【作者】 刘辉；

【导师】 杨眉； 彭建珩；

【作者基本信息】 重庆大学 ， 工程硕士（生物工程领域）（专业学位）， 2017， 硕士

【摘要】 光动力疗法(PhotodynamicTherapy,PDT)作为一种新的治疗癌症的方法,已经成为除手术切除、放射治疗、化学治疗外的第四种治疗手段。光敏剂在光动力学治疗中处于核心位置,也是其发展最难突破的要素。近年来,研究者们不断尝试,将PDT与各种治疗方案联合使用,其中以联合化疗方案备受重视。目前,无论是单一化学药物使用还是简单的药物复配联合使用,均不可避免地存在对正常细胞的毒副作用。为了降低药物的毒副作用,提升抗癌效果,采用具有特定功能的药物载体系统被认为是极具前景的方式,也是目前的研究热点。然而,受不同载体材料的局限性,药物载体也存在如亲和性不高、包药量小以及载药过程中漏药等问题,并且载体材料功能单一,不能配合化学药物起到协同治疗作用。基于此,论文合成了一种光动力药物载体微囊,并同时装载化疗药物,实现以药载药的新型组合治疗方式。论文首先合成了一种新型多烯基类的卟啉类光敏剂。在此基础上,通过界面聚合法,利用苯基卟啉上的多个不饱和双键与甲基丙烯酸二甲氨基乙酯上的不饱和双键加聚反应合成聚合物微囊,同时将阿霉素包合于微囊空腔内,制备出同时具有光疗和化疗双重作用的新型药物载体。目前,关于此类具备高药物包封率和能与包合药物起协同作用的药物载体在国内外尚未有报道。通过研究此类药物载体的制备工艺,并将其用于肿瘤细胞的协同治疗,以期为后续工作提供一些基础研究参考。论文主要工作与结果如下:(1)利用对羟基苯甲醛、吡咯进行化学反应,制备得到5,10,15,20-四(4-羟基苯基)卟啉(H2THPP)。在此基础上,利用甲基丙烯酰氯取代修饰,进一步合成了5,10,15,20-四(4-甲基丙烯酰氧苯基)卟啉(TMaBPP)。(2)设计了界面聚合体系,使得甲基丙烯酸二甲氨基乙酯(DM)与TMaBPP通过界面聚合反应,在不同水相条件下,分别制备了空心微囊poly(TMaBPP-DM)和装载了阿霉素(DOX)的载药微囊DOX@poly(TMaBPP-DM)。最后,通过工艺条件优化,使微囊的载药率、包封率提高,并且减小了尺寸分布,均一了粒径。红外光谱结果显示油水相单体在相界面加聚反应成功;光学显微镜及电子显微镜结果进一步表明了聚合物微囊制备成功,并且成功实现了DOX的包封;采用荧光分光光度法测定载药率与包封率的结果表明微囊密封效果好以及载药量可控;接触角测试结果证明了微囊的亲水效果良好;而粒度仪分析结果表明了微囊尺寸的可控性,可以得到从纳米级到微米级范围的尺寸,能够满足多种使用场景要求。(3)设计了药物释放体系,分别探究了载药微囊DOX@poly(TMaBPP-DM)在油相和水相中释放DOX的情况。在油相体系中考查了温度、激光照射条件下,DOX从微囊内的水相环境释放到微囊外部油相环境中的释放行为;在水相体系中,温度、pH值、激光照射条件下,考查了DOX从微囊外部油相到周围水相环境的释放行为。两种界面释放实验结果表明载药微囊DOX@poly(TMaBPP-DM)的药物释放情况可受到温度、pH值及激光照射多种因素的影响,实验结果为有效保存载药微囊及有效利用载药微囊进行相关细胞实验研究提供了参考依据。(4)以人永生化表皮细胞HaCaT和皮肤黑色素瘤细胞A375为考察对象,通过MTS比色法,考察DOX、poly(TMaBPP-DM)、DOX@poly(TMaBPP-DM)在不同浓度,激光剂量和不同联合给药方式条件下对细胞的毒性作用。细胞实验结果表明:有光照条件下,poly(TMaBPP-DM)在低浓度下,具备对A375较高的光毒性,有效地体现了光敏剂的光疗作用;而无光照条件下,poly(TMaBPP-DM)对HaCaT细胞降低了毒副作用,但表现出了对A375细胞更强的暗毒性。联合给药实验结果表明,DOX@poly(TMaBPP-DM)(包合形态)较poly(TMaBPP-DM)加DOX(混合形态)而言,不仅明显降低了对HaCaT细胞毒副作用,还增加了对A375细胞的杀伤效果。金氏公式分析结果表明,载药微囊较单独化疗或光疗具备协同效应。更多还原

