(315e) Oral Delivery of High Isoelectric Point Therapeutic Proteins Using pH-Responsive Nanoscale Complexation Hydrogels

Protein therapeutics represent a rapidly growing pharmacological sector, with over 200 FDA-approved products¹. However, due to poor stability and large molecular weight, protein administration is almost exclusively limited to injection. These frequent injections can be very painful and are often associated with fear and major side effects at the injection site. Achieving oral delivery is ideal and would almost certainly improve patient compliance but is fraught with challenges. Efforts to develop delivery platforms have been limited by the conditions in the gastrointestinal tract, which greatly reduce the activity of the proteins, and thus, their bioavailability is negligible if delivered in an unprotected state². Environmentally responsive polymer carriers, such as those that swell and collapse in response to changes in pH, have been explored for the development of an oral delivery platform for protein drugs. These pH-responsive carriers rely on charge interactions for the complexation behavior that facilitates protein loading and release³. This characteristic presents a difficulty with proteins that exhibit a high isoelectric point, as the protein is likely to remain bound to the anionic hydrogel carrier at the pH of the small intestine instead of being repelled. This interaction must be overcome to allow protein release and thus, therapeutic efficacy. The aims of our work were to explore strategies for increasing release of high isoelectric point therapeutic proteins from anionic hydrogel nanoparticles under neutral conditions.

Copolymeric nanoparticle systems containing acrylamide, itaconic acid, and crosslinked with N,N’-methylenebisacrylamide were synthesized via inverse emulsion polymerization, a protocol adapted from Zhong et al⁴. Analysis with FTIR and potentiometric titration confirmed the expected chemical composition. Nanogel swelling was characterized with dynamic light scattering and zeta potential was measured with electrophoretic light scattering. With dynamic light scattering, it was confirmed that the synthesized nanogels exhibit the expected swelling behavior with increasing pH values, swelling from a hydrodynamic diameter of approximately 85 nm at pH 3 to 105 nm at pH 9 in 1X PBS. Electrophoretic light scattering demonstrated that both formulations exhibit a negative zeta potential, with the value becoming more negative with increasing pH values. Cytochrome C was used as a model high isolectric point protein and the nanoparticles exhibited loading efficiencies of this protein up to 95%. Minimal protein release at pH 4 was observed while more than 50% of the protein was released within 5.5 hours at pH 7.4. In vitro studies using Caco-2 cells were performed to analyze the cytocompatibility of the nanoparticles at concentrations up to 5mg/mL. Further testing on protein transport studies across the epithelium barrier and implementation of strategies to enhance protein uptake after delivery to the intestinal lumen must be explored. The synthesized nanogels show promise for oral delivery applications of high isoelectric point proteins.

This work was supported by NIH grant number R01 EB022025 and the Cockrell Family Regents Chair in Engineering (UT Austin). F.A.C.V. was supported by the Conacyt/ConTex Fellowship, and the Cockrell School of Engineering Fellowship (UT Austin). H.F.O. was supported by the National Science Foundation Graduate Research Fellowship and the Archie W. Straiton Endowed Graduate Fellowship in Engineering #2 (UT Austin).

References

1. Fosgerau, K. & Hoffmann, T. Drug Discovery Today 20, 122–128 (2015).
2. Gupta, S. et al. Drug Delivery 20, 237–246 (2013).
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4. Zhong, J.X., et al.J Biomed Mater Res Part A 106A, 1677– 1686 (2018).