In Vitro Phototoxicity Assay: A PDT-based Method to Evaluate the Phototoxic Potential of a Photosensitizer in Lung Cancer Cells

1. Synthesis of Hyaluronic Acid-Ceramide (HACE)

1. Solubilize 12.21 mmol of hyaluronic acid (HA) oligomer and 9.77 mmol of tetra-n-butylammonium hydroxide (TBA) in 60 ml of double-distilled water (DDW). Stir for 30 min.
2. To synthesize the DS-Y30 linker, dissolve 8.59 mmol of DS-Y30 ceramide and 9.45 mmol of triethylamine in 25 ml of tetrahydrofuran (THF). Mix with 8.59 mmol of 4-chloromethylbenzoyl chloride in THF. Stir for 6 hr at 60 °C.
3. Dissolve the synthesized 8.10 mmol of HA-TBA and 0.41 mmol of DS-Y30 linker in a mixture of THF and acetonitrile (4:1, v/v). Stir for 5 hr at 40 °C.
4. Remove impurities by filtering with a filter agent, and eliminate the organic solvent by vacuum evaporation. Purify the product using a dialysis membrane (molecular weight cut-off: 3.5 kDa) and lyophilize.

2. Preparation of the Nanoparticles

1. Dissolve 1 mg of HB and 1 mg of paclitaxel in 0.5 ml of dimethyl sulfoxide (DMSO) and blend with 0.5 ml of DDW by vortex-mixing for 5 min. Then, solubilize HACE in that mixture by vortex-mixing for a further 5 min.
2. To eliminate the solvent, heat at 70 °C for 4 hr under a gentle stream of nitrogen gas.
3. Resuspend the film composed of HACE, HB, and paclitaxel with 1 ml of DDW. Filter with a syringe filter (0.45 µm pore size) to remove unencapsulated drugs.

3. In Vitro Phototoxicity

1. Uptake of nanoparticles in lung cancer cell lines
   1. Prepare RPMI-1640 medium-containing 10% (v/v) fetal bovine serum and 1% (w/v) penicillin-streptomycin.
   2. Seed A549 cells in 24-well cell culture plates at a density of 1 × 10⁵ cells/well (triplicates for each group). Incubate for 24 hr at 37 °C in a humidified 5% CO₂ and 95% air atmosphere.
   3. After cell attachment, remove the medium and wash the cells by adding 1 ml of phosphate-buffered saline (PBS).
   4. Dissolve the nanoparticles in PBS to a final concentration of 2 µM/ml HB. Then, incubate the cells with 1 ml of PBS, empty NPs, HB-NPs, or HB-P-NPs in each well for 4 hr in the dark.
   5. Remove all of the solution and wash the cells by adding 1 ml of cold PBS. Repeat the washing step once more. Add fresh culture medium.
2. Cell viability assay
   1. Place the cell culture plate under the PDT fiber (with 1 cm of distance from the PDT fiber to the well). Wear laser safety glasses and illuminate the cells with a PDT laser (630 nm, 400 mW/cm²) in the dark for various periods of time: 0, 5, 10, 20, 30, and 40 sec (0, 2, 4, 8, 12, and 16 J/cm²). Then, incubate the cells for 24 hr in the dark.
   2. Aspirate the medium and wash the cells by adding 1 ml of cold PBS. Repeat the washing step once more.
   3. Add 10 µl of cytotoxicity measuring solution to each well. Incubate in the dark for 2 hr.
   4. Measure the absorbance at 450 nm using a microplate reader.