Integrin family consists of 24 different hetero-dimerized transmembrane receptors which play an important roles in many physiological and pathological processes including cell survival, growth, differentiation, migration, inflammatory responses, platelet aggregation, tissue repair and tumor invasion (1). There are two major targets for angiogenesis imaging with Positron Emission Tomography (PET); vascular endothelial growth factor receptor (VEGFR) and integrin αvβ3. VEGFR is the most significant and potent stimulator of angiogenesis and integrin αvβ3, a heterodimeric cell surface receptor, plays a significant role in angiogenesis by allowing cells to interact with the extracellular matrix, contributing to the migration of endothelial cells.

In our present study we select a short peptide, containing eight amino acids domain and having affinity towards the αvβ3 integrin receptors over expressed in angiogenesis, and introduce various modifications and then been labeled with 99mTc, ⁶⁸Ga, ^131/123I and ¹⁸F.

All needed reagents and solvents were purchased and used with no further purifications unless it’s necessary. The peptide used were purchased from GenScript and used without further purifications. Reaction progress was monitored using both radio-analytical thin-layer chromatography (Radio-TLC) and high pressure liquid chromatographic (Radio-HPLC). Radio-HPLC analyses were carried out on semi-preparative Phenomenex C-18, 250 mm x 10 mm.

Radiolabeled peptide was detected with the eluting of 0.065% TFA in 100% water (v/v) [C] and 0.05% TFA in 100% MeCN(v/v) [D] at 2 ml/min 0 min 90% [C], 10% [D], 25-30min 20% [C], 80% [D], 40min 90% [C] 10% [D]. Radio-HPLC was used to calculate the radiochemical yields. Sodium Citrate Buffer (PH=7.5) was the Radio-TLC mobile phase.

Synthesis of DOTA- Peptide Conjugate.

• 9.97 E-7 mol of the chosen peptide dissolved in 30µl DMSO.
• 1.18 E-6 mol of (DOTA- NHS-ester) dissolved in 20 µl DMSO.
• 5 µl of triethylamine (TEA).
• React at room temperature for 2 Hours.

Synthesis of Radiolabeling.

• 60 μg of conjugated DOTA-Peptide.
• 100 μl of HEPES buffer PH=4.
• 50 μl of ⁶⁸Ga solution (2 mCi [74 MBq]).
• Allowed the solution to react for 30 min at 80°C.

Radio-HPLC chromatogram shows a single peak (Fig.1) indicating a single radiolabeled product [⁶⁸G-DOTA-Peptide]. High purity was detected with the Radio-TLC (Fig. 3).

Fig .1: Radio-HPLC chromatogram of the Radiolabeled [⁶⁸G-DOTA-Peptide], the ration time of product was 8.4 min.
 

Fig .2: Free ⁶⁸Ga on Radio-TLC system, RF= 0.9, H₂O is the mobile phase.

Fig .3: Radiolabeled product [⁶⁸Ga-DOTA-Peptide] on Radio-TLC system, RF= 0.00, H₂O is the mobile phase.

[⁶⁸G-DOTA-Peptide] was prepared in good radiochemical yield and high radiochemical purity.

The product might be a promising for noninvasive imaging of integrin αvβ3 expression which will be a possible target for early cancer diagnosis.

This work was supported by King Abdualaziz City for Science & Technology (AT-29-15).

[1] Niu G, Chen X. Why, “integrin as a primary target for imaging and therapy”, Theranostics. 2011; 1: 30-47.

[2] M. Lourdes Ponce, Suguru Hibino, Agata M. Lebioda, Mayumi Mochizuki, Motoyoshi Nomizu, and Hynda K. Kleinman, “Identification of a Potent Peptide Antagonist to an Active Laminin-1 Sequence That Blocks Angiogenesis and Tumor Growth”, Cancer Research 63, 5060–5064, August 15, 2003.

[3] Mohammed Al-Qahtani and Yousif Al-Malki, (2016), “[⁶⁸Ga]Radiolabelling of oligopeptide that has a pet imaging potential”, presented at the International Conference on Radioanalytical and Nuclear Chemistry (RANC-2016), April 10 – 15, Budapest, Hungary, Book of Abstracts, P 208.

[4] Mohammed Al-Qahtani and Yousif Al-Malki, (2016), “[⁶⁸Ga]Radiolabelling of short peptide that has a pet imaging potential”, presented at the ¹⁸th European Symposium on Radiopharmacy and Radiopharmaceuticals, April 07 – 10, Salzburg, Austria, Book of Abstracts, P 100.