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.

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.

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.

•Therapeutic effect of systemic temozolomide is limited by the blood brain barrier.•Temozolomide can be loaded onto a novel hydrogel matrix.•Hydrogels locally deliver TMZ to the tumor mass and bypass the BBB.•The hydrogels tested are not toxic in vitro or in vivo.•The efficacy of TMZ in glioblastoma models is enhanced by hydrogel delivery.The current treatment of glioblastoma multiforme (GBM) is limited by the restricted arsenal of agents which effectively cross the blood brain barrier (BBB). For example, only a fraction of temozolomide (TMZ) administered systemically is available for therapeutic effect because of the BBB and the instability of TMZ under physiologic conditions. A novel approach to overcome this obstacle is to bypass the BBB and locally deliver chemotherapeutic agents directly to the tumor mass. We have explored the loading of TMZ into a novel hydrogel matrix, which can be delivered in liquid form and then solidifies in situ and releases chemotherapy as the matrix dissolves. Here, we tested the effect of amphiphilic diblock copolypeptide hydrogels (DCHs) of 180-poly-lysine and 20-poly-leucine (K180L20) on TMZ using Glioblastoma models. In both the in vitro model, which involved treatment of a human glioblastoma GSC line suspended as neurospheres, and in vivo using an orthotopic glioma xenograft mouse model, we found that K180L20 could safely enhance the efficacy of TMZ. This technique may offer the opportunity to ‘coat’ the inner lining of the cavity following glioma resection with a slow-release TMZ and potentially decrease recurrence. Future studies in larger animals are needed to delineate this effect.

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.

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.

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.

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.

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.

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.

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.

Polypeptide-based formulations that undergo liquid to hydrogel transitions upon change in temperature have become desirable targets since they can be mixed with cells or injected into tissues as liquids, and subsequently transform into rigid scaffolds or depots. Such materials have been challenging to prepare using synthetic polypeptides, especially when reversible gelation and tunable physical properties are desired. Here, we designed and prepared new nonionic diblock copolypeptide hydrogels (DCH) containing hydrophilic poly(γ-[2-(2-methoxyethoxy)ethyl]-rac-glutamate) and hydrophobic poly(l-leucine) segments, named DCHEO, and also further incorporated copolypeptide domains into DCHEO to yield unprecedented thermoresponsive DCH, named DCHT. Although previous attempts to prepare nonionic hydrogels composed solely of synthetic polypeptides have been unsuccessful, our designs yielded materials with highly reversible thermal transitions and tunable properties. Nonionic, thermoresponsive DCHT were found to support the viability of suspended mesenchymal stem cells in vitro and were able to dissolve and provide prolonged release of both hydrophilic and hydrophobic molecules. The versatility of these materials was further demonstrated by the independent molecular tuning of DCHT liquid viscosity at room temperature and DCHT hydrogel stiffness at elevated temperature, as well as the DCHT liquid to hydrogel transition temperature itself.

We report the design and synthesis of poly(S-alkyl-l-homocysteine)s, which were found to be a new class of readily prepared, multiresponsive polymers that possess the unprecedented ability to respond in different ways to different stimuli, either through a change in chain conformation or in water solubility. The responsive properties of these materials are also effected under mild conditions and are completely reversible for all pathways. The key components of these polymers are the incorporation of water solubilizing alkyl functional groups that are integrated with precisely positioned, multiresponsive thioether linkages. This promising system allows multimodal switching of polypeptide properties to obtain desirable features, such as coupled responses to multiple external inputs.

Glycosylated peptides and proteins are ubiquitous in nature and display a wide range of biological functions including mediation of recognition events, protection from proteases, and lubrication in eyes and joints. Similarly, synthetic glycopolypeptides are also expected to show great potential as biomedical materials (e.g. scaffolds for tissue repair and drug carriers), as well as serve as valuable tools for probing carbohydrate–protein interactions. Although block copolypeptides and other complex polypeptide architectures have been known for some time, the synthesis of complex and well-defined glycopolypeptide materials, until recently, has been challenging. This article reviews the many advances and accomplishments made in the past few years toward development of strategies and methods for the preparation of synthetic glycopolypeptides via ring opening polymerization.

