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[目的]检测三种纳米银颗粒在不同质量浓度、不同时间、不同溶液下的银离子释放动力学,并探讨纳米银释放银离子对细胞毒性作用的影响。[方法]将无包被20 nm、50 nm纳米银(nano-Ag20和nano-Ag50)和聚乙烯吡咯烷酮包被的20 nm纳米银(nano-Ag20PVP)颗粒分散于pH3.6醋酸盐缓冲液、pH6.0醋酸盐缓冲液和pH7.0去离子水中,配制成银质量浓度为0.2、0.5、1.0 g/L的悬液,37℃孵育后超速离心加超滤法分离出银离子,检测银离子含量。将nano-Ag20PVP分散于细胞培养液中,配制成银质量浓度为0、20、40、80、160 mg/L的染毒液,37℃孵育24 h和48 h后检测银离子含量。用nano-Ag20PVP悬液染毒A549和Hep G2细胞,银质量浓度为0.625、2.5、5、10 mg/L的硝酸银溶液作对照,MTT法比较细胞活力。[结果]nano-Ag20PVP分散较好,nano-Ag20和nano-Ag50有团聚现象。不同的pH条件下,三种纳米银的银离子含量与孵育时间呈正相关。相同pH条件和质量浓度下,nano-Ag20PVP比nano-Ag20释放更多银离子(P<0.05);在pH7.0的1.0 g/L组,nano-Ag50的银离子比率高于nano-Ag20(P<0.05)。20 mg/L和160 mg/L nano-Ag20PVP染毒液中的银离子比率分别为0.02%和0.09%,孵育24 h和48 h后仅在160 mg/L组检测到比率分别为0.01%和0.02%的银离子。A549细胞和Hep G2细胞暴露nano-Ag20PVP染毒液24 h和48 h后,细胞内银含量和银离子比率都与染毒液的银质量浓度呈正相关;暴露24 h后,Hep G2细胞内的银含量高于A549细胞(P<0.05),暴露48 h后,Hep G2细胞内的总银含量低于A549细胞(P<0.05);暴露24 h和48 h后,Hep G2细胞内的银离子比率低于A549细胞(P<0.05)。暴露于10~160 mg/L的nano-Ag20PVP 24 h和48 h后,Hep G2细胞的存活率高于A549细胞(P<0.05);暴露于5、10 mg/L的硝酸银24 h和48 h后,Hep G2细胞的存活率高于A549细胞(P<0.05);在相同质量浓度(5、10 mg/L)下,nano-Ag20PVP染毒组的细胞存活率高于硝酸银组(P<0.05)。[结论]有包被的纳米银比无包被的纳米银容易释放银离子。nano-Ag20PVP进入细胞后能进一步释放银离子,后者在纳米银诱导的细胞毒性中发挥重要作用。纳米银对细胞的毒性作用由纳米银颗粒及其释放的银离子共同作用所致。
[Objective] The research aimed to detect the kinetics of silver ion release of three kinds of nano-silver particles under different concentrations, different times and different solutions, and discuss the influence of silver nanoparticles released by silver nanoparticles on the cytotoxicity. [Method] 20 nm nano-Ag20PVP particles coated with 20 nm, 50 nm nano-Ag (nano-Ag20 and nano-Ag50) and polyvinylpyrrolidone were dispersed in pH 3.6 acetate buffer , PH6.0 acetate buffer and pH7.0 deionized water to prepare a suspension with silver mass concentration of 0.2, 0.5, and 1.0 g / L. The silver ions were separated by ultracentrifugation and ultrafiltration after incubation at 37 ° C., Detection of silver ion content. The nano-Ag20PVP was dispersed in the cell culture medium to prepare silver nitrate solution at 0, 20, 40, 80 and 160 mg / L, and the silver ions were detected at 37 ℃ for 24 and 48 h. A549 and Hep G2 cells were treated with nano-Ag20PVP suspension, and silver nitrate solutions with silver concentrations of 0.625, 2.5, 5 and 10 mg / L were used as controls, and cell viability was compared by MTT assay. [Result] The dispersion of nano-Ag20PVP was better and the aggregation of nano-Ag20 and nano-Ag50 was observed. Under different pH conditions, the silver ion content of the three kinds of silver nanoparticles was positively correlated with the incubation time. At the same pH condition and mass concentration, nano-Ag20PVP released more silver ions than nano-Ag20 (P <0.05). At 1.0 g / L pH7.0, the ratio of silver ions to nano-Ag50 was higher than that of nano- P <0.05). The silver ion ratios in 0.02 mg / L and 160 mg / L nano-Ag20PVP were 0.02% and 0.09%, respectively, and the ratios were 0.01% and 0.02% in 160 mg / L group after incubation for 24 and 48 h % Of silver ions. After exposed to nano-Ag20PVP in A549 and Hep G2 cells for 24 h and 48 h, the intracellular silver content and silver ion ratio were positively correlated with the silver concentration in the solution. After 24 h exposure, the silver content in Hep G2 cells (P <0.05). After exposure for 48 h, the total silver content in Hep G2 cells was lower than that in A549 cells (P <0.05). After exposure for 24 h and 48 h, the silver ion ratio in Hep G2 cells was lower In A549 cells (P <0.05). After exposure to 10 ~ 160 mg / L nano-Ag20PVP for 24 h and 48 h, the survival rate of Hep G2 cells was higher than that of A549 cells (P <0.05). Exposure to 5, 10 mg / L silver nitrate for 24 h and 48 h h, the survival rate of Hep G2 cells was higher than that of A549 cells (P <0.05). The cell viability of nano-Ag20 PVP group was higher than that of silver nitrate group (P <0.05) under the same concentration of 5 and 10 mg / L <0.05). [Conclusion] The silver nanoparticles coated with silver nanoparticles can release silver ions more easily than the non-coated silver nanoparticles. After entering the cells, nano-Ag20PVP can further release silver ions, which play an important role in the silver-induced cytotoxicity. The toxic effects of nano-silver on the cells caused by the interaction of nano-silver particles and the release of silver ions.