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

- Photodynamic therapy or PDT is a type of light therapy utilizing photosensitizers-- molecules activated by lights of specific wavelengths. An excited photosensitizer converts molecular oxygen present in cells to reactive oxygen species that cause cell death. To begin the assay, prepare a desired nanoparticle-based drug delivery system carrying the appropriate concentration of the photosensitizer, alone or in combination with a test drug.

Add the preferred concentration of this nanoparticle suspension into a culture of lung cancer cells. Incubate the culture in the dark for an appropriate time to facilitate cellular uptake of nanoparticles. Replace the suspension with the desired culture medium. Subsequently, position a photodynamic therapy fiber laser above the culture at an optimal distance facilitating uniform illumination. Irradiate the cells at an appropriate wavelength for the desired time and incubate overnight.

Replace the media with a suitable cytotoxicity measuring solution. Use a microplate reader to measure cell viability and quantify cytotoxic effects of the experimental treatment. Irradiated cells containing the photosensitizer show decreased viability. In the following protocol, we will perform an in vitro phototoxicity analysis of hypocrellin B, a photosensitizer, and paclitaxel, an anticancer drug, encapsulated in hyaluronic acid-ceramide nanoparticles in A549 lung cancer cells.

- To begin, synthesize hyaluronic acid-ceramide as described in the accompanying text protocol. Purify the product using a 3.5 kilodalton dialysis membrane and lyophilize it.

Next, prepare hyaluronic acid-ceramide nanoparticles by themselves, hyaluronic acid-ceramide nanoparticles along with the photosensitizer, and hyaluronic acid-ceramide nanoparticles with an anticancer agent and the photosensitizer.

Seed the lung cancer cell line A549 into the wells of a 24-well cell culture plate at a density of 1 times 10 to the fifth cells per well, and incubate the cells for 24 hours. The next day, remove the medium and wash the cells with 1 milliliter of PBS.

Then dissolve the prepared nanoparticles in PBS to a final concentration of 2 micromoles per milliliter of the photosensitizer, hypocrellin B, by vortexing.

Into four wells each, add 1 milliliter of PBS, 1 milliliter of the empty hyaluronic acid-ceramide nanoparticles, 1 milliliter of the nanoparticles containing the photosensitizer, and 1 milliliter of the nanoparticles containing both the photosensitizer and the anticancer drug.

When finished, cover the plate and incubate it at 37 degrees Celsius for four hours in the dark to allow for nanoparticle uptake into the cells. Then remove all of the solutions and wash the cells by adding 1 milliliter of cold PBS.

Repeat the washing step once more, and then add fresh culture medium. Next, place the 24-well culture plate under a photodynamic therapy fiber and position the fiber 1 centimeter from the first well.

- The distance from the photodynamic therapy fiber to the cells was the critical factor in this study. It was important to find out the optimal distance which can cover the whole cell with the light.

- Then put on safety glasses and turn out the lights. Illuminate the cells with the laser in the dark for between 5 and 40 seconds to activate highly reactive singlet oxygen and other free radicals in the photosensitizer-containing nanoparticles.

Following light exposure, incubate the cells in the dark for 24 hours. Next, aspirate the culture medium and wash the cells with 1 milliliter of cold PBS. Repeat the wash step once more and then add 100 microliters of media and 10 microliters of cytotoxicity measuring solution to each well.

Incubate cells in the solution for two hours in the dark and then move all the samples to a 96-well plate. Measure the absorbance at 450 nanometers using a microplate reader.