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

Overview

Photodynamic therapy (PDT) is a non-invasive and non-surgical method for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light. In this protocol video, PDT-based method is used to evaluate the phototoxic potential of a potential photosensitizer in lung cancer cells.

Protocol

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.

Materials

Name	Company	Catalog Number	Comments
oligo hyaluronic acid	Bioland Co., Ltd.
DS-Y30 (ceramide 3B; mainly Noleoyl-phytosphingosine)	Doosan Biotech Co., Ltd.
adipic acid dihydrazide	Sigma Aldrich	A0638
N-(3-dimethylaminopropyl)-N′- ethylcarbodiimide	Sigma Aldrich	39391
4-(chloromethyl)benzoyl chloride	Sigma Aldrich	270784
Paclitaxel	Taihua Corporations
syringe filter	Sartorius Stedim Biotech GmbH	17762	15 mm, RC, PP, 0.45 µm
RPMI-1640	Gibco Life Technologies, Inc.	11875
Penicillin–streptomycin	Gibco Life Technologies, Inc.	15070
Fetal bovine serum	Gibco Life Technologies, Inc.	16140071
Celite (Filter agent)	Sigma Aldrich	6858	See step 1.4