Prevention of diabetes by pDNA encoded with IL-10

通过编码 IL-10 的 pDNA 预防糖尿病

• 批准号: 7694184
• 负责人: Jagdish Singh
• 金额: $ 7.18万
• 依托单位: NORTH DAKOTA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7694184
• 关键词: 18 year old; 20 year old; Affect; Amines; Animal Model; Autoimmune Process; Biocompatible; Biological Assay; Blood Glucose; Buffers; Cell Nucleus; Cells; Characteristics; Charge; Chromatography; Confocal Microscopy; Coumarins; DNA delivery; Development; Diabetes Mellitus; Diabetes prevention; Electron Microscopy; Embryo; Emulsions; Ensure; Enzyme-Linked Immunosorbent Assay; Gel; Gel Chromatography; Gene Delivery; Glycolic-Lactic Acid Polyester; Goals; Histology; Human; IL10 gene; In Vitro; Individual; Insulin; Insulin-Dependent Diabetes Mellitus; Interferons; Interleukin-10; Kidney; Measurement; Mediating; Methacrylates; Methylmethacrylate; Molecular Weight; Mus; Newly Diagnosed; Nuclear; Pancreas; Plasmids; Play; Polymers; Population; Prevention; Proteins; Research; Role; Serum; Shapes; Solvents; Surface; System; T-Lymphocyte; Techniques; Testing; Th1 Cells; Therapeutic; Tissues; Toxic effect; Transfection; United States; Weight; alpha benzopyrone; ammonium bromide; biomaterial compatibility; cell mediated immune response; chronic autoimmune disease; copolymer; cytokine; density; evaporation; extracellular; gel electrophoresis; gene delivery system; gene therapy; in vivo; light microscopy; light scattering; monomer; nanoparticle; nanoparticulate; non-viral gene delivery; plasmid DNA; poly(DL-lactide); prevent; protective effect; public health relevance; surfactant; tertiary amine; vector; zeta potential

DESCRIPTION (provided by applicant): In the United States, 20.8 million people (7% of the population) suffer from diabetes. About 75% of all newly diagnosed cases of type I diabetes occurs in individuals younger than 18 years of age. Type 1 diabetes is a chronic autoimmune disease affecting 0.3% of world's population. It results from selective destruction of pancreatic 2-cells. The major goal of the proposed research is to develop a nanoparticulate gene delivery vector for highly efficient nuclear delivery of plasmid encoding interleukin-10 (IL-10) for prevention of type 1 diabetes. We hypothesize that the combination of cationic polymer and poly (lactide-co-glycolide) (PLGA) in the presence of a cationic surfactant will produce nanoparticles with high positive zeta potential that will facilitate efficient loading of negatively charged plasmid DNA encoding IL-10 gene on the surface; and the positively charged nanoparticles loaded with plasmid DNA are biocompatible and can efficiently transfect the cells and express the protein both in vitro and in vivo. To test our hypotheses, we plan to study the following specific aims: (1). To synthesize methacrylate copolymers using monomers, 2- dimethyl amino ethyl methacrylate (DMAEMA) and methylmethacrylate (MMA) with increasing molar ratio of DMAEMA. The copolymers will be characterized for weight average molecular weight by gel permeation 1 chromatography and number average molecular weight by H NMR. (2). To prepare cationic nanoparticles using a blend of cationic polymer and PLGA by double emulsion solvent evaporation technique, using cetyl trimethyl ammonium bromide as a cationic surfactant. The nanoparticles will be characterized for size, shape, charge density, plasmid loading efficiency, buffering ability, and structural integrity of plasmid DNA by dynamic light scattering, electron microscopy, zeta potential measurement, UV spectrophotometer, titrimetric, and gel electrophoresis, respectively. (3). To study the cellular internalization in Human Embryonic Kidney (HEK 293) cells by confocal microscopy, using cationic nanoparticles loaded with coumarin 6. In vitro transfection efficiency of cationic nanoparticles in HEK 293 cells will be studied, using a therapeutic plasmid encoding IL-10. The expression of and IL-10 will be quantified by enzyme-linked immunosorbent assay. (4). To evaluate in vitro and in vivo in mice biocompatibility of cationic nanoparticles, using an MTT assay and light microscopy, respectively. (5). To study the efficiency of cationic nanoparticles to deliver plasmid encoding Interleukin-10 in vivo in mice and its ability to prevent the onset of type 1diabetes. The proposed study will contribute towards the development of a high efficiency and low toxicity non-viral gene delivery vehicle in order to deliver plasmid encoding IL-10 gene for prevention of type 1 diabetes. PUBLIC HEALTH RELEVANCE: Type 1 diabetes is a chronic autoimmune disease affecting 0.3% of world's population. It results from selective destruction of pancreatic 2-cells mediated by T lymphocytes which leads to gradual reduction in body's ability to produce insulin. The gene delivery vectors need special features to overcome extracellular and intracellular barriers, and ensure efficient DNA delivery to the nucleus. The use of cationic polymer and cationic surfactant will synergistically enhance the positive zeta potential of nanoparticles and their transfection efficiency. The study would be conducted in vitro and in vivo in animal model. The proposed study will contribute towards the development of a high efficiency and low toxicity non-viral gene delivery vehicle in order to deliver plasmid encoding IL-10 gene for prevention of type 1 diabetes.

