Administration Of Liposome-encapsulated Hemoglobin Research Articles

Nanovesicular liposome-encapsulated hemoglobin (LEH) prevents multi-organ injuries in a rat model of hemorrhagic shock

The goals of resuscitation in hemorrhagic shock are to correct oxygen deficit and to maintain perfusion pressure to the vital organs. We created liposome-encapsulated hemoglobin (LEH) as a nanoparticulate oxygen carrier (216±2nm) containing 7.2g/dl hemoglobin, and examined its ability to prevent the systemic manifestations of hemorrhagic shock (45% blood loss) in a rat model. We collected plasma after 6h of shock and LEH resuscitation, and determined the circulating biomarkers of systemic inflammation and functions of liver, gut, heart, and kidney. As is typical of the shock pathology, a significant increase in the plasma levels of cardiac troponin, liver function enzymes, soluble CD163 (macrophage activation), and creatinine, and the liver/gut myeloperoxidase activity was observed in the hemorrhaged rats. The plasma levels of TNF-α, IL-6, IL-1α, CINC-1, and IL-22 also increased after hemorrhagic shock. LEH administration prevented the hemorrhagic shock-induced accumulation of the markers of injury to the critical organs and pro-inflammatory cytokines. LEH also decreased the plasma levels of stress hormone corticosterone in hemorrhaged rats. Although saline also reduced the circulating corticosterone and a few other tissue injury markers, it was not as effective as LEH in restraining the plasma levels of creatinine, alanine transaminase, CD163, TNF-α, IL-6, and IL-1α. These results indicate that resuscitation with nanoparticulate LEH creates a pro-survival phenotype in hemorrhaged rats, and because of its oxygen-carrying capacity, LEH performs significantly better than saline in hemorrhagic shock.

Efficacy of liposome-encapsulated hemoglobin in a rat model of cerebral ischemia.

Use of liposome-encapsulated hemoglobin (LEH) for oxygen delivery in the treatment of cerebral ischemia has been studied previously and its expected benefits confirmed. However, the relationship between the timing of administration and the efficacy of LEH in cerebral ischemia has not been studied in detail. We therefore investigated the therapeutic time window of LEH by using a rat model of cerebral ischemia, as well as evaluating the contribution of oxygen delivery to the efficacy of LEH. Dose-dependent effects and the therapeutic time window of LEH were studied using models of transient and permanent middle cerebral artery occlusion (MCAO), respectively, in SD rats. LEH was intravenously administered at 0.5 h after the onset of ischemia in the transient MCAO model and at 0.5, 2, 4, or 6 h in the permanent MCAO model. Efficacy of LEH treatment was evaluated using the infarct volume, which was examined with 2,3,5-triphenyltetrazolium chloride staining and estimated by integrating the unstained areas in serial sections of cerebral tissue. Effects of oxygen delivery by LEH were examined immunohistochemically with pimonidazole to stain for areas of low oxygen tension in the tissue. LEH treatment dose-dependently reduced the cerebral infarct volume, which was especially significant in the cortical region at doses of over 60 mg hemoglobin (Hb)/kg. In rats with permanent MCAO, LEH administration at a dose of 300 mg Hb/kg at 0.5 h and 2 h after the onset of cerebral ischemia significantly reduced cerebral infarct volume. Furthermore, immunohistochemical staining with pimonidazole showed that the areas of cerebral tissue that were hypoxic and had abnormal histological structure were reduced after LEH treatment. These results indicated that LEH is efficacious in the treatment of cerebral infarction secondary to MCAO and that oxygen delivery to ischemic cerebral tissues by LEH administered early after the onset of cerebral ischemia contributes to this effect.

Liposome-encapsulated hemoglobin alleviates hearing loss after transient cochlear ischemia: An experimental study in the gerbil

The effects of liposome-encapsulated hemoglobin (LEH), an artificial oxygen carrier, were experimentally investigated in gerbils in the context of alleviation of hearing loss after transient cochlear ischemia. Animals were randomly assigned to receive 2mL/kg of either LEH (P50O2=15mmHg) or saline 1h after the experimental induction of 15min of ischemia. Sequential recordings of auditory brainstem response (ABR) showed that administration of LEH prevented hearing loss due to cochlear ischemia. The mean ABR threshold at 32kHz on day 1 was 21±7dB in the LEH group (n=6) and 45±6dB in the saline group (n=6). Thereafter, hearing impairment gradually improved up to day 7 in both groups. The animals were then subjected to histological study, which revealed that there was more substantial loss of the inner hair cells, but not the outer hair cells, in the saline group as compared to the LEH group. These results suggest that LEH is an efficient agent with regard to protection against hearing loss and underlying hair cell damager due to ischemic insult.

