School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.
Colloids Surf B Biointerfaces. 2010 Sep 1;79(2):326-33. doi: 10.1016/j.colsurfb.2010.04.004. Epub 2010 Apr 24.
While formulating proteins into solid particles prior to microsphere preparation is regarded as an effective way to stabilize such macromolecules, the protein particles may still contact the aqueous continuous phase and be re-dissolved. Dissolved proteins may not only leak into the aqueous continuous phase (resulting in reduced loading efficiency), but also contact water-oil (the hydrophobic polymer solution) interfaces, factors known to denature proteins. To avoid dissolution of solidified protein particles, we developed a microencapsulation procedure involving a hydrophilic "oil" (hO) continuous phase to which the hydrophobic solution of the controlled-release polymer was dispersed. The hydrophilic "oil" phase was a glycerol-based liquid mixed with ethylene glycol and polyvinyl alcohol solution to adjust viscosity and surface tension. This non-water hydrophilic continuous phase is immiscible with the hydrophobic polymer solution yet unable to dissolve pre-formulated protein particles. After the embryonic microspheres loaded with the protein particles were formed in this hydrophilic "oil" phase, the formulation was transferred into a cold ethanol bath where the microspheres were immediately hardened due to extracting the organic solution by ethanol. This method was examined by microencapsulating bovine serum albumin (BSA) and beta-galactosidase (beta-gal) into polylactide-co-glycolide (PLGA) microspheres for encapsulation efficiency, release kinetics and bioactivity preservation. As measured using size exclusion chromatography (SEC-HPLC), up to 90% added BSA was encapsulated in microspheres, and the release kinetics of the protein was adjusted by selecting surfactants used in microencapsulation emulsification. The assay of enzymatic activity of beta-galactosidase in hydrolysis of o-nitrophenyl-beta-d-galactopyranoside (ONPG) indicated that over 90% of the protein recovered from the microspheres was active.
在制备微球之前将蛋白质制成固体颗粒被认为是稳定这些大分子的有效方法，但是蛋白质颗粒仍可能与水连续相接触并重新溶解。溶解的蛋白质不仅可能泄漏到水连续相中（导致载药量降低），而且还可能与水-油（疏水性聚合物溶液）界面接触，这两个因素已知会使蛋白质变性。为了避免固化蛋白质颗粒的溶解，我们开发了一种微囊化程序，涉及亲水性“油”（hO）连续相，将疏水性控释聚合物溶液分散在其中。亲水性“油”相是一种甘油基液体，与乙二醇和聚乙烯醇溶液混合以调节粘度和表面张力。这种非水亲水性连续相与疏水性聚合物溶液不混溶，但不能溶解预成型的蛋白质颗粒。在该亲水性“油”相中形成负载有蛋白质颗粒的胚胎微球后，将制剂转移到冷乙醇浴中，由于乙醇提取了有机溶液，微球立即变硬。该方法通过将牛血清白蛋白（BSA）和β-半乳糖苷酶（β-gal）包封到聚乳酸-共-羟基乙酸（PLGA）微球中进行了检验，以评估包封效率、释放动力学和生物活性保持。通过尺寸排阻色谱（SEC-HPLC）测量，高达 90%添加的 BSA 被包封在微球中，并且通过选择用于微囊化乳化的表面活性剂来调整蛋白质的释放动力学。β-半乳糖苷酶水解邻硝基苯-β-d-半乳糖吡喃糖苷（ONPG）的酶活性测定表明，从微球中回收的超过 90%的蛋白质是有活性的。
