纳米银的银离子释放及其对细胞的毒性作用
Release of silver ions from silver nanoparticles and their cytotoxicity
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摘要:
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HepG2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HepG2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，HepG2细胞内的银含量高于A549细胞（P < 0.05），暴露48 h后，HepG2细胞内的总银含量低于A549细胞（P < 0.05）；暴露24 h和48 h后，HepG2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，HepG2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，HepG2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。
Abstract:Objective To test the silver ion release kinetics of three kinds of silver nanoparticles under different conditions (such as mass concentration, time, and solution) and assess the cytotoxicity of released silver ions.
Methods Uncoated 20nm, uncoated 50nm, and polyvinylpyrrolidone coated 20nm silver nanoparticles (nano-Ag20, nano-Ag50, and nano-Ag20PVP) were dispersed in different pH buffers such as pH3.6 acetate buffer, pH6.0 acetate buffer, and pH7.0 deionized water to prepare 0.2, 0.5, and 1.0 g/L suspensions. After incubation at 37℃, silver ions were separated by ultracentrifugation and ultrafiltration for detection. Nano-Ag20PVP were dissolved in cell culture to prepare solutions of 0, 20, 40, 80, and 160mg/L concentrations, then incubated at 37℃, and followed by silver ion detection after 24 and 48h, respectively. A549 and HepG2 cells were exposed to nanoAg20PVP suspensions, with silver nitrate solution used as positive control, to determine cell viability by MTT assay.
Results Higher degree of dispersion was found in nano-Ag20PVP than in nano-Ag20 or nano-Ag50. In all designed pH conditions, the silver ion content of three kinds of silver nanoparticles was positively correlated with mass concentration and time. Under same pH and mass concentration conditions, the silver ion ratio of nano-Ag20PVP was always higher than that of nano-Ag20 (P < 0.05). At pH7.0, the silver ion ratio of nano-Ag50 was higher than that of nano-Ag20 (P < 0.05) in the 1.0 g/L group. The silver ion ratios of the 20 mg/L and the 160 mg/L nano-Ag20PVP solutions were 0.02% and 0.09%, respectively. After incubation for 24 h and 48h, the silver ion ratios of the 160mg/L nano-Ag20PVP were 0.01% and 0.02%, respectively. After treatment for 24h and 48h with nanoAg20PVP, both silver content and silver ion ratio in A549 and HepG2 cells were positively associated with the mass concentration of nano-Ag20PVP. After 24 h with nano-Ag20PVP treatment, the content of silver in HepG2 cells was higher than that in A549 cells (P < 0.05), but it was lower after 48 h (P < 0.05). After treatment for 24 h and 48 h with nano-Ag20PVP, the contents of silver ion in HepG2 cells were lower than those in A549 cells (P < 0.05). After treatment for 24 h and 48 h with 10-160 mg/L nano-Ag20PVP, the cell viabilities of HepG2 cells were higher than those of A549 cells (P < 0.05). After treatment for 24 h and 48 h with 5 and 10 mg/L silver nitrate, the cell viabilities of HepG2 cells were higher than those of A549 cells (P < 0.05). At the same mass concentration (5 and 10 mg/L), the cell viability of the nano-Ag20PVP group was higher than that of the silver nitrate group (P < 0.05).
Conclusion Coated silver nanoparticles release more silver ions than uncoated silver nanoparticles. Nano-Ag20PVP could still release silver ions in cells, which plays an important role in the cytotoxicity induced by silver ions in selected cell lines. Silver nanoparticles and released silver ions jointly induce cytotoxicity.
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