2.11. Cellular Uptake of Liposomes

3 × 10⁴ cells/cm² of HUVEC and 9 × 10⁴ cells/cm² of HCT-116 were seeded in 6-well plates. After 24 h, cells were washed with PBS, and the culture medium was replaced with fresh medium containing 0.2 mg/mL of fluorescent liposomes. After 3 h of incubation, cells were washed with PBS, detached by trypsin/EDTA solution (Sigma-Aldrich), resuspended with PBS and centrifuged at 230× g for 10 min at 4 °C. Then the supernatant was discarded, and cell pellets were resuspended in PBS containing 1 mg/mL of trypan blue, to quench extracellular fluorescence due to possible not internalized liposomes [45]. Cells were analyzed by cytofluorimetry, using a BD FACSCanto II and BD FACSDiva™ software (BD Biosciences). The mean fluorescence intensity (MFI) values of control cells (non-incubated with liposomes), due to cell autofluorescence, were considered “background” and were subtracted from the MFI of liposome-treated cells. The values were then normalized with the corresponding fluorescent intensity of liposome suspensions (0.2 mg lipids/mL, in PBS), measured by spectrofluorimetry with a PerkinElmer EnSpire microplate reader. Thereafter the normalized values were divided by 10³ to optimize the format for graphical representation. The experiments were performed in three independent replicates and averages and standard deviations of normalized values were reported as arbitrary units (A.U.).