长效降血糖GLP-1衍生物纳米颗粒的制备及生物学活性研究

发布时间：2018-06-02 02:22

本文选题：GLP-1衍生物 + γ-聚谷氨酸　；参考：《华东师范大学》2015年硕士论文

【摘要】：糖尿病是当今威胁人类健康的全球性疾病与难题,因而新型糖尿病治疗药物的研发是目前新药研发中重要的一环。胰高血糖素样肽-1(GLP-1)作为肠促将血糖素,不仅具有葡萄糖依赖性的刺激胰岛素分泌和抑制胰高血糖素分泌功能,降低了低血糖的危险,还具有促进胰岛β细胞数目增加、抑制胃酸分泌和胃排空等功能,因而成为Ⅱ型糖尿病治疗药物的研究热点。然而天然GLP-1由于二肽基肽酶Ⅳ(DPPⅣ酶)降解和肾清除等作用,体内半衰期仅1-2min,不能直接作为临床药物使用。为抑制DPPⅣ酶降解,我们将GLP-1的第8位丙氨酸A替换成为甘氨酸G,并将两分子该GLP-1衍生物通过一分子赖氨酸共价相连,形成同源二聚体体结构,形成新型GLP-1衍生物,命名DLG3312。实验证明,DLG3312在小鼠体内降血糖作用时间达10-12h,其活性与上市药物利拉鲁肽(Victoza)相当。然而,DLG3312仍需一天注射一次,开发更为长效的GLP-1受体激动剂从而降低注射频率、提高患者顺应性仍是目前的研究方向。本课题主要设计思路为：以γ-聚谷氨酸(γ-PGA)为水溶性骨架,苯丙氨酸乙酯(L-PAE)为疏水基团,制备两性聚合物γ-PGA-PAE。γ-PGA-PAE在水中自组装形成纳米颗粒(NPs)的同时将DLG3312包裹进入其内部核心,形成DLG3312-纳米颗粒(DLG3312-NPs),从而达到以下目的：(1)通过包裹DLG3312形成DLG3312-NPs的形式增加药物直径从而抵御肾小球清除作用；(2)通过纳米载体外壳抑制DPPⅣ酶对DLG3312的降解,以增加体内稳定性；(3)通过体内γ-谷氨酰转肽酶(y-GTP)等酶类对载体多聚氨基酸的逐步降解达到药物缓释的目的；(4)通过注射给药在动物体内达到长效降血糖活性。本课题的研究内容及研究结果如下：1、γ-PGA-PAE的合成首先利用枯草芽孢杆菌DL发酵制备大分子γ-PGA,再采用高温酸解的方式将γ-PGA降解形成小分子γ-PGA,利用水溶性酰胺反应缩合剂EDC.I将γ-PGA的羧基(-COOH)与L-PAE的氨基(-NH2)间形成肽键相连,形成γ-PGA-PAE。核磁共振检测表明,得到的产物中既含有γ-PGA的特征峰,又含有L-PAE的特征峰,表明成功合成了两性聚合物γ-PGA-PAE。2、纳米药物载体(NPs)的制备及表征将溶于二甲基亚砜(DMSO)中的γ-PGA-PAE滴加于水溶液中,使其在水相中自组装,通过原子力显微镜和透射电镜观测到圆形颗粒结构,尺寸约100nm。纳米粒度仪检测结果表明颗粒平均直径为105.9 nm,PdI系数0.078,说明制得的颗粒为单分散体系,且颗粒均一；同时测得粒子的Zeta电势为+29.3mV,接近临界值绝对值30 mV,而高Zeta电势表明粒子具有较高的稳定性。3、DLG3312-NPs的制备及表征将溶于DMSO的γ-PGA-PAE滴加于DLG3312水溶液中,使其自组装形成颗粒,其后离心分离沉淀和上清,通过SDS-PAGE分析发现,DLG3312溶液中DLG3312含量减少,并出现在颗粒沉淀中,说明y-PGA-PAE能将DLG3312包裹进入其颗粒核心区域形成DLG3312-NPs。通过透射电镜表征,发现DLG3312-NPs同样为100nm左右的颗粒结构；纳米粒度仪检测表明,其颗粒平均直径为106.7nm,PdI系数为0.069,呈单分散体系,且颗粒分布均一；其Zeta电势为+33.4mV,高于绝对值30mV,高电位使其能够在溶液中稳定存在。4、NPs与DLG3312-NPs制备条件优化在探索γ-PGA分子量大小对形成的NPs的影响时发现,不同分子量——30.0-60.OkD和265.0 kD的γ-PGA形成的NPs平均粒径相差较小,分别为109.7nm(γ-PGA 30.0-60.0kD)和105.4nm(γ-PGA 265.0 kD),但后者颗粒分布均一,PdI系数仅为0.079,前者分布较不均一,PdI系数达0.287,提示较大分子量的γ-PGA更适于制备NPs。通过探索γ-PGA降解时间、L-PAE接入率、γ-PGA-PAE和DLG3312浓度对DLG3312包裹率的影响发现,在γ-PGA降解9min、L-PAE接入率80%、γ-PGA-PAE浓度30mg/ml和DLG3312溶液浓度2.5mg/ml时包裹率最高,可达到78.4±2.2%。疏水基团接入率会对两性聚合物产生的颗粒理化性质产生重要影响。通过调节L-PAE的投入量,合成L-PAE接入率在54.1-95.2%之间的γ-PGA-PAE,对制备的DLG3312-NPs表征发现,其颗粒平均直径在106-147nm范围内变化,L-PAE实际接入率62.5%的γ-PGA-PAE产生的DLG3312-NPs最稳定、粒径分布最为均一。5、DLG3312-NPs的短期生物学活性研究健康KM鼠、STZ诱导的Ⅱ型糖尿病模型鼠(STZ-T2DM小鼠)、自发性Ⅱ型糖尿病db/db小鼠的腹腔糖耐量实验表明,DLG3312-NPs能够显著提高3种实验鼠的腹腔糖耐量,并且具有一定的剂量依赖效应,表明DLG3312-NPs保留了DLG3312的生物学活性。3种实验鼠的DLG3312-NPs单次给药实验表明,DLG3312-NPs的体内活性时间可达48h,显著延长了DLG3312的10-12h的降血糖活性时间。6、DLG3312-NPs的长期生物学活性研究两种Ⅱ型糖尿病模型鼠——STZ-T2DM小鼠和db/db小鼠体内的长期生物学活性研究结果基本一致：DLG3312-NPs能够平稳的控制两种模型鼠的血糖处于较低的状态,并且能够显著降低空腹血糖、抑制饮水饮食,并呈现一定的剂量依赖效应。糖化血红蛋白(HbA1c)检测表明,DLG3312-NPs能够显著降低两种模型鼠的HbA1c水平,其中STZ-T2DM小鼠降低2.20±0.67%,db/db小鼠降低2.43±0.32%；此外,DLG3312-NPs还能够改善两种模型鼠的脂代谢水平。两日给药一次的DLG3312-NPs与一日给药一次的DLG3312和Victoza相比：DLG3312-NPs血糖控制更为平稳,而在空腹血糖降低、饮食饮水控制、脂代谢改善等方面,DLG3312-NPs要稍优于DLG3312和Victoza,或者两者基本相当。在HbA1c水平方面,DLG3312组下降1.95±0.79%(STZ-T2DM小鼠)、2.37-0.66%(db/db小鼠),Victoza组下降1.73±0.62%(db/db小鼠),表明,DLG3312-NPs血糖控制效果要略优于DLG3312和Victoza o胰腺组织切片结果表明,DLG3312-NPs、DLG3312和Victoza均能一定程度上促进其胰岛p细胞生长,效果基本相当。7、DLG3312-NPs对肝肾功能的影响检测连续给药8周的STZ-T2DM小鼠的肾功能和肝功能发现,DLG3312-NPs给药组各血液指标与健康C57小鼠相比不存在差异,这表明DLG3312-NPs对肝肾功能影响小,提示DLG3312-NPs毒性低,具有良好的生物安全性。检测连续给药6周的db/db小鼠的肝功能和肾功能血液指标,并进行进行肝脏、肾脏的组织切片HE染色分析,结果同样表明DLG3312-NPs对肝肾功能影响小,提示DLG3312-NPs具有良好的生物安全性,毒性低。综上,本研究运用两性聚合物γ-PGA-PAE包裹DLG3312制备了DLG3312-NPs,其平均直径106.7nm,PdI0.069,Zeta电势+33.4mV,通过条件优化其包裹率可达78.4±2.2%。生物学活性研究表明：DLG3312-NPs在3种实验鼠中体内活性时间均可达48h,而DLG3312仅为10-12h；两日一次给药的DLG3312-NPs在HbA1c和空腹血糖降低、饮水进食控制等方面要略优于一日一次给药的DLG3312和Victoza。目前,运用γ-PGA-PAE对GLP-1衍生物进行纳米化包裹在国内外尚未见报道,本研究的探索性工作将为GLP-1研发成为新型的Ⅱ型糖尿病治疗药物奠定基础。
