الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Gold nanoparticles capped with citrate have been prepared via chemical methods and characterized by UV-vis absorption spectroscopy and TEM. The prepared AuNPs showed absorption band due to surface plasmon resonance band at 522 nm and the TEM images indicated that the particles are spherical with average size of 35 nm and surface charges of (-33.6 mV) using Zetasizer Nano ZS (Malvern Instruments). Silver nanoparticles had shown absorption band at 405 nm with spherical shape as indicated by TEM. The size (22.11nm) and surface charge (-9.45 mV) was measured by Zetasizer Nano ZS (Malvern Instruments).
It was clear by inverted microscope that AuNPs (35nm) and AgNPs (22nm) had no morphological alteration on HepG2 cells.
The TEM images had revealed uptake of AuNPs and AgNPs by HepG2 cells with a wide distribution in the cytoplasm especially around the nucleus. The internalization of both types of metal nanoparticles into cell nucleus had been showed by TEM images too.
By colorimetric viability assays; treating HepG2 for 48h with AuNPs induce viability of 89.4% of cell treated with 12.5 μM, while cells viability decreased to 75% after treatment with 100 μM. In case of AgNPs viability of HepG2 cells was 78% after cell treatment with 10 μM and then decreased to 46% after treatment of cells with 100 μM for 48h. Treatment of WISH cells with AuNPs and AgNPs, for 48h, resulting in (96% and 97% cellular viability) respectively. Statistical analysis using SPSS version 23 had revealed no significant difference between cells treated either with AuNPs or AgNPs with probability value p (1.45). While there was a significant difference; p (0.00), between the effect of AuNPs and that of AgNPs on HepG2 cells.
Flow cytometric results had supported the DNA fragmentation results. It declared the accumulation of cells treated with AuNPs in G2/M phase (8.54%) compared to (6.78%) of untreated cells, which getting higher in G0/G1phase, measuring (76.52%) for AuNPs treated cells, while untreated cells evaluated for (65.14%), which leading to decrease in cells entering into S phase than untreated cells (20.94% , and 28.08%) respectively. Silver nanoparticles treated cells revealed a highly accumulation of cells in S phase, (44.83%) for treated and (28.08%) for untreated cells, reflecting the decreasing in G2/M phase (3.05% in case of AgNPs treated cells compared to untreated cells which had count for (6.78%), and G0/G1 phase (52.12%) for treated and (65.14%) for untreated cells. It is now clear the difference in cell response toward each type of nanoparticles, indicating mechanistic difference between both metallic nanoparticles.
DNA fragmentation analysis which was carried out to investigate the genotoxic effects of AuNPs and AgNPs revealed a DNA fragmentation which represented late apoptotic effect was observed after 24 hrs. of cell exposure with high concentration of AuNPs (100 μM, 35.8 mg/L), and AgNPs (100 μM, 9.71 mg/L), while much less significant changes were seen at lower concentration both nanoparticles (10 μM).
As it is well known there is a direct co-relation between the cell cycle progression with its regulating check points and regulatory proteins such as p53 with its subsequent induction proteins (Bax,Bak). Therefore it wasn’t weird to combine the results of flow cytometry with that of RT-PCR in order to achieve a consideration about the mechanism of nanoparticles inside the cells.
Semiquantitative expression of some apoptotic genes was performed by One Step RT-PCR assay. Results showed that there was difference in expression level of p53 between both nanoparticles compared to untreated cells, where the cells treated with AgNPs showed increased in the expression level of P53 compared to cells treated with AuNPs and untreated cells. The same figure was observed for bax and bak genes. This indicates that both metallic nanoparticles behave differently on the same cell type.
AuNPs stop the cell cycle in G1 phase preventing entrance into S phase, and slightly elevation of p53 expression may indicate AuNPs as a mutagenic agent, that would cause cell arrest rather than apoptosis.
AuNPs are able to cause DNA strand breaks which detected by DNA fragmentation assay. Furthermore, it was documented that AuNPs able to induce pyrimidines and purines oxidative damage inhibiting cell proliferation.
Oxidative stress had been found to be one of the toxic effects of gold nanoparticles.
The apoptotic mechanisms of AgNPs can be attributed to release Ag ions from the AgNPs and/or nanoparticles deposited inside the cells and detailed mechanisms mostly include reactive oxygen species (ROS) generation leading to apoptosis. Moreover disruption of energy metabolism and mitochondria mediated DNA fragmentation leading also to programmed cell death involved p53 dependent gene transcription of pro-apoptotic proteins.
Moreover the elevated induction of p53 is not depends only on damaged DNA but moreover on the integrity of cellular nucleolus which related mainly to the cellular protein integrity and hence the endoplasmic reticulum (ER) integrity which may suggest a lethal effect of AgNPs on ER. That may explain the reduction in casepase-3 as ER induces apoptosis is actually through casepase-12.
Though the genotoxic effects of gold and silver nanoparticles on HepG2 cells, those nanoparticles had reveled their safety effect on normal cell line (WISH), that primarily suggesting their antitumor activity, but still lot of investigations areas to be studied.