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Ardipradja K.,Vascular Biotechnology Laboratory | Ardipradja K.,Atherothrombosis and Vascular Biology Laboratory | Ardipradja K.,Austin Hospital | Ardipradja K.,University of Melbourne | And 16 more authors.
Nuclear Medicine and Biology | Year: 2014

Introduction: Activated platelets are key players in thrombosis and inflammation. We previously generated single-chain antibodies (scFv) against ligand-induced binding sites (LIBS) on the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. The aim of this study was the construction and characterisation of a novel 18F PET radiotracer based on this antibody. Methods: ScFvanti-LIBS and control antibody mut-scFv were reacted with N-succinimidyl-4-[18F]fluorobenzoate (S[18F]FB). Radiolabeled scFv was incubated with in vitro formed platelet clots and injected into mice with FeCl3 induced thrombus in the left carotid artery. Clots were imaged in the PET scanner and amount of radioactivity measured using an ionization chamber and image analysis. Assessment of vessel injury as well as the biodistribution of the radiolabeled scFv was studied. Results: After incubation with increasing concentrations of 18F-scFvanti-LIBS clots had retained significantly higher amounts of radioactivity compared to clots incubated with radiolabeled 18F-mut-scFv (13.3±3.8 vs. 3.6±1 KBq, p<0.05, n=9, decay corrected). In the in vivo experiments we found an high uptake of the tracer in the injured vessel compared with the non-injured vessel, with 12.6±4.7% injected dose per gram (ID/g) uptake in the injured vessel and 3.7±0.9% ID/g in the non-injured vessel 5minutes after injection (p<0.05, n=6). Conclusions: Our results show that the novel antibody radiotracer 18F-scFvanti-LIBS is useful for the sensitive detection of activated platelets and thrombosis. Advances in knowledge and implications for patient care: We describe the first 18F variant of a scFvanti-LIBS against activated platelets. This diagnostic agent could provide a powerful tool for the assessment of acute thrombosis and inflammation in patients in the future. © 2014 Elsevier Inc.


Meier S.,Albert Ludwigs University of Freiburg | Putz G.,University Hospital Freiburg | Massing U.,Tumor Biology Center Freiburg | Massing U.,Andreas Hettich GmbH and Co KG | And 10 more authors.
Biomaterials | Year: 2015

To detect unstable atherosclerotic plaques early and noninvasively would be of great clinical interest. Activated platelets are an interesting molecular target for detecting early lesions or unstable plaques. We therefore developed an MRI contrast agent consisting of magnetoliposomes (ML) linked to an antibody (anti-LIBS) specifically targeting the ligand-induced binding site of the activated GPIIb/IIIa receptor of platelets. ML were prepared by dual centrifugation (DC). ML pegylation up to a total PEG content of 7.5mol% positively influenced the stability and amount of entrapped SPIOs, and also reduced SPIO-membrane interactions, while higher PEG contents destabilized PEG-ML. Stable anti-LIBS-ML with high amounts of entrapped SPIOs (~86%, ~0.22mol Fe/mol liposomal lipid) and high MRI sensitivity (relaxivity r2=422s-1mM-1 and r2*=452s-1mM-1) were obtained by coupling anti-LIBS to ML in a two-step post-insertion technique. We confirmed specific binding to the GPIIb/IIIa receptor's activated conformation on activated human platelets and cell lines expressing activated GPIIb/IIIa receptor exvivo. The immuno-ML obtained in this study constitute an important step towards developing a potentially human-compatible MRI contrast agent for the timely detection of plaque rupture by targeting activated platelets. © 2015 Elsevier Ltd.


PubMed | Atherothrombosis and Vascular Laboratory, Vascular Biotechnology Laboratory, Germany; Andreas Hettich GmbH & Co KG, Albert Ludwigs University of Freiburg and University Hospital Freiburg
Type: | Journal: Biomaterials | Year: 2015

To detect unstable atherosclerotic plaques early and noninvasively would be of great clinical interest. Activated platelets are an interesting molecular target for detecting early lesions or unstable plaques. We therefore developed an MRI contrast agent consisting of magnetoliposomes (ML) linked to an antibody (anti-LIBS) specifically targeting the ligand-induced binding site of the activated GPIIb/IIIa receptor of platelets. ML were prepared by dual centrifugation (DC). ML pegylation up to a total PEG content of 7.5mol% positively influenced the stability and amount of entrapped SPIOs, and also reduced SPIO-membrane interactions, while higher PEG contents destabilized PEG-ML. Stable anti-LIBS-ML with high amounts of entrapped SPIOs (86%, 0.22mol Fe/mol liposomal lipid) and high MRI sensitivity (relaxivity r2=422s(-1)mM(-1) and r2()=452s(-1)mM(-1)) were obtained by coupling anti-LIBS to ML in a two-step post-insertion technique. We confirmed specific binding to the GPIIb/IIIa receptors activated conformation on activated human platelets and cell lines expressing activated GPIIb/IIIa receptor exvivo. The immuno-ML obtained in this study constitute an important step towards developing a potentially human-compatible MRI contrast agent for the timely detection of plaque rupture by targeting activated platelets.


PubMed | Vascular Biotechnology Laboratory
Type: Journal Article | Journal: Molecular pharmaceutics | Year: 2014

Imaging of activated platelets using an activation specific anti-GPIIb/IIIa integrin single-chain antibody (scFvanti-LIBS) conjugated to a positron emitting copper-64 complex of a cage amine sarcophagine chelator (MeCOSar) is reported. This tracer was compared in vitro to a (64)Cu(II) complex of the scFv conjugated to another commonly used macrocycle, DOTA. The scFvanti-LIBS-MeCOSar conjugate was radiolabeled with (64)Cu(II) rapidly under mild conditions and with higher specific activity than scFvanti-LIBS-DOTA. The utility of scFvanti-LIBS-MeCOSar as a diagnostic agent was assessed in vivo in a mouse model of acute thrombosis. The uptake of scFvanti-LIBS-(64)CuMeCOSar in the injured vessel was significantly higher than the noninjured vessel. Positron emission tomography (PET) was used to show accumulation of scFvanti-LIBS-(64)CuMeCOSar with high and specific uptake in the injured vessel. ScFvanti-LIBS-(64)CuMeCOSar is an excellent tool for highly sensitive in vivo detection of activated platelets in PET and has the potential to be used for early diagnosis of acute thrombotic events.

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