Study On The Multinucleation Effect Of Amorphous Silica Nanoparticles And The Relevant Mechanisms

The widespread application of silica nanoparticles creates various sources forpotential human exposure. It is possible for silica nanoparticles to enter human bodythrough various means. Therefore, information focuses on the safety and hazards ofsilica nanoparticles are urgently needed. In our previous study, we have found the invitro and in vivo toxic effect of silica nanoparticles, and reported that silicananoparticles could induce multinucleation in HepG2cells for the first time. Whilethe formation and the ending of multinucleated cells are still unknown. Therefore, thepurpose of this study is to clearify the multinucleation effect and relative mechanismsof amorphous silica nanoparticles.1. Characterization of silica nanoparticlesTwo sizes of amorphous silica nanoparticles (Nano-Si64and Nano-Si46) wereinvestigated in our study. The TEM images showed that silica nanoparticles weremostly spherical with uniform size and well dispersed. The average sizes of these twoparticles are63.88±10.35nm and46.15±5.53nm, respectively. The result ofICP-AES manifested that the purity of both Nano-Si64and Nano-Si46is higher than99.9%. Dynamic light scattering (DLS) technique and zeta electric potentialgranulometer were employed to detect the hydrodynamic sizes and zeta potential ofthe two silica nanoparticles in different dispersion medium. Result showed that twoparticles possessed uniform hydrodynamic sizes and well dispersibility.2. Dose dependent and size related cytotoxicity of silica nanoparticlesTo investigate the potential cytotoxicity of the silica nanoparticles, cell viabilitywas detected after L-02cells were treated with two sizes of silica particles (Nano-Si64and Nano-Si46) at concentrations of1-200μg/mL for24h. Both two methods, cellcounting kit (CCK-8) and trypan blue staining, showed substantially the same trend,the cytotoxicity of the silica nanoparticles was dose dependent and size related, thecell viability of L-02cells decreased with the particle dose increased, and smaller silica nanoparticle exhibited stronger toxic effects.3. Cellular uptake and intracellular distribution of silica nanoparticlesFCM and ICP-AES were used to detect the Si content of L-02cells afterexposure to10、20、50μg/mL of two silica nanoparticles. Results indicated that the Sicontent elevated with the silica dose increased, and smaller particle entered the cellsmore easily. The cellular uptake and intracellular distribution of silica nanoparticleswere observed by transmission electron microscope (TEM). TEM images showed thatsilica nanoparticles could enter L-02cells through endocytosis or penetrating the cellmembrane directly; Particles dispersed in cytoplasm with individual or cluster form,and some were found in lysosomal and mitochondria.4. Morphological change of L-02cells induced by silica nanoparticlesMorphological change was detected after L-02cells were treated with10、20、50μg/mL of two silica nanoparticles24h. Morphological observations under phasecontrast microscope showd that, cells of negative control group were mostlypolygonal with little particles in cytoplasm. But in the silica nanoparticle treatedgroup, with the increase of silica dose, the morphological change of L-02cellsbecame more and more significant. Photograph of Giems staining showed that cells intreated group exhibited decrease of cell number decrease and disappearece of cellconnection; In50μg/mL silica treated group, chromatin margination and cell deathcould also be observed. Sccaning electron microscope (SEM) images manifested thatsilica nanoparticles induced cell membrane microvilli rupture and cell membranedamage. TEM observation indicated that both two silica nanoparticles could result inthe injury of many subcellular structures, such as Mitochondria, lysosomes andendoplasmic reticulum.5. Oxidative damage of L-02cells induced by silica nanoparticlesThe result of FCM showed that intracellular ROS of10、20、50μg/mL treatedgroups increased following the silica dose increasing after treated with silicananoparticles for24h. Colorimetry was introduced to detect the activity of SOD,GSH-PX and the content of MDA. Results indecated that with the silica doseincreased, the oxidative stress of L-02cells elevated obviously, and the smallar silicananoparticles exhibited higher oxidative damage effect. 