叶酸偶联2-ME白蛋白纳米粒的制备及性质的初步研究

发布时间：2018-06-01 01:37

  本文选题：2-甲氧基雌二醇 + 牛血清白蛋白　； 参考：《郑州大学》2014年硕士论文

【摘要】：2-甲氧基雌二醇(2-methoxyestradiol,2-ME)是一种甾体激素类化合物，属新型激素类抗肿瘤药物，对快速生长的肿瘤细胞具有相对较高的特异性，且不会对骨髓和造血器官产生毒性。由于2-ME是一种难溶性药物，胃肠道吸收差，血浆消除率快，生物利用度低等特点，限制了其在临床上的开发应用。因此本文以牛血清白蛋白(BSA)作为药物载体，小分子叶酸(FA)作为靶向因子，抗肿瘤药物2-甲氧基雌二醇(2-ME)作为模型药物，构建成以叶酸受体介导的2-ME白蛋白纳米粒肿瘤靶向给药系统(2-ME-FA-BSANPs)，旨在提高2-ME的水溶性和生物利用度，减少给药剂量，降低药物毒副作用。 1.2-ME-FA-BSANPs的制备及相关性质的考察。 首先采用去溶剂化-化学交联法制备2-ME白蛋白纳米粒(2-ME-BSANPs)，通过单因素考察白蛋白浓度、2-ME浓度、交联剂戊二醛用量、乙醇与水比例、溶液pH等条件，确定了2-ME-BSANPs制备工艺为：BSA浓度为40mg/ml；2-ME浓度为4mg/ml；溶液pH值为10.0；乙醇与水的比例为3:1。进一步利用叶酸活性酯与白蛋白纳米粒表面活性氨基进行偶联反应，制备叶酸偶联的2-ME白蛋白纳米粒靶向给药系统(2-ME-FA-BSANPs)，使其具有主动靶向作用，并提高白蛋白纳米粒的肿瘤细胞摄取率。 测定2-ME-FA-BSANPs平均粒径和电位分别为(217.5±5.1) nm和(-37.59±1.39) mV；平均包封率与载药量分别为(82.50±5.80)%和(7.50±0.50)%；扫描电镜图显示:2-ME-FA-BSANPs粒径分布均匀，呈球形且表面光滑；体外释药结果显示：原料药2-ME释放较快，在12h内累积释放量达85%左右；相对应的2-ME-FA-BSANPs释放缓慢平稳，在96h内累积释放量达85%左右，表明靶向给药系统具有一定的缓释特性；将靶向制剂制备成冻干粉，经筛选选择3%的蔗糖作为其冻干保护剂；考察稳定性结果显示：在4℃和室温条件下冻干粉可稳定保存6个月，表明该靶向制剂具有较好的稳定性。 2.2-ME-FA-BSANPs体外抗肿瘤活性与靶向性的研究。 采用MTT法考察靶向给药系统对SMMC-7721肿瘤细胞存活率的影响。在2-ME-FA-BSANPs作用时间24h、48h和72h，最大浓度为40μmol/l时，对应的肿瘤细胞抑制率分别为50.58%、68.53%和75.00%，相同条件下2-ME组的肿瘤细胞抑制率分别为25.28%、30.31%和35.37%，结果表明在不同时间、不同浓度作用下，2-ME-FA-BSANPs组对肿瘤细胞的抑制率均高于2-ME组(P0.05)。 采用荧光显微镜法与细胞流式分析法考察2-ME-BSANPs和2-ME-FA-BSANPs的靶向性。结果显示：相比于2-ME-BSANPs组，，2-ME-FA-BSANPs组的荧光强度明显增强，且在作用4h时2-ME-FA-BSANPs组进入细胞内的比例为99.8%，表明靶向给药系统在叶酸介导作用下可以有效、快速的进入肿瘤细胞内部，对肿瘤细胞具有较高的靶向作用；细胞周期研究结果表明所制备靶向给药系统主要将SMMC-7721肿瘤细胞阻滞在G2/M期，且不改变原料药2-ME的作用机制；细胞凋亡研究结果表明，与2-ME组相比，靶向给药系统2-ME-FA-BASNPs能诱导更多肿瘤细胞发生凋亡。 3.2-ME-FA-BSANPs体内药代动力学的研究。 本研究以Sprague-Dawle(SD)大鼠为动物模型，采用HPLC法测定2-ME在大鼠血浆中的浓度，研究靶向给药系统的体内药代动力学特征。结果显示，2-ME-FA-BSANPs和2-ME组的消除半衰期t1/2β分别为：(124.61±12.00) min和(22.17±6.00) min；两组平均滞留时间MRT分别为(181.03±5.20) min和(22.66±2.54)min；两组AUC分别为(290.53±10.10)(μg/ml) min和(112.99±5.50)(μg/ml) min。结果表明，2-ME-FA-BSANPs靶向给药系统的消除半衰期、平均滞留时间与AUC均显著高于2-ME组(P0.05)，显著改善药物2-ME半衰期短，生物利用度低等缺点，达到了提高药物疗效，降低毒副作用的目的。
[Abstract]:2 - methoxyestradiol ( 2 - methoxyestradiol , 2 - ME ) is a kind of steroid hormone compound . It is a new kind of steroid antitumor drug . It has relatively high specificity for rapidly growing tumor cells , and has no toxicity to bone marrow and hematopoietic organs .

Preparation and related properties of 1.2 - ME - FA - BSANPs .

2 - ME albumin nanoparticles ( 2 - ME - BSANPs ) were prepared by de - solvation - chemical crosslinking method . The preparation of 2 - ME - BSANPs was determined by single factor analysis of albumin concentration , 2 - ME concentration , glutaraldehyde dosage of crosslinking agent , ratio of ethanol to water , pH of solution , etc . The preparation process of 2 - ME - BSANPs was as follows : BSA concentration was 40mg / ml ;
2 - ME concentration of 4mg / ml ;
the pH value of the solution is 10.0 ;
The ratio of ethanol to water is 3 : 1 . The folic acid active ester is further used for coupling reaction with the surface active amino group of the albumin nanoparticle surface active amino group , and the folic acid - coupled 2 - ME albumin nanoparticle targeted administration system ( 2 - ME - FA - BSANPs ) is prepared so as to have an active targeting effect and improve the tumor cell uptake rate of albumin nanoparticles .

The mean particle size and potential of 2 - ME - FA - BSANPs were ( 217.5 卤 5.1 ) nm and ( - 37.59 卤 1.39 ) mV , respectively .
The average entrapment efficiency and the drug loading were ( 82.50 卤 5.80 ) % and ( 7.50 卤 0.50 ) % , respectively .
SEM images show that the particle size distribution of 2 - ME - FA - BSANPs is uniform , spherical and smooth surface .
The results of in vitro drug release showed that the drug substance 2 - ME was released rapidly , and the cumulative release was about 85 % in 12h ;
The corresponding 2 - ME - FA - BSANPs released slowly and steadily , and the cumulative release rate was about 85 % within 96 h , indicating that the targeted drug delivery system had certain sustained release characteristics .
preparing the targeted preparation into lyophilized powder , and screening to select 3 % sucrose as its freeze - drying protective agent ;
The results showed that the freeze - dried powder can be stably stored for 6 months at 4 鈩

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