Abstract
Owing to their ability to efficiently deliver biological cargo and sense the intracellular milieu, vertical arrays of high aspect ratio nanostructures, known as nanoneedles, are being developed as minimally invasive tools for cell manipulation. However, little is known of the mechanisms of cargo transfer across the cell membrane-nanoneedle interface. In particular, the contributions of membrane piercing, modulation of membrane permeability and endocytosis to cargo transfer remain largely unexplored. Here, combining state-of-the-art electron and scanning ion conductance microscopy with molecular biology techniques, it is shown that porous silicon nanoneedle arrays concurrently stimulate independent endocytic pathways which contribute to enhanced biomolecule delivery into human mesenchymal stem cells. Electron microscopy of the cell membrane at nanoneedle sites shows an intact lipid bilayer, accompanied by an accumulation of clathrin-coated pits and caveolae. Nanoneedles enhance the internalization of biomolecular markers of endocytosis, highlighting the concurrent activation of caveolae- and clathrin-mediated endocytosis, alongside macropinocytosis. These events contribute to the nanoneedle-mediated delivery (nanoinjection) of nucleic acids into human stem cells, which distribute across the cytosol and the endolysosomal system. This data extends the understanding of how nanoneedles modulate biological processes to mediate interaction with the intracellular space, providing indications for the rational design of improved cell-manipulation technologies.
Highlights
The intimate molecular biology techniques, it is shown that porous silicon nanoneedle arrays concurrently stimulate independent endocytic pathways which contribute to enhanced biomolecule delivery into human mesenchymal stem cells
Electron microscopy of the cell membrane at nanoneedle sites shows an intact interface established with the cell membrane has enabled nanoneedles to sense proteins,[13] metabolites,[14] and lipids[15] in the intracellular milieu as well as to stimulate and record action potentials of large lipid bilayer, accompanied by an accumulation of clathrin-coated pits and arrays of individual excitable cells.[7,16,17]
These events contribute to the nanoneedle-mediated delivery of nucleic acids into human stem cells, which distribute across the cytosol and the endolysosomal system
Summary
Owing to their ability to efficiently deliver biological cargo and sense the nanoneedles,[3,4,5,6,7,8,9] have demonstrated a broad versatility to efficiently sense[2] and intracellular milieu, vertical arrays of high aspect ratio nanostructures, deliver[10] to the cell interior. Nanoneedles enhance the internalization of biomolecular markers of Nanoneedle arrays promise to be a safe and endocytosis, highlighting the concurrent activation of caveolae- and clathrinmediated endocytosis, alongside macropinocytosis. These events contribute to the nanoneedle-mediated delivery (nanoinjection) of nucleic acids into human stem cells, which distribute across the cytosol and the endolysosomal system. Effective platform for nucleic acid delivery that compares favorably with microinjection and electroporation, thanks to its ease of use, high throughput, elevated biocompatibility, and efficient delivery
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