High-throughput experimentation for discovery of biodegradable polyesters.

The consistent rise of plastic pollution has stimulated interest in the development of biodegradable plastics. However, the study of polymer biodegradation has historically been limited to a small number of polymers due to costly and slow standard methods for measuring degradation, slowing new material innovation. High-throughput polymer synthesis and a high-throughput polymer biodegradation method are developed and applied to generate a biodegradation dataset for 642 chemically distinct polyesters and polycarbonates. The biodegradation assay was based on the clear-zone technique, using automation to optically observe the degradation of suspended polymer particles under the action of a single Pseudomonas lemoignei bacterial colony. Biodegradability was found to depend strongly on aliphatic repeat unit length, with chains less than 15 carbons and short side chains improving biodegradability. Aromatic backbone groups were generally detrimental to biodegradability; however, ortho- and para-substituted benzene rings in the backbone were more likely to be degradable than metasubstituted rings. Additionally, backbone ether groups improved biodegradability. While other heteroatoms did not show a clear improvement in biodegradability, they did demonstrate increases in biodegradation rates. Machine learning (ML) models were leveraged to predict biodegradability on this large dataset with accuracies over 82% using only chemical structure descriptors.

Cradle-to-gate life cycle assessment of Spirulina bioplastic produced via plasticization with glycerol.

Bioplastics have been used as alternatives to conventional petroleum-based plastics to lessen the burdens on marine and terrestrial environments due to their non-biodegradability and toxicity. However, recent studies have shown that not all bioplastics may be environmentally friendly. Microalgae, such as Spirulina that do not require arable land, have been identified as a potential bioplastic source. In this study, cradle-to-gate life cycle assessment (LCA) was carried out in openLCA program using the Agribalyse database, to evaluate the environmental impacts of Spirulina bioplastic, formed from plasticization of Spirulina powder with glycerol. Two processes were created for the inventories of (i) Spirulina powder and (ii) Spirulina bioplastic, where the output of the former served as an input for the latter. The extruded bioplastic sheets were food-grade and could be used as edible packaging materials. The bioplastic was also compared to conventional plastics and it was found that the energy consumption was 3.83 ± 0.26 MJ/kg-bioplastic, which was 12% and 22% higher than that of LDPE and PVC plastic films, respectively. The impacts on the environment showed that the chemical growth medium (Zarrouk medium) and electricity were the main contributors in most of the categories. Compared to the PVC and LDPE films, the Spirulina bioplastic's impacts on the aquatic ecosystems were 2-3 times higher. The global warming potential of the Spirulina bioplastic was 1.99 ± 0.014 kg CO2 eq, which was 23% and 47% lower than that of LDPE and PVC films, respectively. Sensitivity analysis was carried out by changing the electricity source and using alternative growth media. Except for the case of switching to solar energy, the results for other cases did not differ significantly from the base case scenario. Future studies were suggested to identify different greener alternatives to the growth medium as well as different energy mixes for more environmentally benign solutions.

BigSMARTS: A Topologically Aware Query Language and Substructure Search Algorithm for Polymer Chemical Structures.

Molecular search is important in chemistry, biology, and informatics for identifying molecular structures within large data sets, improving knowledge discovery and innovation, and making chemical data FAIR (findable, accessible, interoperable, reusable). Search algorithms for polymers are significantly less developed than those for small molecules because polymer search relies on searching by polymer name, which can be challenging because polymer naming is overly broad (i.e., polyethylene), complicated for complex chemical structures, and often does not correspond to official IUPAC conventions. Chemical structure search in polymers is limited to substructures, such as monomers, without awareness of connectivity or topology. This work introduces a novel query language and graph traversal search algorithm for polymers that provides the first search method able to fully capture all of the chemical structures present in polymers. The BigSMARTS query language, an extension of the small-molecule SMARTS language, allows users to write queries that localize monomer and functional group searches to different parts of the polymer, like the middle block of a triblock, the side chain of a graft, and the backbone of a repeat unit. The substructure search algorithm is based on the traversal of graph representations of the generating functions for the stochastic graphs of polymers. Operationally, the algorithm first identifies cycles representing the monomers and then the end groups and finally performs a depth-first search to match entire subgraphs. To validate the algorithm, hundreds of queries were searched against hundreds of target chemistries and topologies from the literature, with approximately 440,000 query-target pairs. This tool provides a detailed algorithm that can be implemented in search engines to provide search results with full matching of the monomer connectivity and polymer topology.

