Shen Yu-Bin, Guan Yi-Xin, Yao Shan-Jing
College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
Int J Pharm. 2015 Jul 15;489(1-2):226-36. doi: 10.1016/j.ijpharm.2015.05.004. Epub 2015 May 6.
Supercritical fluid assisted atomization introduced by a hydrodynamic cavitation mixer (SAA-HCM) was used to prepare micrometric particles of a labile protein, i.e., trypsin from aqueous solution without use of any organic solvents. The trypsin particles precipitated had various morphologies under different process conditions, with particle diameters ranging from 0.2 to 4 μm. FTIR, SDS-PAGE, CD and fluorescence spectra were performed to analyze the structural stability of the protein, and trypsin retained above 70% of the biological activity. Besides, chitosan was selected as the polymer carrier in an effort to prepare trypsin composite microparticles via SAA-HCM process. The influences of chitosan molecular weight, polymer/protein ratio and solution concentration on the particle morphology and size distribution were investigated in detail. Non-coalescing spherical composite microparticles with a narrow particle distribution (0.2-3 μm) could be obtained. The SAA-HCM prepared particles were amorphous as demonstrated by XRD and had a loading efficiency about 90%. The protein release profiles of the composite microparticles were evaluated using both the immersion condition and a Franz diffusion cell. Finally, the distribution of the protein within the particles was characterized through CLSM analysis of FITC-labeled trypsin-loaded chitosan microparticles. The SAA-HCM process is demonstrated to be a protein-friendly and promising technique for production of protein and polymer/protein composite particles formulations from aqueous solutions for drug delivery systems.
利用由水力空化混合器引入的超临界流体辅助雾化法（SAA-HCM），在不使用任何有机溶剂的情况下，从水溶液中制备了一种不稳定蛋白质——胰蛋白酶的微米级颗粒。在不同工艺条件下，沉淀出的胰蛋白酶颗粒具有多种形态，粒径范围为0.2至4μm。通过傅里叶变换红外光谱（FTIR）、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、圆二色光谱（CD）和荧光光谱对蛋白质的结构稳定性进行了分析，结果表明胰蛋白酶保留了70%以上的生物活性。此外，选择壳聚糖作为聚合物载体，通过SAA-HCM工艺制备胰蛋白酶复合微粒。详细研究了壳聚糖分子量、聚合物/蛋白质比例和溶液浓度对颗粒形态和尺寸分布的影响。可以获得具有窄粒径分布（0.2-3μm）的不聚结球形复合微粒。如X射线衍射（XRD）所示，通过SAA-HCM制备的颗粒为无定形，负载效率约为90%。使用浸泡条件和弗兰兹扩散池对复合微粒的蛋白质释放曲线进行了评估。最后，通过对异硫氰酸荧光素（FITC）标记的载胰蛋白酶壳聚糖微粒进行共聚焦激光扫描显微镜（CLSM）分析，表征了蛋白质在颗粒内的分布情况。结果表明，SAA-HCM工艺是一种对蛋白质友好且有前景的技术，可用于从水溶液中制备用于药物递送系统的蛋白质以及聚合物/蛋白质复合颗粒制剂。
