Jae Joon Koh

Water-Soluble and Low Molecular Weight Chitosan-Based Plasmid DNA Delivery

AbstractPurpose. Chitosan, a natural cationic polysaccharide, is a candidate non-viral vector for gene delivery because of its high positive charges and low cytotoxicity. In this study, low molecular weight chitosan (LMWC, molecular weight of 22 kDa) was characterized and evaluated as a gene carrier. Methods. Plasmid/LMWC complex was analyzed in 1% agarose gel electrophoresis. To confirm that the LMWC protected plasmids from nuclease, DNase I protection assays were performed. pSV-β-galactosidase plasmid/LMWC complex was transfected into 293T cells and transfection efficiency was evaluated by β-galactosidase assay. Cytotoxicity of LMWC was determined by MTT assay. Results. Unlike high molecular weight chitosan (HMWC), LMWC is highly water soluble, and can form complex with plasmids in physiological buffer. The plasmid DNA was completely retarded at a weight ratio of 1:2 (plasmid:LMWC) in 1% agarose gel. DNase I protection assay showed that plasmids were protected from DNase I over 60 min. The most efficient transfection was obtained at a weight ratio of 1:3 (plasmid:LMWC). The transfection efficiency of LMWC was significantly higher than naked DNA and higher than poly-L-lysine (PLL). MTT assay showed that LMWC was less cytotoxic than PLL. Conclusions. LMWC is non-toxic and has higher transfection efficiency than PLL. Therefore, LMWC will be useful in the development of safe gene carriers.

Controlled release of insulin from injectable biodegradable triblock copolymer

Protein drug delivery has become an important area of research due to the large number of recombinant proteins that are now being investigated for therapeutic applications. However, proteins have very short in vivo half-lives and they require multiple injections to achieve the desired therapeutic effect. One of the way to increase the therapeutic efficiency of these polypeptides is encapsulating them in a sustained dosage form that is capable of releasing the macromolecule continuously and at a controlled rate (1). Typically, proteins have been loaded into the polymers like poly(lactic acid) (PLA), poly(lactic acid-co-glycolic acid) (PLGA) and PLGA-PEG-PLGA (2,3). Fabrication of drug delivery systems using such polymers involves the use of organic solvents or heat that results in protein denaturation and loss of bioactivity. Many problems in loading proteins can be overcome by the use of polymeric hydrogels such as the Poloxamer series which have sol to gel transition characteristics and are soluble in water (4). However, they are not considered an optimal system for the delivery of protein drugs because Poloxamers are toxic and are not biodegradable. For an effective injectable formulation and controlled release of insulin, a water soluble, biodegradable triblock copolymer of poly((D,L-lactide-co-glycolide)-b-ethylene glycolb-(D,L-lactide-co-glycolide) was used in this study as a new injectable implant system that possesses both thermosensitivity and biodegradability (5,6). The copolymer is a free flowing sol below 15°C in aqueous solutions and forms a high viscosity gel at body temperature. The known gelling polymer, Poloxamer formed the gel, which is water soluble and dissolved in a few days at most. However, the ReGelt hydrogel system is a water insoluble gel that can maintain its integrity for more than one month (7). Therefore, it is applicable for an injectable long-term drug delivery system(8,9). Drug release from the hydrogel is affected by several factors such as pore size, degradability, size, hydrophobicity, concentration of a drug, and the presence of specific hydrogel-drug interactions. Initially, the release mechanism from a biodegradable hydrogel is limited by the drug’s diffusivity. Then, a combination of diffusion and degradation processes controls the drug’s release from the polymeric matrix (10). In this study, human insulin was used as a target drug. Diabetes mellitus is a serious pathological condition responsible for major health care problems all around the world costing billions of dollars annually. In the United States, it represents the fourth leading cause of death. Diabetes mellitus also leads to severe complications such as kidney disease, retinopathy, neuropathy, leg or foot amputations and heart disease (11). As a consequence of poor oral bioavailability and current lack of alternative delivery routes, insulin is presently administered parenterally. The subcutaneous route, requiring single or multiple daily injections, is the mainstay of conventional insulin therapy (12). In this study, we designed the sustained release system, which provides basal line insulin release for duration of over several weeks by one injection. Human insulin was entrapped in the hydrogel in order to sustain its release in a subcutaneous insulin delivery system. We tried to modify the association states of insulin by zinc in order to inhibit the initial burst effect and obtain constant release rate. At otherwise equivalent conditions, insulin associates from monomer and dimer to hexamer with increasing zinc concentration (13). Insulin samples with different zinc contents exhibit different release profile due to association-state differences within the hydrogel.

