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

项目摘要 外体是细胞外小泡的一个子集，直径在30到200纳米之间，由 大多数真核细胞。它们将生物活性的货物，如脂类、蛋白质和核酸，运送到另一个 目标单元格。最近已经做出了巨大的努力来开发高度特异的基于外显子的 药物输送系统。然而，仍然存在根本性的挑战：1)目前的外切体技术 浓缩和分离不能达到足够的产量或纯度；2)药物载药效率 外切体是非常有限的；3)外切体的生产尚未达到足够高的产量 用于临床测试，甚至进一步开发。为了应对这些挑战，拟议的项目利用 一种三维(3D)压电支架(PES)，用于刺激外切体的产生，一种脂质- 高效载药的手性石墨烯量子点(GQD)膜穿透性研究 (&gt；70%产率)和具有离子径迹非对称纳米孔的可扩展切向流超滤组件 高产量(85%)、高产量(240毫升/小时)的膜(ANM)分离外切体 和纯洁。三个模块的集成构成了一个高吞吐量、高纯度、可扩展的 治疗性外切体连续生产的集成平台。该项目基于PI Wang关于GQD促进药物进入体内的初步实验和计算结果 Exosome的效率比目前任何方法都要高，她在纳米复合材料方面的丰富经验 对于3D组织培养，联合皮常的ANM外显子分离技术用于液体活检和HIS 高通量交流纳米纤维电纺专利技术。我们将通过以下方式实现目标 具体目标如下：1)开发高通量外切体生产生物反应器组件 刺激性PES。2)研制高效手性GQD外体载药模块。3) 开发和建立连续的ANM分离纯化模块。拟议中的项目 包含了几种创新的外切体生产、装载和分离方法，如果成功和 集成，可以在当前设计的基础上提高治疗外切体的生产率，并实现 治疗性外切体的持续生产。这种集成的生产流水线提供了一个 创新用于药物输送的高通量、高纯度外切体制造平台。他们会 还可以方便地放大任意大小的反应堆或处理来自多个 反应堆。只需添加更多相同的平行线即可扩展到更高的吞吐量 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.