A novel nanobiotechnological oxygen and carbon dioxide carrier with enhanced antioxidant properties: polyhemoglobin -superoxide dismutase-catalase-carbonic anhydrase

ABSTRACTIntroduction: The shortage of donor blood gives rise to demand for blood substitute. The most successful blood substitute, polymerized hemoglobin (PolyHb), has been approved for routine clinical use in South African and Russia. However, PolyHb is only an oxygen carrier but not a complete red blood cells (RBCs) substitute. RBCs have three main functions: 1) Oxygen transport; 2) anti-oxidant enzyme activities; 3) Carbonic dioxide transport. Thus, transfusion of PolyHb in severe ischemia conditions could give rise to ischemia reperfusion injuries. This is due to the lack of anti-oxidant enzyme activities in the PolyHb. In addition, recently researches show that elevation of tissue pCO2 in severe hemorrhagic shock is related to increase in mortality. This suggests that CO2 transport is also a vital function of any RBC substitutes. This thesis presents the design and detailed evaluation of a novel blood substitute, Polyhemoglobin-superoxide dismutase- catalase- carbonic anhydrase (Poly-[Hb-SOD-CAT-CA]). This has all of the three main functions of RBC.Method: The enzyme for CO2 transport, carbonic anhydrase (CA) and the anti-oxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) are crosslinked together with Hb by nanobiotechnology to form Poly-[Hb-SOD-CAT-CA]. After optimizing the crosslink procedure, detailed in vitro and in vivo analysis has been carried out to fully evaluate this new complex. The in vitro studies include the test of enzymes activity and kinetics, molecular distribution, temperature stability, storage stability, P50 as well as screening test using complement activation. Two lethal shock models are used. One is 60 min shock and the other is outside the 60 min safety window, 90 min shock. These in vivo studies evaluate the effects of Poly-[Hb-SOD-CAT-CA] on maintaining mean artery pressure (MAP), lowering of the elevated tissue pCO2 as well as recovery of ST-elevation. In the 90 min shock model, the effects on biochemistry and histology of the heart, intestine, kidney and liver have also been analyzed. Result: After crosslinking, Poly-[Hb-SOD-CAT-CA] retains significant enzymes activity and proper molecular distribution. The MetHb decreases to an acceptable content after optimization. The sample could be kept for a long time (128days) in the solution form at 4℃ and -80℃ and still maintain its molecular distribution and main part of enzyme activities. Lyophilization extends the storage time to 320 days and these freeze-dried samples show good temperature stability even for 30 minutes to 3 hours at the pasteurization temperature of 65℃. The oxygen carrying ability of this blood substitute is maintained before and after storage. In vitro screening test shows that Poly-[Hb-SOD-CAT-CA] does not cause complement activation.Studies on the 60 min shock model shows that Poly-[Hb-SOD-CAT-CA] could maintain the MAP at 80mmHg and also facilitate the recovery of the ST-elevation. Unlike PolyHb, Poly-[Hb-SOD-CAT-CA] also effectively lowers the elevated tissue pCO2. When extending beyond the 60 min…