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

All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

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 vivo anticancer efficacy in tumor-bearing mice

1. Lung cancer-induced mouse model
   1. Prepare 1 × 10⁶ A549 cells in 0.1 ml RPMI-1640 medium; keep it in ice.
   2. Anesthetize the mice with an i.p. injection of a xylazine and a mixture of tiletamine and zolazepam (1:2, 1 ml/kg). Confirm proper anesthetization by gently pinching a small fold of mouse skin. Use vet ointment on the eyes to prevent dryness while under anesthesia.
      NOTE: If no movement is observed, the animal is sufficiently anesthetized to start the experiments.
   3. Inject the cells subcutaneously into the left flanks of BALB/C male nude mice (6 – 7 weeks old, 20 – 22 g).
   4. Keep observing the mice until they start to move around the cage.
      NOTE: Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone surgery to the company of other animals until it has fully recovered. Keep the mice under specific pathogen-free (SPF) conditions.
   5. Measure the tumor size with calipers every day. Calculate the tumor volume (mm³) as (length × width²) / 2. When the tumor size reaches approximately 200 mm³ in volume, start the experiment.
2. Anticancer efficacy study
   1. Randomly divide the mice into 4 groups (n = 10 for each group).
   2. Anesthetize the mice with an i.p. injection of a xylazine and a mixture of tiletamine and zolazepam (1:2, 1 ml/kg). Confirm proper anesthetization by gently pinching a small fold of mouse skin. Use vet ointment on the eyes to prevent dryness while under anesthesia.
      NOTE: If no movement is observed, the animal is sufficiently anesthetized to start the experiments.
   3. Dissolve the nanoparticles in PBS to a final concentration of 2 mg/ml HB. Inject PBS, free HB, HB-NPs, or HB-P-NPs via the tail vein (2 mg/kg as HB) twice on days 0 and 7.
   4. Keep observing the mice until they start to move around the cage.
      NOTE: Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone surgery to the company of other animals until fully recovered. Keep the cages dark and under specific SPF conditions.
   5. 24 hr after each injection, anesthetize the mice with an i.p. injection of a xylazine and a mixture of tiletamine and zolazepam (1:2, 1 ml/kg). Confirm proper anesthetization by gently pinching a small fold of mouse skin. Use vet ointment on the eyes to prevent dryness while under anesthesia.
      NOTE: If no movement is observed, the animal is sufficiently anesthetized to start the experiments.
   6. Place the tumor site under the PDT fiber (with 1 cm of distance from the PDT fiber to the tumor). Wear laser safety glasses, turn off the switch, and illuminate the tumor with a PDT laser (630 nm, 400 mW/cm²) for 500 sec (200 J/cm²) twice on days 1 and 8.
   7. Keep observing the mice until they start to move around the cage. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone surgery to the company of other animals until fully recovered.
   8. Keep the cages under SPF conditions. Maintain the cages in the dark for 24 hr after laser treatment.
   9. Visually monitor the tumor volume and the changes at the tumor site every day. Measure the tumor size with calipers, and calculate the volume as (length × width²) / 2 (mm³). Take pictures of the tumor sites every day to check for tumor surface alterations after PDT.
   10. On day 16, sacrifice five mice per group by terminal anesthesia using isoflurane.
   11. With forceps and scissors, cut the outer skin and expose the tumors. Carefully harvest them. Fix them in 10% formalin, embed them in paraffin, and stain them with hematoxylin and eosin (H&E) for the histological analysis.
   12. After 45 days of monitoring, sacrifice the remaining mice by terminal anesthesia using isoflurane.