Biological Effects Of Four Kinds Of Nanoparticles On Human Gastric Carcinoma Cell Line BGC-823

Objective:To prepare the activated carbon nanoparticles(ACNP) of specific particle size and investigate the effects of ACNP on the human gastric carcinoma cell line BGC-823 in vitro.The silicon dioxide nanoparticles(nano-SiO₂),titanium dioxide nanoparticles(nano-TiO₂) and zinc oxide nanoparticles(nano-ZnO) were also used to investigate the effects of nanoparticles with different chemical composition on the human gastric carcinoma cell line BGC-823 in vitro.Methods:ACNP were prepared by ball-mill grinding and floating-filtrating method. The particle size and morphology of ACNP were studied by atomic force microscopy (AFM) and transmission electron microscopy(TEM).The particle sizes and morphology of nano-SiO₂,nano-TiO₂ and nano-ZnO were studied by TEM.BGC-823 cells were treated with ACNP,nano-SiO₂,nano-TiO₂ and nano-ZnO at various concentrations for different durations.The inhibitory effects of the nanoparticles on the proliferation of BGC-823 cells were observed by MTT assay.The lactate dehydrogenase(LDH) leakages in the culture medium were determined with LDH activity detection kit.LDH leakage reflects the integrality of cell membrane.The alterations in morphology were observed by Methyl Green-Pyronin staining and TEM. The apoptotic rate and the cell cycle of BGC-823 cells were examined by flow cytometry(FCM).After being treated with ACNP,nano-SiO₂,nano-TiO₂ for 24 hours, the mitochondrial membrane potential(MMP) was labeled by rhodamine123(Rh123) and examined by FCM.Dihydrorhodamine123(DHR123) was used as a reactive oxygen species(ROS) capture.The mean fluorescent intensity(MFI) of Rh123 which was the product of intracellular oxidation was examined by FCM,and the level of ROS was thus measured.Results:ACNP,nano-SiO₂,and nano-ZnO particles were spherical.The average diameters of ACNP,nano-SiO₂,and nano-ZnO were(34±6)nm,(30±5)nm and(50±10) nm,respectively.Nano-TiO₂ particles were nearly spherical.The average diameter of nano-TiO₂ was(20±5) nm.ACNP,nano-SiO₂,nano-TiO₂ and nano-ZnO significantly inhibited the proliferation of BGC-823 cells in dose-and time-dependent manners.The 50%inhibitory concentrations(IC₅₀) of ACNP after 48 and 72 hours were 1.18 and 0.87 mg/ml,respectively,while those of nano-SiO₂ after 24,48 and 72 hours were 1.26,0.95 and 0.68 mg/ml,those of nano-ZnO after 24,48 and 72 hours were 39.52,30.71 and 20.61μg/mL,respectively.The IC₅₀ of nano-TiO₂ after 72 hours was 0.88mg/ml.The comparison of the cytotoxicities of four kinds of nanoparticles was nano-ZnO＞nano-SiO₂＞ACNP＞nano-TiO₂.At 24 hours after treated with 0.1mg/ml and 0.2mg/ml ACNP,the LDH leakages were not higher than that of the control group. However,the LDH leakages at concentrations of 0.4 mg/ml and 0.8 mg/ml were higher than that of the control group(P＜0.05).At 48 hours after treated with ACNP,the LDH leakages of four ACNP groups were significantly higher than that of the control group. The LDH leakages of nano-SiO₂(0.1mg/ml to 0.8mg/ml) groups significantly increased in a dose-dependent manner.At 24 hours after treated with 0.1 mg/ml nano-TiO₂,the LDH leakage was not higher than that of the control group.As the dose of nano-TiO₂ increased and the time prolonged,the LDH leakages of nano-TiO₂ groups increased.The LDH leakages of nano-ZnO(12.5μg/ml to 100.0μg/ml) groups significantly increased in a dose-dependent manner.The cells treated with 0.1mg/ml ACNP for 24 hours exhibited morphological characteristics of apoptosis including condensation of the cytoplasm,condensation and fragmentation of the nuclear chromatin.The cells treated with 0.2 mg/ml nano-SiO₂,0.2mg/ml nano-TiO₂ or 12.5μg/ml nano-ZnO for 24 hours exhibited morphological characteristics of necrosis including disintegration of the plasma membranes,nuclear condensation and nuclear lysis.Mitochondrial damage was observed in the cells treated with ACNP,nano-SiO₂, nano-TiO₂ or nano-ZnO.These nanoparticles could enter the cells.At 24 hours after treated with 0.1 mg/ml and 0.2 mg/ml ACNP,the apoptotic rates were(5.01±1.16)% and(8.21±1.63)%,respectively,both of which were significantly higher than that of the control group(2.48±0.58)%.The necrotic rates were not higher than that of the control group.At 24 hours after treated with nano-SiO₂,nano-TiO₂ or nano-ZnO,the necrotic rates significantly increased in a dose-dependent manner while the apoptotic rates were not higher than that of the control group.In ACNP group,the percentage of cells at S phase was higher than that in control group,while the percentage of G₀/G₁-phase cells decreased significantly after 24 hours exposure.The typical "Sub-G₁ peak"(apoptotic peak) was also detected in ACNP group.In nano-ZnO group,the percentage of S-phase cells increased while the percentage of G₀/G₁-phase cells decreased significantly after 24 hours exposure.In nano-SiO₂ and nano-TiO₂ groups,the percentage of S-phase cells increased after 48 hours exposure.After treated with 0.1, 0.2,0.4 mg/ml ACNP for 24 hours,the MMP of BGC-823 cells decreased in a dose-dependent manner.After treated with 0.1,0.2,0.4 mg/ml nano-SiO₂ or nano-TiO₂ for 24 hours,the MMP of BGC-823 cells decreased in a dose-dependent manner,too. After treated with 0.1 mg/ml and 0.2 mg/ml ACNP for 24 hours and incubated with 5μmol/L DHR123 for 1 hour,the MFI of oxidized Rh123(or the cellular ROS levels) were 101.11±3.63 and 82.21±2.70,respectively,both of which were significantly higher than that of the control group(62.18±2.05)(P＜0.05).After treated with 0.1, 0.2,0.4 mg/ml nano-SiO₂ for 24 hours,the cellular ROS levels deceased in a dose-dependent manner.After treated with 0.1 mg/ml and 0.2 mg/ml nano-TiO₂ for 24 hours,the cellular ROS levels were higher than that of the control group(P＜0.05).Conclusion:ACNP could inhibit proliferation,cause S-phase arrest and induce apoptosis of BGC-823 cells which demonstrated strong cytotoxicity in vitro.ACNP may induce oxidative stress in BGC-823 cells by overproduction of ROS,and then induce apoptosis of BGC-823 cells via activating mitochondrial signal transduction pathway. Furthermore,Fine activated carbon particles entered the nuclei of tumor cells,which would damage DNA,inhibit DNA replication,and induce cell apoptosis.Nano-SiO₂, nano-TiO₂ and nano-ZnO could directly induce necrosis of BGC-823 cells via disintegrating plasma membranes.In conclusion,nanoparticles with different chemical composition could induce cytotoxicity in BGC-823 cells.The biological effects of nanoparticles may be attributed to particle size and shape,surface characteristics, chemical composition,and some other more factors.