Porous Silicon Nanoneedles Modulate Endocytosis to Deliver Biological Payloads

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.

Cellular Deformations Induced by Conical Silicon Nanowire Arrays Facilitate Gene Delivery

“…Figure a illustrates two distinct patterns of how SiNWs interact with GPE86 cells. An individual SiNW is shown to breach the cell membrane and further indent the nucleus (Figure a‐i); the second SiNW is engulfed by a continuous intact plasma membrane, accompanied by increased membrane curvatures (red arrows), similar to clathrin‐coated pits and caveolae reported in recent studies (Figure a‐ii) 23a,31. Figure b manifests SiNW insertion into the cytoplasm of a L1.2 cell; Figure c displays the curving plasma membrane of Jurkat cells, with accumulation of multiple membrane invaginations and endocytic vesicles (red arrows) along the SiNWs.…”

“…Combined with FIB‐SEM imaging showing the membrane curvatures along SiNWs (Figure a,c), our results suggest that caveolar bulbs could accumulate during SiNW interfacing, which attenuates the increase in membrane tension and protects the plasma membrane from rupture . Caveolae are potential players enabling CavME and downstream cell signaling during cell–SiNW interaction …”

“…The observations by both confocal and FIB‐SEM suggest that direct penetration through membrane and nucleus might not be the prevalent, at least not the only, mechanism behind SiNW‐mediated intracellular delivery. To understand whether other mechanisms, such as endocytosis,23a,31 are involved, we investigated the role of two endocytic markers: clathrin heavy chain (CHC), a clathrin coat protein involved in CME; and caveolin‐1 (CAV‐1), a key protein for CavME. Plasmids tagged with a Cy3 fluorescence dye (Cy3‐gWiz‐GFP) were coated onto flat Si and SiNWs.…”

Immunogold FIB‐SEM: Combining Volumetric Ultrastructure Visualization with 3D Biomolecular Analysis to Dissect Cell–Environment Interactions

“…The ability to rapidly overview the cell–material interface over large areas by SEM imaging enables informed and accurate cell selection prior to milling. Such approaches have provided unprecedented insight into the ultrastructural changes that can occur as cells interface with a material; [12–15] however, conventional FIB-SEM still does not provide any biomolecular information (e.g., protein localization). This limitation can be partially addressed by correlative techniques, [16] in which samples are sequentially imaged by optical microscopy and FIB-SEM, and then superimposed to generate a reconstructed map.…”

Probing the Ultrastructure of Spheroids and Their Uptake of Magnetic Nanoparticles by FIB–SEM

“…The images in Figure E–H display the different phases occurred during the endocytosis of LMNVs. Phase I represents the first contact between the nanovectors and the outer membrane (contact) followed by the formation of membrane invaginations (phase II) and then the formation of specific (electron‐dense [ 26 ] ) clathrin‐coated pits (phase III). Figure 4H shows the final LMNVs internalization and their inclusion into vesicles (phase IV).…”