Davies Owen R, Head Laura, Armitage David, Pearson Elizabeth A, Davies Martin C, Marlow Maria, Stolnik Snjezana
School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom.
Langmuir. 2008 Jul 15;24(14):7138-46. doi: 10.1021/la703735n. Epub 2008 Jun 18.
In this paper, we describe surface modification of poly( D,L-lactide- co-glycolide) (PLG) microspheres, intended for DNA vaccine application, with two functionalities: a steric stabilizing component, provided by poly(vinyl alcohol) (PVA) and a cationic component, aimed at subsequent DNA surface loading. The cationic functionality arises from polycations, such as PEI, poly( L-lysine), trimethyl chitosan, and (dimethylamino)ethyl methacrylate, introduced into the water phase of classical oil-in-water (o/w) solvent evaporation method of PLG microsphere fabrication. By systematic evaluation of production variables, a system was produced with balanced properties in terms of microsphere size appropriate for uptake by antigen presenting (e.g., dendritic) cells, colloidal stability, and relatively high DNA loading. The polycation (PEI) molecular weight and preparation concentration were both found to increase the surface polycation content and DNA binding capacity; however, they lead to an increased tendency for aggregation, particularly when the microsphere size was decreased. DNA loading of almost 100% efficiency was achieved under optimized conditions in physiologically acceptable buffers, resulting in a surface DNA loading appropriate for vaccine purposes. A further increase in surface DNA loading was however associated with an increase in the particles negative potential, indicating the surface presence of DNA charges not neutralized by the polycation and hence potentially not protected from in vivo enzymatic degradation. The internalization of surface-loaded DNA into the target cells was confirmed by monitoring fluorescent DNA after the microspheres were endocytosed by the cells in culture.
在本文中，我们描述了用于DNA疫苗应用的聚（D，L-丙交酯-共-乙交酯）（PLG）微球的表面改性，其具有两种功能：一种是由聚乙烯醇（PVA）提供的空间稳定成分，另一种是阳离子成分，旨在随后进行DNA表面负载。阳离子功能源自多阳离子，例如聚乙烯亚胺（PEI）、聚（L-赖氨酸）、三甲基壳聚糖和甲基丙烯酸（二甲氨基）乙酯，它们被引入到PLG微球制备的经典水包油（o/w）溶剂蒸发法的水相中。通过对生产变量的系统评估，制备出了一种在微球尺寸、适合抗原呈递（如树突状）细胞摄取、胶体稳定性和相对较高的DNA负载量方面具有平衡性能的体系。发现聚阳离子（PEI）的分子量和制备浓度均会增加表面聚阳离子含量和DNA结合能力；然而，它们会导致聚集倾向增加，尤其是当微球尺寸减小时。在生理可接受的缓冲液中，在优化条件下实现了近100%效率的DNA负载，从而获得了适合疫苗用途的表面DNA负载量。然而，表面DNA负载量的进一步增加与颗粒负电位的增加相关，这表明表面存在未被聚阳离子中和的DNA电荷，因此可能无法免受体内酶降解的影响。通过监测培养细胞摄取微球后荧光DNA的情况，证实了表面负载的DNA被内化到靶细胞中。
