Name: An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group (Video) | JoVE Journal
Uploaded: 2016-10-10T15:00:00.000+00:00
Duration: 7 min 23 s
Description: Learn an efficient radiolabeling method for gold nanoparticles using a 125I-labeled azide prosthetic group and copper-free click chemistry. This protocol achieves high radiochemical yield and purity for imaging probe synthesis.

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5 minutes

This work was supported by grants from the National Research Foundation of Korea, funded by the government of the Republic of Korea, (Grant nos. 2012M2B2B1055245 and 2012M2A2A6011335) and by the RI-Biomics Center of Korea Atomic Energy Research Institute.

Caution: The oxidized form of radioactive iodine is quite volatile and must be handled with adequate lead shields and lead vials. All radiochemical steps should be carried out in a well-ventilated charcoal-filtered hood, and the experimental procedures need to be monitored by radioactivity detection devices.
1. Preparation of Chemicals and the Reverse Phase Cartridge for the Synthesis of the 125I-labeled Azide
<ol>
	<li>Preparation of reagents in solution
	<ol>
		<li>Dissolve 1 mg of the azide precursor (2) in 150 &#181;l absolute ethanol (Figure 1). 
		NOTE: A detailed synthetic procedure for the azide precursor (2) was reported in the previous paper22.</li>
		<li>Dissolve 1 mg chloramine T in 20 &#181;l of 1x phosphate buffer saline (pH = 7.4).</li>
		<li>Dissolve 2 mg sodium metabisulfite in 20 &#181;l H2O.</li>
	</ol>
	</li>
	<li>Preparation of the cartridge
	<ol>
		<li>Wash the tC18 cartridge with 10 ml absolute ethanol followed by 10 ml H2O. Do not dry the matrix of the cartridge with air.</li>
	</ol>
	</li>
</ol>
2. Radiosynthesis of the 125I-labeled Azide Prosthetic Group
<ol>
	<li>Radioiodination reaction of the precursor
	<ol>
		<li>Add the azide precursor solution (1 mg in 150 &#181;l of absolute ethanol) and acetic acid (10 &#181;l) to a 1.5 ml microcentrifuge tube.</li>
		<li>Add 150 MBq of [125I]NaI in 0.1 M NaOH (50 &#181;l) to the reaction mixture.</li>
		<li>Add a chloramine T solution (1 mg in 20 &#181;l of 1x phosphate buffer saline) and close the microcentrifuge tube containing reaction mixture.</li>
		<li>Incubate the reaction mixture at room temperature for 15 min until the radioiodination reaction is completed.</li>
		<li>Add a sodium metabisulfite solution (2 mg in 20 &#181;l H2O) to the reaction mixture to quench the radioiodination reaction.</li>
		<li>Withdraw 0.2 &#181;l of the crude product and then dilute it with 100 &#181;l of solution (H2O/CH3CN, 1:1) for HPLC analysis. 
		NOTE: For all HPLC experiments, use 0.1% formic acid containing H2O (solvent A) and 0.1% formic acid containing acetonitrile (solvent B) as eluents.</li>
		<li>Analyze the diluted crude product by using a reverse-phase analytical radio-HPLC (C18 reverse-phase column; flow rate: 1 ml/min; eluent gradient: 20% solvent B for 0-2 min, 20-80% solvent B for 2-22 min, 80-100% solvent B for 22-23 min, and 100% solvent B for 23-28 min; retention time: 16.4 min) (Figure 2).</li>
	</ol>
	</li>
	<li>Purification of the crude product with a preparative HPLC 
	NOTE: Provide enough lead shielding around HPLC parts such as the injector, column, detector, collection vials, and the container in which the effluent is collected.
	<ol>
		<li>Withdraw the entire reaction mixture into an HPLC vial. Rinse the reaction tube with acetonitrile (0.5 ml) and add the rinse into the same injection vial. Dilute the collected solution with H2O (1 ml).</li>
		<li>Inject the crude product onto a preparative radio-HPLC (C18 reverse-phase column; flow rate: 10 ml/min; eluent gradient: 20% solvent B for 0-2 min, 20-80% solvent B for 2-22 min, 80-100% solvent B for 22-23 min, and 100% solvent B for 23-28 min).</li>
		<li>Collect the radioactive peak representing the 125I-labeled azide (1) (tR under these HPLC conditions is 17.8-18.8 min) in a glass test tube (Figure 2).</li>
		<li>Measure the radiochemical yield of the fraction using a radioactivity dose calibrator according to the manufacturer&#39;s protocol.</li>
		<li>Inject the purified product onto an analytical radio-HPLC using the same HPLC conditions for determining the radiochemical purity of the product.</li>
