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

Figures & data

Table I. Composition of test liquids.

Test liquids	Type	Hb gm/dl	SOD Unit/dl	CAT Unit/dl	CA Unit/dl	P50 mmHg
Lactated Ringer's solution	solution	0	0	0	0	N/A
Blood	7 μm	15	112,500	750,000	1,875,000	26.6
PolyHb	solution	10	0	0	0	18.2
PolySFHb	solution	10	70,000	470,000	950,000	19.15
PolyHb-SOD-CAT	solution	10	195,000	3,500,000	0	20.5
PolySFHb-SOD-CAT-CA	solution	10	195,000	3,500,000	1,360,000	19.92

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).

Table II. Histology of organs 60 min after reinfusion.

	Heart	Intestine	Kidney	Liver
Ringers lactated solution	Sarcoplasmic condensation; Contraction bands	Damaged villi as well as gland	Cytoplasmic vacuoles; tubular dilatation; necrosis	Sinusoidal dilatation; hepatocyte necrosis and congestion
PolyHb	Band contraction	Injured villi and mild injured gland	Disorder in the structure of tubular and cell necrosis	Eosinophilic change and nuclear loss
Blood	Sarcoplasmic condensation and eosinophilic changes	Injured villi but intact gland	Congestion and necrotic cells	Mild hepatocyte necrosis and congestion
PolySFHb	Eosinophilic changes	No damage	Tubular epithelial cell swelling	Mild congestion
PolyHb-SOD-CAT	No injury signals	No damage	Tubular epithelial cell swelling	Mild congestion
PolyHb-SOD-CAT-CA	No injury signals	No damage	Tubular epithelial cell swelling	Mild congestion

Table III. Effects of storage time and temperature on stability.

Stability at different temperatures
Donor red blood cells
Maximal time of storage allowed
	20–25C	4C
	1 day?	42 days
Poly-[Hb-SOD-CAT-CA] solution
T1/2 of enzyme activities:
	20–25C	4C
CAT	172 days	380 days
SOD	92 days	198 days
CA	51 days	231 days
Poly-[Hb-SOD-CAT-CA] lyophilized:
Day 320 % enzyme activity
	20–25C	4C
CAT	73% (day 320)	85% (day 320)
SOD	67% (day 320)	76% (day 320)
CA	73% (day 320)	85% (day 320)

Table IV. Comparison of polyHb-SOD-CAT-CA with RBC.

Properties	RBCs	PolyHb-SOD-CAT-CA
Oxygen transport	7 microns	Solution: better perfusion
Carbon dioxide transport	7 microns, enzymes in RBC	Solution + Enhanced enzymes
Oxygen radicals removal	7 microns, enzymes in RBC	Solution+ Enhanced enzymes
Storage	1 day, unless refrigerated: 42 days	> 320 days room temperature
Blood groups	Yes, unless O	None
Heat sterilization	Cannot	Yes for lyophilized form
Platelet-like function	None	Possible
Availability	Limited	No limit
Circulation time	Donor RBC, 30 days	1–3 days
Potential applications	Better for elective long-term RBC replacement	As nanobiotherapeutic for emergency uses