【作者】 王萍；

【导师】 杨桦；

【作者基本信息】 吉林大学 ， 物理化学， 2015， 硕士

【摘要】 癌症，又称为恶性肿瘤，当前为止已成为威胁人类安康和性命的主要因素之一。截至到2013年，若按照死亡率排名，肺癌排在第一位。引起肺癌的原因有很多，吸入污染物和抽烟都是肺癌的主要诱因，而且随着工业的快速发展带来的空气污染加剧以及人类生活方式的快速变化，肺癌的发病率和死亡率都有增高的趋势。由于肿瘤散布在肺部组织中，通过外科手术难以完全切除，目前化疗或者放射性治疗是传统治疗肺癌的方法，但存在副作用大、对机体伤害大、复发率高等缺陷，特别对已发生转移的患者很难达到良好的远期治疗效果。肺部癌症治疗的一个关键因素是将治疗性药物或者基因运载到肺中，比较常见的运载方式是采用静脉注射通过体循环将药物或基因运载到肺中，然而，静脉注射的方式存在多个缺点，如毒性大、体循环过程中药物或基因浓度降低、药物或基因易降解且难以到达靶器官等缺点。肺部给药系统（Pulmonary Drug DeliverySystem, PDDS）指能将药物通过呼吸道传递到肺部，从而产生局部或全身治疗效果的给药系统。与静脉给药相比，肺部给药具有其独特的优势，该给药途径可以使药物或基因在肺部肿瘤组织中快速达到高浓度水平并减少在其它正常组织内的分布，能够实现定量，且对正常组织毒副作用低。此外，肺部组织拥有比较大的吸收表面积，从而避免初级代谢降解。本论文研究的主要目的是制备出几种细胞毒性低且适合气管肺部喷雾给药和体内应用的高分子药物/药物共传递载体，为肺转移癌的治疗提供一种新的治疗方法，同时为高分子载体在临床上应用提供基础研究。本文采用聚谷氨酸载体为基础，制出结构简单、制备过程容易、有效且实用的高分子纳米载体。载体模型分别是：线性载体聚乙二醇-聚谷氨酸（mPEG-b-PLG）与超支化载体聚乙二醇-聚乙烯亚胺-聚谷氨酸（mPEG-OEI-PLG），然后将小分子化疗药物阿霉素（DOX）与顺铂（CDDP）以复合与络合的方式担载到载体上，从而制备出高分子纳米载药颗粒。纳米载药颗粒通过气管喷雾注射器直接喷射到肺中，随后，化疗药DOX和CDDP在肿瘤细胞低pH内涵体环境下缓慢释放，并进入到细胞核中发挥药效；高分子纳米载体发生降解代谢出体外，最终对肺转移癌起到抑制和治疗的作用。本论文通过上述研究，得出如下结论：（1）以mPEG-NH2为大分子引发剂，从而引发BLG-NCA开环聚合，得到mPEG-b-PBLG，最后再脱掉苄基保护基团得到mPEG-b-PLG。然后将小分子化疗药物DOX和CDDP分别通过静电复合与络合担载到mPEG-b-PLG上。分别制备出单载体系：mPEG-b-PLG-DOX和mPEG-b-PLG-CDDP以及共载体系：mPEG-b-PLG-DOX-CDDP。结果发现，在不同pH条件下，共载体系mPEG-b-PLG-DOX-CDDP中的DOX和CDDP在肿瘤酸性条件下释放的较多，中性条件释放较少，并且比单载体系释放的慢；激光共聚焦结果进一步说明了mPEG-b-PLG-DOX-CDDP材料在肿瘤环境下，DOX断裂，不断发挥药效；MTT研究结果表明，共载体系能显著抑制肿瘤细胞的生长；体内抑瘤实验研究结果表明，共载体系与单载体系相比，能明显抑制肿瘤的生长，且H&E染色结果表明，对脏器基本没有损伤作用，说明肺部给药能有效提高药物浓度，且降低毒副作用；体内分布结果显示，与静脉给药相比，采用肺部给药的方式使得药物载体系统在肺部沉积多且时间长；DOX在肺部组织聚集情况分析结果表明，经肺部给药后，DOX在肿瘤部位比在正常组织内富集的多，说明药物在肿瘤部位能很好地发挥作用，而对正常组织影响不大。（2）以mPEG-NHS与OEI600按1：1的摩尔比反应，将mPEG接到OEI6上，之后以mPEG-OEI为引发剂，从而引发BLG-NCA开环聚合，得到mPEG-OEI-PBLG，通过脱保护得到氨基酸多臂共聚物（mPEG-OEI-PLG）。以mPEG-OEI-PLG为载体，分别复合DOX与络合CDDP。体外释放行为结果表明，在不同pH下，mPEG-OEI-PLG中的DOX释放行为不同，尤其是在酸性条件下，释放的较多；激光共聚焦结果显示，不同于游离的小分子化疗药DOX（DOX通过渗透的方式进入细胞膜），纳米载药颗粒是通过内吞的方式进入细胞膜的，这样更能有效延长药物的聚集时间。共载体系抑瘤效果评价表明，共载体系肺部图片瘤明显减少甚至消失，肺部重量明显降低；H&E染色结果表明，对其它脏器基本没有损伤作用。且治疗过程中的体重曲线表明，各个治疗组的体重无差别，说明该给药方式和药物具有安全性；DOX和CDDP在体内的分布情况结果显示，尾静脉给药后，大部分药物分布在肝脏、肾脏部位，药物在肺部分布的量很少。24h后，大部分药物被排出体外，而肺部给药后，大部分药物沉积在肺部不断的发挥作用，且沉积的时间比较长。7d后，肺部仍然能检测到可见的荧光信号。药物在肺部组织聚集情况分析表明，经肺部给药后，药物和基因均在肿瘤部位比在正常组织内富集的多。更多还原

【Abstract】 Cancer, also called malignancy, is one of the major threats to human health andlife nowadays. Lung cancer ranks the first of the mortality rate among all kinds ofcancers since of2013. There are many causes of lung cancer. On the one hand,because of the serious air pollution, a large number of carcinogenic substances areinhaled into the lungs ofhumans that could induce cancer. and smoking is alsoaimportant factor for lung cancer. In particular, with the rapid development ofindustrial as well as the rapid changes of the way of human life, the morbidity andmortality of lung cancer has increased trend.The tumor in the lung tissue can not be completely removed by conventionaltherapy of surgery, chemotherapy or radiation therapy, meanwhile, the damage tothe body, the side effects, the defects such as recurrence rate