Semisynthetic supra plasma expanders: a new class of therapeutics to improve microcircualtion in sickle cell anaemia

Figures & data

Figure 1 Schematic representation of EAF conjugation of Alb with tempo.

Figure 2 Experimental protocol to induce vaso-occlusion in NY1DD.

Table 1. Solution properties of semisynthetic active plasma expanders.

	Molecular radius (nm)	COP (mmHg)	Viscosity (cP)
Alb T12	4.0	10	1.0
EAF P5K6 Alb T12a	7.9	44.8	2.0
EAF P5K6 Alb (4 gm%)	7.8	44.8	2.3
EAF P5K6 Hb (4 gm%) ^b	6.5	50.2^c	2.2
EAF P3K6 Hb (6 gm%)^d	5.4	75.0	2.1

^aThe COP and viscosity were measured using a 4 gm% (protein basis) solution in PBS, pH 7.4, at room temperature; ^bThe data come from the references [11] and [25]; ^cThe COP and viscosity were measured using a 5.5 gm% (protein basis) solution in PBS, pH 7.4, at room temperature, and the data come from reference [26]; ^dThe COP and viscosity were measured using a 6 gm% (protein basis) solution in PBS, pH 7.4, at room temperature.

Figure 3 Representative images of venules from transgenic sickle cell mice (NY1DD) treated with Alb T12, EAF P5K6 Alb T12 and PEG Alb in hypoxia/reoyxgenation. (A) Wild type (B) NY1DD mice (C) NY1DD – untreated H/R (D) NY1DD mice treated with Alb T12 (E) NY1DD mice treated with EAF P5K6 Alb T12 (F) NY1DD mice treated with EAF P5K6 Alb T12.White arrow (→) indicate the blood flow direction and black arrow head indicates leukocytes.

Figure 4 (A) NY1DD sickle mice receiving Alb T12, P5K6 Alb T12 and P5K6 Alb at the onset of reoxygenation showed marked reduction in leukocyte adhesion, with P5K6 Alb T12 having a normalizing effect. (B) NY1DD mice receiving P5K6 Alb and P5K6 Alb T12 at the onset of reoxygenation show marked reduction in leukocyte emigration compared with untreated NY1DD mice. *p< .03 vs. wild type; ⁺p< .00001 vs. normoxic NY1DD mice; ^#p< .0001 vs. untreated NY1DD mice subjected to hypoxia-reoxygenation.

Table 2. The influence of PEG Alb and PEG Alb T12 on haemodynamic parameters of sickle (NY1DD) mice subjected active plasma expansion therapy using Hypoxia-Reoxygenation system.

	Normoxia		Hypoxia-reoxygenation (sickle mice)
Parameter	Wild type (C57BL mice)	Sickle mice	Untreated	Alb T12	EAF P5K6 Alb T12	EAF P5K6 Alb
Diameter, nm	28.7 ± 1.3^a	28.4 ± 1.5	28.4 ± 1.4	26.6 ± 0.91	28.6 ± 1.6	27.6 ± 1.4
Red cell vel, mm/s	6.3 ± 0.6	2.5 ± 0.3*	1.7 ± 0.2**	1.4 ± 0.22	6.1 ± 0.7***	5.0 ± 0.7***
Wall shear rate, s^−1	852.5 ± 102	472.4 ± 39*	211.5 ± 17**	280.8 ± 45	819.8 ± 27.5***	733.8 ± 23.7***
Volumetric flow, nL/s	2.9 ± 0.48	1.1 ± 0.19*	0.65 ± 0.08**	0.51 ± 0.08	2.8 ± 0.64***	1.7 ± 0.12***

Values are mean ± SE. ^aFour to six venules were examined for haemodynamic parameters in each mouse (n = 3–4 each experimental group). *p< .005–.0001 vs. normoxic wild type controls. **p< .05–.023 vs. respective normoxic values for NY1DD mice. ***p< .004–.00001 vs. untreated NY1DD mice subjected to hypoxia-reoxygenation.

Figure 5 Effect of Alb T12, P5K6 Alb T12, and P5K6 Alb on wall shear rate in transgenic sickle cell mice (NY1DD) and wild type (C57BL/6J). Values are mean ± SE. Four to six venules were examined for each mouse (n = 3–4 each experimental group); ⁺p< .005–.0001 vs. normoxic wild type controls; **p< .05–.023 vs. respective normoxic values for NY1DD mice; ^#p< .004–.00001 vs. untreated NY1DD mice subjected to hypoxia-reoxygenation.

Figure 6 The intravital microscopic images of venules from transgenic sickle cell mice (NYIDD) subjected to hypoxia-reoxygenation protocol and treated with EAF P3K6 Hb after hypoxia.

Figure 7 The response of cremaster arterioles (A3) to topical application of SNP (10⁻⁶ M) after pretreatment with 150 μL of 4 g% EAF P5K6 Alb T12.

Figure 8 Exploded view of schematic representation of the structure of (A) Alb tempo, (B) EAF-Alb-tempo and (C) EAF PEG Alb-tempo.

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