Multifunctional cross-linked hemoglobin conjugates for protection of pancreatic beta cells

The isolation of beta-islets from the pancreas and their microencapsulation in a polymeric matrix render them susceptible to hypoxic and hypoxia-induced free radical stresses, causing enhanced apoptosis in vitro and at the site of transplantation. This research project focused on low p50 hemoglobin (Hb) conjugates with the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), for Hb's protection from free radicals and from the combined oxidative and hypoxic stress for beta-islets. This dissertation is an account of the enhanced cell viability of beta-cells for an advancement toward a biohybrid artificial pancreas. A poly(ethylene glycol) cross-linker used in an optimized Hb:PEG molar ratio (1:10) dramatically reduced methemoglobin formation. The addition of antioxidant enzymes with hemoglobin (Hb-SOD-CAT) inhibited methemoglobin formation during storage at 4 °C for a month and also when subjected to external oxidative stress. The size of conjugates obtained by cross-linking was also optimal for coencapsulation with beta-cells, thus preventing the diffusional loss of Hb conjugates from the microcapsules. The addition of poly(ethylene glycol) cross-linked Hb-SOD-CAT conjugates with low p50 characteristics and antioxidant properties showed enhanced protective effects on pancreatic cells (RINm5F cells) when incubated iv in hypoxic (1% oxygen) conditions, as higher oxygen amounts are released in hypoxic condition. Combined hypoxic and free radical (hypoxia-induced) stress conditions are encountered during islet transplantation. RINm5F cells that had been incubated in hypoxia and challenged with oxidants (hydrogen peroxide and superoxide anion) showed low intracellular free radical activity on addition when optimized Hb-SOD-CAT conjugates was added. These optimized Hb-SOD-CAT conjugates also help maintain glucose-induced insulin secretion from pancreatic beta-islets in combined oxidative and hypoxic stress experiments. In summary, the potential of Hb-SOD-CAT conjugates to reduce the dysfunction of beta-islets associated with free radical and hypoxic stress has been demonstrated. It is likely that antioxidant enzymes will play a significant role in the protection of hemoglobin in these conjugates and consequently will enhance cell viability. Consequently, the use of Hb-SOD-CAT conjugates in a biohybrid artificial pancreas consisting of an oxygen carrier, antioxidants, and pancreatic beta-islets in microcapsules has the potential to provide a better solution for long-term insulin secretion.