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;">C18 reverse-phase cartridge</td>
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			<td height="23" style="height:23px;width:231px;">0.22-&#956;m sterile filter</td>
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			<td height="21" style="height:21px;width:231px;">Radio-TLC scanner</td>
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			<td style="width:119px;">AR2000</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>251275</td>
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			<td height="84" style="height:84px;width:231px;">HPLC</td>
			<td style="width:187px;">Waters</td>
			<td style="width:119px;">-</td>
			<td style="width:239px;">Waters 1525 system containing binary pump, photo diode array (Waters 2998), radioactivity detector (Raytest, Gabi)</td>
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			<td height="23" style="height:23px;width:231px;">C-18 colunm</td>
			<td style="width:187px;">Develosil</td>
			<td style="width:119px;">HG31546150W</td>
			<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>
			<td></td>
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		<tr height="21">
			<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>
			<td style="width:187px;">Perkin Elmer</td>
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			<td style="width:239px;">1480 Wizard 3</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>
			<td style="width:119px;">-</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 ...

This method can help answer key questions in the preparation of the Gallium 68 labeled RGD-Peptide and the method for the biological evaluation of its analogs. The main advantage of this technique is that it is a simple systemic protocol for the development of a new radiometal-labeled peptide. Demonstrating the procedure will be Ki-Hye Jung, a post-doc from our institute.Begin by using 0.5 molar hydrochloric acid to elute radiolabeled gallium trichloride from a radiolabeled gallium germanium generator. Purge the compound in five milliliter reaction vial with nitrogen gas at 80 degrees Celsius. Followed by the addition of 100 micrograms of arginine glycine acetate or RGD peptide in one molar sodium acetate to the dried compound.Heat the reaction mixture at 80 degrees Celsius for five minutes before allowing the mixture to cool to room temperature. To purify the crude product with high performance liquid chromatography, add the solution to a C18 column and pass the solution through a C18 reverse-phased cartridge. Wash the cartridge with two milliliters of saline and elute the radiolabeled peptide with 0.7 milliliters of 95%ethanol followed by the removal of the solvent at 80 degrees Celsius under nitrogen gas for 20 minutes.Reconstitute the purified peptide in PBS and filter the radiolabeled product through a sterile 0.22 micron sterile strainer. Formulate the peptide in one milliliter of sterile saline solution and spot one microliter of peptide solution onto an instant thin layer chromatography plate. Then, develop the plate in the chamber containing aqueous 0.1 molar citric acid until nine centimeters away from the spot to determine the radio chemical yield.To assess the in vivo cellular uptake of the peptide, treat one times 10 to the sixth human glioblastoma cells per well in six well plates with 111 kilobecquerel of radiolabeled RGD-peptide per well at 37 degrees Celsius for 30, 60, 90 or 120 minutes in triplicate. At the end of the incubation, wash the wells two times with two milliliters of PBS per wash, and harvest the cells with 0.25%Trypsin and 0.02%EDTA in PBS at 37 degrees Celsius for three to five minutes. Then, transfer the cells from each well into 15 milliliter conical tubes to measure the radiolabeled peptide uptake on a gamma counter.To assess the serum stability of the radiolabeled RGD-peptide, add 500 microliters of freshly prepared mouse serum, 500 microliters of human serum, and 500 microliters of PBS to 50 microliters of each sample and incubate the mixtures at 37 degrees Celsius for up to two hours. Then spot one to two microliters of each sample onto an instant thin layer chromatography plate after 30, 60, 90 and 120 minutes of incubation and develop the plates as demonstrated. To determine the lipophilicity of the peptide, add 10 microliters of the radiolabeled RGD-peptide to an octanal PBS system and mix the vials generously for five minutes at room temperature.Then collect the peptide by centrifugation and harvest 100 microliters of sample from each layer for measuring with a gamma counter. To generate radiolabeled tumor models, load five times 10 to the sixth human glioblastoma cells in 100 microliters of PBS into one half inch insulin syringe equipped with a 28 gauge needle per BALB/c nude mouse to be injected, and deliver the tumor cells subcutaneously into the left flank of each recipient. For in vivo quantification of the peptide by positron emission tomography, or PET, place the head a tumor-bearing, anesthetized mouse into the PET gantry and intravenously administer 7.4 megabecquerels of radiolabeled RGD-peptide solution in 200 microliters of PBS into each xenograft mouse model via the tail vein over one minute while performing a PET scan in the list mode.For ex vivo biodistribution analysis, inject 0.37 megabecquerels of radiolabeled RGD-peptide in 200 microliters of PBS into the tail vein of a xenografted, tumor-bearing animal, and harvest the tissues of interest at 30, 60, 90 and 120 minutes post-injection. Then weigh the tissues and measure their radioactivity with a gamma counter. After chelation, reaction impurities can be successfully removed by high performance liquid chromatography as demonstrated.A greater than 99%radiochemical purity of the radiolabeled RGD peptide can be achieved with a specific activity at the end of the synthesis between 90 and 130 megabecquerels per nanometer. PET analysis demonstrates an initial high uptake in the major organs, including the liver, kidney, heart, muscle, as well as within the tumor. During the later stages of analysis, the tumor region can be clearly visualized with the tumor to muscle ratio remaining unchanged, indicating the kinetic stability of the peptide.Ex vivo biodistribution analysis reveals that the accumulated radioactivity in the tumor decreases over time as expected from the in vivo PET findings. While attempting this procedure, it's important to remember that, although our methodology cannot replace the delicate biological evaluation process, it can be used to considerably be use the time and cost of a traditional drug development practices. Don't forget that working with the radioactive materials can be extremely hazardous and precautions, such as using the proper protective equipment like a personal dosimeter and thermoluminescent dosimeter should always be taken while performing these procedures.

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