pH-SENSITIVE LIPOSOMES AS ANTI-AMASTIGOTE AGENTS

MORILLA MJ; FRANK F; MONTANARI J; CAZORLA S; MALCHIODI E; ROMERO EL; PETRAY P

Rosario (Argentina)

Congreso; XX Reunión Anual de la Sociedad Argentina de Protozoología; 2004

Sociedad Argentina de Protozoología

The main obstacle for eliminating intracellular parasites is that hydrophilic or high molecular weigh drugs can not reach the cytoplasm where they have to exert their action, unless a specific way of transport exist. In this context, we designed and prepared pH-sensitive liposomes loaded with the hydrosoluble, low molecular weigh trypanocidal drug etanidazole (ETZ). pH-sensitive delivery strategy is based on the change of the liposomal membrane phase induced by the low pH in the endosomes/lisosomes, responsible of the concomitant delivery of the ETZ to the cytoplasm. ETZ was loaded in pH-sensitive large unilamellar vesicles (LUV) (dioleoyl-phosphatidylethanolamine: cholesteryl hemisuccinate, 6:4, mol:mol) prepared by extrusion through 200 nm policarbonate membranes followed by 5 freezethaw cycles. The non-encapsulated ETZ was removed by gel permeation chromatography. The resulting LUV-ETZ had a mean diameter of 380 ± 60 nm. The concentration of the resulting liposomal suspensions determined by HLPC was 0.51 mg ETZ /ml, at a 14% w/w drug/total lipid ratio. The endocytosis and intracellular fate of pH-sensitive liposomes loaded with the fluorophore/quencher pair HPTS/DPX was studied by fluorescence microscopy. We also determined the anti-amastigote activity (aa) in J774 murine macrophages infected with Trypanosoma cruzi amastigotes. Upon treatment with LUV-ETZ, no change in viability of healthy or infected cells was registered. The results showed that upon capture, a fast delivery of the liposomal aqueous content into the cytoplasm of non-infected macrophages as well as on infected macrophages occurred. On the other hand, we founded 77% aa after 2 h treatment with LUV-ETZ whereas the treatments with empty liposomes rendered nearly 0% aa. Due to the pharmacokinetic characteristics of ETZ, such high ETZ concentration along two hours used± 60 nm. The concentration of the resulting liposomal suspensions determined by HLPC was 0.51 mg ETZ /ml, at a 14% w/w drug/total lipid ratio. The endocytosis and intracellular fate of pH-sensitive liposomes loaded with the fluorophore/quencher pair HPTS/DPX was studied by fluorescence microscopy. We also determined the anti-amastigote activity (aa) in J774 murine macrophages infected with Trypanosoma cruzi amastigotes. Upon treatment with LUV-ETZ, no change in viability of healthy or infected cells was registered. The results showed that upon capture, a fast delivery of the liposomal aqueous content into the cytoplasm of non-infected macrophages as well as on infected macrophages occurred. On the other hand, we founded 77% aa after 2 h treatment with LUV-ETZ whereas the treatments with empty liposomes rendered nearly 0% aa. Due to the pharmacokinetic characteristics of ETZ, such high ETZ concentration along two hours usedTrypanosoma cruzi amastigotes. Upon treatment with LUV-ETZ, no change in viability of healthy or infected cells was registered. The results showed that upon capture, a fast delivery of the liposomal aqueous content into the cytoplasm of non-infected macrophages as well as on infected macrophages occurred. On the other hand, we founded 77% aa after 2 h treatment with LUV-ETZ whereas the treatments with empty liposomes rendered nearly 0% aa. Due to the pharmacokinetic characteristics of ETZ, such high ETZ concentration along two hours used in vitro could never be sustained in vivo. On the contrary, the aa resulting from LUV-ETZ in vitro could easily be reproduced in vivo. Hence, our results point to LUV-ETZ as potential vehicles capable of delivering high amounts of ETZ to infected cells.could never be sustained in vivo. On the contrary, the aa resulting from LUV-ETZ in vitro could easily be reproduced in vivo. Hence, our results point to LUV-ETZ as potential vehicles capable of delivering high amounts of ETZ to infected cells.in vitro could easily be reproduced in vivo. Hence, our results point to LUV-ETZ as potential vehicles capable of delivering high amounts of ETZ to infected cells.