【作者】 朱赛杰；

【导师】 裴元英； 姜嫣嫣；

【作者基本信息】 复旦大学 ， 药剂学， 2010， 博士

【摘要】 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,首先以聚酰胺-胺树枝状聚合物(Polyamidoamine dendrimer, PAMAM)为聚合物骨架材料,以聚乙二醇(Polyethylene glycol, PEG)为高分子修饰剂,制备不同PEG化程度的PEG-PAMAM,然后将模型药物阿霉素(Doxorubicin, DOX)通过酸敏感的顺式乌头酸酐(Cis-acotonic anhydride, CA)和非酸敏感的丁二酸酐(Succinic anhydride)共价结合到PAMAM表面,制备PEG-PAMAM-DOX偶联物纳米载药系统,通过考察PEG化程度和DOX偶联方式对载药系统体内外活性的影响,优选出PEG化程度最高的,并通过顺式乌头酸酐偶联DOX的PPCD 32/1作进一步研究。为进一步提高PPCD的抗肿瘤活性,以相同分子量的双功能PEG(MAL-PEG-NHS)代替单功能PEG,通过控制投料比制备与PPCD 32/1的PEG化程度和载药量相近的主动靶向给药系统RGD-PPCD,考察偶联RGD多肽对PPCD的体内外活性的影响,并探讨其可能的机理。第一章的主要内容是PEG-PAMAM-DOX的合成和表征。首先以分子量为5000的MeO-PEG-NHS和4代PAMAM为起始原料,制备三种PEG化程度的PEG-PAMAM,分别为PEG-PAMAM 4/1、16/1和32/1。通过1H-NMR峰面积归一化法计算得到每个PAMAM分子表面共价结合了3.8、12.9和20.1个PEG分子。分别采用TLC、UV、1H-NMR、FTIR、GPC等方法对三种PEG-PAMAM进行结构确证和表征。接着通过酸酐的氨解反应分别制备DOX的顺式乌头酸酐衍生物(CAD)和丁二酸酐衍生物(SAD),两者经过活化后分别与三种PEG化程度的PEG-PAMAM偶联,制备PPCD和PPSD。采用UV和GPC等方法对PPCD和PPSD(统称PEG-PAMAM-DOX)进行表征,并测定粒径和Zeta电位。各PEG-PAMAM-DOX的载药量在6.18-18.18%(wt.%),每个PAMAM分子表面偶联约14个DOX分子,游离CAD/SAD的含量小于0.6%(mol%),粒径均在纳米级的范围内,Zeta电位随PEG化程度增加而降低,且PPSD的电位高于PPCD。第二章对PEG-PAMAM-DOX的体内外活性进行评价。酸敏感释放实验表明PPCD具有酸敏感特性,DOX的释放程度和速度均随着PEG化程度的增加以及pH值的降低而增加,PPSD则在各pH条件下均没有DOX释放；以Cathespin B模拟溶酶体的酶环境,释放结果表明PPCD和PPSD均不能通过酶解释放游离DOX。各PEG-PAMAM和PEG-PAMAM-DOX的溶血毒性均显著降低。以SKOV-3和B16细胞为肿瘤细胞模型,PPCD对两种细胞的毒性高于PPSD,且PPCD的细胞毒性随PEG化程度的增加而增加。两种细胞对PPCD和PPSD的摄取均随PEG化程度的增加而降低。SKOV-3细胞对PEG-PAMAM-DOX的摄取机理研究结果表明PEG-PAMAM-DOX主要通过与细胞膜上的负电荷发生静电相互作用,然后经网格蛋白介导的内吞摄取。细胞内分布研究证明了PPCD能在溶酶体酸性环境中释放游离DOX进入细胞核,PPSD虽然也定位于溶酶体,但不能释药。第二章的第二节进一步考察了各PEG-PAMAM-DOX的组织分布和药效学。首先采用活体荧光成像实验初步考察各PEG-PAMAM-DOX在荷SKOV-3肿瘤组织的分布,结果发现肿瘤蓄积随PEG化程度的增加而增加,PPSD的蓄积高于PPCD。采用FLD-HPLC法测定在各组织中游离和结合的DOX浓度。DOX与PEG-PAMAM偶联后,在血液中的滞留时间显著延长,主要表现为AUC, T1/2β和MRT显著增加,Vc和CL大大减小；组织分布特性也发生显著改变,以总DOX计,PPSD 4/1、PPSD 16/1、PPSD 32/1、PPCD 4/1、PPCD 16/1和PPCD 32/1在肿瘤部位的AUC分别为DOX溶液剂组的16.6、51.4、75.8、11.6、21.9和37.5倍。以游离DOX计,PPCD在肿瘤部位AUC大于PPSD, PPCD 32/1和16/1的游离DOX的AUC大于DOX溶液剂组,分别达到35.0和15.5h·μg/g。各PEG-PAMAM-DOX均能有效抑制肿瘤生长,延长小鼠的生存时间,其中PPCD 32/1具有最强的抗肿瘤活性,接种21天后的抑瘤率达到87.4%,平均生存时间和中位生存期最长,生存期延长率最大。第三章对抗肿瘤活性最大的PPCD32/1进一步用RGD多肽修饰,制备主动靶向载药系统。首先通过分子模拟的方法对所设计的环状多肽RGDyC的受体亲和力进行评估；在此基础上通过双功能的MAL-PEG-NHS分别偶联靶向头基RGDyC和载体PAMAM,制备RGD-PEG-PAMAM；最后将CAD偶联到PAMAM表面得到RGD-PPCD。表征结果表明,RGD-PPCD中每个PAMAM分子表面偶联有21.2个PEG分子、16.8个RGDyC分子和14.2个CAD分子,粒径为17.02±0.13nm, Zeta电位为2.31±0.15mV。RGD-PPCD与PPCD具有相近的PEG化程度、载药摩尔数、粒径和Zeta电位,可用于进一步研究考察偶联RGD对PPCD体内外活性的影响。第四章评价了RGD-PPCD的体内外活性。体外释放研究证明了偶联RGD对酸敏性几乎没有影响。以人脐静脉内皮细胞HUVEC作为肿瘤新生血管内皮模型,SKOV-3和B16细胞为肿瘤细胞模型,进行细胞水平的研究。偶联RGD进一步增强了PPCD对三种细胞的毒性,HUVEC、SKOV-3和B16细胞的IC50值分别从8.24μM、20.58μM和15.64μM降低至3.58μM、11.04μM和4.37μM。与PPCD相比,HUVEC细胞对RGD-PPCD的总摄取增加了1.57倍；SKOV-3细胞的总摄取和内吞分别增加了1.32倍和1.58倍；B16细胞的总摄取和内吞分别增加了1.38倍和1.85倍。摄取机理研究结果表明,RGD-PPCD主要通过非特异性的静电相互作用和特异性的整合素αvβ3与RGD多肽相互识别与三种细胞接触,然后主要经网格蛋白介导的内吞被细胞摄取,内吞过程均为肌动蛋白和微管蛋白参与的细胞运动,此外,RGD与整合素αvp3形成的配体受体复合物也对内吞起到促进作用。对RGD-PPCD的细胞内定位研究结果表明,三种细胞摄取RGD-PPCD后均能将其递送至溶酶体,且RGD-PPCD能在溶酶体的弱酸性条件下释放游离DOX,进入细胞核。采用活体荧光成像考察RGD-PPCD在SKOV-3荷瘤裸鼠的组织分布,48h后RGD-PPCD在肿瘤组织的荧光强度为PPCD的1.52倍(P<0.05),游离的RGDyK能显著降低RGD-PPCD在肿瘤部位的荧光强度(P<0.05),证实了RGD-PPCD在肿瘤蓄积的特异性。B16荷瘤小鼠的体内药动学实验结果表明,RGD-PPCD的滞留时间比PPCD稍短。RGD-PPCD在肿瘤组织总DOX和游离DOX的AUC分别是PPCD的1.46倍和2.36倍；另一方面,RGD-PPCD除了能引起肿瘤细胞凋亡外,还能显著引起肿瘤新生血管内皮细胞的凋亡,以上因素的综合作用可能使RGD-PPCD比PPCD具有更高的抗肿瘤活性,平均生存时间,中位生存期和生存期延长率相应增加。HE染色和TUNEL法的结果表明,RGD-PPCD相对于DOX溶液剂表现出良好的安全性,没有心脏毒性,对其他正常组织也几乎无损伤。更多还原

