Exploiting in vitro potential and characterization of surface modified Zinc oxide nanoparticles of Isodon rugosus extract: Their clinical potential towards HepG2 cell line and human pathogenic bacteria

Abstract

Little is known about biogenically synthesized Zinc oxide nanoparticles (ZnONPs) from Isodon rugosus. Synthesis of metal oxide NPs from essential oil producing medicinal plants results in less harmful side effects to the human population as compared to chemically synthesized NPs. In this article, we report biogenic synthesis of ZnONPs from in vitro derived plantlets and thidiazuron (TDZ) induced callus culture of Isodon rugosus. Synthesized NPs were characterized using UV-spectra, XRD, FTIR, SEM and EDX. Furthermore, the NPs were evaluated for their potential cytotoxic (against HepG2 cell line) and antimicrobial (against drug resistant Staphylococcus epidermidis, Bacillus subtilis, Klebsiella pneumoniae and Pseudomonas aeruginosa) activities. Pure crystalline ZnONPs with hexagonal and triangular shapes were obtained as a result of callus extract (CE) and whole plant extract (WPE), respectively. ZnONPs showed potent cytotoxic and antimicrobial potential. The antimicrobial and cytotoxic activities of ZnONPs were found to be shape and surface bound phytochemicals dependent. CE mediated hexagonal ZnONPs showed superior anti-cancer and antimicrobial activities as compared to WPE mediated triangular shaped ZnONPs. It is concluded that biogenic ZnONPs have incredible potential as theranostic agents and can be adopted as useful drug delivery system in next generation treatment strategies.

Keywords: Isodon rugosus; ZnONP; characterization; nanotoxicity; pH; surface adaptation.

Table 1. Atomic percentage of zinc and oxygen in ZnONPs as obtained from EDX analysis

Table 2. Antibacterial activity of CE, WPE, C-ZnO NPs and W-ZnO NPs against two Gram-positive (Staphylococcus epidermidis, Bacillus subtilis) and two Gram-negative (Klebsiella pneumoniae, Pseudomonas aeruginosa) bacteria

Figure 1. (A) Thidiazuron induced callus from stem explant, (B) Estimation of phenolic and flavonoid content in callus extract, (C) UV-Vis spectral analysis of ZnONPs, (D) Optical observation of biogenically synthesized ZnONPs using Isodon rugosus extract, (à) Thidiazuron induced callus extract, (bʹ) reaction mixture of callus derived ZnONPs, (ć) plant extract, (é) reaction mixture of plant derived ZnONPs. After completion of the reaction, presence of white precipitate indicates the synthesis of ZnONPs.

Figure 2. XRD and SEM analysis of ZnONPs where (A) XRD image of C-ZnONPs, (B) XRD image of W-ZnONPs, (C) SEM image of C-ZnONPs and (D) SEM image of W-ZnONPs

Abbreviations: C-ZnONPs: callus derived naoparticles, W-ZnONPs whole plant derived nanoparticles

Figure 3. Figure 3: EDX analysis ZnONPs showing the purity of zinc and oxygen ions in the samples (A) C-ZnONPs, (B) W-ZnONPs

Figure 4. FTIR spectra of powdered samples of (A) C-ZnO NPs, (B) W-ZnO NPs

Figure 5. Cytotoxic effects of ZnONPs and extracts on HepG2 cells upon 24 hour treatment with 200 µg/ml concentration. Untreated cells, DMSO (solvent) and Doxorubicin (34 µM) were included as controls. Microscopic images of HepG2 cells (treated and untreated). Magnification = 200X, Scale = 100 µm. (A) Callus extracts (CE). (B) Callus derived ZnONPs (C-ZnONPs). (C) Untreated HepG2 cells. (D) Whole plant extracts (WPE). (E) Whole plant derived ZnONPs (W-ZnONPs). (F) DMSO 1% (negative control). (G) Doxorubicin 34 µM (positive control)

Figure 6. Percentage viabilities and inhibition of cells relative to untreated control (Mean ± SD). Each sample was studied in triplicates and experiment was performed twice.

Figure 7. Tentative graphical representation of ZnONPs cytotoxicity towards HepG2 cell line. ZnONPs induce cytotoxicity by morphological changes, loss of membrane integrity, cell shrinkage, and reduced cell density which are characteristic features of apoptotic cell formation.

Figure 8. Antibacterial evaluation of Plant derived ZnONPs and extract (A-WPE) and Callus derived (B-CE) ZnONPs and extract analysed by disc diffusion method, showing zone of inhibition against two Gram-positive (Staphylococcus epidermidis, Bacillus subtilis) and two Gram-negative bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa). Zone of inhibition were measured in mm, taking sample concentration 10 mg/mL. Notes: Values (mean ± SD) indicates of three experiments

Abbreviations: Abs, amoxicillin antibiotic; WPE, whole plant extract; W-ZnONPs, whole plant derived zinc oxide nanoparticles; CE, callus extract; C-ZnONPs, callus derived ZnONPs; Zn acetate, zinc acetate dihydrate salt; SD, standard deviation

Figure 9. Diagrammatic representation of ZnONPs cytotoxicity towards human pathogenic bacterial strains depending upon many factors such as shape (transformation of particles as function of pH), production of ROS, dysfunction of bacterial membrane, effect of oxidative stress on DNA, protein, mitochondria (cell death), electrostatic forces, charge surface area, ultimately causing death of microbes