Biomedical subjects

Sung Wan Kim

Publications and source records attributed to Sung Wan Kim.

At least 19 recordsLinked to original sources

PubMed2007

Reducible poly(amido ethylenimine) directed to enhance RNA interference.

Designing synthetic macromolecular vehicles with high transfection efficiency and low cytotoxicity has been a major interest in the development of non-viral gene carriers. A reducible poly(amido ethylenimine) (SS-PAEI) synthesized by addition copolymerization of triethylenetetramine and cystamine bis-acrylamide (poly(TETA/CBA)) was used as a carrier for small interference RNA (siRNA). Poly(TETA/CBA) could efficiently condense siRNA to form stable complexes under physiological conditions and perform complete release of siRNA in a reductive environment. When formulated with VEGF-directed siRNA, poly(TETA/CBA) demonstrated significantly higher suppression of VEGF than linear-polyethylenimine (PEI) (L-PEI, 25kDa) in human prostate cancer cells (PC-3). After 5h of transfection, substantial dissociation and intracellular distribution of siRNA was observed in the poly(TETA/CBA) formulation, but not in the L-PEI formulation. The triggered release of siRNA by reductive degradation of poly(TETA/CBA) in the cytoplasm may affect the RNAi activity by increasing cytoplasmic availability of siRNA. These results suggest that the rational design of non-viral carriers should involve considerations for intracellular dissociation and trafficking of a nucleic acid drug to maximize its effect, in conjunction with formation of stable complexes under physiological conditions.

Aziridines↗

PubMed2006

A hypoxia-inducible gene expression system using erythropoietin 3' untranslated region for the gene therapy of rat spinal cord injury.

Many neurologic disorders are accompanied by ischemic injury during the pathologic process. To develop a controllable and injury-specific gene therapy system for the neurologic disorders, we constructed a hypoxia inducible plasmid with the erythropoietin (Epo) 3' untranslated region (UTR), which can enhance the stability of target mRNAs in response to hypoxia. The Epo 3' UTR was inserted at the 3' flanking region of luciferase gene in pSV-Luc, resulting in the construction of pSV-Luc-EpoUTR. In pEpo-SV-Luc-EpoUTR, the Epo enhancer was inserted into the upstream of the SV40 promoter to increase the hypoxia inducibility. The plasmids were evaluated in N2a mouse neuroblastoma cells under hypoxic conditions and in a rat spinal cord injury (SCI) model. The results showed that the Epo 3' UTR alone showed a three-fold increase in luciferase activity in hypoxic N2a cells as well as in the rat SCI model when compared to the sham control. In contrast, the Epo 3' UTR showed no effect on the luciferase activity in the presence of the Epo enhancer, probably because the Epo enhancer was more sensitive to hypoxia and showed a dominant effect. However, the Epo enhancer itself showed high level of luciferase activity even in normoxia (about five to eight-folds increase), while the Epo 3' UTR did not show enhanced background activity. Immunohistochemical staining showed expression of luciferase from pSV-Luc-EpoUTR both in neurons and astrocytes around the injured spinal cord of rat. These results suggest that the Epo 3' UTR could provide a specific and safe system for the hypoxia-inducible gene therapy of the neurologic disorders including SCI.

3' Untranslated Regions↗

PubMed2006

Reverse thermal gelation of aliphatically modified biodegradable triblock copolymers.

A simple aliphatic modification demonstrated how to turn a water-soluble biodegradable triblock copolymer synthesized from PEG, L-lactide, and epsilon-caprolactone into a thermoreversible polymer of which aqueous solution underwent a sol-to-gel phase transition upon a mild temperature rise. Thermogelling behavior of the aliphatically modified polymer was dependent on the degree of aliphatic modification and polymer concentration. When the polymer solutions were subcutaneously injected into rats, immediate depot formation has been observed. The polymeric gel depots have lasted for two weeks in vivo. This aliphatically modified thermogelling polymer can find applications in drug delivery.

Animals↗

PubMed2006

Dexamethasone conjugated poly(amidoamine) dendrimer as a gene carrier for efficient nuclear translocation.

