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基于巨噬细胞靶向生物纳米胶囊的纳米载体构建

Construction of a Macrophage-Targeting Bio-nanocapsule-Based Nanocarrier.

作者信息

Li Hao, Somiya Masaharu, Tatematsu Kenji, Kuroda Shun'ichi

机构信息

Department of Biomolecular Science and Reaction, The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan.

出版信息

Methods Mol Biol. 2020;2059:299-313. doi: 10.1007/978-1-4939-9798-5_16.

Abstract

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.

摘要

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。

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