin Ricinus communis agglutinin (RCA120) from a complex protein mixture. RCA120 and ELP glycoconjugate interactions were evaluated using laser scanning confocal microscopy and dynamic light scattering. Due to the thermoresponsive nature of the ELP glycoconjugates, it was found that heating a mixture of galactose-functionalized ELP and RCA120 in complex media selectively yielded a phase separated pellet of ELP-RCA120 complexes. Based on these results, ELP glycoconjugates show promise as designer biopolymers for selective protein binding and sorting.

Biomaterials hold promise for therapeutic applications in the central nervous system (CNS). Little is known about molecular factors that determine CNS foreign body responses (FBRs) in vivo, or about how such responses influence biomaterial function. Here, we probed these factors in mice using a platform of injectable hydrogels readily modified to present interfaces with different physiochemical properties to host cells. We found that biomaterial FBRs mimic specialized multicellular CNS wound responses not present in peripheral tissues, which serve to isolate damaged neural tissue and restore barrier functions. We show that the nature and intensity of CNS FBRs are determined by definable properties that significantly influence hydrogel functions, including resorption and molecular delivery when injected into healthy brain or stroke injuries. Cationic interfaces elicit stromal cell infiltration, peripherally derived inflammation, neural damage and amyloid production. Nonionic and anionic formulations show minimal levels of these responses, which contributes to superior bioactive molecular delivery. Our results identify specific molecular mechanisms that drive FBRs in the CNS and have important implications for developing effective biomaterials for CNS applications.

BackgroundHost defense peptides are a family of endogenous short peptides that are found in all living beings and play a critical role in innate immunity against infection.MethodsA nonsystematic review of host defense peptides was conducted with specific interest in properties and applications relevant to plastic and reconstructive surgery.ResultsIn addition to their direct antimicrobial actions against pathogens, including multidrug-resistant bacteria, they also demonstrate important functions in immunomodulation, tumor cell lysis, and tissue regeneration. These properties have made them a topic of clinical interest for plastic surgeons because of their potential applications as novel antibiotics, wound healing medications, and cancer therapies. The rising clinical interest has led to a robust body of literature describing host defense peptides in great depth and breadth. Numerous mechanisms have been observed to explain their diverse functions, which rely on specific structural characteristics. However, these peptides remain mostly experimental, with limited translation to clinical practice because of numerous failures to achieve acceptable results in human trials.ConclusionsDespite the broad ranging potential of these peptides for use in the field of plastic and reconstructive surgery, they are rarely discussed in the literature or at scientific meetings. In this review, the authors provide a summary of the background, structure, function, bacterial resistance, and clinical applications of host defense peptides with the goal of stimulating host defense peptide-based innovation within the field of plastic and reconstructive surgery.

The preparation and characterization of a new epoxide containing polypeptide, poly(5,6-epoxy-l-norleucine), via postpolymerization modification of poly(l-homoallylglycine) is described. Addition of thiols to the epoxide groups in poly(5,6-epoxy-l-norleucine) was studied as a means to prepare side-chain functional polypeptides. The solution properties of the derivatized polypeptides were studied in water and compared to similar thioether containing functional polypeptides prepared via different routes. Subtle differences in side-chain linkage chemistry were found to influence polypeptide solubility, chain conformation in solution, and thermoresponsive behavior. Poly(5,6-epoxy-l-norleucine) was found to be useful as a readily prepared intermediate that can be reacted with thiols to give a variety of functional polypeptides.

The preparation of new diblock copolypeptide hydrogels derived from homologs of l-methionine, that is, l-homomethionine and l-6-(methylthio)-l-norleucine is described. Compared to l-methionine residues, use of l-methionine homologs allow improved copolymerization with l-leucine residues to give well-defined block copolypeptides. These copolypeptides are subsequently modified using robust thioether alkylation reactions employing a variety of functional epoxides, which yield samples capable of forming transparent, self-healing hydrogels in water. The facile variation of different functional epoxides for postpolymerization modification is found to allow predictable functionalization and tuning of hydrogel properties by the modification of simple precursors.

