The overall goals of the following experiment are to demonstrate the synthesis of por foursomes using a high pressure extruder and the efficiency of por foursome enabled photothermal therapy in treating tumors in a subcutaneous xenograft tumor model. This is achieved by conjugating pyro four by alpha to phospholipid to form the pyro feel four by lipid as a second step pyro four by lipids self-assemble into por foursomes by high pressure extrusion and are characterized for quality control. Next por fivesomes are injected into animals bearing subcutaneous tumors to demonstrate their efficacy for photothermal therapy Upon laser irradiation, the results show that por fivesomes are nanoparticles with high homogeneity and highly quenched fluorescence, and that por fivesomes are a potent photothermal agent based upon the 100%survival rate of the in vivo animal study.
Por foursomes are the first organic nanoparticles to serve as efficient photothermal enhancers while maintaining the drug loading capacity and biocompatibility of conventional liposomes and suitable for multimodal imaging and image guided therapy. So implications of this technique extend towards therapy and diagnosis of tumors Prior to this procedure. Pyro four by lipid were previously synthesized according to the text protocol.
Separately dissolve pyro four by lipid polyethylene glycol and cholesterol in chloroform. Record the concentrations of each solution in the fume hood at room temperature. Prepare the por foursome lipid in a burro silicate test tube for a total lipid weight of five milligrams per tube.
Combine the porphyrin lipid PEG 2000 PE and cholesterol for a fractional molar concentration of 65 5 and 30%respectively. Next, briefly drive a lipid films under a stream of nitrogen gas and then drive them under a vacuum for an hour. The lipid films can now be stored at negative 20 degrees Celsius under Argonne for future hydration and extrusion.
To begin the hydration and extrusion at one milliliter of PBS to each lipid film tube, freeze each test tube completely in liquid nitrogen. Then thaw them in a 65 degree Celsius water bath once no longer frozen. Vortex each tube three times for 10 seconds with five seconds of reheating in the bath between vortex Uses.
Repeat the freeze thaw vortex cycles five or more times until no large aggregates can be observed in the hydrated solution. Finally, leave the por foursome suspension on ice. Next, assemble a 10 milliliter high pressure extruder with two stacked 100 nanometer polycarbonate filters.
Connect the extruder to a regulated circulating water bath at 65 degrees Celsius. Then connect the extruder system to a nitrogen tank to provide pressure. Now use a nine inch glass pipette to transfer the suspension to the extruder chamber.
Dial up the initial extrusion pressure to between 200 and 300 PSIG. Then open the valve gradually until there is sufficient por foursome flow. Do not apply more than 800 PSIG after the first extrusion pass.
Close the pressure control valve and open the pressure relief valve. Refill the extruder with the fresh solution and repeat the process at least 10 times to ensure that the final extruded solution is totally homogenous.Ous. Determine the concentration of the final por foursome solution by measuring the absorption of a diluted sample in methanol using a UV vis spectrometer.
Next, dilute the por foursome solution one part to 60 in PBS. Then take three measurements of the distribution of por foursomes via dynamic light scattering. Perform 15 independent runs and average the results.
Lastly, diagnose the fluorescence quenching of por foursome by comparing the fluorescence emission of quenched PHY at one millimolar in PBS to unquenched por fivesome at one millimolar in 1%Triton X 100. Store the por foursome solution protected from light with foil and under argon at four degrees Celsius culture. The KB cells in RPMI 1640 with 10%FBS harvest the KB cells at 20, 000 cells per milliliter in PBS and keep them on ice to an ISO fluoride anesthetized 20 gram nude mouse inject 100 microliters of cells subcutaneously into the right flank.
10 days later, the resultant tumor will be four to millimeters across and ready to treat. Start the treatment by injecting 750 nanomole of the por foursomes through the tail vein. 24 hours later, laser irradiate the por foursomes in an enclosed laser safety room wearing laser safety goggles, the laser fiber and diffuser should be secured by a stand.
Set the laser power at low level and turn on the laser. Adjust the height of the laser fiber to achieve in a radiation area, nine millimeters in diameter. Use a power meter to calibrate the laser power to 750 milliwatts for 1.18 watts per square centimeter.
Then turn the laser off while anesthetizing the animal with ISO fluorine gas. When the animal does not respond to a toe pinch, document the tumor temperature using a tightly focused infrared thermal camera. Take a pre PTT image as well as a regular white light image.
Next turn on the laser to 750 milliwatts. Put the tumor in the center of the laser beam for one minute and measure the tumor's temperature every five seconds. After a minute, turn off the laser, stop the isof fluorine gas and wait until the animal recovers from the anesthesia before returning it to its cage.
Going forward every two days, photograph the tumor and measure its diameter with calipers. When it reaches 10 millimeters in diameter, humanely sacrifice the animal. Using this protocol, pyro four bide was conjugated to phospholipid as a stable lipid monomer and self-assembled to form por foursomes by high pressure extrusion.
The resulting por foursomes will have a high homogeneity of size distribution with a peak around 120 nanometers. They also have two main absorbance peaks at 410 nanometers and 677 nanometers. The fluorescence of porphyrin was quenched as high as 99.8%when the nano structure is intact.
Using the outlined in vivo PTT protocol, por foursomes with laser irradiation caused tumor temperature to increase by over 35 degrees Celsius after one minute of irradiation. While the laser alone induced a temperature increase of less than 10 degrees Celsius. Following the PTT irradiation, the tumors usually turned whitish because of the potent thermal effect.
Thermal ablation resulted in dark brownish escar on tumors and a 100%reduction of tumor volume. ES scars were obvious 24 hours post-treatment and they gradually recovered during the following two weeks. Using por foursomes, subcutaneous tumors were completely eliminated without recurrence.
By comparison. Tumors injected with ssom alone or treated with laser alone continued to grow. After watching this video, you should have a good understanding of how to synthesize por prisms using high pressure extruder and how to perform laser irradiation after roone administration for photothermal therapy.