描述（由申请人提供）：在美国，有2080万人（占人口的7％）患有糖尿病。在所有已有新诊断的I型糖尿病病例中，约有75％发生在18岁以下的个人中。 1型糖尿病是一种慢性自身免疫性疾病，影响了世界人口的0.3％。它是由选择性破坏胰腺2细胞而引起的。拟议的研究的主要目标是开发一种纳米标志性的基因输送载体，用于高效地编码白介素-10（IL-10）的质粒递送，以预防1型糖尿病。我们假设在存在阳离子表面活性剂的情况下，阳离子聚合物和聚（乳酸 - 糖糖苷）（PLGA）的组合将产生具有较高阳性ZETA电位的纳米粒子在表面；并且带有质粒DNA的正电纳米颗粒具有生物相容性，可以有效地转染细胞并在体外和体内表达蛋白质。为了检验我们的假设，我们计划研究以下特定目标：（1）。使用单体2-二甲基氨基乙基丙烯酸乙酯（DMAEMA）和甲基丙烯酸甲酯（MMA）合成甲基丙烯酸酯共聚物，并随着DMAEMA的摩尔比增加。共聚物将通过凝胶渗透1色谱法和H NMR的平均分子量数量的平均分子量来表征。 （2）。使用双乳液溶剂蒸发技术，使用阳离子聚合物和PLGA的混合物来制备阳离子纳米颗粒，使用锡基三甲基溴化铵作为阳离子表面活性剂。通过动态光散射，电子显微镜，ZETA电位测量，紫外分光光度计，滴定度和凝胶电泳，将对纳米颗粒的尺寸，形状，电荷密度，质粒载荷效率，缓冲能力和结构完整性进行表征。 （3）。为了研究人类胚胎肾脏（HEK 293）细胞的细胞内在化，使用加载的阳离子纳米颗粒，加载了用香豆素6。 IL-10和IL-10的表达将通过酶联免疫吸附测定法进行量化。 （4）。分别使用MTT分析和光显微镜评估阳离子纳米颗粒的小鼠生物相容性的体外和体内。 （5）。为了研究阳离子纳米颗粒的效率，在小鼠中在体内递送属属核糖-10及其预防1糖尿病型发作的能力。拟议的研究将有助于发展高效率和低毒性非病毒基因递送载体，以便提供编码IL-10基因的质粒，以预防1型糖尿病。 公共卫生相关性：1型糖尿病是一种慢性自身免疫性疾病，影响了世界人口的0.3％。它是由T淋巴细胞介导的胰2细胞的选择性破坏导致人体产生胰岛素能力的逐渐降低的。基因递送向量需要特殊的特征来克服细胞外和细胞内屏障，并确保有效的DNA递送到细胞核。阳离子聚合物和阳离子表面活性剂的使用将协同增强纳米颗粒及其转染效率的正Zeta潜力。该研究将在动物模型中在体外和体内进行。拟议的研究将有助于发展高效率和低毒性非病毒基因递送载体，以便提供编码IL-10基因的质粒，以预防1型糖尿病。