Exosomes facilitate mRNA and siRNA delivery using cationic liposomes 2X3-DOPE to rat heart mesenchymal cells <i>in vitro</i>

BACKGROUND: The delivery of nucleic acids to mesenchymal stem cells, which are used as model objects in in vitro experiments or as therapeutic agents in regenerative medicine and oncology, is an actively developing area of research. Existing non-viral delivery systems either have low effectiveness or highly toxic to mesenchymal stem cells. Therefore, the development of new carriers has become an urgent priority. AIM: To demonstrate the feasibility of delivering model messenger RNA and small interfering RNA to rat heart mesenchymal stem cells (MSCs) in vitro using original cationic liposomes 2X3-DOPE (1:3 molar ratio) and to evaluate the influence of exosomes incorporated into hybrid nanoparticles with 2X3-DOPE on the efficiency of RNA delivery. METHODS: Exosomes were isolated using a standard ultracentrifugation technique followed by characterization of the obtained vesicles through Western blotting, transmission electron microscopy, atomic force microscopy, and hydrodynamic diameter measurement using dynamic light scattering. Small interfering RNA was chemically synthesized; whereas messenger RNA was obtained by in vitro transcription. Complexes of liposomes or hybrid nanoparticles with RNA were prepared by mixing; the properties of the resulting particles were assessed using dynamic light scattering and atomic force microscopy. To evaluate the efficiency of RNA delivery to rat heart mesenchymal stem cells derived from both healthy and ischemic myocardium, we used fluorescence microscopy, laser scanning confocal microscopy, and flow cytometry. RESULTS: Complexes of cationic liposomes 2X3-DOPE (1:3 molar ratio) with messenger RNA and 2X3-DOPE modified with DSPE-PEG2000 (0.62 mol%) complexed with small interfering RNA were successfully prepared and characterized. It was demonstrated that 2X3-DOPE is ineffective for messenger RNA delivery to rat cardiac mesenchymal stem cells; whereas hybrid nanoparticles incorporating exosomes based on these liposomes exhibited up to 40% transfection efficiency. In addition, 2X3-DOPE modified with DSPE-PEG2000 (0.62 mol%) was effective for small interfering RNA delivery to rat cardiac mesenchymal stem cells, achieving up to 90% transfection efficiency; whereas the use of hybrid nanoparticles based on this formulation resulted in 100% transfected cells with more than a twofold increase in small interfering RNA in the cells as indicated by the average fluorescence intensity. CONCLUSION: Cationic liposomes 2X3-DOPE (1:3 molar ratio) modified with DSPE-PEG2000 (0.62 mol%) are promising vehicles for small interfering RNA delivery to mesenchymal stem cells, both independently and in combination with exosomes. Exosomes integrated in hybrid nanoparticles based on 2X3-DOPE improve the transfection efficiency of both messenger RNA and small interfering RNA in rat cardiac mesenchymal stem cells in vitro.