Modification of insulin molecule for controlled delivery at basal level from triblock copolymers

修饰胰岛素分子以从三嵌段共聚物控制基础水平递送

• 批准号: 8876907
• 负责人: Jagdish Singh
• 金额: $ 34.8万
• 依托单位: NORTH DAKOTA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8876907
• 关键词: American; Animal Model; Autoimmune Diseases; Biocompatible; Biological Assay; Blood; Body Temperature; Body Weight; Bolus Infusion; Bromides; Cerebrum; Charge; Child; Chitosan; Circular Dichroism; Complex; Development; Diabetes Mellitus; Diffuse; Diffusion; Drug Delivery Systems; Drug Formulations; Environment; Enzyme-Linked Immunosorbent Assay; Equilibrium; Exhibits; Foreign Bodies; Forms Controls; Gel; Gel Chromatography; Glucose; Goals; Health; Hepatic; Hydrogels; Implant; In Situ; In Vitro; Injection of therapeutic agent; Insulin; Insulin Antibodies; Insulin-Dependent Diabetes Mellitus; Length; MALDI-TOF Mass Spectrometry; Measures; Modification; Molecular Weight; Non-Insulin-Dependent Diabetes Mellitus; Patients; Pharmaceutical Preparations; Physiological; Polyacrylamide Gel Electrophoresis; Polymers; Principal Investigator; Production; Property; Proteins; Quality of life; Rattus; Research; Sampling; Serum; Site; Sodium Dodecyl Sulfate; Solutions; Spectroscopy, Fourier Transform Infrared; Stream; Subcutaneous Injections; System; Techniques; Temperature; Testing; Therapeutic; Transition Temperature; Water; Weight; Zinc; absorption; aqueous; basal insulin; base; biodegradable polymer; biomaterial compatibility; controlled release; copolymer; diabetic; diabetic rat; dimer; glucose output; improved; in vivo; insulin dimers; insulin secretion; meetings; monomer; novel; novel strategies; poly(lactic acid); poly(lactic acid-ethylene glycol); programs; public health relevance; response; subcutaneous; therapeutic protein; young adult

 DESCRIPTION (provided by applicant): There are two types of insulin secretion in healthy people- basal and stimulated. The basal insulin is secreted continuously between meals and throughout the night at a rate of 0.5-1 unit/h. Although the basal insulin level is low, it modulats the rate of overnight hepatic glucose and glucose output during prolonged periods between meals. This allows for sufficient glucose level for cerebral energy production at bedtime. There is not a single controlled release delivery system available to deliver insulin continuously for longer duration after a single subcutaneous injection to meet the needs of the basal insulin. Burst release is a major issue in controlled delivery of therapeutics from polymeric formulations. In this application, we are proposing to modify the insulin molecule in order to eliminate the burst release. We propose a novel approach of modifying insulin molecule by utilizing the combination of distinctive properties of insulin (self-association in presence of zinc, and abilityto interact with cationic polymers such as chitosan) and subsequent addition of the complex into aqueous solution of thermo sensitive polymer for controlled delivery of insulin. Thermo sensitive polymer is solution in water at room temperature and turns into implant at body temperature at the site of injection. The proposed delivery system would circumvent the problems associated with insulin burst release by reducing the diffusion of zinc insulin-chitosan complex from the polymeric hydrogel matrix due to its larger size and help to stabilize the protein inside the delivery system, while providing controlled release of insulin at basal level. The long-term goal of this project is to develop novel controlled release delivery systems which can deliver insulin a basal level in a conformationally and chemically stable and biologically active form for longer duration (~3 months) after a single subcutaneous injection in patients with type 1 diabetes. We propose to study three specific aims: (1)To synthesize temperature sensitive poly (lactic acid)-poly (ethylene glycol)- poly(lactic acid) (PLAPEG-PLA) triblock copolymers with varying PLA and PEG chain lengths and characterize their critical gel concentration, gel transition temperature, weight average molecular weight by gel permeation chromatography (GPC), and number average molecular weight by 1H NMR. In situ gel-forming controlled delivery systems of insulin will be prepared, using the above copolymers and zinc-insulin-chitosan complex, and will be studied for the in vitro release of insulin. Further, the stability of insulin in the released samples and in the delivery systems will be investigated using Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE), native PAGE and Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry techniques. (2). To evaluate the biocompatibility of polymeric delivery systems in vitro by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and in vivo in rats by histological analysis. (3). To study in vivo absorption and bioactivity of insulin from the delivery systems in the streptozoticin (STZ) induced diabetic rats by measuring serum insulin and glucose levels, respectively. The anti-insulin antibody in serum will be assessed by ELISA in order to rule out the possibility of foreign body response. STZ induced diabetes leads to reduction in the body weight. Therefore the change in body weight in diabetic rats before and after induction of diabetes, as well as after insulin treatment will be evaluated. The proposed study will contribute significantly to the development of a novel delivery system to deliver insulin at a controlled rate for three months after a single subcutaneous injection in order to improve patients' quality of life, and decrease the long term complications associated with diabetes.

