A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.
The overall goal of this protocol is to provide an efficient and rapid synthetic method for radioactive iodine labeled gold nano particles using the strain promoted copper free click reaction. This method can help answer key questions in the radiochemistry field such as the synthesis of radio isotope labeled about three years as well as regular imaging probes for pad or spec imaging studies. The main advantage of this technique is that one to five iodine labeled with golden particles can be efficiently synthesized the using of azide prosthetic group with excellent radiochemical yield and radiochemical purity.
Visual demonstration of this method is critical as the radio evaluation steps are difficult to learn. Since such procedures require a suitable facility and handle radio labeling experience. Along with the study, radio student Ha Eun Shim will be demonstrating the procedure.
To perform the radio iodination reaction, add a precursor solution to a 1.5 mL tube. Add 10 microliters of acetic acid to the precursor solution at room temperature. Then add 150 mega bekarel of iodine 125 in 50 microliters, 0.1 molar sodium hydroxide to the reaction mixture.
Add a chloramine T solution and close the microcentrifuge tube containing the reaction mixture. Incubate the reaction mixture at room temperature for fifteen minutes until the radio iodination reaction is complete. Then add a sodium metabisulfite solution to the reaction mixture to quench the radio iodination reaction.
Withdraw 0.2 microliters of the crude product and then dilute it with 100 microliters of 50%aceto nitryl in water for high performance liquid chromatography, or HPLC analysis. Analyze the diluted crude product by using a reversed phase analytical radio HPLC. Transfer the entire reaction mixture to an HPLC vial.
Rinse the reaction tube with 0.5 mL of aceto nitryl and add the rinse into the same injection vial. Dilute the collected solution with one mL of water. To purify the crude product with reparative HPLC, inject the crude product onto a preparative radio HPLC.
Collect the radioactive peak representing the iodine 125 labeled azide in a glass test tube. Measure the radio chemical yield of the fraction using a radioactivity dose calibrator according to the manufacturer's protocol. Then inject the purified product onto an analytical radio HPLC using the same HPLC conditions for determining the radio chemical purity of the product.
To perform solid phase extraction of the product, dilute the fraction containing the desired product with 40 mL of pure water. Add the diluted solution into a preconditioned TC18 cartridge. Wash the cartridge with an additional 15 mL of water.
Elute the product trapped in the cartridge with 2 mL of acetone into a 10 mL glass vial that is protected by a lead shield. Measure the radioactivity of the eluted product using radioactivity dose calibrator according to the manufacturer's protocol. After evaporating the acetone dissolve the residue with 100 to 200 microliters of DMSO for the next radio labeling step.
Perform the synthesis of DBCO group modified gold nanoparticles as described in the text protocol. Prepare a concentrated solution of the DBCO group modified gold nanoparticles by using centrifugation. And adjust the concentration of the gold nano particles to two micro molar.
Add 4.1 mega bekarel of iodine 125 labeled azide in 5 microliters of DMSO to a suspension of 50 microliters of 2 micro molar gold nanoparticles. Incubate the resulting reaction mixture at 40 degrees Celsius for 60 minutes. Withdraw a 0.2 microliter alloquot from the crude product and apply it onto a silica coded thin layer chromatography or TLC plate.
Develop the TLC plate using ethyl acetate as a mobile phase. Place the TLC plate on a radio TLC scanner and run the scanner to monitor the radio labeling reaction according to the manufacturer's protocol. Purify the reaction mixture containing the iodine 125 labeled gold nanoparticles by centrifugation.
Decant the supernatant and add pure water for resuspension of the gold nanoparticle pellets. Withdraw a 0.2 microliter alloquot from the purified product and apply it onto a silica coated TLC plate. Develop the TLC plate using ethyl acetate as the mobile phase.
Place the TLC plate on a radio TLC scanner and run the scanner to determine the radiochemical yield and radiochemical purity of the iodine 125 labeled gold nanoparticles, according to the manufacturer's protocol. Representative results of the iodine 125 labeled azide prosthetic group are shown here. A 75%radiochemical yield was determined by using a radioactivity dose calibrator.
The analytical radio HPLC result shows excellent radiochemical purity of the product. Representative results of the iodine 125 labeled gold nanoparticle are shown here. Radio TLC analysis presents that both radiochemical yield and purity of the purified gold nano particle were more than 95%Once mastered this technique can be done in three hours if it is performed properly.
The implications of this technique extended toward preparation of various molecular probes for nuclear imaging therapeutic purposes because the labeling chemistry in the present method is highly efficient and straightforward. Don't forget that working with radioactive iodine can be extremely hazardous and the precautions such as lighter bricks or lighter shoes should always be taken while performing this procedure.
