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（由申请人提供）：健康人的胰岛素分泌有两种类型——基础型和刺激型。基础胰岛素在两餐之间和整个晚上以 0.5-1 单位/小时的速率持续分泌。尽管基础胰岛素水平较低，但它可以调节夜间肝葡萄糖的速率和长时间两餐之间的葡萄糖输出。这使得睡前大脑有足够的葡萄糖水平产生能量。没有单一的控释递送系统可以在单次皮下注射后持续较长时间地连续递送胰岛素以满足基础胰岛素的需要。突释是聚合物制剂控制递送治疗的一个主要问题。在此应用中，我们建议修改胰岛素分子以消除突发释放。我们提出了一种修饰胰岛素分子的新方法，该方法结合了胰岛素的独特性质（锌存在下的自缔合以及与阳离子聚合物（如壳聚糖）相互作用的能力），然后将复合物添加到热敏聚合物的水溶液中以控制胰岛素的输送。热敏聚合物在室温下溶于水，并在注射部位在体温下转化为植入物。所提出的递送系统将通过减少锌胰岛素-壳聚糖复合物从聚合水凝胶基质中的扩散（由于其尺寸较大）来避免与胰岛素爆发释放相关的问题，并有助于稳定递送系统内的蛋白质，同时提供基础水平的胰岛素控制释放。该项目的长期目标是开发新型控释递送系统，该系统可以在 1 型糖尿病患者单次皮下注射后，以构象和化学稳定且具有生物活性的形式在较长持续时间（约 3 个月）内递送基础水平的胰岛素。我们建议研究三个具体目标：（1）合成具有不同PLA和PEG链长的温度敏感的聚乳酸-聚乙二醇-聚乳酸（PLAPEG-PLA）三嵌段共聚物，并通过凝胶渗透色谱（GPC）表征其临界凝胶浓度、凝胶转变温度、重均分子量和数均分子量 通过 1H NMR 测定分子量。将使用上述共聚物和锌-胰岛素-壳聚糖复合物制备胰岛素原位凝胶控释系统，并研究胰岛素的体外释放。此外，将使用傅里叶变换红外光谱（FTIR）、圆二色性（CD）、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS PAGE）、非变性PAGE和基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱技术来研究释放样品和递送系统中胰岛素的稳定性。 （2）。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑 (MTT) 测定体外和通过组织学分析在大鼠体内评价聚合物递送系统的生物相容性。 （3）。通过分别测量血清胰岛素和血糖水平，研究链脲佐菌素 (STZ) 诱导的糖尿病大鼠体内胰岛素输送系统的吸收和生物活性。血清中的抗胰岛素抗体将通过ELISA进行评估，以排除异物反应的可能性。 STZ 诱发的糖尿病会导致体重减轻。因此，将评估糖尿病大鼠在糖尿病诱导前后以及胰岛素治疗后的体重变化。拟议的研究将对新型输送系统的开发做出重大贡献，该系统可在单次皮下注射后以受控速率输送胰岛素三个月，以改善患者的生活质量，并减少与糖尿病相关的长期并发症。

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

In Vitro and in Vivo Optimization of Phase Sensitive Smart Polymer for Controlled Delivery of Rivastigmine for Treatment of Alzheimer's Disease.

用于控制卡巴拉汀递送以治疗阿尔茨海默病的相敏智能聚合物的体外和体内优化。

• DOI: 10.1007/s11095-020-2757-6
• 发表时间: 2020
• 期刊: Pharmaceutical research
• 影响因子: 3.7
• 作者: Lipp,Lindsey;Sharma,Divya;Banerjee,Amrita;Singh,Jagdish
• 通讯作者: Singh,Jagdish