新型Bola两亲性表面活性剂的合成及其携载抗肿瘤药物的pH敏感囊泡载体的研究

The aim of this work was to design the synthesis of a novel Bola-amphiphilic surfactant which has amino acids as polar head group and pH-responsiveness. It is constructed to be pH-sensitive vesicles carrying anticancer drug as an alternative to single head surfactant in the conventional vesicles. This carrier is supposed to improve the stability of drug as well as the targeting of cancer cells and the pH-sensitive release of drug. Firstly select the amino acids of biocompatibility and pH-responsiveness as polar groups and alkyl diamine as hydrophobic group to synthesis the two sides of hydrophobic chain which connect the two polar head groups of Bola-form surfactants. Discuss the effect and regulation of the types of amino acid, length of alkyl carbon chain and the different binding between amino acids and alkyl diamine to the characteristics and property of self-assembled vesicles, clarify the formation mechanism of vesicles. Then pH -sensitive vesicle drug delivery system has been constructed. The vesicles formed by Bola-form surfactant have a certain pH-responsiveness in solution and other pH-sensitive lipid materials were added to them for common regulation on their isoelectric point, at the same time anti-cancer drugs were loaded to this pH-sensitive vesicles. A new pH-sensitive vesicle drug delivery system which is tumor-targeting using Bola-form surfactant as major capsule materials was designed. The evaluation on cancer cell targeting was performed from animal and cell level. Our topic clarify the mechanism and regulation to explain the relationships among carrier structure, property and function through a variety of advanced methods in fields covering pharmaceutics, physical chemistry, and cellular biology which have strong scientificity and novelty.

本课题旨在设计合成一种新型的以氨基酸为极性头的具有pH响应的Bola型两亲表面活性剂，替代传统的单极性头表面活性剂，构建携载抗肿瘤药物的pH敏感囊泡载体，在提高载体对药物稳定性的同时，实现该载体对肿瘤细胞的靶向性和药物的pH敏感释放。首先选用生物相容性和具pH响应的氨基酸为极性基团，烷基二胺为疏水基团合成疏水链两端连接两个极性头的Bola型表面活性剂，探讨氨基酸种类，烷基碳链长度以及两者的不同键合方式对自组装囊泡形态和性质的影响规律，阐明囊泡形成机制；随后构建pH敏感囊泡给药系统，以合成的Bola型表面活性剂，或加入其它的pH敏感材料共同调节其等电点，并将抗肿瘤药物载入该囊泡中，设计出全新的以Bola型表面活性剂为主要囊材的pH敏感囊泡给药系统，并从动物水平和细胞水平验证其对肿瘤细胞的靶向性。本课题运用先进研究手段，采用药剂学、物理化学、细胞生物学等多学科交叉，具有极强的科学性和新颖性。

本课题旨在以烷基二胺和一系列氨基酸为主要原料，设计合成一种新型的以氨基酸为极性头的具有pH响应的Bola型两亲表面活性剂，替代传统的单极性头表面活性剂，构建携载抗肿瘤药物的pH敏感囊泡载体，在提高载体对药物稳定性的同时，实现该载体对肿瘤细胞的靶向性和药物的pH敏感释放。.分别以Boc保护的氨基酸（赖氨酸、丝氨酸、脯氨酸等）和不同碳链长度的烷基二胺经缩合和脱保护反应，合成了一系列Bola型两亲表面活性剂。采用质谱、红外和核磁等对其结构进行确证。并测定其在水溶液中的pKa、临界胶束浓度、电导率等物理化学性质。.以5-氟尿嘧啶（5-Fu）为模型药物，合成的Bola型表面活性剂和常用的非离子表面活性剂为复合膜材制备纳米囊泡，并探讨各种因素对囊泡形成的影响。以粒径和包封率为考察指标，选用薄膜分散-超声波粉碎的方法制备囊泡，分别采用单因素考察和正交设计实验对5-Fu囊泡的处方和制备工艺进行筛选优化。最终确定囊泡处方：5-Fu（1 mg/mL）、1,12-二赖氨酸十二烷（DADL）（20 mM）和Tween 60（5 mg/mL）。最优制备工艺为：将处方量的5-Fu溶解于pH 7.4 PBS中，超声使其完全溶解，加入处方量DADL和Tween 60，涡旋20 min，100 W45℃水浴下超声波粉碎2 min。.5-Fu囊泡外观为半透明溶液，有淡蓝色乳光；粒径为30 nm左右；透射电镜照片显示，囊泡为球形，大小均匀，分散性较好。显微红外测定显示DADL囊泡以直链型构象排列而形成单层膜，DAOP囊泡则以“U”型构象排列而形成膜层。.采用动态透析法对5-Fu囊泡的在不同pH介质中的体外释放行为进行考察。结果显示在pH 7.4 PBS中，囊泡中药物的释放缓释， 在pH 6.5和pH 5.8 PBS释放介质中，药物的释放速度加快，表明其具有一定的pH敏感特性。.采用MTT法测定了5-Fu游离药物、空白囊泡和5-Fu载药囊泡对HepG2细胞的细胞毒性，得到其IC50值。5-Fu游离药物和5-Fu载药囊泡均对肿瘤细胞有一定杀伤力，细胞杀伤力依赖药物浓度，随着浓度升高杀伤力增强。空白囊泡载体对细胞抑制率不明显，存活率在80%以上，证明载体的生物安全性较好。.采用接种鼠源S180腹水瘤的昆明小鼠，考察了载药囊泡的体内抗肿瘤活性。结果显示5-Fu囊泡肿瘤抑制率高于市售注射剂，具有一定的肿瘤靶向性。