Li Hao, Somiya Masaharu, Tatematsu Kenji, Kuroda Shun'ichi
Department of Biomolecular Science and Reaction, The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan.
Methods Mol Biol. 2020;2059:299-313. doi: 10.1007/978-1-4939-9798-5_16.
The construction protocol of bio-nanocapsule (BNC)-based nanocarriers, named GL-BNC and GL-virosome, for targeted drug delivery to macrophages is described here. First, genes encoding the Streptococcus sp. protein G-derived C2 domain (binds to IgG Fc) and Finegoldia magna protein L-derived B1 domain (binds to Igκ light chain) are prepared by PCR amplification. Subsequently, the genes encoding hepatic cell-specific binding domain of hepatitis B virus envelope L protein are replaced by these PCR products. The expression plasmid for this fused gene (encoding GL-fused L protein) can be used to transform Saccharomyces cerevisiae AH22R cells. To obtain GL-BNC, the transformed yeast cells are disrupted with glass beads, treated with heat, and then subjected to IgG affinity column chromatography followed by size exclusion column chromatography. In addition, GL-BNCs can be fused with liposomes to form GL-virosome. The targeted delivery of GL-BNC and GL-virosome to macrophages can be confirmed by in vitro phagocytosis assays using the murine macrophage cell line RAW264.7.
本文描述了用于向巨噬细胞靶向递送药物的基于生物纳米胶囊（BNC）的纳米载体（命名为GL-BNC和GL-病毒体）的构建方案。首先，通过PCR扩增制备编码源自链球菌属蛋白G的C2结构域（与IgG Fc结合）和源自巨大芬戈尔德菌蛋白L的B1结构域（与Igκ轻链结合）的基因。随后，用这些PCR产物替换编码乙肝病毒包膜L蛋白肝细胞特异性结合结构域的基因。该融合基因（编码GL融合L蛋白）的表达质粒可用于转化酿酒酵母AH22R细胞。为了获得GL-BNC，将转化的酵母细胞用玻璃珠破碎，加热处理，然后进行IgG亲和柱层析，接着进行尺寸排阻柱层析。此外，GL-BNC可与脂质体融合形成GL-病毒体。使用小鼠巨噬细胞系RAW264.7进行的体外吞噬试验可证实GL-BNC和GL-病毒体向巨噬细胞的靶向递送。
