Flow cytometry and confocal laser scanning microscopy (CLSM) analysis

To evaluate the ability of selective delivery to tumor cells and intracellular release of nanoparticles in tumor cells, flow cytometry and CLSM were used. NR-loaded nanoparticles were prepared by encapsulating fluorescent probe NR (0.2% loading by weight) into HSV nanoparticles and HV nanoparticles using the same preparation method described in “Preparation of PTX-loaded nanoparticles” section. In order to investigate whether the nanoparticles were specifically taken up through HA receptor-mediated endocytosis, human non-small lung cancer A549 cells (CD44 overexpression) were chosen and the cells were incubated with 100-fold excess of free-HA polymer for 2 hours prior to the addition of NR-loaded nanoparticles. In flow cytometry analysis, all the samples were washed with PBS three times, then harvested by trypsinization, and collected in PBS to measure the fluorescence intensity at 560/620 nm using a flow cytometer (BD FACS Calibur; BD Biosciences, San Jose, CA, USA). In CLSM analysis, after incubation with NR-loaded nanoparticles, the culture media were subsequently removed and the cells were rinsed with PBS three times, followed by the addition of Hoechst 33342 (10 μg mL⁻¹) to stain the cell nuclei. The samples were then observed using a confocal microscope (Olympus FV1000; Olympus Corporation, Tokyo, Japan) at the excitation and emission wavelengths of 352 and 455 nm for Hoechst 33342 and 488 and 530 nm for NR, respectively.