Reconstitution of Leishmania plasma membrane to understand the photodynamic effect

Abstract

INTRODUCTION Leishmaniasis is an important neglected disease. Photodynamic therapy (PDT) has been used to fight cutaneous leishmaniasis showing good results. However, PDT mechanisms in Leishmania parasites are not yet completely clarified. OBJECTIVES In this work, our objective was to develop a protocol to produce giant plasma membrane vesicles (GPMVs) from Leishmania amazonensis promastigotes to understand the mechanisms of action of methylene blue (MB)-mediated PDT on the cell membrane of parasites. MATERIALS AND METHODS For membrane extraction, several techniques were tested. The osmotic shock was the technique that presented the best yield and effectiveness. Phosphate and protein measurements were performed to confirm membrane extraction. For the growth of GPMVs, the best technique was electroforming using different frequencies and voltages in 4 cycles. Reconstituted GPMVs were observed by phase-contrast light microscopy. Subsequently, PDT was applied to GPMVs dispersed in an aqueous solution containing 50 μM MB and we verified the changes in permeability before and after exposure to light. The same processwas applied to giant unilamellar vesicles (GUVs) with lipid compositions similar to the parasite membrane. DISCUSSION AND RESULTS The electroforming technique with the protocol developed in this work made it possible to obtain GPMVs froma promastigote membrane isolate of L. amazonensis. The membrane isolation technique was effective to extract the parasite's membrane while preserving lipids and proteins. In GUVs we observe an increase in the area during PDT in different compositions and loss of contrast. The GPMVs showed a loss of contrast as well as the GUVs but did not show an increase in area. CONCLUSION This factor could be explained by the high degree of complexity of the membrane, which contains membrane proteins in addition to containing lipids.