 描述（由申请人证明）：健康人群中有硫蛋白的分泌，在餐点和夜晚以0.5-1的速度刺激了对于脑部胰岛素的单一渗透率，对于持续的contimenterial，我们在此应用中，我们正在胰岛素分子提议，以消除单个胰岛素的unger，我们正在胰岛素胰岛素中的单一含量。释放，我们通过胰岛素F锌的独特特性和与胰岛素递送的能力相互作用，提出了分子的方法。支撑递送系统将通过从较大的SIRP稳定蛋白质的delide系统，同时在基础水平的长期目标中释放蛋白质，从而使胰岛素爆发释放相关。该项目是开发释放详细信息，可以在1型糖尿病患者中以稳定和生物活跃的流行形式（〜3个月）提供胰岛素的基础级别（〜3个月）。 （1）合成温度敏感的聚（乳酸） - 聚（乙二醇） - pory（乳酸 - Pla）的Tribolock共聚物，具有变化的PLA和PEG Chaints，并呈现出临界凝胶浓度，凝胶过渡，凝胶过渡温度，凝胶的平均分子体重，凝胶浓度平均分子体重。凝胶渗透色谱法（GPC）和数量平均重量通过1H NMR。释放的样品和输送系统将使用傅立叶变换py（FTIR），圆二色性（CD），十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS），天然页面和基质的激光时间脱氧/电离时间脱氧/电离时间-Flight（MALDI-TOF）（2）。通过测量血清胰岛素和葡萄糖水平，链二酶（STZ）的糖尿病大鼠将在血清中进行抗胰岛素跟随糖尿病诱导前后糖尿病大鼠身体身体的身体变化，该支撑研究将为开发带来一种新型的输送系统，以在单次亚皮下注射后三个月以受控速率进行。并减少与糖尿病有关的长期并发症。

项目成果

Synthesis and Characterization of Fatty Acid Grafted Chitosan Polymer and Their Nanomicelles for Nonviral Gene Delivery Applications.

• DOI: 10.1021/acs.bioconjchem.7b00505
• 发表时间: 2017-11-15
• 期刊: Bioconjugate chemistry
• 影响因子: 4.7
• 作者: Sharma D;Singh J
• 通讯作者: Singh J

Cell Penetrating Peptide Conjugated Chitosan for Enhanced Delivery of Nucleic Acid.

• DOI: 10.3390/ijms161226142
• 发表时间: 2015-12-04
• 期刊: International journal of molecular sciences
• 影响因子: 5.6
• 作者: Layek B;Lipp L;Singh J
• 通讯作者: Singh J

The Role of Cell-Penetrating Peptide and Transferrin on Enhanced Delivery of Drug to Brain.

细胞穿透肽和转铁蛋白在增强药物向大脑输送中的作用。

• DOI: 10.3390/ijms17060806
• 发表时间: 2016
• 期刊: International journal of molecular sciences
• 影响因子: 5.6
• 作者: Sharma,Gitanjali;Lakkadwala,Sushant;Modgil,Amit;Singh,Jagdish
• 通讯作者: Singh,Jagdish

Chitosan-zinc-insulin complex incorporated thermosensitive polymer for controlled delivery of basal insulin in vivo.

• DOI: 10.1016/j.jconrel.2012.07.035
• 发表时间: 2012-10-28
• 期刊: Journal of controlled release : official journal of the Controlled Release Society
• 作者: Oak M;Singh J
• 通讯作者: Singh J

Smart Thermosensitive Copolymer Incorporating Chitosan-Zinc-Insulin Electrostatic Complexes for Controlled Delivery of Insulin: Effect of Chitosan Chain Length.

• DOI: 10.1080/00914037.2019.1655750
• 发表时间: 2020
• 期刊: International journal of polymeric materials
• 作者: Sharma D;Arora S;Singh J
• 通讯作者: Singh J