The overall goal of this procedure is to assess the heating properties of gold nanoparticles in a 13.56 megahertz radio frequency electric field, in vitro and in vivo. This is accomplished by first assessing the heating rate of concentrated gold nanoparticles and their buffer in a quartz vet. The second step of the procedure is to perform in vitro toxicity studies of gold nanoparticles with cultured cancer cells.
The third step is to perform in vivo studies of gold nanoparticles in an ectopic mouse cancer model. Ultimately, results can show whether or not the investigated nanoparticle could be an effective agent to use with RF mediated hyperthermia through analysis of the nanoparticles in suspension, in vitro and in vivo. This method can help answer key questions in a field of nanoparticle mediated non-invasive radiofrequency hypothermia, such as is heating due to the particles themselves and can it be used for therapeutic purposes?
Generally, individuals new to this method will struggle because there are not many well-established protocols for performing these type of experiments. Demonstrating the procedure will be branded Cisneros, a research assistant, Stuart Core, a postdoctoral scientist, and Leila Green, another postdoctoral scientist. As an example, start with a commercially available 500 milliliter bottle of gold nanoparticles with a five nanometer diameter.
Take about 125 milliliters of the stock gold nanoparticle solution and split it between six 50 kilodalton centrifuge filter tubes, centrifuge the nano particles at 3000 RPM for two minutes and five seconds. Remove the supernatant being the filtered buffer and refill each filter. With more stock solution.
Continue centrifusion and removing the buffer and reloading the tubes until all of the nanoparticle solution has been filtered. After the last addition, replace the filtered buffer with a similar volume of deionized water and repeat the process approximately eight more times after loading the filtered buffer sample to the RF machine. Using the infrared camera software create boxes over the QVE to capture the heating profile After eight washes, the heating data of the five nanometer gold nanoparticle filtrate is approaching that of deionized water.
UV vis analysis can also be used to monitor contaminant absorption.Peaks. Shown here is a clean spectrum. Once the buffer contaminants have been fully removed, we suspend the nanoparticles in 0.5 milliliters of deionized water in the filter tubes that's removing the nanoparticles from the filter.
Then combine all six suspensions into one 15 milliliter tube. Now, analyze the sample using UV VIS and zeta potential for stability data. Use I-C-P-O-E-S and or ICP mass spec to determine the nanoparticle concentration.
SEM and or TEM analysis can also be used to obtain morphological data using a Kansas RF system or derivations of load, a 1.3 milliliter syringe quartz vete, and set the RF electric field to be about 90 kilovolts per meter in air where the Q vete would reside. For a standard saline sample with 0.9%sodium chloride, the electric field would be reduced to about 1.1 kilovolts per meter. These are the approximate conditions used to allow comparisons to be made between different RF systems.
Now, load a clean quartz vete with 1.3 milliliters of purified gold nanoparticle suspension at a concentration of 1000 milligrams per liter. Expose the sample to the RF field for a period of 120 seconds or until the sample hits 70 degrees Celsius. Over exposure can lead to electrical arcing or rapid boiling.
Capture the thermal imaging data as well as control areas using an IR camera and the associated software. Repeat this procedure three times on the same sample. Now filter the sample through another 50 kilodalton centrifuge filter to extract the gold nanoparticles from the water buffer.
Then re-expose the buffer alone to the RF field. Again, three times the difference in the hrs between the gold nanoparticle colloid and the filtrate determines the HR due to the gold nanoparticles themselves. Finally, we suspend the remaining gold nanoparticles from the filter in 1.3 milliliters of water for further experiments.
These in vitro studies can be applied to any type of cancer cell type that forms 2D monolayers. In this example, human hepatocellular carcinoma derived Hep three B cells will be used plate about 50, 000 cells. In three wells of a 12 well plate with one milliliter of growth.
Media continue loading cells until three plates are filled for nanoparticle studies, and three plates are filled as controls. Incubate the plates at 37.5 degrees Celsius for 24 hours. The next day introduce 0.1 milliliter of a 1000 milligram per liter gold nanoparticle solution into each well of the three nanoparticle plates to the control plates.