Liposome-Encapsulated Hemoglobin Improves Energy Metabolism in Skeletal Muscle Ischemia and Reperfusion in the Rat

The effect of liposome-encapsulated hemoglobin (LEH) was tested in a rodent model of limb ischemia and reperfusion--causing local reperfusion injury and a cascade of systemic responses. Intracellular pH (pHi) and phosphocreatine (PCr)/inorganic phosphate (Pi) ratio were serially monitored using ³¹P-nuclear magnetic resonance spectroscopy with a 2-cm solenoid coil on a rodent hind limb. After baseline measurements, the right hind limb underwent ischemia for 70 min, followed 10 min later by intravenous administration of LEH (10 mL/kg, n = 6), homologous red blood cells (RBCs, n = 6), saline (n = 6), or no treatment (n = 6). Reperfusion was then observed for an additional 60 min. While pHi decreased precipitously after the onset of ischemia and even following reperfusion, LEH-treated rats had significantly milder intracellular acidosis compared with all other groups during ischemia, and after reperfusion as well throughout the observation with the saline-treated rats. In contrast, the PCr/Pi ratio decreased regardless of treatment after ischemia until reperfusion, when the ratio returned toward normal or the energy status improved only in the LEH-treated rats, while the ratio remained depressed in the control animals receiving RBC, saline, or no treatment. Morphological studies 7 days later revealed a tendency toward suppressed mononuclear cell infiltration with preservation of muscular mass and structure in the LEH-treated rats. LEH treatment after early limb ischemia appeared to improve intracellular energy metabolism and eventually preserve skeletal muscle in a rodent model of limb ischemia and reperfusion.

Effect of Liposome-Encapsulated Hemoglobin on Antigen-Presenting Cells in Mice

Liposome-encapsulated hemoglobin (LEH) is removed from the circulation and degraded in the reticuloendothelial system, including dendritic cells (DCs) and macrophages. Therefore, LEH at a large dose may overload the system, cause a competitive inhibition in antigen-presenting activity, and impair the immune response of the host. Changes in cellularity of immunocompetent cells were monitored serially up to 4 weeks by flow cytometry in wild-type mice receiving 20 mL/kg of LEH, syngeneic red blood cells (RBCs), or saline. DCs were collected from the host spleen 1, 7, and 28 days after receiving the solution and were cocultured with naïve cluster of differentiation 4 T cells from T-cell receptor transgenic mice in the absence or presence of third-party antigens. After LEH administration, the cellularity of DCs and macrophages in the recipient spleen remained unchanged from control mice receiving RBCs or saline. While subset populations and costimulatory molecule expressions were different, DCs from LEH-administered mice expressed high levels of interleukin-2 production and helper T-cell activation in response to a third-party antigen and superantigens, as did the DCs from control mice receiving RBCs or saline. The results suggest that 20 mL/kg of LEH does not greatly alter antigen-presenting activity to third-party antigens.

Liposome-Encapsulated Hemoglobin Ameliorates Impairment of Fear Memory and Hippocampal Dysfunction After Cerebral Ischemia in Rats

Liposome-encapsulated hemoglobin (LEH) has been developed as a blood substitute. In spite of its size (1/30 - 1/40 of erythrocytes), LEH has an oxygen-carrying capacity comparable to erythrocytes. Thus, LEH is expected to carry oxygen into vital organs via collateral routes during ischemia induced by vascular embolism. In the present study, we examined the therapeutic effects of LEH on behavioral impairments in rats after four-vessel occlusion (4VO) for 30 min. In the open-field test, locomotor activity in 4VO rats did not alter 7 days after ischemia. However, in the contextual fear conditioning (CFC) test, the freezing rate was significantly decreased in 4VO rats, although no behavioral changes in the Y-maze test and elevated plus-maze test were observed. Phosphorylation of the cyclic AMP response element-binding protein (CREB) in the hippocampal CA1 region after the CFC test was attenuated. These 4VO-induced impairments were significantly alleviated by the administration of LEH (5 ml/kg, i.v.) during occlusion. Moreover, LEH did not alter hippocampal blood flow and tissue oxygen pressure during 4VO, but it did suppress hyperoxia after ischemia-reperfusion. These findings suggest that LEH, an artificial oxygen carrier, could be a novel therapeutic agent for brain dysfunction after acute cerebral ischemia.

