Harnessing the perinuclear actin cap (pnAC) to influence nanocarrier trafficking and gene transfection efficiency in skeletal myoblasts using nanopillars

Abstract

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.

Introduction

Gene transfection is a technique that introduces genetic material into target cells to change the properties of the cell. This technique can change cell fate, including triggering apoptosis of cancer cells [1,2], eliciting the immune response [3], producing growth factors and/or cytokines [4], disease treatment [5,6] and so on. Due to the enormous potential of gene transfection, researchers have made a lot of effort to enhance the efficiency of this process. Gene delivery can be achieved using different approaches, including microinjection, a gene gun, electroporation, and nanocarriers [7,8]. Among these methods, non-viral vectors have been suggested as promising candidates in clinical aspects due to lower costs and better safety profiles [7,9].

Gene transfection in cells grown on tissue culture polystyrene (TCPS) is very different from the process in vivo. Gene transfection in tissues is more complex and challenging than in vitro. The main reasons include high cell densities, low cell proliferation rates, and the complex cell behavior occurring on the extracellular matrix (ECM) that can affect endocytosis and intracellular trafficking. Gene transfection of adherent cells can potentially reveal the complex mechanisms involved in in vivo transfection since the majority of the cells are anchor-dependent. Cell morphology or, more accurately, cytoskeleton distribution may be essential factors in vitro and in vivo gene transfection [10].

Substrate properties, including chemistry, stiffness, and topography, play a vital role in regulating cell behavior [11], which in turn, changes gene transfection efficiency [12]. For substrate chemistry, Segura et al. found that the endocytosis pathway and intracellular tension were affected by type I collagen (COLI) or fibronectin (FN) coatings. The transfection of mesenchymal stem cells (MSCs) was inhibited on COLI surfaces but promoted on the FN surfaces [13]. Pannier et al. reported that the transfection of cells on hydrophilic carboxylic surfaces (i.e., -COO⁻) was increased, whereas on a hydrophobic methyl surface (i.e., -CH₃) it decreased. Higher cell densities, more cell spreading with ellipsoid morphologies, and enhanced quantities of focal adhesions leading to higher transfection efficiency were found on carboxylic surfaces [14]. Substrate stiffness can regulate the cytoskeleton resulting in an enhancement of the internalization efficiency of plasmids in human adipose-derived stem cells (hASCs) [15]. The greater existence of well-aligned hASCs actin fibers on the stiffer substrates can increase the endocytosis of Lipofectamine 2000. Nanotopographies with a high aspect ratio can increase cell spreading and the nuclear volume of human primary fibroblasts through focal adhesion rearrangement. This type of cell morphology is suitable for gene transfection [16]. Well-spread and elongated hMSCs on micropatterns have been shown to enhance uptake of Lipofectamine 2000 accelerating DNA synthesis leading to a higher transfection efficiency [17]. These studies suggested that strategies based on optimsing the properties of substrates can affect non-viral gene delivery into cells.

Nevertheless, the specific role of the cytoskeleton played in gene transfection is not fully understood. Previously, we have shown that nanogrooves can modulate cytoskeleta of cells, which in turn changes nucleus shape and subsequent gene transfection efficiency [18]. In this study, we further investigated the effect of the cytoskeleton on intracellular trafficking using two types of nanocarriers; cationic polymer-based reagents (i.e., jetPRIME and jetPEI) and a lipid-based reagent (i.e., Lipofectamine 3000). Various nanopillars fabricated using colloidal lithography were used for harnessing the properties and the cytoskeleton and focal adhesions, and the shape of the nucleus. The results showed that cytoskeletal distribution is one of the pivotal factors in gene transfection. This study provides useful information on in vitro gene transfection, which is potentially applicable to in vivo applications.