Effect on Human Vascular Endothelial Cells of Au Nanoparticles Synthesized from Vitex mollis

Abstract

A green method for synthesizing gold nanoparticles is proposed using hydroethanolic extract of Vitex mollis fruit (Vm extract) as a reducer and stabilizer. The formation of gold nanoparticles synthesized with Vm extract (AuVmNPs) was monitored by measuring the ultraviolet-visible spectra. The morphology and crystalline phase were determined using scanning electron microscopy, X-ray diffraction, and high-resolution transmission electron microscopy. Synthesized nanoparticles were generally spherical, and the size distribution obtained by transmission electron microscopy shows two populations with mean sizes of 12.5 and 22.5 nm. Cell viability assay using MTT and cellular apoptosis studies using annexin V on human umbilical vein endothelial cells (HUVECs) and the human mammary epithelial cell line (MCF10A) indicate that AuVmNPs have low toxicity. Cell migration tests indicate that AuVmNPs significantly inhibit HUVEC cell migration in a dose-dependent manner. The evaluation of the localization of AuVmNPs in HUVECs using confocal laser scanning microscopy indicates that nanoparticles penetrate cells and are found in the cytosol without preferential distribution and without entering the nucleus. The inhibitory effect on cellular migration and low toxicity suggest AuVmNPs as appropriate candidates in future studies of antiangiogenic activity.

Figure 1

(A) Antioxidant capacity (DPPH) and (B) UV–vis spectrum of Vitex mollis extract.

Figure 2

(A) UV–vis spectra of synthesized gold nanoparticles with Vitex mollis extract. The original products and those subjected to the cleaning process are exposed. A vial with AuVmNPs is shown in the inset. The figure in (B) corresponds to the kinetics of gold nanoparticle formation. The curve is constructed sensing the intensity of the resonance plasmon at 540 nm over time.

Figure 3

(A) AuVmNPs’ and Vitex mollis extract’s FTIR spectra. (B) AuVmNPs’ size distribution by DLS. (C) AuVmNPs’ zeta potential.

Figure 4

AuVmNPs’ XRD pattern.

Figure 5

(A) AuVmNPs’ TEM micrograph at low magnification. (B) AuVmNPs’ size distribution estimated by TEM. (C) AuVmNPs’ HRTEM micrograph. (D) Electron diffraction pattern corresponding to the selected square region on (C). The image on (E) corresponds to theoretical reconstruction by inverse FFT from (D). Different crystalline planes can be determined.

Figure 6

(A) AuVmNPs’ SEM micrograph by secondary electrons and (B) micrograph obtained on transmission-SEM mode. (C) AuVmNPs’ EDS spectrum corresponding to the nanoparticle group shown in (D).

Figure 7

MTT cell viability assay at different concentrations of Vm extract, AuVmNPs, and CAuNPs on (A) HUVECs and (B) MCF10A. One-way ANOVA with the Tukey test. *p < 0.05 compared with the cell control, and ^#p < 0.05 for AuVmNPs vs CAuNPs.

Figure 8

Representative dot plots of apoptotic effects of NPs on HUVECs for (A) untreated control, (B) 70 °C treated as an apoptotic control, (C) phytic acid, (D) Vitex mollis extract (100 μg/mL), (E) AuVmNPs (100 μg/mL), and (F) CAuNPs (100 μg/mL). (G) Apoptotic rates of HUVECs with different treatments and concentrations for 24 h determined by annexin V assay using a flow cytometer. One-way ANOVA with the Tukey test. *p < 0.05 compared with the cell control. No significant differences were found between AuVmNPs and CAuNPs.

Figure 9

Representative dot plots of apoptotic effects of NPs in MCF10A cells for (A) untreated control, (B) UV light treated as an apoptotic control, (C) phytic acid, (D) Vitex mollis extract (100 μg/mL), (E) AuVmNPs (100 μg/mL) and (F) CAuNPs (100 μg/mL). (G) Apoptotic rates of MCF10A cells with different treatments and concentrations for 24 h determined by annexin V assay using a flow cytometer. One-way ANOVA with the Tukey test. *p < 0.05 compared with the cell control, and ^#p < 0.05 for AuVmNPs vs CAuNPs.

Figure 10

(A) Representative scratch assay of HUVEC cells treated with Vitex mollis extract (100 μg/mL) and AuVmNPs (200 μg/mL). (B) Results of mobility inhibition of HUVEC cells treated with AuVmNPs at different concentrations. One-way ANOVA with the Tukey test. *p < 0.05 compared with the positive control.

Figure 11

(A) Bright-field image of HUVEC cells without treatment and (B) confocal image where emission from nuclei is shown in blue and actin fibers in green. (C) Bright-field image of HUVEC cells treated with AuVmNPs (50 μg/mL) at 24 h and (D) confocal image where the AuVmNPs are observed in red.

Figure 12

(A–D) Orthogonal projections and (E) 3D image reconstruction of AuVmNP cellular internalization in HUVECs by confocal microscopy.