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ABSTRACT: Mutations in the p53 tumor suppressor gene are the most common molecular genetic abnormalities to be described in lung cancer. However, there have been few reports of nonviral vector-mediated p53 gene delivery in lung cancer. A new formulation of cationic solid lipid nanoparticles (SLNs) for gene delivery was produced by the melt homogenization method with slight modification, and the SLNs were formulated by mixing tricaprin (TC) as a core, 3beta[N-(N', N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), dioleoylphosphatidylethanolamine (DOPE) and Tween 80 in various ratios. Plasmid DNA (pp53-EGFP)/SLNs complexes were transfected into human non-small cell lung cancer cells (H1299 cells) and transfection efficiency was determined by FACS analysis. The gene expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. The cellular growth inhibition and apoptosis of treated cells with pp53-EGFP/SLNs complexes were assessed by trypan blue exclusion assay and annexin V staining, respectively. In vivo biodistribution of plasmid DNA was investigated by PCR and RT-PCR. The transfection efficiency of SLN1 (TC:DC-Chol:DOPE:Tween 80=0.3:0.3:0.3:1), which showed the highest transfection efficiency among the SLN formulations, was higher than that of commercially available Lipofectin. The SLNs-mediated transfection of the p53 gene resulted in efficient high levels of wild-type p53 mRNA and protein expression levels in H1299 cells. The efficient reestablishment of wild-type p53 function in lung cancer cells restored the apoptotic pathway. Taken together, our results reveal that cationic SLN-mediated p53 gene delivery may have potential for clinical application as a nonviral vector-mediated lung cancer therapy due to its effective induction of apoptosis and tumor growth inhibition.
European Journal of Pharmaceutics and Biopharmaceutics 04/2008; 68(3):545-54. · 4.27 Impact Factor
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ABSTRACT: Previously we have formulated a new cationic emulsion, composed of 3beta [N-(N',N'-dimethylaminoethane) carbamoyl] cholesterol and dioleoylphosphatidyl ethanolamine, castor oil and Tween 80, and it efficiently delivered plasmid DNA into various cancer cells with low toxicity. Chitosan is a natural cationic polysaccharide and is able to form polyelectrolyte complexes with DNA, in which the DNA is condensed and protected against nuclease degradation. Based on these facts, chitosan was used as a condensing agent to enhance the transfection efficiency of cationic emulsion-mediated gene delivery vehicle. The particle size, zeta potential and transmission electron micrographs of DNA/emulsion complexes were observed before and after condensation by chitosan. In vitro transfection efficiency of naked or precondensed DNA/emulsion (pcDNA/E) complexes was investigated in human hepatoma cells (HepG2) using flow cytometer, confocal microscope and western blot. In addition, in vivo gene transfer was also evaluated as GFP mRNA expression by reverse transcriptase-polymerase chain reaction. The size of transfection complexes was reduced after the condensation of DNA by chitosan. Moreover, when the pcDNA/E complexes were administered into the mice, the GFP mRNA expression was prolonged in liver and lung until day 6. These results suggest that the use of chitosan enhance the in vitro transfection efficiency and extend in vivo gene transfer.
Biomaterials 06/2005; 26(14):2147-56. · 7.40 Impact Factor
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ABSTRACT: Cationic liposome has been studied as one of the most promising non-viral gene delivery systems. However, it has major drawbacks such as the formation of large aggregates at higher concentrations and the instability in the serum due to cationic lipid. As an alternative gene delivery system, cationic emulsion was formulated and transfection efficiency was evaluated in vitro and in vivo, in comparison with cationic liposome. Cationic emulsion was prepared with varying compositions of 3 beta [N-(N',N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), dioleoylphosphatidyl ethanolamine (DOPE), caster oil and Tween 80. Cationic liposome was prepared with DC-Chol and DOPE. The particle size of all the DNA/lipid complexes varied from 150 to 230 nm. The in vitro transfection efficiency of plasmid DNA was assessed by the expression of green fluorescent protein as a reporter. Of various formulations, cationic emulsion E2 (DC-Chol/DOPE/Castor Oil/Tween 80 = 0.3:0.3:0.3:0.15) and cationic liposome L3 (DC-Chol/DOPE = 0.6:0.3) showed improved transfection. DNA/E2 complexes exhibited higher transfection efficiencies (17.39+/-0.58%) in comparison with DNA/L3 complexes (11.47+/-0.59%). DNA/E2 complexes also showed a better physical stability and a stronger serum resistance than DNA/L3 complexes. Moreover, the cytotoxicity of DNA/E2 complexes was comparable to that of DNA/L3 complexes. When DNA/lipid complexes were intravenously administered, DNA/E2 complexes showed a prolonged circulation in blood and mRNA expression in various tissues compared with DNA/L3 complexes. These results suggest that cationic emulsion E2 could be a potential gene delivery system in clinical approaches because of enhanced in vivo gene transfer with low toxicity.