Strengthening and Toughening of Protein-Based Thermosets via Intermolecular Self-Assembly.

As proteins are abundant polymers in biomass sources such as agricultural feedstocks and byproducts, leveraging them to develop alternatives to synthetic polymers is of great interest. However, the mechanical performance of protein materials is not suitable for most target applications. Constructing copolymers with proteins as hard domains and rubbery polymers as soft domains has been shown to be a promising strategy for improving mechanical properties. Herein, it is demonstrated that toughening and strengthening of protein copolymers can be advanced further by thermal treatment, leading to mechanical enhancements that generalize across a variety of different protein feedstocks, including whey, serum, soy, and pea proteins. The thermal treatment induces a rearrangement of protein structure, leading to the formation of intermolecular ß-sheets. The ordered intermolecular structures in the hard domains of thermosets greatly improve their mechanical properties, providing simultaneous increases in strength, toughness, and modulus, with little sacrifice in fracture strain. Analogous to crystalline structures, the formation of intermolecular ß-sheet structures also leads to reduced hygroscopicity. This is a valuable contribution, as practical applications of natural polymer-based plastics are frequently hindered by the materials' humidity sensitivity. Therefore, this work demonstrates a simple yet versatile strategy to improve the materials' performance from a wide range of protein feedstocks, as well as signifies the implications of protein structural assembly in materials design.

Mechanism Dictates Mechanics: A Molecular Substituent Effect in the Macroscopic Fracture of a Covalent Polymer Network.

The fracture of rubbery polymer networks involves a series of molecular events, beginning with conformational changes along the polymer backbone and culminating with a chain scission reaction. Here, we report covalent polymer gels in which the macroscopic fracture "reaction" is controlled by mechanophores embedded within mechanically active network strands. We synthesized poly(ethylene glycol) (PEG) gels through the end-linking of azide-terminated tetra-arm PEG (Mn = 5 kDa) with bis-alkyne linkers. Networks were formed under identical conditions, except that the bis-alkyne was varied to include either a cis-diaryl (1) or cis-dialkyl (2) linked cyclobutane mechanophore that acts as a mechanochemical "weak link" through a force-coupled cycloreversion. A control network featuring a bis-alkyne without cyclobutane (3) was also synthesized. The networks show the same linear elasticity (G' = 23-24 kPa, 0.1-100 Hz) and equilibrium mass swelling ratios (Q = 10-11 in tetrahydrofuran), but they exhibit tearing energies that span a factor of 8 (3.4 J, 10.6, and 27.1 J·m-2 for networks with 1, 2, and 3, respectively). The difference in fracture energy is well-aligned with the force-coupled scission kinetics of the mechanophores observed in single-molecule force spectroscopy experiments, implicating local resonance stabilization of a diradical transition state in the cycloreversion of 1 as a key determinant of the relative ease with which its network is torn. The connection between macroscopic fracture and a small-molecule reaction mechanism suggests opportunities for molecular understanding and optimization of polymer network behavior.

PolyDAT: A Generic Data Schema for Polymer Characterization.

