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

Abstract

A low p50 hemoglobin (Hb) (p50 indicates O(2) tension at which Hb is half-saturated)-based oxygen carrier conjugated to antioxidant enzymes via dicarboxymethylated poly(ethylene glycol) (PEG) linker may have the beneficial effect in protecting pancreatic beta cells from severe hypoxia at transplantation sites. In this study, the oxygen dissociation curves, Hill plots, Bohr Effect, and oxygen content of Hb conjugates were measured. The protective effect due to incubation of Hb-conjugates (Hb/PEG molar ratio 1:10) with pancreatic beta cells (RINm5F) against hypoxia (6%, 3%, and 1% oxygen) was evaluated by an MTT assay and confocal microscopy. Quantitatively, Hb conjugates with antioxidant enzymes offered statistically significant protection (p<0.01, increased viability ∼80%) from hypoxia compared to control cells in 1% oxygen environment. Confocal images also showed that the low p50 Hb conjugates with antioxidants protected RINm5F cells from hypoxia.

Poly(ethylene glycol) cross‐linked hemoglobin with antioxidant enzymes protects pancreatic islets from hypoxic and free radical stress and extends islet functionality

“…Further, the oxygen supplying capability of hemoglobin to RINm5F cells was shown to be superior in low partial oxygen environment when antioxidant enzymes were cross-linked with Hb(Nadithe and Bae 2011). To some extent, islets may be protected in free radical environment with only SOD and CAT only but not when hypoxic condition is prevailing.…”

“…And also Hb-Hb alone will not function for extended time period as free radical assault will damage hemoglobin. Islets incubated with Hb-SOD-CAT conjugates and exposed to H 2 O 2 and superoxide anion showed minimal uptake of PI which could be due to direct protection of hemoglobin and islets from free radical damage by antioxidant enzymes followed by higher oxygen supply to islets by virtue of hemoglobins low p50 design (Nadithe and Bae 2011). These results strongly suggest that Hb-SOD-CAT may be an appropriate strategy to help prevent hypoxia-induced graft failure in conditions such as encapsulated islet transplantation(de Groot et al 2003; de Groot et al 2004; Ricci et al 2005) and ischemia re-perfusion injury(Chang 2010).…”

“…Experimental groups used optimized hemoglobin conjugates ratio of 1:10 Hb:PEG selected following our previous studies showing low methemoglobin (Nadithe and Bae 2010) and efficient oxygen carrier in hypoxic conditions(Nadithe and Bae 2011). The following groups were selected for evaluating the protective activity of Hb-conjugates: (1) RINm5F cells or islets with medium only, (2) RINm5F cells or islets with medium containing either 1 mM H 2 O 2 or 1 mM X and 10 mU/mL XO, (3) RINm5F cells or islets with medium containing 0.1 mM conjugated Hb-Hb plus 1 mM H 2 O 2 or 1 mM X and 10 mU/mL XO and (4) RINm5F cells or islets with medium containing 0.1 mM hemoglobin conjugated with antioxidants (Hb-SOD-CAT) plus 1 mM H 2 O 2 or 1 mM X and 10 mU/mL XO.…”

“…To overcome the problem of free radical damage to hemoglobin, we synthesized a dicarboxymethylated poly(ethylene glycol) (PEG) (supplemental figure 1) which was used to link antioxidant enzymes ( i.e ., superoxide dismutase (SOD) and catalase (CAT)) to hemoglobin (Hb), creating a Hb conjugate system (Hb-SOD-CAT). Previous in vitro studies showed hemoglobin was effectively protected from free radicals in the conjugate system(Nadithe and Bae 2010) and the low p50 design (p50 indicates O 2 tension at which hemoglobin is half-saturated) helped release of higher amounts of oxygen when exposed to low partial oxygen pressures or hypoxic stress(Nadithe and Bae 2011). In this research, we further tested the robustness and effectiveness of SOD and CAT in protecting RINm5F cells or islets and hemoglobin in normoxia (21%) or hypoxia (6% or 1%) and free radical-rich incubation conditions that were relevant to an in vivo transplantation(Carlsson et al 2000; Veriter et al 2011).…”

Tissue Engineering Sites for Pancreatic Islet Transplantation

“…Increasing the oxygen tension in the islet graft has been shown to improve islet survival [157][158][159][160][161][162][163] and monitoring the graft oxygen tension is desirable [164][165][166]. Strategies to increase local oxygen tension include: incorporation of hemoglobin [167][168][169], calcium peroxide-based oxygen generating particles [170], [171], perfluorocarbons [172], or a nearby gas phase oxygen reservoir [59], [173]. Hypoxia in islets often leads to intracellular generation of reactive oxygen species.…”