Effects of Liposome‐Encapsulated Hemoglobin on Human Immune System: Evaluation in Immunodeficient Mice Reconstituted With Human Cord Blood Stem Cells

As preclinical evaluation in animals does not necessarily portray human responses, liposome-encapsulated hemoglobin (LEH), an artificial oxygen carrier, was tested in immunodeficient mice reconstituted with human hematopoietic stem cells (cord blood-transfused NOD/SCID/IL-2R(gammanull)[CB-NOG] mice). Changes in immunocompetent T-cell and B-cell composition in peripheral blood, spleen, and bone marrow were examined 2 and 7 days after 10 mL/kg of intravenous administration of LEH, empty liposome (EL), or saline using immunohistochemical and flow cytometrical techniques in wild-type mice and CB-NOG mice. Responses to intraperitoneal administration of toxic shock syndrome toxin-1 (TSST-1) under the absence or presence of LEH (10 mL/kg) were also determined 4 h and 3 days later in terms of lymphocyte composition and IL-2 plasma level in wild-type as well as CB-NOG mice. When liposome (LEH or EL) was administered to wild-type or CB-NOG mice, the composition of B-cells and T-cells in the spleen or peripheral blood failed to show any consistent or significant changes. The responses to a bacterial antigen (TSST-1) measured by IL-2 production were comparable regardless of the presence or absence of LEH in wild-type as well as in CB-NOG mice. Cellularity, distribution, and maturation of these human cells in peripheral blood, spleen, and bone marrow were comparable among the groups. The results suggest that simple LEH administration may not change immune cellularity, and LEH presence may not largely affect the early T-cell response to bacterial enterotoxins in murine as well as in reconstituted human immune systems.

Complement-mediated acute effects of liposome-encapsulated hemoglobin.

Recent studies on liposome-encapsulated hemoglobin (LEH) have indicated that this potential blood substitute can activate the complement (C) system of rats, pigs and man. The reaction can involve both the classical and the alternative pathways, and is mediated, in part, by the binding of natural anti-lipid antibodies to the lipid membrane of liposomes. The significance of these discoveries lies in the fact that C activation appears to be the primary cause of the acute physiological, hematological and laboratory changes that have been observed previously in rats and pigs following the administration of LEH or liposomes, which changes include pulmonary vasoconstriction with decreased cardiac output. In light of the proposed use of LEH as an emergency blood substitute, the latter impairment of cardiopulmonary function may warrant particular circumspection as it could aggravate the clinical state of trauma patients who are prone to develop respiratory distress partly as a consequence of C activation by the injury. Our studies on rats and pigs suggest that the above acute side effects of LEH, including the cardiopulmonary distress, can be efficiently inhibited with soluble complement receptor type I, a specific inhibitor of C activation.

Effects of liposome-encapsulated hemoglobin on phorbol ester-induced superoxide production and expression of costimulatory molecules by monocytes in vitro.

The effects of Neo Red Cell (NRC), a liposome-encapsulated hemoglobin (LEH), on the phorbol ester-induced superoxide production and the expression of costimulatory molecules by human peripheral monocytes were investigated. The treatment of human mononuclear cells with NRC caused the potentiation of superoxide production in response to PMA. The longer incubation (20 h) resulted in a decrease in the PMA-induced superoxide production, which was in parallel to a decrease in the viability of the monocytes. A flow cytometric analysis showed that a slight expression of CD80 (B7-1) on monocytes was induced by NRC treatment, whereas the constitutive expressions of CD86 (B7-2) and CD54 (ICAM-1) were unchanged. The activation of monocytes with interferon-gamma (IFN-gamma) induced the expressions of CD80, CD86, and CD54 under all conditions tested, but NRC treatment tended to decrease the IFN-gamma-induced expression of CD54 on monocytes. These results suggest that the administration of LEH may modify the functions of human monocytes.

