Development of Ag-ZnO/AgO Nanocomposites Effectives for Leishmania braziliensis Treatment

Abstract

Tegumentary leishmaniasis (TL) is caused by parasites of the genus Leishmania. Leishmania braziliensis (L.b) is one of the most clinically relevant pathogens that affects the skin and mucosa, causing single or multiple disfiguring and life-threatening injuries. Even so, the few treatment options for patients have significant toxicity, high dropout rates, high cost, and the emergence of resistant strains, which implies the need for studies to promote new and better treatments to combat the disease. Zinc oxide nanocrystals are microbicidal and immunomodulatory agents. Here, we develop new Ag-ZnO/xAgO nanocomposites (NCPs) with three different percentages of silver oxide (AgO) nanocrystals (x = 49%, 65%, and 68%) that could act as an option for tegumentary leishmaniasis treatment. Our findings showed that 65% and 68% of AgO inhibit the extra and intracellular replication of L.b. and present a high selectivity index. Ag-ZnO/65%AgO NCPs modulate activation, expression of surface receptors, and cytokine production by human peripheral blood mononuclear cells toward a proinflammatory phenotype. These results point to new Ag-ZnO/AgO nanocomposites as a promising option for L. braziliensis treatment.

Keywords: Leishmania braziliensis; leishmaniasis; nanocomposites; silver doped; silver oxide; therapeutics; zinc oxide.

Figure 1

Characterization of Ag-ZnO/AgO Nanocomposites. (A) X-ray patterns of ZnO nanocrystals and Ag-ZnO/AgO Nanocomposites (ZnO:5Ag, ZnO:9Ag and ZnO:11Ag). Inset shows the alterations of the ZnO and AgO main peaks due to the Ag concentration. (B) Representation of nanocomposite formed by Ag-doped ZnO nanocrystals and AgO nanocrystals. (C) Scanning microscopy images of ZnO nanocrystals and Ag-ZnO/AgO Nanocomposites (a) ZnO (b) ZnO:5Ag (c) ZnO:9Ag and (d) ZnO:11Ag and a magnification was performed to visualize the dimensions of the rods.

Figure 2

Evaluation of parasitic proliferation when treated with different nanoformulations. The inhibition of proliferation of L.b treated with the different nanomaterials was evaluated by counting the parasites in a Neubauer chamber after fixation with ice-cold 0.2% PFA every 24 h for 5 days. The results are expressed as the mean ± SEM (standard error of the mean), and two-way ANOVA of multiple comparisons was applied, with values of p < 0.05 (*) being considered significant when compared to cell culture without treatment (medium). Cells untreated (medium) and treated with anti-CD3 and anti-CD28 stimuli were used as negative and positive controls, respectively. (A) ZnO nanocrystals at a dose of 25 μg/mL reduced the proliferation of L.b. promastigotes from day 4, while in the treatment with ZnO:5Ag (B) NCPs, there was no significant difference on any of the days analyzed. (C) ZnO:9Ag NCPs reduced L.b parasite proliferation from the third day of treatment from the lowest dose tested. (D) Treatment with ZnO:11Ag NCPs caused a decrease in the proliferation of promastigote forms from day 4 at a dose of 25 μg/mL and on day 5 at a dose of 12.5 μg/mL.

Figure 3

ZnO:9Ag and ZnO:11Ag NCPs reduce L. braziliensis intracellular amastigotes. (A) Intracellular L.b. amastigotes were evaluated by staining with DAPI (blue) and IgG anti-Leishmania+anti-IgG-FITC (green) and counting at least 200 cells/treatment. White arrows indicate intracelular amaastigores. Representative images: Left panel—Non-treated culture; Right panel—ZnO:11Ag—50 μg/mL. (B) RAW 264-7 were infected with L.b. (MOI 10:1) for 12 h and treated with different nanoformulations at doses of 50, 25 and 12.5 μg/mL for 72 h. The results are expressed as the mean ± SEM. The Mann–Whitney test was applied, and values of p < 0.05 (*) were considered significant when compared to non-treated cultures. Culture without treatment (medium). Cells treated with Amphotericin B (2 µg/mL) were used as treatment control.

Figure 4

Effect of nanoformulations on the expression of CD69, annexin-V and CD73 molecules in T lymphocytes. (A) Representative images of gate strategy based on (left to right): removal of cell aggregate, isolation of Lymphocytes based in size and cell complexity, isolation of CD4+ and CD8+ T cells and determination of cell marker expression (%).The results are expressed as the mean fluorescence intensity ± SEM. The unpaired T test (CD69 and annexin) and the Mann–Whitney test (CD73) were used for comparison with untreated cells (medium), with p values <0.05 (*) being considered significant. Cells stimulated with anti-CD3 and anti-CD28 were used as positive controls. (A) Flow Cytometry analysis strategy. (B) From the dose of 12.5 μg/mL, all tested nanomaterials increased the expression of CD69 in CD4+ T cells, with ZnO:9Ag and ZnO:11Ag being the most efficient NCPs. (C) Cell activation of CD8+ CD69+ T lymphocytes was significantly higher in all treatments and doses, with the exception of treatment with ZnO nanocrystals at 6.25 μg/mL. (D) All nanoformulations at a dose of 25 μg/mL increased annexin-V labeling in CD4+ T cells when compared to the untreated control group. (E) In CD8+ cells, it was seen that only ZnO:9Ag increased annexin production. (F) ZnO:9Ag NCPs at a dose of 12.5 μg/mL increased the expression of CD73 in CD4+ T cells. (G) In CD8+ T lymphocytes, ZnO nanocrystals, ZnO:9Ag and ZnO:11Ag NCPs at a dose of 12.5 μg/mL increased the expression of this molecule.