We have synthesized new phosphonate-containing α-amino acid N-carboxyanhydride (NCA) monomers and used these to prepare well-defined phosphonate-containing polypeptides and block copolypeptides. These NCAs were found to undergo living polymerization to high molecular weights with narrow chain length distributions. The methods described here demonstrate that phosphono polypeptides are also readily incorporated into block copolymers with controlled architecture and composition. One polymer, poly(l-phosphonohomoalanine) (poly(Pha)), is an isosteric analogue of poly(l-phosphoserine) and was found to display a pH-responsive conformational change. The pH-responsive α-helical conformation of poly(Pha) is unprecedented and may prove to be useful in the preparation of stimuli-responsive block copolypeptides containing poly(Pha) segments.

Reagents were developed to allow chemoselective tagging of methionine residues in peptides and polypeptides, subsequent bioorthogonal functionalization of the tags, and cleavage of the tags when desired. This methodology can be used for triggered release of therapeutic peptides, or release of tagged protein digests from affinity columns.

We sought to modify adenoviral (Ad) particles by incorporating the advantageous characteristics of non-viral gene delivery vehicles to complement the viral vectors. α-Amino acid-N-carboxyanhydride chemistry was used to synthesize homopolypeptides and diblock copolypeptides that possess well-defined secondary structures. Using cryo-electron and fluorescence microscopy, we showed that these polypeptides can coat the surfaces of Ad particles in a non-covalent manner to modify their transduction properties. The coated Ad particles were found to bind to and be internalized by cells. In contrast to reports using covalently PEGylated Ad particles, we found that our physically coated Ad hybrid complexes facilitate gene transfer both in vitro and in vivo. We showed that our polypeptide coating was able to shield the Ad particles from the neutralizing effect of antibodies and mitigate the binding of blood coagulation factor (Factor X) in vitro. The coating also reduced the antigenicity of Ad in immunocompetent mice. The biodistribution of the systemically administered hybrid complexes mirrored the behavior of both viral and non-viral vectors, exhibiting liver tropism as well as enhanced lung transduction. These data demonstrated that our non-covalent modification was able to alter Ad's interactions with cells and organs with retention of transduction efficiency. Advantages such as facile coating of the Ad vector, design flexibility and ease of attaching ligands to the polypeptides make this system potentially useful as a platform for adding functionalities to Ad to target cancer metastasis.

We report a method for the synthesis of soluble, well-defined, azide-functionalized polypeptides via living polymerization of new azide-containing amino acid N-carboxyanhydride (NCA) monomers. Homo and diblock azidopolypeptides were prepared with controlled segment lengths using (PMe3)4Co initiator and were subsequently modified by reaction with functional alkyne reagents. The azide groups were found to be quantitatively converted to the corresponding triazole derivatives, and the functionalized polymers were obtained in high yield. This methodology provides a facile and straightforward method for preparation of functional and side-chain reactive, high molecular weight polypeptides.

We describe the preparation and assembly of glycosylated amphiphilic diblock copolypeptides, where the hydrophilic glycosylated segments adopt either α-helical or disordered conformations. In this study, glycosylated amphiphilic diblock copolypeptides were prepared using poly(L-leucine), poly(L), as the hydrophobic segment, and poly(α-D-galactopyranosyl-L-lysine), poly(α-gal-K), or poly(α-D-galactopyranosyl-L-cysteine sulfone), poly(α-gal-CO2), as the hydrophilic segment. The poly(α-gal-K) and poly(α-gal-CO2) segments are known to be fully α-helical (>90% at 20 °C) and fully disordered in water, respectively. We found that block copolypeptides containing galactosylated hydrophilic segments of either α-helical or disordered conformation give different assembly morphologies, where the disordered glycopolypeptide segments favor vesicle formation and also present sugar residues that can bind to biological targets.