IntroductionSurgical resection and systemic chemotherapy with temozolomide remain the mainstay for treatment of glioblastoma. However, many patients are not candidates for surgical resection given inaccessible tumor location or poor health status. Furthermore, despite being first line treatment, temozolomide has only limited efficacy.MethodsThe development of injectable hydrogel-based carrier systems allows for the delivery of a wide range of chemotherapeutics that can achieve high local concentrations, thus potentially avoiding systemic side effects and wide-spread neurotoxicity. To test this modality in a realistic environment, we developed a diblock copolypeptide hydrogel (DCH) capable of carrying and releasing paclitaxel, a compound that we found to be highly potent against primary gliomasphere cells.ResultsThe DCH produced minimal tissue reactivity and was well tolerated in the immune-competent mouse brain. Paclitaxel-loaded hydrogel induced less tissue damage, cellular inflammation and reactive astrocytes than cremaphor-taxol (typical taxol-carrier) or hydrogel alone. In a deep subcortical xenograft model of glioblastoma in immunodeficient mice, injection of paclitaxel-loaded hydrogel led to local tumor control and improved survival. However, the tumor cells were highly migratory and were able to eventually escape the area of treatment.ConclusionsThese findings suggest this technology may be ultimately applicable to patients with deep-seated inoperable tumors, but as currently formulated, complete tumor eradication would be highly unlikely. Future studies should focus on targeting the migratory potential of surviving cells.

Multicomponent interpenetrating network hydrogels possessing enhanced mechanical stiffness compared to their individual components were prepared via physical mixing of diblock copolypeptides that assemble by either hydrophobic association or polyion complexation in aqueous media. Optical microscopy analysis of fluorescent-probe-labeled multicomponent hydrogels revealed that the diblock copolypeptide components rapidly and spontaneously self-sort to form distinct hydrogel networks that interpenetrate at micron length scales. These materials represent a class of microscale compartmentalized hydrogels composed of degradable, cell-compatible components, which possess rapid self-healing properties and independently tunable domains for downstream applications in biology and additive manufacturing.

The synthesis of a homologous series containing five new nonionic sulfoxide containing polypeptides was described. Sulfoxide groups bestowed water solubility for all homologues, which allowed their use as a model for study of helix-coil transitions in water while avoiding contributions from charged groups or phase separation. Polypeptides were found to adopt chain conformations in water that were dependent on distance of sulfoxides from chain backbones, overall side-chain lengths, and solvent. These results allow preparation of polypeptide segments with different chain conformations without changing chemical functionality for potential use in structural studies and functional applications.

Abstract Introduction Surgical resection and systemic chemotherapy with temozolomide remain the mainstay for treatment of glioblastoma. However, many patients are not candidates for surgical resection given inaccessible tumor location or poor health status. Furthermore, despite being first line treatment, temozolomide has only limited efficacy. Methods The development of injectable hydrogel-based carrier systems allows for the delivery of a wide range of chemotherapeutics that can achieve high local concentrations, thus potentially avoiding systemic side effects and wide-spread neurotoxicity. To test this modality in a realistic environment, we developed a diblock copolypeptide hydrogel (DCH) capable of carrying and releasing paclitaxel, a compound that we found to be highly potent against primary gliomasphere cells. Results The DCH produced minimal tissue reactivity and was well tolerated in the immune-competent mouse brain. Paclitaxel-loaded hydrogel induced less tissue damage, cellular inflammation and reactive astrocytes than cremaphor-taxol (typical taxol-carrier) or hydrogel alone. In a deep subcortical xenograft model, of glioblastoma in immunodeficient mice, injection of paclitaxel-loaded hydrogel led to a high local concentration of paclitaxel and led to local tumor control and improved survival. However, the tumor cells were highly migratory and were able to eventually escape the area of treatment. Conclusions These findings suggest this technology may be ultimately applicable to patients with deep-seated inoperable tumors, but as currently formulated, complete tumor eradication would be highly unlikely. Future studies should focus on targeting the migratory potential of surviving cells.