【Abstract】 This study is based on the combined use of pharmaceutics, macromolecular chemistry, pharmacology and molecular biology. For the construction of this polymeric drug delivery system, Polyamidoamine (PAMAM) dendrimer was used as the scaffold which was first modified with Polyethylene glycol (PEG) to produce PEG-PAMAM conjugates with different PEGylation degree. Doxorubicin (DOX) was then conjugated to each PEG-PAMAM either by acid sensitive cis-aconityl linkage or acid insensitive succinic linkage to produce the final product of PPCD and PPSD conjugates, respectively. By evaluating the in vitro and in vivo antitumor activity, PPCD 32/1 which had the highest PEGylation and acid sensitive linkage was found to be the most potent product. To further improve the antitumor activity of PPCD 32/1, RGD peptide was used to modify this polymeric drug delviery system via bifunctional MAL-PEG-NHS. By controlling the feed ratio of starting materials, RGD-PPCD was synthesized with similar PEGylation degree and drug laoding with PPCD 32/1. Subsquent evaluation was performed to study the effects of RGD modification on the antitumor activity of PPCD 32/1 and the underlying mechanism.The first chapter focused on the synthesis and characterization of PEG-PAMAM-DOX conjugates. With MeO-PEG-NH2 (M.W.4834) and G4 PAMAM denderimer as the starting materials, PEG-PAMAM conjugates with three different PEGylation degree were prepared, namely PEG-PAMAM 4/1,16/1 and 32/1. The conjugated number of PEG molecules per PAMAM dendrimer was calculated to be 3.8,12.9 and 20.1 respectively. TLC, UV,’H-NMR, FTIR, GPC were used to confirm the conjugation between PEG and PAMAM dendrimer. DOX was then converted to its cis-aconityl and succinic derivatives by ammonolysis reaction to produce CAD and SAD respectively. The two derivatives were subsequently activated and conjugated to PEG-PAMAM with different PEGylation degree to obtain the final products of PPCD and PPSD conjugates. The conjugated number of DOX molecule was controlled to be about 14, DOX loading percentage was in the range of 6.18-18.18% by weight, and the content of free CAD/SAD was less than 0.6% by mole. The particle of PEG-PAMAM 4/1 increased significantly from -7nm to -80 nm after conjugation of CAD/SAD, which might be due to the aggregation of these particles, while PEG-PAMAM 16/1 and 32/1 displayed slight increase in particle size after DOX conjugation, with 16/1 larger than 32/1. The Zeta potential of PEG-PAMAM-DOX decreased with increasing PEGylation degree, and PPSD showed higher Zeta potential than PPCD conjugates at the same PEGylation degree.The second chapter was about the in vitro and in vivo evaluation of PEG-PAMAM-DOX conjugates. In the first section, we first evaluated the in vitro release of DOX from PPCD and PPSD conjugates. PPCD was characteristic of acid sensitive DOX release, with DOX release increasing with increasing PEGylation and