We have developed a facile, scalable method for preparation of enzyme-responsive copolypeptide vesicles that requires no protecting groups or expensive components. We designed amphiphilic copolypeptides containing segments of water-soluble methionine sulfoxide, MO, residues that were prepared by synthesis of a fully hydrophobic precursor diblock copolypeptide, poly(l-methionine)65-b-poly(l-leucine0.5-stat-l-phenylalanine0.5)20, M65(L0.5/F0.5)20, followed by its direct oxidation in water to give the amphiphilic MO derivative, MO65(L0.5/F0.5)20. Assembly of MO65(L0.5/F0.5)20 in water gave vesicles with average diameters of a few micrometers that could then be extruded to nanoscale diameters. The MO segments in the vesicles were found to be substrates for reductase enzymes, which regenerated hydrophobic M segments and resulted in a change in supramolecular morphology that caused vesicle disruption and release of cargos.

Here, we report a method for synthesis of cylindrical copolypeptide brushes via N-carboxyanhydride (NCA) polymerization utilizing a new tandem catalysis approach that allows preparation of brushes with controlled segment lengths in a straightforward, one-pot procedure requiring no intermediate isolation or purification steps. To obtain high-density brush copolypeptides, we used a “grafting from” approach where alloc-α-aminoamide groups were installed onto the side chains of NCAs to serve as masked initiators. These groups were inert during cobalt-initiated NCA polymerization and gave allyloxycarbonyl-α-aminoamide-substituted polypeptide main chains. The alloc-α-aminoamide groups were then activated in situ using nickel to generate initiators for growth of side-chain brush segments. This use of stepwise tandem cobalt and nickel catalysis was found to be an efficient method for preparation of high-chain-density, cylindrical copolypeptide brushes, where both the main chains and side chains can be prepared with controlled segment lengths.

Conformation-switchable glycopolypeptides were prepared by the living polymerization of glycosylated l-cysteine-N-carboxyanhydride (glyco-C NCA) monomers. These new monomers were prepared in high yield by coupling of alkene-terminated C-linked glycosides of d-galactose or d-glucose to l-cysteine using thiol–ene “click” chemistry, followed by their conversion to the corresponding glyco-C NCAs. The resulting glycopolypeptides were found to be water-soluble and α-helical in solution. Aqueous oxidation of the side-chain thioether linkages in these polymers to sulfone groups resulted in disruption of the α-helical conformations without loss of water solubility. The ability to switch chain conformation and remain water-soluble is unprecedented in synthetic polymers, and allows new capabilities to control presentation of sugar functionality in subtly different contexts.

We report the development of a new “click”-type reaction for polypeptide modification based on the chemoselective alkylation of thioether groups in methionine residues. The controlled synthesis of methionine polymers and their alkylation by a broad range of functional reagents to yield stable sulfonium derivatives are described. These “methionine click” functionalizations are compatible with deprotection of other functional groups, use an inexpensive, natural amino acid that is readily polymerized and requires no protecting groups, and allow the introduction of a diverse range of functionality and reactive groups onto polypeptides.

We have prepared stable nano- and microscale emulsion droplets using block copolypeptide surfactants that have been functionalized with biotin. We have varied the biotin density on the droplet surfaces by mixing biotinylated with non-biotinylated surfactants, and used these to optimize the level of biotinylation for binding of NeutrAvidin. Using fluorescence microscopy and dynamic light scattering we have verified specific binding of NeutrAvidin to the biotinylated droplets and developed conditions for specific binding of biotinylated ligands to the NeutrAvidin coated droplets. These experiments outline a process for preparation of a soft, nano- through microscale sized droplet platform capable of specifically conjugating and displaying biotinylated molecules.

The preparation of new glycosylated-l-lysine-N-carboxyanhydride (glyco-K NCA) monomers is described. These monomers employ C-linked sugars and amide linkages to lysine for improved stability without sacrificing biochemical properties. Three glyco-K NCAs were synthesized, purified, and found to undergo living polymerization using transition metal initiation. These are the first living polymerizations of glycosylated NCAs and were used to prepare well-defined, high molecular weight glycopolypeptides and block and statistical glycocopolypeptides. This methodology solves many long-standing problems in the direct synthesis of glycopolypeptides from N-carboxyanhydrides relating to monomer synthesis, purification, and polymerization and gives polypeptides with 100% glycosylation. These long chain glycopolypeptides have potential to be good mimics of natural high molecular weight glycoproteins.

