【作者】 沈鸣；

【导师】 方晓玲； 王建新；

【作者基本信息】 复旦大学 ， 药剂学， 2011， 博士

【摘要】 纳米制剂已被证明可以降低癌症化疗的毒副作用,提高治疗效果,自20世纪后期开始,纳米技术在肿瘤治疗中的研究和应用突飞猛进,纳米材料作为肿瘤药物的载体也得到了空前的重视,纳米药物制剂在肿瘤治疗和诊断方面显现出巨大的前景。但传统纳米给药系统存在的缺点也是显而易见的,如药物泄露、载药量低、靶向性不足等。如何更好地实现抗癌药物的肿瘤组织靶向性,是药学界亟待解决的重要问题之一。聚酰胺胺(PAMAM)是一种树枝状聚合物,表面有大量的官能团可以用来进行修饰；内部存在着较大的孔腔,可以包埋药物分子；分子为真正纳米级,单分子即可形成纳米系统,能够避免纳米系统在体内解聚而导致的药物泄漏。但PAMAM也存在纳米给药系统的共性问题,包括易被网状内皮系统(RES)吞噬,无法保证给药系统在生物环境中的稳定性,以及对肿瘤组织的靶向性差等。针对以上问题,根据肿瘤部位的病理特点：血管通透性增加、酸性环境以及某些肿瘤的细胞膜高度表达叶酸受体的特点,本课题设计了一种新型的肿瘤靶向载体材料。以PAMAM为核心,利用其分子中的大量空穴提高载药量,并可避免药物泄漏；在PAMAM端基上连接pH敏感材料聚甲基丙烯酸酯(PMA),使药物在肿瘤部位pH环境中释放,而在正常组织中不释放；外部连接聚乙二醇(PEG),以避开网状内皮系统(RES)的识别,达到长循环的目的,同时通过EPR效应增加在肿瘤部位的分布；以叶酸(FA)为端基进行修饰,使给药系统可主动靶向于肿瘤细胞。通过纳米材料的pH敏感靶向、纳米粒的被动靶向和叶酸介导的主动靶向的有机结合,提高纳米给药系统的肿瘤靶向性。为合成目标聚合物,首先通过酰胺化反应连接FA和PEG；再通过酯化反应将小分子引发剂BDAT连接在PEG的另一端,得到大分子引发剂FA-PEG-BDAT;通过可逆-链断裂转移自由基(RAFT)聚合反应,将甲基丙烯酸酯类单体(MA)聚合在PEG上,形成嵌段聚合物；将BDAT端氨解,暴露出巯基；在PAMAM的端氨基上连接4-戊烯酸(PA),使端基变成双键；通过巯基和双键的连接,将线形聚合物和PAMAM核心组装在一起,得到目标聚合物。通过对各步骤产物的结构鉴定和表征,证明合成路线可行,合成条件与产物结构的关系可控。根据课题设计目的,为制备在肿瘤部位(pH<6)释放药物,而在正常组织中(pH≈7.4)不释放药物的聚合物,以在不同pH介质中的溶胀性质为初筛指标,考察了甲基丙烯酸-N,N-二甲基氨基乙酯(DMAEMA)与2-羟基甲基丙烯酸乙酯(HEMA)、甲基丙烯酸正丁酯(BuMA)、甲基丙烯酸己酯(HMA)和甲基丙烯酸乙基己酯(EHMA)等一系列单体制备的pH敏感共聚物,筛选得到了具有较好pH敏感溶胀性的DMAEMA与BuMA、HMA、EHMA的共聚物。又以其包载紫杉醇(PTX)的纳米粒在不同pH介质中的释放行为为指标,确定最终的pH敏感嵌段的单体种类和比例为DMAEMA-BuMA50:50.通过对连接水溶性PEG嵌段后对聚合物pH敏感性的影响试验考察,发现连接PEG后,纳米粒对PTX的释放速度有所降低,但pH敏感性在考察范围内没有明显差异,确定了PEG分子量为5000。通过释放可逆性实验,比较了最终目标聚合物为树枝状FA-PEG-PMA-PAMAM和中问产物线形材料的pH敏感释放性能进行了考察和比较,结果表明树枝状材料制备的纳米粒对环境pH的响应速度较快,释放的可逆性优于线形材料纳米粒,验证了本文设计思路的合理性和可行性。通过比较实验,确定了纳米粒的制备方法为乳化-溶剂蒸发法。采用正交实验优化了工艺参数,制备得到的纳米粒包封率>95%,载药量>4.6%,粒径约为100nm,分布范围窄。通过考察在不同pH条件下粒径的变化,发现当介质的pH由7.4降为5.0时,纳米粒的粒径增大。采用抗巨噬细胞吞噬试验评价了纳米粒的长循环性能。结果显示FA-PEG-PMA-PAMAM纳米粒的抗巨噬细胞吞噬能力显著强于PAMAM纳米粒；连接PEG的材料抗吞噬能力均强于未连接PEG的材料；线形材料的抗吞噬能力强于树枝状材料；经修饰的PAMAM强于PAMAM本身;叶酸的连接对抗吞噬能力没有显著影响。从而在细胞层面.上证明了制备的纳米给药系统具有长循环性能。选择对叶酸敏感的肿瘤细胞KB细胞,通过摄取实验评价了纳米粒对肿瘤细胞的靶向性。结果显示含叶酸的纳米粒在缺叶酸培养基和正常培养基中的摄取比率均较无叶酸纳米粒高,趋势为FA-PEG-PMA≈FA-PEG-PMA-PAMAM> PEG-PMA≈PEG-PMA-PAMAM> PMA≈PMA-PAMAM>PAMAMA;同种类型的球形和线性聚合物制备的纳米粒之间摄取的差异没有显著性。用小鼠成纤维细胞L929细胞评价了FA-PEG-PMA-PAMAM的细胞相容性,结果显示,当载体材料浓度<625μg/ml时对L929细胞不显示毒性。对KB的体外细胞毒实验表明,在缺叶酸环境中,载PTX的FA-PEG-PMA-PAMAM纳米粒显示较强的细胞毒作用。PTX浓度为10μg/ml,相当于载体材料浓度200μg/ml,因此给药系统在安全范围内对肿瘤细胞具有较强的杀伤作用。采用液质联用串联质谱法(LC-MS/MS)测定了PTX的血药浓度,以紫杉醇注射液和载PTX的FA-PEG-PAMAM纳米粒作为对照,对载PTX的FA-PEG-PMA-PAMAM纳米粒在大鼠体内的药动学行为和荷KB瘤裸鼠体内的组织分布进行了考察,以研究纳米粒的长循环和肿瘤靶向性。结果显示,大鼠体内纳米粒的分布和消除半衰期以及体内滞留时间均高于紫杉醇注射液,并有统计学意义,说明纳米粒在体内具有长循环性。FA-PEG-PMA-PAMAM纳米粒的AUC>FA-PEG-PAMAM纳米粒>紫杉醇注射液,提示由于FA-PEG-PMA-PAMAM的pH敏感释药特性使PTX在体内的滞留时间延长。组织分布结果验证了纳米粒的长循环和靶向特征。纳米粒给药后在考察的各组织器官中的分布较慢；虽然在肝、肺、肾中的分布仍较游离药组高,但在脾中的分布两者无差异；纳米粒组PTX在肿瘤中的分布显著高于游离药物组。相对于紫杉醇注射液,FA-PEG-PMA-PAMAM纳米粒的靶向效率≈3,相对于FA-PEG-PAMAM纳米粒,靶向效率≈1.8,说明FA-PEG-PMA-PAMAM纳米粒的靶向性强。结合药动学结果,推测原因为FA-PEG-PMA-PAMAM纳米粒的pH敏感释放性能,使其在正常组织对包载的PTX有保护作用,因而回到血循环中再度分布到肿瘤组织的机会增加。通过荷KB瘤裸鼠模型的药效学考察,验证了FA-PEG-PMA-PAMAM纳米粒对肿瘤的抑制作用。分别以对肿瘤体积、瘤重和脏器指数为指标,比较了荷瘤小鼠分别给予生理盐水、紫杉醇注射液和载PTX的FA-PEG-PMA-PAMAM纳米粒的药效学。结果显示在每次1mg/kg,给药3-15次的剂量下,纳米给药系统可显著抑制荷KB瘤裸鼠肿瘤的生长,抑瘤作用显著高于紫杉醇注射液。给药15次后,心、肝、脾、肺、肾的组织切片显示无明显毒性。小鼠急性毒性试验结果显示：载体材料的LDso为35.23mg/kgo兔血管刺激性实验表明空白纳米粒基本无血管刺激性。以上研究结果表明,本课题设计合成的载体材料具有天然靶向性、物理靶向性、长循环性质和主动靶向性,基本达到了本文研究的目的,以该材料制备的纳米粒可显著提高抗肿瘤药物的体内肿瘤靶向性,增强PTX对肿瘤细胞的杀伤和抑制作用。本课题实验结果为肿瘤的靶向药物治疗提供了新的思路。更多还原

