In Vitro Cellular Uptake

To visualize the in vitro uptake and internalization of nanoparticles, LNSs and LNPs were labeled with fluorescent rhodamine labeled lipid (please see Table 2) at 0.5 wt. % of lipid content. HepG2 cells were grown to ~80% confluence on glass coverslips (12 x 12 mm; Fisher Scientific, Texas, USA). Prior to the addition of 200 µg/ml of fluorescently-labeled LNSs or LNPs, the medium was replaced with serum-free medium. After 1 h incubation at 37°C, cells were washed three times with PBS and fixed with 4% (w/v) paraformaldehyde (PFA). Coverslips with fixed cells were mounted onto glass slides with Dako^® mounting media and examined using an Olympus Fluoview 1000 confocal microscope (Olympus Imaging Co., Tokyo, Japan). For 3D spheroid culture, HepG2 cells were obtained from monolayer culture using the protocol described previously (Saw et al., 2017a).

Rhodamine labeled lipid used in LNSs and LNPs for in vitro uptake experiment and in vivo Biodistribution analysis.

*for in vitro experiments, rhodamine labeled lipid was used at 0.5 wt. %; for in vivo BioD analysis, rhodamine labeled lipid was used at 2 wt. %.