Long term safety and immunological effects of a nanobiotherapeutic, bovine poly-[hemoglobin-catalase-superoxide dismutase-carbonic anhydrase], after four weekly 5% blood volume top-loading followed by a challenge of 30% exchange transfusion

Figures & data

Figure 1. The rats are divided into 3 groups each receives 4 weekly infusion of one of the following samples: lactated ringer’s solution (LR) as control group, bovine polySFHb (Phe) and bovine poly-[Hb-CAT-SOD-CA] (PHE) with enhanced enzyme activity. This is followed on week 5 by a 30% exchange infusion.

Figure 2. Body weight changes of the rats during the four-week Top-loading infusion period. The body weight is measured in gram (g). Each figure shows a group of rats injected with corresponding sample (control LR, PHe, PHE). The body weight in rats injected with control (LR) are 158 ± 3.7 g, 227 ± 7 g, 288 ± 17 g, 348 ± 20 g. The body weight in rats injected with bovine PHe are 183 ± 9 g, 248 ± 15 g, 313 ± 9 g, 366 ± 25 g. The body weight in rats injected with bovine PHE are 198 ± 4 g, 257 ± 5 g, 318 ± 9 g, 367 ± 9 g. There are no significant differences from the control group.

Figure 3. BUN urea, creatinine and CK levels. All data are in Table 1. No significant statistical differences from the control groups.

Figure 4. Albumin, total bilirubin, ALT and AST levels. All data are in Table 1. No significant statistical differences from the control groups.

Table 1. Biochemistry test.

		Lactacted ringer's		poly(SFHb)		poly(SFHb-CAT-SOD-CA)		Lactacted ringer's		poly(SFHb)		poly(SFHb-CAT-SOD-CA)
		After 4 weekly IV		After 4 weekly IV		After 4 weekly IV		After 30% transfusion		After 30% transfusion		After 30% transfusion
tests	Units	Average	SD	Average	SD	Average	SD	Average	SD	Average	SD	Average	SD
Total Protein	g/L	57	1.52752523	57.3333333	1.1055416	58.8333333	1.06718737	57	2.23606798	60	0	60.75	0.8291562
Albumin	g/L	31.8333333	1.21335165	31.6666667	1.1055416	32.8333333	1.46249406	31.5	1.11803399	33	0.70710678	32.5	0.8660254
Albumin/Globulin ratio		1.25	0.05	1.25	0.05	1.25	0.07637626	1.225	0.08291562	1.25	0.05	1.15	0.08660254
Glucose	mmol/L	10.7333333	0.81785628	12.1666667	1.18133634	11.4	0.56273143	10.975	1.30838641	10.9	0.6670832	10	0.53851648
BUN Urea	mmol/L	5.71666667	0.63879226	4.86666667	0.74311656	5.2	0.76376262	4.725	0.25860201	4.8	0.41833001	4.775	1.29687123
Creatinine	µmol/L	17.8333333	0.372678	18.1666667	1.06718737	21.1666667	1.57233019	20.25	1.08972474	19.75	1.47901995	20.75	4.96865173
Total Bilirubin	µmol/L	4.33333333	1.80871659	4.33333333	1.13437475	3.16666667	0.89752747	3	0.70710678	3	0.70710678	4.75	1.29903811
ALT	U/L	50.5	6.92218655	45.6666667	6.62486897	53.5	2.5	45.5	4.55521679	39.5	3.04138127	45.75	3.49106001
AST	U/L	69.3333333	3.81517438	67.3333333	4.30761599	73.5	1.25830574	61.5	2.87228132	66.25	4.49305019	82	10.2469508
Alkaline Phosphatase	U/L	266.833333	29.1342677	228.666667	20.0305323	280.833333	33.3637361	192	19.9499373	181.75	12.2142335	203.5	41.8897362
CK	U/L	115.333333	12.9314432	151.333333	17.9412622	138.166667	27.9846188	96	10.7470926	116.25	5.62916512	135.75	18.6731759
Cholesterol	mmol/L	2.04833333	0.2663592	2.3	0.59205856	1.94833333	0.25458572	1.67	0.18881208	2.4325	0.24200981	1.9125	0.3647859
Sodium	mmol/L	135	3.05505046	141	2.94392029	145.666667	1.79505494	141.75	0.4330127	141.25	0.8291562	142.5	0.8660254
Potassium	mmol/L	10.0166667	0.90446424	4.58333333	0.26718699	4.88333333	0.22669118	4.6	0.45276926	4.95	0.28722813	5.225	0.21650635
Chloride	mmol/L	100	2.23606798	104	0.81649658	106	1	102.75	1.29903811	103.5	1.11803399	104.5	1.11803399
Calcium	mmol/L	2.51166667	0.05842849	2.65	0.05446712	2.70666667	0.04384315	2.595	0.025	2.665	0.04609772	2.72	0.01
Phosphorus	mmol/L	2.46333333	0.09012337	2.66666667	0.12762793	2.70833333	0.11964067	2.3725	0.0973075	2.385	0.22655022	2.5625	0.13479151
Magnesium	mmol/L	0.75333333	0.04606758	0.69333333	0.05088113	0.71833333	0.0157233	0.715	0.02598076	0.6825	0.02384848	0.6775	0.02277608

Figure 5. Ouchterlony double diffusion test. Positive control of BSA showing BSA and anti-BSA antibody precipitation.

Figure 6. Ouchterlony double diffusion test on rats after 4 weekly 5% top-loading infusion with lactated ringer’s solution as control, Bovine PHe or PHE (6 rats/group) showing no antigen-antibody precipitations.

