| 内容提要: |
we demonstrate that the capsid formed by Aquifex aeolicus lumazine synthase (AaLS) can act as a nanocarrier for Sec. A previously reported variant of AaLS (AaLS-IC), which contains a single cysteine per subunit that projects into the capsid interior, was modified by reaction with selenocystine to generate a selenosulfide conjugate between the capsid and selenocysteine (AaLS-IC-Sec). Each AaLS-IC-Sec capsid contains approximately 48 selenocysteines, and efficient loading requires placement of the reactive cysteine inside of the protein capsid. A protein with the cysteine exposed to the outside cannot form the unique protein-drug conjugate by thiol-diselenide exchange reaction which is a significant advantage for developing AaLS-IC as a drug delivery system. Atmospheric oxygen are able to oxyidize reduced selenocystine very quickly and efficiently which can also benefit to the loading process. Encapsulated Sec can be quantitatively released from the capsid by common reducing agents, such as GSH or DTT, with a half-time of ~3 h. This property should be useful for the triggered release of selenocysteine in cancer cells, which tend to have higher levels of glutathione, compared to healthy cells. For four of the six cell lines tested, cell viability measurements indicated that AaLS-IC-Sec can go into the cells and release Sec in a toxic form. Fluorescence microscopy experiments revealed an intriguing correspondence between the cytotoxicity of AaLS-IC-Sec and the intracellular trafficking of the AaLS capsid. Cells that do not take up the capsid are insensitive to AaLS-IC-Sec. Moderately and highly sensitive cells accumulate the AaLS capsid in the cytoplasm and nucleus, respectively. This observed phenomenon can help us further modify AaLS-IC with intracellular targeting properties. Taken together, this study provides a promising foundation for the development of novel systems for the delivery of Sec into cells. |