Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of ⁶⁸Ga-Labelled Nano Molecules

Our research focuses on radiopharmacy, so producing new radiopharmaceuticals for both animals and humans. In the end, we want to visualize aspects of a certain disease, in our case, cancer, and this way help treatment in the clinic. We are now going for more than just imaging.

Instead, we develop theranostics, compounds that can be used both for imaging and for therapy simply by switching the radioactive isotope that is built into the tracer. The primary technology used in the field of nuclear medicine is the positron emission tomography, short, PET, which is available for both small laboratory animals and for the clinical practice. Using PET, we can quantify and localize the accumulation of a radioactive tracer inside a body, and this in turn enables us to assess the feasibility of new tracers in small laboratory animal disease models for their feasibility in the clinical use.

The main experimental challenge is to have a radiopharmaceutical that is suitable for an intravenous injection. Additionally, there's a challenge in the animal model. On the one hand, we want an animal model that is mimicking the human situation.

And on the other hand, we want an animal model that is reproducible and predictable, and this is a difficult combination. In the future, our lab will continue to focus in the non-invasive imaging and therapy of breast cancer. Despite several advances in the field of oncology in the past years, there's still a high mortality rate and incidence for breast cancer.

It becomes especially tricky when this type of cancer is metastasized, and using non-invasive molecular imaging, we would like to improve the diagnostic for these patients. And also using radiotherapy, we would like to increase their survival rates. To begin, measure the activity of the radiolabeled nanobody chelator conjugate sample in the micro-centrifuge tube using an activity meter at the correct setting.

Spot a 10 to 15 microliter sample on the pH test strip to measure the pH. Spot one microliter of the radiolabeling reaction mixture on a silica-impregnated TLC strip and allow it to dry for one minute. Fill the TLC chamber with citric acid as a mobile phase and run the strip until the liquid reaches the top of the strip.

Place the TLC strip on a radio-TLC scanner and measure it according to the manufacturer's protocol. Calculate the radiolabeling yield of the reaction by using the radio chromatogram, dividing the integrated area under the curve from RF by the total area under the curve and multiplying by 100. After flooding HPLC lines for 10 minutes with the correct solvent, adjust the setting for measurement using the isocratic flow of a 50-50 mixture of acetonitrile as solvent A and a mixture of 0.9%sodium chloride solution combined with trifluoroacetic acid and citrate solution as solvent B.Measure the UV absorbance at 280 nanometers.

Dilute five microliters of the product with 15 microliters of 0.9%sodium chloride. Inject 15 microliters of the mixture in the analytical radio HPLC. Using the previously described settings, measure the radioactive signal and the UV absorbance of the product at 280 nanometers.

The minor shift in retention times between the UV and gamma channels is due to the HPLC design. Next, determine the radiochemical purity by integrating the peaks in the gamma channel. Integrate all other peaks in the gamma channel and evaluate them as impurities.

Prepare a one-to-20 dilution of the product with a final volume of 150 microliters using certified endotoxin-free water. Insert a disposable endotoxin cartridge commercially prefilled with all necessary reagents in the endotoxin testing reader. Load 25 microliters of the diluted product in each cartridge chamber.

After running the test for 15 minutes, the PTS includes internal positive and negative controls. The product is considered endotoxin-free if the endotoxin content is less than 8.7 endotoxin units per milliliter at maximum volume of 20 milliliters. The HPLC analysis confirmed the chemical and radiochemical purity of the radiopharmaceutical at 98.2%and showed a minor difference in retention time between the radioactive product and the nonradioactive reference compound.

The TLC chromatogram indicated an apparent radiochemical purity of 99.9%for the 68-gallium-labeled nanobody NM02. Begin by filling the syringe with the radioactive tracer depending on the route of administration. Measure the radioactivity of the tracer using the activity meter.

To prepare a 30-gauge catheter, remove the metallic needle shaft from the 30-gauge needle by either cutting it with metal scissors or bending it away from the plastic. Manually insert a 0.3-millimeter diameter PE-10 tube into the part of the shaft removed from the hub. Fill the 30-gauge catheter with 0.9%sodium chloride buffer.

Apply an ophthalmic ointment to the eyes of an anesthetized mouse to prevent drying during sedation. After selecting the vein from the tail, slightly rotate the tail to bring the selected vein into contact with the heating mat and allow it to dilate for one minute. Then turn the mouse onto its lateral position so the selected vein comes upwards.

Fixate the tip and base of the tail with tape. Take the 30-gauge catheter with the dominant hand and gently place the index finger of the non-dominant hand on the selected vein to create a backup of blood inside, forming a small hump. With the dominant hand, place the 30-gauge catheter parallel to the tail facing the hump on the vein.

Move the dominant hand forward to ensure direct venipuncture. Once inside the vein, blood reflux becomes visible in the plastic tube. Fixate the plastic tube to ensure the needle does not move inside the vein during injection.

At the free end of the tube, insert the tracer syringe connected to a 30-gauge needle. Slowly inject the content of the tracer syringe for 10 seconds. While keeping the 30-gauge needle connected to the catheter system, remove only the tracer syringe and connect a syringe with 0.9%sodium chloride buffer to flush the catheter.

Remove the 30-gauge catheter from the tail vein and use a sterile cotton compress to stop the bleeding. Calculate the leftover activity by measuring the activity of the 30-gauge catheter, the buffer syringe, the empty tracer syringe, and the cotton compress using an activity meter. Place the sedated mouse in a ventral position on the animal bed and attach it to the PET scanner.

Ensure that the breathing rate of the mouse during the scan is between 75 and 50 respirations per minute. Fix the mouse behind its neck to the animal bed with medical tape. Set the acquisition time to 45 minutes and select the region of interest for scanning.

Enter the amount of injected activity and the time of injection. Then select 68-gallium as the study isotope and start the scan. The CT image clearly demarcated mouse kidneys and allowed for bone assessment.

The PET image, using a standardized uptake value scale of zero to 5.0, showed notable tumor uptake in the left flank of the mouse and renal clearance of the tracer. The PET-CT fusion image allowed for a comprehensive evaluation of tracer biodistribution. The PET maximum intensity projection revealed urinary bladder activity, reinforcing renal clearance of the tracer.