Development and optimization of pH-responsive oral delivery systems for protein therapeutics
Creators: name: Miller, Matthew Kyle
Publisher: name: The University of Texas at Austin
Year: 2020
Subjects: Oral protein delivery
Drug delivery
pH-Responsive
Polymers
Antibodies

description: A pH-responsive anionic linear polymer was synthesized via controlled radical polymerization using reversible addition-fragmentation chain transfer (RAFT) polymerization. The linear polymer was used to form pH-responsive self-assembled nanoparticles via the nanoprecipitation technique capable of loading antibodies with high efficiency at high weight loading percentages. The linear polymer system and the nanoprecipitation process were optimized using a Design of Experiments approach, resulting in a system capable of maintaining high antibody loading while exhibiting favorable release characteristics in simulated gastric, intestinal, and circulatory conditions. The optimized polymer and nanoparticle system was able to achieve a loading efficiency of 94% with a final weight loading of 32.9% protein compared to polymer. This optimized system showed minimal release in simulated gastric and intestinal conditions, while exhibiting high release at physiological pH, which is ideal for a nanocarrier designed to transit the intestinal epithelium before releasing its payload. The optimized polymer and nanoparticle system were then evaluated in a series of in vitro models to determine their cytocompatibility and transport capabilities. The linear polymer showed no cytotoxicity at concentrations up to 1 mg/mL in two model cell lines using two different assays, which is higher than any concentration likely to be achieved in vivo. Nanoparticles were conjugated with Fc ligands to evaluate if targeting the neonatal Fc receptor (FcRn) on intestinal enterocytes could enhance transport of nanoparticles across the epithelial cell barrier. The nanoparticles were then evaluated in a transepithelial transport model using Caco-2 and HT29-MTX model cell lines. Transepithelial transport was analyzed using an ELISA assay as well as confocal microscopy of fluorescently labeled antibodies. Nanoparticles conjugated with Fc ligands resulted in transepithelial protein transport up to 25x higher than unconjugated nanoparticles or unencapsulated protein based on ELISA results. Confocal microscopy proved inconclusive in differentiating transport between groups, likely due to fluorescence quenching of the FITC probe in acidic intracellular vesicles combined with high levels of background fluorescence. Finally, cell penetrating peptides (CPPs) were evaluated as an alternative means of increasing transepithelial transport of proteins. CPPs were evaluated in a pH-responsive complexation hydrogel system delivering insulin. The crosslinking density and crosslinker type were found to have a strong effect on the loading and release characteristics of CPP-insulin dual loaded microparticles. In an in situ closed-loop intestinal model in rats, the CPP L-PenetraMax showed a promising ability to increase transepithelial transport of insulin, resulting in a decrease in blood glucose levels compared to microparticles loaded with insulin alone. Future work will likely benefit from a combination of CPPs with Fc-conjugated nanoparticle delivery systems.