In Vivo Antitumor Efficacy Analysis of PDT: A Technique to Determine the Phototoxic Potential of a Photosensitizer in Tumor Bearing Mice

- Photodynamic therapy or PDT is a light-based treatment involving photosensitizer molecules. These molecules are activated by lights of specific wavelengths and convert molecular oxygen present in cells to reactive oxygen species that induce cell death. To begin, develop a lung tumor-bearing mouse model by injecting the appropriate concentration of lung cancer cell suspension into the lower flank of an anesthetized mouse. Monitor until a tumor grows to an appropriate volume.

Subsequently, prepare a suspension of the desired photosensitizer loaded in a lung cancer targeted nanoparticle-based delivery system. Inject this suspension into the tail vein of an anesthetized tumor-bearing mouse. Provide sufficient time for the photosensitizer containing nanoparticles to reach the tumor site and enter the cancer cells.

Next, anesthetize the mouse. Position a PDT laser at an optimal distance above the tumor to ensure uniform illumination of its entire volume. Irradiate the tumor at an appropriate wavelength for the desired time. Maintain the mouse within a cage in the dark for 24 hours. Monitor the day-to-day changes to the tumor volume. Irradiated tumor-bearing mice injected with the photosensitizer showed decreased tumor volume. In this protocol, we will analyze the anti-tumor efficacy of hypocrellin B, a photosensitizer, encapsulated in hyaluronic acid ceramide nanoparticles in A549 tumor-bearing mice.

Harvest A549 cells and re-suspend one million cells in 0.1 milliliters of RPMI 1640 medium. Place the re-suspended cells on ice. Next, prepare a BALB/C male nude mouse for injection. Then load the cells into a 1 milliliter syringe equipped with a 26 gauge needle and inject them into the left flank of the mouse. Measure the tumor size with calipers every day.

When the tumor size reaches approximately 200 cubed millimeters in volume, start the experiment by first weighing each mouse. Then dissolve the nanoparticles in PBS to a final concentration of 2 milligrams per milliliter and load one microliters of the suspension per gram of mouse into a 1 milliliter syringe equipped with a 26 gauge needle. Inject the nanoparticles via tail vein injection on days zero and seven.

24 hours after each injection, anesthetize the mouse and place the tumor sire under the photodynamic therapy fiber so that it is one centimeter from the fiber.

It is important to cover the entire tumor surface with the light in order to achieve the best result.

Put on the laser safety glasses, turn off the room switch, and illuminate the tumor with a photodynamic therapy laser for 500 seconds on both days one and eight. Following laser treatment, maintain the mice in their cages in the dark for 24 hours. Visually monitor the tumor volume and the changes at the tumor site every day. Use calipers to measure the tumor's size and take pictures of the tumor sites every day to check for surface alterations.