Size-dependent Cytotoxicity Of Silica Nanoparticles

In this study, we use human hepatoma cell line HepG2 cells study the size- dependent cytotoxicity of amorphous silica nanoparticles. Morphological change of HepG2 cells was observed under light microscopy after Haematoxylin and Eosin (HE) staining. Survival rate of HepG2 cells was measured by Cell Counting Kit(CCK-8) and MTT colorimetry, change of cytomembrane integrity was reflected by LDH kit, alteration of intracellular ROS was determined by fluorescence microscope and flow cytometry after DCF staining, DNA damage was detected by SCGE assay, cell cycle arrest was observed by flow cytometry after PI staining, and then cell apoptosis was examined by AO/EB double fluorescent dye staining and AnnexiⅤn -FITC/PI dying methods. The results of this study were shown as followed:1. Dose-dependent cytotoxicity of silica particles on HepG2 cells The effect of silica particles on cell viability was detected by Cell Counting Kit (CCK-8). After 24 h treatment, Si498 particle displayed a significant inhibitory effect at the concentration of 200μg/mL. Nano-Si68 and Nano-Si43 administration groups were observed reduction of cell viability at the concentration of 100μg/mL compared to the negative control group(P<0.05). And Nano-Si19 particle induced significantly increased in cytotoxicity at 12.5,25,50,100,200μg/mL groups compared to the negative control group(P<0.05).2. Morphological change of HepG2 cells induced by silica particlesMorphological observations under phase contrast microscope show that, cells of negative control group were mostly polygonal with little particles in cytoplasm. But in the administration group, with the decrease of particle size, the morphological change of HepG2 cells became more and more significant. Cell shape changed from polygonal to fusiform or irregular shape, more particulate matter were observed resulting in cell transparency decline, and cell number decreased significantly.Photograph of HE staining showed that, after incubated with 100μg/mL of silica particles, HepG2 cells exhibited morphological changes: cell number decrease, cell turning round and presenting vacuolar degeneration, cell connection disappeared. And trachychromatic chromatin and chromatin margination could also be observed. Compared with the negative control group and Si498 administration group, the rate of binucleated and multinucleated cells increased significantly after treated with three silica nanoparticles.3. Size-dependent cytotoxicity of silica particles on HepG2 cellsCell Counting Kit and MTT colorimetry were performed to examine the viability of HepG2 cells treated with 100μg/mL silica particles. Our results revealed that cell survival rate declined significantly(P<0.05)with the decrease of nanoparticle size, however, in the Si498 administration group there was no obvious change of the cell survival rate compared with counterpart in negative control group.4. Cytomembrane integrity change of HepG2 cells induced by silica particlesLDH kit was used to detect the cytomembrane integrity change of HepG2 cells after exposure to 100μg/mL silica particles for 24 h. The results suggested that four types of silica particles could damage the membrane of HepG2 cells. LDH activity in cell culture medium apparently increased with the decrease of particle size(P<0.05) compared with that in the negative control group.5. Intracellular ROS alteration of HepG2 cells induced by silica particlesThe effects of silica particles on the generation of intracellular oxyradicals were reflected by flow cytometry. HepG2 cells were treated with 100μg/mL silica particles with different size for 24h, it was showed that in the Si498 administration group there was no obvious change of the intracellular ROS compared with the negative control group.But in the nanoparticle administration group, intensity of DHE fluorescence increased with the decrease of particle size(P<0.05).6. DNA damage of HepG2 cells induced by silica particlesDNA damage was measured by the single cell gel electrophoresis (SCGE). Our results showed that after treating HepG2 cells with 100μg/mL silica particles with different size for 24 h, with the decrease of particle size the rate of DNA damage was significantly increased(P<0.05), and the length of comet tail was elevated. It indicated that the rate of DNA damage induced by silica particles was in a size-dependent manner.7. Cell cycle arrest of HepG2 cells induced by silica particlesHepG2 cells were stained with PI and then analyzed the cell cycle by flow cytometry. We analyzed the cell cycle phases by collecting more than 10 000 cells and classified them into G0/G1, S or G2/M phases. The result showed that cells treated with 100μg/mL silica particles for 24h, all four administration group appeared S phase arrest(P<0.05)compared with the negative control group, and what was more, Nano-Si19 administration group also appeared G2/M phase arrest(P<0.05).8. Apoptosis of HepG2 cells induced by silica particlesAO/EB double fluorescent dye staining and FCM with AnnexⅤin -FITC/PI dying methods were performed to examine apoptosis. After treating HepG2 cells with 100μg/mL silica particles with different size for 24 h, The results suggest that all the four silica particles could induce apoptosis in HepG2 cellscompared with the negative control group(P<0.05), and cell apoptotic rate increased obviously as the particle size decreased.In conclusion, silica particles could induce morphological change of HepG2 cells, inhibit cell viability, damage the integrity of cytomembrane, elevate intracellular ROS level, induce DNA damage, cause cell cycle arrest and result in cell apoptosis.