双亲性分子伞的合成表征与穿膜研究
发布时间:2018-05-28 16:13
本文选题:分子伞 + 药物载体 ; 参考:《厦门大学》2008年硕士论文
【摘要】: 细胞膜作为细胞的一道天然屏障,使得许多具有治疗作用的药物和大分子生物活性物质,特别是高亲水性和带电物质,如反义寡聚核苷酸、DNA、蛋白质和多肽等难以通过细胞膜。构建一种药物载体使其能顺利穿透细胞膜并在靶向部位释放,已成为药物设计中亟需解决的问题之一。一种具有双亲性的分子伞载体有望携带上述药物实现跨膜运输,并解决许多载体在治疗过程中存在的穿膜效率低和安全性等问题。本论文的研究内容主要包括设计合成带有绿色荧光的5-羧基荧光素分子伞,考察其穿透细胞膜的能力和体外细胞毒性,探讨分子伞载体可能的穿膜机制。研究结果如下: (一)分子伞的合成与表征 (1)在无水有机溶剂中,胆酸分子与N-羟基琥珀酰亚胺(NHS)在缩合剂N,N′-二环己基碳二亚胺(DCC)的作用下反应得到胆酸-NHS活化酯,然后以二异丙基乙胺(DIPEA)为缚酸碱,在无水二甲基甲酰胺中胆酸-NHS活化酯与亚精胺的两个端氨基偶联,形成含有一个活性氨基(仲胺)的双臂分子伞,即N_1,N_3-亚精胺二胆酸酰胺。 (2)在无水二甲基甲酰胺中,5-羧基荧光素与3-羟基-1,2,3-苯并三嗪-4(3H)-酮(Dhbt)在缩合剂N,N′-二环己基碳二亚胺(DCC)的作用下活化得到5-羧基荧光素的Dhbt活化酯,再以三乙胺为缚酸碱在无水二甲基甲酰胺中与上述双臂分子伞的活性氨基偶联,形成5-羧基荧光素分子伞(5-cf-mu)。 (3)对各步产物如胆酸-NHS活化酯、N_1,N_3-亚精胺二胆酸酰胺和5-cf-mu的结构和分子量分别采用红外光谱法、核磁共振谱法和质谱法进行确定,同时对这三步产物的熔点进行测定;5-cf-mu的纯度采用高效液相色谱法进行分析。结果表明:这三步产物的各种表征结果与其相应分子结构、分子量都完全符合,5-cf-mu的纯度在95%以上。 (二)分子伞的穿膜性能研究 (1)MTT法检测了浓度分别为25,50,100和200μM的5-cf-mu在24和48h内对L929细胞的细胞毒性,结果表明:24 h内上述浓度的5-cf-mu对L929细胞均没有毒性;25μM的5-cf-mu在48 h内也没有毒性,50,100和200μM在48 h呈现少许毒性,但仍有较高的细胞成活率。同时检测了5-cf-mu对Hela细胞在24 h内的细胞毒性,结果表明浓度低于100μM时,分子伞对Hela细胞也没有毒性。上述结果说明分子伞具有良好的生物相容性。 (2)采用荧光显微镜和激光共聚焦显微镜检测了5-cf-mu在Hela细胞和L929细胞内的分布情况。绿色荧光点不仅分布在细胞质中,而且分布在细胞核周围,表明5-cf-mu已经进入了Hela细胞和L929细胞。实验证明分子伞是一种可以实现跨膜运输的载体。 (3)采用流式细胞仪考察了温度、浓度、时间以及ATP抑制剂作用下对5-cf-mu穿透细胞膜的效率的影响。结果表明分子伞的穿膜是一个时间、浓度和温度依赖而非能量依赖的过程。 (三)分子伞的穿膜机制 (1)建立了分子伞穿透细胞膜的数学模型。根据流式细胞仪检测结果,细胞内的平均荧光强度(MFI)与培养时间的平方根(t~(1/2))、初始浓度的平方根(C_o~(1/2))均成很好的线性关系,线性相关系数(R值)分别为0.985和0.994。 (2)分子伞穿透细胞膜的过程符合所建立的数学模型,表明分子伞是以溶解—扩散的方式穿透细胞膜。
[Abstract]:The invention relates to a molecular umbrella carrier with amphipathy , which is expected to carry the drug to realize transmembrane transport and solve the problems of low membrane penetrating efficiency and safety existing in the drug design .
( 1 ) Synthesis and Characterization of Molecular umbrella
( 1 ) In an anhydrous organic solvent , the cholic acid molecule reacts with N - hydroxysuccinimide ( NHS ) under the action of a condensing agent N , N ' - dicyclohexylcarbodiimide ( DCC ) to obtain the cholic acid - NHS activated ester , and then the cholic acid - NHS activated ester is coupled with the two end - amino groups of the spermidine in the anhydrous dimethylformamide to form a double - arm molecular umbrella containing an active amino group ( secondary amine ) , namely , N _ 1 , N _ 3 - spermidine di - cholic acid amide .
( 2 ) 5 - carboxyfluorescein and 3 - hydroxy - 1 , 2 , 3 - benzotriazine - 4 ( 3H ) - one ( dhbt ) are activated under the action of condensing agent N , N ' - dicyclohexylcarbodiimide ( DCC ) under the action of N , N ' - dicyclohexylcarbodiimide ( DCC ) under the action of condensing agent N , N ' - dicyclohexylcarbodiimide ( DCC ) , and then triethylamine is used as acid - binding base to be coupled with the active amino group of the double - arm molecular umbrella in anhydrous dimethylformamide to form a 5 - carboxyfluorescein molecular umbrella ( 5 - cf - mu ) .
( 3 ) The structure and molecular weight of the products such as cholic acid - NHS activated ester , N _ 1 , N _ 3 - spermidine - dicholic acid amide and 5 - cf - mu were determined by infrared spectroscopy , nuclear magnetic resonance spectroscopy and mass spectrometry , respectively . The purity of these three - step products was determined by high performance liquid chromatography . The results showed that all the characterization results of these three products were consistent with their molecular structure and molecular weight , and the purity of 5 - cf - mu was more than 95 % .
Study on the Film Properties of ( II ) Molecular Umbrella
( 1 ) The cytotoxicity of 5 - cf - mu of 25 , 50 , 100 and 200 渭M on L929 cells was detected by MTT assay . The results showed that 5 - cf - mu of 25 渭M had no toxicity to L929 cells .
( 2 ) The distribution of 5 - cf - mu in Hela cells and L929 cells was detected by a fluorescence microscope and a laser confocal microscope . The green fluorescent dots were not only distributed in the cytoplasm but also around the nucleus , indicating that 5 - cf - mu had entered Hela cells and L929 cells .
( 3 ) The effect of temperature , concentration , time and ATP inhibitor on the efficiency of 5 - cf - mu penetrate cell membrane was investigated by flow cytometry . The results showed that the membrane of molecular umbrella was a time , concentration and temperature dependence rather than energy dependence .
( 3 ) Film - penetrating mechanism of molecular umbrella
According to the results of flow cytometry , the mean fluorescence intensity ( MFI ) and the square root of the culture time ( t ~ ( 1 / 2 )) , the square root of the initial concentration ( C _ o ~ ( 1 / 2 )) were linear , and the linear correlation coefficient ( R value ) was 0.985 and 0.994 , respectively .
( 2 ) The process of penetrating the cell membrane by the molecular umbrella accords with the established mathematical model , indicating that the molecular umbrella penetrates the cell membrane in a dissolved - diffusion way .
【学位授予单位】:厦门大学
【学位级别】:硕士
【学位授予年份】:2008
【分类号】:R341
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