Biomaterials 01/2005; 25(27):5893-903. · 7.40 Impact Factor
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ABSTRACT: Cationic lipids have been often used as one of the major components in making most promising non-viral gene delivery systems, whereas sodium cholate, a surfactant so-called edge activator has been used in preparing ultradeformable and ultraflexible liposomes called Transfersomes. Using both a cationic lipid, DOTAP and sodium cholate, a novel formulation of ultradeformable cationic liposome (UCL) has been prepared. The average particle size of this formulation was approximately 80 nm. The physical and chemical stabilities at two different temperatures (4 degrees C and 20 degrees C) were also evaluated for 60 days. The ultradeformability of new formulation was also assessed, and it has been proved that the formulation is deformable. In vitro transfection efficiency of plasmid DNA/UCL was assessed by the expression of green fluorescent protein (GFP) in four cell lines, OVCAR-3 (human ovarian carcinoma cells), HepG2 (human hepatoma cells), H-1299 (human lung carcinoma cells) and T98G (human brain carcinoma cells). The optimal ratio of DNA to liposome for maximal transfection efficiency was 1:14 (w/w) in all the cell lines except for the human brain carcinoma cells. The same formulation was tested for in vivo transfection efficiency and its retention time within the organs by applying the DNA/UCL complexes on hair-removed dorsal skin of mice non-invasively. It was found that genes were transported into several organs for 6 days once applied on intact skin.
Biomaterials 02/2004; 25(2):305-13. · 7.40 Impact Factor
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ABSTRACT: Previously we have formulated a new cationic liposome, DDC, composed of dioleoyltrimethylamino propane (DOTAP), 1,2-dioeoyl-3-phosphophatidylethanolamine (DOPE), and cholesterol (Chol), and it efficiently delivered plasmid DNA into ovarian cancer cells. Mutations in the p53 tumor suppressor gene are the most common molecular genetic abnormalities to be described in ovarian cancer. However, there has been so far no report of nonviral vector-mediated p53 gene deliveries in ovarian cancer. In this study, wild-type p53 DNA was transfected into the ovarian cancer cells, using the DDC as a nonviral vector and the expression and activity of p53 gene were evaluated both in vitro and in vivo.
DDC liposomes were prepared by mixing DOTAP:DOPE:Chol in a 1:0.7:0.3 molar ratio using the extrusion method. Plasmid DNA (pp53-EGFP) and DDC complexes were transfected into ovarian carcinoma cells (OVCAR-3 cells) and gene expression was determined by reverse transcription-polymerase chain reaction and Western blot analysis. The cellular growth inhibition and apoptosis of DDC-mediated p53 transfection were assessed by trypan blue exclusion assay and annexin-V staining, respectively. The OVCAR-3 cells treated with DDC/pp53-EGFP complexes were inoculated into female balb/c nude mice and tumor growth was observed.
The transfection of liposome-complexed p53 gene resulted in a high level of wild-type p53 mRNA and protein expressions in OVCAR-3 cells. In vitro cell growth assay showed growth inhibition of cancer cells transfected with DDC/pp53-EGFP complexes compared with the control cells. The reestablishment of wild-type p53 function in ovarian cancer cells restored the apoptotic pathway. Following the inoculation of DDC/pp53-EGFP complexes, the volumes of tumors in nude mice were significantly reduced more than 60% compared to the control group.
The DDC-mediated p53 DNA delivery may have the potential for clinical application as nonviral vector-mediated ovarian cancer therapy due to its effective induction of apoptosis and tumor growth inhibition.
Gynecologic Oncology 09/2003; 90(2):265-72. · 3.89 Impact Factor
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ABSTRACT: In this study, we modified cationic liposomes either by polyethylene glycol (PEG)-grafting or PEG-adding methods, and compared the physical properties of transfection complexes and transfection efficiency in-vitro and prolonged circulation in-vivo. The PEG-grafted transfection complexes were prepared by mixing plasmid DNA with PEG-grafted cationic liposomes, which were composed of DSPE-PEG 2000 and cationic lipids. The PEG-added transfection complexes were prepared by adding DSPE-PEG 2000 to the mixture of cationic liposomes and plasmid DNA. The particle sizes of the PEG-modified transfection complexes (approximately 200 nm) changed a little over 4 weeks compared with the conventional transfection complexes. In the presence of serum, the transfection efficiency of the conventional transfection complexes was lowered whereas the transfection efficiency of the PEG-modified transfection complexes was maintained. Moreover, the transfection efficiency of the conventional transfection complexes was significantly reduced when they were stored. However, the transfection efficiency was stable for the PEG-modified transfection complexes, even after two weeks of storage. Of the in-vitro transfection efficiencies, there was no difference between PEG-grafted and PEG-added transfection complexes. When the conventional, PEG-grafted, and PEG-added transfection complexes were administered into mice by the tail vein, the PEG-added transfection complexes showed a prolonged circulation of plasmid DNA compared with other transfection complexes. These results suggest that the PEG-added transfection complexes could be a useful non-viral vector because of their simplicity in preparation, enhanced stability and prolonged circulation compared with the conventional transfection complexes.
Journal of Pharmacy and Pharmacology 05/2003; 55(4):453-60. · 2.17 Impact Factor
