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Polyethyleneimine‐mediated gene delivery into rat pheochromocytoma PC‐12 cells
Lee, Jung Hwa,Ahn, Hyun Hee,Kim, Kyung Sook,Lee, Ju Young,Kim, Moon Suk,Lee, Bong,Khang, Gilson,Lee, Hai Bang Wiley (John WileySons) 2008 Journal of tissue engineering and regenerative med Vol.2 No.5
<P>In this study, we examined the use of polyethyleneimine (PEI) as a non-viral gene carrier and lipofectamine(trade mark) 2000 as control for rat pheochromocytoma PC-12 cells. The complex formation of PEI and DNA or lipofectamine and DNA was characterized by gel electrophoresis and measurement of particle size and surface charge. A gradual increase in surface charge (from 0.7 to 43 mV) and a gradual decrease in particle size (from 900 to 130 nm) was observed in the PEI-DNA complex with higher PEI concentrations. The cytotoxicity of PC-12 cells for lipofectamine-DNA complex was similar to PEI-DNA complex at N:P charge ratios of 4 and 8. Transfection efficiency was 14% for lipofectamine and 15% for PEI. At low N:P ratio, DNA condenses poorly, so the particle size tends to be large and polydispersed, resulting in poor transfection efficiency. Meanwhile, a high N:P ratio results in high transfection efficiency and cytotoxicity. Transfected PC-12 cells showed the generation of neurites from transfected PC-12 cells in the presence of NGF, indicating the differentiation of PC-12 cells. NGF-differentiated PC-12 cells were transfected by PEI-DNA complex of N:P charge ratio 8. From real-time imaging for transfection, the enhanced green fluorescent protein (EGFP) started to localize in the nuclei of PC-12 cells at 5 h and localized in the cytoplasm from 15 h. Our study demonstrates that PEI or lipofectamine may be applied as an effective gene carrier for PC-12 cells.</P>
Lee, Sang-Jin,Lee, Young-Moo,Khang, Gilson,Kim, Un-Young,Lee, Bong,Lee, Hai-Bang The Polymer Society of Korea 2002 Macromolecular Research Vol.10 No.3
Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a microbial storage polymer with biodegradable properties. In order to improve the cell compatibility of PHBV surfaces, the physicochemical treatments have been demonstrated. In this study, physical method was corona discharge treatment and chemical method was chloric acid mixture solution treatment. The physicochemically treated PHBV film surfaces were characterized by the measurement of water contact angle, electron spectroscopy for chemical analysis, and scanning electron microscopy (SEM). The water contact angle of the physicochemically treated PHBV surfaces decreased from 75 to 30~40 degree, increased hydrophilicity. due to the introduction of oxygen-based functional group onto the PHBV backbone chain. The mouse NIH/3T3 fibroblasts cultured onto the physicochemically treated PHBV film surfaces with different wettability. The effect of the PHBV surface with different wettability was determined by SEM as counts of cell number and [$^3$H]thymidine incorporation as measures of cell proliferation. As the surface wettability increased, the number of the cell adhered and proliferated on the surface was increased. The result seems closely related with the serum protein adsorption on the physicochemically treated PHBV surface. In conclusion, this study demonstrated that the surface wettabilily of biodegradable polymer as the PHBV plays an important role for cell adhesion and proliferation behavior for biomedical application.
Khang, Gilson,Rhee, John M.,Shin, Philkyung,Kim, In Young,Lee, Bong,Lee, Sang Jin,Lee, Young Moo,Lee, Hai Bang,Lee, Ilwoo The Polymer Society of Korea 2002 Macromolecular Research Vol.10 No.3
In order to endow with new bioactive functionality from small intestine submucosa (SIS) powder as natural source to poly (L-lactide) (PLA) and poly (lactide-co-glycolide) (PLGA) synthetic biodegradable polymer, porous SIS/PLA and SIS/PLGA as natural/synthetic composite scaffolds were prepared by means of the solvent casting/salt leaching methods for the possibility of the application of tissue engineered bone and cartilage. A uniform distribution of good interconnected pores from the surface to core region was observed the pore size of 40~500 ${\mu}{\textrm}{m}$ independent with SIS amount using the solvent casting/salt leaching method. Porosities, specific pore areas as well as pore size distribution also were almost same. After the fabrication of SIS/PLA hybrid scaffolds, the wetting properties was greatly enhanced resulting in more uniform cell seeding and distribution. Five groups as PGA non-woven mesh without glutaraldehyde (GA) treatment, PLA scaffold without or with GA treatment, and SIS/PLA (Code No.3 ; 1 : 12 of salt content, (0.4 : 1 of SIS content, and 144 ${\mu}{\textrm}{m}$ of median pore size) without or with GA treatment were implanted into the back of nude mouse to observe the effect of SIS on the induction of cells proliferation by hematoxylin and eosin, and von Kossa staining for 8 weeks. It was observed that the effect of SIS/PLA scaffolds with GA treatment on bone induction are stronger than PLA scaffolds, that is to say, in the order of PLA/SIS scaffolds with GA treatment > PLA/SIS scaffolds without GA treatment > PGA nonwoven > PLA scaffolds only with GA treatment = PLA scaffolds only without GA treatment for the osteoinduction activity. The possible explanations are (1) many kinds of secreted, circulating, and extracellular matrix-bound growth factors from SIS to significantly affect critical processes of tissue development and differentiation, (2) the exposure of SIS to GA resulted in significantly calcification, and (3) peri-implant fibrosis due to covalent bonding between collagen molecule by crosslinking reaction. In conclusion, it seems that SIS plays an important role for bone induction in SIS/PLA scaffolds for the application of tissue engineering area.
