【作者】 王晓娟；

【导师】 马建标；

【作者基本信息】 南开大学 ， 高分子化学与物理， 2012， 博士

【摘要】 当今化学疗法即“化疗”主要是指应用抗癌药杀灭体内癌细胞，从而达到治疗癌症的目的，其大多被当作手术的代替性手段以及手术后的铲草除根以及防止癌症复发手段使用。近年来，化疗发展的很快，但传统的抗肿瘤药物普遍存在半衰期短、对癌变组织选择性差、全身毒副作用大等缺点，大大限制了用药剂量和治疗效果，因此，设计良好的抗癌药物传递系统是肿瘤治疗过程中迫切需要解决的难题。抗肿瘤药物的理想载体应该具有以下的功能特点：良好的生物相容性和生物可降解性，载药量高，循环时间长，具有肿瘤特异性。聚氨基酸类高分子材料因其良好的生物相容性、生物可吸收性及化学结构匹配性，在生物医用高分子领域有着无法比拟的优点和广泛的应用前景。特别是聚天冬氨酸，具有合成简单，成本较低，良好的生物相容性、生物降解性和可吸收性，易于功能化修饰等诸多优点。且在体内能够被逐渐吸收，其代谢产物对人体无毒，因而不会对周围组织、肝肾、血红细胞等产生毒副作用。因此聚天冬氨酸及其衍生物，被广泛用于药物载体的制备研究。关于聚天冬氨酸类载体已有很多报道，但是大部分的聚合物是通过N-羧酸酐（NCA）开环聚合方法合成得到聚(α-L-天冬氨酸)，这种方法制备的聚合物结构清晰明确，但合成步骤比较复杂，材料成本也相对提高。且一般认为聚-氨基酸二级结构过于规整，链段柔韧性差，不利于药物的扩散和释放。因此需要继续探索更为简便易行的可以实现肿瘤的特异性识别或富集或释放的响应性化学结构。本实验室以聚(α,β-L-天冬酰胺)为基本材料，设计具有环境刺激响应性能的生物降解高分子材料，并对其应用进行探索研究。1）利用酸敏感的腙键将亲水的聚乙二醇和疏水性阿霉素键DOX合到聚天冬酰胺主链上，使得聚合物具有两亲性，再利用侧链键合的阿霉素与剩余游离阿霉素之间的疏水相互作用及-叠合作用将更多的阿霉素物理包埋到胶束内核。研究表明，通过调整DOX/Polymer的投料比，可以有效提高DOX的载药量，最高可达38%。该系列的纳米粒子稳定性好，体外实验表明，载有阿霉素的纳米粒子对阿霉素的释放具有良好的pH响应性。细胞结果表明载有阿霉素的纳米粒子保持了和自由阿霉素盐酸盐相近的药效。2）以3-氨丙基咪唑和水合肼为亲核试剂，进攻聚琥珀酰亚胺进行开环反应得到氨丙基咪唑接枝聚天冬酰胺IM-g-PAHy。再利用醛化聚乙二醇PEG-CHO与肼基之间高效的反应形成腙键，得到侧链具有pH响应性官能团的两亲性聚合物mPEG/IM-g-PAHy，然后再将DOX通过腙键连接到聚合物骨架上，成功制备了得到了具有核可溶胀性的pH响应性阿霉素载药体系mPEG/IM-g-(PAHy-DOX)载药纳米粒子，并对其进行pH响应性、胶束形貌及释放动力学研究。研究发现，mPEG/IM-g-(PAHy-DOX)载药纳米粒子具有很好pH响应性，在pH7.4条件下，仅有少量释放，而在pH5.0条件下，最大释放量明显增加，且随着咪唑含量的增加，DOX的释放速度和释放量具有很大增加。但值得指出的是，随着聚合物中咪唑接枝度的增加，体系载药量也会随之降低。3）生理条件下，利用腙键化学交联聚（α,β-L-天冬氨酸）衍生物原位制备水凝胶，成胶时间快，不需要加入任何小分子催化剂或交联剂。通过扫描电镜观察，水凝胶的内部均可以观察到致密、连贯的网络孔状结构。在模拟条件（pH=7.4和5.0，37℃）下，对聚（α,β-L-天冬氨酸）衍生物水凝胶进行降解行为研究发现，随着时间的推移水凝胶质量明显降低，28天降解35%-45%，说明该水凝胶具有很好的降解性能。以亲水性盐酸阿霉素为模型药物，研究了药物在该水凝胶中的装载和释放行为。在酸性环境pH时，阿霉素的释放速度的明显高于中性环境下的释放速度。根据Rigter-Peppas模型分析释放行为发现，在pH7.4生理条件下，阿霉素的释放遵循Fick扩机制，而在pH5.0酸性条件下，阿霉素在最初的释放过程中遵循无规扩散机制，即Fick扩散和高分子链的松弛协同作用结果。通过Hep G2细胞在水凝胶上的生长情况发现，该水凝胶支架有利于细胞的生长增殖。4）设计了以-天冬氨酸为基本材料，对其聚合物侧链进行改性，制备了具有两性离子特性的聚氨基酸，作为具有pH敏感性的表面抗蛋白非特异性吸附材料，并对聚氨基酸的性质及等电点对抗蛋白质非特异性吸附能力的影响进行了系统的研究。通过缩合反应制备得到的侧链部分接枝组氨酸HIS的聚（α,β-L-天冬氨酸）HIS-PAsp/PAsp，通过透过率、表面电势、酸碱滴定测试，证明其具有典型的两性离子特征。将其应用到无污染表面试验中可以看出，经静电吸附将HIS-PAsp/PAsp聚合物修饰到表面材料后纤维蛋白原的吸附量明显降低，且降低的程度与表面修饰的聚合物浓度有关。该材料有望成为一种很好的阳离子表面修饰材料。通过氨基基团对聚琥珀酰亚胺（PSI）的开环反应引入弱碱性基团是一种效率高，条件可控的聚氨基酸衍生物制备方法。试验中制备了一系列具有不同等电点的两性离子叔胺化聚（α,β-L-天冬氨酸）DMPA-PAsp/Pasp。通过吸附试验证明，经静电吸附将不同等电点聚合物DMPA-PAsp/PAsp修饰到表面材料后对纤维蛋白原和白蛋白的吸附量均明显降低，且降低的程度与表面修饰的聚合物浓度有关。因此该系列聚合物可根据材料表面所带电荷的不同，吸附具有不同等电点的聚合物，因而起到很好的阳离子表面修饰作用。更多还原

【Abstract】 Poly(amino acid)s have been extensively studied in biomedical applications,due to their unique regular secondary structures, rigid backbone, biocompatibility,biodegradability and various side groups for further functionalization. Most ofpolypeptides reported so far were prepared by ring-opening polymerization of aminoacid N-carboxyanhydrides (NCAs). However, the NCA approach has a costdisadvantage and a production problem because the pendant reactive groups carriedby the amino acids need to be protected prior to polymerization and deprotectedunder harsh conditions after