载蛋白药物双层微球的制备和性质研究
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摘要
近年来,多肽和蛋白质类药物的缓释或控释给药系统发展很快,尤其倾向于使用以生物可降解材料为载体制备毫微球或微球长效注射剂。多肽及蛋白类药物微球给药系统具有缓释长效作用,因而成为生物技术类药物制剂研究的热点。目前,各种微球的制备方法容易破坏多肽及蛋白质类药物的稳定性,加上多肽及蛋白质自身结构复杂,造成蛋白质活性丧失,因此,在制备多肽及蛋白质类药物微球时,如何根据药物不同特征选择合适的辅料和制备方法,设计出安全、有效和稳定的多肽和蛋白质药物给药系统是制药业面临的问题。
双层微球是近年来发展较快的一种微球制剂形式,它不仅克服了传统单层微球的一些缺点,比如减少了单层微球的突释,改变释放模式使其更符合人体的需要:而且其制备方法简单,相对温和,对蛋白药物制剂的制备非常适合。
针对上述问题,本研究制备了载蛋白药物的双层微球,具体内容如下:
(1)采用天然高分子壳聚糖和透明质酸钠作为载体,利用乳化凝聚方法制备壳聚糖—透明质酸钠(CHS—HA)双层微球,通过扫描电镜、粒径分析、Zeta电位分析仪以及傅立叶红外对其性质进行表征;探讨制备过程中的多个因素对于微球制备的影响。CHS-HA微球形态圆,表面光滑,平均粒径为12.19±1.37μm,内核多孔,外层致密;制备过程中的外水相pH值、三聚磷酸钠的浓度、搅拌时间以及表面活性剂的添加都是影响微球制备的重要因素;外水相体积和透明质酸钠浓度也对微球形态具有明显的影响;
(2)以胰岛素为模型药物,制备了载胰岛素壳聚糖—透明质酸钠双层微球,考察胰岛素的包封率和微球载药量,并对双层微球进行正交设计,优化实验方案。通过微球包封率和载药量的正交设计结果得出优化条件为:外水相体积为40mL:pH为6.0,透明质酸钠浓度为0.025%,搅拌时间为3h:此时,微球的包封率为(76.46±1.52)%,载药量为(31.28±1.91)%。
(3)考察了两种微球的体外释放,进行机制探讨,并进行数学模型拟合。同时,通过放射免疫法测定了释放介质中的胰岛素的含量,反映胰岛素的活性。双层微球的体外释放分为快速释放相和缓慢释放相两相,分别符合零级动力学方程模型;胰岛素的放射免疫测定结果证明胰岛素在微球制备、储存、释放过程中活性保持良好。
In recent years, protein drugs delivery systems have been developed fast, especially for long-acting injectable microsphere made with biodegradable biomaterials. Because of their sustained release property, those microspheres preparation has been a hot field in pharmaceutics. Now, the activity of protein is ready to denature in the preparation process, so how to choose the proper biomaterials and preparation method based on the characterization of the protein drugs and how to prepare a safe, steady and effective preparation is an important challenge.
Aim at upper questions, a chitosan- hyaluronate(CHS-HA) double-walled microsphere was fabricated in this work. The detailed process was followed:
CHS-HA double-walled microspheres were prepared by emulsification-coacervation method. Tripolyphosphate (TPP) was the ion crosslinker. The individual effect of pH, volume, concentration of outer aqueous phase, andsurfactant on microspheres morphology and fabrication were tested by Zeta (ζ)potential, Scanning electron microscopy (SEM) and Fourier-transform infrared spectrometry (FT-IR). TPP concentration and pH of outer aqueous phase were crucial to microspheres fabrication, when TPP concentration was higher than 8%, spherical and smooth microspheres could be formed. The optimal pH for microspheres formation ranged from 6.0 to 7.0. Microspheres containing both span80 and tween80 in process were less shapely compared with those containing span80 only. In addition, volume and HA concentration of outer aqueous phase also affected the microspheres morphology.
Insulin as a model protein drug was encapsulated in CHS-HA double-walled microphers and the encapsulation efficiency and loading efficiency were measured. Further an orthogonal design was achieved based on those results and the most perfect conditions were chose. Through the orthogonal design, the chosen factors were: the volume of the outer aqueous phase was 40mL; the pH of the outer aqueous phase was 6.0, the concentration of HA was 0.025%, and the stirring time was 3h;
The release profile in vitro was detected and mimicked with mathematics equation. The mechanism of release was expounded. Meantime, Insulin amount was measured by radio-immunity assay (RIA), and detect the activity of Ins in the process of microsphere preparation, storage and release. The release profile had two phases, the fast release phase and the slow release phase and was according with the first kinetics equation. RIA results showed that Insulin bioactivity has no obvious loss in the process of microsphere preparation, storage and release.
