Microencapsulation of hemoglobin in liposomes using a double emulsion, film dehydration/rehydration approach.

A double emulsion, film dehydration/rehydration approach was developed for encapsulation of hemoglobin (Hb) at high concentration in liposomes. The liposome-encapsulated Hb (LEH) membrane was formulated to contain either phosphatidylinositol (PI) or polyethyleneglycol phosphatidylethanolamine (PEG-PE) along with partially hydrogenated egg-PC, cholesterol, and alpha-tocopherol in a molar ratio of 0.1:1:1:0.02, respectively. The methods introduced in this study followed a multi-step procedure. First, a primary emulsion of Hb in organic solvent containing dissolved lipids was formed. Next, the emulsion was dispersed into an aqueous continuous phase to form a water-in-oil-in-water type double emulsion. Other than the lipids noted above, no surfactants were used in this system. The double emulsion was then converted to LEH by the following steps: evaporating the organic solvent; dehydrating the water to form a dry, thin Hb-lipid film; rehydrating the film in Hb solution to form the LEH; reducing the size of the LEH using 'microfluidization' i.e., high pressure/hydrodynamic shear; and lastly washing the down-sized LEH in buffer. Physico-chemical properties of the model LEH were measured, including oxygen content, encapsulated Hb concentration, oxygen affinity and cooperativity, vesicular size distribution, viscosity, and stability. The suitability of LEH prepared in this manner as a red blood cell substitute was shown using continuous isovolemic exchange transfusion techniques in a small animal model: clearance, efficacy and acute toxicity were evaluated.

Hemoglobin multiple emulsion as an oxygen delivery system.

Multiple emulsion technology provides a mechanism for the encapsulation and in vivo delivery of drugs, proteins, and other materials which would otherwise be degraded, cleared rapidly, or toxic to the host. These feasibility studies were performed to evaluate a prototype Hb multiple emulsion as a stable oxygen delivery system. A concentrated solution of hemoglobin (Hb) was encapsulated in the form of a Hb-in-oil-in-water (Hb/O/W) multiple emulsion. Studies using mineral oil demonstrated that Hb multiple emulsions have several important characteristics that are compatible with utility as a blood substitute. These include: satisfactory rheological properties and good hydrodynamic stability compared to whole blood, high encapsulation concentration of Hb and high encapsulation efficiency with little met-hemoglobin generation, and satisfactory oxygen affinity and cooperativity compared to whole blood. Isovolemic exchange transfusions of Hb/O/W multiple emulsion can support life in rats whose hematocrit has been reduced to levels (5% or lower) that are incompatible with survival, and induces no acute toxicity. These results are consistent with the utility of Hb/O/W as an oxygen-carrying red blood cell substitute or organ perfusion media.

Effect of hirudin on platelet deposition to an artificial surface during low-stress shear flow of whole blood.

The effect of hirudin, a known inhibitor of thrombin, was evaluated for whole blood samples in terms of platelet deposition/adhesion to a non-biological test surface (tetrafluoroethylene-propylene copolymer), adenosine diphosphate (ADP) release and reduction in platelet count during laminar shear flow for a shear rate to 5680 s-1 (corresponding to a shear stress of about 150 dynes/cm2). Experiments were done in a cone-and-plate viscometer for samples of whole blood with and without the addition of hirudin. Whole blood samples containing hirudin showed about a 50% reduction in platelet surface coverage compared with blood samples not containing hirudin. Results on low-stress, shear-induced release of ADP showed that for shear rates of 2860 s-1 and above there was an increase in ADP release for the blood samples not containing hirudin compared with the hirudin-treated samples. However, no differences in haemoglobin leakage from red blood cells as well as residual platelet count following shear were observed between both types of blood samples.

Liposome-encapsulated hemoglobin: a synthetic red cell.

Liposome-encapsulated hemoglobin (LEH) is being developed at the Naval Research Laboratory as a universally transfusable oxygen-carrying blood replacement. A chemical engineering scale-up feasibility study has been completed recently. We report here the development of an encapsulation method which produces liters of phospholipid/cholesterol liposomes containing at least 16 g% hemoglobin in a few hours. The 0.2 micron liposomes are produced with a Microfluidizer TM (Microfluidics Corp., Newton, MA) adapted for this purpose, and then washed and sterile filtered using a Pellicon (Millipore, Bedford, MA) tangential flow filtration device. Previously, production limitations and lack of sterility have been serious barriers to toxicity testing for all the researchers engaged in related investigations. The biophysical properties of the LEH thus produced are ideal for use as a blood substitute, resembling those of red blood cells. The oxygen-binding affinity of LEH can be maintained at the level of fresh whole blood for many weeks by co-encapsulation of pyridoxal-5-phosphate. The circulation persistence time of liposomes is a function of the type of phospholipid. We have developed a formulation which has a circulation persistence time of 15-20 hours. The LEH oxygen binding characteristics, circulation half-life and its lipid composition dependence, scale-up preparation method, and a sterilization method are presented.

Effects of blood storage on rheology and damage in low-stress shear flow.

