透明质酸修饰山柰酚纳米结构脂质载体的制备及对非小细胞肺癌细胞NCI-H1299的抑制作用

Preparation of kaempferol nanostructured lipid carrier modified with hyaluronic acid and its inhibitory effect on non-small cell lung cancer cells NCI-H1299

目的:制备以相对分子质量为200000与1300000的透明质酸(hyaluronic acid,HA)修饰的山柰酚纳米结构脂质载体,并考察其对NCI-H1299细胞的抑制作用。方法:建立山柰酚含量的测定方法并进行方法学考察,采用熔融超声法制备山柰酚纳米结构脂质载体(KA-NLC),在此基础上以静电吸附法制备透明质酸修饰的山柰酚纳米结构脂质载体(HA-KA-NLC),通过EdU染色法、克隆形成试验、Hoechst染色法、细胞划痕试验、Transwell法分别考察所得制剂对NCI-H1299细胞增殖、凋亡、迁移和侵袭的抑制作用。采用激光共聚焦显微镜观察NCI-H1299细胞在1,2,4 h对不同制剂的摄取情况,同时采用Westen blot法考察所得制剂对上皮间质转化(epithelial-mesenchymal transition,EMT)过程中的关键性蛋白表达的影响,初步探讨其对NCI-H1299的干预机制。结果:相对于KA-NLC,经过HA修饰后的HA-KA-NLC在抑制肿瘤细胞增殖、凋亡、迁移和侵袭的作用更为突出,且有更为良好的摄取效果和EMT干预作用,而以200000相对分子质量修饰后的KA-NLC相对于1300000相对分子质量修饰所得制剂在全面抑制NCI-H1299方面有着更为突出的表现。结论:HA_(20)-KA-NLC对NCI-H1299有较强的抗肿瘤作用,可以用于开发优良的靶向制剂,在未来治疗NSCLC中具有一定的应用前景。

OBJECTIVE To prepare kaempferol nanostructured lipid carriers modified by hyaluronic acid(HA)with molecular weight of 200000 and 1300000,and to investigate its inhibitory effect on NCI-H1299 cells.METHODS The determination method of kaempferol content was established and the methodology was investigated.The nanostructured lipid carriers of kaempferol(KA-NLC)were prepared by molten ultrasonic method.On this basis,hyaluronic acid modified kaempferol nanostructured lipid carriers(HA-KA-NLC)were prepared by electrostatic adsorption method.The inhibitory effects on proliferation,apoptosis,migration and invasion of NCI-H1299 were investigated by EdU staining,cloning formation assay,Hoechst staining assay,cell scratch assay and Transwell assay,respectively.The uptake of different preparations by NCI-H1299 cells at 1,2 and 4 h was observed by laser confocal microscopy.Meanwhile,the effect of preparations on the epithelial-mesenchymal transition was investigated by Westen blot assay,and the intervention mechanism of NCI-H1299 was discussed.RESULTS Compared with KA-NLC,HA-KA-NLC modified by HA hadmore prominent effect on inhibiting tumor cell proliferation,apoptosis,migration and invasion,and hadbetter uptake effect and EMT intervention effect.Compared with 1300000,KA-NLC modified with 200000 molecular weight hada more outstanding performance in overall inhibition of NCI-H1299.CONCLUSION HA_(20)-KA-NLC has a strong anti-tumor effect on NCI-H1299,which can be used to develop excellent targeted preparations,and has certain application prospects in the treatment of NSCLC in the future.