Nuclear membrane is one of the main barriers in polymer-mediated intracellular gene delivery. It was previously reported that glucocorticoid receptor dilated the nuclear pore and translocated into nucleus when it bound to its ligand, glucocorticoid. This suggests that the transport of DNA into nucleus may be facilitated by glucocorticoid. In this study, a glucocorticoid, dexamethasone, was conjugated to polyamidoamine (PAMAM) dendrimer and the effect of the conjugation was investigated. The PAMAM-Dexamethasone (PAM-Dexa) was synthesized by the one-step reaction using Traut's reagent. PAM-Dexa/plasmid DNA complex was completely retarded at a 1/1 weight ratio (polymer/DNA) in a gel retardation assay. PAM-Dexa protected DNA from DNase I for more than 60 min. PAM-Dexa/plasmid DNA complex showed the highest transfection efficiency to 293 cells at a 0.8/1 weight ratio. At this ratio, PAM-Dexa had higher transfection efficiency than PAMAM. Especially in the presence of serum during the transfection, the transfection efficiency of PAM-Dexa was higher than that of PAMAM or PEI by one order of magnitude. In addition, more PAM-Dexa/DNA complexes were observed in the nucleus region than PAMAM/DNA from the confocal microscopy studies. These results indicated that the technique with dexamethasone might be useful for the gene delivery using polymeric gene carriers and the development of efficient polymer vectors.

Active Transport, Cell Nucleus↗

PubMed2006

An efficient GLP-1 expression system using two-step transcription amplification.

Glucagon-like peptide 1 (GLP-1) is an insulinotropic protein. It was reported that the continuous infusion of GLP-1 normalized the blood glucose level in type 2 diabetes animal model. However, the short half-life of GLP-1 has limited its application in clinical settings and prompted us to develop a GLP-1 gene therapy system. Our previous results showed that the delivery of pbeta-GLP-1 using polyethylenimine (PEI) reduced the blood glucose level effectively. However, the glucose level was not completely normalized. In the present study, the more efficient GLP-1 expression system was developed using two-step transcription amplification (TSTA). To evaluate the TSTA system, pbeta-Gal4-p65 and pUAS-Luc were constructed. The pUAS-Luc/pbeta-Gal4-p65 system showed the highest transfection efficiency at a 2:1 pUAS-Luc/pbeta-Gal4-p65 weight ratio. In addition, the transgene expression by the TSTA system was at least 4 times higher than pbeta-Luc. To apply the TSTA system to the GLP-1 expression plasmid, pUAS-GLP-1 was constructed. The pUAS-GLP-1/pbeta-Gal4-p65 system showed higher mRNA level than pbeta-GLP-1. In addition, the level of GLP-1 by the pUAS-GLP-1/pbeta-Gal4-p65 system was more than 4 times higher than pbeta-GLP-1. Therefore, the TSTA GLP-1 expression system may be useful to develop gene therapy system for type 2 diabetes.

Cell Line↗

PubMed2006

Hypoxia-inducible gene expression system using the erythropoietin enhancer and 3'-untranslated region for the VEGF gene therapy.

Gene therapy with the vascular endothelial growth factor (VEGF) gene is a potential treatment for many disorders or injuries with ischemia. However, unregulated expression of VEGF may induce pathological angiogenesis, promoting tumor growth, diabetic proliferative retinopathy and rupture of atherosclerotic plaque. Therefore, the effective regulation of the gene expression is one of the requirements for the VEGF gene therapy. In this research, we evaluated the hypoxia-inducible gene expression system with the erythropoietin (Epo) enhancer and the Epo 3'-untranslated region (UTR). The luciferase plasmids were constructed with the Epo enhancer (pEpo-SV-Luc), the Epo 3'-UTR (pSV-Luc-EpoUTR) or both (pEpo-SV-Luc-EpoUTR). The polyethylenimine/plasmid complexes were transfected to 293 or A7R5 cells and the cells were incubated under normoxia or hypoxia. The results showed that the Epo enhancer or Epo 3'-UTR increased the target gene expression under hypoxia. pEpo-SV-Luc-EpoUTR showed the highest luciferase expression. The VEGF expression plasmid with the Epo enhancer and 3'-UTR was also constructed. The VEGF expression by pEpo-SV-VEGF-EpoUTR showed the highest specificity of the gene expression in the hypoxic cells. The results suggest that the VEGF plasmid with the Epo enhancer and the Epo 3'-UTR may be useful for gene therapy for ischemic diseases.

