PCL-b-PEG-b-PCL聚合物载药体系的构建及抗肿瘤研究

发布时间：2018-06-30 10:18

本文选题：聚己内酯-b-聚乙二醇-b-聚己内酯 + 聚合物胶束　；参考：《北京协和医学院》2015年博士论文

【摘要】：恶性肿瘤已成为导致人类死亡的第二大原因,仅次于心血管疾病导致的死亡率。化学治疗是治疗转移性恶性肿瘤的重要手段之一。但传统化疗对肿瘤组织和细胞缺乏选择性杀灭作用,常规治疗剂量即可对正常组织器官产生显著毒副作用,导致患者不能耐受,降低药物疗效。为了提高抗肿瘤药物的靶向性和生物利用率,降低毒副作用,改善治疗效果,纳米药物载体已成为肿瘤化疗研究的热点领域,其中代表性的给药系统有脂质体、纳米粒、纳米乳、聚合物胶束、聚合物囊泡等。两亲性聚合物能够在不同条件下自组装成聚合物胶束、聚合物囊泡等不同结构的药物载体。本文以具有良好生物相容性和可生物降解性的两亲性三嵌段共聚物—聚己内酯-b-聚乙二醇-b-聚己内酯(PCL-b-PEG-b-PCL)为载体材料,通过分子自组装用不同亲水/疏水链段的PCL-b-PEG-b-PCL分别研制出聚合物胶束和聚合物囊泡这两种药物载体,同时,进一步研制出基于PCL-b-PEG-b-PCL的磷脂-聚合物杂化纳米粒作为第三种药物载体。以目前临床使用最广泛的抗肿瘤药物紫杉醇和阿霉素为模型药物,研制载紫杉醇聚合物胶束、叶酸靶向载紫杉醇磷脂-聚合物杂化纳米粒、双重载药(亲水内腔载阿霉素、疏水双分子膜层载紫杉醇)聚合物纳米囊泡,研究其作为抗肿瘤药物载体的有效性。本文的主要研究内容如下：一、两亲性三嵌段共聚物PCL-b-PEG-b-PCL形成不同载体的研究两亲性聚合物可以自组装形成球状胶束、柱状胶束、蠕虫状胶束、聚合物囊泡等不同结构,亲水链段的质量比或体积比、共聚物分子量及制备方法是决定自组装形成不同载体结构的关键参数。对两亲性三嵌段共聚物PCL-b-PEG-b-PCL,目前尚无研究文献报道其形成聚合物胶束和聚合物囊泡所需亲水疏水嵌段比及分子量。本文合成了一系列质量可控、结构准确的不同分子量、不同比例亲水疏水链段的聚合物,已经初步确定了PCL-b-PEG-b-PCL聚合物自组装形成囊泡的亲水部分与疏水部分的比例及形成囊泡的规律。研究表明采用薄膜水化超声分散法时,两亲性三嵌段共聚物PCL-b-PEG-b-PCL (PCEP484)中PEG质量百分数约为50%时,能形成具有核-壳结构的聚合物纳米胶束；两亲性三嵌段共聚物PCL-b-PEG-b-PCL (PCEP888)中PEG质量百分数约为33%时,能形成具有明显双分子膜层结构的聚合物囊泡。二、载紫杉醇聚合物胶束的研制及其抗肿瘤研究开发一种能有效在疏水性内核包载抗肿瘤药物紫杉醇(paclitaxel, PTX)的聚合物胶束药物载体,并能用于静脉全身给药,避免临床紫杉醇注射剂使用聚氧乙烯蓖麻油作为增溶剂所引发的过敏反应及毒副作用。本文以PCL-b-PEG-b-PCL (PCEP484)为载体材料,采用薄膜水化超声分散法制备出可用于静脉注射的载紫杉醇聚合物胶束(paclitaxel-loaded polymeric micelles, PTX-PM)。扫描电镜及透射电镜显示所研制的PTX-PM呈球形,大小均匀,具有明显的核壳结构。紫杉醇依靠疏水作用有效载入疏水性链段PCL形成的疏水性内核中,其载药量为28.98%,药物包封率为94.36%,具有较高的载药量与包封率。亲水性PEG链段包裹在疏水性内核外围形成具有明显冠状结构胶束表面,能使聚合物胶束能很好地分散于水相并具有长循环特性。载药聚合物胶束的平均粒径为93nm,多分散系数为0.19,非常有利于静脉全身给药。差示扫描量热分析研究表明将紫杉醇制成缓释纳米粒后其结晶状态发生了变化,以无定型状态存在于聚合物胶束中。在1M水杨酸钠中的体外释放研究表明PTX-PM具有缓释紫杉醇效果,无药物突释,前5天为零级释放动力学模型(R=0.99)。MTT法细胞毒性研究表明空白聚合物胶束对HepG2肝癌细胞及EMT-6乳腺癌细胞均无毒性,在相同紫杉醇含量下,PTX-PM的细胞毒性低于市售紫杉醇/聚氧乙烯蓖麻油注射剂(Taxol(?)),并具有时间和剂量依赖性,说明紫杉醇包封于聚合物胶束中后其活性并没有降低,而且可以随着紫杉醇从聚合物胶束中逐渐释放出来而在更长的时间内作用于癌细胞。大鼠药代动力学研究表明,与Taxol(?)相比,所研制的载药聚合物胶束明显延长了紫杉醇在血液中的循环时间及消除半衰期,显著提高了生物利用度。体内抗肿瘤活性研究表明,PTX-PM对小鼠EMT-6乳腺癌具有明显抑制作用,相同给药剂量下其抑瘤效果优于Taxol(?)(肿瘤抑制率：85.79% vs 63.37%,p0.05)。上述研究表明所研制的PTX-PM高效低毒,能明显延长紫杉醇在血液中的循环时间,具有EPR被动靶向作用,是一种有潜力的可用于肿瘤治疗的紫杉醇缓控释载药体系。三、叶酸靶向载紫杉醇磷脂-聚合物杂化纳米粒的研制及其抗肿瘤研究磷脂-聚合物杂化纳米粒(lipid-polymer hybrid nanoparticles, LPNPs)是一类基于脂质体和聚合物纳米粒发展的新型药物载体,本文开发了一种新型具有叶酸靶向的载紫杉醇磷脂-聚合物杂化纳米粒(PTX-FLPNPs)。以两亲性共聚物PCL-b-PEG-b-PCL (PCEP484)、甲氧基聚乙二醇二硬脂酰磷脂酰乙醇胺(mPEG2000-DSPE)及偶联叶酸的聚乙二醇二硬脂酰磷脂酰乙醇胺(DSPE-PEG(2000)Folate)为载体材料,通过薄膜水化超声分散法制备出以叶酸为靶向的载紫杉醇磷脂-聚合物杂化纳米粒(PTX-FLPNPs)。透射电镜显示PTX-FLPNPs呈球形,大小均匀,具有“核-壳-壳”的结构,疏水性链段PCL及药物PTX通过自组装形成内核结构,磷脂的DSPE形成内层壳结构,两亲性共聚物及磷脂中的亲水性链段PEG形成外层壳结构。激光共聚焦显微镜进一步确证了所制备的罗丹明标记磷脂PTX-FLPNPs具有“核-壳-壳”的结构及明显的磷脂单分子层结构。粒径分析表明,PTX-LPNPs(无叶酸靶向分子)与PTX-FLPNPs(叶酸靶向)都具有较小的分散系数,表明采用该方法能制备出均一粒径的磷脂-聚合物杂化纳米粒。与PTX-LPNPs粒径及zeta电位相比,PTX-FLPNPs的平均粒径略有所增大(279.9 nm vs 271.5 nm),具有更高的负zeta电位值(-17.5 mV vs-14.2 mV),间接表明了FLPNPs外壳具有叶酸靶向分子。紫杉醇投药量为30%时,PTX-FLPNPs和PTX-LPNPs的载药量大于27%,药物包封率大于90%,都具有较高的载药量与药物包封率。体外释放研究表明,紫杉醇从PTX-FLPNPs和PTX-LPNPs中的释放均具有缓释效果,无明显的药物突释。细胞吞噬的定性及定量研究结果表明,通过叶酸受体介导作用,靶向修饰的磷脂-聚合物杂化纳米粒能有效进入叶酸受体高表达的肿瘤细胞内。CCK8法细胞毒性研究表明空白FLPNPs对H1299肺癌细胞及EMT-6乳腺癌细胞均无细胞毒性,在相同紫杉醇剂量下,PTX-FLPNPs与PTX-LPNPs的细胞毒性均低于Taxol(?),而PTX-FLPNPs对细胞的杀伤力显著高于PTX-LPNPs (p0.05),进一步说明通过叶酸的主动靶向性,可提高药物在肿瘤细胞中的浓度,有效杀伤肿瘤细胞。体内抗肿瘤活性研究表明,采取瘤内注射的方式,PTX-FLPNPs对小鼠EMT-6乳腺癌具有与紫杉醇注射剂类似的抑瘤效果(p0.05),但PTX-FLPNPs毒性低于紫杉醇注射剂。PTX-FLPNPs的抑瘤效果优于PTX-LPNPs (65.78% vs 48.38%, p0.05),表明通过叶酸受体介导的靶向作用增加了肿瘤细胞对纳米粒的摄取进而增强了抑瘤作用。