Effects of Cell-Adhesive Ligand Presentation on Pentapeptide Supramolecular Assembly and Gelation: Simulations and Experiments.

The extracellular matrix (ECM) is a complex, hierarchical material containing structural and bioactive components. This complexity makes decoupling the effects of biomechanical properties and cell-matrix interactions difficult, especially when studying cellular processes in a 3D environment. Matrix mechanics and cell adhesion are both known regulators of specific cellular processes such as stem cell proliferation and differentiation. However, more information is required about how such variables impact various neural lineages that could, upon transplantation, therapeutically improve neural function after a central nervous system injury or disease. Rapidly Assembling Pentapeptides for Injectable Delivery (RAPID) hydrogels are one biomaterial approach to meet these goals, consisting of a family of peptide sequences that assemble into physical hydrogels in physiological media. In this study, we studied our previously reported supramolecularly-assembling RAPID hydrogels functionalized with the ECM-derived cell-adhesive peptide ligands RGD, IKVAV, and YIGSR. Using molecular dynamics simulations and experimental rheology, we demonstrated that these integrin-binding ligands at physiological concentrations (3-12 mm) did not impact the assembly of the KYFIL peptide system. In simulations, molecular measures of assembly such as hydrogen bonding and pi-pi interactions appeared unaffected by cell-adhesion sequence or concentration. Visualizations of clustering and analysis of solvent-accessible surface area indicated that the integrin-binding domains remained exposed. KYFIL or AYFIL hydrogels containing 3 mm of integrin-binding domains resulted in mechanical properties consistent with their non-functionalized equivalents. This strategy of doping RAPID gels with cell-adhesion sequences allows for the precise tuning of peptide ligand concentration, independent of the rheological properties. The controllability of the RAPID hydrogel system provides an opportunity to investigate the effect of integrin-binding interactions on encapsulated neural cells to discern how hydrogel microenvironment impacts growth, maturation, or differentiation.

Peptide Stereocomplexation Orchestrates Supramolecular Assembly of Hydrogel Biomaterials.

Stereocomplexation, or specific interactions among complementary stereoregular macromolecules, is burgeoning as an increasingly impactful design tool, exerting exquisite control of material structure and properties. Since stereocomplexation of polymers produces remarkable transformations in mechanics, morphology, and degradation, we sought to leverage stereocomplexation to tune these properties in peptide-based biomaterials. We found that blending the pentapeptides l- and d-KYFIL triggers dual mechanical and morphological transformations from stiff fibrous hydrogels into less stiff networks of plates, starkly contrasting prior reports that blending l- and d-peptides produces stiffer fibrous hydrogels than the individual constituents. The morphological transformation of KYFIL in phosphate-buffered saline from fibers that entangle into hydrogels to plates that cannot entangle explains the accompanying mechanical transformation. Moreover, the blends shield l-KYFIL from proteolytic degradation, producing materials with comparable proteolytic stability to d-KYFIL but with distinct 2D plate morphologies that in biomaterials may promote unique therapeutic release profiles and cell behavior. To confirm that these morphological, mechanical, and stability changes arise from differences in molecular packing as in polymer stereocomplexation, we acquired X-ray diffraction patterns, which showed l- and d-KYFIL to be amorphous and their blends to be crystalline. Stereocomplexation is particularly apparent in pure water, where l- and d-KYFIL are soluble random coils, and their blends form ß-sheets and gel within minutes. Our results highlight the role of molecular details, such as peptide sequence, in determining the material properties resulting from stereocomplexation. Looking forward, the ability of stereocomplexation to orchestrate supramolecular assembly and tune application-critical properties champions stereochemistry as a compelling design consideration.

Biomaterials via peptide assembly: Design, characterization, and application in tissue engineering.

A core challenge in biomaterials, with both fundamental significance and technological relevance, concerns the rational design of bioactive microenvironments. Designed properly, peptides can undergo supramolecular assembly into dynamic, physical hydrogels that mimic the mechanical, topological, and biochemical features of native tissue microenvironments. The relatively facile, inexpensive, and automatable preparation of peptides, coupled with low batch-to-batch variability, motivates the expanded use of assembling peptide hydrogels for biomedical applications. Integral to realizing dynamic peptide assemblies as functional biomaterials for tissue engineering is an understanding of the molecular and macroscopic features that govern assembly, morphology, and biological interactions. In this review, we first discuss the design of assembling peptides, including primary structure (sequence), secondary structure (e.g., α-helix and ß-sheets), and molecular interactions that facilitate assembly into multiscale materials with desired properties. Next, we describe characterization tools for elucidating molecular structure and interactions, morphology, bulk properties, and biological functionality. Understanding of these characterization methods enables researchers to access a variety of approaches in this ever-expanding field. Finally, we discuss the biological properties and applications of peptide-based biomaterials for engineering several important tissues. By connecting molecular features and mechanisms of assembling peptides to the material and biological properties, we aim to guide the design and characterization of peptide-based biomaterials for tissue engineering and regenerative medicine. STATEMENT OF SIGNIFICANCE: Engineering peptide-based biomaterials that mimic the topological and mechanical properties of natural extracellular matrices provide excellent opportunities to direct cell behavior for regenerative medicine and tissue engineering. Here we review the molecular-scale features of assembling peptides that result in biomaterials that exhibit a variety of relevant extracellular matrix-mimetic properties and promote beneficial cell-biomaterial interactions. Aiming to inspire and guide researchers approaching this challenge from both the peptide biomaterial design and tissue engineering perspectives, we also present characterization tools for understanding the connection between peptide structure and properties and highlight the use of peptide-based biomaterials in neural, orthopedic, cardiac, muscular, and immune engineering applications.

Rapidly Assembling Pentapeptides for Injectable Delivery (RAPID) Hydrogels as Cytoprotective Cell Carriers.

Low cell survival after syringe injection hampers the success of preclinical and clinical cell transplantation trials. During syringe injection, cells experience mechanical forces that lead to cell-membrane disruption and decreased viability. To improve cell survival, we designed rapidly assembling pentapeptides for injectable delivery (RAPID) hydrogels that shear-thin, protect cells from extensional flow, form fibers, and provide mechanical properties similar to native tissue. We found that 1.5 wt % RAPID hydrogels mitigate the damaging effects of extensional flow, resulting in significantly greater cell viability (of common laboratory cell lines, primary cells, and human cells) than cells injected in PBS.