3' Untranslated Regions↗

PubMed2006

Non-viral adiponectin gene therapy into obese type 2 diabetic mice ameliorates insulin resistance.

Synthetic polymer vectors are attractive for gene delivery due to their potential safety and versatility. However, due to the low efficiency, most of the successful applications of polymeric vectors are focused on the therapeutic genes whose products have biological effects at low concentrations. Adiponectin is one of the abundant circulating proteins and possesses diverse effects including anti-hyperglycemic and anti-atherogenic properties. In this study, we performed the adiponectin gene delivery using a mini-circle DNA complexed with a polymeric carrier, polyethylenimine, into diet induced obese C57BL/6J mice. The mini-circle DNA showed much higher adiponectin expression than the conventional plasmid in vitro and in vivo. This strategy achieved a sufficient blood level of adiponectin and the parameters related with insulin resistance were normalized. The mini-circle DNA will be useful for the increased efficiency of polymeric vectors and adiponectin gene therapy which is applicable to the treatment of type 2 diabetes.

AMP-Activated Protein Kinases↗

PubMed2006

Current status of polymeric gene delivery systems.

Gene therapy provides great opportunities for treating diseases from genetic disorders, infections and cancer. To achieve successful gene therapy, development of proper gene delivery systems could be one of the most important factors. Several non-viral gene transfer methods have been developed to overcome the safety problems of their viral counterpart. Polymer-based non-viral gene carriers have been used due to their merits in safety including the avoidance of potential immunogenecity and toxicity, the possibility of repeated administration, and the ease of the establishment of good manufacturing practice (GMP). A wide range of polymeric vectors have been utilized to deliver therapeutic genes in vivo. The modification of polymeric vectors has also shown successful improvements in achieving target-specific delivery and in promoting intracellular gene transfer efficiency. Various systemic and cellular barriers, including serum proteins in blood stream, cell membrane, endosomal compartment and nuclear membrane, were successfully circumvented by designing polymer carriers having a smart molecular structure. This review explores the recent development of polymeric gene carriers and presents the future directions for the application of the polymer-based gene delivery systems in gene therapy.

Animals↗

PubMed2006

Cholesteryl oligoarginine delivering vascular endothelial growth factor siRNA effectively inhibits tumor growth in colon adenocarcinoma.

Vascular endothelial growth factor (VEGF) is a multifunctional angiogenic growth factor that is a primary stimulant of the development and maintenance of a vascular network in the vascularization of solid tumors. It has been reported that a blockade of VEGF-mediated angiogenesis is a powerful method for tumor regression. RNA interference represents a naturally occurring biological strategy for inhibition of gene expression. In mammalian systems, however, the in vivo application of small interfering RNA (siRNA) is severely limited by the instability and poor bioavailability of unmodified siRNA molecules. In this study, we tested the hypothesis that a hydrophobically modified protein transduction domain, cholesteryl oligo-d-arginine (Chol-R9), may stabilize and enhance tumor regression efficacy of the VEGF-targeting siRNA. The noncovalent complexation of a synthetic siRNA with Chol-R9 efficiently delivered siRNA into cells in vitro. Moreover, in a mouse model bearing a subcutaneous tumor, the local administration of complexed VEGF-targeting siRNA, but not of scrambled siRNA, led to the regression of the tumor. Hence, we propose a novel and simple system for the local in vivo application of siRNA through Chol-R9 for cancer therapy.

Adenocarcinoma↗

PubMed2006

Anti-angiogenic inhibition of tumor growth by systemic delivery of PEI-g-PEG-RGD/pCMV-sFlt-1 complexes in tumor-bearing mice.

