Fig. 2

Cellular uptake of N-7DHC-lipos. (a) Fluorescence microscopy comparing uptake of N-7DHC-lipos and 7DHC-lipos by H1299 cells at 4 h. Scale bars, 50 μm. (b) Mean fluorescence intensity (MFI) of DiR in cells, analyzed by Image J based on microscopy results of (a). (c, d) Flow cytometry comparing uptake of N-7DHC-lipos and 7DHC-lipos by NTSR1-positive H1299 cells (c) and NTSR1-negative Hcc827 cells (d) (n = 3 biologically independent samples). (e) Competitive binding curve of N-7DHC-lipos (right) and neurotensin (NTS) (left) (n = 3 biologically independent samples). (f) Inhibition of DiR-labeled N-7DHC-lipos uptake by endocytosis inhibitors, evaluated with H1299 cells using flow cytometry (n = 3 biologically independent samples). (g) Fluorescence microscopy analysis of N-7DHC-lipos fusion with cell membranes, tested with live H1299 cells. N-7DHC-liposomes were loaded with DiL, embedded within the lipid layers, and calcein, encapsulated inside the nanoparticles. An increase in intracellular calcein fluorescence and a decrease in colocalization of the two dyes indicate fusion of N-7DHC-lipos to cell membranes and release of the encapsulated contents into the cytosol. Scale bars, 50 μm. (h) Statistical analysis of the change in colocalization between DiL and calcein, based on the microscopy results from (g) and analyzed by Image J. The experiment was repeated three times with similar results. Data are presented as mean ± SD. Statistical difference was evaluated using two-tailed Student’s t-test in (b-d), and one-way ANOVA in (f, h). *p < 0.05, ***p < 0.001, ****p < 0.0001; ns, p > 0.05