【作者】 唐国涛；

【导师】 裴元英； 姜嫣嫣；

【作者基本信息】 复旦大学 ， 药剂学， 2009， 博士

【摘要】 恶性肿瘤已经成为威胁人类健康和生命的最主要疾病之一,其发病率每年新增近1千万例。近几年对肿瘤生理深入的探索,促进了肿瘤诊断和治疗的发展,其死亡率有所下降。目前肿瘤的治疗手段包括手术治疗,放疗和化疗等,放化疗在杀死肿瘤细胞的同时也能杀死正常细胞,给病人带来严重的毒副作用。因此研制更有效地靶向肿瘤细胞的给药系统显得尤为迫切。被动靶向释药系统成功利用肿瘤的生理特征使载药系统通过EPR效应在肿瘤部位蓄积。药物聚合物纳米载体系统由于其良好的可调控性、易于表面修饰、较高的药物包封率、较强的药物保护作用和适于大规模生产,已成为研究热点之一。虽然被动靶向释药奠定了靶向给药临床治疗的基础,但仍存在难以克服一些问题,如并不是所有的肿瘤都能体现出EPR效应,肿瘤细胞的多药耐药现象使部分药物不能有效地进入肿瘤细胞等等。要克服这些困难,则需要使载药系统主动地靶向肿瘤细胞,主动靶向可以通过与细胞表面特定的受体相结合的配体或抗体来实现。本课题通过药剂学、高分子材料学、药理学、分子生物学等手段的交叉应用,以喜树碱（CPT）为模型药物,转铁蛋白（Tf）为靶向头基,首先制备载CPT的主动靶向隐形纳米载药系统,将聚乙二醇（PEG）修饰的被动靶向与Tf修饰的主动靶向作用相结合,有效递送药物到达肿瘤部位。本课题以琥珀酸酐为连接剂,首先合成了琥珀酰喜树碱酯,并用N-羟基琥珀酰亚胺（NHS）活化。用带有双功能基团的NHS-PEG-MAL分子上的NHS端与PAMAM上的氨基相连,合成了三种不同PEG化程度的PAMAM-PEG(PP₁₆,PP₃₂和PP₄₈),通过¹H NMR计算PAMAM所连接的PEG数量并测定了粒径和Zeta电位。喜树碱琥珀酸活化酯与PAMAM表面剩余的氨基共价连接,合成了CPT-PAMAM-PEG纳米载药系统(CPP₁₆,CPP₃₂和CPP₄₈)。药物的体外释放显示出缓释的特性（120小时内累积释放75%）。用Traut’s试剂将转铁蛋白巯基化,合成了转铁蛋白修饰的主动靶向载药纳米复合物CPT-PAMAM-PEG-Tf(CPP₁₆T,CPP₃₂T和CPP₄₈T),采用bradford法测得平均每个PAMAM复合物连接1分子转铁蛋白。连接转铁蛋白后复合物的粒径增加了近一倍,Zeta电位由正负。转铁蛋白修饰的CPT复合物较CPT原料药和未经转铁蛋白修饰的复合物对KB,K562和S180细胞显示出更强的抗肿瘤活性。细胞摄取实验显示转铁蛋白修饰的PAMAM-PEG复合物在KB、K562细胞中的摄取量分别为未经Tf修饰复合物的3.38～3.46倍和2.52～2.86倍。实验显示转铁蛋白修饰的PAMAM-PEG复合物在KB细胞中的摄取具有浓度依赖性,且转铁蛋白可以明显抑制复合物的摄取,说明其通过细胞表面高表达的转铁蛋白受体介导的内吞进入细胞。以异硫氰酸罗丹明B（RBITC）为荧光标记物,合成了带荧光标记物的RPP和RPPT。采用各种内吞抑制剂对复合物的内吞机制进行了初步探讨,结果显示RPP₄₈和RPP₄₈T都是由细胞表面的网格蛋白介导内吞,RPP₄₈T以细胞表面的转铁蛋白受体介导的内吞为主。用Lysotracker green对细胞溶酶体进行染色,激光共聚集显微镜观察表明复合物内吞后经溶酶体途径转运。采用Hochest和PI双染对低浓度CPT复合物诱导肿瘤细胞凋亡的研究发现,CPT,CPP₄₈和CPP₄₈T 12h诱导细胞早期凋亡率分别为0.76%,2.41%和4.63%,24h分别为4.75%,16.02%和30.91%。说明CPP₄₈T被肿瘤细胞摄取最多,诱导凋亡的效果最好。大鼠体内药动学显示复合物中PEG化程度越大,复合物在体内的半衰期和平均滞留时间越长,AUC也越大,但是经转铁蛋白修饰的复合物较未经Tf修饰的复合物的半衰期短。S180荷瘤小鼠体内的组织分布结果表明CPP₄₈T给药后药物在肿瘤内的达峰浓度最高,分别是CPT和CPP₄₈的8.4和2.7倍,AUC分别是CPT和CPP₄₈的28.7和3.6倍。CPP₄₈T的相对靶向效率最高（28.7）,显示CPP₄₈T的肿瘤靶向性最强。对S-180荷瘤小鼠的药效研究结果表明,CPP₄₈T具有最强的抗肿瘤活性,其肿瘤抑制率为65.7%,分别是CPT和CPP₄₈的1.81和1.27倍,显出主动靶向与被动靶向相合的优势。荷瘤小鼠的体重变化和小鼠小肠的组织病理切片显示其没有明显的毒副作用。肿瘤组织切片免疫组化结果显示,CPP₄₈T肿瘤细胞增殖最少,细胞凋亡最明显,表明CPP₄₈T具有最强的抗肿瘤活性。更多还原