Polymers are stochastic materials that represent distributions of different molecules. In general, to quantify the distribution, polymer researchers rely on a series of chemical characterizations that each reveal partial information on the distribution. However, in practice, the exact set of characterizations that are carried out, as well as how the characterization data are aggregated and reported, is largely nonstandard across the polymer community. This scenario makes polymer characterization data highly disparate, thereby significantly slowing down the development of polymer informatics. In this work, a proposal on how structural characterization data can be organized is presented. To ensure that the system can apply universally across the entire polymer community, the proposed schema, PolyDAT, is designed to embody a minimal congruent set of vocabulary that is common across different domains. Unlike most chemical schemas, where only data pertinent to the species of interest are included, PolyDAT deploys a multi-species reaction network construct, in which every characterization on relevant species is collected to provide the most comprehensive profile on the polymer species of interest. Instead of maintaining a comprehensive list of available characterization techniques, PolyDAT provides a handful of generic templates, which align closely with experimental conventions and cover most types of common characterization techniques. This allows flexibility for the development and inclusion of new measurement methods. By providing a standard format to digitalize data, PolyDAT serves not only as an extension to BigSMILES that provides the necessary quantitative information but also as a standard channel for researchers to share polymer characterization data.

Effect of Protein Surface Charge Distribution on Protein-Polyelectrolyte Complexation.

Charge anisotropy or the presence of charge patches at protein surfaces has long been thought to shift the coacervation curves of proteins and has been used to explain the ability of some proteins to coacervate on the "wrong side" of their isoelectric point. This work makes use of a panel of engineered superfolder green fluorescent protein mutants with varying surface charge distributions but equivalent net charge and a suite of strong and weak polyelectrolytes to explore this concept. A patchiness parameter, which assessed the charge correlation between points on the surface of the protein, was used to quantify the patchiness of the designed mutants. Complexation between the polyelectrolytes and proteins showed that the mutant with the largest patchiness parameter was the most likely to form complexes, while the smallest was the least likely to do so. The patchiness parameter was found to correlate well with the phase behavior of the protein-polymer mixtures, where both macrophase separation and the formation of soluble aggregates were promoted by increasing the patchiness depending on the polyelectrolyte with which the protein was mixed. Increasing total charge and increasing strength of the polyelectrolyte promote interactions for oppositely charged polyelectrolytes, while charge regulation is also key to interactions for similarly charged polyelectrolytes, which must interact selectively with oppositely charged patches.

Non-isocyanate urethane linkage formation using l-lysine residues as amine sources.

Bio-based polyurethane materials are broadly applied in medicine as drug delivery systems. Nevertheless, their synthesis comprises the use of petroleum-based toxic amines, isocyanates and polyols, and their biocompatibility or functionalization is limited. Therefore, the use of lysine residues as amine sources to create non-isocyanate urethane (NIU) linkages was investigated. Therefore, a five-membered biscyclic carbonate (BCC) was firstly synthetized and reacted with a protected lysine, a tripeptide and a heptapeptide to confirm the urethane linkage formation with lysine moiety and to optimize reaction conditions. Afterwards, the reactions between BCC and a model protein, elastin-like protein (ELP), and ß-Lactoglobulin (BLG) obtained from whey protein, respectively, were performed. The synthesized protein materials were structural, thermally and morphologically characterized to confirm the urethane linkage formation. The results demonstrate that using both simple and more complex source of amines (lysine), urethane linkages were effectively achieved. This pioneering approach opens the possibility of using proteins to develop non-isocyanate polyurethanes (NIPUs) with tailored properties.

Predicting Protein-Polymer Block Copolymer Self-Assembly from Protein Properties.

