Cell-targeted Gold Nanoparticles for Photo-excitation fo Retinal Ganglion Cells

摘要 视网膜神经节细胞(RGC)以动作电位的形式向大脑传递视觉信号， 轴突的传播依赖于RGC电压门控钠通道(NAV)的激活。在……里面 光感受器退行性疾病，如老年性黄斑变性(AMD)，视网膜内神经元 在许多情况下，包括视网膜节细胞在内的细胞保持完好，能够产生动作电位反应。因此，RGC 代表了旨在恢复晚期AMD及相关视网膜视力的方法的合理目标 疾病，通过绕过不起作用的视杆和视锥感光细胞，建立直接的RGC 对光的反应。在最近对背根神经节细胞(一种被广泛研究为 模型动作电位产生细胞类型)，在海马片制备中，我们已经证明了金 纳米颗粒(AuNPs)与细胞靶向生物分子结合可实现强大的光诱导NAV激活 以及由此产生的动作电位产生。细胞靶向AuNP技术的基本特征是：(I) 吸收光的AuNPs的功能化以将它们定位在导航系统或其附近；(Ii)基于AuNP 等离子体对毫秒/亚毫秒闪光的吸收，AuNP辐射的光能 产生局部的、瞬时的、去极化的电容电流的无损热脉冲 质膜；以及(Iii)引起的激活，即相邻NAV的通道开放，从而起作用 这种去极化可能会引发。在这一应用中，我们提出了探索性研究来应用该AuNP 大鼠活体眼内视网膜节细胞的观察。该项目的目标是建立AuNP治疗条件， 在体内实现强大的AuNP介导的RGC光响应。在大鼠的视杆和视锥中 视网膜神经节细胞的光感受器信号已经被药物抑制，我们将在玻璃体内输送AuNP 设计用于与RGC导航系统的紧邻区域结合的连接物。在使用AuNP治疗后 结合物，我们将使用活体记录与RGC相关的视网膜电信号(ERG) 活动和视觉诱发电位(VEP)以分析RGC电生理反应的特性 到AuNP光激发。这项研究将涉及AuNP的大小/结构的变化，AuNP的- 共轭RGC靶向成分，以及AuNP的持续时间和能量[(强度)x(持续时间)]- 兴奋的闪光。伴随体内实验的将是AuNP的全光刺激/记录- 大鼠离体视网膜介导性动作电位及体内、外处理视网膜的组织学分析 使用AuNPs。体外视网膜实验的结果将指导体内治疗条件的选择 对活体数据进行系统的调查和解释。领导研究的将是 David R.Pepperberg博士(大学伊利诺伊州芝加哥分校)和Francisco Bezanilla博士(大学芝加哥)。

ABSTRACT Retinal ganglion cells (RGCs) convey visual signals to the brain in the form of action potentials, the initiation and axonal propagation of which depend on the activation of RGC voltage-gated sodium channels (NaVs). In photoreceptor degenerative diseases such as age-related macular degeneration (AMD), inner retinal neurons including RGCs in many cases remain intact and capable of generating action potential responses. RGCs thus represent a logical target for approaches aimed at restoring vision in advanced-stage AMD and related retinal diseases, by bypassing the nonfunctioning rod and cone photoreceptors and establishing direct RGC responsiveness to light. In a recent study of dorsal root ganglion cells (a non-retinal neuron widely studied as a model action-potential-generating cell type) and in hippocampal slice preparations, we have shown that gold nanoparticles (AuNPs) conjugated with a cell-targeting biomolecule enable robust light-induced NaV activation and resulting action potential generation. Essential features of the cell-targeted AuNP technique are: (i) functionalization of the light-absorbing AuNPs to localize them at or near the NaVs; (ii) upon the AuNP’s plasmon absorption of a millisecond/submillisecond light flash, AuNP radiation of the light energy as a nondamaging pulse of heat that creates a localized, transient, depolarizing capacitive current across the plasma membrane; and (iii) resulting activation, i.e., channel opening, of neighboring NaVs and thus action potential initiation by this depolarization. In this application we propose exploratory research to apply this AuNP approach to RGCs in the living eye of the rat. The project’s goal is to establish AuNP treatment conditions that achieve robust AuNP-mediated RGC photo-responsiveness in vivo. In rats for which rod and cone photoreceptor signaling to RGCs has been suppressed pharmacologically, we will intra-vitreally deliver AuNP conjugates designed for binding to the immediate vicinity of the RGC NaVs. Following treatment with the AuNP conjugates, we will employ in vivo recording of electroretinographic (ERG) signals associated with RGC activity, and of visual evoked potentials (VEPs), to analyze properties of RGC electrophysiological responses to AuNP photo-excitation. The research will involve variation of the size/structure of the AuNP, of the AuNP- conjugated RGC-targeting component, and of the duration and energy [(intensity) x (duration)] of AuNP- excitatory flashes. Accompanying the in vivo experiments will be all-optical stimulation/recording of AuNP- mediated action potentials in isolated rat retina, and histological analysis of retinas treated in vivo and in vitro with AuNPs. Results of the in vitro retina experiments will guide the selection of in vivo treatment conditions to be systematically investigated and facilitate interpretation of the in vivo data. Leading the research will be David R. Pepperberg, PhD (Univ. of Illinois at Chicago) and Francisco Bezanilla, PhD (Univ. of Chicago).