Expanding the molecular language of protein liquid-liquid phase separation.

Understanding the relationship between a polypeptide sequence and its phase separation has important implications for analysing cellular function, treating disease and designing novel biomaterials. Several sequence features have been identified as drivers for protein liquid-liquid phase separation (LLPS), schematized as a 'molecular grammar' for LLPS. Here we further probe how sequence modulates phase separation and the material properties of the resulting condensates, targeting sequence features previously overlooked in the literature. We generate sequence variants of a repeat polypeptide with either no charged residues, high net charge, no glycine residues or devoid of aromatic or arginine residues. All but one of 12 variants exhibited LLPS, albeit to different extents, despite substantial differences in composition. Furthermore, we find that all the condensates formed behaved like viscous fluids, despite large differences in their viscosities. Our results support the model of multiple interactions between diverse residue pairs-not just a handful of residues-working in tandem to drive the phase separation and dynamics of condensates.

Genetically Fused Resilin-like Polypeptide-Coiled Coil Bundlemer Conjugates Exhibit Tunable Multistimuli-Responsiveness and Undergo Nanofibrillar Assembly.

Peptide-based materials are diverse candidates for self-assembly into modularly designed and stimuli-responsive nanostructures with precisely tunable compositions. Here, we genetically fused computationally designed coiled coil-forming peptides to the N- and C-termini of compositionally distinct multistimuli-responsive resilin-like polypeptides (RLPs) of various lengths. The successful expression of these hybrid polypeptides in bacterial hosts was confirmed through techniques such as gel electrophoresis, mass spectrometry, and amino acid analysis. Circular dichroism spectroscopy and ultraviolet-visible turbidimetry demonstrated that despite the fusion of disparate structural and responsive units, the coiled coils remained stable in the hybrid polypeptides, and the sequence-encoded differences in thermoresponsive phase separation of the RLPs were preserved. Cryogenic transmission electron microscopy and coarse-grained modeling showed that after thermal annealing in solution, the hybrid polypeptides adopted a closed loop conformation and assembled into nanofibrils capable of further hierarchically organizing into cluster structures and ribbon-like structures mediated by the self-association tendency of the RLPs.

Genetically Fusing Order-Promoting and Thermoresponsive Building Blocks to Design Hybrid Biomaterials.

The unique biophysical and biochemical properties of intrinsically disordered proteins (IDPs) and their recombinant derivatives, intrinsically disordered protein polymers (IDPPs) offer opportunities for producing multistimuli-responsive materials; their sequence-encoded disorder and tendency for phase separation facilitate the development of multifunctional materials. This review highlights the strategies for enhancing the structural diversity of elastin-like polypeptides (ELPs) and resilin-like polypeptides (RLPs), and their self-assembled structures via genetic fusion to ordered motifs such as helical or beta sheet domains. In particular, this review describes approaches that harness the synergistic interplay between order-promoting and thermoresponsive building blocks to design hybrid biomaterials, resulting in well-structured, stimuli-responsive supramolecular materials ordered on the nanoscale.

Controlled Release of Drugs from Extracellular Matrix-Derived Peptide-Based Nanovesicles through Tailored Noncovalent Interactions.

Elastin-collagen nanovesicles (ECnV) have emerged as a promising platform for drug delivery due to their tunable physicochemical properties and biocompatibility. The potential of nine distinct ECnVs to serve as drug-delivery vehicles was investigated in this study, and it was demonstrated that various small-molecule cargo (e.g., dexamethasone, methotrexate, doxorubicin) can be encapsulated in and released from a set of ECnVs, with extents of loading and rates of release dictated by the composition of the elastin domain of the ECnV and the type of cargo. Elastin-like peptides (ELPs) and collagen-like peptides (CLPs) of various compositions were produced; the secondary structure of the corresponding peptides was determined using CD, and the morphology and average hydrodynamic diameter (∼100 nm) of the ECnVs were determined using TEM and DLS. It was observed that hydrophobic drugs exhibited slower release kinetics than hydrophilic drugs, but higher drug loading was achieved for the more hydrophilic Dox. The collagen-binding ability of the ECnVs was demonstrated through a 2D collagen-binding assay, suggesting the potential for longer retention times in collagen-enriched tissues or matrices. Sustained release of drugs for up to 7 days was observed and, taken together with the collagen-binding data, demonstrates the potential of this set of ECnVs as a versatile drug delivery vehicle for longer-term drug release of a variety of cargo. This study provides important insights into the drug delivery potential of ECnVs and offers useful information for future development of ECnV-based drug delivery systems for the treatment of various diseases.