Protein-polymer bioconjugate self-assembly has attracted a great deal of attention as a method to fabricate protein nanomaterials in solution and the solid state. To identify protein properties that affect phase behavior in protein-polymer block copolymers, a library of 15 unique protein-b-poly(N-isopropylacrylamide) (PNIPAM) copolymers comprising 11 different proteins was compiled and analyzed. Many attributes of phase behavior are found to be similar among all studied bioconjugates regardless of protein properties, such as formation of micellar phases at high temperature and low concentration, lamellar ordering with increasing temperature, and disordering at high concentration, but several key protein-dependent trends are also observed. In particular, hexagonal phases are only observed for proteins within the molar mass range 20-36 kDa, where ordering quality is also significantly enhanced. While ordering is generally found to improve with increasing molecular weight outside of this range, most large bioconjugates exhibited weaker than predicted assembly, which is attributed to chain entanglement with increasing polymer molecular weight. Additionally, order-disorder transition boundaries are found to be largely uncorrelated to protein size and quality of ordering. However, the primary finding is that bioconjugate ordering can be accurately predicted using only protein molecular weight and percentage of residues contained within ß sheets. This model provides a basis for designing protein-PNIPAM bioconjugates that exhibit well-defined self-assembly and a modeling framework that can generalize to other bioconjugate chemistries.

Engineering Elastin-Like Polypeptide-Poly(ethylene glycol) Multiblock Physical Networks.

Hybrids of protein biopolymers and synthetic polymers are a promising new class of soft materials, as the advantages of each component can be complementary. A recombinant elastin-like polypeptide (ELP) was conjugated to poly(ethylene glycol) (PEG) by macromolecular coupling in solution to form multiblock ELP-PEG copolymers. The hydrated copolymer preserved the thermoresponsive properties from the ELP block and formed hydrogels with different transition temperatures depending on salt concentration. Small angle scattering indicates that the copolymer hydrogels form sphere-like aggregates with a "fuzzy" interface, while the films form a fractal network of nanoscale aggregates. The use of solutions with different salt concentrations to prepare the hydrogels was found to influence the transition temperature, the mechanical properties, and the size of the nanoscale structure of the hydrogel without changing the secondary structure of the ELP. The salt variation and the addition of a plasticizer also affected the nanoscale structure and the mechanical characteristics of the films.

Nucleopore-Inspired Polymer Hydrogels for Selective Biomolecular Transport.

Biological systems routinely regulate biomolecular transport with remarkable specificity, low energy input, and simple mechanisms. Here, the biophysical mechanisms of nuclear transport inspire the development of gels for recognition and selective permeation (GRASP). GRASP presents a new paradigm for specific transport and selective permeability, in which binding interactions between a biomolecule and a hydrogel lead to faster penetration of the gel. A molecular transport theory identifies key principles for selective transport: entropic repulsion of noninteracting molecules and affinity-mediated diffusion of multireceptor biomolecules through a walking mechanism. The ability of interacting molecules to walk through hydrogels enables enhanced permeability in polymer networks. To realize this theoretical prediction in a novel material, GRASP is engineered from a poly(ethylene glycol) network (entropic barrier) containing antibody-binding oligopeptides (affinity domains). GRASP is synthesized using simultaneous bioconjugation and polycondensation reactions. The elastic modulus, characteristic pore size, biomolecular diffusivity, and selective permeability are measured in the resulting materials, which are applied to regulate the transport of equally sized molecules by preferentially transporting a monoclonal antibody from a polyclonal mixture. Overall, this work presents rationally designed, nucleopore-inspired hydrogels that are capable of controlling biomolecular transport.

Improved Ordering in Low Molecular Weight Protein-Polymer Conjugates Through Oligomerization of the Protein Block.

The self-assembly of protein-polymer conjugates incorporating oligomers of a small, engineered high-affinity binding protein, rcSso7d.SA, is studied to determine the effect of protein oligomerization on nanoscale ordering. Oligomerization enables a systematic increase in the protein molar mass without changing its overall folded structure, leading to a higher driving force for self-assembly into well-ordered structures. Though conjugates of monomeric rcSso7d.SA are found to only exist in disordered states, oligomers of this protein linked to a poly( N-isopropylacrylamide) (PNIPAM) block self-assemble into lamellar nanostructures. Conjugates of trimeric and tetrameric rcSso7d.SA are observed to produce the strongest ordering in concentrated solution, displaying birefringent lamellae at concentrations as low as 40 wt %. In highly concentrated solution, the oligomeric rcSso7d.SA-PNIPAM block copolymers exhibit ordering and domain spacing trends atypical from that of most block copolymers. Fluorescent binding assays indicate that oligomerized protein blocks retain binding functionality and exhibit limits of detection up to three times lower than that of surface-immobilized protein sensors. Therefore, oligomerization of the protein block in these block copolymers serves as an effective method to improve both nanoscale ordering and biosensing capabilities.

