Role of actin cytoskeleton in cargo delivery mediated by vertically aligned silicon nanotubes

Abstract

Nanofabrication technologies have been recently applied to the development of engineered nano–bio interfaces for manipulating complex cellular processes. In particular, vertically configurated nanostructures such as nanoneedles (NNs) have been adopted for a variety of biological applications such as mechanotransduction, biosensing, and intracellular delivery. Despite their success in delivering a diverse range of biomolecules into cells, the mechanisms for NN-mediated cargo transport remain to be elucidated. Recent studies have suggested that cytoskeletal elements are involved in generating a tight and functional cell–NN interface that can influence cargo delivery. In this study, by inhibiting actin dynamics using two drugs—cytochalasin D (Cyto D) and jasplakinolide (Jas), we demonstrate that the actin cytoskeleton plays an important role in mRNA delivery mediated by silicon nanotubes (SiNTs). Specifically, actin inhibition 12 h before SiNT-cellular interfacing (pre-interface treatment) significantly dampens mRNA delivery (with efficiencies dropping to 17.2% for Cyto D and 33.1% for Jas) into mouse fibroblast GPE86 cells, compared to that of untreated controls (86.9%). However, actin inhibition initiated 2 h after the establishment of GPE86 cell–SiNT interface (post-interface treatment), has negligible impact on mRNA transfection, maintaining > 80% efficiency for both Cyto D and Jas treatment groups. The results contribute to understanding potential mechanisms involved in NN-mediated intracellular delivery, providing insights into strategic design of cell–nano interfacing under temporal control for improved effectiveness.

The influence of dysfunctional actin on polystyrene-nanotube-mediated mRNA nanoinjection into mammalian cells

“…Interestingly, the untreated PSNTs were slightly compressed vertically after cell interfacing, in comparison to the actin inhibitor treated samples. Furthermore, the observation of broken SiNTs in our previous study treated by actin inhibitors were absent here, 14 where most of the PSNTs remained intact after cell interfacing. This is possibly due to the flexible nature of PSNTs to withstand force exerted by the cytoskeleton network that caused the buckling of SiNTs.…”

“…SiNT arrays have shown mRNA delivery efficiency of ∼87% for untreated cells, ∼85% for post-interface Cyto D-treated cells, and ∼81% for post-interface Jas-treated cells. 14 By contrast, PSNT-mediated delivery has shown delivery efficiencies of ∼83%, ∼46%, and ∼68% for untreated cells, post-interface Cyto D- and Jas-treated cells, respectively. Both SiNTs and PSNTs showed similar delivery efficiency for untreated cells based on Cy5 detection, but PSNTs showed limited GFP expression, possibly due to modulation of two independent input parameters: NT geometry and stiffness.…”

“…The SiNT arrays were fabricated by using e-beam lithography (EBL) and reactive ion etching (DRIE) as described in our previous work. 14 A thin film of octafluorocyclobutane (C 4 F 8 )—Teflon-like polymer—was deposited on the SiNT master as an anti-adhesive layer via plasma polymerization (Fig. 1a(i)).…”

“…The advancement of nanofabrication alongside with nanobiotechnology in recent years has enhanced the engineered nanobio cellular interfaces and widened their applications. [1][2][3][4][5][6] In particular, vertically aligned nanoneedles (NNs) of semiconducting, inorganic, and polymeric materials-such as nanowires, [7][8][9][10] nanostraws, 11,12 nanotubes (NTs), [13][14][15][16] and their electroactive analogues, [17][18][19] -have been shown to be promising tools for complex cellular manipulation, such as mechanotransduction, [20][21][22][23][24][25] biosensing, 26,27 immunomodulation, 28 in vivo and ex vivo gene editing, [29][30][31][32] intracellular delivery, [33][34][35][36][37][38][39] and even cellular reprogramming. [40][41][42] Fabrication complexity [43][44][45][46]…”

Engineering Efficient CAR‐T Cells via Electroactive Nanoinjection

“…[1,10,20,21] The drawbacks associated with viral and BEP approaches have inspired the development of nanoscale technologies that have shown to efficiently negotiate plasma membrane barriers with minimal perturbation and toxicity to the cells. [22][23][24][25] These include high-aspect-ratio vertically configured nanostructure arrays-nanowires, [26][27][28][29][30][31][32][33][34] nanostraw, [35][36][37][38][39][40][41][42][43][44][45][46][47] and nanotubes [48][49][50][51] (NWs, NSs, and NTs). The unique topological morphologies of these high-aspect-ratio structures allow for intracellular access and delivery of targeted cargos by generating highly localized stress on the cell membrane (mechanical forces), with minimal impact on cell viability.…”