Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

This protocol holds great promise for improving post-nanomedicine delivery and the anti-cancer efficacies of nanoparticles in cancer treatment. This technique synergizes magnetic, acoustic, and optical responsivenesses into one nanotherapeutic platform for the control and targeted delivery of nanomedicine, and facilitates the combination of photothermal and magnetic hyperthermia therapy. Demonstrating the procedure will be Siyu Wang, a magnetic, acoustic, and optical-triple-responsive microbubbles for magnetic hyperthermia and photothermal combination cancer therapy fellow from my laboratory.

For nanoparticle shelled microbubble formation, uniformly disperse magnetic iron oxide nanoparticles in deionized water to generate a 10 milligram per milliliter stock solution and load the nanoparticle solution into an ultrasonic cleaning machine for 20 minutes. At the end of the sonication, add 150 microliters of deionized water, 150 microliters of 10 millimolar sodium dodecyl sulfate, and 400 microliters of the sonicated iron oxide nanoparticle solution in a 1.5 milliliter centrifuge tube. Next, fix a homogenizer with a scaffold in an ice bath and place the nanoparticle solution into the ice bath.

Immerse the homogenizer probe in the nanoparticle solution and homogenize the suspension for three minutes at 20, 000 revolutions per minute. At the end of the homogenization, allow the solution to stabilize for 12 hours at room temperature before placing the tube into a magnetic holder to adsorb the nanoparticle shelled microbubbles to the tube wall. Replace the supernatant with one milliliter of fresh deionized water three times to wash the nanoparticle shelled microbubbles.

After the last wash, slightly shake the tube and transfer 10 microliters of the nanoparticle shelled microbubbles onto a clean glass slide. Use a fluorescence microscope and a 20X magnification to image the nanoparticle shelled microbubbles. After imaging, open the image in the microscope software and use the ruler to set a red line with the same length as the ruler.

Click set and scale to enter the length of the ruler and draw lines of the same lengths at the diameters of at least 200 individual microbubbles. Then click report and view report. To measure the acoustic response of the microbubbles, dilute 200 microliters of the nanoparticle shelled microbubbles in 800 microliters of deionized water in a 1.5 milliliter tube and connect the function generator, amplifier, impedance matching, and homemade focus transducer.

Place the transducer in the center of the bottom of the artificial cuboid sink and connect the hydrophone with an oscilloscope to monitor the output ultrasound intensity. Add enough deionized water to submerge the transducer and adjust the function generator to the sweep mode. Tune in the frequency range from 10 to 900 kilohertz and set the amplitude to 20 voltage peak to peak.

Use the amplifier to adjust the power of the ultrasound to 0.1%and the cycle duration to four seconds with a one-second time interval. Place the tube of nanoparticles into the scaffold on the top of the homemade focus transducer and attach the magnet to the bottom of the tube. Turn on the function generator and the amplifier power.

After five 25-second ultrasound cycles, switch off the function generator and remove the magnet. Then replace the nanoparticle solution with one milliliter of deionized water and repeat the ultrasound and treatment. To set up the laser for optical treatment of the microbubbles, first turn on the laser power supply.

After several minutes, fix a fiber-coupled 808 nanometer laser diode onto a retort stand and use an optical fiber to direct the laser beam to the sample stage. Use a convex lens to focus on the sample stage to achieve a six millimeter diameter light spot and measure the power output with the laser power meter. Then adjust the power to one watt per square centimeter.

To perform a photothermal measurement, prepare one milliliter volumes of different concentrations of the iron oxide nanoparticles in individual 1.5 milliliter centrifuge tubes and place the first tube at the focused region of the laser beam. Record the baseline temperature of the sample and turn on the laser and infrared thermal imaging camera. Irradiate the sample continuously for 10 minutes while recording the temperature in real time.

Then turn off the laser and the camera and wait for the temperature of the region to return to the baseline before measuring the other sample concentrations in the same manner. For a magnetic hyperthermia measurement in an aqueous solution, prepare different iron oxide nanoparticle dilutions as demonstrated and place one dilution in the center of a water cold magnetic induction copper coil. Turn on the alternating magnetic field and the infrared thermal imaging camera and continuously induce the sample for 10 minutes while recording the temperature in real time.

At the end of the treatment, turn off the alternating magnetic field and the camera. When the temperature of the copper coil has returned to baseline, measure the next sample. Nanoparticle shelled microbubbles typically demonstrate a spherical shape with an average diameter of about 5.41 micrometers.

Although the microbubbles remain intact for up to a year, a stepwise release of iron can be achieved by increasing the number of ultrasound cycles. Iron oxide nanoparticle-mediated photothermal measurement in aqueous solution reveals a rapid increase in iron oxide nanoparticle temperature over time with a 30 degree Celsius temperature increase achieved upon 10 minutes of exposure to near infrared laser light at a five milligram per milliliter iron concentration. Compared to the control group, no differences in morphology or live cell number are observed when breast cancer cell lines are incubated with a high concentration of iron, suggesting a good bioavailability of the iron oxide nanoparticles.

Upon irradiation, the nanoparticle-treated cancer cells became rounded in shape and demonstrated decreased viability indicating apoptosis. Five minutes after irradiation, the temperature of the gelatin injection areas rapidly increases by about 20 degrees Celsius. When exposed to alternating magnetic field therapy, thermal imaging of different concentrations of iron oxide nanoparticle reveals an alternating magnetic field response characteristic of nanoparticle shelled microbubbles.

Further, wholesale imaging of mice exposed to alternating magnetic field therapy reveals significant rapid temperature changes within the area of interest. During the nanoparticle solution agitation, make sure that the homogenizer probe remains completely immersed within the solution. This protocol can also achieve and improving the penetration into tumor tissues to address the challenges of nanomedicine delivery in cancer treatment.