6. Muitinucleated effect of silica nanoparticles and relative mechanismsThe rate of multinucleated cells was calculated after L-02cells were treated with10、20、50μg/mL of two silica nanoparticles24h. Results showed that themultinucleated effect of silica nanoparticles was also in a dose dependent and sizerelated way, that is the rate of multinucleated cells increased significantly as silicadose increased, and smaller silica nanoparticles induced more multinucleated cells.In the observation of cell fusion, real-time inverted phase contrast microscopeand laser scanning confocal microscope were used to detect cells with or with outfluorescently labeled. However, we haven’t found the phenomenon of cell fusion.In the observation of abnormal mitosis of L-02cells, Actin tracker green,Tubulintracker green and Hoechst33258were used to lable microfilament, microtubules andnucleus. Results of real-time inverted phase contrast microscope and laser scanningconfocal microscope showed that both two silica nanoparticles could induce abnormalchromosome alignment in mitosis metaphase, abnormal chromosome segregation inmitosis anaphase and abnormal cytokinesis in mitosis telophase.DNA damage induced by silica nanoparticles could lead to cell proliferationdisorders G2/M phase arrest, these might also result in the formation of multicleatedcells. Thus, we detected the content of relative regulation proteins in cell proliferationand cell cycle. Results of western blot showed that the content of p-MEK1/2andp-ERK1/2, proteins associated with cell proliferation, decreased with silica doseincreased; the content of Cdc25C, cyclin B1, Cdc2and Cdc20, proteins associatedwith G2/M cell cycle and mitosis, decreased with silica dose increased. Therefore,leading to cell proliferation disorders G2/M phase arrest could be one of the reasonsof the multinucleated effect of silica nanoparticles.7. Injury of liver and muitinucleated effects produced by silica nanoparticles inICR miceTo obtain the LD50of64nm SNPs in ICR mice, doses and interval weredesigned according to the Dixon up-and-down method. The LD50of64nm silicananoparticles was calculated using the formula provided by Dixon’s up-and-downmethod, and the entire estimate for the LD50=262.45±33.78mg/kg. For acutetoxicity study of silica nanoparticles,0,29.5,103.5and177.5mg/kg were chooseedas experimental dose, and14d was choosed as observation period. For sub-acute toxicity study of silica nanoparticles,20mg/kg was chooseed as experimental dose,after five consecutive injections, the cumulative dose was100mg/kg,2w,4w and8w were choosed as observation period.Images of pathological examination showed that lymphocytic infiltration andgranuloma formation appeared in the liver of ICR mice after silica naoparticles treatedacutely and subacutely. In the acute toxicity study, the numbers and sizes ofgranulomas in liver increased in a dose-dependent way. While the sub-acute toxicitystudy, the numbers and sizes of granulomas reached a peak in4w, and obviouslydecreased in8w.After ICR mice treated with silica nanoparticles acutely and subacutely, the rateof multinucleation of hepatocytes was calculated. Results showed that silicanaonoparticles did not increased the number of multinucleated cells in liver. Contrary,in the acute toxicity study, the number of multinucleated cells decreased significantlyin177.5mg/kg treated group; in the acute toxicity study, the number ofmultinucleated cells declined gradually. This phenomenon might be related to theliver injury induced by silica nanoparticles.In further study, we used imunohistochemistry to preliminary investigated themechanism of liver injury. Results showed that silica nanoparticles did not lead to theDNA damage of cells in both Liver parenchyma and granuloma. However, apoptosisof cells in granuloma was observed in the177.5mg/kg treated group of acute toxicitystudy and in2w and4w observation periods.The findings of the above in vivo experiments indicated that acutely andsubacutely exposure to silica nanoparticles through intravenous administration couldlead to lymphocytic infiltration and granuloma formation in the liver of ICR mice. Inthe acute toxicity study, the numbers and sizes of granulomas in liver increased in adose-dependent way. In the sub-acute toxicity study, the most serous granulomas wasobserved in4w, and has been restored in8w, but this not indicated the injury of liverwas recoverd.