Investigation The Mechanism Of Cytotoxicity Of Silver Nanoparticles Based On Biomices Technologies

Due to the increasing number of biomedical applications for nanoparticles, their toxicity has gained great attention and has been investigated at different levels with a variety of methods. However, little has been known about the interactions between nanoparticles and living cells until now. More importantly, it’s unable to discover the molecular mechanism of nanoparticles toxicity systematically with animal experiments, cytological experiments and traditional molecular biology methods. The rapid developments in biomics technologies (gene expression profile microarray, proteomics technology, SOLiD sequencing, etc) enable researchers for the comprehensive and systematic analysis of the molecular mechanisms of nanoparticle toxicity. Until recently, nanoparticle-induced changes in mRNA, protein or microRNA expression profiles were studied separately using a single biomics method at a time. Thus, these studies probed only one aspect of nanoparticle-induced toxicity instead of systematical toxicity mechanism.Silver nanoparticles (SNPs) present strong and broad-spectrum antimicrobial activity, and have been widely applied in medical applications. However, the toxic effects of SNPs are still largely unrevealed due to the complexity of their influence toward biological systems. Till now, few studies have investigated the influence of SNPs on mRNA and protein expression profile. While to our knowledge, SNP-induced changes in microRNA expression profile in cells have not been published. Furthermore, comprehensive studies on biomaterial-cell interactions using three biomics approaches have not been reported. The aim of this thesis is to investigate the regulatory mechanism of microRNA in SNPs-induced human dermal fibroblast (HDF) toxicity by joint analysis of gene, proteome and miRNA expression profiles.The main conteins are as follows:1. SNPs are prepared by reduction of silver nitrate with sodium borohydride. A series of SNPs solution are obtained by changing the kind of solvent and the concentration of sodium borohydride. The results from the UV-visible spectrophotometric and transmission electron microscopy analysis reveal that both the concentration of the reducing agent and the type of solvent will affect the size and shape of SNPs. SNPs with a relatively uniform shape and size are obtained when redistilled water is used as the solvent, and most of the particles had generally spherical morphology. To obtain SNPs with a consistent size (approximately 20 nm), SNPs are prepared in the redistilled solvent using sodium borohydride and silver nitrate with the concentration of 2 mM.2. MTT assay, real-time cell electronic sensing system measurement, optical microscopy, fluorescence microscopy and scanning electron microscopy are used to analyze the influence of cell proliferation and morphology SNPs to HDFs. Flow cytometer technology is also applied to exam the effects of SNPs on cell cycle and apoptosis. The uptake of SNPs by the cells is determined using an atomic absorption spectrometer. Results show that after HDFs are treated with SNPs at lower concentrations (1,10,50,100μM), all of the cytotoxicity grade is 0. However, at higher concentrations (200 and 300 μM), the cytotoxicity grade is 1 after 48 h, and the regular arrangement of HDFs are changed. Although 200μM SNPs show no cytotoxicity and do not affect the cell morphology after 1,4 and 8h, the cell cycle are arrested in DNA synthesis phase, and the influence of SNPs on cell cycle and apoptosis are significant after 8h. In addition, increasing amount of intracellular SNPs over time after the treatment of 200 μM SNPs are observed.3. Proteomics technology of 2D-DIGE and mass spectrometry are used to study the protein expression profile in HDFs after 200μM SNP-treatment for 1,4 and 8 h, and 25 unique functional proteins are identified which are differentially expressed in all 3 time points, with 19 up-regulated,4 down-regulated and 2 up and down regulated proteins. Cluster & Treeview, Gosurfer, GenMAPP, Ingenuity Pathway Analysis are applied for cluster, Gene Ontology (GO), biological pathway and protein-protein interaction network analysis. Results show that 25 differentially expressed proteins involved in a wide range of GO Functional terms,31 biological pathways and 4 protein-protein networks. SNPs may affect cell signaling transduction, cytoskeleton, cell adhesion, cell energy metabolism, mRNA processing, etc, inhibit cell proliferation and induce cell apoptosis finally.4. SOLiD sequencing technology is utilized to analyze microRNA expression profiles in HDFs after 200μM SNP-treatment, and 59,143 and 142 microRNAs are found differentially expressed at 1,4 and 8 h respectively, with 246 in total. The potential target genes of the differentially expressed microRNAs are predicted using miRanda, PicTar and TargetScan, simultaneously.1747,2928 and 2667 target