Gregory A. Hudalla

Gradated assembly of multiple proteins into supramolecular nanomaterials

Biomaterials displaying precise ratios of different bioactive protein components are critical for applications ranging from vaccines to regenerative medicine, but their design is often hindered by limited choices and cross-reactivity of protein conjugation chemistries. Here, we describe a strategy for inducing multiple different expressed proteins of choice to assemble into nanofibers and gels with exceptional compositional control. The strategy employs novel “βTail” tags, which allow for good protein expression in bacteriological cultures, yet can be induced to co-assemble into nanomaterials when mixed with additional β-sheet fibrillizing peptides. Multiple different βTail fusion proteins could be inserted into peptide nanofibers alone or in combination at predictable, smoothly gradated concentrations, providing a simple yet versatile route to install precise combinations of proteins into nanomaterials. The technology is illustrated by achieving precisely targeted hues using mixtures of fluorescent proteins, by creating nanofibers bearing enzymatic activity, and by adjusting antigenic dominance in vaccines.

An Approach to Modulate Degradation and Mesenchymal Stem Cell Behavior in Poly(ethylene glycol) Networks

A simple, sequential approach for creation of hydrolytically degradable poly(ethylene glycol) (PEG) hydrogels has been developed and characterized. The chemistry involves an initial step growth polymerization reaction between PEG-diacrylate and dithiothreitol (DTT) to form acrylate-terminated (-PEG-DTT-)n PEG chains, followed by photocross-linking to form a hydrogel network. Varying the extent of step growth polymerization prior to photocross-linking allowed for control over the equilibrium swelling ratio, degradation, and erosion of PEG hydrogels. Hydrogel degradability had a significant effect on behavior of human mesenchymal stem cells (hMSCs) encapsulated within PEG hydrogels, both in the presence and absence of an RGDSP cell adhesion ligand. In particular, enhanced network degradability resulted in enhanced hMSC viability and spreading during in vitro culture. Comparison of degradable and nondegradable hydrogels with similar physical properties (e.g., equilibrium swelling ratio) demonstrated that hMSC viability and spreading were dependent on network degradability. This study demonstrates that hydrolytically degradable PEG hydrogels can be formed via a sequential step growth polymerization and photocross-linking process and the resulting materials may serve as promising matrices for 3-dimensional stem cell culture and tissue engineering applications.

A Self-Adjuvanting Supramolecular Vaccine Carrying a Folded Protein Antigen

This work illustrates a strategy for the design of molecularly defined immunotherapies, using a blend of supramolecular peptide self-assembly and active site-directed protein capture.

Surfaces That Sequester Serum-Borne Heparin Amplify Growth Factor Activity

Surfaces presenting a heparin-binding peptide can non-covalently sequester heparin from culture supplements, such as fetal bovine serum. In turn, sequestered, serum-borne heparin can non-covalently localize growth factors at the cell-material interface, resulting in amplified growth factor bioactivity.

Adaptable poly(ethylene glycol) microspheres capable of mixed-mode degradation.

A simple, degradable poly(ethylene glycol) (PEG) microsphere system formed from a water-in-water emulsion process is presented. Microsphere network degradation and erosion were controlled by adjusting the number of hydrolytically labile sites, by varying the PEG molecular weight, and by adjusting the emulsion conditions. Microsphere size was also controllable by adjusting the polymer formulation. Furthermore, it is demonstrated that alternative degradation and erosion mechanisms, such as proteolytic degradation, can be incorporated into PEG microspheres, resulting in mixed-mode degradation. Owing to the adaptability of this approach, it may serve as an attractive option for emerging tissue engineering, drug delivery and gene delivery applications.

Controllably degradable β-sheet nanofibers and gels from self-assembling depsipeptides

Self-assembled peptide materials have received considerable interest for a range of applications, including 3D cell culture, tissue engineering, and the delivery of cells and drugs. One challenge in applying such materials within these areas has been the extreme stability of β-sheet fibrillized peptides, which are resistant to proteolysis, degradation, and turnover in biological environments. In this study, we designed self-assembling depsipeptides containing ester bonds within the peptide backbone. Beta-sheet fibrillized nanofibers were formed in physiologic conditions, and two of these nanofiber-forming depsipeptides produced hydrogels that degraded controllably over the course of days-to-weeks via ester hydrolysis. With HPLC, TEM, and oscillating rheometry, we show that the rate of hydrolysis can be controlled in a straightforward manner by specifying the amino acid residues surrounding the ester bond. In 3D cell cultures, depsipeptide gels softened over the course of several days and permitted considerably more proliferation and spreading of C3H10T1/2 pluripotent stem cells than non-degradable analogs. This approach now provides a reliable and reproducible means to soften or clear β-sheet fibrillized peptide materials from biological environments.

Engineering galectin-glycan interactions for immunotherapy and immunomodulation.

Galectins, a 15-member family of soluble carbohydrate-binding proteins, are receiving increasing interest as therapeutic targets for immunotherapy and immunomodulation due to their role as extracellular signals that regulate innate and adaptive immune cell phenotype and function. However, different galectins can have redundant, synergistic, or antagonistic signaling activity in normal immunological responses, such as resolution of inflammation and induction of antigen-specific tolerance. In addition, certain galectins can be hijacked to promote progression of immunopathologies, such as tumor immune privilege, metastasis, and viral infection, while others can inhibit these processes. Thus, eliciting a desired immunological outcome will likely necessitate therapeutics that can precisely enhance or inhibit particular galectin–glycan interactions. Multivalency is an important determinant of the affinity and specificity of natural galectin–glycan interactions, and is emerging as a key design element for therapeutics that can effectively manipulate galectin bioactivity. This minireview surveys current molecular and biomaterial engineering approaches to create therapeutics that can stabilize galectin multivalency or recapitulate natural glycan multivalency (i.e. “the glycocluster effect”). In particular, we highlight examples of using natural and engineered multivalent galectins for immunosuppression and immune tolerance, with a particular emphasis on treating autoimmune diseases or avoiding transplant rejection. In addition, we present examples of multivalent inhibitors of galectin–glycan interactions to maintain or restore T-cell function, with a particular emphasis on promoting antitumor immunity. Finally, we discuss emerging opportunities to further engineer galectin–glycan interactions for immunotherapy and immunomodulation.

Self-Assembled Peptide and Protein Nanofibers for Biomedical Applications

Abstract Spontaneous organization of small-molecule building blocks into highly ordered structures, commonly referred to as “self-assembly,” is receiving increasing interest for fabricating functional biomaterials. Self-assembly is ubiquitous in nature. Understanding the molecular interactions that mediate self-assembly in nature enables rational design of synthetic biomolecules, which can organize into biomaterials with specific nanoscale arcitechtures. In this chapter, we survey recent advances in designing peptides and proteins that self-assemble into high-aspect ratio fibers with nanoscale features.