Title : Metals against leishmaniasis: Unlocking the potential of biodegradable metal nanoparticles against parasitic diseases
Abstract:
In this work, MgO and ZnO biodegradable nanoparticles (BNPs) are investigated as potential therapeutics for the treatment of leishmaniasis. This is a vector-borne infectious disease resulting from infection by protozoan Leishmania spp. parasites, and is among the top ten neglected tropical diseases. Clinically, it manifests in three main forms: cutaneous leishmaniasis (CL), mucocutaneus leishmaniasis (ML) [extensive scarring and facial disfiguration leading to stigmatization and social rejection] and visceral leishmaniasis (VL) which affects internal organs [especially the spleen, liver, and bone marrow] and can be life threatening. Traditional treatment is based on pentavalent antimonial compounds, which were developed more than 50 years ago and present toxicity and several adverse effects for the patient.1-9 Therefore, a new effective treatment is urgently needed.
In the human host, Leishmania parasites thrive inside dendritic cells and macrophages of the immune system.1,2 In their amastigote form they are known acidophyles, thriving in the acidic environment (pH 4.5-5.2) of the parasitophorous vacuole. MgO and ZnO nanoparticles, through their degradation, can increase the pH of the physiological media:
Mg+2H2O →Mg(OH)2+H2 Zn+2H2O →Zn(OH)2+H2
Thus, upon reaching the parasitophorous vacuole of an infected macrophage, these BNPs can increase the pH of the environment killing the parasite in the amastigote form.
To achieve this goal, Zn and Mg BNPs underwent surface functionalization with 3-aminopropyltriethoxysilane (APTES), to increase their dispersibility in physiological media. The physicochemical properties of these particles were characterized by transmission and scanning electron microscopy, dynamic light scattering and inductively coupled plasma-optical emission spectroscopy. The viability of THP-1 human monocytes was assessed against a range of concentrations of functionalized and non-functionalized BNPs at 24h, 48h and 72h. Similarly, the IC50 of the BNPs against Leishmania braziliensis and L. major was determined at the same time points. For instance, it was observed that MgO nanoparticles did not significantly affect THP-1 derived macrophage’s viability up to concentrations of 500 ?g/ml, independently of the incubation time, while the IC50 for
L. braziliensis was of 265.4, 193.1 and 116.3 ?g/ml for 24h, 48h and 72h, respectively. Considering this, the effect of BNPs on infected macrophages was assessed for effective concentrations. A reduction of about 20% of infection was observed when infected macrophages were treated with MgO nanoparticles at a concentration of 250 ?g/ml.
Lastly, the mechanism of action of the used nanoparticles was accessed at specific times. BNPs internalization by infected macrophages was showed through TEM and two fluorescent probes, Lysotracker and MitoTracker Red, were used in the staining of lysosomes and mitochondria. Markers for ROS production, protein oxidation, cytotoxicity and M1/M2 macrophage polarization, in infected and non-infected macrophages, both before and after treatment with BNPs, were identified by the analysis of genetic and protein expression, by RTqPCR and Western Blot respectively, as well as the secretion of specific cytokines, determined by ELISA. This allowed the establishment of a comprehensive model of the cellular uptake, intracellular path and metabolic pathways that result in the anti-Leishmania mechanism of the studied BNPs.
