A Low-Backpressure Single-Cell Point Constriction for Cytosolic Delivery Based on Rapid Membrane Deformations

摘要

Mechanically deforming biological cells through microfluidic constrictions is a recently introduced technique for the intracellular delivery of macromolecules possibly through transient membrane pores induced in the process. The technique is attractive for research and clinical applications mainly because it is simple, fast, and effective while being free of adverse effects often associated with well-known techniques that rely on field- or vector-based delivery. In this nascent approach, an utmost and crucial role is played by the constriction, often in rectangular profile, and it squeezes cells only in one dimension. The results achieved suggest that the longer the constriction is the higher the delivery performance. Contrary to this view, we demonstrate here a unique constriction profile that is highly localized (point) and yet returns comparably effective delivery. Point constrictions are of a semiround geometry, forcing cells in both dimensions while introducing very little backpressure to the system, which is a silicon-glass platform wherein constrictions are arranged in series along an array of channels. The influence of the constriction size and count as well as treatment pressure on delivery performance is presented on the basis of the flow-cytometric analyses of HCT116 cells treated using dextran as model molecules. Delivery performance is also presented for common mammalian cell lines including NIH 3T3, HEK293, and MDCK. Moreover, the versatility of the platform is demonstrated in gene knockdown experiments using synthetic siRNA as well as on the delivery of proteins. Target proteins in some cells exhibit nondiffusive distribution profile raising the plausibility of mechanisms other than transient membrane pores.