decreasing pH value, while PPSD was acid insensitive with no DOX release at any pH condition. Cathespin B, a major protease in lysosomes, had no effect on drug release of PEG-PAMAM-DOX, suggesting the stability of amide bond between PAMAM and CAD/SAD. When evaluating the heamolytic toxicity of these conjugates, we found significantly reduced heamolytic toxicity after conjugation of PEG and CAD/SAD as compared with G4 PAMAM, which ensured their further i.v. administration. Cellular level experimented were carried out using SKOV-3 and B16 cells as the model cell lines. The cytotoxicity of PEG-PAMAM conjugates decreased with increasing PEGylation, while PPCD conjugates showed the opposite trend, which might be due to the release of free DOX. For the same reason, PPCD were more cytotoxic than PPSD. As to cellular uptake, both cell lines showed decreased uptake of PEG-PAMAM-DOX with increasing PEGylation degree. Further investigation on the internalization mechanism by SKOV-3 cells indicated that PEG-PAMAM-DOX mainly interacted with plasma membrane by electrostatic interaction, and then was internalized by clathrin-mediated endocytosis. The results of intracellular distribution by confocal laser microscopy showed that the slightly acid condition in lysosomes triggered DOX release from PPCD 32/1, and enable the following entry into nucleus, while PPSD 32/1 release no free DOX although it was also located in lysosomes.In the second section, we evaluated the biodistribution and in vivo antitumor activity of these PEG-PAMAM-DOX conjugates. In vivo fluorescence imaging was first used to evaluate the tumor accumulation in SKOV-3 tumor bearing nude mice. The results demonstrated that tumor accumulation increased with increasing PEGylation and PPSD conjugates showed higher tumor accumulation than PPCD conjugates. FLD-HPLC method determining the total and free DOX was established to evaluate the biodistribution of these conjugates in B16 tumor bearing mice. After conjugation with PEG-PAMAM conjugates, DOX displayed significant longer blood retention time, with prolonged AUC, T1/2βand MRT, and reduced Vc and CL. Biodistribution profiles of DOX was also significantly changed. Total DOX AUC of PPSD 4/1, PPSD 16/1, PPSD 32/1, PPCD 4/1, PPCD 16/1 and PPCD 32/1 in tumor was increased by 16.6、51.4、75.8、11.6、21.9 and 37.5 times as compared with DOX solution. Significant RES uptake was observed at lower PEGylation degree, which could be improved at higher PEGylation degree. As to free DOX concentration, AUCs in normal tissues were smaller than that of DOX solution. PPCD conjugates displayed more tumor accumulation of free DOX than PPSD conjugates, with the AUCs of PPCD 32/1 and 16/1 even larger than DOX solution, reaching 35.0 and 15.5 h-μg/g, respectively.In the third chapter, we were intended to modify PPCD 32/1, the most powerful PEG-PAMAM-DOX conjugate, with RGD peptides to prepare active targeting drug delivery system. We first evaluated the receptor binding affinity of the newly designed cyclic RGDyC peptide by molecular simulation. With the obtained positive results, we further