【作者】 胡云霞；

【导师】 常津；

【作者基本信息】 天津大学 ， 材料学， 2004， 硕士

【摘要】 本文以聚乳酸-乙醇酸共聚物（PLGA）和自行制备的 O-羧甲基壳聚糖（O-CMC）为原料，分别以 5-氟尿嘧啶为抗癌药物模型，以反义 EGFR 为基因药物模型，采用自身设计的改良复乳法制备了载药和载基因纳米微粒；并在此基础上构建与评价了同载抗癌药物与基因的复合功能纳米药物载体系统。 各种纳米微粒表征结果显示：（1）以 O-CMC 和 PLGA 为原料制得的空载纳米粒子平均粒径为 222.9nm，粒径分布指数为 0.109，ζ电位为 73.46eV，表面氮元素含量为 10.3%，具有明显的核壳结构和良好的表面亲水性；（2）以 5-氟尿嘧啶（5-FU）为抗癌药物模型制得的载药纳米微粒平均粒径为 98.5nm，粒径分布指数为 0.192，粒子表面ξ电位为 61.48eV，表面 N 元素含量为 12.2%，对 5-FU的包载率高达 18.9%；（3）以反义 EGFR 为基因药物模型制得的表面吸附加内部包载基因的纳米微粒平均粒径为 375.3nm，粒径分布指数为 0.263，ζ电位为-10.67eV。 本文用 SEM 动态监测各种纳米粒子降解过程中表面形貌的变化，并连续追踪各种纳米粒子降解过程中的质量损失和降解介质的 pH 变化。载 5-FU 纳米粒子在 PBS 中的释药行为研究表明：（1）前 12 小时的释药动力学符合 Huguchi 方程，具有一级释放特性；（2）在 20 天内的释药动力学符合零级释放特性。作者创新构建的表面吸附加内部包载基因的纳米微粒释药动力学方程也具有零级释放动力学特性。 体外细胞存活率实验和体内动物实验均证实了 PLGA/O-CMC 纳米粒子有较好的生物相容性；MTT 和细胞凋亡实验结果表明载药纳米粒子和载基因纳米微粒对 TJ905 脑胶质瘤细胞增殖有明显的抑制作用；免疫组化实验进一步证实包载基因的纳米微粒能有效地抑制胶质瘤细胞中 EGFR 的表达，从而抑制 TJ905 人脑胶质瘤细胞的增殖。最后用荧光相差显微镜和共聚焦显微镜动态监测了纳米粒子的细胞转染过程，发现纳米粒子可在不同时间内进入细胞浆和细胞核。 本文构建的表面吸附基因且内部同时包载 5-FU 与基因的复合功能纳米微粒对基因的包载量显著提高，并对肿瘤细胞有较高的抑制率，表明复合功能纳米微粒中各组分对肿瘤细胞增殖的抑制具有明显的协同作用。更多还原

【Abstract】 The traditional double emulsion (water-in-oil-in-water) method to preparehydrophilic drug-loaded nanoparticles (NPs) was modified and improved to getsmaller NPs and high encapsulation efficiency of drug; Poly (D, L-lactide-co-glycolicacid) (PLGA) and o-carboxymethyl chitosan (O-CMC) were chosen tomicroencapsulate 5-fluorouracil (5-FU) and antisense-EGFR cDNA by improvedW/O/W method due to their attractive degradation and physicochemical properties;So 5-FU and antisense-EGFR-loaded PLGA/O-CMC NPs were created to realize thecombination of anticancer drug and gene in one kind of biodegradable nanocarrier. The results of characterizing all kinds of nanoparticles showed that: (1) meansize of O-CMC-coated PLGA NPs without drug is 222.9nm, polydispersity is 0.109,Zeta potenial is 73.46eV, N percentage on the surface of NPs is 10.3%, hydrophilicityof NPs is very good; （2）mean size of 5-FU-coated PLGA/O-CMC NPs is 98.5nm,polydispersity is 0.192, Zeta potenial is 61.48eV, N percentage on the surface of NPsis 12.2%,5-FU-loading level is 18.9%; (3) mean size of antisense-EGFR-encapsulatedPLGA/O-CMC NPs while absorbing antisense-EGFR is 375.3nm, polydispersity is0263, Zeta potenial is –10.67eV. SEM was used to study the morphological change of NPs during degradation, andweight loss of NPs and pH change of degradation medium were traced. Researches of5-FU release behavior from NPs showed that (1) release kinetics of 5-FU from NPs inearly 12 hours was coincidence with Huguchi release; （2）release kinetics in 20 dayswas coincidence with Zero-level release. Release kinetics ofantisense-EGFR-encapsulated PLGA/O-CMC NPs while absorbing antisense-EGFRwas accordance with Zero-level release. Cell viability in vitro as well as in vivo demonstrated that biocompatibility ofPLGA/O-CMC NPs was good. Results of MTT and cell apoptosis illustrated that both5-FU-encapsulated NPs and antisense-EGFR-loaded NPs had high cytotoxicity onbrain tumor cells TJ905, and immunohistochemical staining approved thatantisense-EGFR-loaded NPs had inhibitory effect on the expression of EGFR ofhuman gliomas cells. Fluorescence microscope and confocal microscope were used totrace the procedures of cell transfection induced by NPs. It was found that there weresome NPs were in nucleus after 24h. Both 5-FU and antisense-EGFR-encapsulated NPs while absorbing EGFR on thesurface of NPs designed by us creatively had high antisense-EGFR loading level andhigh cytotoxicity on gliomas cells (73.6%), which showed that 5-FU andantisense-EGFR had cooperation effect on the inhibitory effect of gliomas cells.更多还原