[Abstract]:Diabetes is a global disease and difficult problem that threatens human health today. Therefore, the research and development of new type diabetes drugs is an important part of new drug research and development. The glucagon like peptide -1 (GLP-1) is used as an intestinal stimulating hormone, which not only has Glucose dependent stimulation of islet secretion and inhibition of glucagon secretion, but also reduces the secretion of glucagon. The risk of hypoglycemia, the increase of the number of islet beta cells, the inhibition of gastric acid secretion and gastric emptying, has become a hot spot in the study of type II diabetes. However, the natural GLP-1 is only 1-2min due to the degradation of two peptidyl peptidase IV (DPP IV) and renal clearance, which can not be used directly as a clinical drug. In order to inhibit the degradation of DPP IV enzyme, we replace the eighth bit alanine A of GLP-1 into the glycine G, and the two molecule of the GLP-1 derivative is linked together by a molecular lysine, forming a homologous two polymer structure and forming a new GLP-1 derivative. The DLG3312. experiment named DLG3312 has shown that DLG3312 in mice has a time of hypoglycemic action of 10-12h and its survival time. Sex is equivalent to the listed drug lalalu (Victoza). However, DLG3312 still needs to be injected once a day to develop a more long-acting GLP-1 receptor agonist to reduce the injection frequency and improve patient compliance. The main idea is to use gamma polyaline (gamma -PGA) as a water-soluble skeleton, ethyl phenylalanine (L-). PAE) for the hydrophobic group, the amphoteric polymer gamma -PGA-PAE. gamma -PGA-PAE was prepared by self assembly and formed nanoparticles (NPs) in water, and DLG3312 was wrapped into its inner core to form a DLG3312- nanoparticle (DLG3312-NPs), thus achieving the following purpose: (1) increasing the diameter of the drug by wrapping DLG3312 to form DLG3312-NPs to resist the kidney Pellet clearance; (2) the inhibition of DPP IV enzyme degradation by nanoscale carrier to increase the stability of DLG3312 in vivo; (3) the gradual degradation of polyamino acids through the vivo gamma glutamyl transaminopeptidase (y-GTP) and other enzymes can achieve the purpose of drug release; (4) through injection to the animal to achieve long-term hypoglycemic activity. The contents and results of the research are as follows: 1, the synthesis of gamma -PGA by Bacillus subtilis DL was first prepared by Bacillus subtilis DL, and then the gamma -PGA was degraded by high temperature acidolysis, and the carboxyl group (-COOH) of gamma -PGA and the amino group (-NH2) of L-PAE (-NH2) were formed by the water-soluble amide reaction condensation agent EDC.I. -PGA-PAE. NMR detection shows that the obtained products contain both the characteristic peaks of gamma -PGA and the characteristic peak of L-PAE, which indicates that the amphoteric polymer gamma -PGA-PAE.2 is successfully synthesized. The preparation and characterization of the nano drug carrier (NPs) are added to the aqueous solution in the two methyl