Diblock, triblock, and pentablock copolypeptides were designed and prepared for formation of polyion complex hydrogels in aqueous media. Increasing the number of block segments was found to allow formation of hydrogels with substantially enhanced stiffness at equivalent concentrations. Use of similar length ionic segments also allowed mixing of different block architectures to fine-tune hydrogel properties. The pentablock hydrogels possess a promising combination of high stiffness, rapid self-healing properties, and cell compatible surface chemistry that makes them promising candidates for applications requiring injectable or printable hydrogel scaffolds.

The preparation and characterization of a new set of well-defined polypeptides containing N-methylaminooxy side-chain functionality is described. These functional groups enabled the direct coupling of polypeptides with a variety of unmodified reducing saccharides in water to give neoglycopolypeptides in high yields. The use of different polypeptide scaffolds resulted in neoglycoconjugates with tunable chain conformations, hydrophobicity, and charge. These new neoglycopolypeptides were also found to be stable in aqueous media at pH 7.4 and 37 °C for 1 week. The combination of straightforward synthesis using unmodified saccharides, high yields of saccharide conjugation, and conjugate stability makes these polypeptides attractive candidates for development of degradable glycoprotein mimics.

Biomaterials hold promise for diverse therapeutic applications in the central nervous system (CNS). Little is known about molecular factors that determine CNS foreign body responses (FBRs) in vivo , or about how such responses influence biomaterial function. Here, we probed these factors using a platform of injectable hydrogels readily modified to present interfaces with different representative physiochemical properties to host cells. We show that biomaterial FBRs mimic specialized multicellular CNS wound responses not present in peripheral tissues, which serve to isolate damaged neural tissue and restore barrier functions. Moreover, we found that the nature and intensity of CNS FBRs are determined by definable properties. For example, cationic, anionic or nonionic interfaces with CNS cells elicit quantifiably different levels of stromal cell infiltration, inflammation, neural damage and amyloid production. The nature and intensity of FBRs significantly influenced hydrogel resorption and molecular delivery functions. These results characterize specific molecular mechanisms that drive FBRs in the CNS and have important implications for developing effective biomaterials for CNS applications.

Biologically occurring non-canonical di-α-amino acids were converted into new di-N-carboxyanhydride (di-NCA) monomers in reasonable yields with high purity. Five different di-NCAs were separately copolymerized with tert-butyl-l-glutamate NCA to obtain covalently crosslinked copolypeptides capable of forming hydrogels with varying crosslinker density. Comparison of hydrogel properties with residue structure revealed that different di-α-amino acids were not equivalent in crosslink formation. Notably, l-cystine was found to produce significantly weaker hydrogels compared to l-homocystine, l-cystathionine, and l-lanthionine, suggesting that l-cystine may be a sub-optimal choice of di-α-amino acid for preparation of copolypeptide networks. The di-α-amino acid crosslinkers also provided different chemical stability, where disulfide crosslinks were readily degraded by reduction, and thioether crosslinks were stable against reduction. This difference in response may provide a means to fine tune the reduction sensitivity of polypeptide biomaterial networks.

Transected axons fail to regrow across anatomically complete spinal cord injuries (SCI) in adults. Diverse molecules can partially facilitate or attenuate axon growth during development or after injury1-3, but efficient reversal of this regrowth failure remains elusive4. Here we show that three factors that are essential for axon growth during development but are attenuated or lacking in adults-(i) neuron intrinsic growth capacity2,5-9, (ii) growth-supportive substrate10,11 and (iii) chemoattraction12,13-are all individually required and, in combination, are sufficient to stimulate robust axon regrowth across anatomically complete SCI lesions in adult rodents. We reactivated the growth capacity of mature descending propriospinal neurons with osteopontin, insulin-like growth factor 1 and ciliary-derived neurotrophic factor before SCI14,15; induced growth-supportive substrates with fibroblast growth factor 2 and epidermal growth factor; and chemoattracted propriospinal axons with glial-derived neurotrophic factor16,17 delivered via spatially and temporally controlled release from biomaterial depots18,19, placed sequentially after SCI. We show in both mice and rats that providing these three mechanisms in combination, but not individually, stimulated robust propriospinal axon regrowth through astrocyte scar borders and across lesion cores of non-neural tissue that was over 100-fold greater than controls. Stimulated, supported and chemoattracted propriospinal axons regrew a full spinal segment beyond lesion centres, passed well into spared neural tissue, formed terminal-like contacts exhibiting synaptic markers and conveyed a significant return of electrophysiological conduction capacity across lesions. Thus, overcoming the failure of axon regrowth across anatomically complete SCI lesions after maturity required the combined sequential reinstatement of several developmentally essential mechanisms that facilitate axon growth. These findings identify a mechanism-based biological repair strategy for complete SCI lesions that could be suitable to use with rehabilitation models designed to augment the functional recovery of remodelling circuits.

