Novel nanotechnology-based optical stimulation platform for predictive cardiotoxicity assessment

摘要 目前，每个新处方药审批的估计行业平均成本现在超过25亿美元。 导致药物开发成本上升的关键因素之一是Pre-Pre的重点不是最佳的 检测新药候选药物潜在心脏负担的临床研究。认识到这一事实， 生物医学界最近提出了心脏安全性研究的新范式： 体外心律失常分析(CIPA)倡议。它包括从过度简化的HERG专注于心脏毒性的转变 影响人体干细胞动作电位的多离子通道药物效应的筛选 细胞源性心肌细胞(CMS)。基于光学细胞的药物筛选分析可以监测药物的动态变化 干细胞来源的CMS的细胞内钙浓度和细胞膜电位非常适合于 检测有心脏负担的候选药物。由于药物诱导的效应通常依赖于 细胞的激活状态，在药物筛选过程中能否刺激CMS是至关重要的。 我们建议通过提供革命性的基于纳米技术的非侵入性平台来满足这一需求 CMS的光刺激。我们设计了这个光电平台，利用独一无二的 石墨烯是一种“21世纪的神奇材料”。这些特性确保了出色的 石墨烯涂层衬底的生物兼容性，高效将光转换为电能的能力，以及 与光学检测方法兼容。我们的初步结果表明，光可以诱导快速和 石墨烯涂层对干细胞来源CMS收缩和电活动可逆性的影响 底物。此外，我们证实了通过G涂层衬底的光刺激可以实现全光 评价药物使用依赖对CM活性的影响。 简化客户对我们基于石墨烯的光学刺激平台的采用并将其整合 在光学药物筛选分析中，我们建议进行以下活动：a)开发 石墨烯沉积到细胞培养多孔板中的程序，随后制造高质量 石墨烯涂层板材；b)优化电压和钙全光分析的各种参数(包括 双光刺激方案)在定制的显微镜系统上；c)传输优化的全光学分析 到备受好评的高通量和高含量成像仪器，并进行多个 光刺激CMS上的基准化合物。我们预计通过石墨烯对CMS的光学刺激- 基础底物将极大地提高心脏毒性筛选试验的预测价值。建议数 全光学分析将有助于在药物早期正确检测真正的促心律失常风险 发现过程，从而减少了费用和药物开发时间。

ABSTRACT The estimated average industry cost per new prescription drug approval currently now exceeds $2.5 billion. One of the critical factors contributing to the rising costs of drug development is a non-optimal focus of pre- clinical studies for detecting potential cardiac liabilities of novel drug candidates. Recognizing this fact, the biomedical community has recently proposed a new paradigm for cardiac safety studies: the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative. It includes a shift from over-simplified hERG-focused cardiotoxicity screening to evaluation of drug effects on multiple ion channels contributing to action potential in human stem cell-derived cardiomyocytes (CMs). Optical cell-based drug screening assays that monitor the dynamics of intracellular calcium concentrations and the cell membrane potential in stem cell-derived CMs are well-suited for detecting drug candidates with cardiac liabilities. Since drug-induced effects are often dependent on the activation state of cells, it is crucial to be able to stimulate CMs during drug screening. We propose to address this need by offering a revolutionary nanotechnology-based platform for non-invasive optical stimulation of CMs. We designed this optoelectronic platform by taking advantage of the unique properties of graphene, a “wonder material of the 21th century”. These properties ensure excellent biocompatibility of graphene-coated substrates, the ability to efficiently convert light into electricity, and compatibility with optical detection methods. Our preliminary results demonstrate that light can induce fast and reversible changes in contractile and electrical activity of stem cell-derived CMs cultured on graphene-coated substrates. Furthermore, we confirmed that optical stimulation via G-coated substrates can enable all-optical evaluation of use-dependent drug effects on the CM activity. To streamline the customer adoption of our graphene-based optical stimulation platform and incorporation it in into optical drug-screening assays, we are proposing to perform the following activities: a) developing a procedure for graphene deposition into cell culture multi-well plates, following by manufacturing of high-quality graphene-coated plates; b) optimizing the various parameters of voltage and calcium all-optical assays (including a dual-light stimulation protocol) on a customized microscopy system; c) transferring optimized all-optical assays to well-regarded high-through and high-content imaging instruments and performing screening of multiple benchmark compounds on optically stimulated CMs. We expect that optical stimulation of CMs via graphene- based substrates will dramatically increase the predictive value of cardiotoxicity screening assays. The proposed all-optical assays will be instrumental in correctly detecting genuine pro-arrhythmia risks early in the drug discovery process, thus reducing the spending and drug development time.