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: 10223466
• 负责人: Jean-Luc Fraikin
• 金额: $ 82.01万
• 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 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10223466
• 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; detection limit; detection method; 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为 疾病疗法。两者都包含30nm至300nm直径的纳米级颗粒，这些颗粒是在 复杂而异质的生物系统。将这些材料转化为有效和商业上的 可行的产品需要准确测量感兴趣颗粒的浓度和大小（例如， 病毒或电动汽车），以及在研究，开发和生产的各个阶段的任何杂质。 但是，目前尚不可用用于量化这些基本参数的准确工具。最可用的 方法是在相关尺寸范围内的准确测量和在诸如复杂介质中的准确度量 这些应用所需的。其他方法可能需要几天（例如，生物滴度），几乎没有或没有提供 有关颗粒杂质的信息。这些技术充其量通过需要麻烦而产生瓶颈 和昂贵的预定净化；在最糟糕的情况下，他们的测量值为无误 - 重要 对患者安全的影响。因此，对于快速提供的技术而存在至关重要的需求 复杂生物混合物中特定颗粒的精确尺寸和浓度测量。 Spectradyne具有商业化的微流体电阻脉冲传感（MRP），这是一种电力技术 非常适合分析复杂的异质样品。 MRP在工业和 量化电动汽车和病毒的学术界。而MRP准确地测量了复合物中的所有颗粒 样本，它目前无法将感兴趣的颗粒与样品中其他类似大小的颗粒区分开。 Spectradyne将开发Fluor-MRP，这是一种强大的新技术，可以增加单粒子的特异性 MRPS平台的荧光测量。这项新技术将衡量大小， 浓度和溶液中单个颗粒的表型具有前所未有的精度，并且 依次减少生产生物学衍生材料（例如病毒和ev-）的时间和成本 基于疗法。 为了实现这些目标，该项目的第一阶段和第二阶段将达到五个具体目标。在第一阶段的目标中 将生产能够简单的MRP和单一的荧光分析的原型仪器 颗粒。灵敏度和吞吐量将基准测试。在第二阶段的目的中，原型将被优化 用于小粒子检测，对特异性，灵敏度和检测极限进行了彻底评估，并部署了 用于与现实世界中最终用户进行测试。 这项工作的完成将产生易于使用的台式仪器，能够快速准确，并且 在复合物中特异性标记的纳米颗粒表型和杂质的浓度测量值 生物媒体。 Fluor-MRP将在 基因疗法载体和基于EV的治疗的开发和生产。