Effects Of Iron Nanoparticles On The Expression Of Id Genes

Today, the iron oxide magnetic nanoparticles as a promising nanomaterial are increasingly be used in the fields of biomedicine and biology, such as magnetic separation and purification, magnetic transfection, magnetic resonance imaging (MRI), hyperthermia, drug delivery, and tissue repair. As most of these applications require the delivery of large numbers of nanoparticles into the target cells, prior to clinical applications, the potential cytotoxicity of the nanoparticles should be clearly characterized. More and more studies have paid attention to the fate of the nanoparticles internalized into cells, especially their degradation, biocompatibility and the resulted effects on cells. It is valuable to uncover all possible effects of iron nanoparticles on cells at the molecular level.In recent years, we have studied the effects of DMSA coated Fe3O4 magnetic nanoparticles (referred as FeNP) on gene expression of cells using gene expression profiling technologies. In the study, we found a significant effect of FeNP on Id3 gene expression. Because Id genes play an important role in the cell cycle, growth, differentiation, and we decide to deeply study that how FeNP influence expression of Id gene. In this study, we detected the effects of different doses of FeNP on Id genes expression of cells in vivo and in vitro using quantitative PCR, the specific research is as follows:1. This study investigated the cellular effect of various dose of FeNP (50,100 μg/mL) on the expression of Id genes in the multiple cell lines at 24h. The cell lines included mouse macrophage (RAW264.7), mouse hepatoma cells (Hepal-6), human acute monocytic leukemia cells (THP-1), human hepatoma cells (HepG2), human liver cells (HL-7702), and cervical adenocarcinoma cells (HeLa). The results of TEM and zeta potential showed good dispersibility and size uniformity of FeNP. The cell labeling efficiency of FeNP was investigated by observing the intracellular internalization of FeNP with prussian blue staining and measuring the cellular iron loading with colorimetric assay. The results demonstrated the FeNP could label all of the six kinds of cell lines, while the labeling efficiency was signficantly different between the cell lines. At any concentrations of FeNP, RAW264.7 was labeled more effectively than other cells, but with the lowest iron loading labeled in both human liver cells (HepG2 and HL-7702). Quantitative detection of cellular iron loading also showed that the iron loading of cells was in a dose-dependent. CCK-8 cell viability assay demonstrated the cell viability of all the cells were not significantly suppressed by FeNP after exposed to the studied doses for 24h. The DNA microarray results demonstrated that the expression of Idl, Id2, Id3 were almost all significantly down regulated in five cell lines (RAW264.7, Hepal-6, THP-1, HepG2, and HL7702) treated by FeNP, but unobvious for Id4. The qPCR results showed that expression of Id3 gene was significantly down regulated in all cell lines (p<0.0\), whereas the expression of Id1 gene was down regulated in all the cell lines in addition to RAW264.7, and expression of Id2 genes were also significantly down regulated in Hepal-6, HepG2, HL7702 and HeLa cells. According to DNA microarray and qPCR detection results, the expression of Idl, Id2 and Id3 gene were significantly down regulated in three liver cell lines (Hepal-6, HepG2, and HL7702).2. The FeNPs were labeled with a near-infrared fluorophore (IRDye800CW), and the distribution and metabolic clearance process in mice body were observed. Through real-time monitoring, we found the optimal time point for sampling the proper treated liver tissues and collected the liver treated by FeNPs at 24 h as the sample. The results of prussian blue staining revealed that FeNP mainly distributed in the liver and spleen. This study then detecteded the expression of Id genes in the liver tissues of mice that were intravenously injected with the nanoparticle at two doses (2 and 5 mg Fe/kg body weight). The results revealed that the expression of Idl, Id2 and Id3 gene was also significantly down regulated in the liver tissues under each treatment. Another Id gene, Id4, was also significantly regulated in some cells and liver tissues treated with FeNPs. These results indicated that FeNPs exerted a significant effect on the in vitro and in vivo expression of Id genes. This effect suggests that the nanoparticle may disturb the biological processes controlled by this transcription factor family, such as cell growth, differentiation, apoptosis, and tumorigenesis. The results from this study provide new insights into the Id-related cellular effect of iron oxide nanoparticles and the close relationship between iron and the regulation of Id genes.