Sequence-Encoded Differences in Phase Separation Enable Formation of Resilin-like Polypeptide-Based Microstructured Hydrogels.

Microstructured hydrogels are promising platforms to mimic structural and compositional heterogeneities of the native extracellular matrix (ECM). The current state-of-the-art soft matter patterning techniques for generating ECM mimics can be limited owing to their reliance on specialized equipment and multiple time- and energy-intensive steps. Here, a photocross-linking methodology that traps various morphologies of phase-separated multicomponent formulations of compositionally distinct resilin-like polypeptides (RLPs) is reported. Turbidimetry and quantitative 1H NMR spectroscopy were utilized to investigate the sequence-dependent liquid-liquid phase separation of multicomponent solutions of RLPs. Differences between the intermolecular interactions of two different photocross-linkable RLPs and a phase-separating templating RLP were exploited for producing microstructured hydrogels with tunable control over pore diameters (ranging from 1.5 to 150 µm) and shear storage moduli (ranging from 0.2 to 5 kPa). The culture of human mesenchymal stem cells demonstrated high viability and attachment on microstructured hydrogels, suggesting their potential for developing customizable platforms for regenerative medicine applications.

Genetic Fusion of Thermoresponsive Polypeptides with UCST-type Behavior Mediates 1D Assembly of Coiled-Coil Bundlemers.

Thermoresponsive resilin-like polypeptides (RLPs) of various lengths were genetically fused to two different computationally designed coiled coil-forming peptides with distinct thermal stability, to develop new strategies to assemble coiled coil peptides via temperature-triggered phase separation of the RLP units. Their successful production in bacterial expression hosts was verified via gel electrophoresis, mass spectrometry, and amino acid analysis. Circular dichroism (CD) spectroscopy, ultraviolet-visible (UV/Vis) turbidimetry, and dynamic light scattering (DLS) measurements confirmed the stability of the coiled coils and showed that the thermosensitive phase behavior of the RLPs was preserved in the genetically fused hybrid polypeptides. Cryogenic-transmission electron microscopy and coarse-grained modeling revealed that functionalizing the coiled coils with thermoresponsive RLPs leads to their thermally triggered noncovalent assembly into nanofibrillar assemblies.

VEGF-Encoding, Gene-Activated Collagen-Based Matrices Promote Blood Vessel Formation and Improved Wound Repair.

Disruption in vascularization during wound repair can severely impair healing. Proangiogenic growth factor therapies have shown great healing potential; however, controlling growth factor activity and cellular behavior over desired healing time scales remains challenging. In this study, we evaluated collagen-mimetic peptide (CMP) tethers for their capacity to control growth factor gene transfer and growth factor activity using our recently developed gene-activated hyaluronic acid-collagen matrix (GAHCM). GAHCM was comprised of DNA/polyethyleneimine (PEI) polyplexes that were retained on hyaluronic acid (HA)-collagen hydrogels using CMPs. We hypothesized that using CMP-collagen tethers to control vascular endothelial growth factor-A (VEGF-A) gene delivery in fibroblasts would provide a powerful strategy to modulate the proangiogenic behaviors of endothelial cells (ECs) for blood vessel formation, resulting in enhanced wound repair. In co-culture experiments, we observed that CMP-modified GAHCM induced tunable gene delivery in fibroblasts as predicted, and correspondingly, VEGF-A produced by the fibroblasts led to increased growth and persistent migration of ECs for at least 7 days, as compared to non-CMP-modified GAHCM. Moreover, when ECs were exposed to fibroblast-containing VEGF-GAHCM with higher levels of CMP modification (50% CMP-PEI, or 50 CP), high CD31 expression was stimulated, resulting in the formation of an interconnected EC network with a significantly higher network volume and a larger diameter network structure than controls. Application of VEGF-GAHCM with 50 CP in murine splinted excisional wounds facilitated prolonged prohealing and proangiogenic responses resulting in increased blood vessel formation, improved granulation tissue formation, faster re-epithelialization, and overall enhanced repair. These findings suggest the benefits of CMP-collagen tethers as useful tools to control gene transfer and growth factor activity for improved treatment of wounds.

