Immunoliposome Formation via Microfluidic Flow Focusing

脂质体作为生物学和生物医学应用的功能性纳米颗粒的效用目前受到用于其制造的批量生产方法的限制。例如，尽管在脂质体作为药物递送载体的商业化方面已经取得了相当大的进展，但现有的生产方法导致多分散制剂在药物包封水平、血液清除率和细胞摄取方面表现出变化，对药物功效和毒性具有负面影响。我们在这里的目标是发展一个基本的理解的物理过程，驱动脂质体自组装在一个新的微流体过程中，基于两个可混溶的溶剂流的流体动力学聚焦。通过利用在溶剂流之间的亚微米边界处发生的独特相互作用，可以在简单的集成微流体芯片中产生小且均匀的单层脂质体。我们提出了一个计算和实验相结合的努力，以提高我们的理解脂质体自组装过程中的微流体系统，并应用这种理解来优化过程的集成和在线形成功能化的免疫脂质体。预计所提出的努力将导致纳米颗粒竞技场的三个具体进展，即（1）理论和直接的实验评估，导致对脂质体形成过程的更好理解，这是由于微流体系统能够精确地指定在良好限定的层流混合区内的化学和分子分布，（2）将基础物理学与包括通道几何形状和流动条件的系统级参数耦合的多尺度模型，以及（3）应用该模型来证明用于按需生产具有最小多分散性的免疫脂质体的完全集成系统，并且具有可以通过简单调节芯片上流动条件来动态调整的直径。因此，计算和实验相结合的方法将影响我们对脂质体自组装过程的基本理解，同时也导致一个独特的和新颖的工具，用于控制脂质体和immunoliposome production.Broader影响的发展：能够产生脂质体与可调和窄分布的直径在很宽的尺寸范围内具有重要的意义和范围的生物和生物医学应用。高通量方法可直接扩展到大批量生产包封的药物，以及用抗体或其他配体在线修饰脂质体以用于靶向药物递送。由于这些特征，该方法作为在护理点环境中生产个性化药物制剂的简单且低成本的方法提供了很大的希望。 除了药物递送之外，均质脂质体对于免疫测定的应用也具有很大的价值，其中仅当脂质体群体表现出窄的尺寸分布时才能发生受控的信号放大[1]。在该应用中，免疫脂质体内的荧光澄清剂为每个抗体-抗原相互作用提供信号放大，从而实现高灵敏度检测。微流控系统本身提供了一个潜在的基础，为未来开发的集成免疫传感器平台，采用按需形成的免疫脂质体。该技术将应用于一系列生物传感系统，包括便携式和超灵敏的定量诊断测试。最后，该项目将有助于下一代学生在研究生，本科和K-12水平的教育。该项目将为一名博士提供跨学科培训。生物工程专业的学生将接受生物工程，机械工程和化学领域的培训，为这些学科提供坚实的桥梁。本科生也将通过一个既定的NSF赞助的分子和细胞生物工程REU计划被招募到研究项目中，当地高中毕业生将通过一个既定的实习计划参与该项目的选定实验方面。

0966407DeVoeThe utility of liposomes as functional nanoparticles for biological and biomedical applications is presently limited by the bulk production methods used for their manufacture. For example, although considerable progress has been made towards the commercialization of liposomes as drug delivery vehicles, existing production methods result in polydisperse formulations that exhibit variations in drug encapsulation levels, blood clearance rates, and cell uptake, with negative consequences for drug efficacy and toxicity. Our goal here is to develop a fundamental understanding of the physical processes which drive liposome self-assembly in a new microfluidic process, based on the hydrodynamic focusing of two miscible solvent streams. By taking advantage of the unique interactions that occur at the submicron boundary between the solvent streams, small and uniform unilamellar liposomes may be generated in a simple integrated microfluidic chip. We propose a combined computational and experimental effort to improve our understanding of the liposome selfassembly process within the microfluidic system, and apply this understanding to optimize the process for the integrated and in-line formation of functionalized immunoliposomes.Intellectual Merit: The proposed effort is expected to result in three specific advances in the nanoparticle arena, namely (1) theoretical and direct experimental evaluation leading to an improved understanding of the liposome formation process, made possible by the ability of the microfluidic system to precisely specify chemical and molecular distributions within a well-defined laminar mixing zone, (2) a multi-scale model coupling the underlying physics with system-level parameters including channel geometries and flow conditions, and (3) application of this model to demonstrate fully integrated system for the on-demand production of immunoliposomes with minimal polydispersity, and with diameters that may be dynamically tuned by the simple adjustment of on-chip flow conditions. Thus the combined computational and experimental approach will impact our fundamental understanding of the liposome self-assembly process while also leading to the development of a unique and novel tool for controlled liposome and immunoliposome production.Broader Impact: The ability to generate liposomes with tunable and narrowly distributed diameters over a wide size range has important implications for and range of biological and biomedical applications. The high throughput process is directly scalable to large volume production of encapsulated drugs, together with in-line decoration of liposomes with antibodies or other ligands for targeted drug delivery. As a result of these features, the method offers great promise as a simple and low-cost approach to the production of personalized drug preparations in point-of-care settings. Beyond drug delivery, homogeneous liposomes are also of great value for application to immunoassays, where controlled signal amplification can only occur when the liposome populations exhibit a narrow size distribution [1]. In this application, fluorescent encapsulants within the immunoliposomes provide signal amplification for each antibody-antigen interaction, enabling highly sensitive detection. The microfluidic system itself offers a potential base for future development of an integrated immunosensor platform employing on-demand formation of immunoliposomes. The technology will find application in a range of biosensing systems, including portable and ultrasensitive quantitative diagnostic tests. Finally, the project will contribute to the education of next-generation students at the graduate, undergraduate, and K-12 levels. The project will provide interdisciplinary training of one Ph.D. Bioengineering student who will receive training across the fields of bioengineering, mechanical engineering, and chemistry, providing a solid bridge across these disciplines. Undergraduate students will also be recruited to particulate in the research project through an established NSF sponsored Molecular and Cellular Bioengineering REU Program, and local high school seniors will participate in selected experimental aspects of the project through an established internship program.