High-molecular-weight polypeptides with functional aromatic side chains, poly(4-amino-l-phenylalanine), were prepared by the metal-initiated polymerization of the Nα-carboxyanhydride of the corresponding amino acid, which is a microbial derivative of phenylalanine.

Homoallylglycine N-carboxyanhydride, Hag NCA, monomers were synthesized and used to prepare polypeptides containing Hag segments with controllable lengths of up to 245 repeats. Poly(l-homoallylglycine), GHA, was found to adopt an α-helical conformation, which provided good solubility in organic solvents and allowed high yield functionalization of its alkene side-chains via radical promoted addition of thiols. The conformations of these derivatives were shown to be switchable between α-helical and disordered states in aqueous media using thioether alkylation or oxidation reactions. Incorporation of GHA segments into block copolymers with poly(l-methionine), M, segments provided a means to orthogonally modify thioether side-chains different ways in separate copolypeptide domains. This approach allows preparation of functional polypeptides containing discrete domains of oxidized and alkylated thioether containing residues, where chain conformation and functionality of each domain can be independently modified.

Synthetic diblock copolypeptides were designed to incorporate oppositely charged ionic segments that form β-sheet-structured hydrogel assemblies via polyion complexation when mixed in aqueous media. The observed chain conformation directed assembly was found to be required for efficient hydrogel formation and provided distinct and useful properties to these hydrogels, including self-healing after deformation, microporous architecture, and stability against dilution in aqueous media. While many promising self-assembled materials have been prepared using disordered or liquid coacervate polyion complex (PIC) assemblies, the use of ordered chain conformations in PIC assemblies to direct formation of new supramolecular morphologies is unprecedented. The promising attributes and unique features of the β-sheet-structured PIC hydrogels described here highlight the potential of harnessing conformational order derived from PIC assembly to create new supramolecular materials.

Antimicrobial properties of a long-chain, synthetic, cationic, and hydrophobic amino acid block copolymer are reported. In 5 and 60 min time-kill assays, solutions of K100 L40 block copolymers (poly(l-lysine·hydrochloride)100 -b-poly(l-leucine)40 ) at concentrations of 10-100 µg mL-1 show multi-log reductions in colony forming units of Gram-positive and Gram-negative bacteria, as well as yeast, including multidrug-resistant strains. Driven by association of hydrophobic segments, K100 L40 copolymers form viscous solutions and self-supporting hydrogels in water at concentrations of 1 and 2 wt%, respectively. These K100 L40 preparations provide an effective barrier to microbial contamination of wounds, as measured by multi-log decreases of tissue-associated bacteria with deliberate inoculation of porcine skin explants, porcine open wounds, and rodent closed wounds with foreign body. Based on these findings, amino acid copolymers with the features of K100 L40 can combine potent, direct antimicrobial activity and barrier properties in one biopolymer for a new approach to prevention of wound infections.

We report the synthesis of methionine-containing recombinant elastin-like polypeptides (ELPs) of different lengths that contain periodically spaced methionine residues. These ELPs were chemoselectively alkylated at all methionine residues to give polycationic derivatives. Some of these samples were found to possess solubility transitions in water, where the temperature of these transitions varied with ELP concentration, nature of the methionine alkylating group, and nature of the sulfonium counterions. These studies show that introduction and controlled spacing of methionine sulfonium residues into ELPs can be used as a means both to tune their solubility transition temperatures in water using a variety of different parameters and to introduce new side-chain functionality.