	</ol>
	</li>
	<li>Solid phase extraction of the product
	<ol>
		<li>Dilute the fraction containing the desired product (1) with 40 ml pure H2O.</li>
		<li>Add the diluted solution into a preconditioned tC18 cartridge.</li>
		<li>Wash the cartridge with an additional 15 ml H2O.</li>
		<li>Elute the product (1) trapped in the cartridge with 2 ml acetone into a 10-ml glass vial that is protected by a lead shield. Measure the radioactivity of the eluted product using a radioactivity dose calibrator according to the manufacturer&#39;s protocol. 
		NOTE: Dimethyl sulfoxide (DMSO) or absolute ethanol can also be used for elution of the product from the cartridge. Approximately 5-10% of the radioactivity normally sticks to the cartridge, and the remaining radiolabeled product cannot be fully eluted by using excess amounts of organic solvent.</li>
		<li>Evaporate the acetone with a stream of nitrogen or argon gas.</li>
		<li>Dissolve the residue with DMSO (100-200 &#181;l) for the next radiolabeling step.</li>
	</ol>
	</li>
</ol>
3. Synthesis of DBCO-group-conjugated Gold Nanoparticles
<ol>
	<li>Surface modification of 13-nm-sized gold nanoparticles with DBCO-group-containing polyethylene glycol
	<ol>
		<li>Prepare sodium-citrate-stabilized gold nanoparticles (3) (average size = 13 nm) according to a previous report24.</li>
		<li>Add an aqueous solution of Tween 20 (1 mM, 1.5 ml) to the citrate-stabilized gold nanoparticles (10 nM, 15 ml). Shake the solution for 20 min on an orbital shaker.</li>
		<li>Add an aqueous solution of DBCO-group-containing polyethylene glycol thiol (average molecular weight = 5,000, 100 &#956;M, 1.5 ml). Shake the solution for 2 hr on an orbital shaker.</li>
	</ol>
	</li>
	<li>Purification of the DBCO-group-modified gold nanoparticles
	<ol>
		<li>Purify the DBCO-group-modified gold nanoparticles (4) by successive centrifugation (11,400 x g, 15 min x 3).</li>
		<li>Decant the supernatant and add pure water for resuspension of the gold nanoparticle pellets.</li>
	</ol>
	</li>
</ol>
4. Radiolabeling of DBCO-group-modified Gold Nanoparticles via the Copper-free Click Reaction
<ol>
	<li>Synthesis of 125I-labeled gold nanoparticles using the 125I-labeled azide (1)
	<ol>
		<li>Prepare a concentrated solution of DBCO-group-modified gold nanoparticles by using centrifugation (11,400 x g, 15 min), and adjust the concentration of the gold nanoparticles to 2 &#181;M.</li>
		<li>Add 4.1 MBq of the 125I-labeled azide (1) in DMSO (5 &#181;l) to a suspension of gold nanoparticles (4) (2 &#956;M, 50 &#181;l).</li>
		<li>Incubate the resulting reaction mixture at 40 &#176;C for 60 min.</li>
		<li>Withdraw an aliquot (0.2 &#181;l) from the crude product and apply it onto a silica-coated thin-layer chromatography (TLC) plate.</li>
		<li>Develop the TLC plate using ethyl acetate as a mobile phase.</li>
		<li>Place the TLC plate on a radio-TLC scanner and run the scanner to monitor the radiolabeling reaction (Figure 3) according to the manufacturer&#39;s protocol.</li>
	</ol>
	</li>
	<li>Purification of the crude product
	<ol>
		<li>Purify the reaction mixture containing the 125I-labeled gold nanoparticles (4) by centrifugation (11,400 x g, 15 min).</li>
		<li>Decant the supernatant and add pure water for resuspension of the gold nanoparticle pellets.</li>
		<li>Withdraw an aliquot (0.2 &#181;l) from the purified product and apply it onto a silica-coated TLC plate.</li>
		<li>Develop the TLC plate using ethyl acetate as the mobile phase.</li>
		<li>Place the TLC plate on a radio-TLC scanner and run the scanner to determine the radiochemical yield and radiochemical purity of the 125I-labeled gold nanoparticles (4) (Figure 3) according to the manufacturer&#39;s protocol.</li>
	</ol>
	</li>
</ol>

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</ol>

The authors have nothing to disclose.

In general, the observed radiochemical yield of the purified 125I-labeled azide (1) was 75 &#177; 10% (n = 8). The radiolabeling was accomplished with 50-150 MBq of radioactivity, and the radiochemical results are quite consistent. If [125I]NaI (t1/2 = 59.4 d) that underwent radioactive decay for more than a month was used in the radioiodination reaction, the radiochemical yield of 1 was observed to be slightly decreased (53-65%). Therefore, it i...

