基于mPEG-PBLG-SS-DTX的氧化还原敏感载药系统的研究

发布时间：2018-04-29 17:00

本文选题：二硫键 + 氧化还原敏感　；参考：《山东大学》2017年硕士论文

【摘要】：癌症已经成为危害人类身体健康的主要因素之一,在世界范围内引起了广泛关注。目前,在临床上用于治疗癌症的主要方法包括化学治疗、放射治疗、手术治疗等。多西他赛(DTX)是一种在临床上已得到广泛应用的化疗药物,它是紫杉醇的半合成衍生物,是一种广谱抗癌药,对乳腺癌、卵巢癌、前列腺癌、肺癌、头颈癌等癌症均具有治疗作用。但是,由于DTX的水溶性差,在临床应用中,通常在其注射液中加入一定量的吐温-80等增溶剂以增加其溶解性,易引起严重的副反应,如过敏反应等。另外,由于DTX的组织选择性差,容易对正常细胞造成毒副作用。为了增加DTX的溶解性,增加其对肿瘤组织的靶向性,人们近些年研究了多种药物传递系统,如囊泡、纳米粒、聚合物胶束、聚合物-药物结合物胶束等。其中,聚合物胶束是由两亲性嵌段共聚物在水中自组装形成的纳米级别的核壳结构,亲水性嵌段形成聚合物胶束的外壳,而疏水性嵌段组成其疏水性的内核,该疏水内核可以装载疏水性的药物。如果将药物与亲水性或者两亲性嵌段共聚物通过化学键结合,则可形成聚合物-药物结合物胶束。聚合物-药物结合物胶束具有载药量高、稳定性好、药物释放缓慢等优点,具有较好的抗肿瘤效果。另外,可通过环境敏感的化学键将聚合物与药物结合,使聚合物-药物结合物胶束具有氧化还原敏感性、pH敏感性、酶敏感性等等。为了增加DTX的溶解性,降低其毒副作用,本课题将DTX通过一个含有二硫键的连接臂与两亲性嵌段共聚物mPEG-PBLG相连,合成了氧化还原敏感的mPEG-PBLG-SS-DTX嵌段。该嵌段可以在水中自组装形成胶束,并通过对此胶束进行进一步修饰,形成多功能的mPEG-PBLG-SS-DTX/mPEG-FA混合胶束,该混合胶束同时具有氧化还原敏感性和主动靶向性,表现出更好的抗肿瘤效果。本课题的主要研究内容包括以下三点:(1)两种具有不同分子量的 mPEG-PBLG-SS-DTX(mPEG2000-PBLG1750-SS-DTX 和 mPEG5000-PBLG1750-SS-DTX)的合成和表征。以具有不同分子量的mPEG-NH2为大分子引发剂,与γ-Bzl-L-Glu-NCA单体发生开环聚合反应,生成具有不同分子量的两亲性嵌段共聚物mPEG-PBLG(mPEG2000-PBLG1750和mPEG5000-PBLG1750)。然后,通过一个含有二硫键的连接臂将DTX与mPEG-PBLG结合,得到氧化还原敏感的聚合物-药物结合物mPEG-PBLG-SS-DTX。并分别利用1H-NMR和FT-IR确定中间产物和mPEG-PBLG-SS-DTX 的合成成功。(2)氧化还原敏感的mPEG-PBLG-SS-DTX胶束的制备、表征和体外抗肿瘤评价。由于聚合物-药物结合物mPEG-PBLG-SS-DTX具有两亲性,在水溶液中可以自组装形成具有核壳结构的胶束,所以在本实验中我们通过透析法将其制备成胶束。该胶束在TEM下呈现出粒径均一、圆整的球形,利用DLS测得的粒径分别为101.3 ± 1.4和148.9 ±1.4 nm,且粒径布均匀,这两种胶束的Zeta电势分别为-20.1 ± 0.1和-14.5 ± 0.1 mV,表明这两种mPEG-PBLG-SS-DTX胶束具有较好的稳定性。紫外分光光度计法测得的这两种胶束的载药量分别为(13.9 ±0.8)%和(9.2 ± 0.4)%。利用芘探针法测得的CMC均较低,分别为3.98和6.94 μg/mL,这表明这两种胶束即使在血液循环系统中被稀释,仍可以保持结构稳定。该胶束具有氧化还原敏感性,药物的体外释放实验结果表明,在不存在还原性DTT的环境中,120h后,约有10%的DTT从胶束中释放出来,而在DTT存在的条件下,DTX的释放速率明显加快,120 h后,DTX的累积释放量达到40%左右,这说明DTX的释放具有氧化还原敏感性。溶血性实验结果表明,这两种胶束的溶血率均较小(5%),具有较好的血液相容性。在胶束的体外抗肿瘤评价中,以A549和MCF-7/ADR细胞为模型细胞。与DTX相比,mPEG-PBLG-SS-DTX胶束对这两种细胞表现出更好的细胞抑制率,mPEG-PBLG-SS-DTX胶束对MCF-7/ADR细胞24 h的IC50值约为DTX的十五分之一。另外,体外摄取实验结果表明,A549和MCF-7/ADR细胞对mPEG-PBLG-SS-DTX/C-6胶束的摄取效率要强于对C-6的摄取效率,这与细胞毒性实验的结果一致。(3)多功能的mPEG-PBLG-SS-DTX/mPEG-FA混合胶束的制备、表征和体外抗肿瘤评价。本部分选用具有较大载药量的mPEG2000-PBLG1750-SS-DTX与含有主动靶向分子的mPEG-FA混合,通过透析法制备了多功能的mPEG-PBLG-SS-DTX/mPEG-FA混合胶束,该混合胶束不仅具有氧化还原敏感性,还具有主动靶向性。在加入mPEG-FA后,mPEG-PBLG-SS-DTX/mPEG-FA混合胶束呈现出粒径均一、形态圆整的球形,且粒径和载药量分别为129.7±2.1 nm和(9.0 ± 1.7)%。利用芘探针法测得的CMC为5.08 μg/mL,这与mPEG-PBLG-SS-DTX胶束的相似,表明该混合胶束在血液循环系统中可保持结构的稳定。溶血实验结果表明,不同浓度的mPEG-PBLG-SS-DTX/mPEG-FA混合胶束溶液的溶血率均小于5%,说明该混合胶束不会引起红细胞的溶血。药物的体外释放实验结果表明,mPEG-PBLG-SS-DTX/mPEG-FA混合胶束仍然具有氧化还原敏感性。另外,将叶酸受体(FR)低表达的A549细胞和FR高表达的MCF-7细胞作为模型细胞,对mPEG-PBLG-SS-DTX/mPEG-FA混合胶束的抗肿瘤效果进行评价。细胞毒性实验结果表明,对于A549细胞,mPEG-PBLG-SS-DTX/mPEG-FA 混合胶束的抑制率与 mPEG-PBLG-SS-DTX 胶束无明显差别,而对于MCF-7细胞,前者要明显高于后者,这说明mPEG-PBLG-SS-DTX/mPEG-FA混合胶束具有主动靶向性,对FR高表达的肿瘤细胞具有更好的抗肿瘤效果。在细胞摄取实验中,A549细胞对mPEG-PBLG-SS-DTX/C-6 胶束、mPEG-PBLG-SS-DTX/mPEG-FA/C-6 混合胶束和mPEG-PBLG-SS-DTX/mPEG-FA/C-6混合胶束+2 mM FA的细胞摄取效率无明显的差别,而在MCF-7细胞中,mPEG-PBLG-SS-DTX/mPEG-FA/C-6混合胶束进入细胞的效率要明显高于其他两者,这表明mPEG-PBLG-SS-DTX/mPEG-FA/C-6混合胶束可通过FR介导的细胞内吞作用进入细胞,这使其细胞摄取效率得到了显著的提高。细胞凋亡实验展现出了类似的结果,mPEG-PBLG-SS-DTX胶束和mPEG-PBLG-SS-DTX/mPEG-FA混合胶束孵育12 h后,A549细胞的凋亡并无明显差异,而在MCF-7细胞中,后者的凋亡效率要明显高于前者。这些结果均表明,mPEG-PBLG-SS-DTX/mPEG-FA混合胶束具有主动靶向性,对FR高表达的肿瘤细胞表现出更好的抑制效果。
