The overall goal of this experiment is to increase the therapeutic effect of photodynamic therapy in treating lung cancer by using both photosensitizer and anticancer drug-loaded, lung cancer-targeted nanoparticles. This method can help answer key questions in the photodynamic therapy field such as what are the proper agent conditions for in vitro and in vivo studies. Since photodynamic therapies are widely used in the cancer fields, pre-cancer studies with normal photosensitizers or new indications may be performed with this technique.
The implications of this technique extended towards therapy of various diseases such as tuberculosis, ulcers and dermatology fields using different annual modules. To begin, synthesize hyaluronic acid ceramide as described in the accompanying text protocol. Purify the product using a 3.5 kilodalton dialysis membrane and myophalize it.
Next, prepare hyaluronic acid-ceramide nanoparticles by themselves. Hyaluronic acid-ceramide nanoparticles along with a 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 one time 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 one milliliter of PBS. Then, dissolve the prepared nanoparticles in PBS to a final concentration of two micromoles per milliliter of the photosensitizer Hypocrellin B by vortexing. Into four wells each and one milliliter of PBS, one milliliter of the empty hyaluronic acid-ceramide nanoparticles, one milliliter of the nanoparticles containing the photosensitizer and one 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 hour in the dark to allow for nanoparticle uptake into the cells. Then, remove all of the solution and wash the cells by adding one 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 one centimeter from the first well. The distensae 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 five 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 one 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. 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 one-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 weighting each mouse. Then, dissolve the nanoparticles in PBS to a final concentration of two milligrams per milliliter and load one microliter of the suspension per gram of mouse into a one-milliliter syringe equipped with a 26-gauge needle. Inject the nanoparticles via tail vein injection on day zero and seven.
24 hours after each injection, anesthetize the mouse and place the tumor site 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 results. 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 of 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.
Lung cancer cell viability is shown here after four hours of incubation with the nanoparticles followed by light irradiation. Without light, the cells exposed to the nanoparticles containing only photosensitizer showed no sign of toxicity at all, while cells exposed to nanoparticles containing both the photosensitizer and paclitaxel showed about an 81%cell viability. With increasing irradiation time, the cell viability was decreased in both treatment groups with the photosensitizer.
The treatment group also containing paclitaxel showed the greatest amount of phototoxicity. This was also reflected in the tumor model study as the tumor volume in mice treated with these two treatment groups were lower than the controls. In addition, the group treated with the paclitaxel and photosensitizer containing nanoparticles was significantly lower than all the other groups.
Although the tumor size was significantly smaller, the mice treated with nanoparticles having both the photosensitizer and paclitaxel showed severe hemorrhaging and the fastest development of necrosis. After the development, this technique paved the way for laser treats in the field of photodynamic therapy to explore cancer treatment in in vitro and in vivo systems. After watching this video, you should have a good understanding of how to purport photodynaic therapy in vitro and in vivo systems.
