Abstract
Intracellular delivery of nanomaterials into the cells of interest has enabled cell manipulation for numerous applications ranging from cell-based therapies to biomedical research. To date, different carriers or membrane poration-based techniques have been developed to load nanomaterials to the cell interior. These biotools have shown promise to surpass the membrane barrier and provide access to the intracellular space followed by passive diffusion of exogenous cargoes. However, most of them suffer from inconsistent delivery, cytotoxicity, and expensive protocols, somewhat limiting their utility in a variety of delivery applications. Here, by leveraging the benefits of microengineered porous membranes with a suitable porosity, we demonstrated an efficient intracellular loading of diverse nanomaterials to different cell types based on inducing mechanical disruption to the cell membrane. In this work, for the first time, we used ultra-thin silicon nitride (SiN) filter membranes with uniform micropores smaller than the cell diameter to load impermeable nanomaterials into adherent and non-adherent cell types. The delivery performance using SiN microsieves has been validated through the loading of functional nanomaterials from a few nanometers to hundreds of nanometers into mammalian cells with minimal undesired impacts. Besides the high delivery efficiency and improved cell viability, this simple and low-cost approach offers less clogging and higher throughput (107 cell min−1). Therefore, it yields to the efficient introduction of exogenous nanomaterials into the large population of cells, illustrating the potential of these microengineered filters to be widely used in the microfiltroporation (MFP) setup.
Highlights
Operating conditions will trigger temperature increase and induce excessive stress and damage to the target cells as well as delivery biomaterials, limiting their implementation in most of the electroporation p rotocols[19–21]
While this approach offers simplicity and control over the membrane disruption process resulted in minimized cell death, it suffers from clogging issues and cell size dependency owing to specific device geometry lowering its practicality
Ery efficiency and cell viability, including delivery buffer and operational flow rate used to push the cells through the filter membranes
Summary
Operating conditions will trigger temperature increase and induce excessive stress and damage to the target cells as well as delivery biomaterials, limiting their implementation in most of the electroporation p rotocols[19–21]. Despite the success of MFP in cargo delivery with a stated transfection efficiency above 50%, it received much less attention until 2018 when Yen et al developed transmembrane internalization assisted by membrane filtration (TRIAMF) In this method, track-etched membrane filters were deployed to deliver the CRISPR/Cas[9] ribonucleoprotein complex targeting β2-microglobulin gene into the human hematopoietic stem c ells[23]. For the first time, we used SiN microsieves in the MFP setup for highly efficient cytosolic loading of nanomaterials into the mammalian cell lines without compromising the cell viability Utilizing these microengineered filters, we successfully optimized the delivery conditions for fluorescently labeled dextrans up to 2000 kDa. Since microfabricated SiN microsieves are ultra-thin (~ 1 μm), highly biocompatible, and rigid, less pressure and mechanical deformation are induced to the cells while passing through the uniform m icropores[26]. MFP using SiN membranes with regularly spaced pores demonstrates promising characteristics (up to 94.4% delivery efficiency, 98% cell viability, and high throughput ( 107 cell min−1)), which make it an attractive intracellular delivery platform that influences cell-based research
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