Recombinant protein-based injectable materials for biomedical applications.

Injectable nanocarriers and hydrogels have found widespread use in a variety of biomedical applications such as local and sustained biotherapeutic cargo delivery, and as cell-instructive matrices for tissue engineering. Recent advances in the development and application of recombinant protein-based materials as injectable platforms under physiological conditions have made them useful platforms for the development of nanoparticles and tissue engineering matrices, which are reviewed in this work. Protein-engineered biomaterials are highly customizable, and they provide distinctly tunable rheological properties, encapsulation efficiencies, and delivery profiles. In particular, the key advantages of emerging technologies which harness the stimuli-responsive properties of recombinant polypeptide-based materials are highlighted in this review.

Modified hyaluronic acid-collagen matrices trigger efficient gene transfer and prohealing behavior in fibroblasts for improved wound repair.

Growth factor therapy has demonstrated great promise for chronic wound repair, but controlling growth factor activity and cell phenotype over desired time frames remains a critical challenge. In this study, we developed a gene-activated hyaluronic acid-collagen matrix (GAHCM) comprising DNA/polyethylenimine (PEI) polyplexes retained on hyaluronic acid (HA)-collagen hydrogels using collagen mimetic peptides (CMPs). We hypothesized that manipulating both the number of CMP-collagen tethers and the ECM composition would provide a powerful strategy to control growth factor gene transfer kinetics while regulating cell behavior, resulting in enhanced growth factor activity for wound repair. We observed that polyplexes with 50% CMP-modified PEI (50 CP) showed enhanced retention of polyplexes in HCM hydrogels by 2.7-fold as compared to non-CMP modified polyplexes. Moreover, the incorporation of HA in the hydrogel promoted a significant increase in gene transfection efficiency based upon analysis of Gaussia luciferase (GLuc) reporter gene expression, and gene expression could be attenuated by blocking HA-CD44 signaling. Furthermore, when fibroblasts were exposed to vascular endothelial growth factor-A (VEGF-A)-GAHCM, the 50 CP matrix facilitated sustained VEGF-A production for up to 7 days, with maximal expression at day 5. Application of these VEGF-A-50 CP samples stimulated prolonged pro-healing responses, including the TGF-ß1-induced myofibroblast-like phenotypes and enhanced closure of murine splinted wounds. Overall, these findings demonstrate the use of ECM-based materials to stimulate efficient gene transfer and regulate cellular phenotype, resulting in improved control of growth factor activity for wound repair. GAHCM has significant potential to overcome key challenges in growth factor therapy for regenerative medicine. STATEMENT OF SIGNIFICANCE: Despite great promise for growth factor therapies in wound treatment, controlling growth factor activity and providing a microenvironment for cells that maximizes growth factor signaling have continued to limit the success of existing formulations. Our GAHCM strategy, combining CMP gene delivery and a hyaluronic acid-collagen matrix, enabled enhanced wound healing efficacy via the combination of controlled and localized growth factor expression and matrix-mediated regulation of cell behavior. Incorporation of CMPs and HA in the same matrix synergistically enhanced VEGF activity as compared with simpler matrices. Accordingly, GAHCM will advance our ability to leverage growth factor signaling for wound healing, resulting in new long-term treatments for recalcitrant wounds.

