Exploiting in vitro mechanical stimulation of cells to refine the transfection efficiency of non-viral gene delivery vectors

Introduction: Non-viral gene delivery employs positively charged lipidic or polymeric vectors to drive exogenous genetic material into cells (a process called transfection) to alter specific cell functions [1]. Much effort has been made since their first introduction, though the presence of biological barriers that complexes must overcome still reduces their ultimate transfection efficiency (TE) [2]. In this scenario, we propose a new technology to boost the TE of gold standard non-viral polymeric vectors branched polyethyleneimine (bPEI) by modulating the cell behavior with cyclic mechanical strain. Methods: The stimulation device consists of an electro-mechanical actuator controlled by an Arduino microcontroller able to exert equibiaxial cyclic deformation to silicone cell culture chambers through the vertical displacement of a puncher (nominal deformation (NE) range:0-20 %; frequency (f) range: 0-2 Hz, step: 0.1 Hz) (Fig. 1A).The variation of the NE and the resulting strain profile obtained varying the puncher z-axis displacement were assessed through a Finite Element Analysis (FEA) (Abaqus®) and validated experimentally. To evaluate the cell response to mechanical stimulation and its effects on the uptake of non-viral gene delivery complexes, TE of gold standard bPEI/pGL3 complexes on stimulated C2C12 cells (stimulation time (t): 30 min, NE: 5-10 %, f: 0.5-1.5 Hz) was compared to that of statically-transfected cells. Results: The equibiaxial deformation of culture substrates was confirmed by the overlapping of the experimental NE measurements in 2 orthogonal directions (i.e., NE11 and NE22) at different levels of puncher displacement (Fig. 1B). The overlap of experimental and computational NE data assessed the consistency of FEA (Fig.1C). FEA revealed the uniformity of NE distribution in the middle region of culture substrates (corresponding to 1/3 of the total culture chamber) (Fig. 1D). This area was hereinafter considered as the cell-seeding area for further in vitro transfection experiments using bPEI/pGL3 complexes in stimulated conditions. Of note, we found a 4-fold increase of the TE in cyclically stretched cells as compared to unstimulated cells (Figs. 1E-F), and this increase was strain- and frequency-dependent. Conclusion: We developed a versatile stimulation device exerting a homogeneous equibiaxial cyclic strain on 2D cell cultures. Overall, coupling well-defined mechanical cell stimulation cues with chemical vectors was effective in driving gene transfer. References: [1] Bono N et al. Pharmaceutics. 2020:183. [2] Aied A et al. Drug Discov. Today 2013:1090-109