Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.
This method provides some fascinating synthesis of gallium-68 core-doped nanoparticles for hybrid PET/MR molecular imaging. The main advantage of this technique is that thanks to the use of microwave technology, the synthesis is fast and more importantly, completely reproducible. First, dissolve 75 milligrams of iron chloride hexahydrate and 80 milligrams of citric acid trisodium salt dihydrate in nine milliliters of water.
Transfer the mixture to a microwave-adapted flask. Next, load a dynamic protocol in the microwave. Set the temperature to 120 degrees Celsius, the time to 10 minutes, the pressure to 250 psi, and the power to 240 watts.
Add one milliliter of hydrazine hydrate to the reaction mixture. Then start the microwave protocol. In the meantime, rinse a gel filtration desalting column with 20 milliliters of distilled water.
Once the protocol is finished, and the flask cooled to room temperature, pipette 2.5 milliliters of the final mixture onto the column and discard the flowthrough. Following this, add three milliliters of distilled water to the column and collect the nanoparticles in a plastic tube. Add 75 milligrams of iron chloride hexahydrate and 80 milligrams of citric acid trisodium salt dihydrate into a vial.
Elute the gallium-68 generator using the recommended volume and concentration of hydrochloric acid according to the vendor. After the hydrochloric acid injection in the self-shielded generator, four milliliters of gallium-68 chloride is obtained, ready to use without further processing. Add four milliliters of gallium-68 chloride to the microwave-adapted flask.
Then pipette five milliliters of distilled water into the flask and mix well. Now load a dynamic protocol in the microwave. Set the temperature to 120 degrees Celsius, the time to 10 minutes, the pressure to 250 psi, and the power to 240 watts.
Add one milliliter of hydrazine hydrate to the reaction mixture. Then start the microwave protocol. In the meantime, rinse a gel filtration desalting column with 20 milliliters of distilled water.
Once the protocol is finished and the flask cooled to room temperature, pipette 2.5 milliliters of the final mixture onto the column and discard the flowthrough. Then add three milliliters of distilled water to the column and collect the nanoparticles in a glass vial. To measure the hydrodynamic size of the gallium-68 nanoparticles, pipette 60 microliters of the sample into a cuvette and perform three dynamic light scattering measurements per sample.
To assess the colloidal stability of the gallium-68 nanoparticles, incubate 500 microliters of sample in different buffers at 37 degrees Celsius for different times, ranging from zero to 24 hours. At the selected times, transfer 60 microliter aliquots to cuvettes and measure their hydrodynamic size. To obtain a gel filtration radiochromatogram, fractionate the elution from the size exclusion column into 500 microliter aliquots during the gel filtration purification step.
Then measure the radioactivity present in each aliquot using an activimeter. To determine the radiochemical stability, incubate the gallium-68 nanoparticles in mouse serum for 30 minutes at 37 degrees Celsius. After incubation, purify the nanoparticles by ultrafiltration.
Then measure the radioactivity present in the nanoparticles and filtrate. Hydrodynamic size data for the gallium-68 nanoparticles revealed a narrow size distribution and mean hydrodynamic size of 7.9 nanometers. Measurements of five different syntheses proved method reproducibility.
The hydrodynamic size of gallium-68 nanoparticles incubated in different media from zero to 24 hours showed no significant changes, meaning the sample is stable in different buffers and serums. Because of the fast heating achieved using microwave technology, nanoparticles present ultra small core sizes of about four nanometers. Electron microscopy images revealed homogeneous core sizes and the absence of aggregation.
The gallium-68 nanoparticle gel filtration chromatogram shows a main radioactivity peak corresponding to the nanoparticles and a reduced peak corresponding to free gallium-68. The radiolabeling yield was 92%which translates into a specific activity relative to 7.1 gigabecquerel per millimole of iron. An excellent longitudinal value of 11.9 and a modest relaxation value of 22.9 were obtained for five gallium-68 nanoparticle syntheses, yielding an average ratio of 1.9, meaning gallium-68 nanoparticles are ideal for T1-weighted MRI.
MR phantom images at different gallium-68 nanoparticle concentrations show an increase in the iron concentration and positive contrast. An increasing iron concentration implies an increasing gallium-68 concentration, and the PET signal is increasingly intense. The use of microwave technology allows for the reproducible and rapid synthesis of iron oxide nanoparticles for multi-modal imaging.
Following this procedure, we have produced a tracer that can be used for targeted molecular imaging with PET, T1 MRI, or hybrid approaches. After its development, this technique pave the way for researchers to explore the use of hybrid molecular imaging in fields such as oncology and cardiovascular diseases. Don't forget that working with radioactive compounds can be extremely hazardous, and radioprotection precautions should always be taken while performing this procedure.
