Optimization of LNP with cells

FITC-tagged C18-PEG, DSPE-PEG, and Cholesterol-PEG powder were purchased from Nanocs without further purification. The FITC-tagged PEGylated lipids were dissolved in pure anhydrous ethanol at a final concentration of 1 mM, and stored at –80 °C, respectively. Approximately 10⁷ MDA-MB-231 cells were harvested and re-suspended either in 250 μl of Diluent C or 5% human albumin in PBS. 10 nmol of each lipid probe was added into 250 μl of Diluent C before being added to the cell suspension. The samples were mixed gently at 4 °C for 5 min followed by centrifugation at 500g for 5 min to remove redundant lipid probes and fixed with 4% paraformaldehyde at 4 °C for 10 min. Then, cells were re-suspended in 1.5 ml of PBS, stained with DAPI solution at concentration of 1 μg/ml at RT for 10 min, thoroughly rinsed with PBS thrice, and finally re-suspended in 500 μl of PBS. 20 μl of cell suspension were added onto glass cover slips for fluorescent imaging. The pictures were taken under a 40× objective lens using the Olympus microscope IX71. The fluorescence intensities were analysed with ImageJ software.