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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要基于细胞外囊泡（EV）作为生物源药物载体的下一代疗法已经成为治疗多种心血管和呼吸系统疾病的一种非常有前途的途径。尽管人们对基于 EV 的药物开发产生了广泛的兴趣，但越来越明显的是，现有的方法制备治疗性 EV 受到许多限制，这对临床应用构成了重大障碍翻译。除了吞吐量低、处理时间长和劳动密集型操作步骤之外，现有的分离方法存在分离效率差的问题，导致囊泡损失、尺寸偏差和污染所得纳米囊泡药物的可溶性蛋白质的共洗脱。后一个挑战具有特殊意义令人担忧的是，可溶性蛋白质的存在使功效和安全性的解释变得复杂。额外的问题是，虽然封装在 EV 中的 microRNA (miRNA) 是赋予达到治疗效果时，EV 中 miRNA 的内在浓度极低。因此，有效的 EV 疗法需要将外源 miRNA 加载到囊泡中以增加效力。虽然一些电动汽车货运加载技术已经开发出来，其中许多方法需要引入外部电气或声能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处理步骤，这些步骤本身并不可扩展，从而增加了开发速度成本和时间，并对 EV 疗法超越临床前阶段提出了实际挑战。在在这个 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，该方法与临床转化的需求，解决电动汽车生物制造的重大瓶颈并实现用于制备高效 EV 疗法的单步简化工作流程。提议的技术由单个设备组成，将基于尺寸的有效 EV 分离与药物装载集成到可扩展的、自动化且独立的流程。该平台将利用微型水力旋流器技术我们的团队之前开发的该技术有潜力在被动流中隔离 30-150 nm 范围内的 EV 通过微流控芯片。一系列以 1 mL/min 量级的高流速运行的水力旋流器将与集成微流体逆流微透析元件相结合，可实现经过验证的 pH 梯度我们小组开发的基于装载方法来控制电动汽车货物封装。可扩展的平台将使用简单的工作流程，能够在线加载来自任何细胞或生物流体来源的纯化 EV 显着减少治疗性 EV 处理时间和成本。由此产生的系统有望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转化可能影响广泛心血管和呼吸系统疾病治疗的疗法。