At 0.1 milliliter of water into each, well incubate all the plates for another 24 hours. After another 24 hours, aspirate the cell media and wash the cells with PBS to remove any surface bound gold nanoparticles. Then replace the cell media.
The cells are now ready for RF exposure. Place each 12 well cell pack within the RF field and wait until the cells have cooled to 31 degrees Celsius. Once slightly cooled, expose the cells to 3.5 minutes of rf.
The final temperature of the cell media will be about 37 degrees Celsius. Transfer the cells to an incubator and wait 24 hours before analyzing them. When the cells are ready, aspirate the media and replace it with two milliliters of 25%MTT reagent in media.
Then allow the cells to incubate for another four hours. Next, replace the MTT media with two milliliters of DMSO. Place the cell plates on a bench rocker for 10 minutes to allow the DMSO to solubilize the MTT reagents.
Finally, pipette 100 microliter samples from each well to a 96 well plate and optically read the plate at 570 nanometers. These in vivo studies can be applied to any type of cancer that form solid tumors in an orthotopic or ectopic mirroring model. In this example, Hep three B liver cancer cells are used with BPC nude mice.
Begin with approximately 100, 000 cells trypsin ice the cells and produce a solution of 2 million cells per 25 microliters. Add an equal quantity of ice cold matri gel to the cell suspension and mix it thoroughly by repeated aspiration in a pipette. Now inject the mixed suspension at the desired position on each animal's back.
After a few weeks, the tumors will be one half to one centimeter round at this time, begin RF treatment and anesthetize the mice with the solution of ketamine and xylazine by IP injection and keep them warm. Then inject the gold nanoparticles directly into half the tumors using a one cc syringe with a 27 or 30 gauge needle. Use a surgical swab to absorb blood and wipe the injection site with an alcohol wipe.
As a control, inject just PBS into the other half of the tumors. Now, treat half the mice in each group. Mount the mouse to the receiving head of the RF generator and shield non-treated areas with copper tape, including the eyes, ears, and toes.
The most important part of this experiment is proper grounding of your mice. Without proper grounding, charge buildup on the animals can cause thermal or electrodermal injuries. This can be easily prevented.
Position the tumor off the transmission head by at least one centimeter more than the width of the desired treatment site. Now, position the IR thermal camera so that the tumor and treatment area are visible. Turn on the RF for five minutes and record the resulting temperature curve.
If the skin surface temperature rises above 42 degrees Celsius, stop the treatment immediately after the RF exposure. Allow the mice to recover in a warm chamber until they are conscious. Repeat the treatments twice a week for three weeks.
Then euthanize the mice 48 hours after the final RF exposure and process the tumors. The RF heating of the purified gold nanoparticles was determined by the difference in hrs between them and the background deionized water buffer solution. The results were as expected for both five nanometer and 10 nanometer nanoparticles.
The purified gold nanoparticles integrated into human hepatocellular carcinoma derived Hep three B cells. When such cells are exposed to an RF field, they usually become less viable than those exposed to RF without internalized nanoparticles. Figure three depicts an idealized scenario where the gold nanoparticles used have limited toxicity by themselves, but excellent RF initiated cytotoxic properties consult literature for a comprehensive review of in vitro results to date using various types of nanomaterials.
Hep three B.Liver cancer cells were used to generate tumors on the backs of babsi nude mice. These tumors were injected with PBS suspended gold nanoparticles, followed by RF exposure. Treatment post Thomas analysis revealed that the treatment controlled tumor growth and decreased tumor mass.
After watching this video, you should have a good understanding of how to assess nanoparticles for use in RF mediated hypothermia by determining their RF responsive properties and solution in vitro and in vivo. Don't forget that working with high power RF electric fields can be extremely hazardous. Never touch or reach within an active electric field.
Take measurements of the electric field and if necessary, use proper shielding like a Faraday cage. In general, always minimize your exposure to sustained high strength electric fields.
