Nanoparticle Induced Circuit Excitation

描述（由申请人提供）：提出了一组实验来验证和进一步开发一种新的基于纳米颗粒的技术，纳米颗粒诱导电路激励（NICE），用于远程和非侵入性地调节细胞的活性。生物学的一个基本目标是了解复杂生物体中每种细胞类型的作用。细胞功能的最终测试是有选择地打开或关闭活动物体内单一细胞类型的活动，并检查其对生理功能的影响。最近的工具，如光激活离子通道，如通道视紫红质，已经率先在遗传定义的细胞中对膜电位进行外部控制，并为神经科学家的研究建立了一种新的手段。然而，这些光学方法的实际缺点限制了它们的应用，包括需要手术植入侵入性光纤；无法同时刺激多个解剖区域的细胞；以及并行调制多种细胞类型的困难。我们通过使用纳米颗粒通过无线电波远程激活特定的细胞群来解决这一挑战。用链霉亲和素包被的氧化亚铁修饰细胞，细胞在细胞特异性启动子的控制下表达一种生物素受体蛋白。这些细胞也被改造成表达TRPV1，这是一种单组分的温度敏感离子通道，可以检测生理范围内温度的微小变化，并通过构象变化允许分级钙进入。将金属涂层细胞暴露在特定的电磁场中会增加局部温度并激活TRPV1通道，导致Ca2+电流和细胞激活。我们有初步的数据证实了这种方法在体外的有效性，现在我们建议扩展我们的研究，以进一步验证该技术在体外的有效性，并调节体内的功能，如激素释放和神经活动。我们还将建立一种组合激活不同细胞的方法，使用改良的TRPV1和由其他金属制成的纳米颗粒，这些纳米颗粒可以在不同波长下被激发。我们将使用这个工具来检查特定外周和中枢神经系统细胞群在能量代谢中的作用。我们建议分三个阶段发展这种方法：1)在体外验证NICE的安全性和实用性，并通过用不同波长的不同粒子装饰不同的细胞类型来完善方法，以激活不同组合的不同细胞群的集合。2)在体内建立NICE调节激素释放调节糖尿病动物糖代谢的能力。3)表明NICE可用于刺激电兴奋细胞的动作电位来改变行为，并利用NICE研究下丘脑特定群体在（NPY和POMC）中控制食欲的作用。随着时间的推移，NICE可能会被应用于临床，例如，诱导多能干细胞被改造成表达NICE构建物，可以作为自体移植物，实现细胞功能的外部控制。这些应用是遥远的，但并非不可想象的，提出的研究可能形成纳米颗粒临床应用的基础。

DESCRIPTION (provided by applicant): A set of experiments is proposed to validate and further develop a new nanoparticle based technology, Nanoparticle induced Circuit excitation (NICE), for modulating the activity of cells remotely and non-invasively. A fundamental goal of biology is to understand the role of each cell type in a complex organism. The definitive test of cell function is to selectively turn on or off the activity of a single cell type in a living animal and examine the effect on physiological function. Recent tools, such as light activated ion channels such as channel rhodopsin, have pioneered the external control of membrane potential in genetically defined cells and established a new means for investigation by neuroscientists. However, these optical methods have practical disadvantages limiting their application including the need for surgical implantation of invasive fiber optics; the inability to stimulate cells in multiple anatomical regions simultaneously; and the difficulty of modulating multiple cell types in parallel. We address this challenge by using nanoparticles to activate defined cell populations remotely with radiowaves. Ferrous oxide coated with streptavidin is used to decorate cells, which express a biotin acceptor protein under the control of cell specific promoters. These same cells are engineered to also express TRPV1, a single component, temperature-sensitive ion channel that can detect small changes in temperature within the physiological range and by conformational change allow graded calcium entry. Exposing the metal coated cells to a defined electromagnetic field increases the local temperature and activates TRPV1 channels resulting in a Ca2+ current and cell activation. We have preliminary data that confirms the efficacy of this method in vitro and now propose to extend our studies to further validate the technology in vitro and to modulate in vivo functions such as hormone release and neural activity. We will also establish a means for combinatorial activation of different cells using a modified TRPV1 and nanoparticles fabricated from other metals that can be excited at different wavelengths. We will use this tool to examine the roles of specific peripheral and CNS cell populations in energy metabolism. We propose to develop this method in three stages: 1) Validate the safety and utility of NICE in vitro and refine the methodology by decorating different cell types with distinct particles tuned to different wavelengths to activate ensembles of different cell populations in various combinations. 2) Establish the ability of NICE to modify hormone release to regulate glucose metabolism in diabetic animals in vivo. 3) Show that NICE can be used to stimulate action potentials in electrically excitable cells to modify behavior and use NICE to investigate the role of specific hypothalamic populations in (NPY and POMC) to control appetite. In time, NICE may be adapted for clinical uses, e.g: induced pluripotent stem cells engineered to express NICE constructs may act as autografts to enable external control of cell function. These applications are distant but not inconceivable and the studies proposed may form the foundation for the clinical use of nanoparticles.