2.8. Cytotoxicity, Cell Proliferation and ATP Assays

Primary hHSC were seeded (density 26 × 10³/cm²) under basic serum-rich conditions for 24 h, followed by serum deprivation (serum free medium, SFM) for the next 24 h prior to liposomes exposure. Next, cells were treated for 24 h with a range of concentrations (100–12.5 µM) of either Lip-C6 or Lip-G. Cytotoxicity and cell proliferation were assessed by MTT/MTS test (Promega, Southampton, UK) and BrdU ELISA (Sigma Aldrich, Dorset, UK), respectively [43]. Moreover, the Lip-C6 inhibitory effect on cell proliferation was further quantified by employing ATP assay. Cells were seeded at a density of 10,000 cells/well/100 uL in a 96-well plate in culture medium and serum-starved for 24 h followed by incubation with 6.25 µM of Lip-C6 for up to 24 h. Cell lysis was induced using the CellTiter-Glo^® Reagent (Promega, Southampton, UK), according to the manufacturer’s specification. Luminescence was recorded and the intracellular ATP concentration was calculated from an ATP standard curve and normalized to hHSC proliferation, as measured by BrdU assay. All samples were assayed in quadruplicates and according to the manufacturer’s manual and as previously described [43].

For protein analyses, hHSC were exposed to non-cytotoxic doses of Lip-C6 and Lip-G (6.25 and 3.125 µM) for up to 24 h and total protein lysates were analyzed by Western blot, as described below.

In another set of experiments, liposome uptake was monitored in hHSC grown on glass chamber slides. After 24 h of incubation with the same non-cytotoxic doses of 6.25 µM rhodamine-labelled Lip-C6 and Lip-G liposomes, nuclei were counterstained with Hoechst 33342 (1 μM, final concentration) for 10 min, cells were then washed three times in 1× HBSS, and observed under a fluorescence microscope (AxioScopeA1, Carl Zeiss Ltd., Cambridge, UK).