Enhancing non-viral gene delivery through mechanical stimulation of cells

INTRODUCTION: The major drawback in the use of non-viral means for gene delivery purposes stands in the lack of safe and suitable techniques to deliver the exogenous genetic material inside the cells.1 Since their introduction, chemical vectors - i.e., cationic carriers able to self-assemble with anionic nucleic acids (NAs) into nano- and micro-particles in order to overcome cellular barriers - and physical methods - i.e., the application of membrane-disruptive forces to ease the intracellular delivery of NAs - have made strides forward.2-3 However, the poor efficiency of the former and the risk of potential cell damage of the latter are still hampering their widespread application.4 In this context, we propose a novel in vitro gene delivery strategy relying on the delivery of linear polyethylenimine (lPEI, the gold standard non-viral polymeric carrier)5-based polyplexes to mechanically vibrated cells, with the aim of easing the internalization of the genetic cargo, thus improving the transfection efficiency of the non-viral carrier. METHODS: A vibrating platform (Fig. 1A) consisting of a sine wave generator connected to a custom-made mechanical wave driver was used to stimulate HeLa and MG-63 cells. Membrane morphology during and after the application of short vibration loading at different frequencies (i.e., 10, 50, 100, 500 and 1,000 Hz for 5 min) was inspected by means of Scanning Electron Microscopy (SEM). Cells were next transfected with 25 kDa lPEI/pGL3 complexes with an ammine-to-phosphate molar ratio (N/P) of 30 and stimulated for the same period at the above-mentioned frequencies. Transfection efficiency (TE) and cytotoxicity of polyplexes were assessed 24 hrs post-transfection. RESULTS: To shed light on the cell response to vibration stimuli, we evaluated the role of short stimulations at different vibration frequencies on cell membrane morphology. As shown in Fig. 1B, from a stimulation frequency of 100 Hz onward, cells displayed blisters and protrusions all over the surface, probably due to the blebbing phenomenon.7 Moreover, when stimulation was released, cells were able to restore membrane smoothness within one hr. The same threshold (i.e., 100 Hz-vibrations for 5 min) was found to induce an increase in TE of lPEI-based polyplexes with respect to static unstimulated controls and to cells exposed to low-frequency vibrations of 10 and 50 Hz. Besides, viability of transfected cells was not affected by the mechanical stimulation. DISCUSSION & CONCLUSIONS: We herein proposed a novel, cost-effective and non-toxic strategy to boost the transfection efficiency of polymer-based gene delivery vectors on different cell types by means of a short vibration loading. Further investigation on the mechanisms involved in the increase of polyplexes internalization should be performed.