Sub cell gt cell 96 192

To qualitatively determine the amount of poly(I:C) within the ENCPs, samples were loaded in an agarose gel at 1% w/v in Tris Acetate-EDTA buffer (Sigma-Aldrich, MO, USA) before and after the incubation with an excess of heparin for poly(I:C) displacement. Each lane was loaded with 2.5 μg of poly(I:C) and with 1x SYBR®Gold nucleic acid stain (Invitrogen, CA, USA). For the displacement with heparin, 20:1 and 500:1 weight ratios of heparin (Sigma-Aldrich, MO, USA) to poly(I:C) were added for the C12r8 or pArg ENCPs, respectively, and incubated for 30 min at 37°C. Control lanes included a DNA 1 kb ladder (Invitrogen, CA, USA), and free poly(I:C) in the same conditions as the ENCPs. Gels were run for 30 min at 90 V in a Sub-Cell GT cell 96/192 (Bio-Rad Laboratories, CA, USA), evaluated with an UV transilluminator (Molecular Imager® Gel Doc™ XR, Bio-Rad Laboratories, CA, USA) and analyzed with Image Lab™ Software (Bio-Rad Laboratories, CA, USA).
For the release of poly(I:C), ENCPs were incubated in cell culture media, in a 1:1 (v/v) ratio, for 4 or 24 h, prior to been processed as described above. Free poly(I:C) exposed to the same conditions was used as the control.

Cationic SNs were prepared upon addition of the cationic lipid, stearylamine (ST) to the organic phase at a ratio of VitE:SM:PEG:ST 10:1:0.1:1 (w/w). SNs-ST were fully characterized. miRNA was subsequently associated to SNs-ST by the establishment of electrostatic interactions performing a simple incubation. A total of 10 μL of miRNA (10 μg) were incubated with 90 μL of SNs-ST at different miRNA:ST mass ratios (1:10, 1:7.5, 1:5, 1:2.5, and 1:1) to obtain theoretical loadings ranging from 0.8% to 7.6% (w/w) with respect to the total mass of the nanosystems. The association of miRNA was analyzed by agarose gel electrophoresis (1% w/v in Tris-Acetate-EDTA (TAE) Buffer). Briefly, 0.5 μg of nucleic acids labeled with SYBR^® Gold, either in solution, associated to the nanoparticles, or after displacement with an excess of heparin (25-fold heparin with respect to the amount of miRNA for 2 h at 37 °C) were loaded into each well. Gel electrophoresis was run at 100 V, 40 min in a Sub-Cell GT cell 96/192 (Bio-Rad Laboratories Ltd., Deeside, England). Gel images were obtained with a Molecular Imager^® Gel DocTM XR System (UV light 302 nm; Bio-Rad, Madrid, Spain).