Matthew J. Glassman

Solid-State Nanostructured Materials from Self-Assembly of a Globular Protein-Polymer Diblock Copolymer

Self-assembly of three-dimensional solid-state nanostructures containing approximately 33% by weight globular protein is demonstrated using a globular protein-polymer diblock copolymer, providing a route to direct nanopatterning of proteins for use in bioelectronic and biocatalytic materials. A mutant red fluorescent protein, mCherryS131C, was prepared by incorporation of a unique cysteine residue and site-specifically conjugated to end-functionalized poly(N-isopropylacrylamide) through thiol-maleimide coupling to form a well-defined model protein-polymer block copolymer. The block copolymer was self-assembled into bulk nanostructures by solvent evaporation from concentrated solutions. Small-angle X-ray scattering and transmission electron microscopy illustrated the formation of highly disordered lamellae or hexagonally perforated lamellae depending upon the selectivity of the solvent during evaporation. Solvent annealing of bulk samples resulted in a transition toward lamellar nanostructures with mCherry packed in a bilayer configuration and a large improvement in long-range ordering. Wide-angle X-ray scattering indicated that mCherry did not crystallize within the block copolymer nanodomains and that the β-sheet spacing was not affected by self-assembly. Circular dichroism showed no change in protein secondary structure after self-assembly, while UV-vis spectroscopy indicated approximately 35% of the chromophore remained optically active.

Structure and Mechanical Response of Protein Hydrogels Reinforced by Block Copolymer Self-Assembly.

A strategy for responsively toughening an injectable protein hydrogel has been implemented by incorporating an associative protein as the midblock in triblock copolymers with thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) endblocks, producing materials with a low yield stress necessary for injectability and durability required for load-bearing applications post-injection. Responsive reinforcement triggered by PNIPAM association leads to significant increases in the gels elastic modulus as well as its resistance to creep. The performance of these materials is a strong function of molecular design, with certain formulations reaching elastic moduli of up to 130 kPa, effectively reinforced by a factor of 14 over their low temperature moduli, and having stress relaxation times increased by up to a factor of 50. The nanostructural origins of these thermoresponsive enhancements were explored, demonstrating that large micellar cores, high PNIPAM volume fractions, and high densities of associating groups in the protein corona lead to the greatest reinforcement of the gels elastic modulus. Gels with the largest micelles and the highest packing fractions also had the longest relaxation times in the reinforced state. These combined structure and mechanics studies reveal that control of both the micellar and protein networks is critical for making high performance gels relevant for biomedical applications.

Arrested Phase Separation of Elastin-like Polypeptide Solutions Yields Stiff, Thermoresponsive Gels

The preparation of new responsive hydrogels is crucial for the development of soft materials for various applications, including additive manufacturing and biomedical implants. Here, we report the discovery of a new mechanism for forming physical hydrogels by the arrested phase separation of a subclass of responsively hydrophobic elastin-like polypeptides (ELPs). When moderately concentrated solutions of ELPs with the pentapeptide repeat (XPAVG)n (where X is either 20% or 60% valine with the remainder isoleucine) are warmed above their inverse transition temperature, phase separation becomes arrested, and hydrogels can be formed with shear moduli on the order of 0.1-1 MPa at 20 wt % in water. The longest stress relaxation times are well beyond 10(3) s. This result is surprising because ELPs are classically known for thermoresponsive coacervation that leads to macrophase separation, and solids are typically formed in the bulk or by supplemental cross-linking strategies. This new mechanism can form gels with remarkable mechanical behavior based on simple macromolecules that can be easily engineered. Small angle scattering experiments indicate that phase separation arrests to form a network of nanoscale domains, exhibiting rheological and structural features consistent with an arrested spinodal decomposition mechanism. Gel nanostructure can be modeled as a disordered bicontinuous network with interdomain, intradomain, and curvature length scales that can be controlled by sequence design and assembly conditions. These studies introduce a new class of reversible, responsive materials based on a classic artificial biopolymer that is a versatile platform to address critical challenges in industrial and medical applications.

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.