| Rapid development of nanotechnology leads to vast application of engineered nanomaterials in multiple academic and social fields. The direct impact of this new special material to health and environment is of great concern to the population. Engineered nanoparticles may enter the lung when one breathes, penetrate into circulation system through alveoli and accumulate around vital internal organs, such as heart, liver and kidney, causing severe damages to these organs. In this study, human liver cell line HL-7702 cells were used to study liver toxicity of amorphous silica nanoparticles; Rat type I-like alveolar epithelial cell line R3/1 were used to study lung toxicity of nickel particles. Finally, the interrelationship of particle physicochemicalcharacteristics and toxicological effects of nanoparticles are discussed based on particle size, surface area, surface activities and other properties.1. Cytotoxicity of Nano-Si53 nanoparticles on HL-7702 cellsTransmission electron micrographs show that Nano-Si53 particles are uniform in size, evenly distributed, with good monodispersity and spherical shape. The radius of particles is (53±5.1)nm.Morphological observations under phase contrast microscope show that, after treatment, HL-7702 cells exhibit morphological alterations: cell number reduced, cell turning round and flat, cell connection disappeared compared to polygonal shapes of untreated cells.The effect of SiO2 nanoparticles on cell viability was determined by Cell Counting Kit (CCK-8). After 24 h treatment, Nano-Si53 particles induced an increase in cytotoxicity that was significant (P < 0.05, P < 0.001) at 50,100,150,200μg/mL groups compared to the negative control group. During 48 h of normal culture without nanoparticles, cells grew rapidly and their viability was restored to at least 78.68% after 24 h, while after 48 h, cell viability was almost restored to at least 91.05%.To find out the reason of cell viability reduction, HL-7702 cells were stained with fluorescent dye Hoechst 33258 to exam the nuclei changes after treatment of 200μg/mL Nano-Si53 particles. It showed apoptotic features, including nuclear compaction and condensation of chromatin toward the nuclear periphery, and fragmentation of DNA. HL-7702 cells were also stained with Annexin V/PI and subsequently analyzed by flow cytometry to confirm the apoptosis. After treatment of 50,100,150,200μg/mL Nano-Si53 particles for 24 h, moderate cell apoptosis were observed in all groups. An increasing in apoptotic rate was found when compared to untreated cells (P < 0.01, P < 0.001).After stained with Annexin V/PI, laser confocal microscope was also applied to observe HL-7702 cell apoptosis. A similar increase in apoptotic rate was confirmed. But more interestingly, some apoptotic cells, the Annexin V -posotive/PI-negative or Annexin V/PI double positive cells were also binuclear cells, indicating these cells were undergoing proliferation and apoptosis at the same time. In 200μg/mL Nano-Si53 particle treatment group, the binuclear apoptotic rate was increased to 42.85%, suggesting that almost half of apoptotic cells were binuclear cells.To further investigate the formation of binuclear cells, HL-7702 cells were stained with PI to analyze cell cycle by flow cytometry. It showed that cells treated with different doses of Nano-Si53 particles for 24 h predominantly accumulated in the G2/M phase of the cell cycle and cells in G0/G1 phase dramatically decreased with no change of cell numbers in S phase, suggesting that cell cycle arrests in G2/M phase.The effects of Nano-Si53 nanoparticles on the adjusting intracellular Ca2+ were determined by flow cytometry after Fluo-3-AM fluorescent dying. After exposure to different doses of particles, the intensity of Fluo-3 fluorescence increased slowly till 27.62 at 200μg/mL groups, indicating a significant influx of Ca2+ at high dose compared to that of the control (P < 0.01).The mitochondria numbers of HL-7702 cells was measured by flow cytometry after staining with mitochondrial specific fluorescent dye Mito-tracker green. After exposure to different doses of particles, the fluorescence intensity of Mito-tracker green significantly decreased compared to that of the control (P < 0.001), indicating the loss of normal mitochondria.The effects of Nano-Si53 nanoparticles on the generation of intracellular ROS were determined by flow cytometry and laser confocal microscope. After treating HL-7702 cells with 50,100,150,200μg/mL Nano-Si53 particles for 6 h and 24 h showed that the intensity of DCF fluorescence increased dramatically at both time points (P < 0.05, P < 0.001), indicating an over-production of ROS in HL-7702 cells. The effects of Nano-Si53 nanoparticles on the generation of intracellular oxyradicals were determined by flow cytometry. After treating HL-7702 cells with 50,100,150,200μg/mL Nano-Si53 particles for 24 h, it showed that the intensity of DHE fluorescence increased slowly till 53.97 at 200μg/mL group (P < 0.01), indicating an over-production of oxyradicals in HL-7702 cells.In summary, we have demonstrated that silica nanoparticles significantly reduce HL-7702 cell viability; induce cell apoptosis; arrest cell cycle at G2/M phase at 50200μg/mL dosage. The cytotoxicity mechanism involved may be related