【Abstract】 Photodynamic Therapy(PDT)is a new method for cancer treatment,and has become the fourth treatment on cancer besides surgery,radiotherapy,chemical therapy.Photosensitizer which dominates the function of PDT is the hardest bottleneck to breakthrough in development of PDT.In recent years,scholars have been trying to combine PDT with various treatment programs,in which the combined chemotherapy received much attention.Currently,both of simple single and combined medications inevitably display side effects on normal cells.In order to reduce the side effects of drugs,drug delivery system with specific functions is considered as a hotspot and a promising way for tumor therapy.However,owing to limits of carriers materials,drug carriers have some drawbacks,including low affinity,small drug package quantity and drug leakage in process of delivery.With respect to these problems,we developed a novel photodynamic drug carrier microcapsules loaded with chemotherapeutic drugs for synergistic treatment for cancer therapy.In this study,a novel polyene porphyrin photosensitizer was synthesized.The porphyrin is linked to dimethylaminoethyl methacrylate(DM)through the addition polymerization of double bonds,and doxorubicin hydrochloride(DOX)is enclosed in the microcapsule cavity.Thus,a drug carrier having dual therapeutic effect of phototherapy and chemotherapy was prepared.To the best of our knowledge,such drug carriers with high drug encapsulation efficiency and synergistic therapy function have not been reported.After optimized preparation,the microcapsules were utilized for tumour cell therapy,and the results will provide some basic research reference on cancer therapy.The main works are listed as follows:(1)Meso-5,10,15,20-tetrakis(4-hydroxyphenyl)-21 H,23H-porphine(H2THPP)was acquired after a reaction of p-hydroxy benzaldehyde and pyrrole.Subsequently,H2 THPP was modified with methacryloyl chloride to afford meso-5,10,15,20-tetrakis[4-(methacryloyloxy)phenyl]-21 H,23H-porphyrin(TMa BPP)via nucleophilic substitution reaction.(2)Microcapsules loaded with DOX(DOX@poly(TMaBPP-DM))and microcapsules(poly(TMaBPP-DM))were both prepared via interfacial polymerization between TMaBPP and dimethylaminoethyl methacrylate(DM).Under optimized conditions,microcapsules with high drug loading rate,entrapment efficiency and uniform size were synthesized.FT-IR spectrum of poly(TMa BPP-DM)suggested the successful polymerization between TMaBPP and DM.Optical microscope and scanning electron microscope images of DOX@poly(TMaBPP-DM)showed that microcapsules loaded with DOX were synthesized successfully.Excellent drug loading rate and encapsulation efficiency of microcapsules were confirmed by fluorescence spectrophotometer measurement.The contact angle measurement results showed the good hydrophilicity of microcapsules.Particle size analyzer characterization demonstrated the controllability of microcapsule size ranging from nanoscale to microscale.(3)Release behavior of DOX from DOX@poly(TMaBPP-DM)microcapsule in oil and water had been evaluated respectively.In oil,effects of temperature and illumination dose on release of DOX from microcapsule to outside oil solution were studied.Meanwhile,effects of temperature,pH and illumination dose on release of DOX from microcapsule surface to ambient water solution were studied.The results suggested that release behavior of DOX would be affected by temperature,pH and illumination dose,which provided the effective preservation of microcapsules loaded with drug and their utilization on tumour cell experiments.(4)Cell viabilities of human immortalized epidermal cells HaCaT and skin melanoma cells A375 were investigated after incubation of cells and DOX,poly(TMaBPP-DM),DOX@poly(TMaBPP-DM)and DOX-poly(TMaBPP-DM)with different concentrationsand various illumination doses.The results suggested that poly(TMaBPP-DM)showed high phototoxicity with illumination even under low concentration,indicating an excellent phototherapy effect of microcapsule.And poly(TMaBPP-DM)displayed relatively lower toxicity on HaCaT,while higher toxicity on A375 without illumination.In addition,compared to that of poly(TMaBPP-DM)-DOX,DOX@poly(TMaBPP-DM)showed lower toxicity on HaCaT,while higher toxicity on A375,especially with illumination.Furthermore,results obtained by analysis of Jin’s formula suggested that DOX@poly(TMaBPP-DM)display excellent synergy therapy efficiency induced by PDT and chemotherapy of as-prepared drug delivery system.更多还原