pH敏感mPEG-PCL-PGA的合成、表征及在药物传递系统中的应用

发布时间：2018-04-25 20:24

本文选题：mPEG-PCL-PGA + 聚合物囊泡　；参考：《山东大学》2014年硕士论文

【摘要】：高分子合成化学的快速发展使得合成的生物可降解聚合物在生物医药领域得到了广泛的应用。聚酯类聚合物如聚乳酸(PLA)、聚己内酯(PCL)、聚丙交酯-乙交酯共聚物(PLGA)等都是比较常见的合成的聚合物嵌段,这些材料合成简单,对人体毒性较低,具有生物可降解性,因此作为药物传递载体有着较为广阔的应用前景。随着药物传递系统对载体要求的日益提高,近年来聚肽嵌段引起人们的广泛关注,此类聚合物降解后的单体可被生物体利用,且随着外界环境如pH、温度的变化可以改变自身的二级构象,引起嵌段水溶性的改变,引起聚集体结构的聚集-解聚行为或者聚合物聚集体形态的改变。两亲性聚合物由亲水嵌段及疏水嵌段构成,其组成中亲疏水嵌段的比例不同,组装体的形态(胶束、纳米粒、囊泡)也各不相同。聚合物囊泡与细胞结构相似,是由两亲性聚合物自组装形成的完整闭合双层结构。相比于胶束和纳米粒,囊泡的内部为亲水腔,能够将蛋白分子等亲水性药物包裹起来,避免外界环境对药物活性的影响。与具有类似结构的脂质体相比,聚合物囊泡的膜较厚,体系稳定,而脂质体的组成成分磷脂易氧化发生内容物的泄漏。此外,聚合物囊泡的性质可通过改变聚合物嵌段的种类和长度进行调节。盐酸多柔比星是临床上治疗多种肿瘤的一线药物,广泛用于治疗急性白血病、淋巴癌、软组织和骨肉癌、儿童恶性肿瘤及多种成人实体瘤,尤其对乳腺癌和肺癌的治疗效果较为明显,但是应用多柔比星的患者会出现各种各样的副作用如恶心、呕吐、骨髓抑制等,最为严重的是对病人的心脏毒性。为了降低盐酸多柔比星的毒副作用,常将药物包载于载体中进而降低其在正常组织中的分布,使较多的药物运送到肿瘤部位。为了实现药物的肿瘤靶向,可以利用肿瘤部位pH值低的特点,将盐酸多柔比星包载于pH敏感载体中,使其在肿瘤部位能够释放较多的药物。而且已有报道称,将盐酸多柔比星包裹于聚合物囊泡中,能降低其心脏毒性。本课题以mPEG、ε-CL、L-谷氨酸-γ-苄酯-N-羧酸酐为原料合成三嵌段聚合物mPEG-PCL-PGA,根据文献报道选择合适的亲、疏水链段比例,使合成的聚合物在水溶液中可以自发形成囊泡。聚乙二醇(mPEG)作为亲水嵌段能够抵抗血浆蛋白吸附,其伸展在囊泡表面能够赋予囊泡“隐形”的特点,避免网状内皮系统的吞噬,达到长循环的目的；聚己内酯(PCL)作为疏水嵌段,各单体间其通过易水解的酯键相连接,其结构相对柔软,容易被体内酶分解代谢；聚谷氨酸(PGA)嵌段在中性条件下,呈现水溶性的无规卷曲构象(coil),而在酸性条件下则会呈现水溶性差的α螺旋(a-helical)构象,PGA嵌段具有pH敏感性从而赋予其所形成的聚合物囊泡具有pH敏感性,实现在肿瘤部位(pH较低)释药的目的。本论文的主要研究内容如下： 1.三嵌段pH敏感聚合物mPEG-PCL-PGA的合成、表征及聚集行为研究利用开环聚合反应分三大步合成了三嵌段pH敏感聚合物mPEG-PCL-PGA。首先以单甲氧基聚乙二醇为大分子起始物,ε-己内酯为单体,辛酸亚锡为催化剂,开环聚合生成mPEG-PCL;由于一级胺是引发谷氨酸单体聚合最合适的物质,因此将mPEG-PCL末端的羟基与氨基端BOC(叔丁氧羰基)保护的苯丙氨酸通过酯键相连,然后水解掉BOC保护基,露出氨基；以此氨基为引发剂,与谷氨酸活性单体γ-苄基-L-谷氨酸-N-羧酸酐(NCA-BLG)发生聚合,水解掉γ-苄基,得到目标聚合物。聚合物的结构通过核磁共振氢谱与傅里叶红外光谱进行验证；表面张力法测定表面张力等温线,由曲线拐点测出聚合物的临界聚集浓度为3.6mg/mL,此聚合物可以使水的表面张力降到46mN/m。2%磷钨酸溶液负染色透射电子显微镜表明溶液中聚集体是形态比较规整的球形单层囊泡。 2.载药聚合物囊泡的制备、性质及体外释放行为研究本实验采用纳米沉淀法(nanoprecipitation)制备载药聚合物囊泡,负染色法观察载药囊泡的形态；并考察不同药物与聚合物质量比对囊泡的载药量和包封率的影响,根据囊泡稳定性及药物终浓度选择药物/聚合物最适的比例为1.5：10(w/w),此时囊泡的载药量为(10.4±0.5)%,包封率为(78.7±1.4)%；测得囊泡粒径大小为(180.9±7.6)nm,zeta电势为-(38.5±0.3)mV。不同pH条件下的体外释放实验表明,与原料药溶液相比,囊泡中盐酸多柔比星的释放更加缓慢,且在酸性条件下(pH5.0和6.5)较中性条件下药物的释放要快,释放量要高,说明此载药聚合物囊泡具有pH敏感性。 3.