【作者】 刘丹；

【导师】 唐星； 陈大为；

【作者基本信息】 沈阳药科大学 ， 中药制剂学， 2010， 博士

【摘要】 纳米材料与技术是近年来迅速发展起来的前沿科技领域,其在医药领域的发展引起疾病诊断和治疗的革命,特别是在抗肿瘤研究领域中所展现出极大的优越性,预示着它在肿瘤的临床治疗中具有广泛的和十分诱人的前景。PAMAM树状大分子作为一种纳米材料,属于非生物材料,无免疫原性及遗传毒性,以其为药物载体和基因载体对肿瘤进行靶向治疗的临床应用前景广阔。为了更好控制树状大分子包裹药物释放过快,更好发挥主动靶向给药治疗中的优势,维持树状大分子本身固有的与肿瘤细胞亲和性,本课题利用半乳糖(galactose, GAL)修饰的pH敏感聚乙二醇-低聚磺胺二甲氧嘧啶(PEG-b-PSD) PAMAM聚离子复合胶束作为抗癌药物的载体。为达到pH敏感释药及靶向传递,本研究合成了新型靶向辅料半乳糖修饰聚乙二醇-聚磺胺二甲氧嘧啶(LA-PEG-b-PSD),经IR、NMR和GPC等初步鉴定为目的产物。在此基础上,对LA-PEG-b-PSD的溶解度进行测定。PSD的溶解度约为10μg/mL,经过与三种分子量PEG的连接,合成产物的溶解度有明显增加。并且三种合成产物在水中的饱和溶解度随着PEG链长度的增加而略有增加。在大于pH7.0的碱性环境下,溶解度增加更加显著。电位滴定法测得PSD的pKa为7.0,PEG的分子量并没有明显改变PEG-b-PSD及LA-PEGn-b-PSD的pKa。采用溶剂挥发法,制备了载脱盐酸阿霉素树状大分子(PAMAM/DOX):仲用正负电荷吸引的复合方法,制备了乙二醇-低聚磺胺二甲氧嘧啶／树状大分子复合物胶束(LA-PEG-b-PSD/PAMAM)。考察了ζ电位、电荷比、PAMAM/DOX摩尔比、离子强度等对LA-PEG-b-PSD/PAMAM和其载药能力的影响。采用透射电镜观察LA-PEG-b-PSD/PAMAM的形态为类球形,粒径为50nnm。体外不同pH释放实验证明,DOX从LA-PEG-b-PSD/PAMAM中的释放具有pH敏感性。MTT实验结果表明,空白LA-PEG-b-PSD/PAMAM对于HepG2细胞及Hela细胞的细胞毒均小于空白PAMAM。使用家兔红细胞溶血实验证明,所用制剂生物相容性好,无明显的溶血作用。载DOX的LA-PEG-b-PSD/PAMAM及PEG-b-PSD/PAMAM对HepG2细胞的细胞毒作用受到pH值的的影响。且半乳糖的介导作用可提高制剂的细胞毒作用。流式细胞仪、共聚焦显微镜测定发现,LA-PEG-b-PSD/PAMAM复合物具有较好的肝细胞靶向性。利用细胞摄取实验,定量分析了不同条件下DOX被肿瘤细胞摄取的情况。采用荷有H22的荷瘤小鼠为动物模型,考察了LA-PEG-b-PSD/PAMAM/DOX及其参比制剂的体内药效学。与DOX溶液组相比,其余所有参比制剂都表现出更高的抑瘤率,其中LA-PEG-b-PSD/PAMAM/DOX组和PEG-b-PSD/PAMAM/DOX组的抑瘤率增加最为明显,分别为80.87%和66.07%,这一结果说明本课题所设计的pH敏感复合物确实比普通PAMAM和PEG-PAMAM具有更好的靶向效果,再加上半乳糖主动寻靶的作用起到了极明显的抑制肿瘤生长的作用。使用病理组织切片染色,考察了药物对肿瘤的杀伤作用。使用生化试剂盒,测定了肿瘤组织中caspase-8与caspase-3的含量,结果显示,抑瘤率高的制剂组相应的caspase-8与caspase-3的含量也较高。制备了LA-PEG-b-PSD/PAMAM/DOX及其各种参比制剂,在荷有H22的荷瘤小鼠体内靶向性实验发现,LA-PEG-b-PSD/PAMAM改变了阿霉素分布情况,在肿瘤中阿霉素的浓度大于PEG-PAMAM/DOX (P<0.01), Te比为2.91,证明半乳糖介导的pH敏感长循环树状大分子复合物胶束增强了药物在肿瘤组织的靶向性。通过测定大鼠单剂量静脉注射LA-PEG-b-PSD/PAMAM/DOX和其参比制剂,测定了DOX在大鼠体内的药物动力学参数。LA-PEG-b-PSD/PAMAM/DOX组,PEG-b-PSD/PAMAM/DOX组,PEG-PAMAM/DOX组,PAMAM/DOX,DOX组t1/2分别为4.59±1.68 h,6.86±1.09 h,8.66±1.78 h,2.31±0.74h,2.67±1.42h; AUC0-24为271.34±52.29μg·h/mL,433.12±57.47μg·h/mL, 576.52±67.21 h,191.18±34.56 h,125.26±33.21 h。更多还原

