【作者】 屠春来；

【导师】 朱新远；

【作者基本信息】 上海交通大学 ， 高分子化学与物理， 2012， 博士

【摘要】 超分子化学是基于分子间非共价键相互作用而形成分子聚集体的一门新兴学科,主要研究两种或两种以上的分子通过分子间作用力相互结合形成具有一定结构和功能的超分子体系,是当前化学、材料科学和生命科学等多学科交叉的研究前沿。超分子主体分子如杯芳烃、环糊精以及高度支化大分子等是超分子化学的重要研究对象,将这些超分子主体分子引入到高分子领域中,能增加聚合物特性,如两亲性、智能响应性、加工性能等,有助于拓宽应用,开发新型的功能材料。本文在综述前人有关各类超分子主体的聚合改性基础上,将聚乙二醇引入到疏水性的杯芳烃分子和超支化聚酯以及亲水性的环糊精和阳离子型超支化聚合物等体系中,基于配位作用、主客作用和静电作用等非共价相互作用构建了不同功能的超分子体系,并应用于纳米科学和生物医学等领域。主要研究内容和结论概括如下:1.利用聚乙二醇修饰硫代杯[4]芳烃原位可控合成纳米金粒子我们研究了一种简易可控制备纳米金粒子的新方法。首先在对叔丁基硫代杯[4]芳烃这类超分子主体的下沿酚羟基接枝了单链亲水性聚乙二醇。以两亲性的硫代杯芳烃为主体,利用未接枝聚乙二醇的苯酚单元作为还原剂、桥联的硫原子作为金属配位稳定剂,通过简单调节投入的氯金酸和硫代杯芳烃的比例,可控制备金纳米粒子,生成的纳米金粒子能稳定存在于水溶液中。采用核磁共振技术(NMR)和傅里叶变换红外光谱(FTIR)对金纳米粒子合成过程中的氧化还原反应进行了系统表征,结果表明水相条件对该反应是必须的,在还原氯金酸的同时对叔丁基苯酚单元被氧化成了苯醌。采用紫外可见光谱(UV-Vis)、动态光散射(DLS)和透射电子显微镜(TEM)对金纳米粒子的尺寸和形貌进行了表征,结果表明当硫/金摩尔比在2.6以下时,金纳米粒子的尺寸可控,改变摩尔投料比可以方便地得到不同尺寸的金纳米粒子。2.聚乙二醇修饰杯[4]芳烃与二氢卟吩e6的组装及其光动力治疗我们发展了一种基于主客相互作用的光敏剂输送体系。首先,通过对叔丁基杯[4]芳烃上沿去叔丁基化改性和下沿酚羟基聚乙二醇接枝改性,合成了星形聚乙二醇杯[4]芳烃(DC4-PEG)。作为一种水溶性的超分子主体,杯[4]芳烃空腔能够复合疏水性的客体分子,如光敏剂二氢卟吩e6,形成超分子两亲性化合物,并且能够在水相中自组装成胶束。通过核磁滴定法对主客体的复合比例进行了分析,表明两者以1:1的化学计量比进行复合。用TEM和DLS对胶束组装形貌进行了观测,当主客体为1:1时,胶束尺寸较为均一,大小在120 nm左右。用MTT法对光敏剂、主体分子和主客体复合物在HeLa肿瘤细胞中的暗毒性和光毒性进行了评估,发现避光条件下聚乙二醇杯[4]芳烃与二氢卟吩e6的细胞毒性很低,而光照下负载二氢卟吩e6的超分子胶束毒性很大,具有很好的光动力治疗效果。3.聚乙二醇超支化聚轮烷药物输送体系的构建及应用我们合成了一种可还原解离的聚乙二醇超支化聚轮烷作为药物输送的超分子体系。以二丙烯酸寡聚乙二醇酯(PEG-DA)、胱胺(Cys)和?-环糊精(?-CD)为原料,在水相中通过主客复合和迈克尔加成聚合得到超支化聚轮烷(HPRs)。采用NMR和FTIR测试表征了超支化聚轮烷结构,发现随着?-环糊精含量的增加,聚合物支化度降低。广角X射线衍射(WAXD)结果表明HPRs在2θ=7.4o处出现了?-环糊精的管道状内含复合物结晶衍射峰。用MTT法对HPRs在NIH/3T3细胞中的相容性评估表明,HPRs都具有非常低的细胞毒性。HPRs上的?-环糊精具有较大空腔,可以有效负载抗肿瘤药物阿霉素(DOX),其载药量在7%~8%,药物负载效率约为70%~80%。体外还原性环境中HPRs药物释放研究表明,在前15小时释放速率较快,96小时释放率在35%~46%。采用流式细胞仪和激光共聚焦显微镜考察了HPRs载DOX后的细胞内摄行为,发现DOX在15分钟内快速进入细胞核中。最后以HeLa细胞系为研究对象,发现负载DOX的HPRs具有很好的体外抗肿瘤效果,是一类新型的超分子药物输送体系。4.具有pH响应性的聚乙二醇修饰超支化聚酯药物输送体系利用商业化超支化聚酯BoltornTMH40接枝聚乙二醇,构建了pH敏感型药物输送体系。在氯化铟的催化下,超支化聚酯BoltornTMH40末端两个相连羟基与醛基环化缩合形成缩醛结构,从而实现了一步法简单制备以疏水性H40为核、亲水性聚乙二醇为臂的核壳结构单分子胶束。用核磁跟踪测定了缩醛结构在不同pH值下的降解速度,发现缩醛在pH=7.4条件下缓慢降解,而在酸性pH=5.3条件下35小时已降解一半。用MTT法对胶束在NIH/3T3细胞内的毒性进行了评估,发现1 mg/mL聚合物浓度下细胞存活率为80%,细胞毒性较低。以1,6-二苯基-1,3,5-己三烯(DPH)为探针测定了聚合物的临界胶束浓度,结果表明随着PEG含量的降低,临界胶束浓度增大。用TEM研究了胶束负载DOX后的尺寸和形貌,随着PEG含量的增加,球形胶束尺寸减小。用流式细胞仪和激光共聚焦显微镜考察了胶束负载DOX后的细胞内摄行为,发现在2小时细胞孵育时间内,负载DOX的胶束比游离DOX更快进入细胞,最后停留在细胞质中逐步释放DOX。MTT法抑制HeLa细胞增殖结果表明,负载DOX的胶束具有很好的抗肿瘤效果5.阳离子型超支化聚乙二醇的制备及基因转染我们制备了低含氮量的阳离子型超支化聚乙二醇,进而考察了其基因转染行为。将二丙烯酸寡聚乙二醇酯(PEG-DA)和1,8-二氨基-3,6-二氧杂辛烷(EOBEA)进行迈克尔加成聚合,再用二乙胺封端过量的双键,得到不同支化度的阳离子型超支化聚乙二醇(HA-PEGs)。采用NMR、FTIR、示差扫描量热仪(DSC)、尺寸排阻色谱(SEC)等手段对阳离子型超支化聚乙二醇的结构和理化性质进行了分析。用MTT法在COS-7细胞系中评估了HA-PEGs的细胞相容性,发现HA-PEGs具有较低的细胞毒性。作为一种阳离子型聚合物,我们考察了HA-PEGs对电负性质粒DNA的复合和输送行为。采用凝胶电泳(GE)、原子力显微镜(AFM)和圆二色光谱(CD)分析了HA-PEG/DNA静电复合物的压缩行为、组装体形貌和DNA构象变化,发现低电荷密度的HA-PEGs与DNA形成一种比较疏松的复合结构,保留了质粒DNA的二级构象和生物活性。选用COS-7细胞评估了基因转染效率,发现低含氮量的HA-PEGs具有较高的基因转染效率。更多还原