双层微球是近年来发展较快的一种微球制剂形式,它不仅克服了传统单层微球的一些缺点,比如减少了单层微球的突释,改变释放模式使其更符合人体的需要:而且其制备方法简单,相对温和,对蛋白药物制剂的制备非常适合。
针对上述问题,本研究制备了载蛋白药物的双层微球,具体内容如下:
(1)采用天然高分子壳聚糖和透明质酸钠作为载体,利用乳化凝聚方法制备壳聚糖—透明质酸钠(CHS—HA)双层微球,通过扫描电镜、粒径分析、Zeta电位分析仪以及傅立叶红外对其性质进行表征;探讨制备过程中的多个因素对于微球制备的影响。CHS-HA微球形态圆,表面光滑,平均粒径为12.19±1.37μm,内核多孔,外层致密;制备过程中的外水相pH值、三聚磷酸钠的浓度、搅拌时间以及表面活性剂的添加都是影响微球制备的重要因素;外水相体积和透明质酸钠浓度也对微球形态具有明显的影响;
(2)以胰岛素为模型药物,制备了载胰岛素壳聚糖—透明质酸钠双层微球,考察胰岛素的包封率和微球载药量,并对双层微球进行正交设计,优化实验方案。通过微球包封率和载药量的正交设计结果得出优化条件为:外水相体积为40mL:pH为6.0,透明质酸钠浓度为0.025%,搅拌时间为3h:此时,微球的包封率为(76.46±1.52)%,载药量为(31.28±1.91)%。
(3)考察了两种微球的体外释放,进行机制探讨,并进行数学模型拟合。同时,通过放射免疫法测定了释放介质中的胰岛素的含量,反映胰岛素的活性。双层微球的体外释放分为快速释放相和缓慢释放相两相,分别符合零级动力学方程模型;胰岛素的放射免疫测定结果证明胰岛素在微球制备、储存、释放过程中活性保持良好。
In recent years, protein drugs delivery systems have been developed fast, especially for long-acting injectable microsphere made with biodegradable biomaterials. Because of their sustained release property, those microspheres preparation has been a hot field in pharmaceutics. Now, the activity of protein is ready to denature in the preparation process, so how to choose the proper biomaterials and preparation method based on the characterization of the protein drugs and how to prepare a safe, steady and effective preparation is an important challenge.
Aim at upper questions, a chitosan- hyaluronate(CHS-HA) double-walled microsphere was fabricated in this work. The detailed process was followed:
CHS-HA double-walled microspheres were prepared by emulsification-coacervation method. Tripolyphosphate (TPP) was the ion crosslinker. The individual effect of pH, volume, concentration of outer aqueous phase, andsurfactant on microspheres morphology and fabrication were tested by Zeta (ζ)potential, Scanning electron microscopy (SEM) and Fourier-transform infrared spectrometry (FT-IR). TPP concentration and pH of outer aqueous phase were crucial to microspheres fabrication, when TPP concentration was higher than 8%, spherical and smooth microspheres could be formed. The optimal pH for microspheres formation ranged from 6.0 to 7.0. Microspheres containing both span80 and tween80 in process were less shapely compared with those containing span80 only. In addition, volume and HA concentration of outer aqueous phase also affected the microspheres morphology.
Insulin as a model protein drug was encapsulated in CHS-HA double-walled microphers and the encapsulation efficiency and loading efficiency were measured. Further an orthogonal design was achieved based on those results and the most perfect conditions were chose. Through the orthogonal design, the chosen factors were: the volume of the outer aqueous phase was 40mL; the pH of the outer aqueous phase was 6.0, the concentration of HA was 0.025%, and the stirring time was 3h;
The release profile in vitro was detected and mimicked with mathematics equation. The mechanism of release was expounded. Meantime, Insulin amount was measured by radio-immunity assay (RIA), and detect the activity of Ins in the process of microsphere preparation, storage and release. The release profile had two phases, the fast release phase and the slow release phase and was according with the first kinetics equation. RIA results showed that Insulin bioactivity has no obvious loss in the process of microsphere preparation, storage and release.
引文
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