Myco-Nanofabrication of Silver Nanoparticles by Penicillium brasilianum NP5 and Their Antimicrobial, Photoprotective and Anticancer Effect on MDA-MB-231 Breast Cancer Cell Line

Abstract

Currently, the exploration of fungal organisms for novel metabolite production and its pharmacological applications is much appreciated in the biomedical field. In the present study, the fungal strains were isolated from soil of unexplored Yellapura regions. The potent isolate NP5 was selected based on preliminary screening and identified as Penicillium brasilianum NP5 through morphological, microscopic, and molecular characterizations. Synthesis of silver nanoparticles from P. brasilianum was confirmed by the color change of the reaction mixture and UV-visible surface plasmon resonance (SPR) spectra of 420 nm. Fourier transform infrared (FTIR) analysis revealed the functional groups involved in synthesis. Atomic force microscopy (AFM) and transmission electron microscope (TEM) analysis showed aggregation of the NPs, with sizes ranged from 10 to 60 nm, an average particle size of 25.32 nm, and a polydispersity index (PDI) of 0.40. The crystalline nature and silver as the major element in NP5-AgNPs was confirmed by X-ray diffraction (XRD) and energy dispersive X-ray (EDX) analysis. The negative value -15.3 mV in Zeta potential exhibited good stability, and thermostability was recorded by thermogravimetric analysis (TGA). NP5-AgNPs showed good antimicrobial activity on selected human pathogens in a concentration-dependent manner. The MTT assay showed concentration-dependent anticancer activity with an IC₅₀ of 41.93 µg/mL on the MDA-MB-231 cell line. Further, apoptotic study was carried out by flow cytometry to observe the rate of apoptosis. The calculated sun protection factor (SPF) value confirms good photoprotection capacity. From the results obtained, NP5-AgNPs can be used in the pharmaceutical field after successful in vitro clinical studies.

Keywords: MDA-MB-231 breast cancer cells; Penicillium sp.; SPF; anticancer activity; antimicrobial activity; silver nanoparticles.

Figure 1

Morphology of Penicillium brasilianum NP5; (A) Aerial mycelium (1× magnification), (B) Substrate mycelium (1× magnification), (C) SEM image (10 µm).

Figure 2

Phylogenetic tree of Penicillium brasilianum NP5.

Figure 3

Visual observation of the biosynthesis of AgNPs; (A) Penicillium brasilianum NP5 extract, (B) AgNO₃ solution, and (C) Synthesis of silver nanoparticles.

Figure 4

UV-Visible absorption spectrums of Penicillium brasilianum NP5 extract and synthesized AgNPs from Penicillium brasilianum NP5.

Figure 5

FT-IR analysis; (A) Penicillium brasilianum NP5 Extract, (B) Synthesized AgNPs from Penicillium brasilianum NP5.

Figure 6

AFM images of synthesized AgNPs from Penicillium brasilianum NP5; (A) 2D image, (B) Size distribution, and (C) Histogram showing distribution of NP5-AgNPs.

Figure 7

HR-TEM image of synthesized AgNPs by Penicillium brasilianum NP5; (A) Agglomeration of silver nanoparticles, (B) Histogram showing size distribution.

Figure 8

(A) EDX analysis, (B) XRD analysis, (C) Zeta potential, (D) TGA thermogram of the synthesized AgNPs from Penicillium brasilianum NP5.

Figure 9

Antimicrobial activity of synthesized AgNPs from Penicillium brasilianum NP5 against pathogenic microorganisms; (A) E. coli, (B) S. aureus, (C) S. flexneri, (D) B. cereus, (E) C. glabrata, (F) C. albicans, and (G) graph showing the zone of inhibition against selected pathogens. Data are statistically significant at p ≤ 0.01 by one-way ANOVA with Tukey’s B test; error bars are based on three independent experiments (means ± SD, n = 3). Different letters at the same concentration refer to data that are significantly different.

Figure 10

Morphological changes in the MDA-MB-231 cell line after exposure to different concentrations of AgNPs synthesized from Penicillium brasilianum NP5; (A) Untreated, (B) Standard, (C) 12.5 μg/mL, (D) 25 μg/mL, (E) 50 μg/mL, (F) 100 μg/mL, (G) 200 μg/mL of NP5-AgNPs, (H) Graph showing the percentage of cell viability on MDA-MB-231 cancer cells at different concentrations of NP5-AgNPs. Data are statistically significant at p ≤ 0.01 by one-way ANOVA with Tukey’s B test, error bars are based on three independent experiments (means ± SD, n = 3). Different letters at the concentrations refer to the data that are significantly different.

Figure 11

UV absorbance of MTT assay; (A) Only NP5-NPs, (B) NP5-AgNPs with cancer cells, and the difference in the absorbance of treated cancer cells, excluding NPs absorbance.

Figure 12

Flow cytometric analysis of synthesized AgNPs from Penicillium brasilianum NP5 against the MDA-MB-231 cancer cell line. Quadrangular plot of Annexin V/PI expression on MDA-MB-231 cells; (A) Untreated, (B) Standard, (C) NP5-AgNPs treated cells. Cells count on Annexin-V/FITC; (D) Untreated, (E) Standard, (F) NP5-AgNPs treated cells. Scatter plot of forward scatter (FSC) against side scatter (SSC); (G) Untreated, (H) Standard, (I) NP5-AgNPs treated cells.

Figure 13

Graph showing the percentage of live, apoptotic and necrotic cells after the treatment of synthesized AgNPs from Penicillium brasilianum NP5.

Figure 14

Sun protection factor of synthesized AgNPs from Penicillium brasilianum NP5. Data are statistically significant at p ≤ 0.01 by one-way ANOVA with Tukey’s B test, error bars are based on three independent experiments (means ± SD, n = 3). Different letters at the concentrations refer to the data that are significantly different.