不同表面修饰的氧化铁纳米颗粒对A549细胞的毒性及DNA损伤
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摘要
目的:比较不同表面修饰的氧化铁纳米颗粒(IONP_s)对A549细胞的细胞毒性、染色体和DNA损伤及其作用机制的差异。方法:比较相同粒径范围(约5nm)的胺基表面修饰的纳米氧化铁颗粒(Amine-IONP)和聚乙二醇表面修饰的纳米氧化铁颗粒(PEG-IONP)对A549细胞存活率和细胞周期的影响;使用高内涵法检测细胞经IONP_s处理后的胞内活性氧簇(ROS)含量、线粒体膜电位(MMP)和内质网(ER)状态的变化;采用体外胞质分裂法微核试验和彗星电泳评价IONP_s对染色体和DNA完整性的影响。结果:两种纳米氧化铁颗粒处理48h对A549细胞生长抑制率均小于20%。相同浓度条件下,PEG-IONP主要表现为对A549细胞G0/G1期阻滞,自20μg/mL起即明显减少S期细胞比率(P<0.01),320μg/mLPEG-IONP处理24h后可诱导p21与p53表达水平显著升高(P<0.05)。给药48h时,Amine-IONP作用后细胞ROS、MMP及ER水平显著性改变的起始浓度分别为20、20和80μg/mL,而PEG-IONP作用后产生显著性改变的起始浓度分别为40、40和160μg/mL。此外,与PEG-IONP比较,Amine-IONP可在较低浓度条件下诱导微核和彗星拖尾形成(Amine-IONP的起始浓度为20和80μg/mL;而PEG-IONP则为40和160μg/mL)。经氧自由基清除剂乙酰半胱氨酸和叔丁基对羟基茴香醚预处理后,两者导致的胞内ROS含量和尾部DNA百分率均明显降低(P<0.05)。结论:带正电荷的Amine-IONP更易于诱导A549细胞氧化应激及与之有关的DNA损伤;相比之下,PEG-IONP的细胞毒性和遗传毒性较弱,但除氧化损伤外也可通过抑制细胞周期干扰细胞增殖,作为肿瘤诊断试剂具有一定优势。
OBJECTIVE: To compare the induction of cytotoxicity,chromosome aberrations and DNA damage among iron oxide nanoparticles(IONP_s) with different surface-modifications in A549 cells. METHODS:The effects of amine-modified(Amine-IONP) and polyethylene glycol-modified iron oxide nanoparticles(PEG-IONP) of the same particle size range(5 nm) on cell viability and cell cycle were compared in A549 cells;the content of intracellular reactive oxygen species(ROS),mitochondrial membrane potential(MMP) and endoplasmi reticulum stress(ERS) in cells treated with IONP_s were analyzed using high content screening method;the in vitro cytokinesis micronucleus test and Comet assay were performed to evaluate their effects on chromosome and DNA integrity. RESULTS:The growth inhibitory rates of both IONP_s to A549 cells were less than 20% after 48 h. Under the same concentration,PEG-IONP exhibited a more significant effect on the G0/G1 cell cycle arrest(P<0.05):the initial concentration to significantly reduce the cell rate of S phase was 20 μg/mL,and the expression of p21 and p53 was up-regulated by a treatment of 320 μg/mL PEG-IONP for 24 h(P<0.05). After a 48 h treatment,the initial concentrations to show significant effects of Amine-IONP on ROS,MMP and ER were 20,20 and 80 μg/mL,while for PEG-IONP they were 40, 40 and 160 μg/mL respectively. In addition,compared to PEG-IONP,Amine-IONP was able to induce the formation of micronuclei and Comet tail(the initial concentrations for Amine-IONP were 20 and 80 μg/mL;while PEG-IONP were 40 and 160 μg/mL respectively). After the pre-treatment with oxygen free radical scavengers N-acetylcysteine and butylated hydroxyanisole,both IONP_s induced ROS and Comet DNA Tail were significantly inhibited(P<0.05). CONCLUSION:Positively charged Amine-IONP was more effective in inducing oxidative stress and DNA damage in A549 cells;whereas,PEG-IONP with relatively weaker cytotoxicity and genotoxicity interfered with cell proliferation,and showed advantages in the development of tumor diagnostic reagents.