Recent developments in the modification of methionine and other thioether-containing residues in peptides, polypeptides, and proteins are reviewed. Properties and potential applications of the resulting functionalized products are also discussed. While much of this work is focused on natural Met residues, modifications at other side-chain residues have also emerged as new thioether-containing amino acids have been incorporated into peptidic materials. Functional modification of thioether-containing amino acids has many advantages and is a complementary methodology to the widely utilized methods for modification at cysteine residues.

We have designed and prepared a recombinant elastin-like polypeptide (ELP) containing precisely positioned methionine residues, and performed the selective and complete oxidation of its methionine thioether groups to both sulfoxide and sulfone derivatives. Since these oxidation reactions substantially increase methionine residue polarity, they were found to be a useful means to precisely adjust the temperature responsive behavior of ELPs in aqueous solutions. In particular, lower critical solution temperatures were found to be elevated in oxidized sample solutions, but were not eliminated. These transition temperatures were found to be further tunable by the use of solvents containing different Hofmeister salts. Overall, the ability to selectively and fully oxidize methionine residues in ELPs proved to be a convenient postmodification strategy for tuning their transition temperatures in aqueous media.

Targeted killing of cancer cells by engineered nanoparticles holds great promise for noninvasive photothermal therapy applications. We present the design and generation of a novel class of gold nanoshells with cores composed of self-assembled block copolypeptide vesicles with photothermal properties. Specifically, poly(L-lysine)60- block-poly(L-leucine)20 (K60L20) block copolypeptide vesicles coated with a thin layer of gold demonstrate enhanced absorption of light due to surface plasmon resonance (SPR) in the near-infrared range. We show that the polypeptide-based K60L20 gold nanoshells have low toxicity in the absence of laser exposure, significant heat generation upon exposure to near-infrared light, and, as a result, localized cytotoxicity within the region of laser irradiation in vitro. To gain a better understanding of our gold nanoshells in the context of photothermal therapy, we developed a comprehensive mathematical model for heat transfer and experimentally validated this model by predicting the temperature as a function of time and position in our experimental setup. This model can be used to predict which parameters of our gold nanoshells can be manipulated to improve heat generation for tumor destruction. To our knowledge, our results represent the first ever use of block copolypeptide vesicles as the core material of gold nanoshells.

Currently, there is no curative treatment for advanced metastatic prostate cancer, and options, such as chemotherapy, are often nonspecific, harming healthy cells and resulting in severe side effects. Attaching targeting ligands to agents used in anticancer therapies has been shown to improve efficacy and reduce nonspecific toxicity. Furthermore, the use of triggered therapies can enable spatial and temporal control over the treatment. Here, we combined an engineered prostate cancer-specific targeting ligand, the A11 minibody, with a novel photothermal therapy agent, polypeptide-based gold nanoshells, which generate heat in response to near-infrared light. We show that the A11 minibody strongly binds to the prostate stem cell antigen that is overexpressed on the surface of metastatic prostate cancer cells. Compared to nonconjugated gold nanoshells, our A11 minibody-conjugated gold nanoshell exhibited significant laser-induced, localized killing of prostate cancer cells in vitro. In addition, we improved upon a comprehensive heat transfer mathematical model that was previously developed by our laboratory. By relaxing some of the assumptions of our earlier model, we were able to generate more accurate predictions for this particular study. Our experimental and theoretical results demonstrate the potential of our novel minibody-conjugated gold nanoshells for metastatic prostate cancer therapy.

New poly(L-lysine)-b-poly(ethylene glycol) copolypeptides have been prepared, where the side-chain amine groups of lysine residues are modified to contain ortho-amine substituted phenylboronic acid, i.e., Wulff-type phenylboronic acid (WBA), groups to improve their pH responsive, carbohydrate binding properties. These block copolymers form nanoscale complexes with glycosylated proteins that are stable at physiological pH, yet dissociate and release the glycoproteins under acidic conditions, similar to those found in endosomal and lysosomal compartments within cells. These results suggest that WBA modified polypeptide copolymers are promising for further development as degradable carriers for intracellular protein delivery.

