Quantifying Specific Nanoparticle Phenotypes in Complex Biological Fluids by Fluorescence Microfluidic Resistive Pulse Sensing.

通过荧光微流体电阻脉冲传感量化复杂生物流体中的特定纳米颗粒表型。

• 批准号: 10011623
• 负责人: Jean-Luc Fraikin
• 金额: $ 25.2万
• 依托单位: SPECTRADYNE, LLC
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10011623
• 关键词: Academia; Adopted; Adoption; Benchmarking; Biological; Biological Markers; Biology; Caliber; Cell Culture Techniques; Complex; Computer software; Concentration measurement; Coupled; Custom; Detection; Development; Disease; Evaluation; Feedback; Flow Cytometry; Fluorescence; Gene Transduction Agent; Goals; Heterogeneity; Industrialization; Industry; Knowledge; Label; Liquid substance; Measurement; Measures; Mediating; Methods; Microfluidics; Microscope; Modernization; Optical Methods; Optics; Particle Size; Phase; Phenotype; Physiologic pulse; Plaque Assay; Process; Production; Research; Resolution; Sampling; Scheme; Sensitivity and Specificity; Site; Source; Specificity; Speed; System; Techniques; Technology; Testing; Therapeutic; Time; Titrations; Translating; Viral Vector; Virus; Virus Replication; Work; base; biological systems; complex biological systems; cost; design; extracellular vesicles; gene therapy; in vivo; instrument; instrumentation; interest; light scattering; nanoparticle; nanoscale; new technology; particle; patient safety; phase 1 designs; physical property; prevent; prototype; research and development; tool; vector

PROJECT SUMMARY Extracellular vesicle (EV)-based therapies and vector-mediated gene therapies hold enormous promise as disease therapeutics. Both comprise nanoscale particles 30nm to 300 nm in diameter that are produced in complex and heterogeneous biological systems. To translate these materials into effective and commercially viable products requires accurate measurements of the concentration and size of the particles of interest (e.g., virus or EVs), and of any impurities at all stages of research, development and production. However, accurate tools for quantifying these basic parameters are not currently available. Most available methods are incapable of accurate measurements in the relevant size range and in such complex media as required for these applications. Other methods can take days (e.g., biological titer) and provide little or no information about particle impurities. At best these techniques create a bottleneck by requiring cumbersome and costly pre-measurement purification; at worst their measurements are misinterpreted—with important implications for patient safety. A critical unmet need therefore exists for technology that delivers fast and accurate size and concentration measurements of specific particles in complex biological mixtures. Spectradyne has commercialized Microfluidic Resistive Pulse Sensing (MRPS), an electrical technique that is uniquely suited to analyzing complex heterogeneous samples. MRPS is seeing rapid adoption in industry and academia for quantification of EVs and virus. While MRPS accurately measures all particles in a complex sample, it cannot currently distinguish the particles of interest from other similarly-sized particles in the sample. Spectradyne will develop Fluor-MRPS, a powerful new technology that adds the specificity of single-particle fluorescence measurements to the MRPS platform. This new technology will measure the size, concentration, and phenotype of single particles in solution with unprecedented accuracy, and dramatically reduce the time and cost of producing biologically derived materials such as virus- and EV- based therapeutics. To accomplish these goals, five specific aims will be met in Phase I and II of the project. In the Phase I Aims, a prototype instrument will be produced that is capable of simultaneous MRPS and fluorescence analysis of single particles. Sensitivity and throughput will be benchmarked. In the Phase II Aims, the prototype will be optimized for small particle detection, thoroughly evaluated for specificity, sensitivity, and limit of detection, and deployed for beta testing with end users in the real world. Completion of this work will yield an easy-to-use bench top instrument capable of rapid and accurate size and concentration measurements of both specifically labeled nanoparticle phenotypes and impurities in complex biological media. Fluor-MRPS will deliver significant efficiencies and powerful new capabilities in the development and production of gene therapy vectors and EV-based therapeutics.

项目摘要 基于细胞外囊泡（EV）的疗法和载体介导的基因疗法具有巨大的前景， 疾病治疗学两者都包含直径为30 nm至300 nm的纳米级颗粒， 复杂和异质的生物系统。将这些材料转化为有效的和商业化的 可行的产品需要精确测量感兴趣的颗粒的浓度和尺寸（例如， 病毒或EV），以及研究、开发和生产所有阶段的任何杂质。 然而，目前还没有量化这些基本参数的准确工具。大多数可用 方法不能在相关尺寸范围内和在诸如 这些应用程序所需的。其他方法可能需要数天（例如，生物滴度）并提供很少或不提供 有关颗粒杂质的信息。这些技术最多只能通过要求繁琐的 和昂贵的测量前净化;在最坏的情况下，他们的测量结果被误解， 对患者安全的影响。因此，存在一个关键的未满足的需求，即需要一种技术， 精确测量复杂生物混合物中特定颗粒的尺寸和浓度。 Spectradyne已经将微流体电阻脉冲传感（MRPS）商业化，这是一种电气技术， 特别适合分析复杂的异质样品。MRPS在工业中得到了快速采用， 学术界对电动汽车和病毒进行定量。虽然MRPS可以精确测量复合物中的所有粒子， 在样品中，它目前无法将感兴趣的颗粒与样品中的其他类似尺寸的颗粒区分开。 Spectradyne将开发Fluor-MRPS，这是一种强大的新技术， 将荧光测量结果发送到MRPS平台。这项新技术将测量大小， 以前所未有的精度测量溶液中单个颗粒的浓度和表型， 大大减少了生产生物衍生材料如病毒和EV的时间和成本， 基于治疗。 为实现这些目标，将在项目第一和第二阶段实现五个具体目标。在第一阶段目标中，a 原型仪器将生产，能够同时MRPS和荧光分析的单一 粒子将对灵敏度和吞吐量进行基准测试。在第二阶段的目标，原型将优化 对于小颗粒检测，全面评估特异性、灵敏度和检测限，并部署 用于在真实的世界中与最终用户进行beta测试。 完成这项工作将产生一个易于使用的台式仪器能够快速和准确的大小， 复合物中特异性标记的纳米颗粒表型和杂质的浓度测量 生物媒介Fluor-MRPS将在全球范围内提供显著的效率和强大的新功能， 基因治疗载体和基于EV的治疗剂的开发和生产。