OBJECTIVE: To compare the induction of cytotoxicity,chromosome aberrations and DNA damage among iron oxide nanoparticles(IONP_s) with different surface-modifications in A549 cells. METHODS:The effects of amine-modified(Amine-IONP) and polyethylene glycol-modified iron oxide nanoparticles(PEG-IONP) of the same particle size range(5 nm) on cell viability and cell cycle were compared in A549 cells;the content of intracellular reactive oxygen species(ROS),mitochondrial membrane potential(MMP) and endoplasmi reticulum stress(ERS) in cells treated with IONP_s were analyzed using high content screening method;the in vitro cytokinesis micronucleus test and Comet assay were performed to evaluate their effects on chromosome and DNA integrity. RESULTS:The growth inhibitory rates of both IONP_s to A549 cells were less than 20% after 48 h. Under the same concentration,PEG-IONP exhibited a more significant effect on the G0/G1 cell cycle arrest(P<0.05):the initial concentration to significantly reduce the cell rate of S phase was 20 μg/mL,and the expression of p21 and p53 was up-regulated by a treatment of 320 μg/mL PEG-IONP for 24 h(P<0.05). After a 48 h treatment,the initial concentrations to show significant effects of Amine-IONP on ROS,MMP and ER were 20,20 and 80 μg/mL,while for PEG-IONP they were 40, 40 and 160 μg/mL respectively. In addition,compared to PEG-IONP,Amine-IONP was able to induce the formation of micronuclei and Comet tail(the initial concentrations for Amine-IONP were 20 and 80 μg/mL;while PEG-IONP were 40 and 160 μg/mL respectively). After the pre-treatment with oxygen free radical scavengers N-acetylcysteine and butylated hydroxyanisole,both IONP_s induced ROS and Comet DNA Tail were significantly inhibited(P<0.05). CONCLUSION:Positively charged Amine-IONP was more effective in inducing oxidative stress and DNA damage in A549 cells;whereas,PEG-IONP with relatively weaker cytotoxicity and genotoxicity interfered with cell proliferation,and showed advantages in the development of tumor diagnostic reagents.
引文
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[2]WANG L,JING H,CHEN H,et al.Exerting enhanced permeability and retention effect driven delivery by ultrafine iron oxide nanoparticles with T1-T2 switchable magnetic resonance imaging contrast[J].Acs Nano,2017.11(5):4582-4592.
[3]D'ARIENZO A,SCAGLIONE G,BENNATO R,et al.The prognostic value,in active ulcerative colitis,of an increased intensity of colonic perivisceral fat signal on magnetic resonance imaging with ferumoxil[J].Am J Gastroenterol,2001,96(2):481-486.
[4]FU T,KONG Q,SHENG H,et al.Value of functionalized superparamagnetic iron oxide nanoparticles in the diagnosis and treatment of acute temporal lobe epilepsy on MRI[J].Neural Plast,2016,2016:2412958.doi:10.1155/2016/2412958.
[5]SHI D,MI G,BHATTACHARYA S,et al.Optimizing superparamagnetic iron oxide nanoparticles as drug carriers using an in vitro blood-brain barrier model[J].Int JNanomedicine,2016,11:5371-5379.
[6]KONCZOL M,WEISS A,STANGENBERG E,et al.Cellcycle changes and oxidative stress response to magnetite in A549human lung cells[J].Chem Res Toxicol,2013,26(5):693-702.
[7]WATANABE M,YONEDA M,MOROHASHI A,et al.Effects of Fe O magnetic nanoparticles on A549 cells[J].Int J3 4Mol Sci,2013,14(8):15546-15560.
[8]淡墨,赵继云,齐乃松,等.不同表面修饰的纳米氧化铁颗粒诱导胶质瘤细胞凋亡的差异[J].中国新药杂志,2016(24):2887-2892.
