We crosslink hemoglobin (Hb), superoxide dismutase (SOD), catalase (CAT), and carbonic anhydrase (CA) to form a soluble polyHb-SOD-CAT-CA nanobiotechnological complex. The obtained product is a soluble complex with three enhanced red blood cell (RBC) functions and without blood group antigens. In the present study, 2/3 of blood volume was removed to result in 90-min hemorrhagic shock at mean arterial blood pressure (MAP) of 30 mmHg. This was followed by the reinfusion of different resuscitation fluids, then followed for another 60 min. PolyHb-SOD-CAT-CA maintained the MAP at 87.5 ± 5 mmHg as compared with 3 volumes of lactated Ringer’s solution, 43.3 ± 2.8 mmHg; blood, 91.3 ± 3.6 mmHg; polyHb-SOD-CAT, 86.0 ± 4.6 mmHg; poly stroma-free hemolysate (polySFHb), 85.0 ± 2.5 mmHg; and polyHb, 82.6 ± 3.5 mmHg. PolyHb-SOD-CAT-CA was superior to the blood and other fluids based on the following criteria. PolyHb-SOD-CAT-CA reduced tissue pCO2 from 98 ± 4.5 mmHg to 68.6 ± 3 mmHg. This was significantly (p < 0.05) more effective than lactated Ringer’s solution (98 ± 4.5 mmHg), polyHb (90.1 ± 4.0 mmHg), polyHb-SOD-CAT (90.9 ± 1.4 mmHg), blood (79.1 ± 4.7 mmHg), and polySFHb (77 ± 5 mmHg). PolyHb-SOD-CAT-CA reduced the elevated ST level to 21.7 ± 6.7% and is significantly (< 0.05) better than polyHb (57.7 ± 8.7%), blood (39.1 ± 1.5%), polySFHb (38.3% ± 2.1%), polyHb-SOD-CAT (27.8 ± 5.6%), and lactated Ringer’s solution (106 ± 3.1%). The plasma cardiac troponin T (cTnT) level of polyHb-SOD-CAT-CA group was significantly (P < 0.05) lower than that of all the other groups. PolyHb-SOD-CAT-CA reduced plasma lactate level from 18 ± 2.3 mM/L to 6.9 ± 0.3 mM/L. It was significantly more effective (P < 0.05) than lactated Ringer’s solution (12.4 ± 0.6 mM/L), polyHb (9.6 ± 0.7 mM/L), blood (8.1 ± 0.2 mM/L), polySFHb (8.4 ± 0.1 mM/L), and polyHb-SOD-CAT (7.6 ± 0.3 mM/L). PolyHb-SOD-CAT-CA can be stored for 320 days at room temperature. Lyophilized poly-Hb-SOD-CAT-CA can be heat pasteurized at 68F for 2 h. This can be important if there is a need to inactivate human immunodeficiency virus, Ebola virus, and other infectious organisms.

by venous blood. The remaining 93% of CO 2 diffuse into RBCs where the gas is either bound to hemoglobin in the carbamate form, or is converted by RBC enzyme carbonic anhydrase (CA) into carbonic acid, which in turn dissociates into an H ϩ ion and an HCO 3 Ϫ ion [8, 9]. The conversion of approximately 70% of CO 2 to HCO 3 Ϫ ions in the circulation serves two important physiological functions. Firstly, it provides a more effi cient means of transport of CO 2 from tissue cells to the lungs. Secondly, the HCO 3 Ϫ ions produced act in a buffer system for metabolic acids. In the absence of a catalyst, the conversion of CO 2 to H ϩ and HCO 3 Ϫ is a very slow reaction. Thus, CO 2 and carbonic acid exist in the blood in a ratio of 400:1, and the proportion of CO 2 to HCO 3 Ϫ is 20:1 [10]. The hydration reaction of CO 2 would therefore occur much too slowly to be of physiological importance were it not for the presence of the zinc-containing metalloenzyme carbonic anhydrase (CA) in the RBCs [11, 12]. CA is involved in the crucial process of CO 2 transport by catalyzing the reversible hydration of CO 2 to carbonic acid, H ϩ and HCO 3 Ϫ , thus playing a fundamental role in the maintenance of acid-base balance of the body.While antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) have been crosslinked with PolyHb to produce oxygen carriers that can confer antioxidant properties [13], CA has not been incorporated into a PolyHb-enzyme system in suffi cient amount for the transport of CO 2 . The aim of this study is thus to prepare

