Delivery of Condensed DNA by Liposomal Non-viral Gene Delivery System into Nucleus of Dendritic Cells

Abstract

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...

Effect of Polyethyleneglycol Spacer on the Binding Properties of Nuclear Localization Signal-Modified Liposomes to Isolated Nucleus

“…We used STR-NLS since, in a previous study, we demonstrated that modification of STR-NLS on the surface of liposomes enhanced the gene expression of encapsulated pDNA in primary dendritic cells. 10) Although DSPE-NLS may be more useful in comparing the effect of the PEG spacer, it is plausible that almost all of the STR-NLS and DSPE-PEG-NLS were stably incorporated in the lipid envelope since STR-NLS and DSPE-PEG-NLS were incorporated when lipid film was formed.…”

“…Stearyl NLS (STR-NLS) was synthesized as described previously. 10) JAWS II cells derived from murine dendritic cells were purchased from the American Type Culture Collection (Manassas, VA, U.S.A.). Other chemicals used were commercially available and were reagent grade products.…”

“…In this carrier, pDNA was condensed with a polycation, followed by encapsulation with envelopes composed of lipid bilayers. For the targeting the NPC, the surface of the nuclear envelope was modified with nuclear localization signals derived from SV40 (NLS) or nuclear targeting sugars by incorporating stearylated NLS (STR-NLS) 10) or cholesterolconjugated sugars 11) into the lipid film. As a result, gene expression activity was drastically enhanced, compared to that of the NLS-or sugar-unmodified carriers.…”

Transmission of External Environmental pH Information to the Inside of Liposomes <i>via</i> Pore-Forming Proteins Embedded within the Liposomal Membrane

“…20) In this method, PGA/ Dox is mixed with SUVs, and encapsulation of PGA/Dox results from membrane fusion of SUVs around the PGA/Dox complexes. When PGA/Dox was mixed with SUVs (10 mM DOPE : DCP=9 : 2), the particle sizes of the resulting liposomes were found to be smaller than that of PGA/Dox alone (Table 3), which may be due to tight packing of PGA/Dox by the lipid membrane.…”

“Programmed packaging” for gene delivery

“…Thus, as a third strategy, we modified the NLS or sugars (i.e. maltotripse) that can target nucleus on the envelope structure of the MEND by inserting a lipid derivative of a NLS [64,65]. This design permits the the NLS peptide to be spontaneously oriented in an outward direction from the particle surface.…”