Gene transfection is important in biotechnology and is used to modify cells intrinsically. It can be conducted in cell suspension or after cell adhesion, where the efficiency is dependent on many factors such as the type of nanocarrier used and cell division processes. Anchor-dependent cells are sensitive to the substrate they are attached to and adapt their behavior accordingly, including plasmid trafficking during gene transfection. Previously, it was shown in our group that the cytoskeleton is an essential factor in influencing gene transfection in skeletal myoblasts using nanogrooves as a substrate. In this study, the effect of the cytoskeleton on gene transfection efficiency of skeletal myoblasts was studied using various nanopillars and nanocarriers. Nanopillars with different diameters (200-1000 nm) and depths (200 or 400 nm) were fabricated using colloidal self-assembly and reactive ion etching. All surfaces were treated with oxygen plasma or polydopamine (PD) to further control cell morphology. Plasmid DNA was delivered into cells using jetPRIME or Lipofectamine 3000 nanocarriers. After screening hundreds of images, two distinguishable F-actin distributions were found, i.e., cells with or without a perinuclear actin cap (pnAC). Cells attached to nanopillars, especially the deep pillars, had a smaller spreading area, shorter F-actin, more 3D-like cell nuclei, and a lower percentage of pnAC, which lead to a higher gene transfection efficiency using jetPRIME. On the other hand, cells attached to the shallow nanopillars or flat surfaces had a larger spreading area, longer F-actin, more 2D-like cell nuclei, and a higher percentage of pnAC that facilitates gene transfection using Lipofectamine. The effects of cell density, cytoskeleton (cytoD), and focal adhesions (RGD) on gene transfection were also studied, and the results were consistent with our hypothesis that F-actin distribution is one of the critical factors in gene transfection. In conclusion, pnAC plays a vital role in the intracellular trafficking of nanocarrier/plasmid complexes and this study provides new insights into gene transfection in anchor-dependent cells. STATEMENT OF SIGNIFICANCE: This study provides a new perspective in gene transfection using attached cells where perinuclear actin cap (pnAC) is an essential factor involved in transfection efficiency. A series of nanopillars were used to harness cell and cytoskeleton morphology. Two distinguishable cytoskeletal structures were found including cells with or without pnAC. 2D-like cells with pnAC facilitate gene delivery using liposome-based nanocarriers, while 3D-like cells without pnAC benefit gene delivery using cationic polymer-based nanocarriers. This study reveals the importance of the cytoskeleton during gene transfection that is beneficial in tissue transfection.
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