Long-Circulating Liposomes Codelivering Amphotericin B and Retinoic Acid for Cutaneous Leishmaniasis Treatment

Leishmaniasis is a neglected tropical disease that mainly affects socially vulnerable populations. It is caused by various Leishmania species, and manifests primarily as cutaneous (CL) or visceral leishmaniasis (VL). AmBisome, a commercial formulation of amphotericin B (AmB) with conventional liposomes, is the most effective for VL treatment, but its efficacy is limited in some CL cases and immunocompromised patients. This work evaluated a new PEGylated liposomal formulation (LAmB-RA) coencapsulating AmB and retinoic acid (RA), as an immunomodulator, for CL treatment. LAmB-RA displayed a mean size of 125 nm, a polydispersity index <0.2, and high encapsulation efficiencies for AmB (97.1%) and RA (94.7%). Although SAXS analysis indicates that RA did not induce major structural rearrangements in the liposomal bilayer, comparison of circular dichroism spectra between LAmB-RA and the liposomal AmB formulation without RA (LAmB) revealed slightly different aggregated states of AmB. In addition, LAmB-RA showed a significantly reduced hemolytic activity, compared to LAmB. Pharmacokinetic analysis of the PEGylated formulations showed higher C _max and prolonged plasma exposure of AmB compared to AmBisome. In Leishmania major-infected mice, LAmB-RA significantly reduced lesion size and parasite burden (99%) versus untreated control. In the Leishmania amazonensis model, it markedly inhibited lesion progression and was the only treatment to significantly reduce parasite load in the spleen (by 60%). Additionally, LAmB-RA promoted an increase in the IFN-γ/IL-10 ratio in antigen-stimulated splenocytes compared to LAmB, indicating a Th1-skewed immune response. These results support LAmB-RA as a promising therapeutic strategy for CL, combining prolonged circulation and enhanced efficacy.