Interactions of antileishmanial drugs with monolayers of lipids used in the development of amphotericin B-miltefosine-loaded nanocochleates.

The emergence of strains of Leishmania resistant to existing drugs complicates the treatment of life-threatening visceral leishmaniasis. The development of new lipid formulation (nanocochleates), containing two active drugs: amphotericin B (AmB) and miltefosine (hexadecylphosphocholine, HePC), could increase effectiveness, decrease toxicity and reduce the risk of appearance of resistance. Nanocochleates are cigar-shaped structures of rolled negatively charged lipid bilayers bridged by calcium, prepared from dioleoylphosphatidylserine (DOPS) and cholesterol (Cho) and able to accommodate drugs. To determine the interaction, the orientation and the stability of the amphiphilic drugs in the lipid mixture and the optimal drugs/lipids ratio, the Langmuir film balance and BAM (Brewster angle microscopy) were used. The drugs were mixed with the lipids (DOPS or 9DOPS/1Cho) and spread at the air-water interface. A stability study showed that DOPS maintained HePC at the interface at low molar fraction of HePC; this effect became more marked in the presence of Cho. The fact that HePC can be stably associated with the monolayer at low molar fraction (below 10%) suggests that in the nanocochleates HePC is inserted between the lipid molecules rather than between the bilayers. Phase diagrams and BAM images showed that, even at low pressure, DOPS maintains AmB at low molar fraction (below 10%) in the "erect" rather than the horizontal form at the interface and that the presence of Cho reinforces this effect. These results allowed us to predict the organization and the orientation of these drugs in the nanocochleates and to determine the optimal drugs/lipids ratio.