The overall goal of this protocol is the use of microwave technology for the rapid synthesis and functionalization of iron oxide nanoparticles with a bisphosphonate moiety for fast detection by MRI of atherosclerosis in mice models. The use of microwave technology in the synthesis and functionalization of iron oxide nanoparticles is still limited. However, we demonstrate how high quality nanoparticles and extremely fast protocols can be developed.
The main advantage of this technique is that in extremely short times, high quality functionalized nanoparticles can be produced and used for catheterization of atherosclerosis plaque targeting microcalcifications. To begin this procedure, add 0.5 grams of iron 3-acetylacetenate, 1.4 milliliters of oleic acid, 0.6 milliliters of oleylamine, and 1.19 grams of 1, 2-Hexadodecanediol to a microwave-adapted flask. Add 10 milliliters of phenyl ether carefully down the flask wall using a graduated pipette.
Following this, place the flask in a microwave reactor and start the microwave protocol by loading a dynamic study in the microwave. After the reaction is complete, and the flask has cooled to room temperature, transfer the reaction mixture to an Erlenmeyer flask using a glass pipette and add 10 milliliters of 98 percent ethanol. Place the flask on top of a neodymium niobium boron magnet and remove the supernatant with a glass pipette after 5 minutes.
Next, add 10 milliliters of ethanol to the flask. Sonicate the sample at 40 kilohertz for two minutes at room temperature. When finished, place the sample on the magnet and remove the supernatant.
Disperse the oleic acid coated nanoparticles in 30 milliliters of chloroform and sonicate at 40 kilohertz for five minutes at room temperature. Following sonication, transfer 0.5 milliliters of the nanoparticles in a glass cuvette and add 0.5 milliliters of chloroform. Check the hydrodynamic size in a zetasizer per the manufacturer's instructions.
At this point, dissolve 44.3 milligrams of potassium permanganate and 150.4 milligrams of benzoyl trimethyl ammonium chloride in a three to two mixture of water and chloroform. Add the resultant solution to a 5 milliliter Aliquat of the oleic-acid coated nanoparticles in a microwave-adapted flask. Start the microwave protocol for azelaic acid nanoparticles by setting the temperature to 105 degrees Celsius, the time to nine minutes, the pressure to 250 psi, and the power to 300 watts.
Following this, add 10 milliliters of pH 2.9 phosphate buffer to the flask. After repeating the microwave protocol, and removing the supernatant, add 10 milliliters of water to the flask and transfer the mixture to an Erlenmeyer flask. The most critical step is purification of azelaic acid nanoparticles.
Next, add 5 milliliters of 10 percent sodium bisulphate to the Erlenmeyer flask and sonicate at 40 kilohertz for two minutes at 25 degrees Celsius. After collecting the particles, and removing the supernatant, wash the nanoparticles three more times with 1 percent sodium hydroxide. Redisperse the particles in 5 milliliters of pH 7.2 phosphate buffer.
To check the hydrodynamic size and zeta potential, transfer 0.7 milliliters of the azelaic acid nanoparticles to a disposable, folded capillary cell and place it in the zetasizer for analysis. Add 12 milligrams of EDC and 15 milligrams of sulfo-NHS in a centrifuge tube with a 2 milliliter Aliquat of the azelaic acid nanoparticles. Vortex the mixture at room temperature for 35 minutes.
After placing a magnet below the centrifuge tube to destabilize the nanoparticles, aspirate the supernatant and wash the particles with 1.5 milliliters of 1 millimolar hepes buffer. Then, add 5 milligrams of neridronate and vortex the mixture for two hours. Separate the nanoparticles with a magnet and wash with 1 millimolar hepes buffer.
Finally, disperse the neridronate nanoparticles in 2 milliliters of 1 millimolar hepes buffer. To check the hydrodynamic size and zeta potential, add 0.7 milliliters of the neridronate nanoparticles to a disposable folded capillary cell and place it in the zetsizer for analysis. All nanoparticles presented small hydrodynamic size in a very narrow size distribution.
The particles present excellent crystallinity as shown in the TEM images. Additionally, the same results for hydrodynamic size and distribution were obtained after four repetitions of the synthesis, demonstrating one of the most important aspects of the microwave approach, its reproducability. The calcium ion binding properties due to bisphosphonates present in the neridronate nanoparticles were checked by relaxometry and demonstrated that T2 relaxation time increments linearly with the calcium ion amount and incubation time due to the formation of nanoparticle clusters whereas nanoparticles without calcium ion remained stable.
In vivo MRI experiments were performed in 48-week old apoe knockout mice and at one hour post injection, the signal from the plaque was hypointense in comparison to the basal images. Additionally, the plaque to muscle ratio was significantly different between the basal and one hour post injection images. The signal in the liver was also monitored after injection of the neridronate nanoparticles which were completely cleared from circulation after 20 minutes, confirming the selective accumulation of these nanoparticles towards atherosclerotic plaque.
Ex vivo imaging and histology were performed and imaging of aortas, with and without nanoparticles, showed differences in the signal intensity in agreement with the in vivo experiments. Once mastered, the microwave technology allows for the synthesis characterization and plaque detection in a total time of 3 hours if properly performed. Another advantage of this method is that, with a small modification other imaging techniques, like thermography can be performed, in order to answer additional questions like quantification of atherosclerosis.
After its development, this technique paved the way for researchers in the field of cardiovascular imaging to explore the early diagnosis of atherosclerosis with an experimental setup that can be easily adapted to clinical needs. After watching this video, you should have a good understanding of how to produce hydrophilic iron oxide nanoparticles with microwave and its application in cardiovascular imaging. Don't forget that working with microwave ovens can be hazardous depending on the oven used, and precautions such as pressure control of the cistern should always be taken while performing this procedure.
