Peptides that mimic the bioactivity of growth factors are rapidly emerging as therapeutics for a variety of drug delivery applications including therapeutic neovascularization. Neovascularization requires controlled and sustained delivery of proangiogenic factors to stimulate reperfusion of ischemic tissues. To this end, hydrogel nanoparticles were designed to provide sustained and tunable diffusion-based release of a pro-angiogenic peptide, QK. Inverse phase mini-emulsion polymerization (IPMP) was used to generate crosslinked poly(ethylene) glycol (PEG) hydrogel nanoparticles entrapped with the QK peptide. Peptide release kinetics were tuned through adjustments in nanoparticle crosslink density. This was achieved by altering the mole fraction of the crosslinking agent which allowed for the synthesis of low crosslink density (0.754 mmol cm) and high crosslink density (0.810 mmol cm) nanoparticles. Nanoparticle tracking analysis revealed narrow particle size distributions and similar particle sizes regardless of crosslink density (225 ± 75 nm and 233 ± 73 nm, for low and high crosslink density nanoparticles, respectively). The zeta potential was found to be -26 mV for blank nanoparticles and +4 mV in the case of QK-loaded nanoparticles. The resulting nanoparticle crosslink density impacted both peptide loading as well as release kinetics. In terms of cumulative fractional release and weight of peptide released per mass of nanoparticle, higher crosslink density nanoparticles resulted in slower peptide release kinetics. The IPMP process preserved the QK secondary structure and its bioactivity as confirmed using circular dichroism spectroscopy and a Matrigel tubulogenesis assay, respectively, with released peptide. The presented nanoparticles hold great potential for use as drug delivery carriers for stimulation of therapeutic neovascularization of ischemic tissues.

Intestinal disease or surgical intervention results in local changes in tissue and host-derived factors triggering bacterial virulence. A key phenotype involved in impaired tissue healing is increased bacterial collagenase expression which degrades intestinal collagen. Antibiotic administration is ineffective in addressing this issue as it inadvertently eliminates normal flora while allowing pathogenic bacteria to “bloom” and acquire antibiotic resistance. Compounds that could attenuate collagenase production while allowing commensal bacteria to proliferate normally would offer major advantages without the risk of the emergence of resistance. We have previously shown that intestinal phosphate depletion in the surgically stressed host is a major cue that triggers P. aeruginosa virulence which is suppressed under phosphate abundant conditions. Recent findings indicate that orally administered polyphosphate, hexametaphosphate, (PPi) suppresses collagenase, and biofilm production of P. aeruginosa and S. marcescens in animal models of intestinal injury but does not attenuate E. faecalis induced collagenolytic activity (Hyoju et al., 2017 ). Systemic administration of phosphates, however, is susceptible to rapid clearance. Given the diversity of collagenase producing bacteria and the variation of phosphate metabolism among microbial species, a combination therapy involving different phosphate compounds may be required to attenuate pathogenic phenotypes. To address these barriers, we present a drug delivery approach for sustained release of phosphates from poly(ethylene) glycol (PEG) hydrogel nanoparticles. The efficacy of monophosphate (Pi)- and PPi-loaded NPs (NP-Pi and NP-PPi, respectively) and a combination treatment (NP-Pi + NP-PPi) in mitigating collagenase and biofilm production of gram-positive and gram-negative pathogens expressing high collagenolytic activity was investigated. NP-PPi was found to significantly decrease collagenase and biofilm production of S. marcescens and P. aeruginosa . Treatment with either NP-Pi or NP-Pi + NP-PPi resulted in more prominent decreases in E. faecalis collagenase compared to NP-PPi alone. The combination treatment was also found to significantly reduce P. aeruginosa collagenase production. Finally, significant attenuation in biofilm dispersal was observed with NP-PPi or NP-Pi + NP-PPi treatment across all test pathogens. These findings suggest that sustained release of different forms of phosphate confers protection against gram-positive and gram-negative pathogens, thereby providing a promising treatment to attenuate expression of tissue-disruptive bacterial phenotypes without eradicating protective flora over the course of intestinal healing.

Insufficient vascularization limits the volume and complexity of biomaterials-based tissue engineering approaches. The formation of new blood vessels (neovascularization) is regulated by a complex interplay of cellular interactions with biochemical and biophysical signals provided by the extracellular matrix (ECM) which necessitates the development of biomaterial approaches that enable systematic modulation in matrix properties. Poly(ethylene) glycol-based hydrogel scaffolds were engineered with a range of decoupled and combined variations in integrin-binding peptide (RGD) ligand concentration, elastic modulus and proteolytic degradation rate using free-radical polymerization chemistry. The modularity of this system enabled a full factorial experimental design to simultaneously investigate the individual and interaction effects of these matrix cues on vascular sprout formation in 3D culture. Enhancements in scaffold proteolytic degradation rate promoted significant increases in vascular sprout length and junction number while increases in modulus significantly and negatively impacted vascular sprouting. We also observed that individual variations in immobilized RGD concentration did not significantly impact 3D vascular sprouting. Our findings revealed a previously unidentified and optimized combination whereby increases in both immobilized RGD concentration and proteolytic degradation rate resulted in significant and synergistic enhancements in 3D vascular spouting. The above-mentioned findings would have been challenging to uncover using one-factor-at-time experimental analyses.

Gradients in mechanical properties, physical architecture and biochemical composition exist in a variety of complex tissues, yet 3D in vitro models that enable investigation of these cues on cellular processes, especially those contributing to vascularization of engineered tissues are limited. Here, a photopolymerization approach to create cell‐laden hydrogel biomaterials with decoupled and combined gradients in modulus, immobilized cell adhesive peptide (RGD) concentration, and proteolytic degradation enabling spatial encapsulation of vascular spheroids is reported to elucidate their impact on vascular sprouting in 3D culture. Vascular spheroids encapsulated in these gradient scaffolds exhibit spatial variations in total sprout length. Scaffolds presenting an immobilized RGD gradient promote biased vascular sprouting toward increasing RGD concentration. Importantly, biased sprouting is found to be dependent on immobilized RGD gradient characteristics, including magnitude and slope, with increases in these factors contributing to significant enhancements in biased sprouting responses. Conversely, reduction in biased sprouting responses is observed in combined gradient scaffolds possessing opposing gradients in RGD and modulus. The presented work is the first to demonstrate the use of a cell‐laden biomaterial platform to systematically investigate the role of multiple scaffold gradients as well as gradient slope, magnitude and orientation on vascular sprouting responses in 3D culture.

Tissue response to intestinal injury or disease releases pro-inflammatory host stress signals triggering microbial shift to pathogenic phenotypes.