Summary

Un protocollo ottimizzato per la radiomarcatura efficiente di oro nanoparticelle utilizzando un<sup> 125</sup> Azide gruppo I-etichettati protesico

Published: October 10, 2016
doi:

Summary

Una procedura dettagliata per la sintesi di un azide 125 I-etichettati e la radiomarcatura di dibenzocyclooctyne (DBCO) -gruppo-coniugati, nanoparticelle d'oro 13-nm dimensioni utilizzando una reazione click senza il rame è descritto.

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 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.

Protocol

Attenzione: la forma ossidata di iodio radioattivo è molto volatile e deve essere maneggiato con adeguati schermi di piombo e fiale di piombo. Tutti i passaggi radiochimiche dovrebbero essere eseguite in una cappa carbone filtrata ben ventilata, e le procedure sperimentali devono essere controllati da dispositivi di rilevazione di radioattività. 1. Preparazione della Chimica e della cartuccia Reverse Phase per la sintesi del 125 Azide I-marcato Preparazione dei reagenti in soluzio…

Representative Results

La reazione radioiodination del precursore stannylated (2) è stata effettuata utilizzando 150 MBq di [125 I] NaI, acido acetico, e cloramina T a temperatura ambiente per 15 minuti per fornire il prodotto radiomarcato (1). Dopo la purificazione preparativa HPLC della miscela grezza, il prodotto desiderato è stato ottenuto con 75 ± 10% (n = 8) della resa radiochimica. Analytical HPLC ha rivelato che la purezza radiochimica del prodotto 1…

Discussion

In generale, la resa radiochimica osservato del purificato 125 azide I-marcato (1) è stato 75 ± 10% (n = 8). La radiomarcatura è stata compiuta con 50-150 MBq di radioattività, ed i risultati radiochimiche sono abbastanza coerenti. Se [125 I] NaI (t 1/2 = 59,4 d) sottoposto decadimento radioattivo per più di un mese è stato utilizzato nella reazione radioiodination, la resa radiochimica 1 è stata osservata essere leggermente diminuita (53…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

