Vibration Loading Induces Phenotypic Modifications And Increased Transgene Expression

Purpose/Objectives: One major drawback of non-viral gene delivery means stands in the lack of suitable techniques to deliver the exogenous genetic material inside the cells. Since their introduction, chemical vectors - i.e., cationic carriers able to self-assemble with nucleic acids (NAs) into nano- and micro-particles - and physical methods - i.e., the application of membrane-disruptive forces to ease the intracellular delivery of NAs - have made strides forward. However, the poor efficiency of the former and the risk of potential cell damage of the latter are still hampering their widespread application. In this context, we propose a novel in vitro transfection strategy relying on the delivery of linear polyethylenimine (lPEI)-based polyplexes to mechanically vibrated cells, with the aim of easing the internalization of the genetic cargo, thus improving the transfection efficiency (TE) of the non-viral carrier. *Methodology: A vibrating platform consisting of a sine wave generator connected to a custom-made mechanical wave driver was used to stimulate two cell lines. Membrane morphology during and after the application of 5 min vibration loading at different frequencies from 10 to 1,000 Hz 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. TE and cytotoxicity 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. From a stimulation frequency of 100 Hz onward, cells displayed blisters and protrusions all over the surface, probably due to the blebbing phenomenon. 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. *Conclusion/Significance: We 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.