[Abstract]:Cancer has become one of the major factors that harm the health of human beings and has aroused widespread concern around the world. The main clinical methods for the treatment of cancer include chemical therapy, radiation therapy, and surgical treatment. DTX is a clinically widely used chemotherapeutic drug, it is taxol Semisynthetic derivatives, a broad-spectrum anticancer drug, have therapeutic effects on breast, ovarian, prostate, lung and head and neck cancers. However, due to the poor solubility in water of DTX, a certain amount of Twain -80 is usually added to its injection to increase its solubility and cause serious side effects, such as excessive side effects. In addition, due to the poor tissue selectivity of DTX, it is easy to cause toxic and side effects to normal cells. In order to increase the solubility of DTX and increase the targeting of tumor tissue, a variety of drug delivery systems have been studied in recent years, such as vesicles, nanoparticles, polymer micelles, polymer micelles, polymer micelles and so on. It is a nanoscale nuclear shell structure formed by self assembly of two amphiphilic block copolymers in water. The hydrophilic block forms the shell of the polymer micelle, and the hydrophobic block consists of its hydrophobic core. The hydrophobic core can be loaded with hydrophobic drugs. If drug and hydrophilic or two amphiphilic block copolymers are bonded through chemical bonding, the hydrophobic core can be used as a hydrophobic core. Polymer drug binding micelles can form polymer drug binding micelles. Polymer drug binding micelles have the advantages of high drug loading, good stability and slow release of drugs, and have good antitumor effects. In addition, the polymer can be combined with drugs by environmental sensitive chemical bonds to make the polymer drug binding micelles have redox sensitivity. PH sensitivity, enzyme sensitivity and so on. In order to increase the solubility of DTX and reduce its toxic and side effects, DTX has synthesized a redox sensitive mPEG-PBLG-SS-DTX block by connecting a two parent block copolymer with a two sulfur bond arm, which can form micelles by self assembly in water and through this micelle. Further modification was carried out to form a multifunction mPEG-PBLG-SS-DTX/mPEG-FA mixed micelle. The hybrid micelle had both redox sensitivity and active targeting, and showed better anti-tumor effects. The main contents of this study included the following three points: (1) two kinds of