Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations, including their reliance on exogenous materials or electrical fields, which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30-80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells. When used for the delivery of transcription factors, the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed, its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications.drug delivery | induced pluripotent stem cells | reprogramming | protein delivery | nanoparticle delivery I ntracellular delivery of macromolecules is a critical step in therapeutic and research applications. Nanoparticle-mediated delivery of DNA and RNA, for example, is being explored for gene therapy (1, 2), while protein delivery is a promising means of affecting cellular function in both clinical (3) and laboratory (4) settings. Other materials, such as small molecules, quantum dots, or gold nanoparticles, are of interest for cancer therapies (5, 6), intracellular labeling (7,8), and single-molecule tracking (9).The cell membrane is largely impermeable to macromolecules. Many existing techniques use polymeric nanoparticles (10, 11), liposomes (12), or chemical modifications of the target molecule (13), such as cell-penetrating peptides (CPPs) (14, 15), to facilitate membrane poration or endocytotic delivery. In these methods, the delivery vehicle's efficacy is often dependent on the structure of the target molecule and the cell type. These methods are thus efficient in the delivery of structurally uniform materials, such as nucleic acids, but often ill-suited for the delivery of more structurally diverse materials, such as proteins (16,17) and some nanomaterials (7). Moreover, the endosome escape mechanism that most of these methods rely on is often inefficient; hence, much material remains trapped in endosomal and lysosomal vesicles (18). More effective gene delivery methods, such as viral vectors (19,20), however, often risk chromosomal integration and are limited to DNA and RNA delivery.Membrane poration methods, such as electroporation (21, 22) and sonoporation (23), are an attractive alternative in some applications. Indeed, electroporation has demonstrated its efficacy in a number of DNA (24) and ...