【Abstract】 Supramolecular chemistry is a newly-developed subject based on intermolecular noncovalent interactions and aggregation, concerning self-assembly of molecules into supramolecular system with specific structure and function through weak interactions. It has now become a promising field in interdisciplinary subject of chemistry, materials and life science. Supramoleclar hosts, such as calixarenes, cyclodextrins (CDs) and branched macromolecules, etc., are one of the most important research aspects of supramolecular chemistry. Combination with polymers, they will be endowed with polymeric inherent characteristics such as amphiphility, stimuli-responsiveness and processibility, which realizes.the functionalization of these hosts and extends their applications. In this dissertation, based on the summary of previous researches on various polymer-modified supramolecular hosts, poly(ethylene glycol) (PEG) chain was introduced into hydrophobic calixarene and hyperbranched polyester, or hydrophilic cyclodextrin and hyperbranched polymer. By utilization of various noncovalent interactions such as coordination, host-guest interaction and electrostatic effects, different functional supramolecular systems were constructed. Finally, the applications of these functional supramolecular systems in nano-science and biomedicine were studied. The main results are shown as follows:1. PEGylated thiacalix[4]arene for the in-situ controlled preparation of gold nanoparticles (AuNPs)A facile controlled synthesis of AuNPs under mild conditions was developed. Firstly, a single hydrophilic PEG chain was grafted onto the phenolic group of p-tert-butyl thiacalix[4]arene for the formation of amphiphilic host. Benefiting from the phenolic groups and the sulfur linkages, PEGylated thiacalix[4]arene could be used as both reductant and stabilizer of AuNPs. We investigated the redox reaction and found the phenolic groups of host without PEG chain were oxidized to benzoquinone during the preparation of AuNPs. The size and morphology of AuNPs were characterizated by UV-Vis, DLS and TEM. By simply adjusting the feeding ratio of Au/S, AuNPs with different sizes could be readily obtained in an aqueous phase under mild conditions.2. Supramolecular polymeric micelles by the host-guest interaction of PEGylated calix[4]arene and chlorin e6 for photodynamic therapyA novel photosensitizer delivery system based on supramolecular amphiphilic carrers via host-guest interaction was carried out. Firstly, p-tert-butylcalix[4]arene was modified by removing of tert-butyl groups at the upper rims and grafting PEG chains onto phenolic groups at the lower rims. This star-like PEGylated calix[4]arene acted as a hydrophilic host and encapsulated hydrophobic guest, such as chlorine e6, then they self-assembled into micelles in aqeous solution. The equal molar ratio complexes were comfirmed by NMR titration. The size and morphology of supramolecular micelles were studied by TEM and DLS. Moreover, the cytotoxicity of photosensitizer, host and complexes was assayed by MTT in HeLa cancer cells. This photosensitizer delivery system showed very low cytotoxicity in the dark but high cytotoxicity under photo-irradiation. Thus, these supramolecular polymeric micelles can be ultilized as an efficient delivery system for photodynamic therapy.3. Hyperbranched polyrotaxanes for drug deliveryWe developed a novel supramolecular drug delivery system based on reduction-detachable hyperbranched polyrotaxanes (HPRs). HPRs were synthesized from Micheal addition polymerization of poly(ethylene glycol) diacrylate and cystamine in the presence of ?