Nanohydrocyclones for scalable extracellular vesicle purification and drug loading

用于可扩展细胞外囊泡纯化和药物装载的纳米水力旋流器

• 批准号: 10288742
• 负责人: Don L DeVoe
• 金额: $ 23.01万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10288742
• 关键词: 3D Print; Acoustics; Acute Lung Injury; Address; Biological; Biological Assay; Biomanufacturing; Cardiovascular Diseases; Cardiovascular system; Cells; Clinic; Development; Device Designs; Devices; Drug Carriers; Elements; Encapsulated; Filtration; Fostering; Generations; Harvest; In Situ; Lasers; Liposomes; Lung; Lung diseases; Methods; MicroRNAs; Microdialysis; Microfluidic Microchips; Microfluidics; Miniaturization; Modality; Modeling; Molecular Sieve Chromatography; Myocardial Infarction; Myocardial Ischemia; Natural regeneration; Patient Care; Patients; Performance; Pharmaceutical Preparations; Positioning Attribute; Preparation; Process; Production; Proteins; Pulmonary Fibrosis; Reperfusion Injury; Reporting; Research Personnel; Resources; Route; Safety; Scheme; Small Interfering RNA; Source; Stroke; System; Techniques; Technology; Therapeutic; Therapeutic Agents; Therapeutic Effect; Time; Translations; Ultracentrifugation; Vesicle; Writing; base; clinical translation; cost; design; drug development; extracellular vesicles; improved; interest; lung injury; mesenchymal stromal cell; microdevice; nanoscale; nanovesicle; novel strategies; novel therapeutics; pH gradient; particle; pre-clinical; preclinical efficacy; product development; prototype; repaired; small molecule; success; therapeutic development; wound healing

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.

项目摘要 基于生物学衍生的药物的下一代疗法（EV）具有 成为治疗多种心血管和呼吸系统疾病的一条非常有希望的途径。 尽管对基于EV的药物开发具有广泛的兴趣，但越来越明显的是，现有的方法 制备治疗性电动汽车遭受了许多限制，这些限制构成了临床的重大障碍 翻译。除了低吞吐量，较长的处理时间和劳动密集型的操作步骤外， 建立的分离方法的分离效率不佳，导致囊泡损失，大小偏差和 可溶性蛋白的共同污染了所得的纳米层药物。后一个挑战特别是 令人担忧的是，由于存在可溶性蛋白会使疗效和安全性的解释复杂化。另一个 问题是，虽然封装在电动汽车中的microRNA（miRNA）代表授予的关键组件 治疗效应，电动汽车中miRNA的内在浓度极低。结果，有效的电动汽车疗法 要求将外源miRNA加载到囊泡中以增加效力。而许多电动汽车货物 已经开发了加载技术，其中许多方法需要引入外部电气 或声学能量会损坏囊泡及其货物。此外，现有的电动汽车分离和 加载技术需要多个无法固有可扩展的处理步骤，从而增加开发 成本和时间，并提出将电动汽车治疗剂置于临床前阶段的实用挑战。在 这个R21项目，我们提出了一种新的可扩展方法来进行电动汽车分离和药物加载，该方法与 临床翻译的需求，满足了EV生物制造中的重要瓶颈，并使得 单步精简工作流，用于制备高效力EV治疗。提出的技术 由单个设备组成，将有效的基于尺寸的EV分离和药物加载到可伸缩的， 自动化和独立的过程。该平台将利用微型氢化气囊技术 以前是由我们的团队开发的，该团队有可能在30-150 nm范围内隔离电动汽车以被动流动 - 通过微流体芯片。以高流速运行的一系列氢凝管以1 ml/min的速度运行 结合集成的微流体逆流微透析元素，以实现验证的pH梯度 - 我们小组开发的基于加载方法来控制EV货物封装。可扩展平台将 使用预期的简单工作流程，可以从任何单元或生物流体来源启用纯化的电动汽车的在线加载 大大减少治疗性电动汽车处理时间和成本。最终的系统将进一步改善 囊泡纯度和货物载荷效率，支持新的EV的开发和翻译 具有影响多种心血管和呼吸系统疾病的治疗的治疗剂。