This method is significant because it provides the most robust and reproducible way to deliver magnetic nanoparticle hyperthermia treatment in preclinical models. This technique allows for the precise and consistent delivery of nanoparticle hyperthermia by accurately monitoring and controlling the local environment, animal physiology and thermal dose. When attempting this protocol, keep in mind that nanoparticle heating must remain confined to the target tissue.
It is essential to use abundant and appropriate thermometry to ensure reproducibility and safety. 24 hours after plating B16 F10 murine melanoma cells add magnetic nanoparticles to a final concentration of three milligrams ion per milliliter. Ensure that the nanoparticles are distributed evenly throughout the well by creating a stock solution of media and magnetic nanoparticles in advance.
48 hours after adding the nanoparticles, add 0.5 milliliters of trypsin to each well being treated and gently swirl the plate. Use a microscope to check that the cells are detached. Then, add one milliliter of media to each well and collect all the cells into 1.5 milliliter tubes using a clearly labeled separate tube for each well.
Spin the tubes at 60G for two to three minutes to pellet the cells. Then place tubes in the spacer full of water within the coil. Set the temperature of the water bath to maintain the media and cell pallet at 37 degrees Celsius.
Set up separate fiber optic temperature probes to monitor the temperature within the tube and the water bath. Turn on the chiller and check that the coolant is flowing through the coil. Then turn on the power source and adjust the percent of maximum to the desired field.
Operate the 14 turns solenoid coil powered by a 10 kilowatt generator at 165 kilohertz and 23.87 kiloamperes per meter. Treat the cells until a previously determined protocol thermal dose is reached. After treatment, re-suspend the pelleted cells in the media within the tubes and replate them into new, clearly labeled six-well plates.
After anesthetizing the mouse, clean the tumor with an alcohol wipe. Inject the magnetic nanoparticles into the tumor three hours before AMF treatment. After three hours, anesthetize the mouse again and check for the lack of response to the righting reflexes.
Remove the ear tag or any other metal objects on the mouse and gently place a lubed fiber optic temperature probe into its rectum. Place the catheter into the tumor. Remove the needle.
Then cut the catheter so that it does not stick out of the tumor too much. Insert a three-sensor fiber optic temperature probe into the catheter, which will protect the sensors. Tape the rectal and intratumoral probe to the tail of the animal.
Place the mouse into a 50 milliliter tube which should have a hole near the head where the anesthesia can be connected and delivered. Place the tube within the coil setup and reconnect the anesthesia. Place a fiber optic temperature probe loosely into the tube to measure the temperature of the environment.
Then turn on the chiller and ensure that the coolant is being circulated. Verify that the computer software is displaying the various temperatures and begin recording to allow for a CEM43 calculation to be displayed in real time where the required CEM43 dose should have been previously determined. Turn on the magnet at a low power percentage and ensure that the fiber optic temperature probes are recording temperature changes.
In short, that the core temperature of the animal remains at 38 degrees Celsius, regulating it with the conditioned air jacket. Adjust the strength of the magnetic field by changing the power which controls the temperature level in the tumor. Shut off the AMF once the desired dose is achieved.
Remove the tube from the coil and remove the mouse from the tube. Then, shut down the chiller. Extract the probes and catheters and if necessary, tag the animal with a new metal ear tag.
Monitor the mouse during recovery from anesthesia, ensuring that it resumes normal behavior and that there are no complications. For in vivo hypothermia, it is essential to place as many fiber optical temperature probes as possible in strategic sites for real-time efficacy and safety assessment. These probes make it possible to record temperature throughout the experiment, allowing for accurate dosimetry and thermal history.
Curves generated during an in vivo experiment are shown here, highlighting the capability to closely monitor temperature and adjust the system to maintain tumor temperatures within the desired range. The volcano plots show differential expressed genes following in vitro and in vivo magnetic nanoparticle hyperthermia treatment, demonstrating how molecular techniques can be used to monitor the hyperthermia effects. Accurate monitoring of tumor temperatures, animals core temperature, and thermal dose are necessary for consistent delivery of treatment.
Following hyperthermia, different analyses can be performed to understand the effect and mechanism of the treatment. And other therapies can be implemented for a greater therapeutic effect.