sulfoxide (DMSO), so that it is in the water phase. The structure of circular particles was observed by atomic force microscope and transmission electron microscope. The average diameter of the particle size was 105.9 nm and the PdI coefficient was 0.078. The particles were monodisperse and the particles were homogeneous, and the Zeta potential of the particles was +29.3mV and the absolute value of the critical value was 30 mV. The high Zeta potential shows that the particles have high stability.3. The preparation and characterization of DLG3312-NPs are added to DLG3312 water in DLG3312 solution by DLG3312-NPs. The particles are formed by self assembly and then centrifuged to separate the precipitates and supernatants. The SDS-PAGE analysis shows that the DLG3312 content in DLG3312 solution decreases and appears in the particle precipitation. Y-PGA-PAE can form the DLG3312 package into its core region and form DLG3312-NPs. by transmission electron microscopy. It is found that DLG3312-NPs is also a particle structure of about 100nm. The nano particle size analyzer shows that the average diameter of the particles is 106.7nm, the PdI coefficient is 0.069, and the particle distribution system is uniform, and the Zeta potential is +33.4mV, Higher than the absolute value of 30mV, high potential makes it stable in the solution in the presence of.4, NPs and DLG3312-NPs preparation conditions are optimized to explore the effect of gamma -PGA molecular weight on the formation of NPs, the average particle sizes of NPs with different molecular weights, 30.0-60.OkD and 265 kD, are smaller, respectively, 109.7nm (gamma -PGA) and 105. 4nm (gamma -PGA 265 kD), but the latter particles are evenly distributed, the PdI coefficient is only 0.079, the former is not uniform, and the PdI coefficient is 0.287. It suggests that the larger molecular weight of the gamma -PGA is more suitable for the preparation of NPs. by exploring the degradation time of gamma -PGA, the L-PAE access rate, the effect of gamma -PGA-PAE and DLG3312 concentration on the DLG3312 inclusion rate. The inclusion rate is 80%, the concentration of gamma -PGA-PAE concentration 30mg/ml and DLG3312 solution concentration 2.5mg/ml is the highest, which can reach 78.4 + 2.2%. hydrophobic group access rate will have an important influence on the physical and chemical properties of the particles produced by the amphoteric polymer. By adjusting the input of L-PAE, the L-PAE access rate between 54.1-95.2% is synthesized and the DLG3312-NPs table is prepared. It was found that the average diameter of the particles changed in the range of 106-147nm. The DLG3312-NPs produced by gamma -PGA-PAE with the actual L-PAE access rate of 62.5% was the most stable, the particle size distribution was the most uniform.5. The short-term biological activity of DLG3312-NPs was studied by healthy KM mice, STZ induced type II diabetes model mice (STZ-T2DM mice), and spontaneous type II diabetes db/db mice. The intraperitoneal glucose tolerance test showed that DLG3312-NPs could significantly increase the intraperitoneal glucose tolerance of 3 experimental mice, and had a certain dose dependent effect. It showed that DLG3312-NPs retained the biological activity of DLG3312 and the DLG3312-NPs single administration