We have prepared a new l-lysine-based N-carboxyanhydride monomer containing a photolabile o-nitrobenzyloxycarbonyl protecting group. This monomer was used to prepare poly(l-lysine)-block-poly(oNB-l-lysine) block copolypeptides that formed hydrogels with tunable physical properties and the capability to be degraded by UV irradiation. In these materials, the oNB-lysine residues were found to be excellent surrogates for the hydrophobic residues typically used to form block copolypeptide hydrogels, thus adding functionality without adversely altering self-assembly characteristics. Upon irradiation, full cleavage of the o-nitrobenzyloxycarbonyl groups was observed, resulting in dissolution of the product, poly(l-lysine), and complete hydrogel disruption. When dye molecules were entrapped in the hydrogels, photolysis resulted in release and mixing of these molecules with the surrounding media.

We have developed a facile method for preparation of ionic copolypeptide vesicles that requires no protecting groups or expensive components. We prepared amphiphilic copolypeptides containing segments of water-soluble methionine sulfonium residues. These were derived by aqueous phase, direct alkylation of a fully hydrophobic precursor diblock copolypeptide, poly(L-methionine)65-block-poly(L-leucine0.5-stat-L-phenylalanine0.5)20 (M65[L0.5/F0.5]20), with simple alkylating reagents. Methylation of M65(L0.5/F0.5)20 gave the cationic methyl-methionine sulfonium derivative MM65(L0.5/F0.5)20, and carboxymethylation gave the zwitterionic carboxymethyl-methionine sulfonium derivative MC65(L0.5/F0.5)20. Assembly of MM65(L0.5/F0.5)20 or MC65(L0.5/F0.5)20 in water gave rise to vesicles with average diameters of a few microns, which could then be extruded to nanoscale diameters. While the cationic MM-based vesicles were found to be cytotoxic, the zwitterionic MC-based vesicles were found to possess minimal cytotoxicity.

A series of thermoresponsive polypeptides has been synthesized using a methodology that allowed facile adjustment of side-chain functional groups. The lower critical solution temperature (LCST) properties of these polymers in water were then evaluated relative to systematic molecular modifications in their side-chains. It was found that in addition to the number of ethylene glycol repeats in the side-chains, terminal and linker groups also have substantial and predictable effects on cloud point temperatures (Tcp). In particular, we found that the structure of these polypeptides allowed for inclusion of polar hydroxyl groups, which significantly increased their hydrophilicity and decreased the need to use long oligoethylene glycol repeats to obtain LCSTs. The thioether linkages in these polypeptides were found to provide an additional structural feature for reversible switching of both polypeptide conformation and thermoresponsive properties.

A methodology was developed for efficient, chemoselective transformation of methionine residues into stable, functional homocysteine derivatives. Methionine residues can undergo highly chemoselective alkylation reactions at low pH to yield stable sulfonium ions, which could then be selectively demethylated to give stable alkyl homocysteine residues. This mild, two-step process is chemoselective, efficient, tolerates many functional groups, and provides a means for creation of new functional biopolymers, site-specific peptide tagging, and synthesis of biomimetic and structural analogs of peptides.

Transected axons fail to regrow in the mature central nervous system. Astrocytic scars are widely regarded as causal in this failure. Here, using three genetically targeted loss-of-function manipulations in adult mice, we show that preventing astrocyte scar formation, attenuating scar-forming astrocytes, or ablating chronic astrocytic scars all failed to result in spontaneous regrowth of transected corticospinal, sensory or serotonergic axons through severe spinal cord injury (SCI) lesions. By contrast, sustained local delivery via hydrogel depots of required axon-specific growth factors not present in SCI lesions, plus growth-activating priming injuries, stimulated robust, laminin-dependent sensory axon regrowth past scar-forming astrocytes and inhibitory molecules in SCI lesions. Preventing astrocytic scar formation significantly reduced this stimulated axon regrowth. RNA sequencing revealed that astrocytes and non-astrocyte cells in SCI lesions express multiple axon-growth-supporting molecules. Our findings show that contrary to the prevailing dogma, astrocyte scar formation aids rather than prevents central nervous system axon regeneration.

