A cationic lipid (TRX) having an amidine headgroup was synthesized, and a lipoplex (i.e., plasmid DNA+lipid complex) was prepared from the mixture of TRX and two other neutral colipids. Small-angle X-ray scattering showed that the addition of DNA induced a structural transition from normal to inverted hexagonally packed cylinders. Transmission electron microscopy showed a threadlike micelle was transformed to a spherical micelle about 50-200 nm in diameter by adding DNA. A combination of these results leads to the conclusion that the complexed DNA is hexagonally packed (or condensed) into spherical aggregates. The size and morphology are believed suitable for endocytosis uptake or vesicle fusion. The complex made from pDNA (pEGFP-C1) and TRX was transfected to Hep G2. Flow cytometry and confocal microscopy showed that the present system expressed green fluorescence protein (GFP) more than a conventional transfection reagent. Additionally, TRX was less cytotoxic than other transfection reagents. This paper presents the attractive possibility of the amidine group for a transfection device.
When methyl 4,6-O-(p-nitrobenzylidene)-alpha-D-glucopyranoside (p-NO(2)Glu) was dissolved in water, p-NO(2)Glu molecules self-assembled to form a fiber (elemental fiber), and as a result, the solution became a partially transparent gel. When an equal (or more) amount of DNA was added to the gel, a white and crystalline gel was obtained. Energy-dispersive X-ray spectroscopy coupled with TEM and confocal microscopy suggested that DNA was included in the gel fibers made of p-NO(2)Glu molecules. The results imply that p-NO(2)Glu molecules are self-assembled to form an elemental fiber and these elemental fibers and DNA are twisted together to form higher hierarchic fibers. When the complexed gel made of plasmid DNA (pDNA) and p-NO(2)Glu was added to E. coli T7 S30 extract solution, the pDNA had less expression ability compared with naked one. When we added methyl-beta-cyclodextrin (MbetaCyD), the expression rate was recovered with increasing added amount of MbetaCyD. The present paper shows inclusion and controlled release of DNA from a novel supporting material of DNA and that technology could play an important role in the development of localized approaches to gene therapy.
Schizophyllan is a β-(1→3)-d-glucan existing as a triple helix in water and as a single chain in dimethyl sulfoxide (DMSO), respectively. As we already reported, when a homo-phosphodiester-polynucleotide is added to the schizophyllan/DMSO solution and, subsequently, DMSO is exchanged for water, the single chain of schizophyllan (s-SPG) forms a complex with the polynucleotide. In this paper, we report that phosphorothioate oligonucleotides can form a complex with s-SPG in the same manner as phosphodiester oligonucleotides. We carried out an in vitro antisense assay combining melanoma cell lines and a phosphorothioate antisense oligonucleotide (AS ODN) to depress c-myb mRNA. We found that the AS ODN bound to the complex reduces cell growth more efficiently than that of naked AS ODN by preventing the AS ODN from binding to albumin in the culture medium and being hydrolyzed.
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