A cytoplasm-sensitive peptide vector cross-linked with dynein light chain association sequence (DLCAS) enhances gene expression

Abstract

We previously engineered a novel, non-viral, multifunctional gene vector (STR-CH 2R 4H 2C) containing stearoyl (STR) and a block peptide consisting of Cys (C), His (H), and Arg (R). STR-CH 2R 4H 2C forms a nano-complex with pDNA and is stabilized by electronic interactions and disulfide cross linkages. In blood, pDNA, a cytosol-sensitive gene vector, is released from the complex into the cytosol. The current study aimed to make STR-CH 2R 4H 2C capable of active nuclear localization. The dynein light chain association sequence (DLCAS) was disulfide cross-linked to STR-CH 2R 4H 2C/pDNA through disulfide linkages, and the gene expression ability of this DLCAS cross-linked gene vector was evaluated. We examined the gene transfection efficiency of S-180 cells transfected with the STR-CH 2R 4H 2C/DLCAS/pDNA complex. STR-CH 2R 4H 2C/DLCAS/pDNA showed significantly higher and faster gene expression compared with STR-CH 2R 4H 2C/pDNA. We also evaluated the cellular uptake ability of STR-CH 2R 4H 2C/DLCAS/Cy5-labeled pDNA complex. STR-CH 2R 4H 2C/DLCAS/pDNA showed significantly lower cellular uptake compared with STR-CH 2R 4H 2C/pDNA. This result indicates that high gene expression of STR-CH 2R 4H 2C/DLCAS/pDNA does not facilitate its cellular uptake. In addition, the gene expression of DLCAS/STR-CH 2R 4H 2C/pDNA in S-180 cells pretreated with the tubulin polymerization inhibitor, nocodazole (NCZ), was significantly lower than that in the absence of NCZ. These results indicate that the high transfection efficiency of DLCAS/STR-CH 2R 4H 2C/pDNA is dependent on intra-cellular transport utilizing the microtubule motor protein, dynein. Taken together, our results suggest that DLCAS-modified STR-CH 2R 4H 2C may be a promising gene delivery system.