Degradable polymeric carrier for the delivery of IL-10 plasmid DNA to prevent autoimmune insulitis of NOD mice

Recently, we have reported that biodegradable poly [α-(4-aminobutyl)-L-glycolic acid] (PAGA) can condense and protect plasmid DNA from DNase I. In this study, we investigated whether the systemic administration of pCAGGS mouse IL-10 (mIL-10) expression plasmid complexed with PAGA can reduce the development of insulitis in non-obese diabetic (NOD) mice. PAGA/mIL-10 plasmid complexes were stable for more than 60 min, but the naked DNA was destroyed within 10 min by DNase I. The PAGA/DNA complexes were injected into the tail vein of 3-week-old NOD mice. Serum mIL-10 level peaked at 5 days after injection, and could be detected for more than 9 weeks. The prevalence of severe insulitis on 12-week-old NOD mice was markedly reduced by the intravenous injection of PAGA/DNA complex (15.7%) compared with that of naked DNA injection (34.5%) and non-treated controls (90.9%). In conclusion, systemic administration of pCAGGS mIL-10 plasmid/PAGA complexes can reduce the severity of insulitis in NOD mice. This study shows that the PAGA/DNA complex has the potential for the prevention of autoimmune diabetes mellitus.

Prevention of autoimmune insulitis by delivery of interleukin-4 plasmid using a soluble and biodegradable polymeric carrier

AbstractPurpose. We delivered interleukin-4 (IL-4) plasmid (pCAGGS-IL-4) using the biodegradable polymer, poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA), to prevent autoimmune insulitis in NOD mice. Methods. The pCAGGS-IL-4/PAGA complex was transfected to 293T cells. The expression level of IL-4 was measured by ELISA. The pCAGGS IL-4/PAGA complex was injected once to NOD mice intravenously at the age of 4 weeks. RT-PCR was performed to evaluate the level of the IL-4 mRNA in the liver. At 6 weeks after the injection, the grade of insulitis of the mice was evaluated by double blind methods. Results.In vitro transfecton assays showed that PAGA enhanced the expression of IL-4 in 293T cells. RT-PCR of the liver showed that IL-4 was expressed highest in the complex injected group. In the plasmid/PAGA complex injected group, the prevalence of severe insulitis in NOD mice was markedly improved, suggesting that PAGA enhanced the delivery of IL-4 plasmid. Conclusion. The pCAGGS-IL-4/PAGA complex is an effective system to prevent autoimmune insulitis in NOD mice and applicable for the prevention of autoimmune diabetes.

The Effect of Zinc-Crystallized Glucagon-Like Peptide-1 on Insulin Secretion of Macroencapsulated Pancreatic Islets

This research investigates the use of an insulinotropic factor, glucagon-like peptide-1 (GLP-1), to enhance insulin secretion from islets within a macrocapsule. A zinc-crystallized form of GLP-1 was added to the macrocapsule device to have a longer and more controlled release of the bioactive monomer GLP-1. The type of macrocapsule device used for this study consisted of a hollow fiber (MWCO 100,000 and 1 mm inner diameter) containing rat islets and GLP-1 crystals within a poly(N-isopropylacrylamide-co-acrylic acid) (2 mol% acrylic acid) matrix. When incubating the system in media with a high glucose concentration (300 mg/dL), insulin secretion was enhanced with a >85% increase after an induction period. When the same type of system was used in a dynamic perfusion experiment, similar results were obtained. GLP-1 crystals can be an effective form to be entrapped in a bioartificial pancreas to enhance insulin secretion function, especially at high glucose concentrations.

Systemic administration of TerplexDNA system: pharmacokinetics and gene expression.

AbstractPurpose. The aim of this study is to extend our previous studies to investigate the TerplexDNA synthetic gene carrier system in pharmacokinetics, biodistribution, and gene expression in major organs after systemic administration. Methods. The stability of the TerplexDNA system was analyzed in vitro with a serum incubation assay. The TerplexDNA PK/PD studies were conducted by quantitation of Terplex/radiolabeled DNA [CTP α-32P] complexes after rat-tail vein injection. The effect of the TerplexDNA system on gene expression in mouse major organs was analyzed by measuring luciferase activities after systemic administration. Results. The TerplexDNA gene carrier showed significantly longer retention in the vascular space than naked plasmid DNA alone. At early time points (1 h postvenous injection), the lung was the major organ of the TerplexDNA distribution, followed by the liver as a major distribution organ at later time points (24 h postinjection). The major organs of transgene expression after intravenous injection were the liver and heart. Conclusion. The TerplexDNA system has the potential for in vivo applications due to its higher bioavailability of plasmid DNA in the tissues, and due to its organ specific distribution.