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This study aimed to evaluate the in vivo anti-Leishmania amazonensis activity of a Phospholipase A2 (Asp49-PLA2), isolated from Bothrops jararacussu venom, encapsulated in liposomes as a modified toxin release system. The activity of the liposomes was evaluated in BALB/c mice, previously infected with 1 × 105 of the parasite's promastigotes. The size of the paw lesion in Asp49-PLA2-liposomal-treated animals, after 21 days, was observed as decreasing by 16% relative to the untreated control group and 12% by the Glucantime®-treated animals, which was used as a reference drug. At the end of the treatment, the animals were sacrificed and the paw and lymph node tissues were collected. Part of the collection was used to recover amastigotes and another to quantify cytokines and nitrites. In the group treated with Asp49-PLA2-liposomes the parasitic load was observed to be reduced by 73.5% in the macerated lymph node, compared to the control group. Comparatively, in the paw tissue was observed a reduction of 57.1%. The infected groups treated with Asp49-PLA2-liposomes showed significant production in TNF-α measured in lymph nodes and paw (43.73 pg/mL ± 2.25 and 81.03 pg/mL ± 5.52, respectively) and nitrite levels (31.28 μM ± 0.58 and 35.64 μM ± 5.08) also measured in lymph nodes and paw tissues, respectively, compared to untreated groups. These results indicate that the Asp49-PLA2-loaded liposomes were able to activate the production of some cellular components of the protective TH1 response during the infection, constituting a promising tool for inducing the microbicidal activity of the Leishmania-infected macrophages.Asp49-PLA2--liposomes decreased the progression of paw injury in infected mice.Asp49-PLA2--liposomal treated groups reduced the amount of amastigotes in vivo.Liposomes containing Asp49-PLA2 increased TNF-α and NO production by infected mice.