genes are predicted in HDFs treated with 200μM SNPs for 1,4 and 8 h, respectively, with 3594 totally. Bioinformatics analyses reveal that the predicted microRNA target genes significantly affect 171,272 and 233 GO categories at the third level. They also involve in 120,132 and 129 biological pathways whose function relate to cell adhesion, cytoskeleton, cell apoptosis, cell cycle, inflammatory reaction, etc.5. By comparing differentially expressed proteins and the previously obtained gene expression data, it can be noted that there are significant difference on type, amount and expression pattern. Further analyse show that differentially expressed genes, microRNAs and proteins affect different biological process catalogies and biological pathways. Then, differentially expressed mRNAs and proteins are matched with predicted microRNA targets. 36,429 and 116 biologically significant microRNA-mRNA target pairs,2,1 and 3 microRNA-protein target pairs are identified in HDFs treated with SNPs for 1,4 and 8 h, respectively. Overall,28,186 and 82 target mRNAs/proteins are identified respectively, with a total of 257 different targets.6 differentially expressed microRNAs are found regulate 3 target mRNA-protein pairs by inducing mRNA degradation and repressing protein translation, thus leading to differences in expression patterns. Bioinformatics analysis reveal that the main function of 257 microRNA targets relate to cell communication and metabolism process, and involve in 57 biological pathways. Four key pathways are affected by differentially expressed microRNA, target mRNA-protein pairs simultaneously, namely’Regulation of actin cytoskeleton’,’Signaling of hepatocyte growth factor receptor’,’Insulin signaling’ and ’MAPK signaling pathway’. SNP-induced differentially expressed microRNAs are found regulating the expression of target genes and proteins in these four pathways, leading to HDF toxicity through the destruction of cytoskeleton, reduction of intracellular ATP content and induction of apoptosis.6.4 selected proteins and 8 microRNAs are validated with Western blot and qRT-PCR, to confirm the reliability of proteomics analysis and microRNA sequencing results. Actin cytoskeleton kit, ATP analysis kit and Hoechst staining method are further applied to analyze the role of SNPs on cytoskeleton, ATP synthesis and apoptosis after 1,4,8,24,48 and 72h treatment. The bioinformatics results are verified, SNPs lead to HDF toxicity through the destruction of cytoskeleton, reduction of intracellular ATP content and induction of apoptosis.7. With SOLiD sequencing technology,109,78 and 124 microRNAs are found differentially expressed in HDFs after treated with 200μM gold nanoparticles (GNPs) for 1,4 and 8 h respectively.2403,2044 and 3002 target genes are predicted using miRanda, PicTar and TargetScan, simultaneously, which significantly affect 208,193 and 249 GO categories at the third level and involve in 126,125 and 129 biological pathways. By matching differentially expressed microRNA and the previously obtained gene and protein expression data,132,38 and 137 biologically significant microRNA-mRNA/protein target pairs are identified in HDFs treated with GNPs for 1,4 and 8 h, respectively. Overall,187 target mRNAs/proteins are identified, whose function mainly relate to cell metabolism process, and involve in 71 biological pathways. Two key pathways are affected by differentially expressed microRNA, target mRNA-protein pairs simultaneously, namely ’mRNA processing’ and ’MAPK signaling pathway’. GNP-induced differentially expressed microRNAs are found regulating the expression of target genes and proteins in these two pathways, affecting cell cylce while inhibit cell apoptosis.8. The influence of SNPs and GNPs on HDFs are compared at the cell, protein and microRNA level. Resluts show that, at the cell level, there are great differences between the effects of these two nanoparticles on cell proliferation, cell cycle, cytoskeleton, oxidative stress, energy metabolism, apoptosis, etc. At the protein level, only 5 proteins are found diffecentially expressed in SNPs and GNPs treated HDFs. GO categories in biological process, molecular function, cellular components that contain most diffecentially expressed proteins in SNPs or GNPs treated cell are basically the same. However, SNPs are observed to affect more pathways (31 pathways) than GNPs (24 pathways). At the microRNA level, the kind of differentially expressed microRNA, the function of microRNA targets, the involved biological pathways and the influence on pathways are also different. Compared with SNPs, GNPs impacted the cell cycle, weakened the inhibition of ATP synthesis and damage to the cytoskeleton, suppressed apoptosis, and lastly, did not lead to cytotoxicity. The difference in ROS production by these two nanoparticles might in part explain the different effects on HDFs regarding cytotoxicity:GNPs showed no cytotoxic effects, while SNPs induced cytotoxicity.