胰岛素巯基化透明质酸纳米粒的制备与性质评价
发布时间：2018-08-08 13:34
【摘要】：多肽类药物口服给药的研究一直是国内外药剂学研究者热衷的领域,但这类药物由于自身理化性质等特点,口服生物利用度低。这其中研究者尤为关注的是胰岛素。胰岛素是临床上治疗I型糖尿病的一线药物,但存在的突出问题是药物半衰期短,需长期频繁地注射给药。而胰岛素口服给药是一种患者顺应性好、服用方便的给药方式,符合内源胰岛素的分泌模式。然而,胰岛素直接口服给药时遭遇胃肠道酶降解以及膜透过性差等诸多障碍。生物黏附性聚合物纳米粒可以通过延长胃肠道药物滞留时间和减少胃肠道中消化酶对胰岛素的降解等方式,有效改善胰岛素口服生物利用度。 本研究将生物黏附性聚合物透明质酸与L-半胱氨酸通过酰胺键连接制备了巯基化透明质酸(HA-Cys),其游离巯基含量为(225.7±4.4)μmol·g-1,核磁共振以及红外图谱确证了其结构。体外黏附性实验表明聚合物黏附性质良好。 以HA-Cys为载体,采用超声乳化法,制备了载胰岛素的巯基化透明质酸纳米粒(Ins-HA-Cys-NPs)。通过单因素考察,确定了对Ins-HA-Cys-NPs影响较大的因素为油相、水相体积比,HA-Cys用量以及Ins用量,并通过星点设计-效应面优化法,以包封率、载药量、粒径以及多分散指数为评价指标,优化了纳米粒的制备工艺。最终得到的最优处方为水相、油相体积比为1：4.12,HA-Cys用量为25.95mg, Ins用量为8.04mg。通过激光粒度分析仪测定纳米粒粒径和分布,平均粒径(PS)为(178.5±0.75)nm,多分散系数(PDI)为0.214±0.01,Zeta电位(ZP)为(-38.47±0.46)mV；反相高效液相法测定纳米粒包封率和载药量,包封率(EE)为(48.85±0.66)%,载药量(DL)为(4.79±0.13)%。进行了纳米粒的形态观察、黏附性质、体外释放与抗酶降解特性的研究,评价了胰岛素巯基化透明质酸纳米粒的大鼠口服给药的降血糖效果。实验结果表明,所制得的Ins-HA-Cys-NPs外观圆整,且分布均一；制备的Ins-HA-Cys-NPs具有良好的体外黏附特性,黏蛋白与Ins-HA-Cys-NPs纳米粒混悬液混合后Zeta电位由(-17.32±0.38)mV下降到(-36.06±0.62)mV；与胰岛素溶液相比,Ins-HA-NPs及Ins-HA-Cys-NPs药物释放时间均延长；以胃蛋白酶、胰蛋白酶为代表,研究Ins-HA-Cys-NPs的抗酶解作用,说明Ins-HA-Cys-NPs具有较好的抗酶降解作用；药效学实验中,糖尿病大鼠口服Ins-HA-Cys-NPs后1-12h中,显示出平缓的降血糖效果,相对生物利用度为6.9%。
[Abstract]:The study of oral administration of polypeptide drugs has always been a hot field of pharmacists at home and abroad, but the oral bioavailability of these drugs is low due to their physical and chemical properties. Of these, researchers are particularly concerned about insulin. Insulin is a first-line drug in the treatment of type I diabetes, but the outstanding problem is that the half life of the drug is short and it needs to be injected frequently for a long time. The insulin oral administration is a kind of patient's compliance, the convenient administration way, conforms to the endogenous insulin secretion pattern. However, insulin directly administered orally encountered many obstacles, such as gastrointestinal enzyme degradation and poor membrane permeability. Bioadhesive polymer nanoparticles can effectively improve the oral bioavailability of insulin by prolonging the drug retention time in the gastrointestinal tract and reducing the degradation of insulin by digestive enzymes in the gastrointestinal tract. In this study, mercaptohyaluronic acid (HA-Cys) was prepared by linking the bioadhesive hyaluronic acid with L-cysteine by amide bond. Its free thiol content was (225.7 卤4. 4) 渭 mol g ~ (-1). Its structure was confirmed by NMR and IR spectra. In vitro adhesion test showed that the polymer adhesion was good. The sulfated hyaluronic acid nanoparticles (Ins-HA-Cys-NPs) loaded with insulin were prepared by phacoemulsification with HA-Cys as carrier. By single factor investigation, the oil phase, the volume ratio of water phase HA-Cys and the amount of Ins were determined, and the encapsulation efficiency, drug loading, particle size and polydispersity index were used as the evaluation indexes by the method of star design-effect surface optimization. The preparation process of nanoparticles was optimized. The optimum formulation was water phase, the volume ratio of oil phase was 1: 4.12, HA-Cys was 25.95 mg, and the dosage of Ins was 8.04 mg 路mol ~ (-1) 路min ~ (-1) 路min ~ (-1). The particle size and distribution of nanoparticles were measured by laser particle size analyzer. The average particle size (PS) was (178.5 卤0.75) nm, and the polydispersity coefficient (PDI) was 0.214 卤0.01U Zeta potential (ZP) was (-38.47 卤0.46) MV. The encapsulation efficiency and drug loading capacity of nanoparticles were determined by reversed-phase high performance liquid chromatography (RP-HPLC), the entrapment efficiency (EE) was (48.85 卤0.66), and the drug loading (DL) was (4.79 卤0.13) mm. The effects of oral administration of insulin mercaptohyaluronic acid nanoparticles on hypoglycemia in rats were evaluated by means of morphological observation, adhesion properties, release in vitro and anti-enzymatic degradation of nanoparticles. The results showed that the prepared Ins-HA-Cys-NPs had a round appearance and uniform distribution, and the prepared Ins-HA-Cys-NPs had a good adhesion property in vitro, and the Zeta potential decreased from (-17.32 卤0.38) MV to (-36.06 卤0.62) MV after the mixture of mucin and Ins-HA-Cys-NPs nanoparticles suspension, and the Zeta potential decreased from (-17.32 卤0.38) MV to (-36.06 卤0.62) MV. Compared with insulin solution, the release time of Ins-HA-NPs and Ins-HA-Cys-NPs was prolonged. The antienzymatic hydrolysis of Ins-HA-Cys-NPs was studied by using pepsin and trypsin as the representative. Diabetic rats showed a gentle hypoglycemic effect in 1-12 hours after oral administration of Ins-HA-Cys-NPs, and the relative bioavailability was 6. 9%.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943
