scVEGF microbubble ultrasound contrast agents: a novel probe for ultrasound molecular imaging of tumor angiogenesis.
OBJECTIVE
To develop a novel microbubble (MB) ultrasound contrast agent covalently coupled to a recombinant single-chain vascular endothelial growth factor construct (scVEGF) through uniform site-specific conjugation for ultrasound imaging of tumor angiogenesis.


METHODS
Ligand conjugation to maleimide-bearing MB by thioether bonding was first validated with a fluorophore (BODIPY-cystine), and covalently bound dye was detected by fluorometry and flow cytometry. MBs were subsequently site-specifically conjugated to cysteine-containing Cys-tag in scVEGF, and bound scVEGF was quantified by enzyme-linked immunosorbent assay. Targeted adhesion of scVEGF-MB was investigated with in vitro parallel plate flow chamber assays with recombinant murine VEGFR-2 substrates and human VEGFR-2-expressing porcine endothelial cells (PAE/KDR). A wall-less ultrasound flow phantom, with flow channels coated with immobilized VEGFR-2, was used to detect adhesion of scVEGF-MB with contrast ultrasound imaging. A murine model of colon adenocarcinoma was used to assess retention of scVEGF-MB with contrast ultrasound imaging during tumor angiogenesis in vivo.


RESULTS
Proof-of-principle of ligand conjugation to maleimide-bearing MB was demonstrated with a BODIPY-cysteine fluorophore. Conjugation of BODIPY to MB saturated at 10-fold molar excess BODIPY relative to maleimide groups on MB surfaces. MB reacted with scVEGF and led to the conjugation of 1.2 × 10(5) molecules scVEGF per MB. Functional adhesion of sc-VEGF-MB was shown in parallel plate flow chamber assays. At a shear stress of 1.0 dynes/cm2, scVEGF-MB exhibited 5-fold higher adhesion to both recombinant VEGFR-2 substrates and VEGFR-2-expressing endothelial cells compared with nontargeted control MB. Additionally, scVEGF-MB targeted to immobilized VEGFR-2 in an ultrasound flow phantom showed an 8-fold increase in mean acoustic signal relative to casein-coated control channels. In an in vivo model of tumor angiogenesis, scVEGF MB showed significantly higher ultrasound contrast signal enhancement in tumors (8.46 ± 1.61 dB) compared with nontargeted control MB (1.58 ± 0.83 dB).


CONCLUSIONS
These results demonstrate the functionality of a novel scVEGF-bearing MB contrast agent, which could be useful for molecular imaging of VEGFR-2 in basic science and drug discovery research.
Anderson
Christopher R
CR
Product Development, Targeson Inc, San Diego, CA 92121, USA. crandrsn@gmail.com
Rychak
Joshua J
JJ
Backer
Marina
M
Backer
Joseph
J
Ley
Klaus
K
Klibanov
Alexander L
AL
D000818:Animals; D002448:Cell Adhesion; D003287:Contrast Media / Q000378:metabolism; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000000981:diagnostic imaging / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005240:Feasibility Studies; D005434:Flow Cytometry; D066298:In Vitro Techniques; D051379:Mice; D045423:Microbubbles; D057054:Molecular Imaging / Q000379:methods; D009389:Neovascularization, Pathologic; D013552:Swine; D014463:Ultrasonography; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
D003287:Contrast Media; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2