| The dimercaptosuccinic acid (DMSA) was widely used to coat iron oxide nanoparticles (FeNPs). DMSA-coated FeNPs (DMSA-FeNPs) were considered to be biocompatible, which had various biomedical applications, such as magnetic resonance imaging, drug delivery and hyperthermia. DMSA has been especially used as an orally administered antidote in clinical application for treating heavy metal intoxication. DMSA has low toxicity compared to other dithiols due to its extracellular distribution. However, when used as modification molecule, it can be carried intracellularly into cells by the endocytosis of nanoparticles, and introduces a number of active thiol groups into cells. Its intracellular biocompatibility remains to be adequately elucidated.This study investigated the the gene expression profile of four cell lines treated with DMSA-FeNPs. These four cell lines included mouse macrophage (RAW264.7) and hepatomacells (Hepal-6), and human acutemonocytic leukemia cells (THP-1) and hepatoma cells (HepG2). By combining with the studies at the cellular level, we analyzed the changes of gene expression profile to study the influence of DMSA-FeNPs at the molecular level. The main results of this thesis were as follows:1. The size of DMSA-FeNPs was characterized the with transmission electron microscopy (TEM). The intracellular uptake of DMSA-FeNPs were identified by Prussian blue staining. The cellular iron loading and the thiols on the surfaces of FeNP were measured with a colorimetric assay. The cell viability was determined by CCK-8 assay. The results showed that a number of active thiol groups were carried by DMSA-FeNPs, which had good dispersion in aqueous solution and DMEM. The DMSA-FeNPs had labeled all cells studied with Prussian blue staining. However, the RAW264.7 was labeled more efficiently than other cells at various concentrations of the nanoparticles. In addition, the measurement of cellular iron loading indicated that the content of the intravellular nanoparticles significantly increased with the DMSA-FeNP concentration in cell cultures and the treatment time. The cellular iron concentration reached the saturation state in 24 hours. The cell viability of RAW264.7 were not significantly suppressed by the DMSA-FeNPs at the low dose (≤50 μg/mL), however, it was significantly suppressed by the DMSA-FeNPs at the high dose (100 μg/mL).2. The differentially expressed genes (DEGs) were analyzed in the four mammalian cells treated by the DMSA-FeNPs at various doses for different time intervals in this study. Our results revealed that there were total 365 DEGs, nearly one fourth of which encoded cysteine-rich proteins (CRPs) in RAW264.7 cells, indicating that the nanoparticles were greatly affected the expressions of CRP-coding genes (CRP-DEGs), such as encoding zinc finger protein gene. Additionally, about 26% of CRP-DEGs encoded enzymes in these four mammalian cells, indicating that the nanoparticles greatly affected the expressions of enzyme genes. The results of GO analysis demonstrated that the biological processes of various responses, immune activity and apoptosis were enriched by the CRP-DEGs induced by theDMSA-FeNPs. The molecular functions were mainly related to transition metal ion binding in various cells. According to CRP protein rich in cysteine, we speculated that these effects were mainly related to DMSA coating. To confirm the surmise, we simultaneously detected the expressions of some CRP-DEGs in RAW264.7 cells treated with the DMSA-FeNPs and a PEI-coated magnetite nanoparticle (PEI-FeNP) by using qPCR. The results revealed that the effect mainly came from DMSA which was carried into cells by nanoparticles. In summary, this study firstly reported the cellular effects of DMSA as coating molecules of FeNP, which induced the significantly different expresstion of genes encoding CRP. This study provides new insight into the molecular mechanism why the DMSA-coated nanoparticles resulted in the transcriptional changes of many CRP-coding genes in cells. |
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