基于PEG-PLL-PLGA聚合物纳米药物传输系统靶向逆转肿瘤多药耐药的研究

发布时间：2018-06-17 15:41

本文选题：PEG-PLL-PLGA + 纳米颗粒　；参考：《东南大学》2016年博士论文

【摘要】：第一部分PEG-PLL-PLGA聚合物纳米粒的生物相容性以及安全性研究目的本部分实验研究评估聚乙二醇(PEG)-聚赖氨酸(PLL)-聚乳酸羟基乙酸(PLGA) (PEG-PLL-PLGA)纳米粒(nanoparticles, NPs)的生物相容性及安全性。方法选用可生物降解材料PEG、PLL及PLGA作为合成新型纳米载药系统(nano drug delivery system, NDDS)的原料,通过优化反应条件,制得PEG-PLL-PLGA聚合物纳米粒,并将PEG-PLL-PLGA包载经典化疗药物柔红霉素(doxorubicin,DNR)。采用一系列实验检测该纳米材料的生物相容性,如MTT实验评价PEG-PLL-PLGA对小鼠成纤维细胞L929的细胞毒性；流式细胞术(flow cytometry,FCM)检测细胞内DNR浓度和细胞凋亡率；溶血试验评价纳米粒有无溶血作用；小鼠尾静脉注射DNR-PEG-PLL-PLGA以测定其半数致死量(median lethal dose,LD50)；急性毒性实验采用小鼠眼球取血方法,检测小鼠肝肾功能；病理组织学苏木素-伊红(haematoxylin-eosin, HE)染色检测小鼠主要脏器的病理变化；微核试验(micronucleus, MN)评价其有无致畸及致突变作用。结果成功制备PEG-PLL-PLGA聚合物纳米粒,透射电镜(transmission electron microscope, TEM)下PEG-PLL-PLGA呈球形或近似球形,表面光滑,平均粒径为229+15 nm,多分散系数(polydispersity index, PI)为0.085±0.017(n= 3), Zeta电位为-20.1±1.6 mV；不同浓度的PEG-PLL-PLGA纳米粒的细胞毒性均为1级,均属对细胞无毒性范畴；FCM显示DNR组的相对荧光强度(relative fluorescence intensity, RFI)为4.27+2.06, DNR-PEG-PLL-PLGA组RFI为4.39±1.89,与空白对照组相比,DNR组和DNR-PEG-PLL-PLGA组均能显著增加细胞内DNR浓度(P 0.05), PEG-PLL-PLGA实验组与对照组相比,凋亡率没有显著差异(P0.05)；肉眼观察法显示无溶血发生；LDso为464.4 mg/kg,95%的可信区间为399-541.8 mg/kg,纳米粒具有较广的安全值范围；组织病理学HE染色检测示,DNR-PEG-PLL-PLGA实验组与对照组相比,没有显著的组织结构和细胞的病理变化形态；微核试验证实该纳米材料无致畸或致突变作用。结论PEG-PLL-PLGA在体外和体内均无毒性,具有良好的生物相容性,同时为PEG-PLL-PLGA作为化疗药物传输系统的载体奠定了基础。第二部分PEG-PLL-PLGA聚合物纳米粒的靶向逆转肿瘤多药耐药研究目的本部分实验研究转铁蛋白(transferrin, Tf)修饰的PEG-PLL-PLGA纳米粒共聚合抗肿瘤药物DNR和汉防己甲素(Tetrandrine, Tet)治疗白血病裸鼠移植瘤多药耐药(multidrug resistance, MDR)的疗效及体内靶向性分布。方法选用复乳化溶剂挥发法制备共载DNR和Tet的PEG-PLL-PLGA,利用载体末端羟基二咪唑(carbonyldiimidazole, CDI)基团能自发地与Tf的氨基反应,形成Tf-PEG-PLL-PLGA,采用高速冷冻离心法及凝胶渗透色谱法检测蛋白交联率,合成DNR-Tet-Tf-PEG-PLL-PLGA (PEG-PLL-PLGA缩写成NPs,即DNR-Tet-Tf-NPs)聚合物载药纳米粒。建立白血病耐药细胞株K562/A02裸鼠皮下移植瘤模型,当肿瘤长大至75-150 mm3时,随机分为6组：生理盐水对照组,DNR组,DNR和Tet组,DNR-NPs组,DNR-Tet-NPs组和DNR-Tet-Tf-NPs组,每组6只,采用尾静脉注射方式,隔天给药,观察纳米粒的抗肿瘤作用,并测定肿瘤的生长体积,计算肿瘤抑瘤率。用药14天后处死动物,取各组心脏、肝脏、脾脏、肺脏、肾脏及肿瘤组织行病理学观察。免疫组化检测肿瘤组织中凋亡相关基因Caspase-3, Bax, Bcl-2和Survivin的表达。使用实时荧光定量PCR(quantitative real-time polymerase chain reaction, qRT-PCR)以及免疫蛋白印迹试验(Western blot)检测肿瘤靶向及耐药相关抗体TfR, P-gp, MRP和NF-κB的mRNA及蛋白的表达。高效液相色谱法(high performance liquid chromatography, HPLC)检测DNR在血浆、肿瘤组织和主要脏器的浓度。FCM检测近红外染料(near-infrared fluorescent, NIRF) NIR797是否包载至纳米粒中,近红外活体成像实验检测NIR797-labeled DNR-NPs和NIR797-labeled DNR-Tf-NPs在裸鼠体内的分布情况。结果DNR-Tet-Tf-NPs组肿瘤抑制率最高(P0.05),抑瘤作用最强,同时能够逆转肿瘤MDR,且具有靶向抗肿瘤作用。HE染色显示各组心脏、肝脏、脾脏、肺脏、肾脏及肿瘤组织无明显病理改变。肿瘤组织免疫组化观察显示,与对照组和DNR组相比,Bax和Caspase-3蛋白在DNR-Tet-NPs组和DNR-Tet-Tf-NPs组中表达明显增加,而Bcl-2和Survivin蛋白表达明显减少,并且在DNR-Tet-Tf-NPs组中Bax和Caspase-3蛋白表达最强,Bcl-2和Survivin蛋白表达最弱。实时定量PCR和Western blot检测结果示DNR-Tet-Tf-NPs组能够下调P-gp, MRP, NF-κB的mRNA及蛋白的表达,上调TfR的mRNA及蛋白的表达。HPLC实验结果显示DNR-Tet-Tf-NPs组经裸鼠尾静脉给药4h后,在肿瘤组织中的DNR浓度明显高于DNR-Tet-NPs组。近红外活体成像结果显示,通过主要脏器及肿瘤部位的近红外成像发现,大量的荧光信号主要聚集在肿瘤部位。在注射72 h后,最强的信号到达了裸鼠成瘤的部位。非Tf修饰的NIR797-labeled DNR-NPs组也有相同的成像的结果,但是肿瘤的荧光信号比Tf修饰的NIR797-labeled DNR-Tf-NPs组弱。结论DNR-Tet-Tf-NPs具有良好的逆转耐药及主动靶向能力,为肿瘤的靶向治疗提供了一条新途径。
