2.2.5. Cellular Uptake Analysis by Confocal Laser Scanning Microscopy (CLSM)

First, the pDNA biopharmaceuticals were labelled with FITC, allowing the follow-up of its cellular uptake and intracellular localization. Briefly, 5 µg of pDNA was added to 71 µL of labelling buffer (0.020 g of sodium (di)tetraborate in 1 mL of H₂O) and 2 µL of FITC (100 mg of FITC in 200 µL of sterile DMSO). After this, the solution was stirred for 4 h at RT, protected from light. Finally, 85 µL of 3 M NaCl and 212.5 µL of absolute ethanol was added, to precipitate pDNA-labelled FITC by overnight incubation, at −20 °C.

To evaluate pDNA-polyplexes cellular uptake kinetics, 1 × 10³ cells/well were seeded in complete DMEM-F12 in Ibidi µ-Slide 8-well cell culture treated chambers (Ibidi GmbH, Germany) and cultured overnight. Transfection was performed when 70 % of cellular confluence was achieved. Then, cells were incubated for 10 min with Hoechst 33342^® (1:1000) (Invitrogen^TM Molecular Probes^TM) and, subsequently rinsed 3 times with PBS 1x (pH = 7.4). Transfection was performed for 1 h, with the different prepared pDNA-loaded polyplexes. Following the incubation period, the DMEM-F12 medium was exchanged and 4 % paraformaldehyde in PBS 1x was used for transfected cells fixation (for 20 min, at RT). To enable better visualization, transfected cells were washed three times with PBS 1x. Visualization was finally performed using a Zeiss LSM 710 laser scanning confocal microscope (Carl Zeiss SMT Inc., Jena, German) equipped with a plane-apochromatic 63×/DIC objective.