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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.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.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.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.