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. 2020 Feb 21:8:95.
doi: 10.3389/fbioe.2020.00095. eCollection 2020.

Pleiotropic Functions and Biological Potentials of Silver Nanoparticles Synthesized by an Endophytic Fungus

Radhika Chandankere  1   2 , Jayabaskaran Chelliah  2 , Kamalraj Subban  2 , Vanitha C Shanadrahalli  2 , Amreesh Parvez  1 , Hossain M Zabed  1 , Yogesh C Sharma  3 , Xianghui Qi  1
Affiliations

Pleiotropic Functions and Biological Potentials of Silver Nanoparticles Synthesized by an Endophytic Fungus

Radhika Chandankere et al. Front Bioeng Biotechnol. .

Abstract

In recent years, the biological synthesis of silver nanoparticles (AgNPs) from microorganisms has become an emerging trend for developing biocompatible nanomaterials that finds applications in nano and biomedical sectors. In the present study, we demonstrated a facile, green and eco-friendly method for AgNPs synthesis using the endophytic fungi (Colletotrichum incarnatum DM16.3) isolated from medicinal plant Datura metel and its in vitro antithrombin and cytotoxic activity. At first, biosynthesis of colloidal AgNPs was predicted by visual observation of color change and UV-visible spectra demonstrated specific surface plasmon resonance peak at 420 nm which confirmed the presence of nanoparticles. Microscopic analyses revealed the structure of highly aggregated, spherical and crystalline AgNPs in the diameter range of 5-25 nm. Transform infrared spectroscopy (FT-IR) spectral analysis confirmed the presence of probable biomolecules required for the reduction of silver ions. In vitro evaluation of thrombin activity demonstrates that AgNPs could exert strong inhibition against both thrombin activity (87%) and thrombin generation (84%), respectively. Further, in silico based mechanistic analysis yielded a better insight in understanding the probable amino acids responsible for AgNPs binding with thrombin protein. Similarly, in vitro cytotoxicity of synthesized AgNPs on human epithelial cells using MTT assay did not produce any substantial effects after 24 h exposure which indicates excellent biocompatibility nature, whereas notable toxicity was observed on human cancerous (HeLa) cells at 50 μg/mL (IC50 value). In addition, assessment of AgNPs at 10 μg/mL concentration via crystal violet method on biofilm forming Gram-positive (Vibrio cholerae) and Gram-negative bacteria (Bacillus cereus) revealed inhibition up to 85 and 46%, respectively. Overall, this study showed the possibility of microbially synthesized AgNPs as a potent inhibitor for managing acute thrombosis and highlighted their role for other biomedical applications.

Keywords: Colletotrichum incarnatum; antithrombin potential; biogenic synthesis; endophytic fungus; silver nanoparticles.

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Figures

FIGURE 1
FIGURE 1
(A) Progressive proliferation of conidiogenous cells developments X400. (B) Progressive proliferation accumulation of wall layers may eventually plug the opening X400. (C–D) Polyphialides (more than one conidiogenous locus) X400. (E) Young conidia, mature conidia (insert) X400.
FIGURE 2
FIGURE 2
(A) UV-Vis spectra of AgNPs from C. incarnatum DM16.3 at different times. (B) FTIR spectrum of biosynthesized AgNPs (Inset (a) Erlenmeyer flask with C. incarnatum DM16.3 before exposure and (b) after exposure to AgNO3 ions). (B) FTIR spectrum of biosynthesized AgNPs.
FIGURE 3
FIGURE 3
(A) X-ray diffraction. (B) Energy dispersive X-ray spectra of biosynthesized AgNPs from C. incarnatum DM16.3.
FIGURE 4
FIGURE 4
(A) Transmission electron microscopic image of AgNPs biosynthesized from C. incarnatum DM16.3. (B) Histogram analysis of the particle size distribution.
FIGURE 5
FIGURE 5
(A) SAED patterns of the AgNPs. (B) TGA-DTA thermogram of biosynthesized AgNPs from C. incarnatum DM16.3.
FIGURE 6
FIGURE 6
(A) Atomic force micrograph, and (B) topographical image of biosynthesized AgNPs from C. incarnatum DM16.3.
FIGURE 7
FIGURE 7
(A) Thrombin Inhibitory assay and thrombin generation assay of biosynthesized AgNPs from C. incarnatum DM16.3. (B) Schematic representation showing the mode of action of AgNPs as antithrobotic drug (ATD) in blood coagulation cascade. (a) ATD as a factor Xa (blood coagulating factor) inhibitor. Binding of ATD to Xa neutralizes and inhibits the coagulation factor Xa. Henceforth, reduced conversion of prothrombin to thrombin, and fibrinogen to fibrin in a sequential order (b), which eventually inhibit the blood clotting process.
FIGURE 8
FIGURE 8
Molecular docking of thrombin protein with AgNP and single Ag atom; (A) Orientation of AgNP and thrombin juxtaposed towards the various amino acid residues namely Asp243, Gln239, Ala1, Lys235, Asp125, ASN204 and Tyr208. (B) The docking model shows an interaction of Ag to thrombin through Asp49 and Glu2476. (C) RSDM analysis of molecular dynamics (MD) simulations for thrombin, Ag and AgNP.
FIGURE 9
FIGURE 9
Cell viability assay of AgNPs against normal cells (MCF-12A human breast epithelial cell line) and cancerous cells (HeLa human cervical carcinoma cell line).
FIGURE 10
FIGURE 10
Phytoxicity activities of C. incarnatum DM16.3 derived-AgNPs. (A) Seed germination percentage when treated with AgNPs, distilled water (positive control) and AgNO3 (Negative control), and (B) Normalized root elongation when treated with AgNPs, distilled water (positive control) and AgNO3 (negative control).
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