experiment of.3 experimental mice showed that the activity time of DLG3312-NPs in vivo could reach 48h, and the DLG3312 was significantly prolonged. The hypoglycemic time of 10-12h,.6, and the long-term biological activity of DLG3312-NPs, the results of the long-term biological activity of two type 2 diabetes model mice - STZ-T2DM mice and db/db mice were basically the same: DLG3312-NPs could control the blood sugar of the two model rats in a lower state, and could significantly reduce the blood sugar. Low fasting blood glucose inhibited drinking water diet and showed a dose dependent effect. Glycosylated hemoglobin (HbA1c) detection showed that DLG3312-NPs could significantly reduce the HbA1c level of two model mice, of which STZ-T2DM mice decreased by 2.20 + 0.67% and db/db mice were 2.43 + 0.32%, and DLG3312-NPs could also improve lipid metabolism in two model mice. Level. The one time DLG3312-NPs was compared with one day DLG3312 and Victoza: DLG3312-NPs blood glucose control was more stable, and DLG3312-NPs was slightly better than DLG3312 and Victoza in the decline of fasting blood glucose, drinking water control, and lipid metabolism, or the same. In HbA1c level, the DLG3312 group dropped 1.. 95 + 0.79% (STZ-T2DM mice), 2.37-0.66% (db/db mice), and Victoza group decreased by 1.73 + 0.62% (db/db mice). The results showed that the effect of DLG3312-NPs on blood glucose control was slightly better than that of DLG3312 and Victoza o pancreatic tissue sections. The results showed that DLG3312-NPs, DLG3312 and Victoza could promote the growth of the pancreatic islet cells to a certain extent. The effect of s on the function of liver and kidney detected the renal function and liver function of STZ-T2DM mice after 8 weeks of continuous administration. The blood indexes of DLG3312-NPs administration group were not different from those of healthy C57 mice, which showed that DLG3312-NPs had little effect on liver and kidney function, suggesting that DLG3312-NPs was low toxicity and had good biological safety. The detection of D in D was d. B/db mice liver function and renal function blood index, and carry on the liver, kidney tissue section HE staining analysis, the result also shows that DLG3312-NPs has little effect on liver and kidney function, suggesting that DLG3312-NPs has good biological safety and low toxicity. In summary, this study used two sex polymer gamma -PGA-PAE to package DLG3312 to prepare DLG3312-NPs, The average diameter of 106.7nm, PdI0.069, and Zeta potential +33.4mV was optimized. The biological activity of the inclusion rate of 78.4 + 2.2%. was optimized by conditions. The results showed that the activity time of DLG3312-NPs in the 3 experimental mice could reach 48h, and DLG3312 was only 10-12h; the DLG3312-NPs of the two day administration was reduced to HbA1c and fasting blood glucose, and the drinking water feeding control and so on. DLG3312 and Victoza. are superior to one day, and the use of gamma -PGA-PAE to encapsulate GLP-1 derivatives has not been reported at home and abroad. The exploratory work of this study will lay the foundation for the research and development of GLP-1 as a new type of type 2 diabetes treatment drug.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TQ460.1;TB383.1

【相似文献】