[9]文海若,毛志慧,耿兴超,等.人源HepaRG肝细胞毒性与遗传毒性高通量筛选方法的初步建立[J].药物评价研究,2017,40(11):1550-1558.
[10]文海若,淡墨,齐乃松,等.多细胞系胞质分裂阻滞微核细胞组学试验法的建立与应用[J].癌变·畸变·突变,2015,27(4):304-308.
[11]GUPTA A K,GUPTA M.Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications[J].Biomaterials,2005,26(18):3995-4021.
[12]PAOLINI A,GUARCH C P,RAMOS-LOPEZ D,et al.Rhamnose-coated superparamagnetic iron-oxide nanoparticles:an evaluation of their in vitro cytotoxicity,genotoxicity and carcinogenicity[J].J Appl Toxicol,2016,36(4):510-520.
[13]MALVINDI M A,DE MATTEIS V,GALEONE A,et al.Toxicity assessment of silica coated iron oxide nanoparticles and biocompatibility improvement by surface engineering[J].PLoSOne,2014,9(1):e85835.
[14]DE LIMA R,OLIVERIA J L,MURAKAMI P S K,et al.Iron oxide nanoparticles show no toxicity in the comet assay in lymphocytes:A promising vehicle as a nitric oxide releasing nanocarrier in biomedical applications[C].J Phys:Conf Ser,2013,429:1-8.
[15]HARRIS G,PALOSAARI T,MAGDOLENOVA Z,et al.Iron oxide nanoparticle toxicity testing using high throughput analysis and high content imaging[J].Nanotoxicology,2015,9(Sup1):87-94.
[16]VERMA A,STELLACCI F.Effect of surface properties on nanoparticle-cell interactions[J].Small,2010,6(1):12-21.
[17]LIU H,ZHANG J,CHEN X,et al.Application of iron oxide nanoparticles in glioma imaging and therapy:from bench to bedside[J].Nanoscale,2016,8(15):7808-7826.
[18]GAHARWAR U S,PAULRAJ R.Iron oxide nanoparticles induced oxidative damage in peripheral blood cells of rat[J].JBiomed Sci Eng,2015,08(4):274-286.
[19]NEMMAR A,BEEGAM S,YUVARAJU P,et al.Ultrasmall superparamagnetic iron oxide nanoparticles acutely promote thrombosis and cardiac oxidative stress and DNA damage in mice[J].Part Fibre Toxicol,2015,13(1):22.doi:10.1186/s12989-016-0132-x.
[20]ALARIFI S,ALI D,ALKAHTANI S,et al.Iron oxide nanoparticles induce oxidative stress,DNA damage,and caspase activation in the human breast cancer cell line[J].Biol Trace Elem Res,2014,159(1/2/3):416-424.
[21]ZHANG X,ZHANG H,LIANG X,et al.Iron Oxide Nanoparticles Induce Autophagosome Accumulation through Multiple Mechanisms:Lysosome Impairment,Mitochondrial Damage,and ER Stress[J].Mol Pharm,2016,13(7):2578-2587.
[22]LI X,XU L,SHAO A,et al.Cytotoxic and genotoxic effects of silver nanoparticles on primary Syrian hamster embryo(SHE)cells[J].J Nanosci Nanotechnol,2013,13(1):161-170.
[23]HUANG D M,HSIAO J K,CHEN Y C,et al.The promotion of human mesenchymal stem cell proliferation by superparamagnetic iron oxide nanoparticles[J].Biomaterials,2009,30(22):3645-3651.
[24]XU F,PIETT C,FARKAS S,et al.Silver nanoparticles(AgNPs)cause degeneration of cytoskeleton and disrupt synaptic machinery of cultured cortical neurons[J].Mol Brain,2013,6(1):1-15.
[25]ZELJEZIC D,BJELIS M,MLADINIC M.Evaluation of the mechanism of nucleoplasmic bridge formation due to premature telomere shortening in agricultural workers exposed to mixed pesticides:Indication for further studies[J].Chemosphere,2015,120(2):45-51.