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.

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.

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.

Alginate hydrogels are widely used as biomaterials for cell culture and tissue engineering due to their biocompatibility and tunable mechanical properties. Reducing alginate molecular weight is an effective strategy for modulating hydrogel viscoelasticity and stress relaxation behavior, which can significantly impact cell spreading and fate. However, current methods like gamma irradiation to produce low molecular weight alginates suffer from high cost and limited accessibility. Here, a facile and cost-effective approach to reduce alginate molecular weight in a highly controlled manner using serial autoclaving is presented. Increasing the number of autoclave cycles results in proportional reductions in intrinsic viscosity, hydrodynamic radius, and molecular weight of the polymer while maintaining its chemical composition. Hydrogels fabricated from mixtures of the autoclaved alginates exhibit tunable mechanical properties, with inclusion of lower molecular weight alginate leading to softer gels with faster stress relaxation behaviors. The method is demonstrated by establishing how viscoelastic relaxation affects the spreading of encapsulated fibroblasts and glioblastoma cells. Results establish repetitive autoclaving as an easily accessible technique to generate alginates with a range of molecular weights and to control the viscoelastic properties of alginate hydrogels, and demonstrate utility across applications in mechanobiology, tissue engineering, and regenerative medicine.

Knee osteoarthritis (OA) is characterized by cartilage degeneration and significant reduction in lubrication. One strategy to recover the natural lubrication of the synovial fluid is the injection of hydrogel microspheres. Here, we have fabricated polyethylene glycol (PEG)-based hydrogel microspheres via a modified electrospraying setup. To improve throughout, crosslinking of PEG droplets was delayed until after droplet formation was complete. A custom-synthesized super-lubricious copolymer consisting of adhesive dopamine methacrylate (DMA), zwitterionic sulfobetaine methacrylate (SBMA), and fluorescent rhodamine B was used to dip-coat the PEG microspheres. Super-lubricious PEG microspheres coating reduced coefficient of friction by 57% compared to simulated synovial fluid, indicating beneficial lubrication properties. When injected into C57BL6 mice, PEG microspheres exhibited stability for up to 26 d and did not adversely affect mouse behavior. These super-lubricious PEG microspheres offer great promise to reduce the friction that is a hallmark of progressive OA, potentially mitigating the need for total knee arthroplasty.