Classical Challenges in the Physical Chemistry of Polymer Networks and the Design of New Materials.

Polymer networks are widely used from commodity to biomedical materials. The space-spanning, net-like structure gives polymer networks their advantageous mechanical and dynamic properties, the most essential factor that governs their responses to external electrical, thermal, and chemical stimuli. Despite the ubiquity of applications and a century of active research on these materials, the way that chemistry and processing interact to yield the final structure and the material properties of polymer networks is not fully understood, which leads to a number of classical challenges in the physical chemistry of gels. Fundamentally, it is not yet possible to quantitatively predict the mechanical response of a polymer network based on its chemical design, limiting our ability to understand and characterize the nanostructure of gels and rationally design new materials. In this Account, we summarize our recent theoretical and experimental approaches to study the physical chemistry of polymer networks. First, our understanding of the impact of molecular defects on topology and elasticity of polymer networks is discussed. By systematically incorporating the effects of different orders of loop structure, we develop a kinetic graph theory and real elastic network theory that bridge the chemical design, the network topology, and the mechanical properties of the gel. These theories show good agreement with the recent experimental data without any fitting parameters. Next, associative polymer gel dynamics is discussed, focusing on our evolving understanding of the effect of transient bonds on the mechanical response. Using forced Rayleigh scattering (FRS), we are able to probe diffusivity across a wide range of length and time scales in gels. A superdiffusive region is observed in different associative network systems, which can be captured by a two-state kinetic model. Further, the effects of the architecture and chemistry of polymer chains on gel nanostructure are studied. By incorporating shear-thinning coiled-coil protein motifs into the midblock of a micelle-forming block copolymer, we are able to responsively adjust the gel toughness through controlling the nanostructure. Finally, we review the development of novel application-oriented materials that emerge from our enhanced understanding of gel physical chemistry, including injectable gel hemostats designed to treat internal wounds and engineered nucleoporin-like polypeptide (NLP) hydrogels that act as biologically selective filters. We believe that the fundamental physical chemistry questions articulated in this Account will provide inspiration to fully understand the design of polymer networks, a group of mysterious yet critically important materials.

Kinetic Effects on Self-Assembly and Function of Protein-Polymer Bioconjugates in Thin Films Prepared by Flow Coating.

The self-assembly of nanostructured globular protein arrays in thin films is demonstrated using protein-polymer block copolymers based on a model protein mCherry and the polymer poly(oligoethylene glycol acrylate) (POEGA). Conjugates are flow coated into thin films on a poly(ethylene oxide) grafted Si surface, forming self-assembled cylindrical nanostructures with POEGA domains selectively segregating to the air-film interface. Long-range order and preferential arrangement of parallel cylinders templated by selective surfaces are demonstrated by controlling relative humidity. Long-range order increases with coating speed when the film thicknesses are kept constant, due to reduced nucleation per unit area of drying film. Fluorescence emission spectra of mCherry in films prepared at <25% relative humidity shows a small shift suggesting that proteins are more perturbed at low humidity than high humidity or the solution state.

Three-Dimensional Ordered Antibody Arrays Through Self-Assembly of Antibody-Polymer Conjugates.

