Switchable Coacervate Formation via Amino Acid Functionalization of Poly(dehydroalanine).

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.

Sulfur Switches for Responsive Peptide Materials.

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.

Recovery of walking after paralysis by regenerating characterized neurons to their natural target region.

Axon regeneration can be induced across anatomically complete spinal cord injury (SCI), but robust functional restoration has been elusive. Whether restoring neurological functions requires directed regeneration of axons from specific neuronal subpopulations to their natural target regions remains unclear. To address this question, we applied projection-specific and comparative single-nucleus RNA sequencing to identify neuronal subpopulations that restore walking after incomplete SCI. We show that chemoattracting and guiding the transected axons of these neurons to their natural target region led to substantial recovery of walking after complete SCI in mice, whereas regeneration of axons simply across the lesion had no effect. Thus, reestablishing the natural projections of characterized neurons forms an essential part of axon regeneration strategies aimed at restoring lost neurological functions.

Preparation and stability of pegylated poly(S-alkyl-L-homocysteine) coacervate core micelles in aqueous media.

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.

Controlled Synthesis and Properties of Poly(l-homoserine).

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.

Poly(dehydroalanine): Synthesis, Properties, and Functional Diversification of a Fluorescent Polypeptide.

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.

Active Controlled and Tunable Coacervation Using Side-Chain Functional α-Helical Homopolypeptides.

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.

Design of Thermoresponsive Elastin-Like Glycopolypeptides for Selective Lectin Binding and Sorting.

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.

Foreign body responses in mouse central nervous system mimic natural wound responses and alter biomaterial functions.

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.

Injectable diblock copolypeptide hydrogel provides platform to deliver effective concentrations of paclitaxel to an intracranial xenograft model of glioblastoma.

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 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.

Overview of Host Defense Peptides and Their Applications for Plastic and Reconstructive Surgeons.

BACKGROUND: Host 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. METHODS: A nonsystematic review of host defense peptides was conducted with specific interest in properties and applications relevant to plastic and reconstructive surgery. RESULTS: In 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. CONCLUSIONS: Despite 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.

Modification of Poly(5,6-epoxy-l-norleucine) Gives Functional Polypeptides with Alternative Side-Chain Linkages.

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.

Tunable, Functional Diblock Copolypeptide Hydrogels Based on Methionine Homologs.

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.

Influence of Sulfoxide Group Placement on Polypeptide Conformational Stability.

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.

Versatile N-Methylaminooxy-Functionalized Polypeptides for Preparation of Neoglycoconjugates.

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.

Self-Healing Multiblock Copolypeptide Hydrogels via Polyion Complexation.

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.

Self-Sorting Microscale Compartmentalized Block Copolypeptide Hydrogels.

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.

Influence of Sulfur-Containing Diamino Acid Structure on Covalently Crosslinked Copolypeptide Hydrogels.

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.