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. 2020 Jun 24;5(26):16238-16245.
doi: 10.1021/acsomega.0c02032. eCollection 2020 Jul 7.

Gold-Silver Bimetallic Nanoparticles Reduced with Herbal Leaf Extracts Induce ROS-Mediated Death in Both Promastigote and Amastigote Stages of Leishmania donovani

Dayakar Alti  1 , M Veeramohan Rao  2 , D Narayana Rao  3 , Radheshyam Maurya  1 , Suresh K Kalangi  4
Affiliations

Gold-Silver Bimetallic Nanoparticles Reduced with Herbal Leaf Extracts Induce ROS-Mediated Death in Both Promastigote and Amastigote Stages of Leishmania donovani

Dayakar Alti et al. ACS Omega. .

Abstract

Resistance to antileishmanial drugs such as sodium stibogluconate (SSG), amphotericin B (Amp-B), and miltefosine is on the rise, and alternate strategies for effective treatment have gained importance in recent years. Although nanoparticle (NP)-based composite drugs that have emerged recently have been found to be effective, the associated toxicity limits their usage. Bimetallic NPs produced through reduction with medicinal plant extracts are proposed to overcome the toxicity of the NPs. In the present study, three types of gold-silver bimetallic nanoparticles (Au-Ag BNPs) were synthesized through a single-step reduction process using fenugreek, coriander, and soybean leaf extracts. All of the three types of BNPs exhibited high antileishmanial effects against promastigotes with half-inhibitory concentration (IC50) values in the range of 0.03-0.035 μg/mL. The IC50 values of the BNPs are much lower compared to those of miltefosine (IC50 = 10 μg/mL). The synthesized BNPs induced the reactive oxygen species (ROS)-mediated apoptosis-like death in the promastigotes and could potentiate the antileishmanial activity of macrophages. The intracellular amastigotes were reduced by 31-46% in macrophages. The biogenic BNPs synthesized in this study and their potent antileishmanial activity provide further impetus to the ongoing quest for novel drugs to effectively manage leishmaniasis.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
UV–visible spectra of Au–Ag BNPs for fenugreek, coriander, and soybean; the X-axis represents wavelength (nm), and the Y-axis represents the absorbance of extracts.
Figure 2
Figure 2
FTIR spectra of Au–Ag BNPs for fenugreek, coriander, and soybean; the X-axis represents wavelength (cm–1), and the Y-axis represents the transmission.
Figure 3
Figure 3
(A) TEM images (scale 100 nm) showing regular and sphere-shaped Au–Ag BNPs for fenugreek, coriander, and soybean. (B) Histograms showing the average size of BNPs as 10–12 nm; the X-axis represents the BNP size (nm), and the Y-axis represents the count.
Figure 4
Figure 4
Selected area electron diffraction pattern of Au–Ag BNPs for fenugreek, coriander, and soybean.
Figure 5
Figure 5
Cytotoxicity of BNPs against L. donovani promastigotes; the X-axis represents the concentrations of BNPs between 0.005 and 0.16 μg/mL and of miltefosine between 0.3 and 40 μg/mL, and the Y-axis represents the percentage viability of promastigotes. IC50 of BNPs on promastigotes intercepted with dotted lines.
Figure 6
Figure 6
BNPs induced apoptosis-like features in promastigotes. (A) Light microscopy analysis of morphological alteration in Giemsa-stained promastigotes (scale 10 μm). (B) SEM analysis of morphological aberrations in promastigotes (scale 2000×). (C) Genomic DNA fragmentation as a smear in promastigotes. (D) H2DCF fluorescence intensity measured in the FL1-H channel by a flow cytometer, and intracellular ROS production in promastigotes represented as MFI (insert), “p values” are represented as *p ≤ 0.05 and **p ≤ 0.01.
Figure 7
Figure 7
Effect of BNPs on amastigote growth; light microscopic analysis and counting of intracellular amastigotes present inside the Giemsa-stained THP-1 macrophages (scale 100×) and indicated with arrow marks. The average number of amastigotes per 100 macrophages is shown in the bar graph, and p values are represented as *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.
Figure 8
Figure 8
Intracellular ROS production in macrophages. Flow cytometry analysis of ROS levels represented as MFI on the Y-axis of the bar graph. The comparison was done between infected variables with or without BNP treatment. Uninfected cells and infected cells with miltefosine (2.5 μM or 1 μg/mL) treatment are used as controls. The p values are represented as *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.
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