to mitochondrial injury and cellular oxidative stress.2. Cytotoxicity of Nano-Ni60 on R3/1 cellsMembrane permeability of R3/1 cells was detected by measuring the lactate dehydrogenase (LDH) releasing into cell culture medium after exposure to 0.47, 1.20, 2.40, 4.70, 9.50 and 19.00μg/cm2 of Nano-Ni60 particles for 6 h, 24 h and 72 h. 9.50 and 19.00μg/cm2 of Nano-Ni60 particles could increase the LDH release into culture medium at 3 h and 6 h. At 24 h and 48 h, nanoparticles could significantly increase LDH release in a dose- and time-dependent manner in comparison with untreated group (P < 0.01, P < 0.001).To determine if oxidative stress is involved in cytotoxicity of Nano-Ni60 particles, protein carbonylation, one of the protein oxidative damage, was measured using ELISA method. After treatment R3/1 cells with 0.47, 1.20, 2.40, 4.70, 9.50 and 19.00μg/cm2 of Nano-Ni60 particles for 6 h and 24 h, cell protein carbonylation gradually elevated, which was significant at 19.00μg/cm2 group at both 6 h and 24 h compared to that of untreated group (P < 0.001).To determine the anti-oxidative defense of R3/1 cells, HO-1 expression was measured by Western blotting. After treatment R3/1 cells with 0.47, 1.20, 2.40, 4.70, 9.50 and 19.00μg/cm2 of Nano-Ni60 particles for 6 h, nickel nanoparticles induced over expression of HO-1. 4.70, 9.50 and 19.00μg/cm2 particles treatment could significantly increased HO-1 express by factors of 1.9, 2.0 and 2.4, respectively (P < 0.05, P < 0.001), suggesting the anti-oxidative system was triggered in R3/1 cells.In conclusion, we have demonstrated that nickel nanoparticles significantly enhance R3/1 cell membrane permeability by a dose- and time-dependent manner at 0.4719.00μg/cm2 dosage. The cytotoxicity mechanism involved may be also related to cellular oxidative stress.3. Interrelationship of particle physicochemicalcharacteristics and toxicological effects of nanoparticlesTransmission electron micrographs (TEM) showed that Nano-43 and Nano-Si68 particles are uniform in size, evenly distributed, with good monodispersity and spherical in shape. The radius of particles are (43±4.2) and (68±5.7) nm. Nano-Ni60 particles showed most severe aggregation state under TEM.Nano-Si43 and Nano-Si68 particles had smallest size in pure water, which are 2.3 and 1.4 times bigger than their TEM sizes. In RPMI 1640 cell culture, medium particle sizes of the two particles increased by factors of 11 and 9 compared to the TEM size, suggesting aggregation state in solution. In RPMI 1640 cell culture medium containing 1% FBS, particles sizes reduced to one-fifith and one-fourth of their TEM sizes, respectively, suggesting the modify of particle aggregation caused by serum proteins.Nano-Si43 and Nano-Si68 particles did not increase LDH leaking into culture medium or induce protein carbonylation in 010.5μg/cm2 dosage within 48 h. These particles have no cytotoxicity or oxidative injury to R3/1 cells. When dose increased to 15.8μg/cm2 and 31.6μg/cm2, Nano-Si43 could cause LDH release and protein carbonylation while Nano-Si68 didn't have the same effect untill dose increased to 7.4μg/cm2, indicating smaller particle has more severe toxic effect.The sizes of Ni100300, Ni200 and Nano-Ni60 particles in pure water are 483 nm, 452 nm and 198nm, respectively. They did not change much when particles were dispersed in RPMI 1640 and DMEM cell culture medium containing 1% FBS The modified effect of serum proteins and the type of cell culture medium are not of importance to change aggregation state of these metal nickel particles.Compared to Ni200 particles, Nano-Ni60 nanoparticles increased LDH leaking in R3/1 cells at as early as 3 h and 6 h in the same dosage, and Nano-Ni60 particles could induce protein oxidation at lower dose, the results perfectly match the non cellular ROS activities of the two nickel particles. But at 48 h, Ni200 could increase LDH leaking at dose of 0.47μg/cm2, suggesting other factors may be involved.Compared to Nano-Si68 particles, Nano-Ni60 particle have the same size range, but a much high non-celluar ROS activity, which is 38 times bigger than Nano-Si68 particles. This could be one of the reasons that Nano-Ni60 particles are more toxic on R3/1 cells; the other possible factor is the releasing of metal nickel into cell culture medium.For Rat Alveolar Cells TypeⅡRLE-6TN cells, Ni200 and Nano-Ni60, instead of Ni100300 particles, could causing LDH leaking only at very high dose None of three particles have oxidative injure to cell proteins at any dose and any duration , suggesting nanoparticles may have different effects to different cells.In conclusion, it is a very complicate process when the nanaparticles take effect on cell cytotoxicity. The particle size, surface acotvity and chemical components all contribute to this difference. For amorphous silica nanopaticles, modification of serum proteins and small size of particles may be very important. For metal nickel particles, surface coating and surface activity are key factors. For different metal and nonmetal nanoparticles, chemical composition and transition metal cause different cytotoxicity effects. For different cell types, nanoparticles may have different effects on different cells.
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