We previously investigated the intracellular trafficking properties of our novel poly(l-glutamate)60-b-poly(l-leucine)20 (E60L20) vesicles (EL vesicles) conjugated to transferrin (Tf). In this study, we expand upon our previous work by investigating the drug encapsulation, release, and efficacy properties of our novel EL vesicles for the first time. After polyethylene glycol (PEG) was conjugated to the vesicles for steric stability, doxorubicin (DOX) was successfully encapsulated in the vesicles using a modified pH-ammonium sulfate gradient method. Tf was subsequently conjugated to the vesicles to provide active targeting to cancer cells and a mode of internalization into the cells. These Tf-conjugated, DOX-loaded, PEGylated EL (Tf-DPEL) vesicles exhibited colloidal stability and were within the allowable size range for passive and active targeting. A mathematical model was then derived to predict drug release from the Tf-DPEL vesicles by considering diffusive and convective mass transfer of DOX. Our mathematical model reasonably predicted our experimentally measured release profile with no fitted parameters, suggesting that the model could be used in the future to manipulate drug carrier properties to alter drug release profiles. Finally, an in vitro cytotoxicity assay was used to demonstrate that the Tf-DPEL vesicles exhibited enhanced drug carrier efficacy in comparison to its non-targeted counterpart.

Tuning the lower critical solution temperature (LCST) of temperature-responsive recombinant elastin-like polypeptides has usually been achieved by designing different protein sequences, in terms of amino acid composition and length, implying tedious molecular cloning steps. In the present work, we have explored the chemoselective alkylation of methionine as an easy means to modify elastin repeat side chains and easily modulate the LCST of the polypeptides. Such a versatile synthetic method shall practically be exploited to modulate any properties of recombinant polymers.

Biomaterial vehicles have the potential to facilitate cell transplantation in the central nervous system (CNS). We have previously shown that highly tunable ionic diblock copolypeptide hydrogels (DCH) can provide sustained release of hydrophilic and hydrophobic molecules in the CNS. Here, we show that recently developed non-ionic and thermoresponsive DCH called DCHT exhibit excellent cytocompatibility. Neural stem cell (NSC) suspensions in DCHT were easily injected as liquids at room temperature. DCHT with a viscosity tuned to prevent cell sedimentation and clumping significantly increased the survival of NSC passed through injection cannulae. At body temperature, DCHT self-assembled into hydrogels with a stiffness tuned to that of CNS tissue. After injection in vivo, DCHT significantly increased by three-fold the survival of NSC grafted into healthy CNS. In injured CNS, NSC injected as suspensions in DCHT distributed well in non-neural lesion cores, integrated with healthy neural cells at lesion perimeters and supported regrowing host nerve fibers. Our findings show that non-ionic DCHT have numerous advantageous properties that make them useful tools for in vivo delivery of cells and molecules in the CNS for experimental investigations and potential therapeutic strategies.

Methodology was developed for efficient alkylation of methionine residues using epoxides as a general strategy to introduce a wide range of functional groups onto polypeptides. Use of a spacer between epoxide and functional groups further allowed addition of sterically demanding functionalities. Contrary to other methods to alkylate methionine residues, epoxide alkylations allow the reactions to be conducted in wet protic media and give sulfonium products that are stable against dealkylation. These functionalizations are notable since they are chemoselective, utilize stable and readily available epoxides, and allow facile incorporation of an unprecedented range of functional groups onto simple polypeptides using stable linkages.

Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess low cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs.

We report methods for the synthesis of polypeptides that are fully functionalized with desirable phosphorylcholine, PC, groups. Because of the inherent challenges in the direct incorporation of the PC group into α-amino acid N-carboxyanhydride (NCA) monomers, we developed a synthetic approach that combined functional NCA polymerization with efficient postpolymerization modification. While poly(L-phosphorylcholine serine) was found to be unstable upon synthesis, we successfully prepared poly(L-phosphorylcholine homoserine) with controlled chain lengths and found these to be water-soluble with disordered chain conformations.