四、双重载药聚合物纳米囊泡的研制及其肿瘤靶向初步研究聚合物囊泡具有亲水性内腔及较厚的疏水性双层膜,其独特的结构可以使其同时包载亲水性及疏水性药物,用于肿瘤的协同治疗。本文以PCL-b-PEG-b-PCL (PCEP888)为载体材料,采用薄膜水化超声分散法制备出具有明显双分子膜层类似脂质体结构的聚合物囊泡。疏水性药物紫杉醇(paclitaxel, PTX)依靠疏水性作用被包载进入聚合物囊泡的疏水性膜层中,采用硫酸铵梯度法将亲水性盐酸阿霉素(doxorubicin, DOX)包载进入聚合物囊泡的亲水性内腔中。透射电镜表明双重载药聚合物囊泡(polymersomes loaded with both PTX and DOX, PS-PTX-DOX)不但具有明显的双层膜层结构,亲水性内腔由于DOX的载入还显示出明显的内核。激光共聚焦显微镜进一步确证带有自发荧光的DOX载入亲水性内腔中。当紫杉醇和阿霉素的投药量为10%时,所研制的双PS-PTX-DOX的平均粒径为169.7nm,多分散系数为0.211,粒度较为均一,非常有利于作为全身给药药物载体用于肿瘤的联合化疗。细胞吞噬研究表明,在同样的培养时间和阿霉素剂量下,PS-PTX-DOX在细胞核内的荧光强度远小于阿霉素,表明聚合物囊泡载体具有明显的缓释作用。随着培养时间的延长,所释放的DOX进入细胞核,细胞核内DOX荧光强度增强。PS-PTX-DOX的粒径小于200 nm,可用于静脉全身给药。荷瘤小鼠药物体内分布研究表明,尾静脉注射PS-PTX-DOX后,由于其具有长循环特性,可以延长血液循环时间,并通过EPR效应有效将药物富集于肿瘤部位。
[Abstract]:Malignant tumor has become the second major cause of human death, second only to the mortality caused by cardiovascular disease. Chemical therapy is one of the most important methods for the treatment of metastatic malignant tumors. However, traditional chemotherapy does not kill the tumor tissues and cells selectively. The conventional treatment dose can produce significant toxic and side effects on normal tissues and organs. In order to increase the patient's intolerance and reduce the efficacy of the drug, in order to improve the targeting and bioavailability of antitumor drugs, reduce the side effects and improve the therapeutic effect, nanoscale drug carriers have become a hot field in the research of tumor chemotherapy. The representative drug delivery systems include liposomes, nanoparticles, nanoscale, polymer micelles, and polymer vesicles. Two amphiphilic polymers can be self assembled into polymer micelles, polymer vesicles and other drug carriers under different conditions. In this paper, the two Pro three block copolymers with good biocompatibility and biodegradability, polyhexyl -b- poly (ethylene glycol -b-) polyhexyl (PCL-b-PEG-b-PCL), are used as carrier materials. Polymer micelles and polymer vesicles were developed by PCL-b-PEG-b-PCL with different hydrophilic / hydrophobic segments in the self-assembly. At the same time, the PCL-b-PEG-b-PCL based phospholipid polymer hybrid nanoparticles were developed as the third drug carriers. The most widely used antitumor drug paclitaxel and armilla were made in the present clinic. As a model drug, the preparation of paclitaxel polymer micelles, folic acid targeted paclitaxel phospholipid polymer hybrid nanoparticles, dual drug loading (hydrophilic adriamycin, hydrophobic biolecular paclitaxel) polymer nano vesicles, study its effectiveness as an antitumor drug carrier. The main contents of this paper are as follows: one, two parents Study on the formation of different carriers of the three block copolymer PCL-b-PEG-b-PCL two amphiphilic polymers can form spherical micelles, columnar micelles, vermicular micelles, polymer vesicles and other different structures, mass ratio or volume ratio of hydrophilic chain segments, molecular weight and preparation methods of copolymers are key parameters determining the formation of different carrier structures by self assembly. For two Pro sex three block copolymer PCL-b-PEG-b-PCL, there is no research literature on the hydrophilic block ratio and molecular weight required for forming polymer micelles and polymer vesicles. A series of polymers with different molecular weights and different proportions of