【Abstract】 Nano drug delivery systems have been proved effective in decreasing side effects of tumor chemotherapy. Since late20th century, rapid advances have been developed this technology. Nano-materials, as targeting drug carrier, have also been thrown an unprecedented attention on. Nano-drug formulations in cancer treatment and diagnosis are showing great promise. However, the shortcomings of traditional nano drug delivery systems were obvious, such as drug leakage, low drug loading capacity, poor targeting, et al.Poly(amidoamine)(PAMAM) is a kind of dendrimer with a great number of active groups on its surface and easy to be modified. There are many caves inside it which can embed drug. The molecular can set up an indeed nanoparticle (NP) with it self thus the drug leakage when nanoparticles of multimolecule collapsed could be avoided. As same as other nano drug delivery systems, some problems such as uptaking by reticuloendothelial system (RES), lack of stability in body and poor targeting were also found in PAMAM. It’s important to enhance its tumor targeting capbility.As to make up those shortcomings, we set up to design a new material. According to the pathological feature in tumor:higher vessel permeability, acid and highly expressed foliate receptor, we designed a new material. The new material was designed with PAMAM as the core to improve drug loading capacity as it possesses a large number of caves in the molecule. The end of PAMAM was connected to a pH-sensitive material, polymethacrylate (PMA), so that the drug would be released in the tumor pH environment but not in the normal tissue. The external end of polymethacrylate was connected to polyetheyloxygen (PEG) to avoid recognition by RES to achieve the purpose of long-cycling. As the same time, PEG can increase distribution in tumor through EPR effect. Folic acid (FA) was used as end group to achieve active targeting feature to tumor cells. The material was designed to enhance tumor targeting by the combination of pH-sensitivity, passitive targeting and active tumor targeting of FA.The synthesis began with the combination of FA and PEG. The FA-PEG was then combined with S-isobutyl-S’-(a,a’-dimethyl-a"-acetic acid)trithiocarbonate (BDAT) to be a polymer initiator of reversible addition fragmentation chain transfer (Raft) polymerization. Methyl methacrylate monomers were polymerized to the BDAT end of the initiator to be a two block polymer. Then, the BDAT end of polymer was aminolized to hydrosulfide.4-pentenoic acid (PA) was combined to the terminal amino group of PAMAM to get a double bond end. The double bond end of PAMAM-PA and the