Data are presented on the rheological and hemolytic behavior of whole human blood as it ages while stored at 4 degrees C (as in blood banking practice) up to 26 days. The viscometric properties of steady shear viscosity eta and oscillatory (complex) viscosity eta * = eta' - i eta" reported over ranges of shear rate gamma and radian frequency omega of 33 less than gamma less than 4130 s-1 and 1.5 less than omega less than 48 s -1; data on autologous plasma are given for reference. The Cox-Merz relation, eta (gamma) = [eta *(omega)] omega = gamma, is found to be a good approximation, with eta greater than or equal to [eta *], over the range studied. Release of hemoglobin (Hgb) and lactate dehydrogenase (LDH) into the plasma during shearing is tracked as a function of time for 30 min, and its sensitivity to gamma magnitude is measured. Bloods from four different donors are studied, with primary attention given to one (SSR). For all bloods, the release of both Hgb and LDH increases with storage age, but differences in such aging characteristics between different bloods can be substantial (even when rheological properties are identical). A post-shear incubation at 4 degrees C for one day shows no enhancement of plasma Hgb and LDH levels beyond those expected from normal aging after the shearing experience, demonstrating the absence of significant delayed-action effects as a consequence of shearing trauma.

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.

Low stress shear-induced hemolysis in capillary flow.

An experimental investigation was undertaken to evaluate shear-induced blood damage behavior in capillaries within the clinically relevant low-stress regime (stress less than 300 dynes/cm2). As a result of these studies, it was determined that: blood damage increases most rapidly during the initial seconds of capillary residence time and then increases more slowly thereafter; there is a minimum capillary length through which red blood cells must travel, before any significant leakage of hemoglobin occurs; capillary blood damage correlates with wall shear rate as H alpha gamma . n, where the exponent depends on the blood sample tested; experimental results for the largest capillary tested indicated a blood cell damage mechanism based on 2 competing factors: a) time of shearing and b) shearing intensity level; the theoretical model developed to describe capillary blood damage is in good agreement with the experimental results. The blood damage model is discussed in another report and is based on the simultaneous contribution of surface and bulk hydrodynamic and mass transfer effects.

Surface and bulk effects on platelet adhesion and aggregation during simple (laminar) shear flow of whole blood.

This study attempts to clarify the role of the artificial surface and the fluid bulk on platelet adhesion and aggregation events during simple shear flow of whole blood. The experimental approach involved the shearing of fresh whole blood samples over the shear rate range of 720-5680 s-1, which corresponded to a shear stress maximum of about 150 dyn cm-2. Results on platelet adhesion, measured as surface coverage by platelets, and platelet aggregation, measured in terms of reduction in platelet count and adenosine diphosphate (ADP) release, were determined as a function of the surface to volume ratio (S/V); and artificial surface used. Both shear-induced platelet adhesion and platelet count reduction showed significant variation over the range of S/V employed. The ratios between the three different S/V values used in this system (10:6:4) were about the same as the ratio of the shear rate-averaged results obtained. Also, for shear-induced hemolysis, an increase in the release of hemoglobin from red blood cells was found as S/V was increased, again with ratios between the shear rate-averaged values similar to the ratio of S/V values employed. The shear-induced release of ADP, presumably from platelets and from red blood cells indicated a different dependence of ADP release on S/V than was observed for the other parameters reported. Irreversible platelet aggregation was expected to occur because the amount of ADP that was released as a result of the shear was substantial. Models proposed to explain the experimental results were found to support a surface-controlled mechanism.

Red blood cell effect on platelet adhesion and aggregation in low-stress shear flow. Myth or fact?

Evidence has been accumulating which suggests that red blood cells affect platelet adhesion on nonbiological surfaces and platelet aggregation in the bulk; this in vitro study investigated these red blood cell effects. For samples of either whole blood or suspensions of either ghosts or red blood cells in platelet rich plasma undergoing low-stress simple shear flow, data on hemoglobin and adenosine diphosphate (ADP) released from red blood cells, single platelet reduction (which is a measure of platelet adhesion to nonbiological surfaces and platelet aggregation in the bulk) and percent of surface covered with platelets and platelet aggregates were obtained in a cone-and-plate viscometer for shear rates up to 5680 s-1. The results obtained suggest that red blood cells release a significant fraction of their ADP (2% at 5680 s-1), which is enough to induce platelet aggregation, and contribute about 65% to the total ADP release for a sheared blood sample; ADP released from red blood cells contributes about 60% to single platelet reduction and about 28% to platelet adhesion, whereas ADP from platelets contributes about 8% and 14%, respectively; and the physical effect of red blood cells, which is coupled to the chemical effect, acts to catalyze (enhance) the chemical effects. Based on the results obtained a mechanism was developed to describe both the chemical and physical nature of the red blood cell effect.

Artificial surface effect on red blood cells and platelets in laminar shear flow.

