A novel nanobiotherapeutic poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] with no cardiac toxicity for the resuscitation of a rat model with 90 minutes of sustained severe hemorrhagic shock with loss of 2/3 blood volume

Abstract

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.

Keywords: antioxidant; artificial cells; biotherapeutic; carbon dioxide carrier; carbonic anhydrase; cardiac toxicity; catalase; hemorrhagic shock; inactive infectious agents; ischemia–reperfusion; oxygen carrier; oxygen radicals; pasteurization; resuscitation; storage stability; superoxide dismutase.

Figure 1.

Mean arterial blood pressure. 2/3 volume of blood was removed to result in 90 min of hemorrhagic shock at MAP of 30 mmHg. After this, different fluids were reinfused. Reinfusion with 3 volumes of lactated Ringer's solution increased MAP transiently and fell to 43.3 ± 2.8 mmHg. Blood (Hb 15 g/dl) maintained the MAP at 91.3 ± 3.6 mmHg, while polyHb-SOD-CAT-CA (Hb 10 g/dL) maintained it at 87.5 ± 5 mmHg, polyHb-SOD-CAT at 86.0 ± 4.6 mmHg, polySFHb at 85.0 ± 2.5 mmHg, and polyHb at 82.6 ± 3.5 mmHg.

Figure 2.

Comparison of mean blood pressures. The above individual measurements were pooled together for comparison. Sham control measurement, where no blood loss occurred, was added as control.

Figure 3.. Tissue pCO₂ and plasma lactate. Following the 2/3 volume of blood loss, the tissue pCO₂ level increased steadily from the baseline tissue pCO₂ of 54.7 ± 2.5 mmHg to 98 ± 4.5 mmHg by 90 min after the hemorrhagic shock. Reinfusion with polyHb-SOD-CAT-CA reduced this to 68.6 ± 3 mmHg in 1 h. The effect that the other fluids had on tissue pCO₂ were 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.

Figure 4.

Comparison of pCO₂. The above measurements of each group were pooled together for comparison. Sham control measurement, where no blood loss occurred, was added as control. The results showed that reinfusion with polyHb-SOD-CAT-CA reduced this to 68.6 ± 3 mmHg in 1 h. 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).

Figure 5.

ST elevation. PolyHb-SOD-CAT-CA reduced the ST to 21.7 ± 6.7%. This was significantly (p < 0.05) better than polyHb (57.7 ± 8.7%), blood (39.1 ± 1.5%), polySFHb (38.3% ± 2.1%), and polyHb-SOD-CAT (27.8 ± 5.6%). Lactated Ringer's solution exhibited no significant (p > 0.05) effects (106 ± 3.1%).

Figure 6.

Plasma lactate. PolyHb-SOD-CAT-CA reduced the plasma lactate level from 18 ± 2.3 mM/L to 6.9 ± 0.3 mM/L. It was significantly (p < 0.05) more effective 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).