纳米材料体外细胞毒性试验标准的关键项目研究

目的: 参考ASTM标准,研究纳米材料体外细胞毒性检测方法(MTT试验和LDH试验)中的关键项目指标,为制定纳米材料体外细胞毒性试验标准提供实验依据。方法: 通过全波长扫描获得MTT试验和LDH试验的检测波长;测试阳性对照品APAP的使用剂量和保存条件;通过改变MTT试剂的配制介质,以及DMSO溶解后加入和不加入甘氨酸缓冲液来测试MTT试验条件;考察含不同浓度血清(10%,5%,2.5%)的细胞培养液对LDH试验的背景影响;在确定优化的实验条件下用MTT试验和LDH试验,对某纳米银凝胶产品进行细胞毒性评价。结果: MTT试验和LDH试验在测定吸光度时双波长可分别设定为540~570/680 nm和490/680 nm。APAP作为阳性对照的合适浓度为20~30 mmol·L^-1,该溶液保存条件为-20℃,14 d内有效。血清对LDH试验的干扰与浓度呈正相关,为确保细胞正常生长,该方法中血清浓度确定为10%,血清造成的背景值干扰可从空白组扣除。纳米银凝胶产品MTT试验和LDH试验结果(IC₅₀)分别为26.8 mg·mL^-1和61.8 mg·mL^-1。结论: MTT试验和LDH试验相结合可以在1个实验体系内获得2个检测结果,能够更全面地评价纳米材料可能引起的细胞毒性。本课题确定了纳米材料体外细胞毒性检测方法(MTT试验和LDH试验)的关键项目指标,使该方法具有良好的操作性与可行性。

Objective: To investigate the key factors and parameters of the in vitro cytotoxicity test(MTT assay and LDH assay) for assessing nanomaterials referring to ASTM,so that to provide evidences for establishing standards of nanomaterial's cytotoxicity test.Methods:The detection wavelength of MTT assay and LDH assay were obtained by full-wavelength scanning; the concentration and storage condition of the positive control APAP were tested; the experimental factors of MTT assay were tested by changing the MTT media with or without glycine buffer after adding DMSO; the experimental conditions of LDH assay were measured by changing the concentration of serum(10%,5%,2.5%) in cell culture media.The cytotoxicity of the silver nanoparticle containing hydrogel was assessed by refined MTT assay and LDH assay.Results: The double-wavelength was found at 540-570/680 nm and 490/680 nm,which is suitable for MTT assay and LDH assay respectively,in this experimental system.The proper concentration of positive control APAP was 20-30 mmol·L^-1,which should be stored at-20℃ and will be stable for 14 days.The serum contained in the cell culture media could affect the LDH test with a serum concentration-depended manner,but this kind of interference could be deducted by the blank control.The IC₅₀ of silver nanoparticle-containing hydrogel tested by refined MTT assay and LDH assay were 26.8 mg·mL^-1 and 61.8 mg·mL^-1,respectively.Conclusions:Two assessment points can be obtained in one experiment by combining refined MTT assay and LDH assay,so that the cytotoxicity of nanomaterials can be evaluated more comprehensively.In addition,this study defines the key factors and parameters of the cytotoxicity test(MTT assay and LDH assay) for assessing nanomaterials,and improves the feasibility of the method.