Quercetin-mediated synthesis of graphene oxide-silver nanoparticle nanocomposites: a suitable alternative nanotherapy for neuroblastoma

Abstract

Background: Graphene and graphene-related materials have gained substantial interest from both academia and industry for the development of unique nanomaterials for biomedical applications. Graphene oxide (GO) and silver nanoparticles (AgNPs) are a valuable platform for the development of nanocomposites, permitting the combination of nanomaterials with different physical and chemical properties to generate novel materials with improved and effective functionalities in a single platform. Therefore, this study was conducted to synthesize a graphene oxide-silver nanoparticle (GO-AgNPs) nanocomposite using the biomolecule quercetin and evaluate the potential cytotoxicity and mechanism of GO-AgNPs in human neuroblastoma cancer cells (SH-SY5Y).

Methods: The synthesized GO-AgNPs were characterized using various analytical techniques. The potential toxicities of GO-AgNPs were evaluated using a series of biochemical and cellular assays. The expression of apoptotic and anti-apoptotic genes was measured by quantitative real-time reverse transcription polymerase chain reaction. Further, apoptosis was confirmed by caspase-9/3 activity and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and GO-AgNPs-induced autophagy was also confirmed by transmission electron microscopy.

Results: The prepared GO-AgNPs exhibited significantly higher cytotoxicity toward SH-SY5Y cells than GO. GO-AgNPs induced significant cytotoxicity in SH-SY5Y cells by the loss of cell viability, inhibition of cell proliferation, increased leakage of lactate dehydrogenase, decreased level of mitochondrial membrane potential, reduced numbers of mitochondria, enhanced level of reactive oxygen species generation, increased expression of pro-apoptotic genes, and decreased expression of anti-apoptotic genes. GO-AgNPs induced caspase-9/3-dependent apoptosis via DNA fragmentation. Finally, GO-AgNPs induced accumulation of autophagosomes and autophagic vacuoles.

Conclusion: In this study, we developed an environmentally friendly, facile, dependable, and simple method for the synthesis of GO-AgNPs nanocomposites using quercetin. The synthesized GO-AgNPs exhibited enhanced cytotoxicity compared with that of GO at very low concentrations. This study not only elucidates the potential cytotoxicity against neuroblastoma cancer cells, but also reveals the molecular mechanism of toxicity.

Keywords: apoptosis; autophagy; cell viability; cytotoxicity; graphene oxide–silver nanoparticles nanocomposite; neuroblastoma.

Figure 1

Characterization of GO and GO-AgNP nanocomposite using UV-visible spectroscopy. Notes: (A) GO spectra exhibited a maximum absorption peak at ~230 nm corresponding to the π–π transitions of aromatic C–C bonds. A new peak at 420 nm was observed after deposition of AgNPs on the GO surface; the band at 420 nm in the absorption spectrum of the GO-AgNPs is attributed to the surface plasmons and presence of AgNPs. (B) XRD patterns of GO and GO-AgNPs. In the XRD pattern of GO, a strong, sharp peak at 2θ=11.7° corresponds to an interlayer distance of 7.6 Å. The GO-AgNPs showed distinct reflections in the XRD pattern at 31.8 corresponding to the (111) plane of face-centered cubic Ag. (C) FTIR spectra of GO and GO-AgNPs. Dried powders of GO and GO-AgNPs were diluted with KBr to perform FTIR spectroscopy and spectrum GX spectrometry within the range of 500–4,000 cm⁻¹. (D) SEM images of GO and GO-AgNP dispersions at 500 μg/mL. (E) TEM images of GO and GO-AgNPs. (F) Raman spectroscopy analyses of GO and GO-AgNP nanocomposite. Raman spectra of GO and GO-AgNPs were obtained using a laser excitation wavelength of 532 nm at a power of 1 mW after the removal of background fluorescence. At least three independent experiments were performed for each sample, and reproducible results were obtained. Abbreviations: AgNP, silver nanoparticle; FTIR, Fourier transform infrared; GO, graphene oxide; SEM, scanning electron microscopy; TEM, transmission electron microscopy; XRD, X-ray diffraction.

Figure 2

Effects of GO and GO-AgNPs on cell viability of human ovarian cancer cells. Notes: (A) The viability of human neuroblastoma cancer cells was determined after 24 h exposure to different concentrations of GO (0–50 μg/mL). (B) The viability of human neuroblastoma cancer cells was determined after 24 h exposure to different concentrations of GO-AgNPs (0–10 μg/mL) using the CCK-8 assay. The results are expressed as the mean ± standard deviation of three independent experiments. The treated groups showed statistically significant differences from the control group by the Student’s t-test. *P<0.05. Abbreviations: AgNP, silver nanoparticle; CCK, cell counting kit; GO, graphene oxide.

Figure 3

Effect of GO-AgNPs on proliferation of human neuroblastoma cancer cells. Notes: (A) Cell proliferation was observed in the cells treated with GO (25 μg/mL) for 24 h by a trypan blue exclusion assay. (B) Cell proliferation was observed in the cells treated with GO-AgNPs (5 μg/mL) for 24 h by a trypan blue exclusion assay. The results are expressed as the mean ± standard deviation of three independent experiments. The treated groups showed statistically significant differences from the control group by the Student’s t-test. *P<0.05. Abbreviations: AgNP, silver nanoparticle; GO, graphene oxide.

