(T1230-08-49) Microfluidic Synthesis of pH- and Ca^2+- Responsive PNAG Microparticles

Purpose: Poly(N-acryloyl glycine) (PNAG), a hydrogel-forming material, has shown promising characteristics such as tunable mechanical properties, self-healing properties, biocompatibility, and autolytic behavior. However, its potential as a biomedical release system for biopharmaceutics is limited by the harsh conditions typically required for its synthesis, which can compromise the structure of its drug load, rendering it ineffective or, worse, cause immunogenicity. To address this challenge, the aim of this study was to generate microparticulate structures through microfluidics and investigate the swelling behavior of PNAG. By leveraging the swelling-deswelling mechanism, drug loading might be carried out post-synthesis, under milder conditions, preserving the stability and activity of biopharmaceutics.

Methods: The gelation kinetics of PNAG during polymer synthesis were determined in situ by oscillatory photorheology. Formulations with varying concentrations of monomer and photoinitiators (PI) were investigated across different pH values and presence of Ca2+. Microfluidic droplet generation was achieved using a flow-focusing capillary device, with subsequent crosslinking. The loading with some peptide/proteins as model biopharmaceuticals was performed by swelling.

Results: The monomer concentration had the greatest impact on gelation in photorheology. At high monomer concentration, the effect of different PIs on gel properties was minimal. TPO-nanoparticles (diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide) were identified as the most preferable PI based on the rheological properties of the materials, even at low PI concentration. Next, the effect of pH on gelation was investigated. At neutral and basic pH, stable networks formed only at high monomer concentration, while acidic pH also provided gels at 5 wt.% monomer. During microparticle production in microfluidics, the PI concentration significantly affected droplet formation: high concentrations impeded droplet generation due to a non-ionic surfactant present in the TPO-nanoparticles, while lower PI concentrations were suitable to produce monodisperse droplets. Adding Ca²⁺ before droplet formation resulted in similar particle sizes as without Ca²⁺, while the number of satellite droplets increased. Swelling studies confirmed the ability of the PNAG p<span style="mso-ansi-language: #1000;">articles to increase in size depending on pH and the presence of Ca2+, with degrees of swelling ranging from 10% to 600% depending on the conditions. Swelling substantially increased as the pH of the medium increased. The molecular weight and net charge of peptides/proteins had a strong effect on their loading levels achievable during swelling in the respective peptide/protein solution. Interestingly, a lower loading was observed in the case of higher opposing charge of hydrogel particles and protein, presumably due to strong interaction at the particle surface, hindering further drug molecules from entering the hydrogel's porous network. Loading levels as high as 21 wt.% and release periods over up to 2 weeks were achievable for a peptide drug.

Conclusion: PNAG hydrogel microparticles were successfully synthesized by in situ polymerization in a microfluidic device. The particles exhibited a pH-responsive swelling, which could be used for loading with model biopharmaceuticals. The particles showed high loading levels and extended release periods for a peptide model drug, suggesting their exploration for the local release of a broader range of biopharmaceuticals.