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2.
Sci Rep ; 7(1): 14898, 2017 11 02.
Article in English | MEDLINE | ID: mdl-29097799

ABSTRACT

Platelets can be found on the surface of inflamed and ruptured atherosclerotic plaques. Thus, targeting of activated platelets may allow for molecular imaging of vulnerable atherosclerotic lesions. We here investigated microbubbles (MB) functionalized with the selectin ligand sialyl Lewisa individually (MBsLea) or dually with sLea and an antibody targeting ligand-induced binding sites of the activated GPIIb/IIIa receptor (MBDual). Assessed by in vitro flow chamber, targeted MB exhibited increased adhesion to platelets as compared to MBControl. While MBsLea rolled slowly on the platelets' surface, MBDual enhanced the percentage of firm adhesion. In vivo, MB were investigated by ultrasound in a model of ferric chloride induced non-occlusive carotid artery thrombosis. MBsLea and MBDual revealed a higher ultrasound mean acoustic intensity than MBControl (p < 0.05), however MBDual demonstrated no additional increase in mean signal intensity as compared to MBsLea. The degree of carotid artery stenosis on histology correlated well with the ultrasound acoustic intensity of targeted MB (p < 0.05). While dual targeting of MB using fast binding carbohydrate polymers and specific antibodies is a promising strategy to support adhesion to activated platelets under arterial shear stress, these advantages seem not readily translatable to in vivo models.


Subject(s)
Blood Platelets/pathology , Contrast Media/analysis , Microbubbles , Platelet Activation , Thrombosis/diagnostic imaging , Animals , Antibodies, Immobilized/analysis , Antibodies, Immobilized/metabolism , Blood Platelets/metabolism , CA-19-9 Antigen , Carotid Arteries/diagnostic imaging , Carotid Arteries/metabolism , Carotid Arteries/pathology , Contrast Media/metabolism , Female , Ligands , Mice, Inbred C57BL , Oligosaccharides/analysis , Oligosaccharides/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Selectins/metabolism , Thrombosis/metabolism , Thrombosis/pathology , Ultrasonography
3.
Circ Res ; 109(4): 365-73, 2011 Aug 05.
Article in English | MEDLINE | ID: mdl-21700932

ABSTRACT

RATIONALE: Antibody-targeted delivery of imaging agents can enhance the sensitivity and accuracy of current imaging techniques. Similarly, homing of effector cells to disease sites increases the efficacy of regenerative cell therapy while reducing the number of cells required. Currently, targeting can be achieved via chemical conjugation to specific antibodies, which typically results in the loss of antibody functionality and in severe cell damage. An ideal conjugation technique should ensure retention of antigen-binding activity and functionality of the targeted biological component. OBJECTIVE: To develop a biochemically robust, highly reproducible, and site-specific coupling method using the Staphylococcus aureus sortase A enzyme for the conjugation of a single-chain antibody (scFv) to nanoparticles and cells for molecular imaging and cell homing in cardiovascular diseases. This scFv specifically binds to activated platelets, which play a pivotal role in thrombosis, atherosclerosis, and inflammation. METHODS AND RESULTS: The conjugation procedure involves chemical and enzyme-mediated coupling steps. The scFv was successfully conjugated to iron oxide particles (contrast agents for magnetic resonance imaging) and to model cells. Conjugation efficiency ranged between 50% and 70%, and bioactivity of the scFv after coupling was preserved. The targeting of scFv-coupled cells and nanoparticles to activated platelets was strong and specific as demonstrated in in vitro static adhesion assays, in a flow chamber system, in mouse intravital microscopy, and in in vivo magnetic resonance imaging of mouse carotid arteries. CONCLUSIONS: This unique biotechnological approach provides a versatile and broadly applicable tool for procuring targeted regenerative cell therapy and targeted molecular imaging in cardiovascular and inflammatory diseases and beyond.


Subject(s)
Aminoacyltransferases/metabolism , Bacterial Proteins/metabolism , Cell Movement , Cell Tracking/methods , Contrast Media , Cysteine Endopeptidases/metabolism , Magnetic Resonance Imaging , Magnetite Nanoparticles , Molecular Probe Techniques , Single-Chain Antibodies/metabolism , Thrombosis/pathology , Aminoacyltransferases/biosynthesis , Aminoacyltransferases/genetics , Animals , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Blood Platelets/metabolism , CHO Cells , Chlorides , Cricetinae , Cricetulus , Cysteine Endopeptidases/biosynthesis , Cysteine Endopeptidases/genetics , Disease Models, Animal , Ferric Compounds , Flow Cytometry , Green Fluorescent Proteins/metabolism , Humans , Mice , Mice, Inbred C57BL , Microscopy, Video , Platelet Activation , Recombinant Fusion Proteins/metabolism , Single-Chain Antibodies/biosynthesis , Single-Chain Antibodies/genetics , Thrombosis/chemically induced , Thrombosis/metabolism
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