Therapeutic nanocarriers comprising extracellular matrix-inspired peptides and polysaccharides.

INTRODUCTION: The extracellular matrix (ECM) is vital for cell and tissue development. Given its importance, extensive work has been conducted to develop biomaterials and drug delivery vehicles that capture features of ECM structure and function. AREAS COVERED: This review highlights recent developments of ECM-inspired nanocarriers and their exploration for drug and gene delivery applications. Nanocarriers that are inspired by or created from primary components of the ECM (e.g. elastin, collagen, hyaluronic acid (HA), or combinations of these) are explicitly covered. An update on current clinical trials employing elastin-like proteins is also included. EXPERT OPINION: Novel ECM-inspired nanoscale structures and conjugates continue to be of great interest in the materials science and bioengineering communities. Hyaluronic acid nanocarrier systems in particular are widely employed due to the functional activity of HA in mediating a large number of disease states. In contrast, collagen-like peptide nanocarriers are an emerging drug delivery platform with potential relevance to a myriad of ECM-related diseases, making their continued study most pertinent. Elastin-like peptide nanocarriers have a well-established tolerability and efficacy track record in preclinical analyses that has motivated their recent advancement into the clinical arena.

A 3-D hydrogel based system for hematopoietic differentiation and its use in modeling down syndrome associated transient myeloproliferative disorder.

Induced pluripotent stem cells (iPSCs) provide an extraordinary tool for disease modeling owing to their potential to differentiate into the desired cell type. The differentiation of iPSCs is typically performed on 2-dimensional monolayers of stromal cell or animal tissue derived extracellular matrices. Recent advancements in disease modeling have utilized iPSCs in 3-dimensional (3D) cultures to study diseases such as muscular dystrophy, cardiomyopathy, and pulmonary fibrosis. However, these approaches are yet to be explored in modeling the hematological malignancies. Transient myeloproliferative disorder (TMD) is a preleukemic stage, which is induced in 10-20% of children with trisomy 21 possessing the pathognomonic mutation in the transcription factor GATA1. In this study, we established a synthetic 3D iPSC culture system for modeling TMD via hematopoietic differentiation of customized iPSCs. A chemically cross-linkable PEG hydrogel decorated with integrin binding peptide was found to be permissive of hematopoietic differentiation of iPSCs. It provided a cost-effective system for the generation of hematopoietic stem and progenitor cells (HSPCs) with higher yield of early HSPCs compared to traditional 2D culture on Matrigel coated dishes. Characterization of the HSPCs produced from the iPSC lines cultured in 3D showed that the erythroid population was reduced whereas the megakaryoid and myeloid populations were significantly increased in GATA1 mutant trisomic line compared to disomic or trisomic lines with wild-type GATA1, consistent with TMD characteristics. In conclusion, we have identified a cost-effective tunable 3D hydrogel system to model TMD.

Alteration of Microstructure in Biopolymeric Hydrogels via Compositional Modification of Resilin-Like Polypeptides.

Heterogeneities in hydrogel scaffolds are known to impact the performance of cells in cell-laden materials constructs, and we have employed the phase separation of resilin-like polypeptides (RLPs) as a means to generate such materials. Here, we study the compositional features of resilin-like polypeptides (RLPs) that further enable our control of their liquid-liquid phase separation (LLPS) and how such control impacts the formation of microstructured hydrogels. The evaluation of the phase separation of RLPs in solutions of ammonium sulfate offers insights into the sequence-dependent LLPS of the RLP solutions, and atomistic simulations, along with 2D-nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy (COSY) 1H NMR, suggest specific amino acid interactions that may mediate this phase behavior. The acrylamide functionalization of RLPs enables their photo-cross-linking into hydrogels and also enhances the phase separation of the polypeptides. A heating-cooling protocol promotes the formation of stable emulsions that yield different microstructured morphologies with tunable rheological properties. These findings offer approaches for choosing RLP compositions with phase behaviors that can be easily tuned with differences in temperature to control the resulting morphology and mechanical behavior of the heterogeneous hydrogels in regimes useful for biological applications.

