Magnetic iron oxide nanoparticles (FeNPs) have great potential applications in biomedical field because of good biocompatibility. At present, some iron oxide nanoparticles have been used as magnetic targeting drug carriers, iron supplements, magnetic resonance imaging (MRI) contrast enhancers, cell markers and so on. Therefore, it is significant to study their nanotoxicity. The potential nanotoxicity of FeNPs has been gradually recognized and studied mainly at the cellular level including internalization and the ways into cell, the effects on cell ultrastructure, growth, and oxidative stress. With the development and maturation of GeneChips technology, gene expression of the genome can be detected completly. Cells can promptly regulate its gene expression profile in response to any changes triggered by nanoparticles. Selecting out all different expression genes induced by a nanomaterial at the cell or tissue levels can provide valuable clues to explore any potential toxicity and investigate the relevant molecular mechanism. Therefore, to evaluate changes of gene expression profiles used high-throughput techniques is much helpful to uncover the potential nanotoxicity of nanomaterials. The current studies show that we should systematically research the effects of size, charge, morphology and surface modification on the nanoparticles, explore their potential molecular mechanisms, and develop and apply them.
The nanomaterials are most concentrated in the blood and hepatocytes of the body due to passive targeting after intravenous administration. In this study, two blood cells and two hepatocellular carcinoma cells derived from mouse and the corresponding human were used as the objects of study, respectively, including mouse macrophage RAW264.7, mouse hepatoma Hepa1-6, human acute monocyte THP-1 and human hepatoma HepG2. Four kinds of cells were treated with different doses of dimercaptosuccinic acid (DMSA) modified Fe3O4 nanoparticles (DMSA-FeNPs). Their gene expression profiles were detected by GeneChips. The effect of DMSA-FeNPs on the gene expression profile, the potential molecular mechanisms and possible applications was explored by data in-depth analysis combined with experimental verification. The main works of this study were as follows:
1. The characteristics of DMSA-FeNPs, the cell internalization and viability were detected by transmission electron microscopy (TEM), Malvern particle size analyzer (DLS), infrared spectroscopy, Prussian blue staining, and CCK-8 assay. DMSA-FeNPs had good dispersibility and stability in solution with an average particle size of about 11 nm and the hydrodynamic size of 64 nm observed by TEM and DLS, respectively. Prussian blue staining showed that DMSA-FeNPs were uptaked in four mammalian cell lines. The ability of phagocytosis was the highest for macrophage RAW264.7 cells, while lowest for THP-1 cells. DMSA-FeNPs were transported and located in the cytoplasm with vesicles coating through TEM. DMSA-FeNPs caused the viability of some cells decline when treated with 100 μg/mL, while no obvious effects at dose of 50 μg/mL. Furthermore, the proliferation of RAW264.7 cells with 50 μg/mL treatment was significantly increased. Macrophages are a class of important immune cells that fight against pathogen invasion and activate adaptive immunity, playing a very important role in the development and progression of tumors. This finding provides a basis for studying the immunotherapy of DMSA-FeNPs by activation of macromonocytes.
2. A viable strategy to assess the toxicity of nanomaterials is to evaluate the nanotoxicity with human cells and their mouse equivalents. The toxic effects of DMSA-FeNPs on two mouse cell lines have been detected earlier in our laboratory. The toxicity of DMSA-FeNPs to two equivalent human cell lines (THP-1 cells and HepG2 cells) was appraised at the same two doses (50 and 100 μg/mL) for the same time (24 h). The global profile of each treatment was abtained through GeneChips. Analysis revealed that there were hundreds of DMSA-FeNPs response genes (FeRGs) in the two cell lines, most of which were different, indicating cell type-specific effects. By comparing these FeRGs, annotation functions, and related KEGG pathways, DMSA-FeNPs were found to cause general and cell-specific effects. DMSA-FeNPs induced various responses and inhibited protein translation in THP-1 cells, whereas promoted cell metabolism, growth and migration in HepG2 cells. These effects all showed a dose-dependent, which means that the high dose of DMSA-FeNPs had a greater effect on the cells. The common genes, biological processes and KEGG pathways induced by DMSA-FeNPs were still compared in four cell lines. It is found that Id3 gene was commonly down-regulated in four cell lines.The Id3 gene is a redox-sensitive signal transduction molecule, the protein which encoding can form non-functional dimers with other transcription factors to inhibit transcription. Its significant down-regulation indicated that DMSA-FeNPs might disrupt cellular normal biological processes, such as cell growth, differentiation, apoptosis and tumorigenesis. Therefore, Id3 gene can be used as a universal and sensitive biomarker of DMSA-FeNPs cytotoxicity, which provides new insights into the explotation and identification of nano-toxicity of DMSA-FeNPs.