were very serious. Forinstance, it is difficult to achieve good long-term efficacy especially for patientswith metastasis. A key factor in lung cancer treatment is to carry therapeutic genesor drugs into the lungs, for instance, carrying drugs or genes into the lungs byintravenous administration through the body circulation is the traditional method,however, there are several disadvantages by intravenous injection, such as hightoxicity, lower concentration of drugs or genes during circulation, easilybiodegradable of drugs or genes, difficult to reach the target organ and so on.Compared with intravenous administration, pulmonary administration has its uniqueadvantages, the route of administration can lead to a high concentration level ofdrugs or genes in lung tumor tissue quickly and reduce the distribution in othernormal tissues, and it is possible to achieve quantitative and low toxicity to normal tissues. In addition, lung tissue has a relatively large surface area that can absorb thegene or drug retention in the lungs and avoid primary metabolic degradation.The main purpose of this paper is to prepare polymer drug and drug co-deliverycarriers with low cytotoxicity and high transfection efficiency, which is suitable forspray administration trachea and lungs in vivo, and provides a new treatment forlung metastasis treatment.Based on the polyglutamic acid cationic carrier, we prepared simple structure,easy, effective and practical polymer nanoparticle carrier. The carriers are poly(ethylene glycol)-b-poly(L-glutamicacid)(mPEG-b-PLG) and poly(ethyleneglycol)-polyethyleneimine-poly(L-glutamicacid)(PEG-OEI-PLG). Chemotherapydrugscan be complexed to carriers, and then composite particles are prepared. Thecomposite nanoparticles can be sprayed by a syringe through the trachea, and theninjected directly into the lungs. Subsequently, the Doxorubicin (DOX) andcis-platinum (CDDP) can be released into the nucleus of tumor cells in the form ofconnotation at a low pH. The carrier dissociates and metabolismout to the body andthe drugs inhibit the metastasis and treatment.Through the research above, this paperdraw the concrete points are as follow:(1) Using mPEG-NH2as the macroinitiator, triggered BLG-NCA ring openingpolymerization, abtained mPEG-b-PBLG, at last prepared mPEG-b-PLG afterdeprotection. The chemotherapy drugs (DOX and CDDP) can be complexed tomPEG-b-PLGcarrier, respectively. And the carriers are: mPEG-b-PLG-DOX,mPEG-b-PLG-CDDP and mPEG-b-PLG-DOX-CDDP. The results showed that, theDOX and CDDP from mPEG-b-PLG-DOX-CDDP could be released largely underacidic conditions, while the DOX and CDDP from mPEG-b-PLG-DOX-CDDPcould be released less in acidic or neutral conditions. Moreover, the results ofconfocal laser experiments validated the consistent results of acid-sensitiveproperties of mPEG-b-PLG-DOX-CDDP, as well as the phenomenon that the DOXfracture and play efficacy continuously in tumor tissues. MTT