Figure 5

Nanomaterials promote increased expression of TNF-α receptors in T lymphocytes. Expression levels of TNFR1 (Figure 4A,B) and TNFR2 (Figure 4C,D) were determined by flow cytometry in CD4+ and CD8+ T lymphocytes from treated PBMCs for 72 h. The results are expressed as the mean fluorescence intensity ± SEM. The Mann–Whitney test was applied, and values of p < 0.05 (*) were considered significant when compared to untreated cell culture (medium). Untreated cells (medium) and cells treated with anti-CD3 and anti-CD28 stimuli were used as negative and positive controls, respectively. (A) All treatments and doses (except ZnO:11Ag NCPs at 6.25 μg/mL) upregulated TNFR1 expression in CD4+ T lymphocytes. (B) In CD8+ T cells, all nanoformulations (except ZnO nanocrystals at 6.25 μg/mL) upregulated the expression of TNFR1, with this production being more significant in cells treated with ZnO:9Ag NCPs. (C) CD4+ TNFR2+ T lymphocytes were increased in cultures treated with ZnO nanocrystals (at 12.5 μg/mL and 6.25 μg/mL), ZnO:5Ag NCPs (at 6.25 μg/mL) and ZnO:9Ag NCPs (at 12.5 µg/mL). (D) CD8+ T cells expressed more TNFR2 than the control group in all tested nanoformulations in a dose-dependent manner, with the ZnO:9Ag and ZnO:11Ag NCPs at 12.5 μg/mL having the highest activity.

Figure 6

NPCs differentially impact the expression of CD210 and PD-1 in T lymphocytes. Expression of CD210 (Figure 5A,B) and PD-1 (Figure 5C,D) was performed by flow cytometry on CD4+ and CD8+ T lymphocytes from PBMCs treated for 72 h, and these results are expressed as the mean fluorescence intensity ± SEM. The Mann–Whitney test was applied, and values of p < 0.05 (*) were considered significant when compared to cell culture without treatment (medium). Cells without treatment (medium) and cells treated with anti-CD3 and anti-CD28 stimuli were used as negative and positive controls, respectively. (A) ZnO nanocrystals increased CD4+ 210+ T cells in cultures when ZnO:11Ag NCPs were added. (B) In CD8+ T lymphocytes, all treatments upregulated the expression of CD210. (C) CD4+ T cells treated with ZnO nanocrystals and ZnO:5Ag NCPs increased the expression of PD1, while ZnO:9Ag NCPs downregulated this expression. (D) All tested nanoformulations increased PD-1 expression in CD8+ T lymphocytes but in a dose-dependent manner.

Figure 7

Cytokine production by treated PBMCs. The levels of TNF-α, IL-10, IFN-γ and IL-4 were evaluated by ELISA in the supernatant of cultures of PBMCs treated with different nanoformulations at doses of 12.5 μg/mL and 6.25 μg/mL for 72 h. The results are expressed as the mean ± SEM in picograms per milliliter (pg/mL). The Mann–Whitney test was applied, and values of p < 0.05 (*) were considered significant when compared to cell culture without treatment (medium). Cells without treatment (medium) and cells treated with anti-CD3 and anti-CD28 stimuli were used as negative and positive controls, respectively. (A) TNF-α levels were increased when treated with ZnO nanocrystals, ZnO:9Ag and ZnO:11Ag NCPs at the two doses tested, as well as treatment with ZnO:5Ag at 12.5 μg/mL. (B) The level of IFN-γ was upregulated in ZnO nanocrystals and ZnO:9Ag NCPs at both doses. (C) IL-10 quantification was lower in the treatment with ZnO:5Ag NCPs at 6.25 μg/mL and in both doses in the treatment with ZnO:9Ag and ZnO:11Ag NCPs. (D) For IL-4, only ZnO:11Ag NCPs at 6.25 μg/mL decreased the production of this cytokine. (E) All nanomaterials had increased levels of TNF-α compared to IL-10 levels. (F) ZnO:5Ag at 6.25 μg/mL and ZnO:11Ag (two doses tested) positively regulated IFN-γ levels when compared with IL-4 levels.

Figure 8

ZnO:9Ag and ZnO:11Ag NCPs increased NO production. NO production was evaluated by the modified Griess method in the supernatant of PBMC cultures treated with different nanoformulations at doses of 12.5 μg/mL and 6.25 μg/mL for 72 h. The results are expressed as the mean ± SEM in picograms per milliliter (pg/mL). The Mann–Whitney test was applied, and values of p < 0.05 (*) were considered significant when compared to cell culture without treatment (medium). Cells without treatment (medium) and cells treated with anti-CD3 and anti-CD28 stimuli were used as negative and positive controls, respectively. NO production was increased in cultured cells treated with ZnO:9Ag and ZnO:11Ag NCPs, especially at the lowest dose.