载药聚合物囊泡的体外抗肿瘤实验本实验采用WST-1法研究聚合物材料的毒性,原料药溶液和载药囊泡溶液对人乳腺癌细胞(MCF-7)和人肺癌细胞(A549)的细胞毒性作用。最高浓度聚合物材料(500μg/mL)在各时间点对MCF-7细胞增殖的抑制率如下：(25.7±1.5)%(72h)、(17.1±3.4)%(48h)、(12.8±2.7)%(24h),对A549的抑制率如下：(36.3±1.7)%(72h)、(19.6±3.4)%(48h)、(11.3±2.7)%(24h)；各组对细胞的杀伤力均与浓度和时间成正相关性,各浓度载药囊泡溶液组对两种细胞的杀伤力均比原料药组强,盐酸多柔比星原料药溶液对MCF-7的抑制率为(86.7±4.1)%(72h)、(79.9±7.3)%(48h)、(71.2±4.9)%(24h),而载药囊泡对MCF-7的抑制率为(93.4±3.4)%(72h)、(89.7±2.3)%(48h)、(79.0±3.4)%(24h)；盐酸多柔比星原料药溶液对A549的抑制率为(84.2±2.2)%(72h)、(76.5±6.7)%(48h)、(70.2±3.0)%(24h),载药囊泡的对A549的抑制率为(86.3±0.9)%(72h)、(84.4+7.2)%(48h)、(75.0±2.5)%(24h)。细胞摄取实验表明：MCF-7对盐酸多柔比星原料药溶液及囊泡溶液的摄取无差别,A549对载药囊泡的摄取量高于对原料药的摄取。 4.载药聚合物囊泡的体内药动学研究本实验以Wistar大鼠为实验动物,尾静脉给药,沉淀血浆蛋白后,内标法测定血药浓度。所测各时间点药物浓度数据采用DAS2.0软件处理,以统计矩原理分析各组药动数据得统计矩参数。注射载药囊泡大鼠药动曲线下面积AUC(2081.621),高于原料药组(1591.873)；且在大鼠体内的体内平均滞留时间(MR7)增加了一倍,分别为1.897h(PolyDox)和0.948(Dox)；载药囊泡与盐酸多柔比星的表观分布容积(V)分别为0.015L/kg与0.008L/kg,载药囊泡的的表观分布容积提高一倍多；同时载药囊泡在药物体内的清除率低于盐酸多柔比星原料药。综上所述,本论文合成了pH敏感的三嵌段聚合物mPEG-PCL-PGA,其在水中可形成稳定的聚合物囊泡,用此囊泡包载抗癌药物盐酸多柔比星,减少了药物的毒副作用,提高了药物在体内的浓度并使其较多的释放于肿瘤部位,增强了对肿瘤细胞的杀伤力,为开发安全有效的pH敏感药物载体提供了一定的研究基础。
[Abstract]:The rapid development of synthetic polymer chemistry makes synthetic biodegradable polymers widely used in the field of biological medicine. Polyester polymers such as polylactic acid (PLA), polyhexyl ester (PCL), polylactone - glycolide copolymer (PLGA) are common polymer blocks, which are simple in synthesis and to human toxicity. Because of its low sex and biodegradability, it has a broad application prospect as a carrier of drug delivery. With the increasing requirements of the drug delivery system, polypeptides have attracted wide attention in recent years. The monomer after the degradation of this kind of polymer can be used by the raw body, and with the external environment such as pH, the temperature changes. It can change the two level conformation of its own and cause the change of water solubility in the block, which causes the aggregation and depolymerization of the aggregate structure or the change of the morphology of the polymer aggregates.