mPEG-PBLG-SS-DTX (mPEG2000-PBLG1750-SS-) with different molecular weights. Synthesis and characterization of DTX and mPEG5000-PBLG1750-SS-DTX. Using mPEG-NH2 with different molecular weights as a macromolecular initiator and an open ring polymerization with gamma -Bzl-L-Glu-NCA monomer, two amphiphilic block copolymers with different molecular weights, mPEG-PBLG (mPEG2000-PBLG1750 and mPEG5000-PBLG1750), are produced. Then, a two sulfur bond is used. The joint arm combines DTX with mPEG-PBLG to obtain the redox sensitive polymer drug binding mPEG-PBLG-SS-DTX. and determine the synthesis of intermediate products and mPEG-PBLG-SS-DTX using 1H-NMR and FT-IR respectively. (2) the preparation, characterization and antitumor evaluation of the redox sensitive mPEG-PBLG-SS-DTX micelles. The compound mPEG-PBLG-SS-DTX has two affinity and can be self assembled into a micelle with nuclear shell structure in aqueous solution. So in this experiment, we prepared the micelles by dialysis. The micelles showed a uniform size and round ball under TEM. The size of the particles was 101.3 + 1.4 and 148.9 + 1.4 nm, respectively. The Zeta potential of these two micelles is -20.1 + 0.1 and -14.5 + 0.1 mV, respectively, indicating that the two mPEG-PBLG-SS-DTX micelles have good stability. The dosage of the two micelles obtained by UV spectrophotometer is (13.9 + 0.8)% and (9.2 + 0.4)% respectively. The CMC measured by pyrene probe method are lower, 3.98 and 6.94 mu, respectively. G/mL, which indicates that the two micelles can remain stable even if they are diluted in the blood circulation system. The micelles have redox sensitivity and the drug release in vitro shows that about 10% of DTT released from the micelles in the absence of reductive DTT environment, and the release of DTX under the presence of DTT. After 120 h, the cumulative release of DTX was about 40%, which indicated that the release of DTX was redox sensitivity. The hemolysis results of the two micelles showed that the hemolysis of these two kinds of micelles were smaller (5%) and had better blood compatibility. In the anti tumor evaluation of micelles, A549 and MCF-7/ADR cells were used as model cells. And DTX In contrast, mPEG-PBLG-SS-DTX micelles showed better cell inhibition rates for these two cells, and the IC50 value of mPEG-PBLG-SS-DTX micelles to MCF-7/ADR cells 24 h was about 1/15 of DTX. In addition, the uptake of A549 and MCF-7/ADR cells in vitro showed that the uptake efficiency of A549 and MCF-7/ADR cells to mPEG-PBLG-SS-DTX/C-6 micelles was stronger than that of C-6. This is in accordance with the results of cytotoxicity test. (3) preparation, characterization and in vitro anti-tumor evaluation of multifunction mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles. This part uses a mixture of