Study On Cytotoxicity And Toxic Mechanisms Of Silica Nanoparticles In HepG2 Cells

The research on biological effects and toxicity of nanomaterials is very important to the development of nanotechnology and widely application of nanomaterials. Silica nanoparticles can be used in many fields, especially in biological and medicine fields. At present, some toxicity and related mechanisms of silica nanoparticles have been reported, but it also remains thorough research. Particularly the influence of silica particles for apoptosis and related mechanisms is not system. It is reported that liver is one of the target organ for toxic effects of silica nanoparticles that they can easily accumulate in it. In present study, HepG2 cells was selected as research model, the cytotoxicity and apoptotic mechanisms of silica nanoparticles were discussed. It can provide experimental basis for understanding of the silica nanoparticles toxicity and related mechanisms.1. Characterization of silica nanoparticlesThe TEM images showed that silica nanoparticles were mostly spherical with uniform size and well dispersed. The average size of particle is (43±3.5) nm.The size, size distribution and dispersion of particles after disperse in serum-free DMEM and DMEM with 1%,5% and 10% FBS for 24 h were detected by DLS method. The results showed that the hydrodynamic sizes were (127.76±2.49) nm, (507.10±14.24) nm, (371.44±12.97) nm and (287.42±3.75) nm respectively without ultrasonicification. In addition, the hydrodynamic sizes of particles were (127.76±2.49) nm, (186.32±7.89) nm, (185.34±10.46) nm and (187.68±10.75) nm after ultrasonicification for 5 min, respectively.2. Cellular uptake and intracellular distribution of silica particlesThe cellular uptake was observed by fluorescence microscope showed that red pointlike fluorescence nanoparticles could be found in HepG2 cells after silica nanoparticles exposure for 3 h. TEM images showed that silica nanoparticles could adhere to the cell surface and distribute in cytoplasm, moreover, the particles were found to deposit in some organelles, such as mitochondria, endoplasmic reticulums and lysosomes.3. Cellular uptake mode of silica nanoparticles The images of TEM showed that the silica nanoparticles dispersed in cytoplasm with cluster or individual form. Some particles were found in endocytic vesicles and lysosomes, others dispersed scattering without membrane binding. In addition, the silica nanoparticles were found to entering the cells through penetrating the cell membrane directly.4. Effect of silica nanoparticles on cell viabilityThe effect of silica nanoparticles on cell viability was determined by MTT method. The cell viability of HepG2 cells decreased following the particles dose increasing after silica nanparticles exposure for 3 h and 24 h. After treated with different dose of particles for 24 h, the cell viability decreased significantly compared to that of negative control group (P<0.05).5. Effect of silica nanoparticles on cell morphologyThe images of phase contrast microscope showed that cell number reduced gradually following the particles dose increasing after treated with silica nanoparticles to HepG2 cells for 24 h. Cell morphology changed significantly, presented irregular shape and cellular connection disappear in 200 mg/L particle group. TEM images showed that after treated with silica nanoparticles for 24 h, the cell surface was smooth, no microvili on the membrane. Cytoplasmic cavitation and nucleus edge set were observed. Mitochondrial swelling, cristae rupture or disappear and the number of mitochondria reduced. Endoplasmic reticulum expansion was found after silica nanoparticles exposure.6. Effect of silica nanoparticles on intracellular ROSThe result of FCM showed that intracellular ROS of 25,50 and 100 mg/L groups increased following the particles dose increasing after treated with silica nanoparticles for 3 h and 24 h. However, the intracellular ROS of 200 mg/L group are declined compare to 100 mg/L group in both time points. After 3 h treatment, intracellular ROS of each particle group increased significantly compared to that of negative group (P<0.05). After 24 h treatment, intracellular ROS of 50 and 100 mg/L particle groups increased significantly compared to that of negative group (P< 0.05).7. Effect of silica nanoparticles on intracellular Ca2+The result of FCM showed that intracellular Ca2+ increased following the particles dose increasing after treated with silica nanoparticles for 3 h and 24 h. intracellular Ca2+ of 50,100 and 200 mg/L particle groups increased significantly compared to that of negative group (P<0.05).8. Effect of silica nanoparticles on mitochondrial membrane potentialThe images of laser scanning confocal microscope and the result of FCM showed that MMP decreased following the particles dose increasing after treated with silica nanoparticles for 3 h and 24 h. MMP of 50,100 and 200 mg/L particle groups increased significantly compared to that of negative group (P<0.05).9. Effect of silica nanoparticles on apoptosisThe result of FCM showed that early apoptotic rate, late apoptotic and necrotic rate and total apoptotic rate increased following the particles dose increasing after treated with silica nanoparticles for 3 h and 24 h. Moreover, this effect had dose-dependent manner. After 3 h treatment, the apoptotic rate of each particle group increased significantly compared to that of negative group except late apoptotic and necrotic rate of 25 mg/L particle group (P<0.05). After 24 h treatment, the apoptotic rate of 50,100 and 200 mg/L particle groups increased significantly compared to that of negative group (P<0.05). Early apoptotic rate, late apoptotic and necrotic rate and total apoptotic rate increased following the particles dose increasing after treated with silica nanoparticles for 3 h and 24 h with adding to Z-VAD-FMK.10. Effect of silica nanoparticles on apoptotic protein expressionThe results of western blot showed that after treated with silica nanoparticles for 24 h, the Bcl-2 expression decreased significantly in each particle group compared to that of negative group (P<0.05). The Bax expression did not change obviously, but the ratio of Bax/Bcl-2 increased significantly. The CytC expression increased significantly in 100 and 200 mg/L group compared to that of negative group (P< 0.05). In each particle group, the GRP78, CHOP and AIF expression increased significantly compared to that of negative group (P<0.05). Moreover, The JNK and Caspase-3 expression increased significantly in 50,100 and 200 mg/L group compared to that of negative group (P<0.05).In conclusion, silica nanoparticles dispersed in serum-free DMEM well and the serum in DMEM could influence the size, size distribution and dispersion of particles. In present study, after silica nanoparticles exposure, the particles entered into the cells with different uptake modes. Then the particles could deposit in cytoplasm and some organelles. Moreover, cytotoxicity could be induced by the intracellular particles. The increase of intracellular ROS and Ca2+ can be seen as the major mechanism of cytotoxicity. Mitochondria were also found as one of the target organelles for the toxic effects of silica nanoparticles. These mechanisms together induced cells to apoptosis or necrosis and reduced the cell viability. Mitochondrial mediated pathway, endoplasmic reticulum mediated pathway and AIF related caspase-independent pathway participated in the regulation of cell apoptosis. In addition, the increase of intracellular ROS and Ca2+ were also related to the apoptosis. These various factors regulated the apoptotic process in a "corsstalk" manner.