Experimental
In vitro studies on liposomal amphotericin B obtained by supercritical carbon dioxide–mediated process

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Abstract

Nanotechnology in drug delivery is a rapidly expanding field. Nanosized liposomal preparations are already in use for efficient drug delivery with better therapeutic indices. Existing methods of liposome preparation are limited by problems of scale-up, difficulty in controlling size, and intercalation efficiency. Here we prepare amphotericin B–intercalated liposomes by a novel process where amphotericin B and purified phosphatidyl choline are solubilized in suitable solvent and precipitated in supercritical fluid carbon dioxide (known as a gas antisolvent technique), to obtain microsized particles that are subsequently introduced into a buffer solution. The morphology of liposomes was characterized through a phase-contrast microscope, and the particle size distribution studied by laser technique showed nanosize with a narrow range of size distribution (between 0.5 and 15 μm) and a higher intercalation efficiency. In vitro studies conducted using Aspergillus fumigatus (MTCC 870) strain proved to be efficient in the retardation of the growth of the organism.

Section snippets

Materials

Amp-B (Cal Biochem, La Jolla, CA, an affiliate of Merck KGoA, Darmstad, Germany) and purified phosphatidyl choline prepared in the author's laboratory, by deoiling soylecithin in SCF CO2, fractionation by ethanol, followed by silica gel column chromatography using Gradifrac fraction collector (Pharmacia Biotech, Uppsala, Sweden) as described earlier[16], [17], [18], [19]. Cholesterol extra pure was from Loba Chemie (Mumbai, India), ammonium thiocyanate and ferric chloride from Ranbaxy

Particle morphology by phase-contrast microscopy and SEM

The liposomes prepared by sonication and SCF-CO2-GAS method were observed under the phase-contrast microscope to characterize the morphology, shape, and intercalation efficiency of the liposomes. Liposomes prepared by both the methods had a spherical shape. The intercalated Amp-B was clearly visible with its yellow color attached to the inner bilayer wall of the liposomes (Figure 2, A and B). The size of the liposomes prepared by the SCF-CO2-GAS method is around 0.15 to 3 μm, whereas the

Conclusions

The SCF-CO2-GAS method provides a simpler way to prepare liposomes with a characteristically smaller size and with a better morphology (uniform size and shape) and efficient intercalation of Amp-B. The stability of the liposomes is superior, and a more abundant occurrence of nanosize liposomes is observed using the GAS method for preparation of liposomes. In vitro studies with A. fumigatus MTCC 870 strain demonstrated the efficiency of the liposomes prepared by both the methods. The SCF-CO2-GAS

Acknowledgments

The authors acknowledge the encouragement from Dr. P.K. Ghosh, former Adviser, Department of Biotechnology, New Delhi, Dr. V. Prakash, Director, Central Food Technological Research Institute, Mysore and Mr. K. Anabalangan, Technical Officer, CFTRI, Mysore for the high quality SEM photographs.

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