Highly selective ergosterol binding and impaired redox balance leads to improved antileishmanial efficacy for amphotericin b synthesized silver nanoparticleswith reduced toxicity- In vitro and in vivo studies.

Abstract

We aim to develop a low-cost silver nanoparticle (AgNP)-based delivery of AmB (AmB-AgNP) which can replace the costly AmBisome and toxic Fungizone formulation for applications against visceral leishmaniasis (VL) caused by the parasite Leishmania donovani (LD). Using different molar ratios of AmB and silver nitrate, we have identified a specific NP of ∼110 nm (zeta potential of -36.7 mV and PDI of 0.15) as the ideal antileishmanial agent with increased efficacy than AmB against LD promastigotes and amastigotes. These NPs were characterized by UV-visible, DLS, Zeta potential, FT-IR, DSC, and FE-SEM studies.The uptake of metallic silver by ICP-MS studies indicate that AmB-AgNP is internalized >3.4 and >2.8 fold more than citrate-reduced AgNPs inside the LD and murine macrophage cells, respectively. AmB-AgNPs are less cytotoxic than AmB and show more necrotic mode of death than AmB. Here, production of high amount of recative oxygen speccies, lipid peroxides, protein carbonylations and decreased expression of antioxidant enzymes are also observed. AmB-AgNP LD causes a dose-dependent ergosterol (ERG) depletion which can be reversed by ERG supplementation. Further, ITC studies established selective and enhanced binding efficacy of AmB-AgNPagainst ERG and not choesterol. The selective and enhanced inhibition of the ERG and trypanothione biosynthesis pathway by AmB-AgNP, compared to AmB, was proven by proteomics studies. The rate-limiting enzyme of ERG biosynthesis, HMG-CoA-reductase, was downregulated >9-fold in the presence of AmB-AgNP treatment. The acute toxicity studies on mice showed that AmB-AgNP has a selectivity index of > 6-fold compared to AmB. However, AgNP is <30% less effective than AmB in antileishmanial efficacy with equivalent doses in vivo. The higher selectivity index of AmB-AgNP provides a better therapeutic window than Fungizone, whereas the lost-cost synthesis, compared to AmBisome, makes the AmB-AgNP a viable cheaper delivery option against VL for future investigations.