壳聚糖和油酸钠修饰的超顺磁性四氧化三铁纳米粒子的细胞毒性

Cytotoxicity of superparamagnetic iron oxide nanoparticles modified by chitosan or sodium oleate

目的 研究壳聚糖和油酸钠修饰的超顺磁性四氧化三铁纳米粒子（superparamagnetic iron oxide nanoparticles，SPIONs）的细胞毒性，为临床应用提供实验依据。方法 用透射电子显微镜对壳聚糖SPIONs和油酸钠SPIONs进行形态学观察；用10 μg/mL的壳聚糖SPIONs和油酸钠SPIONs悬液处理体外培养的人肺腺癌细胞A549，普鲁士蓝染色观察2种SPIONs在细胞内的分布；用不同浓度（0、25、50、100和200 μg/mL）的2种SPIONs分别处理A549细胞，MTT法测定细胞生长活性；2种SPIONs染毒A549细胞48 h时，测定上清液中乳酸脱氢酶（LDH）的活性；DAPI染色法观察细胞凋亡情况。结果 （1）2种SPIONs均呈类球形结晶，粒径20~30 nm，此时四氧化三铁纳米粒子之间的热振动能足以克服磁吸引力而呈现超顺磁性。（2）普鲁士蓝染色显示细胞内可见蓝色沉着，说明2种SPIONs均可进入A549细胞内。（3）MTT检测结果显示壳聚糖SPIONs对细胞生长几乎无抑制，而200 μg/mL 油酸钠SPIONs在48 h和72 h时对细胞有明显的抑制作用（P〈0.05）。（4）200 μg/mL壳聚糖SPIONs染毒细胞的上清液中LDH活性稍高于对照组（P〈0.05），而100 μg/mL和200 μg/mL油酸钠SPIONs染毒细胞的上清液中LDH活性均高于对照组（P〈0.05），同时也高于相同浓度壳聚糖SPIONs染毒组（P〈0.05）。（5）DAPI凋亡检测显示，壳聚糖SPIONs染毒细胞与对照组间差异无统计学意义，而油酸钠SPIONs染毒细胞发生明显的细胞核皱缩及胞核碎裂现象，且细胞凋亡率高于对照组（P〈0.05）。结论 壳聚糖SPIONs与油酸钠SPIONs相比具有较低的细胞毒性。

Objective To investigate the cytotoxicity of superparamagnetic iron oxide nanoparticles （SPIONs） modified by chitosan or sodium oleate, so as to provide experimental evidence for future clinical application. Methods The morphology of chitosan-SPIONs or sodium oleate-SPIONs was observed by transmission electron microscope （TEM）. Human lung adenocarcinoma A549 cells were treated with 10 μg/mL chitosan-SPIONs or sodium oleate-SPIONs; prussian blue staining was performed to observe the intracellular distribution of the two modified SPIONs. Chitosan-SPIONs or sodium oleate-SPIONs of different concentrations （0, 25, 50, 100, and 200 μg/mL） were used to treat A549 cells and then MTT assay was used to detect cell proliferation. The activity of LDH in the supernatant of A549 cells was tested 48 h after treatment with chitosan-SPIONs or sodium oleate-SPIONs; furthermore, cell apoptosis was analyzed by DAPI staining. Results （1） Both the chitosan-SPIONs and sodium oleate-SPIONs exhibited structure of spherical crystallization, with a diameter of 20-30 nm. The thermal vibrational energy could overcome the magnetic attraction among the SPIONs, presenting superparamagnetism. （2） Prussian blue staining showed blue deposits in the cells, indicating that the two kinds of SPIONs entered the cytoplasm of A549 cells. （3） MTT assay results showed that chitosan-SPIONs hardly affected cell growth, while sodium oleate-SPIONs （200 μg/mL） inhibited the proliferation of A549 cells after 48 h and 72 h （P〈0.05）. （4） The LDH activity in the supernatant of A549 cells treated with 200 μg/mL chitosan-SPIONs was significantly higher than that in the control group （P〈0.05）, while the LDH activities in 100 μg/mL and 200 μg/mL sodium oleate-SPIONs groups were significantly higher than those in the control group （P〈0.05） and corresponding chitosan-SPIONs group（P〈0.05）. （5） The results of DAPI dye showed no significant difference between the chitosan-SPIONs group and the control group, while the cells treated with sodium oleate-SPIONs displayed typical apoptosis characteristics, such as nuclear shrinkage and fragmentation, with the apoptosis rate being significantly higher than that of the control group（P〈0.05）. Conclusion It is suggested that chitosan-SPIONs have slighter cytotoxicity to A549 cells compared with sodium oleate-SPIONs.