Three-dimensional (3D) ordered arrays of human immunoglobulin G (IgG) were fabricated using well-defined full-length antibody-polymer conjugates (APCs). The conjugates were prepared through a two-step sequential click approach with a combination of oxime ligation and strain promoted alkyne-azide cycloaddition. They were able to self-assemble into lamellar nanostructures with alternating IgG and poly(N-isopropylacrylamide) (PNIPAM) nanodomains. As a proof-of-concept, these materials were fabricated into thin films and their specific binding ability was tested. The nanostructure not only improves the packing density and the proper orientation of the IgG, but also provides nanochannels to facilitate substrate transport.

Biosynthesis of poly(glycolate-co-lactate-co-3-hydroxybutyrate) from glucose by metabolically engineered Escherichia coli.

Metabolically engineered Escherichia coli strains were constructed to effectively produce novel glycolate-containing biopolymers from glucose. First, the glyoxylate bypass pathway and glyoxylate reductase were engineered such as to generate glycolate. Second, glycolate and lactate were activated by the Megasphaera elsdenii propionyl-CoA transferase to synthesize glycolyl-CoA and lactyl-CoA, respectively. Third, ß-ketothiolase and acetoacetyl-CoA reductase from Ralstonia eutropha were introduced to synthesize 3-hydroxybutyryl-CoA from acetyl-CoA. At last, the Ser325Thr/Gln481Lys mutant of polyhydroxyalkanoate (PHA) synthase from Pseudomonas sp. 61-3 was over-expressed to polymerize glycolyl-CoA, lactyl-CoA and 3-hydroxybutyryl-CoA to produce poly(glycolate-co-lactate-co-3-hydroxybutyrate). The recombinant E. coli was able to accumulate the novel terpolymer with a titer of 3.90g/l in shake flask cultures. The structure of the resulting polymer was chemically characterized by proton NMR analysis. Assessment of thermal and mechanical properties demonstrated that the produced terpolymer possessed decreased crystallinity and improved toughness, in comparison to poly(3-hydroxybutyrate) homopolymer. This is the first study reporting efficient microbial production of poly(glycolate-co-lactate-co-3-hydroxybutyrate) from glucose.

Complex Coacervate Core Micelles for the Dispersion and Stabilization of Organophosphate Hydrolase in Organic Solvents.

Organophosphate (OP) nerve agents are a class of chemical warfare agents (CWAs) that exist as bulk stocks in current and past war zones. Thus, a technology that can perform on-site decontamination in a safe and timely fashion is desirable. Here, complex coacervate core micelles (C3Ms) were used to encapsulate organophosphate hydrolase (OPH) and chemostabilize it to maintain activity after exposure to organophosphate simulants ethanol and dimethyl methylphosphonate (DMMP). C3Ms were formed by two polymers-poly(acrylic acid) (PAA) and poly(oligo(ethylene glycol) methacrylate)-b-poly(4-vinyl N-methylpyridyl iodide), (POEGMA-b-qP4VP). Complexes of the coacervate micelles with the enzyme OPH were investigated by small angle neutron scattering (SANS), dynamic light scattering (DLS), and transmission electron microscopy (TEM), demonstrating the formation of micellar structures in solution. The activity of OPH against methyl paraoxon in these C3Ms under aqueous conditions was assayed after heat treatment for 3 days at 37 °C. The OPH in C3Ms retained 88 ± 7% of its initial activity, as compared to the 48 ± 3% activity retained by OPH alone, indicating that the C3Ms were able to stabilize the enzyme to heat treatment. C3Ms transferred into the two organic solvents formed larger structures than inverse micelles formed by the block copolymer alone. The addition of OPH to the C3Ms in organic solvents did not significantly change their structure. The activity of OPH (again, against methyl paraoxon) after 24 h of incubation at 4 °C was measured and compared to that of OPH in C3Ms. While OPH alone retained less than 5% of its activity after this incubation in both solvents, OPH in C3Ms retained 35 ± 3% of its activity in DMMP and 26 ± 1% of its activity in ethanol.

The Effect of Protein Electrostatic Interactions on Globular Protein-Polymer Block Copolymer Self-Assembly.