polymerization to give the functional side groups. Poly(α, β-L-aspartic acid)(α, β-L-PAsp) can be synthesized by thermal polycondensationof L-aspartic acid (L-Asp), which is easily controlled and not costly. It would be agood candidate for preparing polypeptide derivatives.In this thesis, Poly (amino acid)-based amphiphilic copolymer was utilized tofabricate a better micellar drug delivery system (DDS) with improved compatibilityand sustain release of Doxorubicin (DOX). First, Poly (ethylene glycol) monomethylether (MPEG) and DOX were conjugated onto polyasparihyazide, prepared via thereaction of poly (succinimide)(PSI) with hydrazine, to afford an amphiphilicpolymer [PEG-hyd-P (AHy-hyd-DOX)] with acid-liable hydrazone bonds. The DOX,chemically conjugated to the water-soluble polymer of the PAHy throughpH-sensitive hydrazone bond, was designed to supply hydrophobic segments andimprove the compatibility between the core and physically entrapped DOXmolecules. PEG segments grafted to the polymer via hydrazone bond was designedto prolong its lifetime in blood circulation. Free DOX molecules could be entrappedinto the nanoparticles of such an amphiphilic polymer via-interaction betweenthe conjugated and free DOX molecules to obtain a drug delivery system with highDOX load. The drug loading capacity, drug release behavior, and morphology of themicelles were investigated. The micelles’ biological activity was evaluated in vitro.Results showed that drug loading capacity was intensively augmented by adjusting the feed ratio, and the maximum loading capacity was as high as38%. Besides, theDOX-loaded system exhibited a pH-dependent drug release profile in vitro. Thecumulative release of DOX was much faster at pH5.0than that of the release at pH7.4. The DOX-loaded system kept highly antitumor activity for a long time,compared with free DOX. This easy-prepared DDS, with features ofbiocompatibility, biodegradability, high loading capacity and responsiveness to pH,was a promising candidate for the DOX delivery and controlled release.In order to accelerate the rate of DOX release, a facile micelle-formingcopolymer-drug conjugate mPEG/IM-g-(PAHy-DOX) were reported. The DOX,chemically conjugated to the pH-responsive poly (amino acid)-based amphiphilicgraft polymer mPEG/IM-g-PAHy by hydrozone bonds, was designed to have a fasterrelease rate due to the presence of pH-sensitive group of imidazole. The drug loading,drug release, and morphology of the micelles were investigated. Compared with theamphiphilic polymer [PEG-hyd-P (AHy-hyd-DOX)], DOX-loaded system reportedhere has obviously enhanced pH-sensitivity and superior drug release behaviors, atpH5.0, the cumulative release of DOX from mPEG/IM-g-(PAHy-DOX)was muchfaster than that of the release from [PEG-hyd-P (AHy-hyd-DOX)].An injectable poly (α,β-L-aspartic acid)-based hydrogel via hydrazonecrosslinking