VIBRATION-BASED STIMULATION OF CELLS ENHANCES POLYETHYLENIMINE-MEDIATED GENE DELIVERY

Introduction Gene therapy is a promising strategy for the treatment of genetic disorders. In this context, a safe and efficient transfer of genetic material into target cells (i.e. transfection), i.e. the successful introduction of exogenous nucleic acids (DNA or RNA) through the cell membrane, is required [1-2]. Over the past two decades, considerable progress has been made in gene transfer technologies, and thus far, different delivery strategies have been developed. Chemical gene delivery vectors utilize specialized non-viral carriers, such as cationic polymers, to overcome the cellular barriers and ease the transfer of exogenously delivered nucleic acids into cells [3-4]. In this light, gene transfer mediated by cationic polymer/DNA complexes (polyplexes) exhibit high transfection efficiencies both in vitro and in vivo [5]. As an alternative to vector-mediated gene delivery, physical methods (e.g. electroporation, sonoporation, magnetofection) relies on the use of different kinds of mechanical forces to induce the relatively quick and transient destabilization of the plasma membrane barrier, hence enabling the nucleic acids to enter the cell [6]. Herein, we propose a novel in vitro transfection strategy relying on the combined use of a physical method and a chemical vector. Specifically, mechanical stimulation in the form of vibrations was used to induce a transient cell membrane poration in order to promote linear polyethylenimine (lPEI)/pDNA polyplex uptake by cells, and enhance transfection efficiency. Results and Discussion In this work a novel, inexpensive, easy-to-use mechanical stimulation device (Fig. 1-A), modified ad-hoc because not meant to stimulate cells, was used for cell culture experiments to induce transient cell membrane poration. We thus evaluated the role of different stimulation frequencies (i.e. 10, 50, 100, 500 and 1,000 Hz for 5 min) on the morphology of HeLa (human epithelial ovarian carcinoma cells) and MG-63 (human fibroblast osteosarcoma) cells by means of a Scanning Electron Microscopy (SEM) inspection. As shown in Fig. 1-B, unstimulated, 10 Hz- and 50 Hz-stimulated cells shared very similar morphologies and a marked smooth surface. Notably, when stimulated at 100, 500 and 1,000 Hz, SEM analysis revealed the presence of pores and blisters covering the whole cell surface (Fig. 1-B). Such protrusions found on the cell surface as the consequence of the high frequency-stimulation (i.e. from 100 Hz onward), may be related to the blebbing phenomenon, that is a defensive mechanism exploited by cells once subjected to some stressors [7]. We also examined the kinetics of cell membrane recovery after poration and we found that cells were able to restore membrane integrity and recover their original morphology within 1 hour when stimulation was released. Taken together, these observations pointed out that the application of mechanical stimulation was not detrimental to cells and proved that the vibration-based cell stimulation from 100 Hz onward led to a transient cell membrane destabilization, and ultimately to the formation of membrane pores. Dynamic in vitro transfection experiments were performed as well. Cells were challenged with lPEI/pGL3 complexes (DH = 113±4 nm; surface charge (Zp) = +17±5 mV), then stimulated at different frequencies (10, 50, 100, 500 and 1,000 Hz for 5 min) and for different times (500 Hz for 5, 30 and 60 min). Transfection efficiency (TE) in stimulated cells was evaluated 24 hr-post transfection and compared to statically transfected cells. As reported in Fig. 1-C and 1-D, high-frequencies vibrational stimulation (from 100 Hz onward) dramatically enhanced TE of lPEI/DNA complexes, leading to a ≈50- and ≈100- fold increase in transgene expression in MG-63 and HeLa cells, respectively, irrespective of the duration of the stimulation. Besides, low-to-negligible cytotoxic effects were found in both cells lines. These findings mirrored SEM results, since from 100 Hz onward, a change in cell morphology was observed, which ultimately resulted in enhanced cell uptake of polyplexes. We can speculate that cell blebbing induced through mechanical stimulation, together with the consequent cell repair mechanism, accounts for increased polyplex internalization, and for the consequent increase in TE. It is worthy of note that the same behavior was found in both HeLa and MG-63 cell lines, demonstrating that such stimulation effects were cell-type independent. Conclusion We herein demonstrated that the application of a vibration-based stimulation to cells at suitable frequencies (i.e. 100 Hz onward) dramatically enhanced the TE of lPEI-based DNA delivery, as compared to standard static transfection conditions, coupled with low-to-negligible cytotoxicity. Additional investigation on the precise mechanism(s) and pattern(s) involved in cell membrane perturbation and recovery are required to shed light on how mechanical vibrations enhance polyplex internalization