mPEG2000-PBLG1750-SS-DTX with a larger dose and a mPEG-FA containing active target molecules, and the multifunction mPEG-PBLG-SS-DTX/mPEG-FA is prepared by dialysis method. The mixed micelles not only have redox sensitivity, but also have active targeting. After adding mPEG-FA, the mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles show a uniform particle size and round shape, and the particle size and drug loading are 129.7 + 2.1 nm and (9 + 1.7)% respectively. The CMC of the pyrene probe is 5.08 mu g/mL, which is with M The similarity of PEG-PBLG-SS-DTX micelles shows that the mixed micelles can maintain the stability of the structure in the blood circulation system. The hemolysis test results show that the hemolysis rate of the mixed micelle solution of different concentrations is less than 5%, indicating that the mixed micelle does not cause hemolysis of the red cells. The results of the drug release in vitro show that the mixture micelle does not cause hemolysis. MPEG-PBLG-SS-DTX/mPEG-FA mixed micelles still have redox sensitivity. In addition, the antitumor effect of mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles was evaluated by using A549 cells with low expression of folic acid receptor (FR) and MCF-7 cells with high expression of FR as model cells. The cytotoxicity test results showed that for A549 cells, mPEG-PBLG-SS-DTX. The inhibition rate of /mPEG-FA mixed micelles was not significantly different from that of mPEG-PBLG-SS-DTX micelles, but for MCF-7 cells, the former was significantly higher than that of the latter, which indicated that mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles had active targeting and had better anti-tumor effects on FR highly expressed tumor cells. In cell uptake experiments, A549 cells were to mPEG-PBLG There was no significant difference in the cell uptake efficiency of -SS-DTX/C-6 micelles, mPEG-PBLG-SS-DTX/mPEG-FA/C-6 mixed micelles and mPEG-PBLG-SS-DTX/mPEG-FA/C-6 mixed micelles, +2 mM FA, but in MCF-7 cells, the efficiency of mPEG-PBLG-SS-DTX/mPEG-FA/C-6 mixed micelles into cells was significantly higher than that of both of them, which indicated that mPEG-PBLG-SS-DTX/mPEG-FA/C-6 The mixed micelles can enter cells through the cell endocytosis mediated by FR, which significantly improves the efficiency of cell uptake. Apoptosis experiments show similar results. After incubation of 12 h micelles and mPEG-PBLG-SS-DTX/mPEG-FA micelles of mPEG-PBLG-SS-DTX micelles, there is no significant difference in the apoptosis of A549 cells, but in MCF-7 cells. The results showed that the mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles had active targeting and showed a better inhibitory effect on the tumor cells with high expression of FR.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943

【参考文献】