The strain-promoted copper-free click reaction between azides and cyclooctynes has been extensively applied to the efficient bioorthogonal labeling of a wide range of biomolecules, nanomaterials, and living subjects1-7. Due to the excellent site-specificity and rapid reaction rate of this conjugation reaction, it has also been used to synthesize radiolabeled tracers. A few 18F-labeled azide or DBCO prosthetic groups have been prepared for in vitro labeling of various cancers targeting peptides and antibodies, as well as for in vivo pre-targeted imaging of tumors8-13. In addition to these examples, the same conjugation reaction was applied to the metal-radioisotope-labeling of nanomaterials for positron emission tomography (PET) imaging studies14-16.
For several decades, radioactive iodines have been used for biomedical research and clinical trials through PET imaging (124I), single-photon emission computed tomography (SPECT) imaging (123I, 125I), and thyroid cancer treatment (131I)17-21. Therefore, an efficient method for radioactive iodine labeling is fundamentally important for various investigations, including molecular imaging studies, analysis of organ distribution of biomolecules, biomarker identification, and drug development. A copper-free click reaction strategy could be used in radioactive iodine labeling. However, this application has not been investigated as extensively as 18F-labeled biomolecules22-23. Here, we will provide a step-by-step protocol for the synthesis of an 125I-labeled azide for radiolabeling of DBCO-group-derived molecules. The procedures in the present report will include radioiodination of the stannylated precursor, purification steps with HPLC, and solid phase extraction. We also demonstrate efficient radiolabeling of DBCO-group-modified 13-nm-sized gold nanoparticles using the 125I-labeled azide. The detailed protocol in this report will help synthetic chemists understand a new radiolabeling methodology for the synthesis of radiolabeled products.

<table><tbody><tr><td>Chloramine T trihydrate</td><td>Sigma</td><td>402869</td><td></td></tr><tr><td>[&lt;sup&gt;125&lt;/sup&gt;I]NaI in aq. NaOH</td><td>Perkin-Elmer</td><td>NEZ033A010MC</td><td></td></tr><tr><td>Sodium metabisulfite&amp;nbsp;</td><td>Sigma</td><td>S9000</td><td></td></tr><tr><td>Formic acid</td><td>Sigma</td><td>251364</td><td></td></tr><tr><td>Sep-Pak tC18 plus cartridge</td><td>Waters</td><td>WAT036800</td><td></td></tr><tr><td>Dimethyl sulfoxide&amp;nbsp;</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Acetone</td><td>Sigma</td><td>650501</td><td></td></tr><tr><td>Ethanol</td><td>Sigma</td><td>459844</td><td></td></tr><tr><td>Gold(III) chloride trihydrate</td><td>Sigma</td><td>520918</td><td></td></tr><tr><td>Tween 20&amp;nbsp;</td><td>Sigma</td><td>P1379</td><td></td></tr><tr><td>DBCO PEG SH (MW 5,000)</td><td>NANOCS</td><td>PG2-DBTH-5k</td><td></td></tr><tr><td>TLC silica gel 60 F&lt;sub&gt;254&lt;/sub&gt;</td><td>Merck</td><td></td><td></td></tr><tr><td>Analytical HPLC</td><td>Agilent</td><td>1290 Infinity</td><td>Model number</td></tr><tr><td>Preparative HPLC</td><td>Agilent</td><td>1260 Infinity</td><td>Model number</td></tr><tr><td>Analytical C18 reverse-phase column</td><td>Agilent</td><td>Zorbax Eclipse XDB-C18</td><td></td></tr><tr><td>Preparative C18 reverse-phase column</td><td>Agilent</td><td>PrepHT XDB-C18</td><td></td></tr><tr><td>Radio TLC scanner</td><td>Bioscan</td><td>AR-2000</td><td>Model number</td></tr><tr><td>Radioisotope dose calibrator</td><td>Capintec, Inc</td><td>CRC -25R dose calibrator</td><td>Model number</td></tr></tbody></table>

an optimized protocol for the efficient radiolabeling of gold nanoparticles by using a 125i-labeled azide prosthetic group

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

The radioiodination reaction of the stannylated precursor (2) was carried out using 150 MBq of [125I]NaI, acetic acid, and chloramine T at room temperature for 15 min to provide the radiolabeled product (1). After preparative HPLC purification of the crude mixture, the desired product was obtained with 75 ± 10% (n = 8) of radiochemical yield. Analytical HPLC revealed that the radiochemical purity of the 125I-labeled product was more ...

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.

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

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.

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Research

JoVE Journal

Chemistry

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Learn an efficient radiolabeling method for gold nanoparticles using a 125I-labeled azide prosthetic group and copper-free click chemistry. This protocol achieves high radiochemical yield and purity for imaging probe synthesis.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a ¹²⁵I-labeled Azide Prosthetic Group

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