Riferimenti

  1. Jewett, J. C., Bertozzi, C. R. Cu-free Click Cycloaddition Reactions in Chemical Biology. Chem. Soc. Rev. 39, 1272-1279 (2010).
  2. Debets, M. F., et al. Bioconjugation with Strained Alkenes and Alkyne. Acc. Chem. Res. 44, 805-815 (2011).
  3. Sletten, E. M., Bertozzi, C. R. From Mechanism to Mouse: A Tale of Two Bioorthogonal Reactions. Acc. Chem. Res. 44, 666-676 (2011).
  4. Koo, H., et al. Bioorthogonal Cu-Free Click Chemistry in vivo for Tumor-Targeted Delivery of Nanoparticles. Angew. Chem. Int. Ed. 51, 11836-11840 (2012).
  5. Chang, P. V., et al. Copper-Free Click Chemistry in Living Animals. Proc. Natl. Acad. Sci. U. S. A. 107, 1821-1826 (2010).
  6. Bostic, H. E., Smith, M. D., Poloukhtine, A. A., Popik, V. V., Best, M. D. Membrane Labeling and Immobilization via copper-free Click Chemistry. Chem. Commun. 48, 1431-1433 (2012).
  7. Someya, T., Ando, A., Kimoto, M., Hirao, I. Site-Specific Labeling of RNA by Combining Genetic Alphabet Expansion Transcription and Copper-Free Click Chemistry. Nucl. Acids Res. 43, 6665-6676 (2015).
  8. Lee, S. B., et al. Mesoporous Silica Nanoparticle Pretargeting for PET Imaging Based on a Rapid Bioorthogonal Reaction in a Living Body. Angew. Chem. Int. Ed. 52, 10549-10552 (2013).
  9. Sachin, K., et al. F-18 Labeling Protocol of Peptides Based on Chemically Orthogonal Strain-Promoted Cycloaddition under Physiologically Friendly Reaction Conditions. Bioconjugate Chem. 23, 1680-1686 (2012).
  10. Evans, H. L., et al. Copper-Free Click – A Promising Tool for Pre-targeted PET Imaging. Chem. Commun. 48, 991-993 (2012).
  11. Campbell-Verduyn, L. S., et al. Strain-Promoted Copper-Free "Click" Chemistry for 18F Radiolabeling of Bombesin. Angew. Chem. Int. Ed. 50, 11117-11120 (2011).
  12. Arumugam, S., Chin, J., Schirrmacher, R., Popik, V. V., Kostikov, A. P. 18F]Azadibenzocyclooctyne ([18F]ADIBO): A Biocompatible Radioactive Labeling Synthon for Peptides using Catalyst Free [3+2] Cycloaddition. Bioorg. Med. Chem. Lett. 21, 6987-6991 (2011).
  13. Bouvet, V., Wuest, M., Wuest, F. Copper-Free Click Chemistry with the Short-Lived Positron Emitter Fluorine-18. Org. Biomol. Chem. 9, 7393-7399 (2011).
  14. Satpati, D., Bauer, N., Hausner, S. H., Sutcliffe, J. L. Synthesis of [64Cu]DOTA-ADIBON3-Ala-PEG28-A20FMDV2 via Copper-Free Click Chemistry for PET Imaging of Integrin αvβ6. J. Radioanal. Nucl. Chem. 302, 765-771 (2014).
  15. Lee, D. E., et al. Facile Method To Radiolabel Glycol Chitosan Nanoparticles with 64Cu via Copper-Free Click Chemistry for MicroPET Imaging. Mol. Pharmaceutics. 10, 2190-2198 (2013).
  16. Zeng, D. 64Cu Core-Labeled Nanoparticles with High Specific Activity via Metal-Free Click Chemistry. ACS Nano. 6, 5209-5219 (2012).
  17. Jeon, J., et al. Radiosynthesis and in vivo Evaluation of [125I]2-4(iodophenethyl)-2-Methylmalonic Acid as a Potential Radiotracer for Detection of Apoptosis. J. Radioanal. Nucl. Chem. 308, 23-29 (2016).
  18. Adam, M. J., Wilbur, D. S. Radiohalogens for Imaging and Therapy. Chem. Soc. Rev. 34, 153-163 (2005).
  19. Jeon, J., et al. Radiosynthesis of 123I-Labeld Hesperetin for Biodistribution Study of Orally Administered Hesperetin. J. Radioanal. Nucl. Chem. 306, 437-443 (2015).
  20. Kil, K. E., et al. Development of [123I]IPEB and [123I]IMPEB as SPECT Radioligands for Metabotropic Glutamate Receptor Subtype. ACS Med. Chem. Lett. 5, 652-656 (2014).
  21. Chen, M. K., et al. The Utility of I-123 Pretherapy Scan in I-131 Radioiodine Therapy for Thyroid Cancer. Thyroid. 22, 304-309 (2012).
  22. Jeon, J., et al. Efficient Method for Iodine Radioisotope Labeling of Cyclooctyne-Containing Molecules using Strain-Promoted Copper-Free Click Reaction. Bioorg. Med. Chem. 23, 3303-3308 (2015).
  23. Choi, M. H., et al. Synthesis and Evaluation of an 125I-Labeled Azide Prosthetic Group for Efficient and Bioorthogonal Radiolabeling of Cyclooctyne-Group Containing Molecules using Copper-Free Click Reaction. Bioorg. Med. Chem. Lett. 26, 875-878 (2016).
  24. Kim, Y. H., et al. Tumor Targeting and Imaging Using Cyclic RGD-PEGylated Gold Nanoparticle Probes with Directly Conjugated Iodine-125. Small. 7, 2052-2060 (2011).

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Citazione di questo articolo
Jeon, J., Shim, H. E., Mushtaq, S., Choi, M. H., Park, S. H., Choi, D. S., Jang, B. An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group. J. Vis. Exp. (116), e54759, doi:10.3791/54759 (2016).

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