by venous blood. The remaining 93% of CO 2 diffuse into RBCs where the gas is either bound to hemoglobin in the carbamate form, or is converted by RBC enzyme carbonic anhydrase (CA) into carbonic acid, which in turn dissociates into an H ϩ ion and an HCO 3 Ϫ ion [8, 9]. The conversion of approximately 70% of CO 2 to HCO 3 Ϫ ions in the circulation serves two important physiological functions. Firstly, it provides a more effi cient means of transport of CO 2 from tissue cells to the lungs. Secondly, the HCO 3 Ϫ ions produced act in a buffer system for metabolic acids. In the absence of a catalyst, the conversion of CO 2 to H ϩ and HCO 3 Ϫ is a very slow reaction. Thus, CO 2 and carbonic acid exist in the blood in a ratio of 400:1, and the proportion of CO 2 to HCO 3 Ϫ is 20:1 [10]. The hydration reaction of CO 2 would therefore occur much too slowly to be of physiological importance were it not for the presence of the zinc-containing metalloenzyme carbonic anhydrase (CA) in the RBCs [11, 12]. CA is involved in the crucial process of CO 2 transport by catalyzing the reversible hydration of CO 2 to carbonic acid, H ϩ and HCO 3 Ϫ , thus playing a fundamental role in the maintenance of acid-base balance of the body.While antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) have been crosslinked with PolyHb to produce oxygen carriers that can confer antioxidant properties [13], CA has not been incorporated into a PolyHb-enzyme system in suffi cient amount for the transport of CO 2 . The aim of this study is thus to prepare

Even though erythrocytes transport both oxygen and carbon dioxide, research on blood substitutes has concentrated on the transport of oxygen and its vasoactivity and oxidative effects. Recent study in a hemorrhagic shock animal model shows that the degree of tissue PCO 2 elevation is directly related to mortality rates. We therefore prepared a novel nanobiotechnological carrier for both O 2 and CO 2 with enhanced antioxidant properties. This is based on the use of glutaraldehyde to crosslink stroma free hemoglobin (SFHb), superoxide dismutase (SOD), catalase (CAT) and carbonic anhydrase(CA)to form a soluble PolySFHb-SOD-CAT-CA. It was compared to blood and different resuscitation fluids on the ability to lower elevated tissue PCO 2 in a 2/3 blood volume loss rat hemorrhagic shock model. Sixty minutes of sustained hemorrhagic shock at 30 mm Hg resulted in the increase of tissue PCO 2 to 95 mm ± 3 mmHg from the control level of 55 mm Hg. Reinfusion of whole blood (Hb 15 g/dL with its RBC enzymes) lowered the tissue PCO2 to 72 ± 4.5 mmHg 60 minutes after reinfusion. PolySFHb-SOD-CAT-CA (SFHb 10 g/dL plus additional enzymes) was more effective than whole blood in lowering PCO 2 lowering this to 66.2 ±3.5 mmHg. Ringer's Lactated solution or polyhemoglobin lowered the elevated PCO2 only slightly to 87 ± 4.5 mmHg and 84.8 ± 1.5 mmHg, respectively. Moreover, ST-elevation for whole blood (Hb 15 g/dL) and PolySFHb-SOD-CAT-CA (Hb 10 g/dL) was respectively 12.8% ± 4% and 13.0% ± 2% of the control 60 minutes after reinfusion. Both are significantly better than those in the Ringer's lactated group and the PolyHb group. In conclusion, this novel approach for blood substitute design has resulted in a novel nanobiotechnological carrier for both O 2 and CO 2 with enhanced antioxidant properties.