Figure 4

Effect of GO and GO-AgNPs on cytotoxicity in human neuroblastoma cancer cells. Notes: (A) The cells were treated with GO (25 μg/mL), GO-AgNPs (5 μg/mL), and DOX (1 μg/mL) for 24 h, and LDH activity was measured at 490 nm using the LDH cytotoxicity kit (B) The cells were treated with GO (25 μg/mL), GO-AgNPs (5 μg/mL), and DOX (1 μg/mL) for 24 h, and the dead-cell protease levels were determined by a CytoTox-Glo™ cytotoxicity assay. The results are expressed as the mean ± standard deviation of three independent experiments. The treated groups showed statistically significant differences from the control group by the Student’s t-test. *P<0.05. Abbreviations: AgNP, silver nanoparticle; Con, control; DOX, doxorubicin; GO, graphene oxide; LDH, lactate dehydrogenase.

Figure 5

Effect of GO and GO-AgNPs on mitochondrial dysfunction in human neuroblastoma cancer cells. Notes: (A) The cells were treated with GO (25 μg/mL), GO-AgNPs (5 μg/mL), and DOX (1 μg/mL) for 24 h, and the mitochondrial membrane potential was determined using the cationic fluorescent indicator JC-1. (B) The cells were treated with GO (25 μg/mL), GO-AgNPs (5 μg/mL), and DOX (1 μg/mL) for 24 h, and the mitochondria copy number was determined by real-time PCR. *P<0.05. Abbreviations: AgNP, silver nanoparticle; Con, control; DOX, doxorubicin; GO, graphene oxide; PCR, polymerase chain reaction.

Figure 6

Effect of GO and GO-AgNPs on ROS generation and MDA levels in human neuroblastoma cancer cells. Notes: (A) The cells were treated with GO (25 μg/mL), GO-AgNPs (5 μg/mL), and DOX (1 μg/mL) for 24 h. Relative fluorescence of DCF was measured at the excitation wavelength of 485 nm and emission wavelength of 530 nm using a spectrofluorometer. (B) The cells were treated with GO (25 μg/mL), GO-AgNPs (5 μg/mL), and DOX (1 μg/mL) for 24 h. After incubation, the cells were harvested and washed twice with an ice-cold PBS solution. The cells were collected and disrupted by ultrasonication for 5 min on ice. The concentration of MDA was measured on a microplate reader at a wavelength of 530 nm. The results are expressed as the mean ± standard deviation of three independent experiments. The treated groups showed statistically significant differences from the control group by the Student’s t-test. *P<0.05. Abbreviations: AgNP, silver nanoparticle; Con, control; DCF, 2′,7′-dichlorofluorescein; DOX, doxorubicin; GO, graphene oxide; MDA, malondialdehyde; PBS, phosphate-buffered saline; ROS, reactive oxygen species.

Figure 7

Effect of GO and GO-AgNPs on expression of apoptotic and anti-apoptotic genes in human neuroblastoma cancer cells. Notes: The cells were treated with GO (25 μg/mL), GO-AgNPs (5 μg/mL), and DOX (1 μg/mL) for 24 h. Relative mRNA expression was analyzed by qRT-PCR in human neuroblastoma cancer cells after the treatments. The results are expressed as the mean ± standard deviation of three independent experiments. The treated groups showed statistically significant differences from the control group by the Student’s t-test. *P<0.05. Abbreviations: AgNP, silver nanoparticle; Con, control; DOX, doxorubicin; GO, graphene oxide; mRNA, messenger RNA; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

Figure 8

Effect of GO and GO-AgNPs on caspase-9/3 activity in human neuroblastoma cancer cells. Notes: (A) The cells were treated with GO (25 μg/mL), GO-AgNPs (5 μg/mL), and DOX (1 μg/mL) for 24 h with and without caspase-9 inhibitor. The concentration of p-nitroanilide released from the substrate was calculated from the absorbance at 405 nm. (B) The cells were treated with GO (25 μg/mL), GO-AgNPs (5 μg/mL), and DOX (1 μg/mL) for 24 h with and without caspase-3 inhibitor. The concentration of p-nitroanilide released from the substrate was calculated from the absorbance at 405 nm. The results are expressed as the mean ± standard deviation of three independent experiments. The treated groups showed statistically significant differences from the control group by the Student’s t-test. *P<0.05. Abbreviations: AgNP, silver nanoparticle; Con, control; DOX, doxorubicin; GO, graphene oxide.

Figure 9

Effect of GO and GO-AgNPs on apoptosis in human neuroblastoma cancer cells. Notes: The cells were treated with GO (25 μg/mL) (A), GO-AgNPs (5 μg/mL), and DOX (1 μg/mL) (B) for 24 h. Apoptosis of human neuroblastoma cancer cells after a 24-h treatment was assessed by the TUNEL assay; the nuclei were counterstained with DAPI. Representative images show apoptotic (fragmented) DNA (red staining) and the corresponding cell nuclei (blue staining). The images are ×100 magnification. Abbreviations: AgNP, silver nanoparticle; Con, control; DAPI, 4′,6-diamidino-2-phenylindole; DOX, doxorubicin; GO, graphene oxide; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 10

GO- and GO-AgNP-induced accumulation of APs and autophagic vacuoles. Notes: The cells were treated with (A) control, (B) GO (25 μg/mL), (C) GO-AgNPs (5 μg/mL), and (D) DOX (1 μg/mL) for 24 hours and processed for TEM. GO-AgNPs-treated cells in C showed many multi-vesicular and membrane-rich APs and significant accumulation of autophagic vacuoles compared with that of GO and Con (red arrows). Abbreviations: AgNP, silver nanoparticle; AP, autophagosome; AV, autophagic vacuoles; C, cytoplasm; Con, control; DOX, doxorubicin; GO, graphene oxide; M, mitochondria; N, nucleus; TEM, transmission electron microscopy.