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Preparing a 68Ga-labeled Arginine Glycine Aspartate (RGD)-peptide for Angiogenesis

Published: January 7th, 2019



1Division of Applied RI, Korea Institute of Radiological and Medical Sciences

The αvβ3 integrin is a type of adhesion protein that is highly expressed on activated endothelial cells undergoing angiogenesis. Thus, evaluating the integrity of the integrin is of great interest in oncology. Here, we introduce a method to prepare 68Ga-labeled radiopeptides and a method to assess its biological effectiveness.

The αvβ3 integrin is a heterodimeric adhesion molecule involved in tumor cell migration and angiogenesis. The integrin is overexpressed in angiogenic tumor endothelial cells, where it typically has a low concentration. This specific expression of αvβ3 makes it a valid biomarker for antiangiogenic and imaging drugs. As a functional imaging modality, positron emission tomography (PET) provides information about biochemical and physiological changes in vivo, due to its unique high sensitivity at the nanomolar scale. Hence, radiometal-based PET radiopharmaceuticals have received great attention for the non-invasive quantification of tumor angiogenesis. This paper provides a systemic protocol to prepare a new radiometal-labeled peptide for the evaluation of angiogenesis. This protocol contains information about radiochemical reliability, lipophilicity, cell uptake, serum stability, and pharmacokinetic properties. The 68Ga-RGD-peptide is one of the representative PET ligands toward αvβ3 integrin. Here, we introduce a protocol to prepare a 68Ga-RGD-peptide and the evaluation of its biological efficacy.

Angiogenesis is a biological process that is characterized by the development of new blood vessels. Among many angiogenetic factors, αvβ3 integrin is associated with invasiveness, because the integrin is highly expressed in angiogenic tumor vessels but is absent in normal tissue1.

Radiolabeled receptor-binding peptides with the arginine glycine aspartate (RGD) domain, which has a high affinity toward αvβ3 integrin receptors, are considered promising angiogenesis imaging agents2,3,....

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All animal experiments were conducted in compliance with the Guidelines for the Care and Use of Research Animals under protocols approved by the Korea Institute of Radiological and Medical Sciences Animal Studies Committee. All reagents and solvents were purchased and used without further purification. NOTA-RGD-peptides were prepared according to literature methods15.

CAUTION: 68Ga emits both positron and gamma rays. All experiments, including direct or indir.......

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The chelation of 68GaCl3 with the NOTA-RGD-peptide was straightforward, and the radiolabeling yield was 99%. Reaction impurities were successfully removed as shown in Figure 2. The radiochemical purity of 68Ga-RGD-peptide was greater than 99%, and specific activity at the end of the synthesis was 90 - 130 MBq/nmol (Figure 3).

The cell up.......

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In the present study, we introduced a protocol to prepare a radiopeptide targeting αvβ3 integrin and its biological evaluation. Traditional drug development involves a complicated procedure. It requires a large quantity of reference material and a relatively long evaluation time. Although the suggested methodology cannot replace the delicate evaluation process, this system can be used for screening purposes. This proposed system would considerably reduce the time and cost.

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This work was supported by a Nuclear Research and Development Program of the National Research Foundation of Korea (NRF) grant funded by the Korean government (No. 2017M2A2A6A02019904).


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Name Company Catalog Number Comments
68Ga/68Ge generator ITG Company - 10 mCi 
Hydrogen chloride solution Sigma-aldrich 84429
Sodium acetate Sigma-aldrich S2889
C18 reverse-phase cartridge Waters WAT020515
0.22-μm sterile filter Milllipore SLGV033RS
Radio-TLC scanner Bioscan AR2000
ITLC paper Agilent SGI001
Citric acid Sigma-aldrich 251275
HPLC Waters - Waters 1525 system containing binary pump, photo diode array (Waters 2998), radioactivity detector (Raytest, Gabi)
C-18 colunm Develosil HG31546150W ODS-HG-5, 150 ⅹ 4.6 mm, 5μm
Acetonitrile J.T. Baker 14-650-359
Trifluoroacetic acid Sigma-aldrich 302031
Dulbecco's modified Eagle media  Thermo fisher scientific 11965092
fetal bovine serum Thermo fisher scientific 16000044
T175 flasks  Corning CLS431080
Trypsin-EDTA (0.25%) Thermo fisher scientific 25200072
penicillin-streptomycin Thermo fisher scientific 15240112
γ-counter Perkin Elmer - 1480 Wizard 3
Insunlin syringe Becton Dickinson 326105
Synringe pump Harvard Apparatus 70-4500
micro-PET/CT Siemens Inveon -

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