Self-assembled peptide-based biomaterials provide versatile platforms for biomedical uses, featuring customizable physicochemical properties, biocompatibility, and dynamic capabilities. This self-assembly process is primarily dictated by primary sequence features, such as hydrophobicity, length, and charge, leading to the formation of fibrils and hydrogels. Amphipathic peptides, with alternating polar and hydrophobic residues, are especially effective in forming supramolecular nanofibers stabilized by π-π interactions and hydrogen bonds. Chemical modifications on aromatic side chains are promising for controlling assembly morphology, stability, and biological activity. However, the influence of these substituents on peptide packing and immunogenicity remains relatively unexplored. Herein, we examine the effect of substituents on benzyl groups attached to short amphipathic peptides. By introducing different electron-donating and withdrawing groups at the para-position of benzyl rings and modifying the chain length connecting the backbone to the aromatic moiety, we observe notable effects on fibril formation, molecular packing, and immunogenicity both in vitro and in vivo. Our results show that subtle chemical modifications are practical tools for designing tailored peptide nanomaterials with promising potential in vaccine delivery, tissue engineering, and regenerative medicine.

Microgels are increasingly recognized as versatile building blocks for granular cell scaffolds, offering advantages over bulk hydrogels for a variety of biomedical applications. While existing methods for scaffold fabrication often require multistep processes involving separate microgel formation and assembly, here we introduce a streamlined, one-pot approach that achieves microgel formation and scaffold assembly in minutes. This developed method is robust, reproducible, user-friendly, and requires no specialized equipment, making it broadly accessible. Specifically, aqueous two-phase separation (ATPS) was utilized to form ~2 μm gelatin methacrylate (GelMA) microgels in sodium sulfate salt solution, which rapidly “clicked” to form macroporous scaffolds under UV light. Various parameters were modulated to observe the effect on scaffold formation including timing of UV exposure, salt concentration, photoinitiator concentration, and polymer concentration. Our results indicated a mechanically stable scaffold able to quickly imbibe water due to its interconnected macropores. U-87 glioblastoma, NIH 3T3 fibroblast, and ATDC5 chondrocyte cells were successfully encapsulated within these granular scaffolds and exhibited an elongated morphology at 24 h and > 90% viability over 14-21 days of culture. The ability to produce microgel scaffolds containing living cells in one step opens new routes to the production of cell-laden porous scaffolds.

Morquio A syndrome is a rare genetic disorder where deficiency in N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme prevents breakdown of glycosaminoglycans (GAGs). Recombinant human GALNS (rhGALNS) is currently administered by intravenous infusion, but the treatment is costly and time-consuming and provides limited efficacy. Patient quality of life could be improved by an injectable sustained rhGALNS release device that would eliminate weekly multi-hour infusions. Polyethylene glycol (PEG) hydrogels can be employed as a hydrophilic, tunable, non-toxic, and biodegradable drug delivery system for the sustained release of rhGALNS, as explored by us previously. Here, we investigated the stability of rhGALNS in various buffers mimicking the in vivo environment that would be encountered by the enzyme, inside of and outside the PEG hydrogels. rhGALNS activity was reduced 85% by reversible inhibition in phosphate-buffered saline (PBS), representing interstitial fluid and plasma. Buffer exchanging into acidic buffer representing the lysosome recovered this loss. However, incubation in PBS for 3 days resulted in an irreversible loss of 85%. There were no significant changes in rhGALNS hydrodynamic radius upon activity loss, suggesting structural integrity. Such activity loss makes sustained delivery impractical without additional stabilization, such as confinement within the hydrogel. rhGALNS activity was retained upon encapsulation, and the average specific activity of rhGALNS released from a hydrogel decreased only 20% over 7 days. These results show that the activity of rhGALNS was better retained within the hydrogel than in buffer alone, potentially enabling sustained release for rhGALNS or other enzymes unstable in physiological conditions with our hydrogel delivery device.

Sustained local delivery of biologics hydrogel carriers is a promising approach to enhance protein safety and efficacy. The addition of nanoparticles to polymeric hydrogels has been shown to further improve the retention and delivery kinetics of biologics. Specifically, nanoparticles with high surface area, such as nanosilicates, have shown potential for complexing with biologics to enable highly tunable release profiles. Here, LAPONITE® XLG nanosilicate (NS) was examined due to its platelet-like structure with negatively charged faces and positively charged edges. Our previous results have shown NS to greatly slow the release of model proteins from poly(ethylene glycol) (PEG) hydrogels due to NS-protein complexation. This work aims to determine the structure and stability of several NS-protein complexes, as well as protein activity and structure upon complexation. Binding affinity assays revealed a strong correlation between affinity and protein charge, with positively charged proteins being more attracted to NS. Proteins were shown to unfold in the presence of NS in solution, leading to a partial loss in bioactivity. However, this unfolding was determined to be temporary, as proteins released from PEG-NS hydrogels recovered secondary structure and bioactivity. Binding to NS also provided some protection against protein denaturant guanidine thiocyanate. Through understanding the interactions between proteins and NS, this study paves the way for the application of these NS-protein complexes as tunable, sustained-release delivery devices.

Peptides and peptidomimetics that self-assemble through LLPS have recently emerged as vital building blocks for creating functional biomaterials, thanks to their unique physicochemical properties and dynamic nature. One of life’s most distinctive features is its selectivity for chiral molecules. To date, coacervates comprised of d-amino acids have not been reported. Here, we demonstrate that histidine-rich repeats of (GHGXY) (X = L/V/P) and their enantiomers undergo LLPS, paving the way for improved coacervate stability. Through a series of biophysical studies, we found that the droplet size can be tuned based on L, V, or P substitution, and molecular cargo between 600 and 150 000 Da is efficiently recruited in a bioactivity-preserving aqueous environment during phase separation. Mechanistic studies reveal that the droplets enter cells energy-dependent endocytic pathways, exhibit composition-selective fusion properties, and effectively deliver molecular therapeutics across various cell types. Finally, we demonstrate that the coacervates enhance antigen presentation to CD4 and CD8 T cells, resulting in robust proliferation and the production of functional cytokines. Our study outlines the development and characterization of enantiomeric peptide coacervates as promising vaccine delivery vehicles with tunable physicochemical properties.