Gyeong-Won Jeong

Anticancer effect of gene/peptide co-delivery system using transferrin-grafted LMWSC.

A series of ternary complex was designed to deliver psiRNA-bcl2 and (KLA)4 peptide into cancer cells for cancer therapy. The delivered psiRNA-bcl2 induced gene-silencing in a nucleus of cancer cells, while (KLA)4 peptide inhibited cancer growth via mitochondrial apoptosis, indicating that the ternary complexes exerted very strong synergistic effects on cancer growth suppression by acting on psiRNA-bcl2 and (KLA)4 peptide simultaneously. The ternary complexes having a targeting-ligand, transferrin (TfP), were found to be especially effective at binding to the TfP receptor rich cancer cells, HCT119. The plasmid DNA (pDNA) in ternary complexes was completely condensed at various content of LMWSC-PEG-TfP (32-64 times more than pDNA) and released into cells. pDNA in the complexes was protected from DNase present on the exterior of cells. The size (165-248 nm) of ternary complexes with LMWSC-PEG-TfP was increased, but surface charges (3-4.5 mV) were decreased. These results likely occurred because the free amine-group of LMWSC decreased in response to conjugated transferrin. Moreover, transfected ternary complexes with LMWSC-PEG-TfP were not expressed in the normal cells (HEK293), but were over expressed in HCT119 cells. These findings indicate that the ternary complexes can be specifically targeted to HCT119 cancer cells. The useful complexes for gene and peptide delivery had high anticancer activities via a synergistic effect due to co-operative action of psiRNA and (KLA)4 peptide in HCT119 cells.

Simple nanophotosensitizer fabrication using water-soluble chitosan for photodynamic therapy in gastrointestinal cancer cells

The polysaccharide chitosan has abundant cationic amine groups, and can form ion-complexes with anionic molecules such as the strong photosensitizer chlorin e6 (Ce6). In this study, water-soluble chitosan (WSC) was used to fabricate Ce6-incorporated nanophotosensitizers (Abbreviated as ChitoCe6 nanophotosensitizer) via a self-assembling process. This was accomplished by dissolving WSC in pure water and then directly mixing the solution with solid Ce6 causing ion complex formation between WSC and Ce6. The resulting nanophotosensitizer was spherical in shape and had a particle size of less than 300nm. The photodynamic effect of ChitoCe6 nanophotosensitizer was evaluated using gastrointestinal (GI) cancer cells. At in vitro study using SNU478 cholangiocarcinoma cells, ChitoCe6 nanophotosensitizer showed improved Ce6 uptake by tumor cells, reactive oxygen species production, and cellular phototoxicity. An in vivo study using SNU478-bearing nude mice showed that the ChitoCe6 nanophotosensitizer efficiently accumulated in the tumor tissue and inhibited tumor growth more than treatment with Ce6 alone. Furthermore, ChitoCe6 nanophotosensitizer was also efficiently absorbed through tissue layers in an ex vivo study using porcine bile duct explants. ChitoCe6 nanophotosensitizer showed enhanced photosensitivity and photodynamic effects against cancer cells in vitro and in vivo. We present ChitoCe6 nanophotosensitizer as a promising candidate for photodynamic therapy of GI cancer.

Evaluation of disulfide bond-conjugated LMWSC-g-bPEI as non-viral vector for low cytotoxicity and efficient gene delivery

For efficient gene delivery, non-viral vectors should have high cellular uptake, excellent endosomal escape, and the ability to rapidly release the gene into the cytoplasm. Here, we developed a disulfide bond-conjugated bioreducible LMWSC-g-bPEI (LCP) composed of low molecular-weight water soluble chitosan (LMWSC), bPEI, and cystamine (Cys). The developed LCP had advantages such as low toxicity, great endosomal escape, and rapid release of pDNA into the cytoplasm. The polyplexes with LCP showed higher uptake into the nucleus and greater transfection efficiency than that without disulfide bond. Moreover, LCP polymer and polyplexes with LCP indicated lower cytotoxicity than bPEI 25kDa. In addition, a gel retardation assay and particle size were analyzed to demonstrate the reduction-sensitive gene delivery system. Besides, intracellular uptake pathway of polyplexes was investigated by various endocytosis inhibitor and confirmed to internalization into cell via macropinocytosis. These results suggest that bioreducible LCP is a superb non-viral vector for efficient gene delivery.

