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

The advancement of nanofabrication technologies has transformed the landscape of engineered nano-bio interfaces, especially with vertically aligned nanoneedles (NNs). This enables scientists to venture into new territories, widening NN applications into increasingly more complex cellular manipulation and interrogation. Specifically, for intracellular delivery application, NNs have been shown to mediate the delivery of various bioactive cargos into a wide range of cells-a physical method termed "nanoinjection". Silicon (Si) nanostructures demonstrated great potential in nanoinjection, whereas the use of polymeric NNs for nanoinjection has rarely been explored. Furthermore, the underlying mechanism of interaction at the cell-NN interface is subtle and multifaceted, and not fully understood-underpinned by the design versatility of the NN biointerface. Recent studies have suggested that actin dynamic plays a pivotal role influencing the delivery efficacy. In this study, we fabricated a new class of NNs-a programmable polymeric nanotubes (NTs)-from polystyrene (PS) cell cultureware, designed to facilitate mRNA delivery into mouse embryonic fibroblast GPE86 cells. The PSNT delivery platform was able to mediate mRNA delivery with high delivery efficiency (∼83%). We also investigated the role of actin cytoskeleton in PSNTs mediated intracellular delivery by introducing two actin inhibitors-cytochalasin D (Cyto D) and jasplakinolide (Jas)-to cause dysfunctional cytoskeleton, via inhibiting actin polymerization and depolymerization, respectively (before and after the establishment of cell-PSNT interface). By inhibiting actin dynamics 12 h before cell-PSNT interfacing (pre-interface treatment), the mRNA delivery efficiencies were significantly reduced to ∼3% for Cyto D-treated samples and ∼1% for Jas-treated sample, as compared to their post-interface (2 h after cell-PSNT interfacing) counterpart (∼46% and ∼68%, respectively). The added flexibility of PSNTs have shown to help withstand mechanical breakage stemming from cytoskeletal forces in contrast to the SiNTs. Such findings will step-change our capacity to use programmable polymeric NTs in fundamental cellular processes related to intracellular delivery.