[Abstract]:The biocompatibility and safety of PEG - PLL - PLGA nanoparticles ( PEG - PLL - PLGA ) nanoparticles ( PEG - PLL - PLGA ) nanoparticles ( PEG - PLL - PLGA ) nanoparticles were evaluated . The biocompatibility of the nano - material was tested by a series of experiments , such as MTT assay to evaluate the cytotoxicity of PEG - PLL - PLGA on mouse fibroblast L929 ;
Flow cytometry ( FCM ) was used to detect the concentration and cell apoptosis rate in the cells .
Hemolysis test was used to evaluate the hemolytic effect of nanoparticles .
The median lethal dose ( LD 50 ) was determined by intravenous injection of dnr - PEG - PLL - PLGA in tail vein of mice .
In acute toxicity experiment , mouse eyeball blood taking method was used to detect the liver and kidney function of mice .
The pathological changes of the main organs of mice were detected by pathological histology and eosin ( HE ) staining .
The results showed that PEG - PLL - PLGA nanoparticles were spherical or approximately spherical in PEG - PLL - PLGA polymer nanoparticles with smooth surface , average particle size of 229 + 15 nm , polydispersity index ( PI ) of 0.085 卤 0.17 ( n = 3 ) , zeta potential of - 20.1 卤 1.6 mV .
The cytotoxicity of PEG - PLL - PLGA nanoparticles with different concentrations was grade 1 , which belongs to the non - toxic category of cells .
The relative fluorescence intensity ( rfi ) was 4.27 + 2.06 , the rfi of rfi in dnr group was 4.27 + 2.06 , rfi of dnr - peg - pll - PLGA group was 4.39 卤 1.89 . Compared with the control group , there was no significant difference in the apoptosis rate ( P0.05 ) .
naked eye observation showed no hemolysis ;
LDso is 464.4 mg / kg , 95 % confidence interval is 399 - 541.8 mg / kg , and the nanoparticles have a wide range of safety values ;
Tissue pathology HE staining showed that there was no significant difference in structure and pathological changes compared with the control group .
In vitro and in vivo , PEG - PLL - PLGA nanoparticles ( PEG - PLL - PLGA ) modified with PEG - PLL - PLGA nanoparticles were used to study the antitumor effect of tumor . The expression of P - gp , MRP and NF - 魏B mRNA and protein in nude mice was detected by high performance liquid chromatography ( HPLC ) .
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.53

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