In general, foreign substances can not enter the nucleus of non-dividing cells, in which nuclear membrane does not disappear by cell division, since nuclear transfer is strictly controlled by precise machinery. For example, nuclear proteins are recognized by importin a in the cytoplasm with a nuclear localization signal (NLS) attached to the nuclear proteins, and importin b then combines with the NLS/importin a. The complex binds to the nuclear pore complex (NPC), and passes through the NPC into nucleus. 1) Therefore, it is difficult to transfer foreign plasmid DNA (pDNA) into the nucleus of a non-dividing cell by means of an artificial gene delivery system such as cationic liposomes or cationic polymers. [2][3][4][5][6] Many researchers have attempted to artificially transfer pDNA utilizing nuclear transport machinery, i.e., the nuclear transfer of electrostatic complexes between NLS and DNA, 7-10) NLS-conjugated DNA, etc. [11][12][13][14][15][16] We also synthesized NLS conjugated linear pDNA and evaluated its nuclear transfer by a microinjection technique, but, no increase in the nuclear transport of pDNA was observed.17) This result suggests that a positively charged NLS is not able to function due to electrostatic interactions with the negatively charged DNA. Therefore, it is possible that steric flexibility in the NLS is necessary for an interaction with importin.In this study, we attempted to transfer pDNA condensed by a cationic peptide protamine into the nucleus of non-dividing mouse bone marrow derived dendritic cells (BMDC) using an NLS-modified liposomal non-viral gene delivery system (NLS-DMEND), of which NLS peptide was presented on the surface to retain its flexibility. We evaluated intracellular trafficking and the gene expression of the NLS-DMEND to evaluate its nuclear transfer capabilities. In addition, we examined the effect of metabolic inhibitors to confirm the energy dependency of the nuclear transfer of pDNA by the NLS-DMEND. Preparation of Bone Marrow-Derived Dendritic Cells (BMDC) of Mice BMDC were prepared based on a previously reported method 19,20) with some modifications. BM cells were treated with antibodies and rabbit complement to remove natural killer cells, B-lymphocytes, T-lymphocytes and granulocytes. The remaining BM cells were then cultured in RPMI 1640 medium containing 50 mM 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, 100 unit/ml penicillin-streptomycin, 10 ng/ml GM-CSF and 10% FCS. On day 2 and day 4, non-adherent cells were removed, and adherent cells were cultured in fresh medium containing GM-CSF. On day 6, non-adherent and loosely adherent cells were used in experiments as immature dendritic cells.
MATERIALS AND METHODS
Materials Dioleoyl phosphatidylethanolamine (DOPE) and N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-DOPE (NBD-DOPEPreparation and Transfection of Octaarginine-Modified Multifunctional Envelope-Type Nano Device (R8-MEND) The multifunctional envelope-type nano device (MEND) encapsulating protamine-condensed pDNA was prepared by the lipid film hydratio...
