Vibropolyfection: coupling polymer-mediated gene delivery to mechanical stimulation to enhance transfection of adherent cells

Abstract

Background With the success of recent non-viral gene delivery-based COVID-19 vaccines, nanovectors have gained some public acceptance and come to the forefront of advanced therapies. Unfortunately, the relatively low ability of the vectors to overcome cellular barriers adversely affects their effectiveness. Scientists have thus been striving to develop ever more effective gene delivery vectors, but the results are still far from satisfactory. Therefore, developing novel strategies is probably the only way forward to bring about genuine change. Herein, we devise a brand-new gene delivery strategy to boost dramatically the transfection efficiency of two gold standard nucleic acid (NA)/polymer nanoparticles (polyplexes) in vitro. Results We conceived a device to generate milli-to-nanoscale vibrational cues as a function of the frequency set, and deliver vertical uniaxial displacements to adherent cells in culture. A short-lived high-frequency vibrational load (t = 5 min, f = 1,000 Hz) caused abrupt and extensive plasmalemma outgrowths but was safe for cells as neither cell proliferation rate nor viability was affected. Cells took about 1 hr to revert to quasi-naïve morphology through plasma membrane remodeling. In turn, this eventually triggered the mechano-activated clathrin-mediated endocytic pathway and made cells more apt to internalize polyplexes, resulting in transfection efficiencies increased from 10-to-100-fold. Noteworthy, these results were obtained transfecting three cell lines and hard-to-transfect primary cells. Conclusions In this work, we focus on a new technology to enhance the intracellular delivery of NAs and improve the transfection efficiency of non-viral vectors through priming adherent cells with a short vibrational stimulation. This study paves the way for capitalizing on physical cell stimulation(s) to significantly raise the effectiveness of gene delivery vectors in vitro and ex vivo. Graphical Abstract

A new microfluidic platform for the highly reproducible preparation of non-viral gene delivery complexes

“…Polyplexes were prepared with l PEI and b PEI transfection reagents because their behavior is extremely well-known. 60–63…”

“…Polyplexes were prepared with lPEI and bPEI transfection reagents because their behavior is extremely well-known. [60][61][62][63] 25 kDa lPEI and the pGLuc plasmid encoding the secreted luciferase enzyme were mixed through the chip following operation mode 2 to give rise to seven polyplex suspensions invariably at N/P 40. L929 fibroblasts were the recipient cells used to assess the transfection behavior of single polyplex preparations and compare the effectiveness of the chip units with each other.…”

Recent developments of polymeric delivery systems in gene therapeutics

“…75 Mechanical loads applied to cells cause specific cellular responses, such as mechanical regulation of membrane transport 76 and cytoskeletal remodeling. 77,78 In this context, cyclic stretching, 79 shear stress, 80 and vibrational loading 81 may improve the transfection efficiency of non-viral gene delivery vectors by enhancing their uptake and intracellular transport. Ponti et al propose a method to enhance intracellular delivery and improve transfection efficiency by using mechanical stimulation to initiate cells.…”

“…A short-lived high-frequency vibrational load ( t = 5 min, f = 1000 Hz) applied vertically to the plane of the cell culture plate induces abrupt and extensive plasma membrane growth, triggering the clathrin-mediated endocytosis pathway that makes it easier for the cells to internalize the polymer gene vector, leading to an increase in the transfection efficiency from 10-fold to 100-fold. 81 This process is safe for the cells, which recover after 1 hour through plasma membrane remodeling.…”

Correction: Vibropolyfection: coupling polymer-mediated gene delivery to mechanical stimulation to enhance transfection of adherent cells

“…Following publication of the original article [ 1 ], the authors would like to replace the references [64] and [65] with the following references: Roffay, Chloé, et al "Passive coupling of membrane tension and cell volume during active response of cells to osmosis." Proceedings of the National Academy of Sciences 118.47 (2021): e2103228118.…”