Effect of Cross-Linked Hemoglobin on Functionality and Viability of Microencapsulated Pancreatic Islets

Abstract

Of many obstacles involved in developing a bioartificial pancreas, which consists of encapsulated and physically immunoprotected islets, for long-term implantation in insulin-dependent diabetic patients, the impaired functionality and decreasing viability of encapsulated islets over time are critical factors in determining the size and longevity of the implant. These factors are closely associated with short oxygen supply to the encaged islets from the implant site. To facilitate oxygen transport to islets in the capsules, we coencapsulated hemoglobin cross-linked with difunctional polyethylene glycol (Hb-conjugate, Hb-C) which is large in size (>100 kDa), thus preventing diffusional loss through the immunoprotecting membrane. The coencapsulation of Hb-C with islets in alginate-poly-L-lysine microcapsules by dissolving Hb-C in an islet-suspended alginate solution at a concentration of 0.25 mM improved the insulin secretion and viability of the islets. At week 0, the islets, coencapsulated with Hb-C, cultured at P(O2) = 40 mmHg (assumed oxygen partial pressure in the most common implant site, the peritoneal cavity), secreted 200% more insulin compared with the control islets without Hb-C at glucose concentrations of both 100 and 300 mg/dL. The Hb-C effect became more significant with time at higher glucose concentrations. After culturing the islets for 8 weeks at 40 mmHg, the insulin secretion was enhanced 200 and 550% at glucose concentrations of 100 and 300 mg/dL as compared with the control, respectively. The results were closely associated with improved viability and suggest that the introduction of Hb-C is an effective approach to maintaining the oxygen supply to encapsulated islets. In addition, Hb-C coencapsulation with pancreatic islets may (1) provide a partial clue to reducing the large size of the biohybrid artificial pancreas, (2) lead to a reduced need for pancreas donation, and (3) prolong the longevity of the biohybrid artificial pancreas in the body.

Synthesis and characterization of hemoglobin conjugates with antioxidant enzymes via poly(ethylene glycol) cross-linker (Hb–SOD–CAT) for protection from free radical stress

“…This solution was then transferred to 100 kDa dialysis tube and dialyzed for 24 hours where only pure hemoglobin was diffused out, which can be further concentrated by using Amicon membranes. The purity of hemoglobin was verified by SDS-PAGE and quantified by using Drabkin's method for quantification [28]. …”

“…by employing previously reported methods [28, 30, 31]. Briefly, the hydroxyl group of PEG was converted to ethyl-protected carboxymethylated PEG by using potassium tertiary butoxide and ethyl bromoacetate.…”

“…Our previous research had shown that crosslinked hemoglobin improved the functionality of encapsulated isolated pancreatic beta cells in transplanted diabetic mice by maintaining oxygen supply to encapsulated cells [28, 29]. However, the shelf-life of hemoglobin was limited because of its conversion to methemoglobin and free radical damage induced by autooxidation of hemoglobin.…”

Hemoglobin Conjugates with Antioxidant Enzymes (Hemoglobin-Superoxide Dismutase-Catalase) via Poly(Ethylene Glycol) Crosslinker for Protection of Pancreatic Beta RINm5F Cells in Hypoxia

“…19 PEG (2 kDa) was also chemically modified as previously described 19 ; potassium tertiary butoxide and ethyl bromoacetate was used to modify the hydroxyl end groups on PEG to ethyl-protected carboxymethylated PEG. Basic hydrolysis of this intermediate PEG compound resulted in mixture of PEG-carboxylic acids.…”

Dynamic Oxygen Enhances Oocyte Maturation in Long-Term Follicle Culture

“…The oxygen gradient across the walls of microcapillaries is steep, reducing the intracapillary levels by half. 17 The Po 2 across the subrenal capsule drops from the peritoneal level of 40 to 14-19 mmHg. 16 In contrast, an abundance of blood vessels are found in the region of the ovary that contains secondary and antral follicles.…”