Richard L. Beissinger

The stabilization of hemoglobin multiple emulsion for use as a red blood cell substitute

Abstract Utilizing emerging multiple emulsion and liquid membrane technology to form stable hemoglobin-in-oil-in-water (Hb/O/W) multiple emulsion (ME) droplets, efforts were aimed at formulating an artificial red blood cell for use as a blood substitute or organ perfusion system, which provides adequate oxygen-carrying capacity. With the development of this methodology, using large-scale fabrication methods, a concentrated Hb solution (to 35 g/100 ml) was emulsified in oil to form microdroplets by using an appropriate nontoxic surfactant in the oil phase, followed by dispersion of the primary emulsion into an outer aqueous phase containing surfactant to form the ME. Of the various oils tested both white (mineral) and tricaproin (triglyceride) showed the highest encapsulation efficiency for hemoglobin. Surfactants were used in the liquid membrane and outer aqueous phases to form and stabilize the multiple emulsion droplets (artificial cells). The Hb encapsulation efficiency and the oxygen content of freshly prepared multiple emulsion were as much as 98% and 14 ml/100 ml ME, respectively. The average diameter of the prepared ME droplets was about 2 to 3 μm. The steady shear viscosity of the ME was similar to that of whole blood over the shear rate range studied. Sensitivity to shear was evaluated for shear rates to 2160 s−1 and showed only very small leakage of Hb from the ME droplets into the outer aqueous phase; also the stability of the “artificial cells” during short-term storage (i.e., less than 30 days)

LIPOSOME MICROENCAPSULATIONS WITHOUT USING ANY ORGANIC SOLVENT

ABSTRACT Liposomes are microscopic vesicles composed of phospholipid bilayers surrounding discrete aqueous compartment. Eventhough phospholipid bilayers are formed spontaneously in water, their conventional preparation methods involve either organic solvents or detergent molecules. Organic solvents may create a serious safety problem during liposome manufacturing especially on a large scale and can cause denaturation of proteins and affect the membrane properties. Liposomes are very promising carriers for protein drugs which have aroused great interests in the field of pharmaceutics in these recent years. The objective of this study was to develop a new dispersion process to prepare liposomes without the usage of organic solvents and detergents. The approach involved the usage of a microfludizer for the extrusion and homogenization of aqueous dispersion of phospholipids, and a modified evaporator in order to increase lipid thin film surface area. The properties of the liposomes prepared by the novel metho...

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.

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.

Efficacy, Physical Properties and Pharmaco-Kinetics of Sterically-Stabilized Liposome-Encapsulated Hemoglobin

We recently reported that hemoglobin (Hb) encapsulated in liposomes (LEH) containing phosphatidyl-inositol (PI) was efficacious in rats. However, liposomes containing PI may temporarily compromise mononuclear phagocytic system (MPS) function. The objective of this study was then to determine whether a polyethylene oxide derivative of phosphatidyl ethanolamine (PEG-PE) would serve as an acceptable substitute for PI in our LEH formulation. In this study we compare the physical properties, pharmacokinetics and efficacy in life support obtained for Hb encapsulated with either PI or PEG-PE phospholipids. Both liposome compositions contained the same matrix lipids, egg derived phosphatidyl choline (PC) and cholesterol, were of similar size and contained the same amount of encapsulated Hb. The liposomes differed only in their phospholipid component, one containing 5 mol% PI and the other an equal amount of the sterically-stabilizing lipid PEG-PE. The physical characteristics of the PI and PEG-PE compositions were remarkably similar: only small amounts of Met-Hb were generated during processing and following 1 month frozen storage, oxygen affinity and cooperativity and steady shear viscosity values for 30% by volume suspensions (in isotonic/isooncotic saline containing albumin) were near the normal values expected for whole blood, incubation in plasma at 37 degrees C resulted in only small amounts of Hb release and shear had very little impact on Hb leakage. Circulation half-lives following 50% isovolemic exchange-transfusion in rats were also similar, about 15-20 hours for either formation. Animals survived following 97% isovolemic exchange-transfusion of both compositions, confirming the efficacy of each.

Evaluation of shear-induced particle diffusivity in red cell ghosts suspensions

The shear-induced particle diffusivity in the red blood cell suspensions was evaluated based on the flow model and experimental results in a rectangular flow chamber. The effective diffusivity (De) of solute in the particle suspensions is equal to the stationary diffusivity (Ds) of the solute plus the shear-induced particle diffusivity (Dp). The effective diffusivity (De) of bovine serum albumin (BSA) in the red blood cell (RBC) ghost suspensions was determined under diffusion-limited conditions using a total internal reflection fluorescence (TIRF) method as a function of suspended RBC ghost volume fractions (0.05-0.7) and shear rates (200-1,000 s,-1). The stationary diffusivity (Ds) of BSA in RBC ghost suspensions was calculated by Meredith and Tobias model. Therefore the shear-induced particle diffusivity undergoing laminar shear flow can be evaluated. The shear-induced RBC ghost diffusivity was ranged from 0.35xl0-7 to 21.2xl0-7 cm2/s and it increased with increasing shear rate. Also the shear-induced RBC ghost diffusivity increased as a particle volume fraction increased as well, up to a particle volume fraction of 0.45. However, for RBC ghost volume fractions above 0.45, the shear-induced particle diffusivity decreased with increasing particle volume fraction. The shear-induced particle diffusivity in RBC ghost suspensions is a function of a particle Peclet number (or shear rate) and particle volume fractions. The dimensionless particle diffusivity (Dρ/a2γ) was investigated as a function of particle volume fraction and these results are in good agreement with the literature values.

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...

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 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.

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.