A Scalable Continuous Production Platform for Large-Scale Manufacturing of Therapeutic Exosomes

项目摘要 外泌体是细胞外囊泡的一个子集，直径在30和200 nm之间，由 大多数真核细胞。它们将生物活性货物，如脂质，蛋白质和核酸，递送到其他细胞。 靶细胞最近在开发高度特异性的基于外泌体的 药物输送系统。然而，仍然存在根本性的挑战：1）目前的外泌体技术 富集和分离不能实现足够的通量或纯度; 2）药物负载效率， 外泌体非常有限;以及3）外泌体的生产尚未达到足够高的通量 用于临床试验甚至进一步开发。为了应对这些挑战，拟议项目利用 三维（3D）压电支架（PES）刺激外泌体的产生，脂质- 用于将药物有效负载到外泌体中的膜穿透手性石墨烯量子点（GQD） （>70%产率）和具有离子轨道不对称纳米孔的可扩展切向流超滤模块 膜（ANM）分离外泌体，产率高（>85%），通量高（每个模块240 mL/小时） 和纯洁三个模块的集成构成了一个高通量、高纯度和可扩展的 用于连续制造治疗性外泌体的集成平台。该项目基于PI Wang的初步实验和计算结果表明，GQD有助于药物加载到 她在纳米复合材料方面的丰富经验， 对于3D组织培养，co-PI Chang的用于液体活检的ANM外泌体分离技术和他的 高通量交流静电纺丝专利技术。我们将通过以下方式实现目标： 1）开发高通量外泌体生产生物反应器模块， 刺激PES。2)开发高效的外泌体手性GQD载药模块。第三章 开发和建立持续的ANM分离纯化模块。拟建项目 包含几种创新的外泌体生产、装载和分离方法，如果成功的话， 整合，可以提高治疗性外泌体生产率超过目前的设计，并实现 连续生产治疗性外泌体。这种集成的生产管道提供了 创新的高通量和高纯度外泌体制造平台，用于药物递送。他们将 还被设计成允许任意尺寸的反应器或来自多个反应器的流出物容易地按比例放大。 反应堆扩展到更高的吞吐量可以通过添加更多相同的并行线来实现。 模块。

Project Summary Exosomes are a subset of extracellular vesicles, with diameters between 30 and 200 nm, secreted by most eukaryotic cells. They deliver bioactive cargos, such as lipids, proteins and nucleic acids, to other target cells. Tremendous effort has been made recently on developing highly specific exosome-based drug delivery systems. Yet, there remain fundamental challenges: 1) current technologies for exosome enrichment and isolation cannot achieve sufficient throughput or purity; 2) drug loading efficiency into exosomes is very limited; and 3) the production of exosomes has yet to reach sufficient high throughput for clinical tests or even further development. To address these challenges, the proposed project utilizes a three-dimensional (3D) piezoelectric scaffold (PES) to stimulate exosome production, a lipid- membrane penetrating chiral graphene quantum dots (GQD) for efficiently load drugs into exosomes (>70% yield) and a scalable tangential-flow ultrafiltration module with ion-track asymmetric nanopore membranes (ANM) to isolate exosomes with high yield (>85%), throughput (240 mL/hour per module) and purity. The integration of three modules constitutes a high-throughput, high-purity, and scalable integrated platform for continuous manufacturing of therapeutic exosomes. The project is based on PI Wang’s preliminary experimental and computational results that GQDs facilitated drug loading into exosomes with higher efficiency than any current method, her extensive experience on nanocomposite for 3D tissue cultures, the co-PI Chang’s ANM exosome isolation technology for liquid biopsy and his patented technology on high-throughput AC nanofiber electrospinning. We will achieve the goal by following specific aims: 1) Develop high-throughput exosome production bioreactor module with stimulating PES. 2) Develop high-efficiency exosome drug loading module with chiral GQDs. 3) Develop and establish continuous ANM isolation and purification module. The proposed project contains several innovated approaches of exosome production, loading and isolation, if successful and integrated, can enhance therapeutic exosome production rate over current design, and achieve continuous production of therapeutic exosomes. Such an integrated production pipeline provides an innovative high-throughput and high-purity exosome manufacturing platform for drug delivery. They will also be designed to allow easy scale up for reactors of arbitrary size or for effluents from multiple reactors. Scaling to higher throughput can be achieved by just adding more parallel lines of the same modules.