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

摘要 目前的治疗剂被设计为抑制子宫肌层收缩或宫颈成熟以延迟子宫内膜的成熟。 自然早产（PTB）的风险。这些方法在减少 PTB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， 改善妊娠结局。成功怀孕是由F-M的调节机制决定的， 确保先天性和适应性免疫细胞通过平衡 炎症，同时重塑子宫组织。免疫失调，主要由胎儿介导 对妊娠相关危险因素（如感染）的炎症反应导致PTB。无法 药物跨越胎儿-母体界面障碍，以治疗母亲和胎儿，以及缺乏适当的 药物转运、代谢变化、致畸性和细胞毒性的测试方法阻碍了PTB药物 发展使用天然存在的抗炎细胞因子白细胞介素（IL）-10， 通过电穿孔（eIL-10）工程化的外泌体（细胞外囊泡，40-160 nm），IL-10穿过细胞外膜。 胎盘屏障，LPS诱导的延迟性PTB，与LPS相比，减少胎儿炎症反应 一个人然而，电穿孔的方法将改变外泌体的向性，并且外泌体的产生将改变外泌体。 装载重组IL-10的外泌体非常昂贵。为了克服这些电穿孔限制，我们 开发了一种替代方法，通过将IL-10掺入细胞内来产生大量富含IL-10的外泌体。 10质粒进入小鼠巨噬细胞（RAW 264.7细胞），并用地塞米松（Dex）刺激它们。Dex 诱导IL-10产生，并将大量包装在外泌体（eIL-10）中。这种方法有 克服了电穿孔的限制，并提高了IL-10在胞外体中的包装效率， 生物起源因此，该方法使外泌体蛋白的改变最小化，并且eIL-10的产生将被抑制。 性价比我们将检验eIL-10将降低上行感染的发生率的假设（E。大肠杆菌） PTB小鼠模型。我们提出以下目标来检验我们的假设。 1)为了表征eIL-10并在体外研究中确定其功能活性， 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期，并可转化为急需的特异性介入治疗。 减少PTB相关感染/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.