叶酸偶联紫杉醇纳米粒的制备及其生物效应研究

Preparation and Biological Effect of Folic Acid Coupling Paclitaxel Nanoparticles

目的以叶酸修饰的生物可降解材料乳酸-羟基乙酸共聚物(PLGA-PEG-FA)为载体,构建叶酸偶联紫杉醇纳米粒〔PLGA-PEG-FA(PTX)-NPs〕并进行评价。研究其对MCF-7细胞的效应并初步探讨其靶向作用机制。方法乳化分散法制备PLGA-PEG-FA(PTX)-NPs,并对纳米粒的形态、粒径、Zeta电位、包封率及载药量进行评价;同法制备无叶酸偶联的紫杉醇纳米粒〔PLGA-PEG(PTX)-NPs〕,分别用荧光物质FITC标记,应用荧光显微镜观察其对MCF-7细胞和A549细胞的靶向作用,以及游离叶酸对PLGA-PEG-FA(PTX)-NPs的干扰作用;MTT法观察PLGA-PEG-FA(PTX)-NPs、PLGA-PEG(PTX)-NPs对MCF-7细胞的生长抑制作用;流式细胞术检测细胞凋亡率。结果制备的PLGA-PEG-FA(PTX)-NPs为均匀球形粒子,粒径为(88.2±6.7)nm,Zeta电位为(56.5±4.2)mV,包封率为(92.96±0.16)%,载药量为(4.84±0.10)%。与PLGA-PEG(PTX)-NPs相比,PLGA-PEG-FA(PTX)-NPs能更有效的被MCF-7细胞吞噬(P<0.05);PLGA-PEG-FA(PTX)-NPs、PLGA-PEG(PTX)-NPs对A549细胞无明显靶向作用;培养基中加入游离叶酸干扰了PLGA-PEG-FA(PTX)-NPs的靶向性。MTT法测得PLGA-PEG-FA(PTX)-NPs抑制MCF-7细胞生长的IC50值(10.72μg·ml^(-1))明显低于PLGAPEG(PTX)-NPs组(17.41μg·ml^(-1),P<0.05),紫杉醇浓度在1~15μg·ml^(-1)时,PLGA-PEG-FA(PTX)-NPs组的生长抑制率在24h明显高于PLGA-PEG(PTX)-NPs组(P<0.05),紫杉醇浓度达到20μg·ml^(-1)时,两组无显著性差异。PLGA-PEG(PTX)-NPs组的48h细胞凋亡率(87.04%)明显高于PLGA-PEG(PTX)-NPs组(64.14%)。结论制备的PLGA-PEG-FA(PTX)-NPs能够靶向抑制MCF-7细胞增殖并促进其凋亡,其靶向性可能是由叶酸受体介导的。

Objective To establish and evaluate the folic acid coupling paclitaxel nanoparticles〔PLGA-PEG-FA（PTX）-NPs〕with biodegradable materials of lactic acid-glycolic acid copolymer modified by FA（PLGA-PEG-FA）as the carrier,and to study the effect and mechanism of PLGA-PEG-FA（PTX）-NPs in targeting treatment of MCF-7cells.Methods The emulsification-dispersion method was used to prepare PLGA-PEG-FA（PTX）-NPs,which were evaluated in terms of the morphology of nanoparticles,particle size,zeta potential,entrapment efficiency and drug-loading rate.The same method was used to prepare paclitaxel nanoparticles without FA 〔PLGA-PEG（PTX）-NPs〕.PLGA-PEG-FA（PTX）-NPs and PLGA-PEG（PTX）-NPs were marked by FITC,and their targeting treatment to MCF-7and A549 was observed with a fluorescence microscope.The interference effect of free folic acid in the cell culture medium to PLGA-PEG（PTX）-NPs was observed with a fluorescence microscope.The growth inhibition was studied by MTT.The apoptosis rate of cells was detected by flow cytometry.Results PLGA-PEG-FA（PTX）-NPs were uniform spherical particle,with a particle size of（88.2±6.7）nm.Zeta potential was（56.5±4.2）mV,the entrapment efficiency was（92.96±0.16）%,and the drug-loading rate was（4.84±0.10）%.Compared to PLGA-PEG（PTX）-NPs,MCF-7cells could devour PLGA-PEG-FA（PTX）-NPs more effectively（P 0.05）.PLGAPEG-FA（PTX）-NPs and PLGA-PEG（PTX）-NPs couldn′t be targeted to A549 cells obviously.The IC50ofPLGA-PEG-FA（PTX）-NPs inhibiting MCF-7cells proliferation（10.72μg·ml^（-1））was significantly lower than PLGA-PEG（PTX）-NPs（17.41-1μg·ml）（P 0.05）.When the final concentration of paclitaxel of the two types of nanoparticles was 1-15μg·ml^（-1）,the growth inhibition rate of MCF-7cells in PLGA-PEGFA（PTX）-NPs group was significantly increased compared to PLGA-PEG（PTX）-NPs after cells were treated for 24h（P 0.05）.When the final concentration of paclitaxel reached 20μg·ml^（-1）,the cell growth inhibition rate of the two groups had no significant difference（P 0.05）.The apoptosis rate of MCF-7cells in PLGA-PEG-FA（PTX）-NPs group（87.04%）was significantly increased compared to PLGA-PEG（PTX）-NPs group（64.14%）after cells were treated for 48h（P 0.05）.ConclusionInhibiting MCF-7cells proliferation constructed can be targeted to inhibit MCF-7cell proliferation and promote their apoptosis,and the targeting treatment may be mediated by folate receptors.