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: 10458751
• 负责人: Don L DeVoe
• 金额: $ 19.14万
• 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 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10458751
• 关键词: 3D Print; Acoustics; Acute Lung Injury; Address; Biological Assay; Biological Products; 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; Respiratory Disease; 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; translational potential; 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.

项目总结 基于细胞外小泡(EVS)作为生物衍生药物载体的下一代治疗药物 被认为是治疗多种心血管和呼吸系统疾病的极有希望的途径。 尽管基于EV的药物开发引起了广泛的兴趣，但越来越明显的是，现有的治疗方法 制备治疗性电动汽车面临着许多限制，这些限制给临床带来了巨大的障碍 翻译。除了吞吐量低、处理时间长和劳动密集型操作步骤外， 已建立的分离方法存在分离效率低的问题，这会导致囊泡丢失、大小偏差和 共洗脱污染所得纳米胶囊药物的可溶性蛋白质。后一项挑战是特别的 令人担忧的是，可溶性蛋白质的存在使对有效性和安全性的解释变得复杂。一项额外的 问题是，虽然封装在电动汽车中的microRNAs(MiRNAs)代表着一个关键组件 治疗效果方面，EVS中miRNA的本征浓度极低。因此，有效的电动汽车疗法 需要将外源miRNA装载到囊泡中以提高效力。而一些电动汽车的货物 加载技术已经发展起来，其中许多方法都需要引入外部电气 或声能，会损坏小泡和它们的货物。此外，现有的电动汽车分离和 加载技术需要多个处理步骤，而这些步骤本身并不是可伸缩的，这增加了开发 成本和时间，并为将电动汽车疗法推向临床前阶段提供了一个实际挑战。在……里面 在R21项目中，我们提出了一种新的可扩展的电动汽车分离和药物加载方法，该方法与 临床翻译需求，解决电动汽车生物制造中的一个重大瓶颈，并使 用于制备高效EV疗法的单步简化工作流程。建议的技术 由单个设备组成，集成了高效的基于尺寸的电动汽车分离和药物加载到可扩展的 自动化、自成一体的流程。该平台将利用微型水力旋流技术 之前由我们的团队开发的，有可能将30-150 nm范围内的电动汽车隔离在被动流动中- 通过微流控芯片。以1毫升/分钟的数量级高流速运行的一系列水力旋流器将 与集成的微流体逆流微渗析元件相结合，实现了经过验证的pH梯度- 基于本课题组开发的控制电动汽车货物封装的装载方法。该可扩展平台将 支持在线加载来自任何细胞或生物流体来源的纯化电动汽车，使用预计将实现的简单工作流程 显著减少治疗性EV处理时间和成本。由此产生的系统预计将进一步改进 囊泡纯度和货物装载效率，支持新型电动汽车的开发和翻译 治疗学有可能影响一系列心血管和呼吸系统疾病的治疗。