本文编号：2171938
[Abstract]:The study of oral administration of polypeptide drugs has always been a hot field of pharmacists at home and abroad, but the oral bioavailability of these drugs is low due to their physical and chemical properties. Of these, researchers are particularly concerned about insulin. Insulin is a first-line drug in the treatment of type I diabetes, but the outstanding problem is that the half life of the drug is short and it needs to be injected frequently for a long time. The insulin oral administration is a kind of patient's compliance, the convenient administration way, conforms to the endogenous insulin secretion pattern. However, insulin directly administered orally encountered many obstacles, such as gastrointestinal enzyme degradation and poor membrane permeability. Bioadhesive polymer nanoparticles can effectively improve the oral bioavailability of insulin by prolonging the drug retention time in the gastrointestinal tract and reducing the degradation of insulin by digestive enzymes in the gastrointestinal tract. In this study, mercaptohyaluronic acid (HA-Cys) was prepared by linking the bioadhesive hyaluronic acid with L-cysteine by amide bond. Its free thiol content was (225.7 卤4. 4) 渭 mol g ~ (-1). Its structure was confirmed by NMR and IR spectra. In vitro adhesion test showed that the polymer adhesion was good. The sulfated hyaluronic acid nanoparticles (Ins-HA-Cys-NPs) loaded with insulin were prepared by phacoemulsification with HA-Cys as carrier. By single factor investigation, the oil phase, the volume ratio of water phase HA-Cys and the amount of Ins were determined, and the encapsulation efficiency, drug loading, particle size and polydispersity index were used as the evaluation indexes by the method of star design-effect surface optimization. The preparation process of nanoparticles was optimized. The optimum formulation was water phase, the volume ratio of oil phase was 1: 4.12, HA-Cys was 25.95 mg, and the dosage of Ins was 8.04 mg 路mol ~ (-1) 路min ~ (-1) 路min ~ (-1). The particle size and distribution of nanoparticles were measured by laser particle size analyzer. The average particle size (PS) was (178.5 卤0.75) nm, and the polydispersity coefficient (PDI) was 0.214 卤0.01U Zeta potential (ZP) was (-38.47 卤0.46) MV. The encapsulation efficiency and drug loading capacity of nanoparticles were determined by reversed-phase high performance liquid chromatography (RP-HPLC), the entrapment efficiency (EE) was (48.85 卤0.66), and the drug loading (DL) was (4.79 卤0.13) mm. The effects of oral administration of insulin mercaptohyaluronic acid nanoparticles on hypoglycemia in rats were evaluated by means of morphological observation, adhesion properties, release in vitro and anti-enzymatic degradation of nanoparticles. The results showed that the prepared Ins-HA-Cys-NPs had a round appearance and uniform distribution, and the prepared Ins-HA-Cys-NPs had a good adhesion property in vitro, and the Zeta potential decreased from (-17.32 卤0.38) MV to (-36.06 卤0.62) MV after the mixture of mucin and Ins-HA-Cys-NPs nanoparticles suspension, and the Zeta potential decreased from (-17.32 卤0.38) MV to (-36.06 卤0.62) MV. Compared with insulin solution, the release time of Ins-HA-NPs and Ins-HA-Cys-NPs was prolonged. The antienzymatic hydrolysis of Ins-HA-Cys-NPs was studied by using pepsin and trypsin as the representative. Diabetic rats showed a gentle hypoglycemic effect in 1-12 hours after oral administration of Ins-HA-Cys-NPs, and the relative bioavailability was 6. 9%.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943
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