Preparation and Characterization of Chitosan Microsphere for Encapsulation of Natural Antioxidant with Effective Protection against ROS

Recently, active oxygen has been seriously induced due to severe exposure to life radiation and medical radiation, and many health functional foods using natural antioxidants have been released to suppress such active oxygen. However, the antioxidant activity of natural antioxidants is reduced due to low bioavailability. In this study, we have developed a chitosan-based microsphere carrier capable of increasing bioavailability of natural antioxidants, and analyzed the physicochemical characteristics of the microsphere carriers to evaluate their applicability as a natural antioxidant carrier. In order to solve the problem of reproducibility of chitosan having a broad molecular weight distribution, chitosan having a narrow molecular weight distribution and accurate molecular weight specification was prepared by using an ultrafiltration membrane. Chitosan microspheres using fractionated chitosan were prepared by spray dry method. The physicochemical properties of the fractionated chitosan and microspheres were analyzed by GPC, H NMR, FTIR and SEM. In addition, to evaluate the applicability of chitosan microspheres in vivo, their cytotoxicity was evaluated by MTT assay. These results suggest that chitosan microspheres can be applied in vivo, and natural antioxidant-encapsulated chitosan microsphere can be used to maximize antioxidant activity by increasing bioavailability.

Anticancer Effect of Intracellular-Delivered Doxorubicin Using a Redox-Responsive LMWSC- g -Lipoic Acid Micelles

To induce a quick-drug release, lipoic acid (LA) was introduced to amine group of low molecular weight water-soluble chitosan (LMWSC) by coupling agent. The disulfide bond (-S-S-) of lipoyl group from LMWSC-grafted lipoic acid (LL) can response to glutathione (GSH) at cytoplasm with reducing environment, where LL can rapidly be disassembled via dissociation of disulfide bond (-S-S-) by GSH. The doxorubicin (DOX)-encapsulated LL (LLDOX) was prepared by dialysis method, which allowed quick release of DOX by destabilization of inner-hydrophobic core of LL when disulfide bond (-S-S-) was dissociated by GSH. To demonstrate a redox-responsive release behavior of LLDOX, its drug release was accomplished under PBS buffer (pH 7.4) and GSH (10 mM) condition. In addition, the particle size and morphology of LL and LLDOX were respectively confirmed by DLS and TEM. The particle sizes of LL30% and LL60% were indicated as 268.8±30.9 and 308.1±11.9 nm, respectively. In addition, particle size of LLDOX was decreased more than that of LL. Also, surface charge of LLDOX was displayed to strong positive charge (LLDOX30%: 12.6±0.5 mV, LLDOX60%: 9.6±1.1 mV). Moreover, their morphological structure has a spherical shape. The cytotoxicity LL and LLDOX was confirmed by using MTT assay. Besides, to investigate the intracellular uptake of DOX from LLDOX against HeLa and AGS cell lines, fluorescence image was observed by using fluorescence microscopy. These results suggest that LL is an excellent as a drug carrier due to high anticancer effect by rapid drug release with redox-responsive effect.

Evaluation of hyaluronic acid-combined ternary complexes for serum-resistant and targeted gene delivery system

Branched polyethylenimine (bPEI) was well known as high transfection agent, which has many amine group. However, utilization of bPEI was limited due to high toxicity. To solve these problems, bPEI was introduced to low molecular weight water-soluble chitosan (LMWSC) with coupling agent. In addition, hyaluronic acid (HA), one of natural anion polymer, was introduced to binary complex of pDNA/bPEI-grafted LMWSC (LMPEI) to target the specific cancer cell and impart the serum resistant. Ternary complexes of pDNA/LMPEI/HA were prepared by electrostatic charge interaction and their binding affinity and DNase protection assay were conducted by gel retardation assay. Particle size of ternary complexes showed that had each 482 ± 245.4 (pDNA/LMPEI2%/HA, 1:16:1, w/w/w) and 410 ± 78.5 nm (pDNA/LMPEI4%/HA, 1:16:2, w/w/w). Moreover, to demonstrate serum-resistant effect of ternary complexes, particle size of them was measured according to incubated time (0-10 h) under serum condition. Transfection assay of ternary complexes showed that their transfection efficiency in CD44-receptor overexpressed HCT116 cell was higher than CD44-receptor negative CT26 cell. Additionally, intracellular uptake of ternary complexes with propidium iodide (PI)-labeled pDNA was observed to confirm targeting effect and cellular internalization by fluorescence microscopy. These results suggest that ternary complexes are superb gene carrier with excellent serum-resistant and high gene transfection.

