Developing IL-10 encapsulated exosomes as novel therapeutics for spontaneous preterm birth

开发 IL-10 封装的外泌体作为自发性早产的新型疗法

• 批准号: 10592047
• 负责人: Ananth kumar Kammala
• 金额: $ 8万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-09 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10592047
• 关键词: 37 weeks gestation; Address; Amniotic Fluid; Animals; Anti-Inflammatory Agents; Bacteria; Bacterial Infections; Biodistribution; Biogenesis; Biological; Birth; CD81 gene; Cd68; Cells; Cervical Ripening; Cervix Uteri; Clinical Trials; Data; Development; Dexamethasone; Dose; Drug Delivery Systems; Drug Transport; Dyes; Electron Microscope; Electroporation; Encapsulated; Engineering; Ensure; Escherichia coli; Euthanasia; Exclusion; Exposure to; Fetal Membranes; Fetal Reduction; Fetal Tissues; Fetus; Future; Hour; Image; Immune; Immune Tolerance; In Vitro; Incidence; Infection; Inflammation; Inflammatory; Inflammatory Response; Interleukin-10; Intervention; Intravenous; Investigational Drugs; Label; Lipopolysaccharides; Macrophage; Maternal-Fetal Exchange; Maternal-Fetal Medicine Units Network; Mediating; Metabolic; Methods; Modeling; Monitor; Morphology; Mothers; Mus; NF-kappa B; Neonatal Mortality; Pathologic; Pharmaceutical Preparations; Physiological; Placenta; Plasma; Plasmids; Pregnancy; Pregnancy Outcome; Premature Birth; Production; Prolonged Pregnancy; Property; Proteins; Recombinants; Risk; Risk Factors; Sampling; Surface; Teratogens; Testing; Therapeutic; Therapeutic Agents; Time; Tissues; Toxic effect; Transcription Coactivator; Translating; Tropism; Ultracentrifugation; United States; Uterine Contraction; Uterus; cervical remodeling; chemokine; cohort; confocal imaging; cost effective; cytokine; cytotoxicity; delivery vehicle; design; drug development; drug testing; effective therapy; efficacy evaluation; efficacy testing; engineered exosomes; exosome; extracellular; extracellular vesicles; fetal; human fetal cells; imaging system; improved; in vivo; in vivo imaging system; innovation; lipophilicity; mouse model; nanoparticle; neonatal morbidity; neonate; novel therapeutics; risk minimization; success; trafficking

ABSTRACT Present therapeutic agents are designed to inhibit the myometrial contractions or cervical ripening to delay delivery in the risk of spontaneous preterm birth (PTB). These approaches have limited success in reducing the overall rate of PTB, which his ~11% in the USA. A novel drug delivery system needs to be designed to improve pregnancy outcomes. Successful pregnancy is determined by regulatory mechanisms at the F-M, ensuring that both innate and adaptive immune cells aptly support fetoplacental development by balancing inflammation while remodeling uterine tissues. Immune dysregulation, primarily mediated by a fetal inflammatory response to pregnancy-associated risk factors (eg, infections), leads to PTB. The inability of drugs to cross feto-maternal interface barriers to treat both the mother and her fetus, as well as lack of proper ways of testing drug transport, metabolic changes, teratogenicity, and cytotoxicity, has hindered PTB drug development. Using, the naturally occurring anti-inflammatory cytokine interleukin (IL)-10, enclosed in exosomes (extracellular vesicles, 40–160 nm) engineered by electroporation (eIL-10), IL-10 crossed the placental barrier, delayed PTB induced by LPS, and reduced fetal inflammatory response compared to LPS alone. However, the method of electroporation will alter the tropism of exosomes, and the production of recombinant IL-10 loaded exosomes is very expensive. To overcome these electroporation limitations, we developed an alternate approach to produce large quantities of IL-10 enriched exosomes by incorporating IL- 10 plasmid into mouse macrophages (RAW 264.7 cells) and stimulating them with dexamethasone (Dex). Dex induces IL-10 production and will be packaged in large quantities in exosomes (eIL-10). This approach has overcome the limitation of electroporation and increased the efficiency of IL-10 packaging in exosomes during biogenesis. Thus, the approach minimizes alterations of exosome proteins, and the production of eIL-10 will be cost-effective. We will test the hypothesis that eIL-10 will reduce the incidence of ascending infection (E. coli) induced PTB mouse model. We propose the following aims to test our hypothesis. 1) To characterize eIL-10 and determine its functional activity in in vitro studies, 2) To determine efficacy and biodistribution of exosomal IL-10 (eIL-10) in in vivo studies. Our innovative approach will prolong pregnancy and can translate into a critically needed specific interventional strategy to decrease infection/inflammation associated with PTB and thereby improve pregnancy outcomes.

抽象的 目前的治疗剂旨在抑制子宫肌层收缩或宫颈成熟以延迟 分娩时存在自发性早产 (PTB) 的风险。这些方法在减少 PTB 的总体发生率在美国约为 11%。需要设计一种新型药物输送系统 改善妊娠结局。成功怀孕取决于 F-M 的调节机制， 确保先天性和适应性免疫细胞通过平衡来适当支持胎儿胎盘发育 重塑子宫组织时的炎症。免疫失调，主要由胎儿介导 对妊娠相关危险因素（例如感染）的炎症反应会导致 PTB。的无能 跨越胎儿-母体界面障碍来治疗母亲及其胎儿的药物，以及缺乏适当的药物 测试药物转运、代谢变化、致畸性和细胞毒性的方法阻碍了 PTB 药物的开发 发展。使用天然存在的抗炎细胞因子白细胞介素 (IL)-10，封装在 通过电穿孔 (eIL-10) 改造的外泌体（细胞外囊泡，40–160 nm），IL-10 穿过 与 LPS 相比，胎盘屏障、LPS 诱导的 PTB 延迟以及胎儿炎症反应降低 独自的。然而，电穿孔的方法会改变外泌体的向性，并且产生 负载重组 IL-10 的外泌体非常昂贵。为了克服这些电穿孔的局限性，我们 开发了一种替代方法，通过掺入 IL-10 来生产大量富含 IL-10 的外泌体。 将 10 质粒导入小鼠巨噬细胞（RAW 264.7 细胞）并用地塞米松 (Dex) 刺激它们。右旋糖酐 诱导 IL-10 产生，并将大量包装在外泌体 (eIL-10) 中。这种方法有 克服了电穿孔的局限性，提高了IL-10在外泌体中的包装效率 生物发生。因此，该方法最大限度地减少了外泌体蛋白的改变，并且 eIL-10 的产生将 性价比高。我们将检验 eIL-10 会降低上行感染（大肠杆菌）发生率的假设 诱导PTB小鼠模型。我们提出以下目标来检验我们的假设。 1) 表征 eIL-10 并确定其在体外研究中的功能活性， 2) 确定外泌体 IL-10 (eIL-10) 在体内研究中的功效和生物分布。 我们的创新方法将延长妊娠时间，并可转化为急需的特定介入治疗 减少与 PTB 相关的感染/炎症的策略 从而改善妊娠结局。