Modulation of transfection efficiency of polymer non-viral vectors by mechanical stimulation of cells

Introduction Since their first introduction, non-viral vectors for gene delivery purposes have made strides forward. Generally, non-viral vectors are cationic lipids or polymers able to self-assemble with nucleic acids into micro/nanoparticles, with the purpose of protecting and driving the genetic material into cells to alter target functions1. The main challenge of current research relies on the design and synthesis of more and more performing non-viral vectors. However, the biological barriers that complexes have to overcome and the cytotoxic effect of such compounds are still hampering their clinical practice2. In this context, we propose an innovative in vitro transfection technology able to dramatically enhance the transfection efficiency of linear polyethyleneimine (lPEI)-based non-viral vectors, that is the gold standard polymeric vector3, on different cell lines, with no detrimental effects. The novelty of such work relies on the manipulation of cells behavior by means of an external vibration-based stimulation, in order to ease the cell/complexes interactions thus increasing the uptake and expression of the gene of interest. Experimental methods The stimulation device (Figure 1A) consists of a sine wave generator, able to produce sinusoidal waves at different frequencies, connected to a mechanical wave driver. The driver converts the input signal into Z-axis displacements (in a range between 100 nm - 1 mm) of the driver arm, equipped with a commercial cell culture plate. In this way, 2D-cell monolayers are subjected to micro-to-nano vibrations depending on the applied frequency. Before transfection, a morphological inspection of cells during and after the application of stimulation was carried out to investigate the cell response to different stimulation patterns. HeLa, MG-63 and L929 cells were then transfected with lPEI/DNA complexes at N/P (i.e., amine-to-phosphate) ratio of 30, then stimulated at different frequencies for short periods (i.e., 5 min). Transfection efficiency and cytotoxicity were assessed 24 hours post transfection. Results and discussions To shed light on the cell response to stimulation, morphological inspection of cells undergoing mechanical loading was carried out. Of note, cells stimulated from 100 Hz onward displayed blisters and protrusions all over their surface (Figure 1B), probably due to the blebbing phenomenon4, that is strictly related to a cytoskeletal reorganization5,6. Indeed, such membrane perturbations were reversed in an hour from the end of the stimulation. Such results highlighted the presence of a stimulation threshold, corresponding to 100 Hz for all the tested cell lines, able to trigger reversible cell membrane rearrangements without detrimental effects. As shown in Figure 1C-D, the transfection efficiency of lPEI-based polyplexes was dramatically increased after cell stimulation at 100 Hz for 5 min, with negligible cytotoxicity for all the tested cell lines, with respect to unstimulated (static) controls. Conclusions We herein demonstrated the efficiency of a novel, simple and versatile transfection strategy aimed at improving cell/complexes interactions through the control of the cell behavior. Indeed, when cells were properly stimulated (i.e., from 100 Hz onward), there was a 10-to-100 fold-increase in the ultimate transfection efficiency of PEI/based polyplexes with respect to unstimulated transfected cells, with no effect on cell viability. Further investigations need to be carried out to get a better insight on the mechanisms of cells/complexes interactions responsible for such outstanding results.