稳定干扰素-α-2b的多糖玻璃体颗粒的研究

Study on Glassy Polysaccharide Particles Stabilizing Interferon-α-2b

目的:在温和条件下,将结构脆弱的干扰素-α-2b制备成可耐受苛刻制剂条件的微粒。方法:通过干扰素在多糖和PEG共溶液中的冷冻相分离作用,制备干扰素分配在多糖分散相的微粒。在显微镜和扫描电镜下观察了颗粒的大小和外观,同时将颗粒置于不同密度的溶剂中考察了密度。用MicroBCA和ELISA法测量了颗粒的包封率和载药量;用SEC-HPLC和细胞病变抑制法初步考察了颗粒制备后蛋白稳定性的变化。结果:颗粒表面光滑圆整,粒径约1～5μm。密度约为1.47～1.48 g/cm^3,说明颗粒结构致密。MicroBCA和ELISA法测量的包封率分别为90%和80%以上,SEC-HPLC法测定蛋白无明显聚集体产生,细胞病变抑制法得出的活性保留率80%左右。结论:冷冻相分离法是一种简单但有效的制备蛋白多糖颗粒的方法。颗粒粒径均一,形态规整。该方法可进一步应用于蛋白缓释及微粉吸入等剂型的研究。

Objective： To prepare interferon-α-2b with fragile structure under mild conditions and to make it be particles which can tolerate harsh preparation conditions. Methods： Interferon-α-2b was loaded in polysaccharide glassy particles through a freezing-induced phase separation of a solution containing the protein, dextran and polyethylene （PEG）, followed lyophilization. The sizes and density of the polysaccharide particles were estimated under a microscope equipped with scale and by suspending in a series of organic solvents having different densities. Protein loading capacity and efficiency were determined using MicroBCA and ELISA methods, and protein integrity was assayed using size-exclusion chromatography （ SEC-HPLC ）. The bioactivities of the interferon-α-2b recovered from glassy particles were determined by WISH/VSV methods. Results： The dextran particles, 1-5μm in diameter and 1.47-1.48 g/cm^3 in density, possessed spherical shape and smooth surfaces. The encapsulation efficiency were 〉90% and 〉80% as determined using MicroBCA and ELISA methods, respectively. SEC-HPLC assay of proteins recovered from the dextran particles showed no increased protein aggregation as compared with original protein sample. WISH/VSV test indicated that the bioactivity retention of the dextran particles was about 80%. Conclusions： Freezing-induced phase separation is an effective and simple method to load structurally delicate proteins into polysaccharide fine particles without protein denaturing. These protein-containing particles may further be incorporated into variety of pharmaceutical dosage forms without protein aggregation or be directly applied as a dosage form such as inhalation powders.