Advancing antileishmanial treatments through innovative nanotechnology in vitro approaches and characterization of novel therapeutic compounds

[eng] Leishmaniasis is a vector-borne disease, caused by the parasite Leishmania spp. and transmitted by sand flies of the genus Phlebotomus or Lutzomyia. There are over 12 million people infected worldwide and around one million new cases each year, which represents a significant global health challenge. Actually, leishmaniasis is the second deadliest parasitic disease in the world after malaria, but it is still considered a neglected tropical disease. The current treatment options of leishmaniasis face challenges, such as long regimens, drug resistance, side effects, issues related to the parenteral administration, limited availability in endemic regions, and high costs. Thus, there is an urgent need to develop new safer treatments with a better patient compliance and more effective than the actual antileishmanial drugs. In this work, three strategies are proposed to overcome some of the challenges mentioned above. Firstly, nanotechnology was applied with the aim of developing nanoformulations to encapsulate high amounts of pentamidine (one of the antileishmanial drugs currently used for clinical purposes) and, using heparin and chondroitin sulfate (CS) as targeting elements, to direct the nanoformulations to the free parasite or to macrophages infected with the parasite. Such specific delivery allowed for (i) increasing the targeting to macrophages when liposomes were coated with either heparin or CS, (ii) significantly reducing the IC50 of the drug on L. infantum and L. pifanoi promastigotes when liposomes were coated with heparin or chondroitin sulfate, (iii) significantly reducing the cytotoxicity on human umbilical endothelial cells, and (iv) obtaining a higher deposition of the nebulized liposomal preparations in the deeper stages of a human lung airway simulator when compared to free pentamidine. The second approach was focused on the search for new drugs with a different mechanism of action than the current antileishmanial drugs. In here, different compounds which interfere with protein aggregation showed some extent of antileishmanial activity. In particular, the bis(styrylpyridinium) salt YAT2150 had a better activity against L. infantum than most of the currently used drugs for the clinical treatment of leishmaniasis, including pentamidine, miltefosine and paromomycin. In relation to the mode of action of YAT2150, a reduction in the overall protein aggregation in a culture of L. infantum promastigotes was observed, according to Thioflavin T analysis. This finding aligns with the proposed mode of action of YAT2150 in P. falciparum determined in a previous study. The encapsulation of YAT2150 in liposomes or immunoliposomes functionalized with an antibody against LPG, significantly increased the selectivity index of the drug above 100. Therefore, the activity of YAT2150 against both Leishmania and Plasmodium represents a promising opportunity for the treatment of co-infections with these parasites. The third strategy was spurred by the urgent need to develop ligands that specifically recognise the parasite, and that could be used, e.g., for the functionalization of drug delivery systems or as diagnostic tools. GP63, is the major surface protein present in Leishmania promastigotes and constitutes one of its main virulence factors, playing a role in the adhesion of the parasite to the macrophage and in the survival of amastigotes. DNA aptamers were developed against a L. infantum mature form of GP63 (LiGP63m), using the systematic evolution of ligands by exponential enrichment (SELEX) method. After 7 SELEX cycles, 5 individual aptamer sequences were selected, showing a high affinity for endogenous LiGP63 within a promastigote lysate, with a binding affinity in the μM range, according to an Aptamer Linked Immobilized Sorbent Assay (ALISA). In conclusion, the aptamers discussed here hold promise for interventions against leishmaniasis as potential diagnostic tools and as targeting molecules for drug delivery nanocarriers.