hydrophilic hydrophobic chain segments have been synthesized, and PCL-b has been preliminarily identified. The proportion of the hydrophilic part and the hydrophobic part of the -PEG-b-PCL polymer and the formation of the vesicles are formed by the self-assembly of the polymer. The study shows that when the PEG mass percentage of the two Pro three block copolymer (PCEP484) is about 50%, the polymer nano micelle with nuclear shell structure can be formed by the film hydration ultrasonic dispersion method; two When the PEG mass percentage of the amphiphilic three block copolymer PCL-b-PEG-b-PCL (PCEP888) is about 33%, it can form a polymer vesicle with an obvious double molecular membrane structure. Two, the development of the paclitaxel polymer micelles and the development of the antitumor research and development of a polymer that can effectively encapsulate the aggregation of paclitaxel (PTX) in the hydrophobic core. The compound micellar drug carrier, which can be used in the whole body of intravenous administration, avoids the allergic reaction and side effects caused by the use of polyoxyethylene castor oil as a solubilizing agent in the clinical taxol injection. In this paper, PCL-b-PEG-b-PCL (PCEP484) was used as the carrier material to prepare paclitaxel poly (paclitaxel) for intravenous injection. Paclitaxel-loaded polymeric micelles (PTX-PM). The scanning electron microscope (SEM) and transmission electron microscopy (SEM) show that the developed PTX-PM has a spherical, uniform size and a clear nuclear shell structure. The paclitaxel can be effectively loaded into hydrophobic core formed by hydrophobic chain segment PCL by hydrophobicity. The drug loading amount is 28.98% and the drug encapsulation rate is 94.36%. High drug loading and encapsulation efficiency. The hydrophilic PEG segment encapsulated on the periphery of the hydrophobic core formed a distinct coronal micellar surface, which could make the polymer micelles dispersed well in the aqueous phase and have a long cycle characteristic. The average particle size of the polymer micelles of the drug carrier was 93nm and the number of polydispersity was 0.19, which was very beneficial to the intravenous administration. The study of scanning calorimetry showed that the crystalline state of the paclitaxel nanoparticles was changed and in the amorphous state in the polymer micelles. The release in vitro of 1M sodium salicylate showed that PTX-PM had the effect of sustained release taxol, no drug release, and the zero order release kinetics model (R=0.99).MTT method for the first 5 days Cytotoxicity studies showed that the blank polymer micelle had no toxicity to HepG2 hepatoma cells and EMT-6 breast cancer cells. Under the same paclitaxel content, the cytotoxicity of PTX-PM was lower than that of the commercial paclitaxel / polyoxyethylene castor oil injection (Taxol (?)), and the time and dose depended on the activity of paclitaxel in polymer micelles. It did not decrease, and could be released with paclitaxel from the polymer micelles to function in the cancer cells for a longer period of time. The pharmacokinetic study of the rodenticide showed that the drug loaded polymer micelles developed in comparison with Taxol (?) significantly prolonged the cycle time and half-life of taxol in the blood and