Hydrogels assembled from symmetric amphiphilic pentablock copolypeptides were investigated using rheology and cryogenic transmission electron microscopy (cryoTEM). Due to the presence of two α-helical hydrophobic segments per chain, the pentablocks have the potential for organized intrachain folding, akin to natural proteins, in addition to intermolecular assembly. We found that variation of the central hydrophilic segment lengths in the copolymers enabled precise tuning of assembly morphology and hydrogel properties. CryoTEM characterization revealed that the hydrogels are nanoporous networks composed of branched and entangled tape-like fibrils, where average fibril length varied with the length of hydrophilic spacer between the hydrophobic domains. The pentablock architecture in these copolymers thus allowed additional control over self-assembled morphology and substantial enhancement of hydrogel properties.

We describe the application of flash column chromatography on silica gel as a rapid and general method to obtain pure α-amino acid-N-carboxyanhydride (NCA) monomers, the widely used precursors for the synthesis of polypeptides, without the need for recrystallization. This technique was effective at removing all common impurities from NCAs and was found to work for a variety of NCAs, including those synthesized using different routes, as well as those bearing either hydrophilic or hydrophobic side chains. All chromatographed NCAs required no further purification and could be used directly to form high molecular weight polypeptides. This procedure is especially useful for the preparation of highly functional and low melting NCAs that are difficult to crystallize and, consequently, to polymerize. This method solves many long-standing problems in NCA purification and provides rapid access to NCAs that were previously inaccessible in satisfactory quality for controlled polymerization. This method is also practical in that it requires less time than recrystallization and often gives NCAs in improved yields.

Water-in-oil-in-water emulsions are examples of double emulsions, in which dispersions of small water droplets within larger oil droplets are themselves dispersed in a continuous aqueous phase1, 2, 3. Emulsions occur in many forms of processing and are used extensively by the foods, cosmetics and coatings industries. Because of their compartmentalized internal structure, double emulsions can provide advantages over simple oil-in-water emulsions for encapsulation, such as the ability to carry both polar and non-polar cargos, and improved control over release of therapeutic molecules4, 5, 6. The preparation of double emulsions typically requires mixtures of surfactants for stability; the formation of double nanoemulsions, where both inner and outer droplets are under 100 nm, has not yet been achieved7, 8, 9. Here we show that water-in-oil-in-water double emulsions can be prepared in a simple process and stabilized over many months using single-component, synthetic amphiphilic diblock copolypeptide surfactants. These surfactants even stabilize droplets subjected to extreme flow, leading to direct, mass production of robust double nanoemulsions that are amenable to nanostructured encapsulation applications in foods, cosmetics and drug delivery.

This article summarizes recent developments in the synthesis of copolypeptides from polymerization of α-amino acid-N-carboxyanhydrides (NCAs), focusing on their development for potential use in biomedical applications. Block and random copolypeptides of controlled dimensions (including: molecular weight, sequence, composition, and molecular weight distribution) can now be prepared. This review summarizes recent efforts to develop polypeptide hydrogels for tissue engineering applications, polypeptide membranes for therapeutic delivery, polypeptide adhesives for use as surgical sealants, antimicrobial polypeptides for treatment against infectious diseases, and immunomodulating polypeptides for treatment of autoimmune disorders. While these materials show great potential, much development is still required before they will be useful in some of these applications. However, the ability to prepare polypeptides with predictable structure and function puts these applications within reach.

There is a long history of man's use of materials derived from peptides and proteins. These natural materials possess sophisticated mechanisms of nanoscale self assembly, which have inspired the design of many synthetic and biosynthetic amino-acid based materials. These materials are attractive since they can have exceptional properties, environmental responsive behavior, biological activity, and can be metabolized. With all of their complexity, peptides and proteins rely primarily on two fundamental modes of self assembly: association of β-strands and the coiling of helices. In this context, a class of recently synthesized and characterized polypeptide materials are reviewed here, which were found to self-assemble by a fundamentally different process. This new mode of assembly was found to give rise to polypeptide hydrogels with a unique combination of properties (e.g. heat stability and injectability) making them attractive for applications in foods, personal care products, and medicine.

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.

Reagents were developed to allow chemoselective tagging of methionine residues in peptides and polypeptides, subsequent bioorthogonal functionalization of the tags, and cleavage of the tags when desired. This methodology can be used for triggered release of therapeutic peptides, or release of tagged protein digests from affinity columns.