甘露糖修饰的共价键连BODIPY的聚合物纳米胶束及其靶向光动力治疗的研究

发布时间：2019-07-06 16:14

【摘要】：将光动力治疗应用于癌症的治疗在生物研究领域备受关注,它主要通过光动力学反应选择性破坏病变组织,从而达到治疗癌症的目的。但光敏剂分子的疏水性和靶向性的缺乏限制了光动力治疗的应用,导致其在生物体内生物利用率低,很难在肿瘤部位集聚。因此,需要寻找光敏剂分子输送载体将光敏剂分子靶向输送至肿瘤部位,以改善光敏剂分子的生物相容性,提高对癌症的治疗效果。在多种纳米载体中,聚合物胶束由于其潜在的应用价值和独特的性能受到科研工作者们的青睐。在聚合物胶束表面修饰上能被肿瘤细胞靶向识别的靶向分子,然后利用其疏水部分负载光敏剂分子,从而克服光敏剂分子在光动力治疗中的缺陷,以达到理想的治疗效果。本研究首先通过两步原子转移自由基聚合(ATRP)反应制备了嵌段共聚物聚丙烯酸叔丁酯-聚甲基丙烯酸缩水甘油酯(Pt BA-b-PGMA),再通过水解反应将聚合物片段Pt BA上的叔丁基脱去生成带有羧基的嵌段共聚物PAA-b-PGMA,接着借助酰胺缩合反应将预先制备的端基位带有氨基的甘露糖修饰在聚合物上,制备了甘露糖修饰的嵌段共聚物Mannose-PAA-b-PGMA,随后在嵌段聚合物片段PGMA上的环氧基团在叠氮钠的条件下发生开环反应,生成了带有叠氮基的甘露糖修饰的嵌段共聚物Mannose-PAA-b-PGMA-N3,最后通过嵌段聚合物上的叠氮基团与预先制备好的带有炔基的光敏剂分子氟硼二吡咯(BODIPY)在抗环血酸钠和五水硫酸铜的条件下发生“click”反应,最终成功制备了表面甘露糖修饰的共价键连BODIPY光敏剂的聚合物纳米胶束(Mannose-PAA-b-PGMA-BODIPY)。利用核磁共振氢谱仪和傅立叶变换红外光谱仪对其进行结构确认;通过透射电镜和动态光散射等考察其理化性质。结果表明:所制备的甘露糖修饰的负载光敏剂的聚合物纳米胶束呈规则球形,粒径分布均一,稳定性良好。通过Agilent Cary Eclipse荧光分光光度计测定单线态氧的生成,检测结果表明:负载光敏剂的聚合物胶束在特定波长光的照射下能够不断产生单线态氧。以人乳腺癌MDA-MB-231细胞和人肾上皮HEK293正常细胞为细胞模型,利用激光共聚焦显微镜对两种细胞的甘露糖受体靶向性和光动力治疗疗效进行考察和评价。结果表明:聚合物胶束能被人乳腺癌MDA-MB-231细胞表面高表达的甘露糖受体特异性识别,并被内吞进入癌细胞内,聚合物胶束对人乳腺癌MDA-MB-231细胞和人肾上皮HEK293正常细胞均无暗毒性,在535 nm LED光照下对人乳腺癌MDA-MB-231细胞具有较好的光动力杀伤作用。最后通过MTT细胞毒性实验评价聚合物光敏胶束在535 nm LED光照下的生物相容性和细胞毒性。结果表明:聚合物胶束具有良好的生物相容性,在535 nm LED的光照下,对肿瘤细胞具有较大的毒性,而对正常细胞无毒性作用。总之,Mannose-PAA-b-PGMA-BODIPY有望成为一种理想的光敏剂,应用于癌症的治疗。
文内图片：

图片说明：负载光敏剂前后的聚合物纳米胶束的透射电镜照片
[Abstract]:The application of photodynamic therapy to the treatment of cancer has attracted much attention in the field of biological research. it mainly destroys the lesion tissue selectively through photodynamic reaction, so as to achieve the purpose of treating cancer. However, the lack of hydrophobicity and targeting of photosensitizers limits the application of photodynamic therapy, which leads to its low bioavailability in vivo, so it is difficult to gather in tumor sites. Therefore, it is necessary to find the carrier of photosensitizer molecular transport to the tumor site in order to improve the biocompatibility of photosensitizer molecules and improve the therapeutic effect on cancer. In many kinds of nano-carriers, polymer micelle is favored by researchers because of its potential application value and unique properties. The targeted molecules which can be targeted by tumor cells are modified on the surface of polymer micelle, and then the hydrophobic part of the molecule is used to load the photosensitizer molecules, so as to overcome the defects of photosensitizer molecules in photodynamic therapy and achieve ideal therapeutic effect. In this study, the block copolymer tert-butylacrylic acid-polyglycidyl methacrylate (Pt BA-b-PGMA) was prepared by two-step atom transfer radical polymerization (ATRP) reaction, and then the tert-butylgroup on the polymer fragment Pt BA was removed by hydrolysis to form a block copolymer PAA-b-PGMA, with carboxy group. Then the pre-prepared mannose with amino group was modified on the polymer by amide condensation reaction. The mannose modified block copolymer Mannose-PAA-b-PGMA, was prepared. The epoxy group on the block polymer fragment PGMA then opened the ring under the condition of sodium azide to form the mannose modified block copolymer Mannose-PAA-b-PGMA-N3, with azide group. Finally, the polymer nano-micelle (Mannose-PAA-b-PGMA-BODIPY) of surface mannose modified covalent BODIPY photosensitizer (Mannose-PAA-b-PGMA-BODIPY) was successfully prepared by the reaction of azide group on block polymer with preprepared photosensitizer fluoroboron dipolypyrrole (BODIPY) under the condition of anti-sodium cyclophosphate and copper sulfate pentahydrate. Its structure was confirmed by nuclear magnetic resonance hydrogen spectrometer and Fourier transform infrared spectrometer, and its physical and chemical properties were investigated by transmission electron microscope and dynamic light scattering. The results show that the polymer nanoparticles modified by mannose are regular spherical, the particle size distribution is uniform and the stability is good. The formation of singlet oxygen was measured by Agilent Cary Eclipse fluorescence spectrophotometer. the results show that the polymer micelle loaded with photosensitizer can continuously produce singlet oxygen under the irradiation of specific wavelength light. Using human breast cancer MDA-MB-231 cells and human renal epithelial HEK293 normal cells as cell models, the targeting and photodynamic effects of mannose receptor in two kinds of cells were investigated and evaluated by laser confocal microscope. The results showed that polymer micelle could be specifically recognized by mannose receptor highly expressed on the surface of human breast cancer MDA-MB-231 cells and swallowed into cancer cells. Polymer micelle had no dark toxicity to human breast cancer MDA-MB-231 cells and human renal epithelial HEK293 normal cells, and had good photodynamic killing effect on human breast cancer MDA-MB-231 cells under 535 nm LED light. Finally, the biocompatibility and cytotoxicity of polymer photosensitive micelle under 535 nm LED light were evaluated by MTT cytotoxicity test. The results showed that the polymer micelle had good biocompatibility. Under the irradiation of 535 nm LED, the polymer micelle had great toxicity to tumor cells, but had no toxic effect on normal cells. In a word, Mannose-PAA-b-PGMA-BODIPY is expected to be an ideal photosensitizer for cancer treatment.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.5;TQ317

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