results showed that,the cell survival rate in the co-delivery system was significantly lower than that inthe single-delivery system, which indicated that a common carrier system can significantly inhibit the growth of tumor cells in the former group. The anti-tumorexperiments in vivo showed that the co-delivery system could significantly inhibittumor growth compared with the single-delivery system, and H&E staining resultsalso showed that this system was harmless to organs, which indicated thatpulmonary administration can effectively improve drug concentration, and reducetoxicity. The distribution results in vivo showed that pulmonary administration canimprove the deposition of co-delivery systems in the lungs and prolong the releasingtime compared with intravenous administration. The gathering appearance of DOXshowed that the drugs and genes gathered more at the tumor site than that in thenormal tissue after pulmonary administration, which indicated that the drugs andgenes play a good role at the tumor site without side effects to normal tissues.(2) According to the molar ratio ofmPEG-NHS and OEI60(1:1), we triggeredBLG-NCA ring opening polymerization using mPEG-NH2as the macroinitiator,abtained mPEG-OEI-PBLG, at last prepared mPEG-OEI-PLG after deprotection.The chemotherapy drugs (DOX and CDDP) can be complexed tomPEG-OEI-PLGcarrier, respectively. The release results in vitro showed that atdifferent pH, DOX from mPEG-OEI-PLG released more especially under acidicconditions. The confocal laser experiments showed that DOX from mPEG-OEI-PLGentered the cells by endocytosis, which is different free DOX. So the accumulationtime could be prolonged. The inhibitory effect of the co-delivery system on thetumor cells were determined, and the results indicated that the co-delivery systemgroup contained a significantly reduced tumor numbers or even absent tumors, andthe lung weights of the co-delivery system group were significantly lower than thatof the PBS group. H&E staining results further indicated the advantages of theco-delivery system, and H&E staining showed no injury to other organs. And thebody weight curves showed no difference for each treatment group, indicating thatthe safety of the drug and the mode of pulmonary administration. The results ofDOX and CDDP distribution in vivo showed that, after intravenous administration,the majority of drug distributed in the liver and kidney, only a small amount of drugsdistributed in lungs, even most of the drugs were released after24h; by contrast, after the pulmonary administration, the large part of the drugs could deposit in lungs,and continuously play efficacy, and it is still possible to detect visible fluorescencesignal in lungs after seven days. The gathering appearance of drugs and genes inlungs indicated that the majority of drugs and genes gathered around the tumortissue instead of the normal tissue, and this may be related to the EPR effect.更多还原