Direct formation of proteo-liposomes by in vitro synthesis and cellular cytosolic delivery with connexin-expressing liposomes
Direct formation of proteo-liposomes by in vitro synthesis and cellular cytosolic delivery with connexin-expressing liposomes
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DOI：
10.1016/j.biomaterials.2009.04.006
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发表时间：
2009-08-01
期刊：
影响因子：
14
通讯作者：
Morita, Ikuo
中科院分区：
文献类型：
--
作者：
Kaneda, Makoto;Nomura, Shin-ichiro M.;Morita, Ikuo
脂质体作为药物载体广泛应用于分子生物学和医学领域。在这里，我们报告了一个新的脂质体-细胞相互作用通过连接蛋白。通过使用无细胞转录/翻译系统，在脂质体的存在下用编码连接蛋白43（Cx43）的质粒制备含有Cx43的脂质体。表达的膜蛋白，Cx43，直接构成的脂质体膜后，在体外合成，导致纯的膜蛋白含有脂质体。将亲水性染料钙黄绿素从表达Cx43的脂质体有效地转移到培养的细胞（表达Cx43）。在存在间隙连接抑制剂（18 β-大黄酸）的情况下以及在其他类型的连接蛋白（Cx 32）表达细胞的情况下，转移被显著阻断。结果表明，钙黄绿素通过连接蛋白介导的途径进入细胞。含有可溶性NEMO结合结构域肽的Cx43脂质体抑制了表达Cx43的细胞中IL-1 β诱导的NF-κ B活化和环加氧酶-2表达的细胞内信号级联，证实了有效的肽转移到细胞中。这是一种新的亲水性分子的直接胞质递送方法。(C)2009爱思唯尔有限公司保留所有权利。
Liposomes are widely utilized in molecular biology and medicine as drug carriers. Here we report a new liposome-cell interaction through connexins. Connexin 43 (Cx43)-containing liposomes were prepared by using cell-free transcription/translation systems with plasmids encoding Cx43 in the presence of liposome. The expressed membrane protein, Cx43, was directly constituted to the liposome membrane upon in vitro synthesis, leading to pure membrane protein-containing liposomes. The hydrophilic dye calcein was efficiently transferred from Cx43-expressing liposomes to cultured cells (Cx43 expressing). The transfer is significantly blocked in the presence of gap junction inhibitor (18 beta-glycyrrhetinic acid) and in the case of the other type of connexin (Cx32)-expressing cell. The results show that calcein entered the cell through connexin-mediated pathway. Cx43 liposomes containing a soluble NEMO-binding domain peptide suppressed the intracellular signaling cascade IL-1 beta-induced NF-kappa B activation and cyclooxygenase-2 expression in Cx43-expressing cells, confirming effective peptide transfer into the cell. This is a new method for direct cytosolic delivery of hydrophilic molecules. (C) 2009 Elsevier Ltd. All rights reserved.