Designing Biocompatible Hydrogel Nanoparticle Emulsions for Regenerative Engineering

Polymeric nanoparticle (NP) emulsions have shown significant promise for regenerative engineering applications as they can be designed as topical and/or injectable formulations for <i>in vivo</i> administration of therapeutic compounds. Hydrophilic therapeutic delivery often requires water-in-oil-in-water (w/o/w) double emulsions which are difficult to produce and have limited storage life because of their inherent instability. Inverse, water-in-oil (w/o) emulsions for encapsulation of hydrophilic therapeutics are far less common as they involve use of organic solvents. Highly biocompatible and biodegradable crosslinked hydrogel NP are perfectly suited for controlled and sustained diffusive-based release of therapeutics, enhanced tissue-targeted delivery and bioavailability of hydrophilic drugs. Hydrogel NP, however, come in contact with cytotoxic organic solvents and surfactants during emulsion polymerization requiring subsequent purification and isolation before being administered directly <i>in vivo</i>. This leads to limitations, including significant drug loss, dosage uncertainty, compromised drug release kinetics, and loss of particle stability, thereby minimizing <i>in vivo</i> therapeutic efficacy. To address these limitations, we have developed novel biocompatible NP emulsions (BCNE) consisting of stable poly(ethylene) glycol (PEG) crosslinked hydrogel NP dispersed in a naturally-derived consumable organic phase that can be formulated as a liquid (or as an ointment) and administered <i>en masse</i>, directly to target tissues. BCNE are synthesized with biocompatible components including PEG diacrylate (PEGDA) crosslinking macromer, N-vinyl pyrrolidone or ethylene glycol comonomers, polyphosphate (PPi) or NaCl as the lipophobe, soybean oil (SO), as the organic phase, with PolyGlycerol PolyRicinoleate (PGPR) emulsifier (FDA recognized as safe for human consumption) and with V50 initiator using inverse phase miniemulsion polymerization. We synthesized BCNE with varying mesh dimensions to accommodate the encapsulation and sustained release of hydrophilic therapeutics of varying size and conformation including PPi (PPi-BCNE), NaCl (NaCl-BCNE) and the α-helical pro-angiogenic peptide QK (QK-BCNE). BCNE NP size measurements (Nanosight LM10, Malvern) yielded mean NP diameters of 111±7.2nm and 96±3.9nm for PPi-BCNE and NaCl-BCNE, respectively. To define mesh dimensions suitable for encapsulation and release of PPi and QK, experimental measurements of hydrogel mass swelling ratio were coupled with theoretical kinetic and thermodynamic interpretations we recently developed to characterize the network architecture of hydrogel networks comprised of macromeric crosslinkers. Our approach accounts for crosslinker size, and its behavior under swelling, both neglected by the Flory-Rehner theory. This allows for quantification of two new hydrogel mesh dimension variables that establish bounds on the maximum size and aspect ratio of therapeutics that can be encapsulated and released from crosslinked hydrogel NP networks. Using this approach to guide our experimental design, QK-BCNE were designed to release QK for 46 days whereby 90% of the encapsulated peptide was retained for 36 days <i>in vitro</i>. <i>In vitro</i> findings indicate that PPi-BCNE completely disperse PAO1 <i>P. aeruginosa</i> biofilms compared to blank BCNE and no treatment controls. Preliminary experiments conducted with e<i>n masse</i> topical application of BCNE in a <i>Staphylococcus aureus</i> C57BL/6 mouse model of burn injury and impaired healing demonstrated incontrovertible evidence that PPi-BCNE improves wound healing compared to blank BCNE treatment. These results highly suggest that BCNE provide a versatile platform whereby hydrogel nanoparticle mesh dimensions can be precisely tuned for enacpsulation of hydrophilic therapeutics of varying size and complex conformation (peptides, growth factors and iRNA) to enable sustained tissue-targeted delivery <i>in vivo</i> for regenerative medicine applications.