Interaction of amphotericin B and its low toxic derivative, N-methyl-N-D-fructosyl amphotericin B methyl ester, with fungal, mammalian and bacterial cells measured by the energy transfer method.

Amphotericin B (AMB) derivative, N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME) retains the broad antifungal spectrum and potency of the parent antibiotic, whereas its toxicity towards mammalian cells is reduced by about two orders of magnitude. The purpose of this work was to find out whether the differences observed in the toxicity of MFAME and native AMB are due to the differential drugs affinity to fungal and mammalian cell membranes. Comparative studies on AMB and MFAME biological activity and their affinity to fungal, mammalian and bacterial cells were performed. The interaction of AMB and MFAME with cells have been studied by fluorescence method based on the energy transfer between membrane fluorescent probe (donor) and the polyenic chromophore of the antibiotic (acceptor) simultaneously present in the cell membrane. The amount of the antibiotic bound to cells was indicated by the extent of fluorescence quenching of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) or 1,6-diphenyl-1,3,5-hexatriene (DPH) by polyenic chromophore of the antibiotic. The results obtained indicate that binding extent and characteristics for both antibiotics are comparable in the three types of cells studied. Dramatically lower toxicity of MFAME as compared to AMB towards mammalian cells is not related to the antibiotic-cell affinity, but rather to different consequences of these interactions for cells, reflected in membrane permeabilization. MFAME is definitely less effective than parent AMB in the permeabilizing species formation in mammalian cell membrane.

Delivery agents for oligonucleotides.

This chapter provides a basic overview of most of the oligonucleotide delivery systems available for an in vitro use. Two major classes are described: systems that act through an endocytosis process (e.g., lipid-based vectors, nanoparticles, and polycations) and systems that by-pass this endocytosis process (e.g., peptides and pore-forming agents). Each technique is briefly described to allow a critical choice of the best delivery systems suitable for specific purposes in cultured cells.

Heat-induced reformulation of amphotericin B-deoxycholate favours drug uptake by the macrophage-like cell line J774.

AIM: Heat treatment of deoxycholate-amphotericin B (AmB-DOC) leads to a therapeutically interesting supramolecular rearrangement (h-AmB-DOC); this reformulation improves the therapeutic index of AmB-DOC by reducing amphotericin B (AmB) toxicity in mammalian cell lines from 3- to 10-fold. Its activity in experimentally induced fungal infection in mice remains unchanged compared with AmB-DOC, whereas its activity is 2.5 times higher in Leishmania donovani-infected mice. This work investigates the in vitro mechanism that allows this improvement. METHODS: In this study, we analysed the role of serum components on the interaction of h-AmB-DOC with two cultured cell lines: murine peritoneal macrophage cells (J774) and kidney epithelial cells (LLCPK1). The methods used were: spectrophotometry for AmB uptake; MTT assay for cell viability; and lactate dehydrogenase release for membrane damage. RESULTS: In the presence of 10% fetal calf serum (FCS), the toxicity of AmB-DOC or h-AmB-DOC for both cell lines was null or weak. Interestingly, in J774 cells, the uptake of AmB in the form of h-AmB-DOC was much higher. In LLCPK1 cells, AmB uptake was more limited in both cases but remained higher with h-AmB-DOC. In the absence of FCS, no toxicity for either cell line was observed with h-AmB-DOC. CONCLUSIONS: These findings confirm the importance of serum proteins in AmB biodistribution and suggest that, in vivo, the reduced toxicity and the improved antileishmanial activity of AmB-DOC after moderate heating may be the result of its increased uptake by macrophages.