• International Journal of Oral Biology
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• v.38 no.4
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• pp.155-160
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• 2013

The major issue in the development of nucleic acid based therapeutics is the inefficient delivery of these agents into cells. We prepared cholesterol conjugated spermine and evaluated its usefulness as a delivery modality for antisense oligonucleotides in HeLa-Luc cells. A 2'-O-methyl antisense oligonucleotide sequence, designed to correct splicing at an aberrant intron inserted into a normal luciferase reporter gene, was used for complex formation with cholesterol conjugated spermine. Effective delivery of this antisense agent into nucleus would results in the expression of a luciferasereporter gene product. The cholesterol-spermine formed stable complexes with the antisense oligonucleotide and showed modest delivery activity. Furthermore, this delivery activity was maintained even in the presence of serum proteins, mimicking in vivo conditions. Cholesterol-spermine thus has potential as a delivery system for antisense oligonucleotides into cells.

• Journal of Microbiology and Biotechnology
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• v.22 no.6
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• pp.866-871
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• 2012

A novel cholesterol-based cationic lipid containing a tri-2-hydroxyethylamine head group and ether linker (Chol-THEA) was synthesized and examined as a potent gene delivery vehicle. In the preparation of cationic liposome, the addition of DOPE as helper lipid significantly increased the transfection efficiency. To find the optimum transfection efficiency, we screened various weight ratios of DOPE and liposome/DNA (N/P). The best transfection efficiency was found at the Chol-THEA:DOPE weight ratio of 1:1 and N/P weight ratio of 10~15. Most of the plasmid DNA was retarded by this liposome at the optimum N/P weight ratio of 10. The transfection efficiency of Chol-THEA liposome was compared with DOTAP, Lipofectamine, and DMRIE-C using the luciferase assay and GFP expression. Chol-THEA liposome with low toxicity had better or similar potency of gene delivery compared with commercial liposomes in COS-7, Huh-7, and MCF-7 cells. Therefore, Chol-THEA could be a useful non-viral vector for gene delivery.

• Biomedical Science Letters
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• v.15 no.4
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• pp.265-272
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• 2009

Recently, mesenchymal stem cells (MSCs) began to be utilized as a vehicle for ex vivo gene therapy based on their plasticity. Effective and safe transfection of therapeutic genes is a critical step for genetic modification of MSCs. Therefore, optimization of in vitro gene delivery into MSCs is essential to provide genetically modified stem cells. In this study, various cationic liposomes, O,O'-dimyristyl-N-lysyl aspartate (DMKD), DMKD/cholesterol, O,O'-dimyristyl-N-lysyl glutamate (DMKE), DMKE/cholesterol, and N-[1-(2,3-dioleoyloxy)]-N,N,N-trimethylammonium propane methyl sulfate (DOTAP)/cholesterol, were mixed with plasmid DNA encoding luciferase (pAAV-CMV-Luc) at varied ratios, and then used for transfection to MSCs under varied conditions. The MSCs were also transfected by electroporation under varied conditions, such as voltage, pulse length, and pulse interval. According to the experimental results, electroporation-mediated transfection was more efficient than cationic liposome-mediated transfection. The best MSC transfection was induced by electroporation 3 times pulses for 2 ms at 200 V with 10 seconds of a pulse interval.

• Bulletin of the Korean Chemical Society
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• v.26 no.8
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• pp.1209-1213
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• 2005

Nuclear membrane is one of the main barriers in intracellular delivery of genetic materials. The previous report showed that glucocorticoid receptor dilated the nuclear pore to 60 nm in the presence of a ligand. It was also suggested that the transport of genetic material to nucleus might be facilitated by glucocorticoid. In this study, the effect of glucocorticoid preincubation in the polymeric gene delivery was investigated. The cells were preincubated with dexamethasone, a potent glucocorticoid, and transfection assays were performed with polyethylenimine (PEI) and polyamidoamine (PAMAM) dendrimer. As a result, the transfection efficiency of PEI or PAMAM to the cells in the presence of dexamethasone was enhanced, compared to the cells without dexamethasone. This effect was not observed in the cells preincubated with cholesterol. The polymer/DNA complex was stable in the presence of dexamethasone. In addition, the cytotoxicities of the polymeric carriers to the cells were observed in the presence of dexamethasone. In conclusion, dexamethasone enhances the transfection efficiency of polymeric carriers and may be useful in the development of polymeric gene carriers.

• Journal of Pharmaceutical Investigation
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• v.28 no.2
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• pp.93-98
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• 1998

We have developed liposomes which can be easily prepared with inexpensive lipid, have enhanced stability, and can efficiently deliver DNA into the COS-l cells, Liposome formulations were prepared using cationic materials such as dimethyldioctadecyl ammonium bromide (DDAB), cetyltrimethyl ammonium bromide(CTAB), We investigated the effect of cationic liposome formulations on in vitro DNA transfection, DDAB-containing liposomes showed increased transfection efficiency which was 3.2-fold as much as that by $Lipofectin^{\circledR}$, but CTAB-containing liposomes were inactive in gene transfection. The effect of colipid of DDAB-containing liposome was also investegated. As a colipid, dioleylphosphatidylethanolamine(DOPE) and cholesterol did altered the transfection efficiency of DDAB-containing liposomes. And increased DDAB concentration lowered the transfection efficiency. The optimum amount of liposomal formulation was $10\;{\mu}M$ for $1\;{\mu}g$ of DNA. In the experiment of stability, DOPE-containing liposomes formulation showed a broad size distribution and separation of two major peaks on a 5th day of preparation, but liposomes containing cholesterol was stable for 10 days. DDAB-containing liposomal DNA delivery system was prepared easily and was stable.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.427.1-427.1
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• 2002