significantly increased the birth rate. The antitumor activity in vivo showed that PTX-PM had obvious inhibitory effect on EMT-6 breast cancer in mice. The tumor inhibition effect of the same dose was better than that of Taxol (tumor inhibition rate: 85.79% vs 63.37%, P0.05). The above studies showed that the developed PTX-PM was highly effective and low toxicity, and could significantly prolong the circulation time of taxol in the blood. EPR passive targeting is a potential drug delivery system for paclitaxel with potential for cancer treatment. Three, folic acid targeting paclitaxel phospholipid polymer hybrid nanoparticles and its anti-tumor research, phospholipid polymer hybrid nanoparticles (lipid-polymer hybrid nanoparticles, LPNPs) is a class based on liposomes and polymerization A novel drug carrier for the development of nanoparticles was developed. A new type of paclitaxel phospholipid polymer hybrid nanoparticle (PTX-FLPNPs) with folic acid targeting was developed. Two amphiphilic copolymer PCL-b-PEG-b-PCL (PCEP484), methoxy polyethylene glycol two stearyl phosphatidyl ethanolamine (mPEG2000-DSPE) and polyethylene glycol two stearyl coupling with folic acid were developed. Phosphatidyl ethanolamine (DSPE-PEG (2000) Folate) was used as carrier material to prepare paclitaxel phospholipid polymer hybrid nanoparticles (PTX-FLPNPs) targeted by folic acid through a thin film hydration ultrasonic dispersion method. Transmission electron microscopy showed that PTX-FLPNPs was spherical, uniform in size, with a nuclear shell shell structure, hydrophobic chain segment PCL and drug PTX passed by self. The inner shell structure of phospholipid DSPE forms the inner shell structure, the two amphiphilic copolymer and the hydrophilic segment PEG in phospholipid form outer shell structure. The laser confocal microscope confirms that the prepared Luo Danming labeled phospholipid PTX-FLPNPs has the structure of "nuclear shell shell" and the obvious phospholipid monolayer structure. The results showed that PTX-LPNPs (no folate targeting molecules) and PTX-FLPNPs (folic acid targeting) had a small dispersion coefficient, indicating that the method could produce a homogeneous particle size phosphatide polymer hybrid nanoparticles. Compared with the PTX-LPNPs particle size and the zeta potential, the average particle size of PTX-FLPNPs increased slightly (279.9 nm vs 271.5 NM), and had a higher negative ZET. The a potential value (-17.5 mV vs-14.2 mV) indirectly indicates that the FLPNPs shell has a folic acid targeting molecule. When the dosage of paclitaxel is 30%, the drug loading of PTX-FLPNPs and PTX-LPNPs is greater than 27%, the drug encapsulation efficiency is greater than 90%, and the drug loading and drug encapsulation efficiency are higher. The release of taxol in vitro shows that paclitaxel is released from PTX-FLPNPs and PTX-LPNPs. Both qualitative and quantitative studies of cell phagocytosis show that the targeting modified phospholipid polymer hybrid nanoparticles can effectively enter the.CCK8 cytotoxicity study in the tumor cells with high expression of folate receptor. The results show that the blank FLPNPs on H1299 lung cancer cells and EMT-6 The cytotoxicity of breast cancer cells was no cytotoxicity. The cytotoxicity of PTX-FLPNPs and PTX-LPNPs was lower than