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

摘要 目前的治疗药物旨在抑制子宫肌层收缩或延缓宫颈成熟 有自然早产(PTB)风险的分娩。这些方法在减少 肺结核的总发病率，在美国约为11%。需要设计一种新的药物输送系统来 改善妊娠结局。成功怀孕是由F-M的调节机制决定的， 通过平衡确保先天免疫细胞和获得性免疫细胞适当地支持胎儿-胎盘发育 炎症同时重塑子宫组织。免疫失调，主要由胎儿 对妊娠相关危险因素(如感染)的炎症反应，导致肺结核。无能为力 药物跨越母胎界面障碍同时治疗母亲和胎儿，以及缺乏适当的 测试药物转运、代谢变化、致畸和细胞毒性的方法阻碍了肺结核药物 发展。使用，自然产生的抗炎细胞因子白介素10，封闭在 电穿孔(eIL-10)工程的胞外体(胞外囊泡，40-160 nm)，IL-10穿过 与脂多糖相比，胎盘屏障、脂多糖诱导的延迟PTB和降低胎儿炎症反应 独自一人。然而，电穿孔的方法将改变外切体的取向，并产生 负载IL-10的重组外切体价格昂贵。为了克服这些电穿孔限制，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的外切体 10质粒导入小鼠巨噬细胞(RAW 264.7细胞)，用地塞米松(Dex)刺激。德克斯 诱导IL-10的产生，并将大量包装在外切体(eIL-10)中。这种方法具有 克服电穿孔的局限性，提高IL-10在外切体中的包装效率 生物发生学。因此，该方法将外体蛋白的变化降至最低，eIL-10的产生将是 性价比高。我们将检验eIL-10将降低上行感染发生率(大肠杆菌)的假设。 诱导PTB小鼠模型。为了验证我们的假设，我们提出了以下几点。 1)为了在体外研究中鉴定eIL-10并确定其功能活性， 2)研究外源IL-10(eIL-10)的体内药效及生物分布。 我们的创新方法将延长妊娠，并可以转化为迫切需要的特定干预 减少与肺结核相关的感染/炎症的策略 从而改善妊娠结局。

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.