Multi-stimuli-responsive, liposome-crosslinked poly(ethylene glycol) hydrogels for drug delivery.

The development of hybrid hydrogels has been of great interest over recent decades, especially in the field of biomaterials. Such hydrogels provide various opportunities in tissue engineering, drug delivery, and regenerative medicine due to their ability to mimic cellular environments, sequester and release therapeutic agents, and respond to stimuli. Herein we report the synthesis and characterization of an injectable poly(ethylene glycol) hydrogel crosslinked via thiol-maleimide reactions and containing both chemically crosslinked temperature-sensitive liposomes (TSLs) and matrix metalloproteinase-sensitive peptide crosslinks. Rheological studies demonstrate that the hydrogel is mechanically stable and can be synthesized to achieve a range of physically applicable moduli. Experiments characterizing the in situ drug delivery and degradation of these materials indicate that the TSL gel responds to both thermal and enzymatic stimuli in a local environment. Doxorubicin, a widely used anticancer drug, was loaded in the TSLs with a high encapsulation efficiency and the subsequent release was temperature dependent. Finally, TSLs did not compromise viability and proliferation of human and murine fibroblasts, supporting the use of these hydrogel-linked liposomes as a thermo-responsive drug carrier for controlled release.

Substrate stiffness directs the phenotype and polarization state of cord blood derived macrophages.

Cord blood (CB) mononuclear cell populations have demonstrated significant promise in biomaterials-based regenerative therapies; however, the contributions of monocyte and macrophage subpopulations towards proper tissue healing and regeneration are not well understood, and the phenotypic responses of macrophage to microenvironmental cues have not been well-studied. In this work, we evaluated the effects of cytokine stimulation and altered substrate stiffness. Macrophage derived from CB CD14+ monocytes adopted distinct inflammatory (M1) and anti-inflammatory (M2a and M2c) phenotypes in response to cytokine stimulation (M1: lipopolysaccharide (LPS) and interferon (IFN-γ); M2a: interleukin (IL)-4 and IL-13; M2c: IL-10) as determined through expression of relevant cell surface markers and growth factors. Cytokine-induced macrophage readily altered their phenotypes upon sequential administration of different cytokine cocktails. The impact of substrate stiffness on macrophage phenotype was evaluated by seeding CB-derived macrophage on 3wt%, 6wt%, and 14wt% poly(ethylene glycol)-based hydrogels, which exhibited swollen shear moduli of 0.1, 3.4, and 10.3 kPa, respectively. Surface marker expression and cytokine production varied depending on modulus, with anti-inflammatory phenotypes increasing with elevated substrate stiffness. Integration of specific hydrogel moduli and cytokine cocktail treatments resulted in the differential regulation of macrophage phenotypic biomarkers. These data suggest that CB-derived macrophages exhibit predictable behaviors that can be directed and finely tuned by combinatorial modulation of substrate physical properties and cytokine profiles.

Regulation of neovasculogenesis in co-cultures of aortic adventitial fibroblasts and microvascular endothelial cells by cell-cell interactions and TGF-ß/ALK5 signaling.

