title: 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(2)R(4)H(2)C) containing stearoyl (STR) and a block peptide consisting of Cys (C), His (H), and Arg (R). STR-CH(2)R(4)H(2)C 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(2)R(4)H(2)C capable of active nuclear localization. The dynein light chain association sequence (DLCAS) was disulfide cross-linked to STR-CH(2)R(4)H(2)C/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(2)R(4)H(2)C/DLCAS/pDNA complex. STR-CH(2)R(4)H(2)C/DLCAS/pDNA showed significantly higher and faster gene expression compared with STR-CH(2)R(4)H(2)C/pDNA. We also evaluated the cellular uptake ability of STR-CH(2)R(4)H(2)C/DLCAS/Cy5-labeled pDNA complex. STR-CH(2)R(4)H(2)C/DLCAS/pDNA showed significantly lower cellular uptake compared with STR-CH(2)R(4)H(2)C/pDNA. This result indicates that high gene expression of STR-CH(2)R(4)H(2)C/DLCAS/pDNA does not facilitate its cellular uptake. In addition, the gene expression of DLCAS/STR-CH(2)R(4)H(2)C/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(2)R(4)H(2)C/pDNA is dependent on intra-cellular transport utilizing the microtubule motor protein, dynein. Taken together, our results suggest that DLCAS-modified STR-CH(2)R(4)H(2)C may be a promising gene delivery system.
authors.affiliation: Laboratory of Pharmaceutics and Drug Delivery, Department of Pharmaceutical Science, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
authors.forename: Ko
authors.initials: K
authors.lastname: Tanaka
authors.forename: Takanori
authors.initials: T
authors.lastname: Kanazawa
authors.forename: Ken
authors.initials: K
authors.lastname: Sugawara
authors.forename: Shogo
authors.initials: S
authors.lastname: Horiuchi
authors.forename: Yuuki
authors.initials: Y
authors.lastname: Takashima
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Okada
mesh_terms: D000818:Animals; D001692:Biological Transport; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000008:administration & dosage*; D004398:Dyneins / Q000008:administration & dosage / Q000737:chemistry*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000008:administration & dosage / Q000737:chemistry*; D051379:Mice; D015739:Nocodazole / Q000494:pharmacology; D010957:Plasmids; D014162:Transfection; D050257:Tubulin Modulators / Q000494:pharmacology
chemical_list: D018076:DNA, Complementary; D050257:Tubulin Modulators; D004398:Dyneins; D015739:Nocodazole