Role of Complement in Rats Injected with Liposome-Encapsulated Hemoglobin

Previous studies have documented that liposome-encapsulated hemoglobin (LEH) can cause a rapid and transient thrombocytopenia following intravenous injection into small animals. The present study evaluated the role of complement during the LEH-induced thrombocytopenia in rats. We have compared changes in platelet levels in the blood, platelet organ distribution, and total hemolytic complement levels following intravenous administration of LEH in control and complement-depleted rats. Changes in platelet organ distribution at various times after LEH administration were monitored by labeling autologous platelets with indium-111 (111In)-oxine and imaging the111In-platelets with a gamma camera after reinjection. Platelet counts were determined by light-scattering methods and by following111In radioactivity at various times after LEH administration. Platelet levels did not significantly change for the complement-depleted rats during the 60 min following an injection of LEH, whereas thrombocytopenia (40% decrease) was noted within 4 min post-LEH-injection for control rats with a gradual return to baseline circulating platelet levels within 60 min. This drop in circulating platelets was correlated with a rapid redistribution of111In-platelets from the circulation to the lungs and liver, whereas complement-depleted rats showed no transient movement of the111In-platelets from the circulation. Baseline complement levels of 21.6 ± 2.2 CH50/ml for control rats and 0.2 ± 0.1 CH50/ml for complement-depleted rats did not significantly change during the 60 min following LEH administration. This study suggests that complement must be present during LEH-induced transient thrombocytopenia, as complement-depleted rats underwent no thrombocytopenia, and that the transient LEH-induced thrombocytopenia may be associated with complement activation.

Complement activation and thromboxane secretion by liposome-encapsulated hemoglobin in rats in vivo: inhibition by soluble complement receptor type 1.

Intravenous administration of liposome-encapsulated hemoglobin (LEH) in rats led to an early (within 15 min) decline of hemolytic complement (C) activity in the plasma along with a significant, parallel rise in thromboxane B2 (TXB2) levels. The TXB2 response was inhibited by co-administration of soluble C receptor type 1 (sCR1) with LEH, as well as by C depletion with cobra venom factor. These observations provide evidence for a causal relationship between LEH-induced C activation and TXB2 release, and suggest that sCR1 could be useful in attenuating the acute respiratory, hematological and hemodynamic side effects of LEH described earlier in the rat.

Liposome-Encapsulated Hemoglobin: A Red Blood Cell Substitute

AbstractA safe and effective red blood cell (RBC) substitute would have broad implications in the practice of emergency medicine, trauma management, surgery, and several other areas of medicine. Several hemoglobin-based RBC substitutes have been developed that can deliver oxygen to peripheral tissues. However, although these RBC substitutes have desirable biophysical properties, their in vivo efficacy is limited by their significant toxicity. In view of the very high doses of blood substitute that are likely to be used clinically, toxicity as well as other safety issues that include hemostasis and thrombosis are critical considerations for the development and ultimate application of RBC surrogates.Recent work conducted in our laboratories has demonstrated that administration of liposome-encapsulated hemoglobin (LEH) in rats was efficacious. Also our results have demonstrated that the replacement of more conventional lipids with the sterically-stabilizing lipid polyethyleneglycol distearoylphosphatidyl-eth...

Liposome-Encapsulated Hemoglobin using Film Hydration Processing to form Artificial Red Blood Cells

Process parameters using a Microfluidizer M110 to produce liposome-encapsulated hemoglobin (LEH) were further studied to examine their effect on hemoglobin (Hb) encapsulation efficiency (yield), steady shear viscosity, mechanical stability, and oxygen delivery. Liposome formulation loading ratios of up to 300 mumol of lipid per ml of Hb solution were evaluated; a maximum yield was obtained at 300 mumol/ml. Liposomes containing encapsulated Hb concentrations as high as 15.5 g/100 ml were prepared. LEH particle size distribution, determined from negatively stained whole mount preparations using transmission electron microscopy, resulted in average vesicle sizes for optimal batches of about 155 nm. Steady shear viscosity of LEH (up to 40% by volume) in an isotonic-isooncotic solution of PBS containing either albumin or dextran were evaluated for shear rates to 2000 s-1. Values obtained were generally higher than those of whole blood at all shear rates tested. Little leakage of Hb from liposomes stored in isotonic PBS was observed as a function of storage time and shear rate. Administration of LEH supported life in rats whose hematocrit had been reduced via isovolemic exchange transfusion to levels well below 5%, which was incompatible with survival when exchange transfusion was performed with the isotonic-isooncotic PBS solution.