strategy was also studied in this thesis. The gelation occurs underphysiological conditions immediately upon mixing of the two aqueous poly(α,β-L-aspartic acid) derivatives specifically derivatized through hydrazone linkageswith aldehyde (PAPDAL) and hydrazide (PAHy) functional groups, respectively,without addition of crosslinker or catalyst. The fast gelation provides this systemwith injectable property. PAsp hydrogels were synthesized in PBS solution andcharacterized by different methods including gel content and swelling, Fouriertransformed infrared spectra, in vitro degradation and biocompatibility experiments.A scanning electron microscope viewed the interior morphology of gel whoseporous three-dimensional structure enabled it to efficiently encapsulate the drugs.Sustained and stable DOX release from the PAsp hydrogel was observed during invitro delivery experiments and exhibited its potentially high application prospect inthe field of protein drug delivery. In addition, a novel zwitterionic polypeptide derivative, denoted as His-PAsp/PAsp,has been successfully synthesized by amidation of Poly (α, β-L-aspartic acid) withL-histidine methyl ester. Turbidity, zeta potential and1H NMR measurements wereused to study the aggregation behaviors of His-PAsp/PAsp under different pH values.The modified polypeptide derivative composed of certain negatively and positivelycharged residues randomly, exhibiting an isoelectric point (IEP) and bearing oppositecharges at pH values far high or below the isoelectric point. Silicon wafer, as a modelsubstrate material, was then coated with zwitterionic polypeptide. The amount ofprotein adsorbed to the coated surface could be controlled, depending on the dose ofthe polypeptide. Since its good biodegradability and superior anti-protein-foulingproperty, this pH-responsive zwitterionic polypeptide is a promising candidate forsurface modification in many biomedical applications, including medical implants,drug delivery carriers, and biosensors.A series of zwitterionic dimethyl aminopropyl amine-grafted poly(α, β-L-asparticacid)s (DMAP-PASP) with pH-dependent charge profiles, were synthesized bysubsequent aminolysis polysuccinimide with3-dimethyl aminopropyl amine andalkali. The zwitterionic polypeptides had an IEP, which could be easily tuned frompH3.8to9.5and showed opposite charges below and above the IEP. Because of theflexibility of controlling pH dependence, it is expected that zwitterionicpolyaspartamide would have a number of biomedical applications, where the abilityto respond to delicate pH changes is required. The zwitterionic polypeptide was ableto inhibit the protein adsorption to the anionic surface at physiological pH. Theseresults warrant further exploration of zwitterionic polypeptides as a stealth coatingfor tumor-targeted nanocarriers to overcome the current limitation of PEG.更多还原