-CD. The structure of HPRs was characterized by NMR, FTIR and WXRD. The host-guest inclusion complexes were comfirmed by the existence of characteristic diffraction peak at 2θ=7.4o. Furthermore, the cytotoxicity of HPRs was assayed by MTT in NIH/3T3 cells. HPRs showed very low toxicity. The hydrophobic drug doxorubicin (DOX) could be encapsulated in the cavities of ?-CD in HPRs, and the drug-loaded efficiency reached 70%~80%. Moreover, the encapsulated drug could be released quickly in a reducing environment. The cellular uptake behaviors of DOX-loaded HPRs were investigated by flow cytometry (FCM) and confocal laser scanning microscopy (CLSM). The loaded DOX could enter into the nucleas in 15 minutes. The proliferation inhibition of Hela cells was evaluated by MTT assay.4. PEGylated hyperbranched polyester for pH-responsive drug delivery carrierWe synthesized a pH-responsive drug delivery carrier by one-step PEGylation of BoltornTM H40. The hydroxyl terminals of H40 could react with aldehyde group to form acetal linkages under InCl3 catalysis. Thus, unimolecular micelles were constructed by hydrophobic H40 core and hydrophilic PEG arms. As a pH-responsive material, the degradation rates of H40-star-PEG were inverstigated by NMR titration under neutral and acidic conditions. These amphiphilic micelles showed low toxicity when they were grafted to many PEG chains. The results were comfirmed by MTT in NIH/3T3 cells. The cell viability after treated with micelles for 24 h was up to 80% under the polymer concentration of 1 mg/mL. Critical micelle concentrations (CMC) of H40-star-PEG were measured by 1,6-diphenyl-1,3,5-hexatriene (DPH) probe. Furthermore, the size and morphology of micelles were studied by TEM. The cellular uptake behaviors of DOX-loaded micelles were investigated by FCM and CLSM. Finally, the proliferation inhibition of HeLa cells was evaluated by MTT assay. It demonstrates that H40-star-PEG is a smart and safe drug carrier.5. Cationic hyperbranched PEG for gene deliveryWe synthesized a low charge-density hyperbranched PEG (HA-PEGs) with different degree of branching by Micheal addition polymerization of poly(ethylene glycol) diacrylate and 2,2’-(ethylenedioxy)-bis(ethylamine). Then, the excess acrylate terminals were end-capped by diethylamine. The structure and properities of HA-PEGs were characterized by NMR, FTIR, DSC and SEC. As a biomaterial consisting of biocompatible PEG, its low cytotoxicity was confirmed by MTT assay in COS-7 cells. Moreover, as a polycation, we studied its DNA condensation ability by agarose gel electrophoresis (GE), atomic force microscope (AFM), and circular dichroism (CD). Since the low charge-density, DNA/HA-PEG complexes maintained a loose structure and the B-type conformation of plasmid DNA remained during the complexation. Suprisingly, HA-PEGs with low nitrogen content displayed high transfection efficiency in COS-7 cells.更多还原