Multichannel Electrosonic Actuation Microarray for Cell-Based Screening

描述(由申请人提供)：高通量、并行传递和将生物相关材料导入细胞仍然是一项艰巨的任务。这项拟议的工作通过将多个电声驱动微阵列(EAMS)集成到单个MIST(多个集成样本治疗)设备中来解决这一问题，该设备优化了并行药物和/或基因/核酸的输送，并通过精确控制生物物理行为(即同时进行声/机械操作和电穿孔)将其导入多个细胞样本。该设备格式与现有的大型电池处理设备兼容。EAM是一种支持微电子机械系统(MEMS)的设备，它通过带有内置电穿孔电极的微型(单个细胞大小的)喷嘴喷射包含生物细胞的样品，从而通过联合机械/电穿孔在细胞膜上打开孔洞，以吸收纳米材料。操作的高超声频率和阵列格式使快速处理大细胞群(高达每秒数百万个细胞)成为可能。一个单一的储罐系统可以容纳大范围的规定体积，从~100 NL到任意大的样本量。通过将EAM排列在一起，MIST可以同时并行和均匀地处理几个不同的样品。经济的制造使设备的流体处理部件可以一次性使用，从而消除了交叉样品污染。MIST适用于基础/应用研究以及诊断和治疗用途。MIST细胞处理的设计、开发和评估将建立在通过表征多模式台式规模原型STEAM(通过电声驱动微阵列进行单一样品处理)所获得的经验的基础上。对EAM治疗过程中某些细胞类型的生物效应的了解不断加深，将有助于向多通道设备的过渡。通过同时筛选合成小干扰RNA(SiRNA)对细胞功能的不同影响，MIST的开发有可能极大地加速癌症治疗的发现。证明我们方法的可行性将为MIST在其他市场的商业化提供机会。第一阶段SBIR的主要目标是将我们的单样本STEAM平台扩展到多样本雾化设备中，并展示多个生物分子在不同细胞样本中的并行传递和转染。为了实现这一目标，(1)将开发一个针对细胞处理进行优化的MIST平台，以及(2)将使用标准细胞分析、共聚焦荧光显微镜和流式细胞术评估MIST在活性和增殖、处理的一致性和转染性方面的安全处理。此外，雾化处理将与传统的脂质体介导和基于电穿孔的转染法进行比较。 公共卫生相关性：MIST(多重综合样本处理)平台的开发将满足目前对高通量、并行输送和将生物相关材料导入细胞的需求。此外，由于该设备格式与现有的大规模细胞处理设备(例如，384孔板系统)兼容，因此它有可能通过高效筛选大的互补DNA(CDNA)和小干扰RNA(SiRNA)文库来加速发现潜在的癌症治疗方法。利用MIST同时转染编码荧光蛋白的多个不同DNA质粒，将证明其在广泛的药物开发和基因治疗应用中的可行性。

DESCRIPTION (provided by applicant): High-throughput, parallel delivery and transfection of biologically relevant material into cells remains a difficult task. The proposed work addresses this issue through integration of multiple Electrosonic Actuation Microarrays (EAMs) into a single MIST (Multiple Integrated Sample Treatment) device that is optimized for parallel drug and/or gene/nucleic acid delivery and transfection into several cell samples via precise control of biophysical action (i.e., concurrent sono/mechanoporation and electroporation). The device format is compatible with existing large-scale cell-handling equipment. The EAM is a microelectromechanical systems (MEMS)-enabled device that ejects a sample containing biological cells through microscopic (of order size of a single cell) nozzles with incorporated electroporation electrodes, thereby opening pores in the cell membrane via combined mechano/electroporation for uptake of nanomaterials. The high ultrasonic frequency of operation and array format enable fast processing of large cell populations (up to millions of cells per second). A single reservoir system can accommodate a wide range of prescribed volumes, from ~100 nL to arbitrarily large sample sizes. By arraying EAM together, MIST enables parallel and uniform treatment of several different samples simultaneously. Economical fabrication enables fluid handling components of the device to be made disposable, which eliminates cross-sample contamination. MIST is suited to basic/applied research, as well as diagnostic and therapeutic uses. Design, development and evaluation of cell treatment by MIST will build on experience gained through characterization of a multimode bench-top-scale prototype, STEAM (Single-sample Treatment via Electrosonic Actuation Microarray). A growing understanding of the bioeffects induced in certain cell types during EAM treatment will facilitate the transition to a multichannel device. MIST development has the potential to greatly accelerate discovery of cancer therapies by enabling simultaneous screening of synthetic small interfering RNA (siRNA) for different effects on cell function. Demonstrating the feasibility of our approach will open opportunities for commercialization of MIST in other markets. The primary objective of this Phase I SBIR is to expand our single-sample STEAM platform into the multi-sample MIST device and to demonstrate parallel delivery and transfection of multiple biomolecules into different cell samples. To achieve this objective, (1) a MIST platform that is optimized for cell treatment will be developed, and (2) safe handling with regard to viability and proliferation, uniformity of treatment, and the transfection capabilities of MIST will be evaluated using standard cell assays, confocal fluorescence microscopy and flow cytometry. In addition, MIST treatment will be compared with conventional lipofectamine-mediated and electroporation-based transfection. PUBLIC HEALTH RELEVANCE: Development of a MIST (Multiple Integrated Sample Treatment) platform will address the current need for high-throughput, parallel delivery and transfection of biologically relevant material into cells. Further, because the device format is compatible with existing large-scale cell handling equipment (e.g., 384-well plate-based systems), it has the potential to accelerate discovery of potential cancer therapies through efficient screening of large complementary DNA (cDNA) and small interfering RNA (siRNA) libraries. Simultaneous transfection of multiple different DNA plasmids encoding fluorescent proteins using MIST, will demonstrate its feasibility in a wide range of drug discovery and gene therapy applications.