Figure 7. Ouchterlony double diffusion test on rats after 30% blood volume exchange transfusion with control (LR), PHe, or PHE (4 rats/group)) showing no antigen-antibody precipitations.

Figure 8. Total IgG levels after four-weekly 5% blood volume top-loading infusions followed by 30% blood volume exchange transfusion. After four-weekly injection-period, the total IgG levels in rat plasma against LR control group, PHe and PHE are 5.42 ± 2.36 mg/ml, 7.88 ± 3.13 mg/ml and 10.41 ± 7.80 mg/ml, respectively; p values by one-way ANOVA is .32 (>.05). After 30% blood volume exchange transfusion, the total IgG concentrations in LR control group, PHe and PHE group are 5.84 ± 1.86 mg/ml, 7.82 ± 3.27 mg/ml and 9.24 ± 5.80 mg/ml; p values by one-way ANOVA is .60 (>.05).

Figure 9. Total IgM levels after four-weekly 5% blood volume top-loading infusions followed by 30% blood volume exchange transfusion. Total IgM levels after top-loading infusion with LR control group, PHe or PHE are 0.56 ± 0.33 mg/ml, 1.24 ± 0.60 mg/ml and 2.28 ± 0.87 mg/ml; p values by one-way ANOVA is .002 (<.05). Total IgM levels after 30% blood volume exchange transfusion with LR control group, PHe or PHE are 0.57 ± 0.22 mg/ml, 1.79 ± 0.75 mg/ml and 2.11 ± 1.33 mg/ml; p values by one-way ANOVA is .14 (>.05). However, if we only compare the control group and PHe group by student’s T-test, the p value is .035 (<.05); compare the control group and PHE by student’s T-test, p value is .093 (>.05).

Figure 10. The excess hemoglobin in the poly-[Hb-CAT-SOD-CA] complex nanoencapsulate the more antigenic enzymes.

Figure 11. The antibodies levels against SOD after 4-weekly Top-loading infusions in group of control (LR), PHe and PHE are 1.92 ± 2.09 ng/ml, 1.30 ± 2.11 ng/ml and 1.28 ± 2.57 ng/ml respectively. p Values by one-way ANOVA is .48 (> .05). The antibodies against bovine SOD in group of control (LR), PHe and PHE are 21.13 ± 7.57 ng/ml, 28.79 ± 15.07 ng/ml and 6.80 ± 7.79 ng/ml; p values by one-way ANOVA is .09 (> .05).

Figure 12. The concentration of antibodies against bovine Hb after four-weekly injection in group of control (LR), PHe and PHE are 0.41 ± 0.09 mg/ml, 0.49 ± 0.13 mg/ml and 0.42 ± 0.08 mg/ml, respectively. The p values by one-way ANOVA is .83 (> .05). After 30% blood volume exchange, the antibodies against Hb in group of control (LR), PHe and PHE are 0.44 ± 0.14 mg/ml, 0.40 ± 0.10 mg/ml and 0.52 ± 0.11 mg/ml, respectively; p values by one-way ANOVA is .47 (> .05).

Figure 13. Antibody against bovine CAT after 4-weekly injections followed by 30% blood volume exchange transfusion. No antibody is detected.

Figure 14. Antibody against bovine CA after four-weekly injections followed by 30% blood volume exchange transfusion. No antibody is detected.

Figure 15. C3a activation test. After four-weekly injections, C3a levels in LR control group, PHe and PHE groups are 144.49 ± 4.39 ng/ml, 150.74 ± 6.38 ng/ml and 153.56 ± 9.90 ng/ml, respectively; p values by one-way ANOVA is .16 (>.05). After four-week top-loading infusions followed by 30% blood volume exchange, C3a levels in LR control group, PHe and PHE groups are 150.74 ± 1.60 ng/ml, 149.70 ± 2.77 ng/ml and 150.50 ± 3.18 ng/ml, respectively; p values by one-way ANOVA is .88 (>.05).

Figure 16. Mean arterial pressures are recorded just before and 5 min after each top-loading infusion. Each figure shows a group of rats injected with corresponding sample. Control (LR): the correlation coefficients between the blood pressure before or after injection are 0. 27 and 0.56, respectively. The p values of the coefficients are 0.73 and 0.44, respectively. PHe: the correlation coefficients between the blood pressure before or after injection are 0.89 and 0.77, respectively. The p values of the coefficients are 0.11 and 0.23, respectively. PHE: the correlation coefficients between the blood pressure before or after injection are 0.16 and 0.30, respectively. The p values of the coefficients are 0.24 and 0.25, respectively.

Figure 17. Mean arterial pressure during the 30% blood volume exchange transfusion. Each figure showed a group of rats infused with one of lactated ringer’s solution (control), PHe, PHE. Control (LR): the correlation coefficients of the blood pressure before and after infusion is 0.13. The p values of the coefficient is .55. PHe: the correlation coefficient between the blood pressure before or after infusion is −0.03. The p values of the coefficient is .87. PHE: the correlation coefficients between the blood pressure before or after infusion are −0.16. The p values of the coefficient is .45. Thus, there are no significant changes.

Figure 18. In anaphylactic reaction both histamine and tryptase are released from the mast cells.

Figure 19. Histamine and tryptase levels tested before and 30 min after 30% blood volume exchange transfusion of LR (control), PHe or PHE. Plasma trptase analysis is more accurate than histamine as shown by the higher SD for histamine. The histamine levels: the p values by one-way ANOVA among the three groups is .62 (>.05). The tryptase levels: comparing the LR control groups and the PHe and PHE groups, the p values by one-way ANOVA is .51 (>.05) before infusion, and the p values of after infusion is .21 (>.05). Thus, there are no significant changes in the histamine or tryptase levels between the control group and the PHe and PHE groups.