可改善药物功效的新型药用材料亚油酸和聚苹果酸修饰的壳聚糖类衍生物研究

项目名称： 可改善药物功效的新型药用材料亚油酸和聚苹果酸修饰的壳聚糖类衍生物研究

项目编号： No.30873204

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 印春华

作者单位： 复旦大学

项目金额： 32万元

中文摘要： 药物载体材料是改善药物功效的给药系统研究的关键，尤其是抗肿瘤药物和基因借助递送载体可提高药物的靶向性和功效、增强基因药物的体内转染效果。本项目设计研究新型亚油酸和聚苹果酸修饰的壳聚糖(LMC)；研究其生物相容性和安全性；以紫杉醇(PTX)为模型药物研究其提高难溶性药物溶解度、抗肿瘤药物靶向性及功效；以绿色荧光蛋白表达质粒(pEGFP)为模式质粒研究经LMC递送增强基因药物体内外转染效果。结果表明：LMC在水中可自组装形成纳米粒，平均粒径为190～350 nm，Zeta电势为-2～-20 mV；LMC具良好的生物相容性和安全性；LMC纳米粒包载难溶性药物PTX的包封率超过70%，载药量达9.9%；叶酸和生物素修饰可选择性增强肿瘤细胞的摄取、对肿瘤组织的靶向性及抑瘤效果。 LMC/pEGFP纳米复合物可显著提高pEGFP抗酶解能力、抑制非特异性蛋白吸附能力、细胞黏附、细胞摄取、细胞质中pEGFP释放量、细胞核质粒分布百分率和体内外基因转染效率。LMC有望成为一种抗肿瘤药物新型给药载体及高效、稳定、安全的非病毒基因递送载体。上述研究为可改善药物功效的药用材料的研究提供理论依据和技术基础。

中文关键词： 两亲性壳聚糖衍生物纳米粒；生物相容性；递送载体；抗肿瘤药物；基因

英文摘要： Drug delivery vehicles are the key factors which decide the improved efficacy provided by drug delivery systems, especially for antitumor drug which can be enhanced the targeting and therapeutic efficacy and for gene that can be enhanced the in vitro and in vivo gene transfection efficiency. In the present study, linoleic acid (LA) and poly(malic acid) double-grafted chitosan (LMC) was prepared through acrylation reaction. FTIR, 1H NMR, XRD, DSC, and elemental analysis confirmed the synthesis of LMC. The biocompatibility and safety of LMC were evaluated. The improvement of solubility of insoluble drug as well as the targeting and therapeutic efficacy of paclitaxel (PTX) loaded LMC nanoparticles was investigated. Model plasmids that expressed green fluorescent protein (pGFP) were encapsulated in LMC nanoparticles to investigate in vitro and in vivo gene transfection efficiency. The LMC with good biocompatibility and safety was obtained. The resultant amphiphilic LMC self-assembled into nanoparticles in water. Average particle size of LMC and its derivatives nanoparticles at pH 7.4 was 190-350 nm, and Zeta potential was -2~-20 mV. Particle size and Zeta potential of the LMC nanoparticles were sensitive towards pH values. Loading efficiency of LMC and its derivatives nanoparticles for PTX was above 70%, and the loading capacity increased with LA substitution degree which reached maximal value of 9.9%. The tumor inhibition rate (TIR) was: Folic acid modified PEG-LMC (FA-PEG-LMC)> Biotin modified PEG-LMC (BT-PEG-LMC)> PEG modified LMC (PEG-LMC)>LMC>PTX solution, with TIR of 82.5% and 80.6% for FA-PEG-LMC and BT-PEG-LMC, respectively. Cellular uptake study of LMC and LMC derivatives nanoparticles were performed in SMMC-7721 cells and HEK-293 cells to evaluate the in vitro tumor targeting effect. The uptake amount of FA-LMC and BT-LMC nanoparticles was 3-5 fold and 2-3 fold of that of LMC nanoparticls and the uptake amount of FA and BT modified LMC nanoparticles in HEK-293 was significantly lower than that of SMMC-7721. LMC nanoparticles were 150-300 nm in diameters, and Zeta potential measurements indicated a positive surface charge at pH 5.5 which enabled the encapsulation of anionic pEGFP. LMC/pEGFP nanocomplexes demonstrated higher pEGFP protection capability as compared to chitosan/pEGFP nanocomplexes. Cell adsorption and cellular uptake percentages were promoted 0.8-3.8 and 1.7-3.2 folds by LMC/pEGFP nanocomplexes compared to chitosan/pEGFP nanocomplexes, respectively. LMC/pEGFP nanocomplexes mediated the highest gene expression, and the transfection percentages reached 34.5% at the weight ratio of 12, which were 8.0-folds higher than those of chitosan/pEGFP nanocomplexes on HEK293 cells. In vivo transfection effiency of LMC/pEGFP nanocomplexes was evaluated through intramuscular administration in mice. The results showed that the highest EGFP expression was mediated by LMC/pEGFP nanocomplexes (36.62±10.66 I/mg), which were 4.2-folds and 2.2-folds higher than those of PEI/pEGFP and chitosan/pEGFP nanocomplexes. Therefore, the LMC nanoparticles could be an effective and safe vehicle for hydrophobic antitumor drugs and gene. These results could provide the theoretical and technical bases for investigation of drug vehicles with improved efficacy.

英文关键词： Amphiphilic chitosan derivative nanoparticles; Biocompatibility; Delivery vehicles; Antitumor Drug; Gene