hydrosulfide of the two block polymer combined through addition. The linear polymer and the PAMAM core assembled to get the aimed polymer. The synthesis route was proved feasible and the structure of product was under control.The nano drug delivery system was designed according to the different pH between tumor and normal tissue. It should release drug rapidly in tumor (pH<6) and not release in normal tissue (pH-7.4). The critical pH point of polymethacrylate depends on the kind and ratio of monomers. So the copolymers of DMAEMA and2-droxyethylmethacrylate (HEMA), n-butylmethacrylate (BuMA), hexyl methacrylate (HMA),2-ethylhexyl methacrylate (EHMA) were screened by swell property in different pH. Then the copolymers were made into NPs loading paclitaxel (PTX) and their release character in different pH was researched. The final monomer kind and ratio of monomers in pH-sensitive block was decided as DMAEMA-BuMA50:50.This article also examined the effect of PEG on the pH sensitivity. When connected with water-soluble PEG, the release rate of PTX decreased. The pH sensitivity changed not so obviously in the scope of this study. Therefore we used PEG (5000) at last.Reversible release experiment was practiced between the dendrite material and the linear mid-product. The results showed that the NP prepared of the dendrite material possessed faster response speed to the changed pH and better reversibility than that of the linear mid-product, thus the design was verified reasonable. The final material was FA-PEG-PMA-PAMAM with the molecular of PEG5000and the pH-sensitive block of DMAEMA-BuMA50:50.The preparation method of PTX loading NP was initially identified as the emulsion-solvent evaporation method. The parameters were determined by orthogonal experiments. The encapsuled ratio (ER) was more than95%and the drug loading (DL) was more than4.6%. The particle size in water was about100nm with a narrow distribution. The particle size increased as the pH changed from7.4to5.0. Anti-macrophage phagocytosis test was used to evaluate the long-circulating character of the drug delivery system. The results showed that the anti-phagocytic ability of the NP of FA-PEG-PMA-PAMAM was significant stronger than that of PAMAM. The introduction of PEG decreased the phagocytosis, phagocytosis of linear material was less than dendrite materials, of modified PAMAM less than of PAMAM itself. In addition, FA showed no significant effect against anti-phagocytosis. The long-circulating feature has been proved on cell level.FA sensitive tumor cell KB was used to evaluate the tumor targeting feature of the NPs. The ratio of uptake ratio in non-folic acid medium and normal medium suggested that the trend of FA-PEG-PMA-FA-PEG-PMA-PAMAM> PEG-PMA-PEG-PMA-PAMAM> PMA-PMA-PAMAM> PAMAMA. There was no significant difference between dendrite and linear materials of the same composition.L929cell