Jetmessenger

DC2.4 (1×10⁵ cells/well) were seeded in a 24-well plate 24 h before transfection. Then, CNEs (10%, 20%, or 30% DOTAP) were incubated with 0.75 μg of enhanced GFP-encoding mRNA (5moU) (Cat#: L-7201, TriLink Biotechnologies, San Diego, CA) to reach a N/P ratio ranging from 1.5 to 54 in a total volume of 50 μL DEPC-treated water respectively for 30 min at 4 °C, while 1 μg of JetMessenger (Polyplus-transfection® SA, New York, USA) was used as positive control. Meanwhile, the transfection efficiency of mRNA-loaded CNEs (30% DOTAP, N/P = 4.5) prepared by electrostatic adsorption or Microfluidic Chip (Micronit, X3550 CH.2, Netherlands) on both BMDCs and DC2.4 was also investigated. Later, 50 μL carrier-mRNA complexes were added to each well containing 450 μL serum-free DMEM medium. Six hours later, the RPMI 1640 medium in the wells was replaced with fresh medium containing 10% FBS. Another 18 h later, cells were washed twice with PBS and imaged under a microscope, with fluorescence pictures captured and further analyzed.

Gelatin GELITA^® MedellaPro^® < 100, porcine gelatin, 228 g Bloom, pharmaceutical-grade was purchased from GELITA^® Deutschland GmbH, Eberbach, Germany. Protamine sulfate was purchased from Sigma-Aldrich, Darmstadt, Germany. JetMessenger (JetM) and JetPrime (JetP) were purchased from Polyplus-transfection^®, Illkirch, France. Branched polyethyleneimine (PEI), M_w~25,000, was purchased from Sigma-Aldrich Darmstadt, Germany. Lipofectin was purchased from Invitrogen, Thermo Fisher Scientific, Darmstadt, Germany. Purified water was obtained from a Milli-Q water purification system (Merck Millipore, Darmstadt, Germany), and is referred to as MQ water. DLin-MC3-DMA was purchased from MedChemExpress (Middlesex County, NJ, USA), Cholesterol was purchased from Sigma-Aldrich Darmstadt, Germany, DSPE-PEG 2000 and DPPC were a kind gift from Lipoid GmbH, Ludwigshafen, Germany.