The two amphiphilic polymers are composed of hydrophilic block and hydrophobic block, and the proportion of hydrophobic block in its composition is different. The morphology of the assembly (micelle, nanoparticle, vesicle) is different. The polymer vesicles are similar to the cell structure, which are formed by the self-assembly of two amphiphilic polymers. Compared with micelles and nanoparticles, vesicles It is a hydrophilic cavity in which the hydrophilic drugs such as protein molecules can be wrapped up to avoid the influence of the external environment on the drug activity. Compared with the liposomes with similar structure, the membrane of the polymer vesicles is thicker and the system is stable, and the composition of the liposomes is easily oxidized by the phospholipids. In addition, the properties of the polymer vesicles are accessible. Adjust the type and length of polymer block.
Doxorubicin is a first-line drug in the clinical treatment of various tumors. It is widely used in the treatment of acute leukemia, lymphadenocarcinoma, soft tissue and bone meat cancer, children's malignant tumors and many adult solid tumors, especially for breast and lung cancer, but patients with doxorubicin will have a variety of side effects such as Nausea, vomiting, bone marrow suppression, and so on, the most serious is the heart toxicity of the patient. In order to reduce the toxic and side effects of doxorubicin hydrochloride, the drug is often loaded in the carrier to reduce its distribution in the normal tissue, so that more drugs are transported to the tumor site. In order to achieve the target of the tumor, the pH value of the tumor site can be used low. It is characterized by carrying doxorubicin in the pH sensitive carrier to release more drugs at the tumor site. And it has been reported that doxorubicin can be wrapped in a polymer vesicle and can reduce its cardiac toxicity.
The three block polymer mPEG-PCL-PGA was synthesized from mPEG, epsilon -CL, L- glutamic acid - gamma benzyl -N- carboxylic anhydride. According to the literature, it was reported that the suitable parent, the proportion of hydrophobic segments, was selected to form the vesicles spontaneously in aqueous solution. Polyethylene glycol (mPEG) was used as a hydrophilic block to resist plasma protein adsorption. The surface of the vesicles can give the vesicles "stealth", avoiding the phagocytosis of the reticuloendothelial system and reaching the goal of a long cycle. Polyhexyl ester (PCL) is used as a hydrophobic block, and each monomer is connected through a hydrolytic ester bond, its structure is relatively soft and easy to be metabolize in vivo, and polyglutamic acid (PGA) block is under neutral condition. The water-soluble random curl conformation (coil) shows a conformation of poor water-soluble alpha helix (A-helical) under acidic conditions, and PGA block has pH sensitivity, thus giving its formed polymer vesicles with pH sensitivity, which can be achieved at the tumor site (lower pH).
The main contents of this paper are as follows:
1. synthesis, characterization and aggregation behavior of three block pH sensitive polymer mPEG-PCL-PGA
The three block pH sensitive polymer mPEG-PCL-PGA. was synthesized by open ring polymerization in three steps. First, monomethoxy polyethylene glycol was used as large molecular initiator, epsilon hexyl ester as monomer, and stannous octanoate was used as the catalyst to produce mPEG-PCL. As the first order amine was the most suitable substance for the polymerization of glutamic acid monomer, the end of mPEG-PCL was made. The phenylalanine protected by the terminal hydroxyl group and the amino terminal BOC (TERT oxo carbonyl) is connected through the ester bond and then hydrolyzed out of the BOC protection group to expose the amino group. The amino group is used as an initiator to polymerize the glutamic acid active monomer gamma benzyl -L- glutamic acid -N- carboxylic anhydride (NCA-BLG) and hydrolyze the gamma benzyl group to get the target polymer. The structure of the polymer passes through the core. The magnetic resonance hydrogen spectrum and Fourier infrared spectrum are verified. Surface tension method is used to determine the surface tension isotherm, and the critical concentration of polymer is measured from the curve point of 3.6mg/mL. The polymer can reduce the surface tension of water to 46mN/m.2% phosphotungstic acid solution negative dyeing transmission electron microscope, indicating that the aggregates in the solution are compared. Regular spherical monolayer vesicles.