Vascular endothelial growth factor (VEGF) is an endogenous mediator of tumor angiogenesis. Blocking associations of the VEGF with its corresponding receptors (Flt-1, KDR/flk-1) have become critical for anti-tumor angiogenesis therapy. Previously, we synthesized PEI-g-PEG-RGD conjugate and evaluated as an angiogenic endothelial polymeric gene carrier. In this study, PEI-g-PEG-RGD/pCMV-sFlt-1 complexes are evaluated in terms of tumor growth inhibition in vivo. Complexes were repeatedly injected systemically via tail vein into subcutaneous tumor-bearing mice. As a result, tumor growth was inhibited in the PEI-g-PEG-RGD/pCMV-sFlt-1 injected group. However, this effect was not identified in PEI-g-PEG/pCMV-sFlt-1 or PEI-g-PEG-RGD/pCMV-GFP control groups. Moreover, the survival rate increased in the PEI-g-PEG-RGD/pCMV-sFlt-1 group compared with the controls group. These results suggest that delivery of pCMV-sFlt-1 using PEG-g-PEG-RGD may be effective for anti-angiogenic gene therapy.

Angiogenesis Inhibitors↗

PubMed2006

PEG conjugated VEGF siRNA for anti-angiogenic gene therapy.

A novel siRNA delivery system based on polyelectrolyte complex (PEC) micelles was introduced in this study. Vascular endothelial growth factor (VEGF) siRNA was conjugated to poly(ethylene glycol) (PEG) via a disulfide linkage (siRNA-PEG). The siRNA-PEG conjugate could form PEC micelles by interacting with cationic polyethylenimine (PEI) as a core forming agent. The VEGF siRNA-PEG/PEI PEC micelles showed greater stability than naked VEGF siRNA against enzymatic degradation. Under a reductive condition similar to cytosolic environment, an intact form of siRNA was released from the siRNA-PEG conjugate by cleavage of the disulfide linkage. The VEGF siRNA-PEG/PEI PEC micelles effectively silenced VEGF gene expression in prostate carcinoma cells (PC-3) up to 96.5% under an optimized formulation condition. They also showed a far superior VEGF gene silencing effect than VEGF siRNA/PEI complexes even in the presence of serum. This study suggests that the siRNA delivery system using VEGF siRNA-PEG/PEI PEC micelles could be potentially applied to RNAi-based anti-angiogenic treatment of cancer in vivo.

Blood Proteins↗

PubMed2006

Novel pH sensitive block copolymer micelles for solvent free drug loading.

Novel pH sensitive biodegradable block copolymers (MPEG-PDLLA-OSM) composed of mono-methoxy poly(ethylene glycol) (MPEG), poly (D,L-lactide) (PDLLA) and sulfamethazine oligomer (OSM) were synthesized via ring-opening polymerization and a dicyclohexyl carboimide (DCC) coupling reaction. These copolymers had a relatively low critical micelle concentration (CMC) due to the strong hydrophobic properties of non-ionized OSM at pH 7.0. Also, the pH sensitive block copolymers showed the micelle-unimer transition due to the ionization-non-ionization of OSM in the pH range (pH 7.2-8.4) above the CMC. Due to the pH sensitive properties of the block copolymer, the hydrophobic drug paclitaxel (PTX) was incorporated into a pH sensitive block copolymer micelle by the pH induced micellization method, without using an organic solvent. The block copolymer micelle prepared by pH induced micellization showed a relatively high PTX loading efficiency, and good stability for 2 d at 37 degrees C. Furthermore, the PTX loaded micelle showed a sustained release of PTX with a small burst in vitro over 2 d. The present results suggest that the pH induced micellization method due to the micelle-unimer transition of the pH sensitive block copolymer would be a novel and valuable drug incorporation tool for hydrophobic and protein drugs, since no organic solvent is involved in the formulation.

Biocompatible Materials↗

PubMed2006

Sulfonamide-based pH- and temperature-sensitive biodegradable block copolymer hydrogels.

