Green SPIONs as a novel highly selective treatment for leishmaniasis: an in vitro study against Leishmania amazonensis intracellular amastigotes

Abstract

The main goal of this work was to evaluate the therapeutic potential of green superparamagnetic iron oxide nanoparticles (SPIONs) produced with coconut water for treating cutaneous leishmaniasis caused by Leishmania amazonensis. Optical and electron microscopy techniques were used to evaluate the effects on cell proliferation, infectivity percentage, and ultrastructure. SPIONs were internalized by both parasite stages, randomly distributed in the cytosol and located mainly in membrane-bound compartments. The selectivity index for intracellular amastigotes was more than 240 times higher compared to current drugs used to treat the disease. The synthesized SPIONs showed promising activity against Leishmania and can be considered a strong candidate for a new therapeutic approach for treating leishmaniases.

Keywords: Leishmania amazonensis; Leishmaniasis; SPIONs; coconut water; nanomedicine.

Figure 1

Bright-field optical microscopy of L. amazonensis promastigotes (A, B) and intracellular amastigotes (C, D) treated with 100 µg/mL of SPIONs for 24 h, after staining with Prussian blue (A–D). (A) The arrows indicate the blue stain characteristic for the reaction with ferrous compounds in the promastigote cytosol. (B) Digital magnification shows that SPIONs are randomly distributed throughout the cytosol. (C) In the case of macrophages infected with intracellular amastigotes, the SPIONs were observed inside the parasitophorous vacuoles. (D) Digital magnification shows the SPIONs (arrows) inside the macrophage cytosol, the parasitophorous vacuoles, and the amastigote cytosol (arrowheads).

Figure 2

Scanning electron microscopy of macrophages infected with L. amazonensis intracellular amastigotes after treatment with 100 µg/mL SPIONs for 24 h. The plasma membrane was gently removed to observe the presence of nanoparticles inside the cells. Panel A shows infected macrophages with some amastigotes (arrowheads) inside the parasitophorous vacuoles (thin arrows). Panel B shows the same macrophage; however, the image was obtained by detecting backscattered electrons, revealing several electron-lucent aggregates (arrows). Digital magnification (highlighted rectangular area) showed electron-lucent aggregates even inside intracellular amastigotes (arrowheads). Panels C and D show the X-ray microanalysis mapping of infected macrophages, indicating the presence of iron in the cytosol (red color in Figure 2D).

Figure 3

Transmission electron microscopy of L. amazonensis promastigotes and intracellular amastigotes treated with 100 µg/mL of SPIONs for 24 h. Electron-dense aggregates of SPIONs (arrowheads) are randomly distributed in both developmental stages. (A) SPIONs (arrowheads) were observed in the promastigote cytosol, closely associated with endoplasmic reticulum profiles and lipid bodies. (B) High-magnification image with SPION aggregates (arrowheads) inside membrane-bound compartments (arrows). (C) SPIONs (arrowheads) are associated with thin filaments inside the flagellar pocket and in the cytosol closely associated with the flagellar pocket membrane. (D) In the macrophages infected with intracellular amastigotes, the SPIONs appear inside the parasitophorous vacuole and in the macrophage and parasite cytosol (arrowheads). In this image, it is also possible to observe the SPIONs surrounded by a membrane (arrows) and an aggregate close to the membrane of the parasitophorous vacuole (large arrow). (E, F) High-magnification images of intracellular amastigotes revealing SPIONs (arrowheads) inside membrane-bound compartments (arrows). The aggregates are formed by smaller individual nanoparticles (small arrow). Figure 3E also shows many lipid bodies, vacuoles, and a multivesicular structure, which are features typically found in treated parasites. F, flagellum; FP, flagellar pocket; LB, lipid body; M, mitochondrion; N, nucleus; and PV, parasitophorous vacuole.

Figure 4

Analysis of the antiproliferative effect in L. amazonensis promastigotes and intracellular amastigotes treated with different concentrations of SPIONs. (A) Growth curve of L. amazonensis promastigotes; the SPIONs were added to the culture medium after 24 h of growth. (B) For intracellular amastigotes, infected macrophages were treated, and the percentage of infection was obtained for each treatment condition; the SPIONs were added to the infected macrophage culture after 24 h of infection. P values for panel B: **** p < 0.0001.

Figure 5

Transmission electron microscopy of L. amazonensis intracellular amastigotes treated with 100 µg/mL of SPIONs for 24 h. Different ultrastructural changes were observed in intracellular amastigotes: (1) many lipid bodies (A–C, thin arrows), (2) increased secretion of extracellular vesicles (A–C, broad arrows), (3) intracellular vacuolization (A–C, arrows), (4) myelin-like figures (A, arrowhead), (5) mitochondrial swelling (C, star), and (6) destroyed amastigotes (D, triangle). F, flagellum; k, kinetoplast; LB, lipid body; M, mitochondrion; and N, nucleus.