Immu mount

Tissue sections or cells were fixed with ice-cold methanol and permeabilized with 1% Triton-X 100 in PBS. Thereafter, the detergent was removed by repeated washing in PBS. Primary antibodies (see Additional file 1) were applied in PBS overnight (4 °C). After the incubation with secondary antibodies (see Additional file 1) and subsequent staining with DAPI, cells were covered with IMMU-MOUNT (Thermo Electron Corporation; Pittsburgh; USA) and a glass coverslip. Images were taken by confocal laser scanning microscopy (Nikon Eclipse Ti-E, Düsseldorf, Germany).

Cellular uptake of DOX-loaded HAssLG nanoparticles was studied with fluorescence microscopy observation and flow cytometry analysis. MDA-MB231 cells were seeded onto a cover glass and incubated overnight in a CO₂ incubator (5 % CO₂ at 37 °C). Then, nanoparticles were treated to cells for 1 h in a CO₂ incubator. One hour before the addition of nanoparticles, the CD44 receptor was blocked by pretreatment of free HA (10 equivalents of nanoparticle contents). Following this, cells were washed with PBS (pH 7.4, 0.1 M) and fixed with 4 % paraformaldehyde. This was fixed with immobilization solution (IMMU-MOUNT, Thermo Electron Corporation, Pittsburgh, PA, USA). Cells were then observed with a confocal laser scanning microscope (CLSM, TCS-SP2; Leica, Wetzlar, Germany).
For flow cytometry analysis of cells, 1 × 10⁶ cells were seeded in each well of six-well plates. Nanoparticles were treated to the cells and incubated for 1 h in a CO₂ incubator. One hour before the addition of nanoparticles, the CD44 receptor was blocked by pretreatment of free HA (10 equivalents of nanoparticle contents). Cells were washed with PBS (pH 7.4, 0.1 M) and trypsinized to harvest cells. The cells were analyzed with flow cytometery (FACScan) at an excitation wavelength of 488 nm and emission wavelength of 522 nm for DOX fluorescence intensity.

5 × 10⁵ cells seeded on the cover glass in 6 well plates were exposed to Ce6 or nanophotosensitizers. Ninety minutes later, cells were washed with PBS twice and fixed with 4% paraformaldehyde solution. They were mounted with immobilization solution (Immu-Mount, Thermo Electron Co., Pittsburgh, PA, USA) to observe cell morphology using fluorescence microscope (Eclipse 80i; Nikon, Tokyo, Japan).

KB cells (5 × 10⁵ cells/well) seeded onto cover glass in 6-well plates were cultured overnight at 37 °C in a 5% CO₂ incubator. Prior to nanophotosensitizer treatment, cells were treated with 5-mM folic acid for 1 h to study folate receptor sensitivity. Cells were washed with PBS and then exposed to Ce6 or nanophotosensitizers (2-μg/mL as a Ce6 concentration) for 1 h. Following this, cells washed with PBS was fixed with 4% paraformaldehyde solution and then mounted with Immu-Mount solution (Thermo Electron Co, Pittsburgh, PA, USA) to observe cells using fluorescence microscope.

Cells grown on coverslips were washed with phosphate-buffered saline (PBS) and fixed by incubation with 4% formaldehyde in PBS for 20 minutes at room temperature. Cells were incubated with 2 mg/L DAPI and 1x phalloidin-633 (SC-363796, Santa Cruz Biotechnology) in PBS to stain for nuclei and Tubulin. Coverslips were mounted onto microscopy slides using Immu-Mount (Invitrogen) and left to dry overnight at 4°C. Images were taken using a Leica TCS SP5 confocal microscope with HCX PLAN Apochromat lambda blue 63.0x1.40 oil UV objective. Samples were scanned using a 405-nm Violet Diode laser for DAPI staining, a 488-nm argon laser for GFP and a 633-nm helium-neon laser for phalloidin. Detector gain (PMT and HyD) and offset (PMT) settings were kept constant within experiments that compared a fusion protein in different conditions.