Poly(ethylene glycol)-Poly(lactic acid) block-copolymer (PEG-PLA) was prepared and characterized using Fourier transform infrared spectrophotometer (FTIR). Glutaraldehyde was used to crosslink stroma-free hemoglobin (SFHb), superoxide dismutase (SOD), catalase (CAT), and carbonic anhydrase (CA) into a soluble complex of PolySFHb-SOD-CAT-CA. PEG-PLA was then used to nanoencapsulated PolySFHb-SOD-CAT-CA by oil in water emulsification. This resulted in the formation of PLA-PEG-PolySFHb-SOD-CAT-CA nanocapsules that have enhanced antioxidant activity and that can transport both O 2 and CO 2 . These are homogeneous particles with an average diameter of 100 nm with good dispersion and core shell structure, high entrapment efficiency (EE%), and nanocapsule percent recovery. A lethal hemorrhagic shock model in rats was used to evaluate the therapeutic effect of the PLA-PEG-PolySFHb-SOD-CAT-CA nanocapsules. Infusion of this preparation resulted in the lowering of the elevated tissue PCO 2 and also recovery of the mean arterial pressure (MAP).

We have prepared a novel nanobiotherapeutic, Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase], which not only transports both oxygen and carbon dioxide but also a therapeutic antioxidant. Our previous study in a severe sustained 90 min hemorrhagic shock rat model shows that it has a hepatoprotective effect. We investigate its hepatoprotective effect further in this present report using an alcohol-damaged primary hepatocyte culture model. Results show that it significantly reduced ethanol-induced AST release, lipid peroxidation, and ROS production in rat primary hepatocytes culture. It also significantly enhanced the viability of ethanol-treated hepatocytes. Thus, the result shows that Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] also has some hepatoprotective effects against alcohol-induced injury in in vitro rat primary hepatocytes cell culture. This collaborate our previous observation of its hepatoprotective effect in a severe sustained 90-min hemorrhagic shock rat model.

No abstract

This is a mini review on the biotechnological aspects of the most extensively developed hemoglobin-based oxygen carriers The emphasis is on the most recent Polyhemoglobin-catalase-superoxide dismutase-carbonic anhydrase (PolyHb-CAT-SOD-CA), which is a nanobiotechnological complex that is being investigated and scaled up with the potential for clinical use as nanobiotherapeutics. Hemoglobin, a tetramer, is an excellent oxygen carrier. However, in the body it is converted into toxic dimers. Diacid or glutaraldehyde can crosslink hemoglobin into polyhemoglobin (PolyHb) and prevent its breakdown into toxic dimers. This has been developed and tested in clinical trials. A bovine polyhemoglobin has been approved for routine clinical use for surgical procedures in South Africa and Russia. Clinical trials with human PolyHb in hemorrhagic shock were effective but with a very slight increase in non-fatal myocardial ischemia. This could be due to a number of reasons. For those conditions with ischemia-reperfusion, one would need an oxygen carrier with antioxidant properties. One approach to remedy this is with prepared polyhemoglobin-catalase-superoxide dismutase (PolyHb-CAT-SOD). Another reason is an increase in intracellular pCO2. We therefore added an enhanced level of carbonic anhydrase to prepare a PolyHb-CAT-SOD-CA. The result is an oxygen carrier with enhanced Carbonic Anhydrase for CO2 transport and enhanced Catalase and Superoxide Dismutase for antioxidant functions. Detailed efficacy and safety studies have led to the industrial scale up towards clinical trial. In the meantime, oxygen carriers are being investigated around the world for use in ex vivo biotechnological fluid for organ preservation for transplantation, with one already approved in France.