【Abstract】 Cancer remains one of the world’s most devastating diseases,with more than 10 million new cased very year.However,mortality has decreased in the past two years, owing to better understanding of tumor biology and improved diagnostic devices and treatments.Current cancer treatments include surgical intervention,radiation and chemotherapeutic drugs,which often also kill healthy cells and cause toxicity to the patient.It would therefore be desirable to develop chemotherapeutics that can either passively or actively target cancerous cells.Passive targeting exploits the characteristic features of tumor biology that allow nanocarriers to accumulate in the tumor by the enhanced permeability and retention（EPR） effect.Polymeric drug delivery system has been one of the hot research fields not only because of its tremendous versatility in polymer matrices allowing for tailoring of the system properties to meet the specific intended need,but also its ease of surface modification, high encapsulation efficiency of the drug,drug protection,control over the release of drugs,and feasibility of scale-up and manufacturing.Although passive targeting approaches form the basis of clinical therapy,they suffer from several limitation because certain tumor do not exhibit the EPR effect and some drugs cannot diffuse efficiently for multiple-drug resistance.One way to overcome these limitations is to programme the nanocarrers so they actively bind to specific cells.This binding cay be achieved by attaching targeting agents such as ligands molecules that bind go specific receptors on the cell surface.In this study,by means of pharmaceutics,macromolecular chemistry, pharmacology and molecular biology,CPT loaded transferrin modified PEG-PAMAM dendrimers were first prepared for effective drug delivery to solid tomor by the combination of passive and active targeting.succinic anhydride as linker,We first synthezised Camptothecin-hemisuccinate. The remain carboxyl group was activated by N-Hydroxysuccinimide.The primary amino groups on the surface of PAMAM were specifically reacted with the NHS groups of the bifunctional PEG derivative.Three different PEG proportion conjugates PP₁₆,PP₃₂ and PP₄₈ were obtained and the average number of PEG chains attached PAMAM were 10.5,20.2 and 28.8 calculated through ¹H NMR spectra.The average diameters of three conjugates were 7.12nm,7.34nm and 8.56nm,respectively and the Zeta potential were 4.84mV,4.43mV and 1.90mV.Camptothecin-SA-NHS ester were reacted with the remains amino groups on the surface of PAMAM,named CPP₁₆, CPP₃₂ and CPP₄₈.The diameter and Zeta potential were also measured by Zetasizer Nano.The release profile of CPT from conjugates showed a slower and sustained fashion（75%of CPT was released within 120 h）.Tf was thiolated using Traut’s reagent and the extent of thiolation was determined by Ellmann’s reagent.The MAL groups of PEG were specifically reacted with the thiol groups of thiolated Tf.The average number of Tf per conjugate was about 1.0 using Braford method.The diameter of CPP₁₆T,CPP₃₂T and CPP₄₈T were lager about 1-fold and the Zeta potential became negative.Tf modified CPT-conjugates displayed the stronger tumor cell cytotoxicity compared with CPT solution and no Tf modified CPT-conjugates in KB,K562 and S180 cells.The mean fluorescence intensity were determined during flow mytometric analysis of the cellular uptake of conjugates against KB cell,K562 cells and S180 cells.The uptake of Tf modified CPT-conjugates were 3.38～3.46 fold in KB cells and 2.52～2.86 fold in K562 cells compared with no Tf modified CPT-conjugates.The uptake of conjugates by KB cells was concentration dependent,which could be inhibited by excess free Tf in the medium,strongly indicating that the uptake mechanism being an energy-driven,transferrin receptor mediated endocytosis process.The endocytosis mechanism of Tf modified conjugates were evaluated.The results of cellular inhibition showed that CPP₄₈ and CPP₄₈T could be taked up by KB cells via clathrin -dependent endocytosis.CPP₄₈T was major mediated via transferrin receptor and Lysosome apparatus were involvement in the cellular distribution.KB cells were incubated with rhodamine labeled conjugates in the presence of lysotracker green,a marker for secondary endosomes and lysosomes,colocalization of rhodamine labeled conjugates in late endosome and lysosome were observed,indicating that cellular transport of conjugate depends on the lysosome process.To measure the apoptosis effect of CPT and conjugates quantificationally, Hochst33342 and PI was used to double stain KB cells.When cells were treated with CPT or conjugates with 1Nm for 12h,the early apoptosis of CPT,CPP₄₈ and CPP₄₈T were 0.76%,2.41%and 4.63%,4.75%,16.02%and 30.91%for 24h,respectively, which was consistent with the above results and indicated that CPP₄₈T induce more apoptosis due to more uptake of CPT,and produce higher cytotoxicity than CPP₄₈ and CPT.The pharmacokinetic study in rats showed that The more PEG chains on surface of the conjugates,the longer t_1/2 of the CPT were observed.However,the half life of conjugates became shorter than that of Tf modified conjugates.The tissue distribution was investigated on S180 tumor bearing mice.CPP₄₈T showed the highest tumor concentration(8.4-fold and 2.7-fold that of CPT solution and CPP₄₈,respectively),largest AUC in tumor(28.7-fold and 3.6-fold that of CPT solution and CPP₄₈,respectively).Good tumor targeting effect of CPP48T was showed by the parameters.Relative targeting efficiency（r_e） was 28.7CPP₄₈T showed the most powerful anti-tumor activity of with the inhibition rate of 65.7%against S180 tumor in mice（1.81-and 1.27-fold that of CPT solution and, respectively）,which was attributed to the double effect from passive and active targeting.No significant side effects were observed by weight changes of the mice and the histological examination of the small intestine.Immunohistochemistry of the tumor tissues showed that CPP₄₈T had highest antitumor effects（inhibiting the tumor cells proliferation and inducing the tumor cells apoptosis）.更多还原