Name: preparing a 68ga-labeled arginine glycine aspartate (rgd)-peptide for angiogenesis
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Preparing a 68Ga-labeled Arginine Glycine Aspartate RGD-peptide for Angiogenesis at JoVE.com

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).

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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In the present study, we introduced a protocol to prepare a radiopeptide targeting &#945;v&#946;3 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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Angiogenesis is a biological process that is characterized by the development of new blood vessels. Among many angiogenetic factors, &#945;v&#946;3 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 &#945;v&#946;3 integrin receptors, are considered promising angiogenesis imaging agents2,3,4,5,6,7. Several radiopharmaceuticals have been created for PET and its biological properties have been validated in various animal models8,9,10,11. In terms of a radionuclide, 68Ga has several advantages over other radioisotopes. Firstly, it has a high accessibility for users and is economically advantageous because a cyclotron is not required. Secondly, 68Ga-based radiopharmaceuticals produce high spatial resolution compared with single-photon emission computed tomography (SPECT), allowing more accurate quantification. Lastly, the 67.71 minutes half-life of 68Ga may be sufficient for the preparation of small peptides or proteins.
To produce a stable complex with 68Ga, many chelators have been developed. Representative chelators are 1,4,8,11-tetraazacyclotetradecanetetraacetic acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), and N,N&#39;-di(2-hydroxybenzyl)ethylenediamine-N,N&#39;-diacetic acid (HBED). NOTA has been reported to form a highly stable complex with 68Ga (log stability constant 30.98)12,13,14.
The purpose of the present study is to provide a concise protocol for the development of a new radiopeptide (Figure 1). As an example, we prepare 68Ga-labeled RGD-peptides and present methods for the biological evaluation of these analogues in a xenograft model.

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			<td height="21" style="height:21px;width:231px;">Sodium acetate</td>
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			<td height="21" style="height:21px;width:231px;">ITLC paper</td>
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			<td height="21" style="height:21px;width:231px;">Citric acid</td>
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			<td>ODS-HG-5, 150 &#8569; 4.6 mm, 5&#956;m</td>
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			<td height="21" style="height:21px;width:231px;">Acetonitrile</td>
			<td style="width:187px;">J.T. Baker</td>
			<td style="width:119px;">14-650-359</td>
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			<td height="21" style="height:21px;width:231px;">Trifluoroacetic acid</td>
			<td style="width:187px;">Sigma-aldrich</td>
			<td style="width:119px;">302031</td>
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			<td height="21" style="height:21px;width:231px;">Dulbecco&#39;s modified Eagle media&#160;</td>
			<td style="width:187px;">Thermo fisher scientific</td>
			<td style="width:119px;">11965092</td>
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			<td height="21" style="height:21px;">fetal bovine serum</td>
			<td style="width:187px;">Thermo fisher scientific</td>
			<td style="width:119px;">16000044</td>
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			<td height="21" style="height:21px;width:231px;">T175 flasks&#160;</td>
			<td style="width:187px;">Corning</td>
			<td>CLS431080</td>
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			<td height="21" style="height:21px;width:231px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:187px;">Thermo fisher scientific</td>
			<td>25200072</td>
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			<td height="21" style="height:21px;width:231px;">penicillin-streptomycin</td>
			<td style="width:187px;">Thermo fisher scientific</td>
			<td style="width:119px;">15240112</td>
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			<td height="21" style="height:21px;width:231px;">&#947;-counter</td>
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			<td height="21" style="height:21px;width:231px;">Insunlin syringe</td>
			<td style="width:187px;">Becton Dickinson</td>
			<td style="width:119px;">326105</td>
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			<td height="21" style="height:21px;width:231px;">Synringe pump</td>
			<td style="width:187px;">Harvard Apparatus</td>
			<td style="width:119px;">70-4500</td>
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			<td height="21" style="height:21px;width:231px;">micro-PET/CT</td>
			<td style="width:187px;">Siemens Inveon</td>
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preparing a 68ga-labeled arginine glycine aspartate (rgd)-peptide for angiogenesis

The &#945;v&#946;3 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 &#945;v&#946;3 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 &#945;v&#946;3 integrin. Here, we introduce a protocol to prepare a 68Ga-RGD-peptide and the evaluation of its biological efficacy.

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...

Watch this Scientific Journal Video about Preparing a 68Ga-labeled Arginine Glycine Aspartate RGD-peptide for Angiogenesis at JoVE.com

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

The &#945;v&#946;3 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.

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

The &#945;v&#946;3 integrin is a heterodimeric adhesion molecule involved in tumor cell migration and angiogenesis. The integrin is overexpressed in ...

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