Novel pH- and temperature-sensitive biodegradable poly(epsilon-caprolactone-co-lactide)-poly(ethylene glycol) (PCLA-PEG) block copolymers were synthesized with oligomeric sulfamethazine (OSM) end groups (OSM-PCLA-PEG-PCLA-OSM). Aqueous solutions of these block copolymers have shown sol-gel transition behavior upon both temperature and pH changes under physiological conditions (37 degrees C, pH 7.4). The sol-gel transition of these block copolymer solutions was fine-tuned by controlling the PEG length, the hydrophobic to hydrophilic block ratio (PCLA/PEG), and the molecular weight of the sulfamethazine oligomer. Since changes in temperature do not induce gel formation in this pH- and temperature-sensitive block copolymer solution, this hydrogel can be employed as an injectable carrier using a long guide catheter into the body. In addition, the pH of the block copolymer solution showed no change following PCLA degradation over 1 month, and no indication of gel collapse was observed on addition of buffer solution. As such, these properties make the OSM-PCLA-PEG-PCLA-OSM hydrogel an ideal candidate for use as an injectable carrier for certain protein-based drugs known to denature in low-pH environments.

Biodegradation, Environmental↗

PubMed2005

Ischemic injury-specific gene expression in the rat spinal cord injury model using hypoxia-inducible system.

STUDY DESIGN: A spinal cord injury and in vitro neural hypoxia models were used to evaluate the hypoxia responsive gene expression. OBJECTIVES: To limit the risk of unwanted overexpression of therapeutic genes, we developed a hypoxia-inducible gene therapy system using the erythropoietin (Epo) enhancer and the RTP801 promoter. SUMMARY OF BACKGROUND DATA: Gene therapy is an emerging therapeutic technique to treat spinal cord injury. However, uncontrolled overexpression of therapeutic genes in nondisease tissues during gene therapy raises a doubt about its safety. Post-traumatic ischemia is an important factor worsening the spinal cord damage, and hypoxia could regulate the gene expressions using a hypoxia-inducible promoter. METHODS: The plasmids, pEpo-SV-Luc and pRTP801-Luc, were constructed. Mouse neuroblastoma cells (N2A) were used to evaluate the hypoxia-inducible gene expression in vitro.- Gene transfection and expression were allowed for 24 hours under normoxia (pO2, 152 mm Hg) or hypoxia (pO2, 7.6 mm Hg). Spinal cord injury was made using clip compression. Plasmids were injected directly into the injured spinal cord immediately following injury. The gene expression was assessed by luciferase assay. RESULTS: pEpo-SV-Luc and pRTP801-Luc showed more than three times higher gene expression in N2A cells under hypoxia than normoxia. The expression level of luciferase in the injured spinal cord was higher than in the normal spinal cord. Immunostaining demonstrated that neurons, astrocytes, and capillary endothelial cells expressed luciferase in the cytoplasm. CONCLUSIONS: The pEpo-SV-Luc and pRTP801-Luc systems are effective in that they induce gene expression specifically in neurons under the hypoxic condition and spinal cord injury.

Animals↗

PubMed2005

Anti-GAD antibody targeted non-viral gene delivery to islet beta cells.

An islet cell targeting polymeric gene carrier was synthesized by conjugating anti-GAD Fab' fragment to PEI via PEG linker (PEI-PEG-Fab'). The Fab' fragment was prepared from a murine monoclonal antibody against glutamic acid decarboxylase (GAD), which has been identified as one of the major auto-antigens expressed in islet cells, and used as a targeting moiety for islet cell targeting. The electrophoretic migration of plasmid DNA (pCMVLuc)/PEI-PEG-Fab' complexes in agarose gel was completely retarded above the N/P ratio of 2. The complexes demonstrated a size of 100-275 nm with an almost neutral surface charge. Confocal microscopy revealed that the PEI-PEG-Fab' complexes showed much higher cellular binding and uptake efficiency compared to PEI-PEG complexes. The PEI-PEG-Fab' showed about 10-fold higher transfection efficiency (relative luciferase activity) than PEI-PEG in GAD-expressing mouse insulinoma cells (MIN6), however the transfection efficiency of PEI-PEG-Fab' reduced to that of PEI-PEG in GAD negative cells (293) and in the presence of competitive free Fab'. Considering the neutral surface charge of its complexes with DNA, and selectivity toward the islet cells expressing a specific antigen, the PEI-PEG-Fab' conjugate could be thought as a potential candidate of the systemic gene therapy for the treatment of type I diabetes.

