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....

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....

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 more .......

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

List of materials used in this article

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 5,000)	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