Silicon‐Nanotube‐Mediated Intracellular Delivery Enables Ex Vivo Gene Editing

Abstract

Engineered nano–bio cellular interfaces driven by vertical nanostructured materials are set to spur transformative progress in modulating cellular processes and interrogations. In particular, the intracellular delivery—a core concept in fundamental and translational biomedical research—holds great promise for developing novel cell therapies based on gene modification. This study demonstrates the development of a mechanotransfection platform comprising vertically aligned silicon nanotube (VA‐SiNT) arrays for ex vivo gene editing. The internal hollow structure of SiNTs allows effective loading of various biomolecule cargoes; and SiNTs mediate delivery of those cargoes into GPE86 mouse embryonic fibroblasts without compromising their viability. Focused ion beam scanning electron microscopy (FIB‐SEM) and confocal microscopy results demonstrate localized membrane invaginations and accumulation of caveolin‐1 at the cell–NT interface, suggesting the presence of endocytic pits. Small‐molecule inhibition of endocytosis suggests that active endocytic process plays a role in the intracellular delivery of cargo from SiNTs. SiNT‐mediated siRNA intracellular delivery shows the capacity to reduce expression levels of F‐actin binding protein (Triobp) and alter the cellular morphology of GPE86. Finally, the successful delivery of Cas9 ribonucleoprotein (RNP) to specifically target mouse Hprt gene is achieved. This NT‐enhanced molecular delivery platform has strong potential to support gene editing technologies.

Emerging Roles of 1D Vertical Nanostructures in Orchestrating Immune Cell Functions

“…[ 36,116,163 ] Using fluorescence staining and confocal imaging, components of multiple endocytic pathways such as clathrin light chain, caveolin‐1, DNM2, and CLTA have also been observed to coassemble at highly curved cell membranes along VNS (Figure 9e). [ 12,36,116,167 ] An inhibition study treated GPE86 cells with nystatin and chloropromazine, finding that nystatin (which suppresses formation of lipid rafts, cholesterol‐enriched domains, and caveolae) caused greater impairment of Si nanotube‐mediated mRNA delivery than chloropromazine (which inhibits clathrin‐mediated endocytosis and interferes with fast endophilin‐mediated endocytosis). [ 12 ] But for primary immune cells, especially for lymphocytes, endocytic pathways are often carefully gated via foreign element detectors such as TLR.…”

“…[ 12,36,116,167 ] An inhibition study treated GPE86 cells with nystatin and chloropromazine, finding that nystatin (which suppresses formation of lipid rafts, cholesterol‐enriched domains, and caveolae) caused greater impairment of Si nanotube‐mediated mRNA delivery than chloropromazine (which inhibits clathrin‐mediated endocytosis and interferes with fast endophilin‐mediated endocytosis). [ 12 ] But for primary immune cells, especially for lymphocytes, endocytic pathways are often carefully gated via foreign element detectors such as TLR. [ 168 ] It was reported that delivery of genetic agents using SiNWs did not activate endocytic or inflammatory pathways.…”

“…Due to the complexity of cellular responses toward physical, mechanical, and biochemical signals, it is highly likely that multiple pathways are invoked in VNS‐mediated intracellular delivery (Figure 9f, Table 1 ). [ 6,8,12,14,111,125,169,170 ] A better understanding of regulation and crosstalk between different pathways would be extremely useful for designing advanced 1D‐VNS platforms to maximize transfection efficiency while inducing minimal cellular impairment.…”

“…[ 2–5 ] In particular, 1D vertical nanostructures (1D‐VNS)—nanowires, nanoneedles, nanopillars, nanotubes, nanosyringes, nanostraws, nanocones, and nanospikes ( Figure a)—have helped tackle various biological problems such as intracellular recording and genetic interrogation. [ 6–16 ] Unlike other high‐aspect‐ratio nanostructures, such as freestanding carbon nanotubes, 1D‐VNS can be rationally designed and synthesized, via top‐down and/or bottom‐up approaches, with defined key parameters—including topological geometry (pitch, diameter, length), chemical composition, doping, and electronic properties. [ 17–21 ] So by well‐controlled programming coupled with selective surface functionality, 1D‐VNS can provide unprecedented spatial and mechanical resolution to enable direct, sensitive, and rapid analysis of biological species and manipulation of cellular activities.…”

High Throughput and Highly Controllable Methods for In Vitro Intracellular Delivery

“…Nanostructure substrates ( Figure 2E) are a group of methods utilizing 1D structures such as nanoneedles, [107,110] nanowires, [111][112][113][114][115][116][117] nanotubes, [118,119] and nanostraws [120][121][122][123] with diameters small enough that when cells are adhered to them they either induce spontaneous penetration, alter membrane permeabilization, or elicit endocytosis for molecular delivery. There is an ongoing debate as to which of these mechanisms or combination of mechanisms is primarily responsible for delivery using nanostructures.…”