섬유아세포에서의 DNA/폴리에틸렌이민 나노복합체를 이용한 유전자 전달
이정화 ( Jung Hwa Lee ),안현희 ( Hyun Hee Ahn ),신유나 ( Yu Na Shin ),김문석 ( Moon Suk Kim ),이봉 ( Bong Lee ),강길선 ( Gil Son Khang ),이일우 ( Il Woo Lee ),이해방 ( Hai Bang Lee ) 한국조직공학·재생의학회 2007 조직공학과 재생의학 Vol.4 No.4
The aim of this study is to evaluate transfection efficiency in NIH/3T3cell using DNA/polyethyleneimine (PEI) complexes. The formation of complexes from DNA and PEI was performed by the adding of the PEI solution to the DNA solution. Gel retardation assay showed the optimal N/P charge ratio where PEI completely binds DNA. The particle size of DNA/PEI complexes measured by dynamic light scattering showed diameters of up to 500 nm. The viability of NIH/3T3 fibroblast cells in the presence of DNA/PEI complexes was evaluated by the MTT assay. After gene transfection, enhanced green fluorescence protein was monitored by flow cytometry. From AFM measurement, DNA/PEI complexes showed round-shape. These results indicated that DNA was sufficiently condensed by PEI to give DNA/PEI complexes. As the relative amount of PEI was increased, its cytotoxicity to NIH/3T3cells increased. By increasing culture time, fluorescence microscopy showed a more green fluorescence cells. Naked DNA showed no EGFP expression, whereas delivery of DNA/PEI complexes to cells showed EGFP expression and resulted in 2-15% transfection. Transfection efficiencies of DNA/PEI complexes increased with increasing N/P charge ratio.
이주영 ( Ju Young Lee ),김경숙 ( Kyung Sook Kim ),이정화 ( Jung Hwa Lee ),황성주 ( Sung Joo Hwang ),이봉 ( Bong Lee ),강길선 ( Gilson Khang ),이해방 ( Hai Bang Lee ),김문석 ( Moon Suk Kim ) 한국조직공학과 재생의학회 2008 조직공학과 재생의학 Vol.5 No.4
To date, considerable attention has been devoted to development of the protein-drug delivery system although protein-drug is limited to extensive utilizations due to the short half-life and denaturalization in physiology condition. Thus, a number of drug delivery systems are developed in order to endow stabilityof protein drugs and to improve their bioefficacy. In this review, we describe the recent trend of protein drug delivery such as protein drugpolymer conjugation, nano-scale carriers, hydrogels, liposome-micelles and microcapsules, important in the understanding of the basic science for protein drug delivery.
Lee, Ha Young,Lim, Nak Hyun,Seo, Jin A.,Yuk, Soon Hong,Kwak, Byung Kook,Khang, Gilson,Lee, Hai Bang,Cho, Sun Hang Wiley Subscription Services, Inc., A Wiley Company 2006 Journal of Biomedical Materials Research Part B Vol. No.
<P>Polyvinylpyrrolidone (PVP)-coated iron oxide nanoparticles were prepared by the thermal decomposition of Fe(CO)<SUB>5</SUB> (iron pentacarbonyl) in one step. X-ray diffraction (XRD), transmission electron microscopy (TEM), electrophoretic light scattering (ELS), infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) together with the variation of the molar ratio of PVP/Fe(CO)<SUB>5</SUB>, solvent, and molecular weight of PVP, were used to characterize the PVP-coated iron oxide nanoparticles. Fifty to hundred nanometer-sized iron oxide nanoclusters with a spherical shape were formed in dimethylformamide (DMF), used as a solvent, and exhibited an enhanced stability in the aqueous media. Their magnetic properties were investigated by superconducting quantum interface device (SQUID). The in vitro cytotoxicity test revealed that the PVP-coated iron oxide nanoparticles exhibited excellent biocompatibility by MTT assay. Magnetic resonance imaging (MRI) effect was observed with the administration of PVP-coated iron oxide nanoparticles through the marginal vein of rabbit, resulting in improved detection of the liver lesions. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006</P>