Flocculation and Algicidal Effect of Harmful Green-Tide according to Molecular-weight of Chitosan

The serious occurrence of green-tides is attributed to global warming and eutrophication. In South Korea, the red clay and chemical-coagulant agent are used to remove these green-tide but these methods have an problem with low removal efficiency and secondary pollution by chemical substance. In this study, to solve these problems, we confirmed flocculation and algicidal efficacy of green-tide using various molecular-weight chitosan which is natural polymer and has a strong positive charge. We investigated the optimal concentration and flocculation efficacy of chitosan against green-tide. The green-tide was harvested from Yongsan river (Jeonnam) and number of green-tide was adjusted to 2×10 cells/mL. The optimal concentration of chitosan was 5 mg/mL. The surface charge and morphological property were analyzed to confirm mechanism of flocculation and algicidal effect between chitosan and green-tide. These result confirmed that green-tide of negative charge was changed to positive charge when chitosan put into green-tide. In addition, morphological observation showed that growth of green-tide was inhibited by treated chitosan which was covered to surface of green-tide. The evaluation of ecosystem stability of chitosan by surval rate of limnobios with green-tide indicated that survival rate of limnobios increased by chitosan treatment. These results suggest that chitosan is a beneficial algicide with no adverse effects on ecosystem health.온난화 현상과 하천의 부영화에 의해 녹조가 심각하게 발생하고 있다. 국내에서는 이러한 녹조를 제거하기 위해 황토 및 화학응집제를 주로 사용하고 있지만, 화학물질로 인한 2차 오염 및 낮은 제거 효율 등의 문제점을 갖고 있다. 본 연구에서는 이러한 문제점들을 해결하기 위해 천연 물질이며 강한 양전하를 가지고 있는 키토산을 이용하여 키토산의 분자량에 따른 녹조의 응집 및 살조 효율을 확인하였다. 녹조 응집효율은 영산강(전남) 지류에서 유해녹조를 채취한 후 개체수를 2×106 cells/mL로 조절하여 키토산의 최적 응집 농도 및 녹조 응집 효율을 규명하였고, 최적 농도는 5 mg/mL임을 확인하였다. 또한 녹조의 응집 및 살조 메카니즘 분석은 표면전하 및 형태학적 특성을 이용하여 분석하였다. 이 결과 키토산이 처리되었을 때 녹조의 표면전하가 음전하에서 양전하로 바뀌는 것을 확인하였다. 아울러 형태학적 관찰 결과 키토산이 녹조 생물의 표면을 감싸면서 녹조생물의 성장을 억제하는 것을 확인하였다. 키토산의 생태계 안정성 평가는 녹조 환경에서 담수 생물의 생존율로 규명하였다. 이 결과 키토산이 처리된 실험군에서 담수 생물의 생존율이 증가하는 것을 확인하였다. 이러한 결과를 통해 항녹조 물질인 키토산이 생태계에 영향을 주지 않고, 유해녹조만을 살조할 수 있는 효과적인 물질임을 확인하였다.

The Bio-diesel Conversion by Transesterification of Extracted Lipid from Harmful Green-Tide

At present, the energy consumption of Korea is increasing due to the increase of population density. Such an increase in energy consumption leads to a rise in oil prices and depletion of fossil fuels, and increases the concentration of carbon dioxide in the atmosphere, thereby causing serious environmental pollution. It is necessary to develop new and renewable energy to overcome this. The purpose of this study is to establish the optimum conditions of lipid-extraction using harmful green algae in the four rivers to substitute a petroleum and fossil fuels, and to investigate the possibility of using them as biodiesel. The antigreen-tide substances developed in the previously reported research papers were treated on the harmful green algae, and the flocculated and floating harmful green algae were collected. After drying, the optimal extraction conditions were confirmed according to the presence of various solvents and the ultrasonic treatment. As a result, in the chloroform/methanol/water mixed solvent, the most lipid was extracted and it was confirmed that it was most suitable for utilization as biodiesel when converted to fatty acid methyl ester (FAME) using extracted lipid under this condition. These results suggest that if the harmful green algae of the four major rivers are recycled, they can be utilized as next generation energy.