Red blood cell (RBC) effects on platelet adhesion to a nonbiologic test surface (tetrafluoroethylene propylene copolymer) and platelet aggregation during laminar shear flow for shear rates to 5,680 s-1 (corresponding to shear stress to 200 dyne/cm2) were investigated. Results on hemoglobin (Hb) and adenosine diphosphate (ADP) release from RBCs, percent decrease of single platelets in the bulk, and percent of test surface covered with platelets were obtained in a cone-and-plate (CP) viscometer for samples of whole blood, suspensions of RBC ghosts in platelet-rich plasma (PRP), and suspensions of RBCs in either PRP or platelet-poor plasma. Results obtained over the shear rate range studied for samples of normal hematocrit indicated that low-stress shearing led to ADP and Hb release from intact RBCs; shear-induced release of ADP from RBCs was about twice that of platelets, and of the total ADP released, the ADP released from RBCs contributed about six times that of the platelets to single platelet reduction in the bulk and about twice that of the platelets to platelet adhesion, ie, coverage of the test surface with platelets. Results obtained for various hematocrits showed that above a threshold hematocrit of about 25% to 35% the RBCs (suspended in PRP) had a greater contribution to ADP release, platelet adhesion, and platelet aggregation than the platelets themselves. Single platelet reduction for samples of RBC ghosts suspended in PRP correlated with shear rate level and not with shear stress.

Liposome-encapsulated hemoglobin processing methods.

An effective and safe red blood cell substitute is being developed based on double emulsion/evaporation techniques followed by high pressure homogenization to form liposome-encapsulated hemoglobin (LEH). Formulations are made up of hydrogenated phosphatidylcholine (PC, soy or egg), cholesterol, phosphatidylinositol (PI), and alpha-tocopherol in a molar ratio of 1:1:0.2:0.02, respectively. Resulting LEH-encapsulated hemoglobin (Hb) concentrations are greater than 80% of precursor Hb solutions. Met-Hb generation accompanying LEH processing appears to be small with only a 3% increase for encapsulated over precursor. These results correspond to an oxygen content for an LEH suspension sample (50% by volume LEH) of 15 volume% oxygen. Oxygen affinity and cooperativity values for LEH suspensions appear to be near the normal values expected for whole blood. The viscosity of LEH suspension samples (50% by volume LEH in phosphate-buffered saline containing 7.5 wt% albumin) were slightly higher than that of whole blood. The effect of shear rate on leakage of encapsulated Hb from LEH was small, i.e. 0.5% or less. Nearly total isovolemic exchange transfusion using a cannulated rat model demonstrates efficacy of LEH suspension samples. There appears to be no difference in rat internal organ weights between rats exchanged with control compared to rats exchanged with LEH. Circulation half-life following 50% isovolemic exchange-transfusion is about 15 to 18 hours.

Reduced spinal reflexes following intrathecal baclofen in the rabbit.

Intrathecal baclofen is effective in reducing polysynaptic spinal reflexes in awake rabbits. A single lumbar infusion of 2.5-5.0 ng drug caused a significant reduction in the crossed extensor response to electrical stimulation of the plantar surface of the hindlimb. This inhibition lasted 5 h or more. At doses of 3 micrograms or less, the forelimbs were unaffected.

Influence of steric stabilization of liposome-encapsulated hemoglobin on Listeria monocytogenes host defense.

Liposome-encapsulated hemoglobin (LEH) products are being investigated as potential blood substitutes. To determine if changes in LEH composition can modify the immune response, red blood cell substitutes based on conventional lipids containing phosphatidylinositol (LEH1) and sterically stabilized lipid vesicles containing polyethylene glycol phosphatidylethanolamine (LEH2) were tested for effects on host resistance. On Day 0, groups of 18 to 20 female CD-1 mice were given an intravenous (i.v.) infectious challenge with a 20% lethal dose of Listeria monocytogenes. Mice received a single i.v. dose of LEH1, LEH2, or albumin vehicle on Day +1 or Day -3 relative to infectious challenge. Mice dosed with LEH1 and LEH2 on Day +1 died rapidly from Listeria infection; but mice dosed with LEH2 lived significantly longer than did mice receiving LEH1. By contrast, when administered on Day -3, LEH1 had no significant effect on host immunity, while LEH2 increased susceptibility to Listeria infection. In addition, LEH1 and LEH2 both caused significant reduction of phagocytic activity as measured by rat alveolar macrophage (AM) ingestion of latex microspheres. AM incubated 4 hr with either LEH1 or LEH2 prior to addition of microspheres ingested fewer beads in a dose-dependent manner. No difference in in vitro phagocytic activity was observed between LEH1 or LEH2. The inability to differentiate LEH formulations based on in vitro phagocytic activity suggests that the in vivo Listeria infection model may be more relevant in discerning the immunotoxicity of the LEH formulations tested.

Liposome-encapsulated hemoglobin as an artificial red blood cell: characterization and scale-up.

Encapsulation methods using high pressure extrusion and homogenization were developed which produce hemoglobin-containing liposomes, approximately one micron or less in diameter with an oxygen-carrying capacity of more than half that of red blood cells (RBCs). These methods were developed for scaling-up liposome-encapsulated hemoglobin (LEH) production. Previously, the lack of adequate scale-up methods has been a serious barrier to full scale efficacy and toxicity testing for all the researchers engaged in such investigations. The scale-up methods and characterization of the resulting LEH preparations are presented.