Microfluidic-Enabled Intracellular Delivery of Membrane Impermeable Inhibitors to Study Target Engagement in Human Primary Cells

Abstract

Biochemical screening is a major source of lead generation for novel targets. However, during the process of small molecule lead optimization, compounds with excellent biochemical activity may show poor cellular potency, making structure-activity relationships difficult to decipher. This may be due to low membrane permeability of the molecule, resulting in insufficient intracellular drug concentration. The Cell Squeeze platform increases permeability regardless of compound structure by mechanically disrupting the membrane, which can overcome permeability limitations and bridge the gap between biochemical and cellular studies. In this study, we show that poorly permeable Janus kinase (JAK) inhibitors are delivered into primary cells using Cell Squeeze, inhibiting up to 90% of the JAK pathway, while incubation of JAK inhibitors with or without electroporation had no significant effect. We believe this robust intracellular delivery approach could enable more effective lead optimization and deepen our understanding of target engagement by small molecules and functional probes.

Cell engineering with microfluidic squeezing preserves functionality of primary immune cells in vivo

“…To address the potential consequences and concerns associated with subjecting cells to electric fields, we characterized the impact of well-established electroporation treatments on a transcriptional, translational, and phenotypic level. We also compared electroporation to a newer-generation microfluidic system, cell squeezing ( 24 – 28 ). We selected two cell types that are common targets for gene engineering: HSCs and T cells.…”

Controllable Cell Deformation Using Acoustic Streaming for Membrane Permeability Modulation

“…[ 1–4 ] Exogenous mechanical stimulation can regulate the growth, [ 5 ] differentiation, [ 6 ] intercellular signaling of cells, [ 7 ] providing important guidance for tissue engineering and regenerative medicine. [ 8,9 ] Besides, controllable force exertion and cell extrusion are demonstrated to have profound effects on transmembrane transportation by producing transient disruptions on the membrane, [ 10,11 ] which can be used for drug targeting [ 12 ] and gene engineering. [ 13 ] However, details on the mechanism of cell deformation process and the relationship between deformation and membrane permeability are still less studied.…”

Recent Developments in Cell Permeable Deubiquitinating Enzyme Activity-Based Probes

“…Each of these approaches also present significant challenges for extended periods of live cell profiling, and are largely inapplicable to whole organism analysis (Shi et al, 2018). CellSqueeze technology, which uses a commercial microfluidics device and pressure system to open membrane pores, may provide an alternative approach to garner higher value from cell-impermeable probes (Szeto et al, 2015;Li et al, 2017). Whether this would improve DUB coverage or better preserve cell integrity remains to be seen, and the method may be difficult to scale.…”