3. Previous study found that the biological processes of response to the virus and the pathway of hepatitis C virus were significantly enriched in THP-1 cells treated with DMSA-FeNPs. The particles had similar particle structure with virus, suggesting that DMSA-FeNPs were recognized like virus, resulting in virus-like cellular immune effects. The effects of DMSA-FeNPs on the immune system of two immune cells (RAW264.7 and THP-1 cells) were investigated through data mining in depth, which is the most important host defense system for cell antivirus. The results show that DMSA-FeNPs triggered the immune response like viral in the two kinds of cells, mainly including 8 innate immunity pathways and 3 adaptive immunity pathways, and producing a large number of different cytokines. Thereamong, almost half of the DEGs were found to be interferon-stimulating genes (ISGs), which are closely related to anti-virus. For example, proteins encoded by Osa1/2/3/L, Mx1/2 are the typical antiviral factors. Many cytokines were induced production. Both the immune response and cytokine products showed dose-dependent and cell-type-dependent effects. The virus-like immunoactivation effect may result from the size of the DMSA-FeNPs, DMSA coating, negative charge, or monodisperse spherical particles similar to that of the virus. Experimental study found that DMSA-FeNPs could activate RAW264.7 cells. DMSA-FeNPs significantly promoted cell viability, mobility and the attack power. RAW264.7 cells surrounded and effectively killed Hepa1-6 cells in vitro. Therefore, this study first reported the systematic immune response of DMSA-FeNPs at the level of gene transcription and the potential application of DMSA-FeNPs itself in tumor immunotherapy, which sheds new insights into its cellular biological effects and potential molecular mechanisms and promotes the design and development of new nanomaterials with good immune properties or useful nanoimmunoassay. Furthermore, the study is developing application of DMSA-FeNPs itself as immunotherapy drug in clinic.
4. DMSA has been widely used in the modification of FeNPs, which can significantly improve the stability, biocompatibility, intracellularization and biological distribution of FeNPs. Nevertheless, a large number of reductive thiol groups were carried into cells once DMSA modified on the surface of FeNPs. The potential cytotoxic remains to be fully elucidated. The study searched the NCBI database with eight keywords containing large amounts of disulfide bonds to find all genes that encoding cysteine-rich protein (CRP) as much as possible. At three time points (4, 24 and 48 h) treated with three doses (30, 50 and 100 μg/mL) of DMSA-FeNPs in RAW264.7 cells, DEGs encoding cysteine-rich proteins (CRP-DEGs) were screened through comparision DEGs with genes coding CRP. The results demonstrate that about quarter of DEGs encoded CRP in each treatment, indicating that DMSA-FeNPs significantly affected the expression of CRP gene (such as gene encoding zinc finger protein). Furthermore, about 31% of all CRP-DEGs encoded enzymes, suggesting that the DMSA-FeNPs significantly affected the expression of the enzyme genes. GO analysis showed that DMSA-FeNPs induced various biological processes, such as response, immunological activity and apoptosis, while the molecular function was mainly related to iron ion binding. The similar effects were found in the three other cell lines, Hepa1-6, THP-1 and HepG2. To confirm the different expression of CRP genes resulted from the modified molecule DMSA, the study simultaneously detected the expression of some typical CRP-DEGs in the cells treated by DMSA-FeNPs, Fe3O4 nanoparticles coating with polyethyleneimine (PEI), and pure DMSA through quantitative polymerase chain reaction (qPCR). The results display that the effect of DMSA-FeNPs on CRP gene expression was mainly caused by DMSA into the cells. This study first reported that the cytotoxic effect of DMSA as surface modifier of DMSA-FeNPs at the gene transcription level, shedding a new molecular mechanism for the significant effects of DMSA-FeNPs on the differential expression of CRP gene. The study also provides a new insight for the biocompatibility evaluation of surface modification molecules of nanomaterials.