Engineering the Intracellular Micro- and Nanoenvironment

描述（由申请人提供） 摘要：细胞内部生化信号传导的定位对于细胞功能至关重要，影响人类健康中的多种重要过程，包括细胞分裂、分化、运动性以及细胞间和细胞内信号传导。因此，生物学家对研究和探索生物分子定位的作用非常感兴趣，并开发了分子生物学工具，以便选择性地干扰细胞内部的局部环境。虽然这些工具，包括基因敲除，基因融合，和光活化配体，已经允许一些本地化效果的调查，一个简单而强大的方法，广泛适用于精确定位的范围内的生物分子是严重缺乏。磁性纳米颗粒可以与一系列感兴趣的生物分子缀合，并且容易被大多数细胞摄取，但是细胞内操作和定位受到在这样的小颗粒上产生可观的力所需的强场梯度的限制。这种限制已经通过使用引起模态行为的磁梯度增强基底（“纳米活性载玻片”）来克服。使用这种方法允许外部磁体的宏观尺度运动到细胞内环境修饰纳米颗粒集合的纳米尺度运动的稳健转换。动态定位的纳米粒子可以通过前所未有的控制来消除各种化学，机械和热信号。实现基于纳米颗粒的细胞内工程的全部潜力，包括基于微杆的细胞内纳米环境的动态控制和大规模平行阵列中细胞内位置的精确扰动，将需要额外的研究和开发。推进领域将包括纳米粒子结合和递送、透明基底开发、细胞图案化和与磁性元件对准，以及开发用于纳米粒子移动的基于磁杆的控制系统。虽然最重要的影响将是在创建一个通用的生物学实验平台，同时开发工具的精密工程的细胞内环境，我们也将使用这些工具的初始开拓性研究的影响，在细胞运动和Ca2+传播内部的细胞定位。这些实验可能最终能够远程控制细胞运动，并通过工程设计其结果来定量了解细胞迁移所需的关键因素。 公共卫生相关性：通过开发简单的工具来精确控制细胞内的纳米颗粒，将有可能理解和控制以前无法探测的细胞行为的各个方面。类似于我们如何通过扰动局部器官系统来研究大规模生理学，这些类型的工具将允许通过扰动亚细胞位置和细胞器在时间和空间上对细胞行为进行更多的数学理解。最终，这种类型的工具将被生物学家广泛使用，以揭示直接影响人类健康的细胞功能的独特方面。

DESCRIPTION (Provided by the applicant) Abstract: Localization of biochemical signaling internal to cells is critical for cellular function, affecting a variety of processes of importance in human health, including cell division, differentiation, motility, and inter- and intracellular signaling. As such, biologists are keenly interested in studying and probing the role of biomolecule localization, and have developed molecular biological tools in order to selectively perturb local environments internal to cells. Although these tools, including gene knockouts, gene fusions, and photoactivated ligands, have allowed some investigation into localization effects, a simple and robust approach that is broadly applicable to precisely localize a range of biomolecules intracellularly is critically lacking. Magnetic nanoparticles can be conjugated to a range of biomolecules of interest and are readily uptaken by most cells, but intracellular manipulation and localization has been limited by the strong field gradients required to create appreciable forces on such small particles. This limitation has been overcome by using magnetic-gradient enhancing substrates ("nano-active slides") that elicit modal behavior. Using this approach allows for the robust transformation of macroscale movements of an external magnet to nanoscale movements of intracellular environment-modifying nanoparticle ensembles. Dynamically localized nanoparticles can then act to transduce a variety of chemical, mechanical, and thermal signals with unprecedented control. Achieving the full potential of nanoparticle-based intracellular engineering, including joystick-based dynamic control of the intracellular nanoenvironment and precision perturbation of intracellular locations in massively parallel arrays will require additional investigation and development. Thrust areas will include nanoparticle conjugation and delivery, transparent substrate development, cell patterning and alignment to magnetic elements, and development of joystick-based control systems for nanoparticle movement. Although the most significant impact will be in creating a general platform for biological experimentation, concurrent with developing tools for precision engineering of intracellular environments we will also use these tools for initial pioneering studies of the effect of localization in cellular motility and Ca2+ propagation internal to cells. These experiments will potentially culminate in the ability to remotely control cell movement and extract quantitative understanding of key factors required for cell migration through engineering its outcome. Public Health Relevance: By developing simple tools for precise control of nanoparticles locally within cells it will be possible to understand and control aspects of cell behavior that could not be probed previously. Similar to how we investigate large-scale physiology by perturbing local organ systems, these type of tools will allow a more mathematical understanding of cell behavior through perturbation of sub-cellular locations and organelles in time and space. Ultimately, this type of tool will be broadly used by biologists to uncover unique aspects of cellular function that directly impact human health.