Preparation, properties and in vitro release behavior of 2. drug loaded polymer vesicles
The drug loaded polymer vesicles were prepared by nano precipitation method (nanoprecipitation). The morphology of the carrier vesicles was observed by negative staining, and the effects of the mass ratio of different drugs and polymers on the drug loading and encapsulation efficiency of the vesicles were investigated. The optimum proportion of the vesicle stability and the final drug concentration of the drug / polymer was 1.5:10 (w/w). The loading capacity of the vesicles was (10.4 + 0.5)%, the encapsulation rate was (78.7 + 1.4)%, the size of the vesicles was (180.9 + 7.6) nm, and the release experiment of zeta electric potential - (38.5 + 0.3) mV. in different pH conditions showed that the release of doxorubicin in the vesicles was more slow compared with the solution of the API, and it was more neutral in acid conditions (pH5.0 and 6.5). The drug release was fast and the release rate was high, indicating that the drug loaded polymer vesicles had pH sensitivity.
In vitro antitumor experiment of 3. drug loaded polymer vesicles
In this experiment, the toxicity of the polymer material, the solution of the drug and the solution of the vesicle on the cytotoxicity of human breast cancer cells (MCF-7) and human lung cancer cells (A549) were studied by WST-1 method. The inhibition rate of the maximum concentration of polymer material (500 mu g/mL) on the proliferation of MCF-7 cells at all time points was as follows: (25.7 + 1.5)% (72h), (17.1 + 3.4)% (48h), (12.8). The inhibition rate of A549 was as follows: (24h) as follows: (36.3 + 1.7)% (72h), (19.6 + 3.4)% (48h) and (11.3 + 2.7)% (24h). The cell killing ability of each group was positively correlated with concentration and time, and the killing force of each concentration of vesicle solution group was stronger than that of the drug group, and the inhibitory rate of doxorubicin hydrochloride on MCF-7 was 86 (86). .7 + 4.1)% (72h), (79.9 + 7.3)% (48h), (71.2 + 4.9)% (24h), and the inhibition rate of drug vesicles to MCF-7 was (93.4 + 3.4)% (72h), (89.7 + 2.3)% (48h), (79 + 3.4)% (24h), and the inhibition rate of doxorubicin hydrochloride was (84.2 + 2.2)% (72h), (48h)% (24h)% (24h)). .9)% (72h), (84.4+7.2)% (48h), (75 + 2.5)% (24h). Cell uptake experiments showed that MCF-7 had no difference in the uptake of doxorubicin hydrochloride and vesicle solution, and the uptake of vesicles by A549 was higher than that of the drug.
Pharmacokinetic study of 4. drug loaded polymer vesicles in vivo
In this experiment, Wistar rats were used as experimental animals. After the tail vein was given, plasma protein was precipitated, the blood concentration was measured by internal standard. The data of drug concentration at each time point were treated by DAS2.0 software. The statistical moment principle was used to analyze the statistical moment parameters of the pharmacokinetic data of each group. The area under the dynamic curve under the dynamic curve of the injection of drug vesicles was AUC (2081.621), higher than the raw material. In the drug group (1591.873), the average retention time (MR7) in the body was doubled, 1.897h (PolyDox) and 0.948 (Dox), and the apparent volume (V) of the drug vesicles and doxorubicin (V) was 0.015L/kg and 0.008L/kg, respectively. The apparent volume of the vesicles carrying the vesicles was more than doubled, and the drug vesicles were loaded in the drug body. The internal scavenging rate is lower than the drug of doxorubicin hydrochloride.
To sum up, this paper synthesizes the pH sensitive three block polymer mPEG-PCL-PGA, which can form a stable polymer vesicle in water and encapsulate the anticancer drug doxorubicin in the water. It reduces the side effects of the drug, increases the concentration of the drug in the body and releases more of the drug in the tumor site, and strengthens the tumor cells. Lethality provides a basis for developing safe and effective pH sensitive drug carriers.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943

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