was used to evaluate the cell compatibility of FA-PEG-PMA-PAMAM. The safe dose was below625μg/ml.KB cell was used to evaluate the cell toxicity of NP. The PTX-NP of FA-PEG-PMA-PAMAM showed stronger toxicity in non-FA DMEM. The toxicy dose is10μg/ml of PTX, equal to200μg/ml of dendrimer when made into nanoparticle.The pharmacokinetics of PTX loading NP in rats and the distribution in KB-bearing nude mice was determined by LC-MS/MS method to verify the long circulating and tumor targeting capability. The experiments were practiced with PTX injection, PTX loaded FA-PEG-PAMAM and PTX loaded FA-PEG-PMA-PAMAM.In the compartment model parameters, distribution and elimination half-life ti/2α, t1/2β and MRT (0-t), MRT (O-∞)of NP were significantly higher than the free PTX., reflecting the longer retention time than free PTX. AUC (0-t) and AUC (0-∞) of FA-PEG-PMA-PAMAM NP were significantly higher than those of free PTX and FA-PEG-PAMAM NP, suggesting the pH sensitive release of FA-PEG-PMA-PAMAM NP prolonged the retention time of PTX.Result of tissue distribution experiment proved tumor targeting feature of FA-PEG-PMA-PAMAM NP. NPs distributed more slowly after injection than the free-PTX. The PTX concentrations of NPs were higher than free PTX in liver, lung and kidney. There was no difference in spleen. The PTX concentrations of NPs were higher than free PTX in tumor. The targeting efficiency of NP was more than 3compared with the free-PTX and about1.8compared with FA-PEG-PAMAM NP, which means the targeting capacity of FA-PEG-PMA-PAMAM is higher than that of FA-PEG-PAMAM. The reason maybe is that the pH sensitive release of FA-PEG-PMA-PAMAM NP could protect the PTX, thus the opportunity of return to the circle and arrive at the tumor increases.The results of pharmacokinetics and distribution experiments proved the long circling and tumor targeting capacity of FA-PEG-PMA-PAMAM.The pharmacodynamic study was practiced in KB tumor subcutaneously imbed nude mice. Through the monitoring of tumor volume, tumor weight and the organ ratio statistics, the results were displayed. The results showed significant inhibition of KB cells in nude mouse tumor growth after3to15times injection of the PTX loading FA-PEG-PMA-PAMAM NP with the dosage of1mg/kg. The inhibition was significantly higher than free PTX. The inhibition ratio was about70%. After administration of15times, there was no obvious injury in heart, liver, spleen, lung and kidney in biopsy.The intravenous injection LD50of mice was35.23mg/kg. The NP showed no obvious irritation in rabbit ear vein after5time injection.The study suggests that the project design and synthesis of the carrier material was successful. The material possesses multi-level tumor targeting character of natural targeting, physical targeting, long circulating propertie and active targeting. It may be a new idea for therapy of FA sensitive tumors.更多还原