LIPOSOME-MEDIATED DELIVERY OF ANTI-SICKLING AGENTS

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We propose to develop a liposome-mediated transport system for the intracellular delivery of red cell membrane impermeable anti-sickling agents into the sickle erythrocyte (RBC). The agents we propose to deliver by this technique include the aromatic amino acids phenylalanine (Phe) and tryptophan (Try) as well as small peptides (Pep) containing these amino acids (L-threonine-L-phenylalanine and L-lysine-L-phenylalanine-L-phenylalanine). These agents are non-toxic and are known to be effective non-covalent inhibitors of hemoglobin S gelation, however, the relatively high intracellular concentrations required to inhibit gelation (approximately 10 mM) coupled with their limited permeability across intact RBC membranes currently makes their therapeutic use impractical. Our research plan involves: (1.) Development of the ideal liposome, in terms of liposome composition and size, and optimizaton of liposome-RBC incubation conditions to effect the maximal encapsulation and subsequent delivery of Phe, Try and Pep (PTP) into sickle RBC, (2.) Systematic evaluation of the anti-sickling properties of PTP in intact RBC and hemolysates, including treatment-induced alterations in: resistance to hypoxia-induced morphological shape changes, hemoglobin oxygen affinity and relative solubility, percent polymer formation, red cell indices, cation leak, cell density and deformability; (3.) Examination of the intracellular retention of exogeneously delivered PTP, as well as the (4.) Effect of PTP-loading on in vitro generation of irreversibly sickled cells and (5.) RBC adhesiveness. We also plan to (6.) look for possible toxic effects of elevated intracellular PTP levels by examining specific RBC metabolic parameters, and (7.) Perform in vivo survival studies using the PTP-loaded RBC, both in animals (rabbits) and in humans. In addition, we propose to (8.) Utilize liposomal-mediated transport to deliver into sickle RBC dimethyl-adipimidate (DMA), which has been shown to possess potent anti-sickling properties but unfortunately simultaneously confers to the treated-cells new antigenic determinates which lead to decreased RBC survival in vivo. Liposomal encapsulation of DMA allowing specific intracellular delivery of this agent may alleviate problems currently associated with its use. The development of a liposome transport system can be applied to any number of therapeutic applications where potentially clinically useful agents have problems associated with their membrane permeability and/or non-specific reactivity.