Adventitial fibroblasts (AFs) are critical mediators of vascular remodeling. However, the contributions of AFs towards development of vasculature and the specific mechanisms by which these cells regulate physiological expansion of the vasa vasorum, the specialized microvasculature that supplies nutrients to the vascular wall, are not well understood. To determine the regulatory role of AFs in microvascular endothelial cell (MVEC) neovasculogenesis and to investigate the regulatory pathways utilized for communication between the two cell types, AFs and MVECs were cultured together in poly(ethylene glycol)-based hydrogels. Following preliminary evaluation of a set of cell adhesion peptides (AG10, AG73, A2G78, YIGSR, RGD), 7.5wt% hydrogels containing 3 mM RGD were selected as these substrates did not initiate primitive tubule structures in 3D MVEC monocultures, thus providing a passive platform to study AF-MVEC interaction. The addition of AFs to hydrogels promoted MVEC viability; however, increasing AF density within hydrogels stimulated MVEC proliferation, increased microvessel density and size, and enhanced deposition of basement membrane proteins, collagen IV and laminin. Importantly, AF-MVEC communication through the transforming growth factor beta (TGF-ß)/activin receptor-like kinase 5 (ALK5) signaling pathway was observed to mediate microvessel formation, as inhibition of ALK5 significantly decreased MVEC proliferation, microvessel formation, mural cell recruitment, and basement membrane production. These data indicate that AFs regulate MVEC neovasculogenesis and suggest that therapeutics targeting the TGF-ß/ALK5 pathway may be useful for regulation of vasculogenic and anti-vasculogenic responses.

Characterizing aggregate growth and morphology of alanine-rich polypeptides as a function of sequence chemistry and solution temperature from scattering, spectroscopy, and microscopy.

The aggregation behavior and stability of a series of alanine-rich peptides, which are included as components of peptide-polymer conjugates, were characterized using a combination of biophysical techniques. Light scattering techniques were used to monitor changes in peptide morphology and size distributions as a function of time and temperature. The results show large particles immediately upon dissolution in buffer. At room temperature, these particles relaxed to reach a mostly monomeric peptide state, while at higher temperatures, they grew to form aggregates. Circular dichroism spectroscopy (CD) was used to monitor temperature- and time-dependent conformational changes as a function of peptide sequence and incubation time. CD measurements reveal that all of the sequences are helical at low temperatures with transitions to non-helical conformation with increased temperature. Samples incubated at room temperature were able to recover their original helicity. At increased temperature, the shorter and longer peptide sequences showed notable changes in conformation, and were not able to recover their original helicity after 72 h. After incubation for up to one week, ß-sheet conformations were observed in these two cases, while only α-helical conformation loss was observed for the peptide of intermediate molecular weight. Transmission electron microscopy measurements reveal the formation of fibrils after 72 h of incubation at 60 °C for all samples, in agreement with the scattering measurements. Additional quenching experiments show that peptide aggregation can be stalled when solutions are cooled to room temperature.

Enhanced wound healing via collagen-turnover-driven transfer of PDGF-BB gene in a murine wound model.

Wound healing is a complex biological process that requires coordinated cell proliferation, migration, and extracellular matrix production/remodeling, all of which are inhibited/delayed in chronic wounds. In this study, a formulation was developed that marries a fibrin-based, provisional-like matrix with collagen mimetic peptide (CMP)/PDGF gene-modified collagens, leading to the formation of robust gels that supported temporally controlled PDGF expression and facile application within the wound bed. Analysis employing in vitro co-gel scaffolds confirmed sustained and temporally controlled gene release based on matrix metalloproteinase (MMP) activity, with ~30% higher PDGF expression in MMP producing fibroblasts as-compared with non-MMP-expressing cells. The integration of fibrin with the gene-modified collagens resulted in co-gels that strongly supported both fibroblast cell recruitment/invasion as well as multiple aspects of the longer-term healing process. The excisional wound healing studies in mice established faster wound closure using CMP-modified PDGF polyplex-loaded co-gels, which exhibited up to 24% more wound closure (achieved with ~2 orders of magnitude lower growth factor dosing) after 9 days as compared to PDGF-loaded co-gels, and 19% more wound closure after 9 days as compared to CMP-free polyplex loaded co-gels. Moreover, minimal scar formation as well as improved collagen production, myofibroblast activity, and collagen orientation was observed following CMP-modified PDGF polyplex-loaded co-gel application on wounds. Taken together, the combined properties of the co-gels, including their stability and capacity to control both cell recruitment and cell phenotype within the murine wound bed, strongly supports the potential of the co-gel scaffolds for improved treatment of chronic non-healing wounds.

