The long-term goal of our research is to apply the unique structural features and lipid interaction properties of apolipoproteins to the treatment of leishmaniasis. Apolipoproteins possess the capacity to transform phospholipid vesicles into discrete, nanometer scale, disc-shaped, complexes termed nanodisks (ND). Studies to date have shown that incorporation of specific hydrophobic drug molecules into ND is feasible. ND loaded with the polyene antibiotic amphotericin B (ampB) effectively inhibits fungal growth in vitro. Furthermore, in vivo experiments in Leishmania major infected mice have revealed that ampB-ND show efficacy at non-toxic doses. To investigate the molecular basis of this effect, pharmacokinetic studies will be performed to determine plasma decay kinetics and tissue distribution of ampB administered as ND. The apolipoprotein component of ND will be subject to chemical and/or genetic alteration to create a molecule that is recognized by the class A scavenger receptor (SR-A) on macrophages. Site-directed mutagenesis of the apolipoprotein component of ND will be performed to remove specific positively charged residues and position negatively charged amino acid residues such that they create a recognition site for SR-A. Chinese hamster ovary cells transfected with SR-A will be employed in binding experiments designed to measure the SR-A binding activity of engineered apolipoproteins. The efficacy of ampB-ND harboring apolipoprotein components targeted to SR-A will be evaluated in a mouse model of leishmaniasis. Studies will be performed to examine the potential enhancement achieved by incorporating ampB with a second hydrophobic anti-leishmanial drug, hexadecylphosphocholine, into ND. The extent to which ampB-ND induce parasite destruction in cells that harbor L. major will be assessed and the effect of ampB-ND exposure on the T cell/cytokine response of human peripheral blood mononuclear cells determined. The results of these experiments should offer new opportunity for the treatment of leishmaniasis, provide further insight into the ligand recognition characteristics of SR-A, and document the utility of ND as vehicles for targeted delivery of hydrophobic drugs. ? ? ?
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