激光共聚焦显微镜法分析载药微球的结构

Structure Analysis of Microspheres by Confocal Laser Scanning Microscopy

目的 验证激光共聚焦显微镜 (CL SM)可否作为研究微球结构的工具 ,并用以获取微球内部结构的信息。方法 微球用复凝聚法制备 ,牛血清白蛋白 (BSA)为模型蛋白药物和聚合物 (明胶与阿拉伯胶 )为成球材料 ,在制备微球前均共价标记了荧光物。应用 CL SM将微球在不同荧光通道下呈像 ,并将微球切割成一系列平行切面分别成像 ,对微球进行三维重建和图像分析。结果 在 CL SM下微球中 BSA与聚合物均呈均匀分布 ,加入BSA不影响聚合物的分布。结论 CL SM可观察载药微球的内部结构 ,而不需事先破坏样品即可获得被包裹药物的定位和成球材料所构成微球的结构资料。

Objective This study was aimed to visualize the polymer distribution within the microspheres, to localize the encapsulated drug, and to evaluate the potential of confocal laser scanning microscope(CLSM)as a characterization tool for microspheres. Methods Microspheres were prepared by complex coacervation. Model protein drugs(BSA) and polymers (gelatin and arabic gum) were covalently labelled with a fluorescent marker prior to microsphere preparation. By applying CLSM, the polymer distribution or changes in polymer matrix composition under the influence of the drug were visualized, and three-dimensional reconstruction and image analysis of the microspheres were performed by scanning different coplanar sections throughout the object. Results A homogeneous distribution of both gelatin and arabic gum throughout the microspheres was observed. The addition of fluorescently labeled BSA as a macromolecular model compound to the coacervate resulted in a homogeneous distribution of BSA within the microspheres; concurrently, the distribution of gelatin and acacia indicated that their deposition was not influenced by the presence of BSA-FITC. Conclusion CLSM allows the inspection of internal microsphere structures without prior sample destruction. It can be used to localize the encapsulated compounds and to detect special structural details of the microsphere composition.