Characterization of bio-nanocapsule as a transfer vector targeting human hepatocyte carcinoma by disulfide linkage modification
Characterization of bio-nanocapsule as a transfer vector targeting human hepatocyte carcinoma by disulfide linkage modification
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DOI：
10.1016/j.jconrel.2006.12.020
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发表时间：
2007-04-23
影响因子：
10.8
通讯作者：
Seno, Masaharu
中科院分区：
文献类型：
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作者：
Nagaoka, Tadahiro;Fukuda, Takayuki;Seno, Masaharu
由重组的乙肝病毒包膜L蛋白组成的生物纳米胶囊(BNC)构成了有效的针对人肝细胞的传递载体。在这里，我们试图提高BNC的稳定性，因为BNC中的L蛋白在4℃长时间储存时，由于随机二硫键桥联而聚集。被膜蛋白在S结构域含有14个半胱氨酸残基。如果取代或去除不必要的半胱氨酸残基，包膜蛋白的聚集可能会被避免，因为自由巯基的不可逆烷基化防止了包膜蛋白的聚集，并提高了包埋效率。在这项研究中，评估了减少S结构域中半胱氨酸残基数量以提高BNC稳定性的可能性。用定点突变替换每个半胱氨酸残基表明，14个半胱氨酸残基中有9个不是获得分泌到培养基中的BNC所必需的。此外，在评估这些突变的组合后，发现有8个替换残留物是可以接受的。用8个半胱氨酸残基替换的突变BNC不仅对胰酶有更强的抵抗力，而且比原始类型的BNC更能有效地将基因导入人肝癌细胞系HepG2。因此，我们证明了S结构域中最少的半胱氨酸残基可以提高BNC的稳定性。(C)2006爱思唯尔B.V.保留所有权利。
The bio-nanocapsules (BNCs) composed of the recombinant envelope L-protein of hepatitis B virus constitute efficient delivery vectors specifically targeting human hepatocytes. Here, we have tried to enhance the stability of the BNCs because the L-proteins in the BNCs were aggregated due to random disulfide bridging when stored for a long period at 4 degrees C. The envelope protein contains fourteen cysteine residues in the S domain. Aggregation of the envelope proteins might be avoided if unessential cysteine residues are replaced or removed because the irreversible alkylation of the free sulfhydryl group protects against the aggregation and enhances the efficiency of encapsulation. In this study, the possibility of reducing the number of cysteine residues in the S domain to enhance the stability of the BNCs was assessed. The replacement of each cysteine residue by site-directed mutation showed that nine of fourteen cysteine residues were not essential to obtaining BNCs secreted into the culture media. Furthermore, upon evaluating the combination of these mutations, it was found that eight residues of replacement were acceptable. The mutant BNCs with replaced eight cysteine residues were not only more resistant against trypsin, but also more effective in transducing genes into human hepatoma-derived HepG2 cells than the original type BNC. Thus, we demonstrated that the minimized number of cysteine residues in the S domain could enhance the stability of the BNCs. (c) 2006 Elsevier B.V. All rights reserved.