Liposomes containing an ASP49-phospholipase A2 from Bothrops jararacussu snake venom as experimental therapy against cutaneous leishmaniasis

Abstract

The aim of this study was to evaluate in vitro anti-Leishmania amazonensis activity of a Phospholipase A2 (Asp49-PLA2) isolated from the venom of Bothrops jararacussu and its encapsulated form. Asp49-PLA2 (2mg/mL) was added to a lipid mixture, solubilized in chloroform and dried under nitrogen flow. The lipid vesicles were formed homogeneously using the extrusion method, physicochemically characterized by their diameters, zeta potentials, encapsulation rate and also submitted to a molecular docking in silico analysis. The activity of Asp49-liposomes was evaluated in vitro against promastigote forms of L. amazonensis and J774 macrophages. Parasite and macrophage viabilities using MTT method were assessed after 48h incubation. L. amazonensis-infected macrophages were also incubated with encapsulated Asp49 and in solution form. The amastigote forms were counted inside the infected macrophages and the culture supernatants were collected for nitrites and TNF-α quantifications. Asp49-PLA2 in solution form displayed an anti-Leishmania concentration-dependent effect. Asp49-liposomes were able to reduce 78% of promastigote forms and preserved 82% of J774 macrophages' viability. After 48h of incubation with nanoencapsulated Asp49-PLA2 there was a significant reduction in the number of amastigotes (55%; p<0.05) compared to the control group. When the macrophages were infected and incubated with Asp49-liposomes a significant increase in the production of nitrites and TNF-α was observed when compared to infected cells alone. The results indicated that the liposomal system achieved in this study is a promising tool to enhance microbicidal activity of the infected macrophages, conferring a biotechnological therapeutic approach against experimental leishmaniasis.