Micromechanical Properties of Microstructured Elastomeric Hydrogels.

Local, micromechanical environment is known to influence cellular function in heterogeneous hydrogels, and knowledge gained in micromechanics will facilitate the improved design of biomaterials for tissue regeneration. In this study, a system comprising microstructured resilin-like polypeptide (RLP)-poly(ethylene glycol) (PEG) hydrogels is utilized. The micromechanical properties of RLP-PEG hydrogels are evaluated with oscillatory shear rheometry, compression dynamic mechanic analysis, small-strain microindentation, and large-strain indentation and puncture over a range of different deformation length scales. The measured elastic moduli are consistent with volume averaging models, indicating that volume fraction, not domain size, plays a dominant role in determining the low strain mechanical response. Large-strain indentation under a confocal microscope enables the visualization of the microstructured hydrogel micromechanical deformation, emphasizing the translation, rotation, and deformation of RLP-rich domains. The fracture initiation energy results demonstrate that failure of the composite hydrogels is controlled by the RLP-rich phase, and their independence with domain size suggested that failure initiation is controlled by multiple domains within the strained volume. This approach and findings provide new quantitative insight into the micromechanical response of soft hydrogel composites and highlight the opportunities in employing these methods to understand the physical origins of mechanical properties of soft synthetic and biological materials.

Manipulation of the dually thermoresponsive behavior of peptide-based vesicles through modification of collagen-like peptide domains.

Materials that respond to temporally defined exogenous cues continue to be an active pursuit of research toward on-demand nanoparticle drug delivery applications, and using one or more exogenous temperature stimuli could significantly expand the application of nanoparticle-based drug delivery formulations under both hyperthermal and hypothermal conditions. Previously we have reported the development of a biocompatible and thermoresponsive elastin-b-collagen-like polypeptide (ELP-CLP) conjugate that is capable of self-assembling into vesicles and encapsulating small molecule therapeutics that can be delivered at different rates via a single temperature stimulus. Herein we report the evaluation of multiple ELP-CLP conjugates, demonstrating that the inverse transition temperature (T t) of the ELP-CLPs can be manipulated by modifying the melting temperature (T m) of the CLP domain, and that the overall hydrophilicity of the ELP-CLP conjugate also may alter the T t. Based on these design parameters, we demonstrate that the ELP-CLP sequence (VPGFG)6-(GPO)7GG can self-assemble into stable vesicles at 25°C and dissociate at elevated temperatures by means of the unfolding of the CLP domain above its T m. We also demonstrate here for the first time the ability of this ELP-CLP vesicle to dissociate via a hypothermic temperature stimulus by means of exploiting the inverse transition temperature (T t) phenomena found in ELPs. The development of design rules for manipulating the thermal properties of these bioconjugates will enable future modifications to either the ELP or CLP sequences to more finely tune the transitions of the conjugates for specific biomedical applications.

Covalent co-assembly between resilin-like polypeptide and peptide amphiphile into hydrogels with controlled nanostructure and improved mechanical properties.

Covalent co-assembly holds great promise for the fabrication of hydrogels with controllable nanostructure, versatile chemical composition, and enhanced mechanical properties given its relative simplicity, high efficiency, and bond stability. This report describes our approach to designing functional multicomponent hydrogels based on photo-induced chemical interactions between an acrylamide-functionalized resilin-like polypeptide (RLP) and a peptide amphiphile (PA). Circular dichroism (CD) spectroscopy, electron microscopy, and amplitude sweep rheology were used to demonstrate that the co-assembled hydrogel systems acquired distinct structural conformations, tunable nanostructures, and enhanced elasticity in a PA concentration-dependent manner. We envisage the use of these materials in numerous biomedical applications such as controlled drug release systems, microfluidic devices, and scaffolds for tissue engineering.