Animals↗

PubMed2005

Folate receptor-mediated intracellular delivery of recombinant caspase-3 for inducing apoptosis.

Recombinant reversed caspase-3 (rev-caspase-3) is a pro-apoptotic gene capable of intracellular autocatalytic processing, which leads to programmed cell death. Folate receptor-specific intracellular delivery of the rev-caspase-3 gene into KB cells over-expressing folate receptors was explored by employing the folate-poly(ethylene glycol)-polyethylenimine (FOL-PEG-PEI) conjugate as a nonviral polymeric carrier. Using luciferase as a reporter gene, the conditions for formulation of DNA/polymer polyplexes were pre-optimized to attain the highest folate receptor-mediated gene transfection efficiency. FOL-PEG-PEI conjugate complexed with rev-caspase-3 plasmid in an optimized condition gave rise to a great increase in expression and activation of exogenous rev-caspase-3 in KB cells when pretreated with doxorubicin. The synthesized conjugate exhibited higher transfection efficiency than other commercially available transfection agents due to a unique mechanism of folate-receptor mediated endocytic gene transfer. The transfected cells showed a significant extent of apoptosis by rev-caspase-3. This study suggests the potential of using folate-receptor-mediated delivery of rev-caspase-3 gene for inducing tumor cell death in a target-specific manner.

Apoptosis↗

PubMed2005

Soluble Flt-1 gene delivery using PEI-g-PEG-RGD conjugate for anti-angiogenesis.

Vascular endothelial growth factor (VEGF), a potent angiogenic molecule specific for vascular endothelial cells, is overexpressed in most tumors and closely associated with tumor growth and metastasis. It has been shown that a soluble fragment of VEGF receptor Flt-1 (sFlt-1) has anti-angiogenic properties by way of its antagonist activity against VEGF. In the present study, we demonstrated that the stable expression of sFlt-1 by endothelial cell targeted non-viral gene delivery inhibited the angiogenesis of endothelial cells. A targeted polymeric gene delivery system, PEI-g-PEG-RGD, was developed by incorporating the alphanubeta3/alphanubeta5 integrin-binding RGD peptide, ACDCRGDCFC (single-letter amino acid code), into the cationic polymer, polyethylenimine (PEI) via a hydrophilic polyethylene glycol (PEG) spacer. The functional analysis of therapeutic gene encoding sFlt-1/carrier complex was performed with an endothelial cell proliferation assay. The complex of sFlt-1 gene with PEI-g-PEG-RGD conjugate efficiently inhibited the proliferation of cultured endothelial cells, representing that expressed sFlt-1 predominantly bound to exogenous VEGF and blocked the binding of VEGF to the full-length Flt-1 receptor. These findings suggest that the combination of targeted gene carrier and sFlt-1 possesses the potential to be an efficient tool for the anti-angiogenic gene therapy to treat cancer.

Angiogenesis Inhibitors↗

PubMed2005

Glucagon-like peptide-1 plasmid construction and delivery for the treatment of type 2 diabetes.

Glucagon-like peptide-1 (GLP-1) is a 30-amino-acid hormone produced by intestinal L cells. It has been proposed that GLP-1 can be used as a new treatment for type 2 diabetes mellitus because it acts to augment insulin secretion and its effectiveness is maintained in type 2 diabetic patients. Despite its many remarkable advantages as a therapeutic agent for diabetes, GLP-1 is not immediately clinically applicable because of its extremely short half-life. One way to overcome this drawback is GLP1 gene delivery, which enables GLP-1 production in the body. In this study, the effect of GLP1 gene delivery was evaluated both in vitro and in vivo using a new plasmid constructed with a GLP1 (7-37) cDNA. The expression of the GLP1 gene was driven by a SV40 promoter/enhancer. To increase the expression level of GLP-1, nuclear factor kappaB binding sites were introduced. The in vitro results showed expression of GLP-1 and in vitro activity of GLP-1, which is a glucose-dependent insulinotropic action. A single systemic administration of polyethyleneimine/pSIGLP1NFkappaB complex into DIO mice resulted in increasing insulin secretion and decreasing blood glucose levels for a duration longer than 2 weeks.

Animals↗