Nanohydrocyclones for scalable extracellular vesicle purification and drug loading

项目摘要 基于细胞外囊泡（EV）作为生物衍生的药物载体的下一代治疗剂具有 作为治疗广泛的心血管和呼吸系统疾病的非常有前途的途径出现。 尽管对基于EV的药物开发有着广泛的兴趣，但越来越清楚的是，现有的用于药物开发的方法并不适用于基于EV的药物。 制备治疗性EV受到许多限制，这些限制对临床应用构成显著障碍。 翻译.除了低通量、长处理时间和劳动密集型操作步骤之外， 已建立的分离方法存在分离效率差的问题，这导致囊泡损失、尺寸偏差， 污染所得纳米囊泡药物的可溶性蛋白质的共洗脱。后一项挑战尤其重要， 这是一个令人担忧的问题，因为可溶性蛋白的存在使疗效和安全性的解释复杂化。额外 问题是，虽然封装在EV中的microRNA（miRNAs）代表了赋予EV 在治疗效果方面，EV中miRNA的固有浓度极低。因此，有效的EV疗法 需要将外源性miRNA加载到囊泡中以增加效力。虽然一些电动汽车货物 加载技术已经发展，其中许多方法需要引入外部电 或声能，可以破坏囊泡和它们的货物。此外，现有的EV分离和 加载技术需要多个处理步骤，这些处理步骤本身不是可伸缩的，增加了开发 成本和时间，并提出了一个实际的挑战，超越临床前阶段的EV治疗。在 在R21项目中，我们提出了一种新的可扩展的EV分离和药物装载方法， 临床转化的需求，解决电动汽车生物制造的重大瓶颈， 用于制备高效EV治疗剂的单步简化工作流程。所提出的技术 包括一个单一的设备，将有效的基于尺寸的EV分离与药物装载集成到一个可扩展的， 自动化和独立的过程。该平台将利用微型水力旋流器技术 我们的团队以前开发的，有可能在被动流中隔离30-150 nm范围内的EV- 通过微流控芯片在大约1 mL/min的高流速下操作的水力旋流器阵列将被 与集成的微流体逆流微透析元件相结合，以实现经过验证的pH梯度- 基于我们的小组开发的装载方法来控制EV货物封装。可扩展平台将 能够使用简单的工作流程从任何细胞或生物流体来源在线加载纯化的EV， 显著减少治疗EV处理时间和成本。由此产生的系统有望进一步完善 囊泡纯度和货物装载效率，支持一类新的EV的开发和翻译 这些药物可能影响广泛的心血管和呼吸系统疾病的治疗。

PROJECT SUMMARY Next-generation therapeutics based on extracellular vesicles (EVs) as biologically-derived drug carriers have emerged as a highly promising route to the treatment of a wide range of cardiovascular and respiratory diseases. Despite the broad interest in EV-based drug development, it is increasingly clear that existing methods for preparing therapeutic EVs suffer from a number of constraints that present a significant barrier to clinical translation. In addition to low throughput, long processing times, and labor-intensive operational steps, established separation methods suffer from poor separation efficiencies that result in vesicle loss, size bias, and co-elution of soluble proteins that contaminate the resulting nanovesicle drug. This latter challenge is of particular concern, as the presence of soluble proteins complicates interpretations of efficacy and safety. An additional issue is that while microRNAs (miRNAs) encapsulated within EVs represent a key component conferring therapeutic effect, the intrinsic concentration of miRNA in EVs is extremely low. As a result, effective EV therapies require that exogenous miRNA be loaded into the vesicles to increase potency. While a number of EV cargo loading techniques have been developed, many of these methods demand to introduction of external electrical or acoustic energy that can damage the vesicles and their cargo. Furthermore, existing EV separation and loading techniques require multiple processing steps that are not inherently scalable, increasing development cost and time, and presenting a practical challenge for moving EV therapeutics beyond the preclinical stage. In this R21 project, we propose a new scalable approach to EV separation and drug loading that is compatible with the needs for clinical translation, addressing a significant bottleneck in EV biomanufacturing and enabling a single-step streamlined workflow for the preparation of high potency EV therapeutics. The proposed technology consists of a single device integrating efficient size-based EV separation with drug loading into a scalable, automated, and self-contained process. The platform will leverage a miniature hydrocyclone technology previously developed by our team that has the potential to isolate EVs in the 30-150 nm range in a passive flow- through microfluidic chip. An array of hydrocyclones operating at high flow rates on the order of 1 mL/min will be combined with integrated microfluidic counterflow microdialysis elements to implement a proven pH-gradient- based loading method developed by our group to control EV cargo encapsulation. The scalable platform will enable in-line loading of purified EVs from any cell or biofluid source, using a simple workflow expected to significantly reduce therapeutic EV processing time and cost. The resulting system is further expected to improve vesicle purity and cargo loading efficiency, supporting the development and translation of a new class of EV therapeutics with the potential to impact treatment of a broad range of cardiovascular and respiratory diseases.