【Abstract】 Nano-materials and technology developed rapidly in recent years. its development in the field of medicine diagnosis and treatment of diseases was important, especially in the field of anti-tumor study. It’s development in clinical treatment of cancer has a broad and very attractive prospect. PAMAM dendrimers used as a drug delivery system was non-toxic, no-immunogenic and biodegradable. PAMAM dendrimers have shown great advantages as carriers for gene delivery and anticancer therapies.In order to enhance the tumor site targetting ability andmaintain the dendrimer inherent affinity with the tumor cells, an pH-sensitive PAMAM complexes modified with lactose were prepared with PAMAM dendrimers and LA-PEG-b-PSD in this paper.Lactose -poly (ethylene glycol)-poly (methacryloyl sulfadimethoxine) diblock copolymer (LA-PEG-b-PSD) were synthesized to produce a novel pH sensitive polymer with a mono-galactoside moiety. The chemical structure was characterized by IR, NMR and GPC. The solubility in water of LA-PEG-b-PSD was investigated, the solubility of PSD was 10μg/mL.An increase in the solubility was observed in LA-PEG-b-PSD, and this increase was affected by the the molecular weight of PEG. Moreover, when the pH of the solution higher than 7.0, the solubility f LA-PEG-b-PSD increased significantly. The apparent pKa values of SD, PSD, PEG-b-PSD and LA-PEG-b-PSD were investigated by potentiometric titration. the pKa of PSD, PEG-b-PSD and LA- PEG-b-PSD was about 6.97,7.04 and 6.95, respectively, which is close to the acidic conditions at the tumor sites.The LA-PEG-b-PSD/PAMAM complexes were prepared with LA-PEG-b-PSD carrying a negative charge and PAMAM dendrimers carrying a positive charge. The DOX was encapsulated in PAMAM by a hydrophobic interaction. The effect ofζpotential, charge ratio (+/-), molar ratio and ionic strength on the preparation of LA-PEG-b-PSD/PAMAM complexes was studied. The morphology of LA-PEG-b-PSD/PAMAM complexes was observed using TEM and the particle size was 50nm. The drug release study in vitro indicated that the release behavior was affected by the pH value.The MTT assay showed that the blank LA- PEG-b-PSD/PAMAM complexes have lower cytotoxicity against HepG2 cells and Hela cells. The hemolysis experiments using rabbit red blood cells indicated that all the formulations were not hemolysis.The LA- PEG-b-PSD/PAMAM/DOX complexes showed higher cytotoxicity against HepG2 cells. Their growth-inhibiting activity was higher than PEG-b-PSD/PAMAM/DOX complexes and PAMAM/DOX at pH 7.4 after 48h incubation, and this advantage was not observed at pH 6.5. The flow cytometry and the confocal laser scanning microscopy were also used to study the pH sensitive and targeting ability of LA- PEG-b-PSD/PAMAM/DOX complexes, the results indivated that the lactose moiety and PSD can enhance the liver tumor cells targetting ability of LA- PEG-b-PSD/PAMAM/DOX complexes. To study the intracellular kineties behavior,the HPLC methord was established to determine the concentration of DOX in HepG2 cells.Using H22 tumor-bearing mice, the anti-tumor efficacy of DOX in different formulations was evaluated. All of the treatment groups produced a significant anti-tumor effect compared with the physiological saline control group (P< 0.05). The treatment with LA-PEG-b-PSD/PAMAM/DOX complexes and PEG-b-PSD/PAMAM/DOX complexes inhibited primary tumor growth compared with other control formulations, the relative tumor inhibition was 80.87% and 66.07% separately.The results indicated that the pH sensitive complexes exhibited better antitumor activity than PAMAM/DOX group and PEG-PAMAM group, furthermore, the LA-PEG-b-PSD/PAMAM/DOX complexes showed higher anti-tumor efficacy than non-lactose PEG-b-PSD/PAMAM/DOX complexes. The pathology of the tumor was investigated to anti-tumor effect of drugs. The expression level of the caspase-8 and caspase-3 was measured by biochemical kits, the results show that the contents of caspase-8 and caspase-3 in the tumor of LA-PEG-b-PSD/PAMAM/DOX complexes group were higher.To test the targetability in KM mice bearing H22 cells, LA-PEG-b-PSD/PAMAM/DOX complexes was prepared. It was found that the DOX distribution was changed after treated with LA-PEG-b-PSD/PAMAM/DOX complexes. The DOX concentration in tumor treated with LA-PEG-b-PSD/PAMAM/DOX complexes was significantly greater than PEG-PAMAM/DOX, the ratio of Te was 2.91. Results suggested the targetability was enhanced when using LA-PEG-b-PSD/PAMAM/DOX complexes. The pharmacokinetics parameters of LA-PEG-b-PSD/PAMAM/DOX complexes and other formulations were studied in wastar rat. For LA-PEG-b-PSD/PAMAM/DOX complexes, PEG-b-PSD/PAMAM/DOX complexes, PEG-PAMAM/DOX, PAMAM/DOX and DOX, the t1/2 value were 4.59±1.68 h,6.86±1.09 h,8.66±1.78 h, 2.31±0.74 h,2.67±1.42h; AUC0-24为271.34±52.29μg·h/mL,433.12±57.47±ng·h/mL, 576.52±67.21 h,191.18±34.56 h,125.26±33.21 h, respectively.更多还原