High Throughput Screening (HTS) allows for the rapid comparison of many different compounds for potential lead candidates in a wide variety of diseases. It is generally separated into two types, biochemical or cell based assays. Biochemical assays rely on simplified systems with a single well defined target that correlate with disease. Alternatively, cell based assays rely on detecting a complex phenotypic change relevant to a disease state. Researchers have found that testing a compound's phenotypic effect should occur as early as possible to minimize wasted investment; however, currently the screening process often begins in biochemical assays. The cell based phenotypic assay is not as commonly used, especially if the target is intracellular, since the assay requires the compounds to be membrane permeable to be tested. As a result, biochemical assays not only progress compounds that are potentially non-specific to their target, but also likely miss many more compounds that have a phenotypic or genotypic response that is undetectable in biochemical assays. If one could remove this bias through the direct introduction of the compound into the intracellular space of living cells, one could effectively enable intracellular biochemical assays that can directly test the compound's activity in the complex environment of the cell. This novel approach to drug discovery could potentially lead to much greater productivity of existing chemical libraries and facilitate identification of drug candidates to hig value targets that have thus far been undruggable. Furthermore, new therapeutic modalities including siRNA, mRNA, peptides and nucleases are often membrane impermeable and therefore must be chemically altered or incorporated into a delivery vehicle for effective delivery Our proposed project could enable the direct intracellular delivery of these materials for high throughput testing of activity while eliminating concerns surrounding delivery vector toxicity and off-target effects. The principle underlying this approach is temporary membrane disruption by rapid mechanical deformation, or squeezing, of cells to facilitate uptake of loading material in the fluid medium. Through the use of microfluidics and a novel workflow aided by robotic precision we present preliminary data to scale this technique to HTS applications. We also share preliminary data demonstrating that our platform is capable of delivering a wide range of materials and cell types. We hypothesize that the proposed HTS platform be applied to screen new drug candidates. The key objectives of this project are to: (1) Develop a modified microfluidic chip and workflow able to interface with existing liquid handlers to deliver membrane impermeable dyes intracellularly in a 384 well plate format and (2) Demonstration of the delivery of small molecules, siRNA and Ab to HeLa cells and primary human T-cells. Our goal is the development of a platform that can dramatically improve our ability to identify and elucidate the phenotypic potential of active compounds.
Our current ability discover new therapeutic agents is limited by our current high throughput screening assays which cannot easily determine the intracellular activity of a potential compound. This thus biases the materials that are passed on for further development and therapeutic use. We propose to break this paradigm and commercialize a promising new, microfluidics-based, delivery platform that relies on the temporary disruption of the cell membrane to facilitate delivery directly into the cell cytoplasm to measure intracellular activity. Our extensive preliminary data indicate that the proposed technology can overcome many of the disadvantages of existing delivery technologies and could be integrated into the high throughput screening robotic workflows.
