Ultradeformable liposomes à la carte: the Last Supper for Leishmania brasiliensis promastigotes

MONTANARI, JORGE A.M.; MORILLA, M.J.; ROMERO, E.L.

Saarbrücken, Alemania

Workshop; 7th Conference and Workshop on Biological Barriers and Nanomedicine – Advanced Drug Delivery and Predictive non vivo Testing Technologies; 2008

Saarland University

With the aim of developing a novel topical treatment against cutaneous leishmaniasis based on a nanocarrier capable of experience locomotion through the nanopores in the stratum corneum to reach the macrophagic population at the inner layers of epidermis, unilamellar ultradeformable Liposomes (UDL), of 100nm diameter, were prepared by resuspending in tris buffer (NaCl 0,9%) a lipid film obtained from evaporation of lipid/surfactant mixtures (Soy phosphatidylcholine and Sodium cholate, 6:1 w/w) in chloroform and chloroform:methanol 1:1 respectively, following sonication and extrusion to reduce lamellarity and size. Different concentrations of UDL were incubated with 5x105 free Leishmania brasilensis promastigotes in BHT medium at 25ºC during 5’, 30’ and 24h. Motility (by optical microscopy) and viability (by the MTT assay) were determined, observing that even at the shortest incubation time leaded to their immovilization and reduced viability. To verify that the lethal effect was due to the endocytic capture of UDL by promastigotes, parasites were incubated with UDL containing fluorescent probes both in their hydrophilic inner space (HPTS) and in their hydrophobic membrane (Rhodamine-PE). After 15 minutes, the parasites were washed by centrifugation and were resuspended in BHT UDL-free medium, fixed over microscope slides, and observed by confocal microscopy. It was found that the parasites were able to endocyte the UDL, being this capture the probable cause of the immovilization of the parasites. On the other hand, the generation of oxidative stress by UDL over cell lines (J774 and Vero) was determined by the glutation assay, and it was found that there was glutation consumption in J774 but not in Vero. Citotoxicity assays by MTT did not show viability diminution for the same concentration over both cell lines. With the aim of finding adequate conditions for the dry storage, UDL were submitted to different drying processes: lyophilization, speed-vac, and vacuum drying, in the presence of different concentrations of trehalose and sucrose as protectants for dehydration. After reconstitution, we evaluated the absence of aggregation (by turbidity determination at 400nm); size maintenance (by dynamic light scattering with a Nanozetasizer); maintenance of ultradeformable properties (by determination of phospholipidic flux extruded at 0,8MPa -low pressure- through a sandwich of 50nm polycarbonate filters); retention of inner aqueous content (by quantification of a colored phthalocyanine after size exclusion chromatography). We found that not all the drying methods were appropriate to preserve UDL. Lyophilization (under different freezing conditions and protectant concentrations) could not effectively preserve UDL, even under the same conditions which lead to successful reconstitution of conventional liposomes. Speed-vac drying was not effective either in order to reconstitute UDL. Nevertheless, the vacuum drying method remarkably provided a successful reconstitution of UDL, but only when protectants were present in 20% w/v concentration. In this method, a 99% of water was evaporated for 72 hours into a vacuum desiccator in the presence of silica gel, with the aim of inducing the formation of a glassy state with a high glass transition. The maintenance of ultradeformability, as it depends on the presence of Sodium cholate in the UDL membrane, showed that the surfactant remained associated to the bilayer. Analysis of symmetric stretching of P-O bond and CH2, asymmetric stretching N-(CH3)3  and vibration of carbonyl bond of the vacuum dried UDL in the presence of 20% w/v trehalose or sucreose by FTIR, showed changes similar to those reported for conventional liposomes lyophilized in the presence of protectants, as hydrogen bounds between protectants and polar heads of phospholipids, interaction of protectants with choline groups, maintenance of the fluid phase of acylic chains, and formation of an intermediate glassy state for the sugar matrix. In sum, the toxicity of UDL against free forms of Leishmania immediately after its capture, the low toxicity and oxidative stress over macrophagic and epithelial cell lines, and the finding of a reproductible method for their dry preservation and reconstitution, become relevant for the use of UDL in the developing of an anti-leishmanic treatment for acute infection stages.