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

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

Saarbrücken, Alemania

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

  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. Different concentrations of UDL were incubated with 5x105 free L.brasilensis promastigotes at 25ºC during 5’, 30’ and 24h. Motility (implying viability) was determined, observing that even the shortest incubation time leaded to immovilization. To verify that the lethal effect was due to the capture of UDL by promastigotes, parasites were incubated with UDL containing fluorescent probes both in their hydrophilic inner space and in their hydrophobic membrane, and observed by confocal microscopy. Parasites were able to endocyte the UDL, being this capture the probable cause of their immovilization. On the other hand, the generation of oxidative stress by UDL over cell lines (J774 and Vero) was determined by the glutation assay, finding that there was glutation consumption in J774 but not in Vero. Citotoxicity assays did not show viability diminution for the same concentration over both cell lines. With the aim of finding adequate conditions for dry storage, UDL were submitted to lyophilization, speed-vac, and vacuum drying, in the presence of different concentrations of protectants for dehydration. After reconstitution, we evaluated the absence of aggregation, the size maintenance, maintenance of ultradeformable properties and retention of inner aqueous content. Lyophilization and Speed-vac drying could not preserve UDL, even under the same conditions which lead to successful reconstitution of conventional liposomes. Nevertheless, the vacuum drying method remarkably provided a successful reconstitution of UDL, but only when protectants were present in 20% w/v concentration and a 99% of water was evaporated into a vacuum desiccator in the presence of silica gel. The maintenance of ultradeformability, as it depends on the presence of Na cholate in the membrane, showed that the surfactant remained associated to the bilayer. Analysis of symmetric stretching of PO bond and CH2, asymmetric stretching N-(CH3)3 and vibration of carbonyl bond of the vacuum dried UDL with 20% w/v trehalose or sucrose by FTIR, showed changes similar to those reported for conventional liposomes lyophilized in the presence of protectants. asymmetric stretching N-(CH3)3 and vibration of carbonyl bond of the vacuum dried UDL with 20% w/v trehalose or sucrose by FTIR, showed changes similar to those reported for conventional liposomes lyophilized in the presence of protectants. was determined, observing that even the shortest incubation time leaded to immovilization. To verify that the lethal effect was due to the capture of UDL by promastigotes, parasites were incubated with UDL containing fluorescent probes both in their hydrophilic inner space and in their hydrophobic membrane, and observed by confocal microscopy. Parasites were able to endocyte the UDL, being this capture the probable cause of their immovilization. On the other hand, the generation of oxidative stress by UDL over cell lines (J774 and Vero) was determined by the glutation assay, finding that there was glutation consumption in J774 but not in Vero. Citotoxicity assays did not show viability diminution for the same concentration over both cell lines. With the aim of finding adequate conditions for dry storage, UDL were submitted to lyophilization, speed-vac, and vacuum drying, in the presence of different concentrations of protectants for dehydration. After reconstitution, we evaluated the absence of aggregation, the size maintenance, maintenance of ultradeformable properties and retention of inner aqueous content. Lyophilization and Speed-vac drying could not preserve UDL, even under the same conditions which lead to successful reconstitution of conventional liposomes. Nevertheless, the vacuum drying method remarkably provided a successful reconstitution of UDL, but only when protectants were present in 20% w/v concentration and a 99% of water was evaporated into a vacuum desiccator in the presence of silica gel. The maintenance of ultradeformability, as it depends on the presence of Na cholate in the membrane, showed that the surfactant remained associated to the bilayer. Analysis of symmetric stretching of PO bond and CH2, asymmetric stretching N-(CH3)3 and vibration of carbonyl bond of the vacuum dried UDL with 20% w/v trehalose or sucrose by FTIR, showed changes similar to those reported for conventional liposomes lyophilized in the presence of protectants. asymmetric stretching N-(CH3)3 and vibration of carbonyl bond of the vacuum dried UDL with 20% w/v trehalose or sucrose by FTIR, showed changes similar to those reported for conventional liposomes lyophilized in the presence of protectants. 5 free L.brasilensis promastigotes at 25ºC during 5’, 30’ and 24h. Motility (implying viability) was determined, observing that even the shortest incubation time leaded to immovilization. To verify that the lethal effect was due to the capture of UDL by promastigotes, parasites were incubated with UDL containing fluorescent probes both in their hydrophilic inner space and in their hydrophobic membrane, and observed by confocal microscopy. Parasites were able to endocyte the UDL, being this capture the probable cause of their immovilization. On the other hand, the generation of oxidative stress by UDL over cell lines (J774 and Vero) was determined by the glutation assay, finding that there was glutation consumption in J774 but not in Vero. Citotoxicity assays did not show viability diminution for the same concentration over both cell lines. With the aim of finding adequate conditions for dry storage, UDL were submitted to lyophilization, speed-vac, and vacuum drying, in the presence of different concentrations of protectants for dehydration. After reconstitution, we evaluated the absence of aggregation, the size maintenance, maintenance of ultradeformable properties and retention of inner aqueous content. Lyophilization and Speed-vac drying could not preserve UDL, even under the same conditions which lead to successful reconstitution of conventional liposomes. Nevertheless, the vacuum drying method remarkably provided a successful reconstitution of UDL, but only when protectants were present in 20% w/v concentration and a 99% of water was evaporated into a vacuum desiccator in the presence of silica gel. The maintenance of ultradeformability, as it depends on the presence of Na cholate in the membrane, showed that the surfactant remained associated to the bilayer. Analysis of symmetric stretching of PO bond and CH2, asymmetric stretching N-(CH3)3 and vibration of carbonyl bond of the vacuum dried UDL with 20% w/v trehalose or sucrose by FTIR, showed changes similar to those reported for conventional liposomes lyophilized in the presence of protectants. asymmetric stretching N-(CH3)3 and vibration of carbonyl bond of the vacuum dried UDL with 20% w/v trehalose or sucrose by FTIR, showed changes similar to those reported for conventional liposomes lyophilized in the presence of protectants. 2, asymmetric stretching N-(CH3)3 and vibration of carbonyl bond of the vacuum dried UDL with 20% w/v trehalose or sucrose by FTIR, showed changes similar to those reported for conventional liposomes lyophilized in the presence of protectants.3)3 and vibration of carbonyl bond of the vacuum dried UDL with 20% w/v trehalose or sucrose by FTIR, showed changes similar to those reported for conventional liposomes lyophilized in the presence of protectants.