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

Summary

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.

Abstract

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 ± 10%, n = 8) and excellent radiochemical purity (>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.

Introduction

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 subjects^1-7. 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 tumors^8-13. In addition to these examples, the same conjugation reaction was applied to the metal-radioisotope-labeling of nanomaterials for positron emission tomography (PET) imaging studies^14-16.

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 biomolecules^22-23. 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.

Protocol

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

1. Preparation of reagents in solution
   1. Dissolve 1 mg of the azide precursor (2) in 150 µl absolute ethanol (Figure 1).
      NOTE: A detailed synthetic procedure for the azide precursor (2) was reported in the previous paper²².
   2. Dissolve 1 mg chloramine T in 20 µl of 1x phosphate buffer saline (pH = 7.4).
   3. Dissolve 2 mg sodium metabisulfite in 20 µl H₂O.
2. Preparation of the cartridge
   1. Wash the tC18 cartridge with 10 ml absolute ethanol followed by 10 ml H₂O. Do not dry the matrix of the cartridge with air.

2. Radiosynthesis of the ¹²⁵I-labeled Azide Prosthetic Group

1. Radioiodination reaction of the precursor
   1. Add the azide precursor solution (1 mg in 150 µl of absolute ethanol) and acetic acid (10 µl) to a 1.5 ml microcentrifuge tube.
   2. Add 150 MBq of [¹²⁵I]NaI in 0.1 M NaOH (50 µl) to the reaction mixture.
   3. Add a chloramine T solution (1 mg in 20 µl of 1x phosphate buffer saline) and close the microcentrifuge tube containing reaction mixture.
   4. Incubate the reaction mixture at room temperature for 15 min until the radioiodination reaction is completed.
   5. Add a sodium metabisulfite solution (2 mg in 20 µl H₂O) to the reaction mixture to quench the radioiodination reaction.
   6. Withdraw 0.2 µl of the crude product and then dilute it with 100 µl 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.
   7. 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).
2. 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.
   1. 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).
   2. 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).
   3. Collect the radioactive peak representing the ¹²⁵I-labeled azide (1) (t_R under these HPLC conditions is 17.8-18.8 min) in a glass test tube (Figure 2).
   4. Measure the radiochemical yield of the fraction using a radioactivity dose calibrator according to the manufacturer's protocol.
   5. Inject the purified product onto an analytical radio-HPLC using the same HPLC conditions for determining the radiochemical purity of the product.
3. Solid phase extraction of the product
   1. Dilute the fraction containing the desired product (1) with 40 ml pure H₂O.
   2. Add the diluted solution into a preconditioned tC18 cartridge.
   3. Wash the cartridge with an additional 15 ml H₂O.
   4. 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'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.
   5. Evaporate the acetone with a stream of nitrogen or argon gas.
   6. Dissolve the residue with DMSO (100-200 µl) for the next radiolabeling step.

3. Synthesis of DBCO-group-conjugated Gold Nanoparticles

1. Surface modification of 13-nm-sized gold nanoparticles with DBCO-group-containing polyethylene glycol
   1. Prepare sodium-citrate-stabilized gold nanoparticles (3) (average size = 13 nm) according to a previous report²⁴.
   2. 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.
   3. Add an aqueous solution of DBCO-group-containing polyethylene glycol thiol (average molecular weight = 5,000, 100 μM, 1.5 ml). Shake the solution for 2 hr on an orbital shaker.
2. Purification of the DBCO-group-modified gold nanoparticles
   1. Purify the DBCO-group-modified gold nanoparticles (4) by successive centrifugation (11,400 x g, 15 min x 3).
   2. Decant the supernatant and add pure water for resuspension of the gold nanoparticle pellets.

4. Radiolabeling of DBCO-group-modified Gold Nanoparticles via the Copper-free Click Reaction

1. Synthesis of ¹²⁵I-labeled gold nanoparticles using the ¹²⁵I-labeled azide (1)
   1. 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 µM.
   2. Add 4.1 MBq of the ¹²⁵I-labeled azide (1) in DMSO (5 µl) to a suspension of gold nanoparticles (4) (2 μM, 50 µl).
   3. Incubate the resulting reaction mixture at 40 °C for 60 min.
   4. Withdraw an aliquot (0.2 µl) from the crude product and apply it onto a silica-coated thin-layer chromatography (TLC) plate.
   5. Develop the TLC plate using ethyl acetate as a mobile phase.
   6. Place the TLC plate on a radio-TLC scanner and run the scanner to monitor the radiolabeling reaction (Figure 3) according to the manufacturer's protocol.
2. Purification of the crude product
   1. Purify the reaction mixture containing the ¹²⁵I-labeled gold nanoparticles (4) by centrifugation (11,400 x g, 15 min).
   2. Decant the supernatant and add pure water for resuspension of the gold nanoparticle pellets.
   3. Withdraw an aliquot (0.2 µl) from the purified product and apply it onto a silica-coated TLC plate.
   4. Develop the TLC plate using ethyl acetate as the mobile phase.
   5. 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's protocol.

Results

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 ...

Discussion

In general, the observed radiochemical yield of the purified ¹²⁵I-labeled azide (1) was 75 ± 10% (n = 8). The radiolabeling was accomplished with 50-150 MBq of radioactivity, and the radiochemical results are quite consistent. If [¹²⁵I]NaI (t_1/2 = 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...

Materials

Name	Company	Catalog Number	Comments
Chloramine T trihydrate	Sigma	402869
[125I]NaI in aq. NaOH	Perkin-Elmer	NEZ033A010MC
Sodium metabisulfite	Sigma	S9000
Formic acid	Sigma	251364
Sep-Pak tC18 plus cartridge	Waters	WAT036800
Dimethyl sulfoxide	Sigma	D2650
Acetone	Sigma	650501
Ethanol	Sigma	459844
Gold(III) chloride trihydrate	Sigma	520918
Tween 20	Sigma	P1379
DBCO PEG SH (MW 5000)	NANOCS	PG2-DBTH-5k
TLC silica gel 60 F254	Merck
Analytical HPLC	Agilent	1290 Infinity	Model number
Preparative HPLC	Agilent	1260 Infinity	Model number
Analytical C18 reverse-phase column	Agilent	Zorbax Eclipse XDB-C18
Preparative C18 reverse-phase column	Agilent	PrepHT XDB-C18
Radio TLC scanner	Bioscan	AR-2000	Model number
Radioisotope dose calibrator	Capintec, Inc	CRC -25R dose calibrator	Model number