conjugated RGDyC to PAMAM dendrimer by bifunctional MAL-PEG-NHS, followed by the coupling of CAD. The final product of RGD-PPCD contained 21.2 molecules of PEG,16.8 molecules of RGDyC and 14.2 molecules of CAD in each PAMAM molecule. The particle size and Zeta potential of RGD-PPCD were 17.02±0.13 nm and 2.31±0.15 mV, respectively. In conclusion, RGD-PPCD had similar PEGylation degree, drug loading, particle size and Zeta potential with PPCD, thus could be used to evaluate the effects of RGD modification on the in vitro and in vivo antitumor activities of PPCD conjugate.In vitro and in vivo evaluation of RGD-PPCD was carried out in the fourth chapter. In the first section, it was demonstrated that RGD modification had no influence on the acid sensitive release of DOX from PPCD conjugates. Besides, RGD-PPCD displayed no heamolytic toxicity which permitted its further i.v. administration. Human Umbilical Vein Endothelial Cells (HUVEC) was included into the cellular level experiments as the endothelial cell model of tumor neovasculature. Cytotoxicity of RGD-PPCD against HUVEC, SKOV-3 and B16 cells was enhanced as compared with PPCD. RGD-PPCD also displayed significant higher cellular uptake than PPCD (P<0.01), with 1.57-fold increase in total uptake by HUVEC cells, 1.32-and 1.58-fold increase in total uptake and internalization respectively by SKOV-3 cells, and 1.38- and 1.85-fold increase in total uptake and internalization respectively by B16 cells. Internalization mechanism studies revealed that RGD-PPCD interacted with plasma membrane both by non-specific electrostatic interaction and specific recognition between integrinαvβ3 and RGD peptides, and subsequently be internalized mainly by clathrin-mediated endocytosis, which was related with the cellular movement involving actin filaments and microtubule cytoskeleton, and also with complex of integrinαvβ3 and RGD peptides. All the three cell lines were able to internalize RGD-PPCD and deliver it to the lysosomes where the acidic condition permitted the release of free DOX and the entry into nucleus.In the second section, we first evaluated the tissue distribution of RGD-PPCD in SKOV-3 tumor bearing nude mice by in vivo fluorescence imaging. Fluorescent intensity of RGD-PPCD in tumor site was 1.52 times higher than that of PPCD 48 h post injection (P<0.05), which could be significatnly reduced but not fully inhibited by free RGDyK. Pharmacokinetics in B16 tumor bearing mice demonstrated that modification of RGD peptides led to reduced t1/2β, AUC and MRT, and increased Vc and CL. RGD-PPCD displayed 1.46- (by total DOX) and 2.36-(by free DOX) fold higher AUC in tumor site than PPCD. On the other hand, RGD-PPCD not only induced apoptosis of tumor cell, but also remarkably induced apoptosis of tumor neovasculature endothelial cell. Accordingly, RGD-PPCD demonstrated higher antitumor activity than PPCD, with greater MST, Median and ILS. The results of HE staining and TUNEL revealed that RGD-PPCD exhibited almost no toxicity to normal organs, which was a great improvement in safeness compared with DOX solution.更多还原