To improve stability and transfection efficiency, a novel combination of cationic emulsion and galactosylated chitosan was developed for targeted gene delivery. Six formulations of cationic liposome and our novel emulsion were prepared for comparison of stability and transfection efficiency. Cationic liposomes composed of 3［N-(N.N dimethylaminoethylene) carbamoyl］ cholesterol (DC-Chol) and dioleyl phophatidyl ethanolamine (DOPE) were prepared by extrusion method and cationic emulsions composed of DC-Chol. DOPE. castor oil, and Tween 80 were prepared by sonication method. (omitted)

• Journal of Microbiology and Biotechnology
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• v.21 no.1
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• pp.93-99
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• 2011

Amongst a number of potential nonviral vectors, cationic liposomes have been actively researched, with both gemini surfactants and bola amphiphiles reported as being in possession of good structures in terms of cell viability and in vitro transfection. In this study, a cholesterol-based diquaternary ammonium gemini surfactant (Chol-GS) was synthesized and assessed as a novel nonviral gene vector. Chol-GS was synthesized from cholesterol by way of four reaction steps. The optimal efficiency was found to be at a weight ratio of 1:4 of lipid:DOPE (1,2-dioleoyl-L-${\alpha}$- glycero-3-phosphatidylethanolamine), and at a ratio of between 10:1~15:1 of liposome:DNA. The transfection efficiency was compared with commercial liposomes and with Lipofectamine, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE-C), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP). The results indicate that the efficiency of Chol-GS is greater than that of all the tested commercial liposomes in COS7 and Huh7 cells, and higher than DOTAP and Lipofectamine in A549 cells. Confirmation of these findings was observed through the use of green fluorescent protein expression. Chol-GS exhibited a moderate level of cytotoxicity, at optimum concentrations for efficient transfection, indicating cell viability. Hence, the newly synthesized Chol-GS liposome has the potential of being an excellent nonviral vector for gene delivery.

• BMB Reports
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• v.33 no.6
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• pp.476-482
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• 2000

In this paper, we report a new cationic lipid composed of L-lysinamide and cholesterol as a potent gene delivery vector. $3{\beta}$[L-Lysinamide-carbamoyl] cholesterol could self-assemble with plasmid DNA forming discrete lipoplexes. From atomic force microscopic images of the complexes, the size distribution was observed to range from 100 to 150 nm in diameter. The transfection efficiency of this amphiphile on different cell lines was evaluated as a micellar solution in the absence of the fusogenic helper lipid, dioleoyl phosphatidyletbanolamine (DOPE). Transfection experiments were performed as a function of charge ratio (lipid/DNA) and transfection time. Cytotoxicity and in vitro transfection efficiency of the amphiphile was demonstrated and compared with those of commercially available Lipofectin and polyethylenimine (PEI).

• Macromolecular Research
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• v.12 no.6
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• pp.573-580
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• 2004

To prepare chitosan-based polymeric amphiphiles that can form nanosized core-shell structures (nanopar­ticles) in aqueous milieu, chitosan oligosaccharides (COSs) were modified chemically with hydrophobic cholesterol groups. The physicochemical properties of the hydrophobized COSs (COSCs) were investigated by using dynamic light scattering and fluorescence spectroscopy. The feasibility of applying the COSCs to biomedical applications was investigated by introducing them into a gene delivery system. The COSCs formed nanosized self-aggregates in aqueous environments. Furthermore, the physicochemical properties of the COSC nanoparticles were closely related to the molecular weights of the COSs and the number of hydrophobic groups per COS chain. The critical aggregation concentration values decreased upon increasing the hydrophobicity of the COSCs. The COSCs effi­ciently condensed plasmid DNA into nanosized ion-complexes, in contrast to the effect of the unmodified COSs. An investigation of gene condensation, performed using a gel retardation assay, revealed that $COS6(M_n=6,040 Da)$ containing $5\%$ of cholesteryl chloroformate (COS6C5) formed a stable DNA complex at a COS6C5/DNA weight ratio of 2. In contrast, COS6, the unmodified COS, failed to form a stable COS/DNA complex even at an elevated weight ratio of 8. Furthermore, the COS6C5/DNA complex enhanced the in vitro transfection efficiency on Human embryonic kidney 293 cells by over 100 and 3 times those of COS6 and poly(L-lysine), respectively. Therefore, hydrophobized chitosan oligosaccharide can be considered as an efficient gene carrier for gene delivery systems.

• Bulletin of the Korean Chemical Society
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• v.33 no.2
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• pp.651-656
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• 2012

Cationic immunoliposomes were prepared by conjugation of Fab' fragments of the recombinant humanized monoclonal antibody (HuCC49) against tumor-associated glycoprotein (TAG)-72 to sterically unilamella liposomes. The cationic immunoliposomes are composed of cationic lipid (O,O'-dimyristyl-N-lysyl aspartate, DMKD), cholesterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)$_{2000}$] (DPPE-PEG-maleimide) with a molar ratio of 0.5:0.47:0.03. Plasmid DNA was effectively condensed by addition of transferrin (Tf) during the formation of anti-TAG-72 PEG-immunolipoplexes (PILPs). These anti-TAG-72 PILPs were able to adhere to the surface of TAG-72-overexepressing LS174T human colon cancer cells more effectively than conventional liposomes, thereby facilitating gene delivery in vitro. Furthermore, intravenous administration of the anti-TAG-72 PILPs into the tumor-carrying mice exhibited efficient localization of the reporter gene in the tumor tissues.