(586e) Oral Delivery of Insulin Bioconjugates Using Intelligent Complexation Hydrogels

Complexation graft copolymers of poly(methacrylic acid-g-ethylene glycol), designated as P(MAA-g-EG), have been shown to be effective in oral delivery of insulin. Their hydrogen bonding complexation/decomplexation characteristics render these responsive hydrogels able to protect the insulin in the harsh, acidic environment of the stomach before releasing the bioactive agent in the small intestine. Further, they can inhibit the activity of Ca2+ dependent proteolytic enzymes, increase the residence time of the drug in the small intestine by mucoadhesion, and reversibly open the tight junctions between the intestinal cells. Transport studies using Caco-2 cells, a widely used in vitro model for intestinal absorption of drugs, have proven the efficacy of the polymeric system. The effectiveness of this system in delivering insulin in vivo was evident from the reported 16% bioavailability of insulin seen in the diabetic rat studies.

Further enhancement in the insulin bioavailability may be obtained by conjugating it to molecules that can recognize specific receptors on the epithelial cells and get transported across the intestinal epithelium. This approach eliminates the side effects associated with prolonged opening of the tight junctions. Since the conjugate complex crosses the cell barrier by the transcellular mechanism and is specific towards the protein of interest, unspecific transport of toxic compounds is avoided. Researchers have investigated receptor-mediated transcytosis of the insulin-transferrin conjugate in Caco-2 cells. Transferrin is a naturally occurring protein that is involved in the uptake of iron by the cells. The iron-bound transferrin binds to the specific receptor on the epithelial cells and the complex is then endocytosed into the cells.

The use of the complexation hydrogel as a delivery vehicle for the insulin conjugates is under investigation. The transferrin molecule (~80 kDa) is significantly bigger than the insulin (~5.8kDa). Hence, as a step towards developing the delivery system, the loading and release profile of the transferrin from the swollen and deswollen polymer microparticles was investigated. The loading efficiency was found to be dependent on the pH of the protein solution. Loading of the microparticles near the iso-electric point of the protein resulted into high loading efficiencies. Further, the effect of loading and release of transferrin from the microparticles on transferrin's iron binding was investigated. The binding of iron to the transferrin is critical to its uptake by the epithelial cells. Understanding the loading and release of transferrin from the complexation hydrogels is important in developing a delivery system for the insulin-transferrin conjugates.

We investigated the use of the complexation hydrogel as a delivery vehicle for the insulin conjugates. We investigated the loading and release profiles of insulin conjugates from P(MAA-g-EG) microparticles. The conjugates were synthesized by coupling the proteins via succinimidyl 3-(2-pyridyldithio)propionate (SPDP), an amine reactive heterobifunctional crosslinker. The conjugates were purified by size exclusion chromatography and protein modifications were confirmed by mass spectroscopy. The insulin: transferrin ratio in the conjugates was also measured to determine the number of insulin molecules coupled to a single transferrin molecule. The loading and release studies were done under acidic pH and neutral pH conditions to mimic the transit of the polymer formulation from stomach into the small intestine. Understanding the loading and release behavior of the conjugates was critical to developing a delivery system for the insulin-transferrin conjugates.

Supported by grants from the National Institutes of Health (EB 000246) and the National Science Foundation (DGE-0333080).