that of Taxol (?) at the same paclitaxel dose, while PTX-FLPNPs was significantly more lethal than PTX-LPNPs (P0.05). It further indicated that the active targeting of folic acid could be used to raise the concentration of high drug in the tumor cells and effectively kill the tumor cells. The study of internal antitumor activity showed that PTX-FLPNPs was similar to Paclitaxel injection in mouse EMT-6 breast cancer by intratumoral injection (P0.05), but the inhibitory effect of PTX-FLPNPs toxicity lower than that of Paclitaxel injection.PTX-FLPNPs was superior to PTX-LPNPs (65.78% vs 48.38%, P0.05), indicating the targeting of folate receptor mediated targeting The action of the tumor cells increased the uptake of the nanoparticles and enhanced the tumor suppressor. Four, the development of the double loaded polymer nanoscale and its tumor targeting preliminary study of the polymer vesicles with a hydrophilic inner cavity and a thicker hydrophobic double layer membrane, and its unique structure can be used to encapsulate hydrophilic and hydrophobic drugs at the same time and be used for swelling. In this paper, PCL-b-PEG-b-PCL (PCEP888) is used as the carrier material to prepare a polymer vesicle with an obvious double molecule membrane like liposome structure by the membrane hydration ultrasonic dispersion method. The hydrophobic drug taxol (paclitaxel, PTX) is loaded into the hydrophobic membrane of the polymer vesicles by hydrophobicity, and the hydrophobic drug is applied to the hydrophobic membrane of the polymer vesicles. The hydrophilic hydrochloric acid (doxorubicin, DOX) was loaded into the hydrophilic cavity of the polymer vesicles by the ammonium sulfate gradient method. The transmission electron microscopy showed that the double carrier polymer vesicles (polymersomes loaded with both PTX and DOX, PS-PTX-DOX) not only have an obvious double layer membrane structure, but also the hydrophilic inner cavity is also shown by the DOX loading. The confocal laser confocal microscope confirmed that the DOX carrying hydrophilic inner cavity with spontaneous fluorescence was further confirmed. When the dosage of paclitaxel and adriamycin was 10%, the average particle size of the double PS-PTX-DOX was 169.7nm, the polydispersity coefficient was 0.211, and the granularity was relatively uniform, which was not often beneficial to the swelling of the drug carrier for the whole body. The cell phagocytosis study showed that the fluorescence intensity of PS-PTX-DOX in the nucleus was much less than that of adriamycin at the same incubation time and doxorubicin dose, indicating that the polymer vesicle carrier has a significant release effect. The release of DOX into the nucleus with the prolongation of the incubation time, the DOX fluorescence intensity in the nucleus enhanced.PS. The particle size of -PTX-DOX is less than 200 nm and can be used for intravenous administration. The distribution of drug in the tumor bearing mice shows that after the injection of PS-PTX-DOX in the tail vein, the blood circulation time can be extended because of its long circulation characteristics, and the drug can be enriched in the tumor site effectively through the EPR effect.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R943;R96

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