Mutation of a superfolder green fluorescent protein (GFP) was used to design GFP variants with formal net charges of 0, -8, and -21, providing a set of three proteins in which the total charge is varied to tune protein-protein interactions while controlling for the protein size and tertiary structure. After conjugating poly(N-isopropylacrylamide) (PNIPAM) to each of these three GFP variants, the concentrated solution phase behavior of these three block copolymers is studied using a combination of small-angle X-ray scattering (SAXS), depolarized light scattering (DPLS), and turbidimetry to characterize their morphologies. The electrostatic repulsion between supercharged GFP suppresses ordering, increasing the order-disorder transition concentration (CODT) and decreasing the quality of the ordered nanostructures as measured by the full width at half-maximum of the primary scattering peak. By contrast, the charge distribution of the neutrally charged GFP results in its largest dipole moment, calculated about the protein's center of mass, among the three GFP variants and a self-complementary Janus-like electrostatic surface potential that enhances nanostructure formation. The different electrostatic properties result in different protein-protein interactions that affect the high temperature morphologies, including the formation of macrophase separated or homogeneous micellar phases and the smaller hexagonal ordering window of the supercharged GFP. Small improvements in the quality of the ordered nanostructures of GFP(-21)-PNIPAM can be achieved through protein-divalent cation interactions. Therefore, varying protein charge and electrostatics is demonstrated as a method of tuning the magnitude and directionality of protein-protein interactions to control self-assembly.

Toughening of Thermoresponsive Arrested Networks of Elastin-Like Polypeptides To Engineer Cytocompatible Tissue Scaffolds.

Formulation of tissue engineering or regenerative scaffolds from simple bioactive polymers with tunable structure and mechanics is crucial for the regeneration of complex tissues, and hydrogels from recombinant proteins, such as elastin-like polypeptides (ELPs), are promising platforms to support these applications. The arrested phase separation of ELPs has been shown to yield remarkably stiff, biocontinuous, nanostructured networks, but these gels are limited in applications by their relatively brittle nature. Here, a gel-forming ELP is chain-extended by telechelic oxidative coupling, forming extensible, tough hydrogels. Small angle scattering indicates that the chain-extended polypeptides form a fractal network of nanoscale aggregates over a broad concentration range, accessing moduli ranging from 5 kPa to over 1 MPa over a concentration range of 5-30 wt %. These networks exhibited excellent erosion resistance and allowed for the diffusion and release of encapsulated particles consistent with a bicontinuous, porous structure with a broad distribution of pore sizes. Biofunctionalized, toughened networks were found to maintain the viability of human mesenchymal stem cells (hMSCs) in 2D, demonstrating signs of osteogenesis even in cell media without osteogenic molecules. Furthermore, chondrocytes could be readily mixed into these gels via thermoresponsive assembly and remained viable in extended culture. These studies demonstrate the ability to engineer ELP-based arrested physical networks on the molecular level to form reinforced, cytocompatible hydrogel matrices, supporting the promise of these new materials as candidates for the engineering and regeneration of stiff tissues.

Self-Assembly of Differently Shaped Protein-Polymer Conjugates through Modification of the Bioconjugation Site.

Self-assembly of protein-polymer block copolymers is an attractive route for preparing biocatalytic materials. To clarify the effect of bioconjugate shape on self-assembly without changing the chemical details of either block, four different conjugation sites are selected on the surface of the model globular protein mCherry at residues 3, 108, 131, and 222 to alter the colloidal shape of the bioconjugate. All four mCherry-b-poly(N-isopropylacrylamide) bioconjugates show qualitatively similar phase diagrams, indicating that self-assembly is robust with respect to changes in conjugation site. However, protein orientation has an effect on the location of the order-disorder transition concentration, and the stability of the disordered micellar phase is shown to be different for each conjugate. Differences in domain spacing also suggest changes in protein orientation within the lamellae.