title: Nanobody-coupled microbubbles as novel molecular tracer.
abstract: Camelid-derived single-domain antibody-fragments (~15kDa), called nanobodies, are a new class of molecular tracers that are routinely identified with nanomolar affinity for their target and that are easily tailored for molecular imaging and drug delivery applications. We hypothesized that they are well-suited for the design of targeted microbubbles (μBs) and aimed to develop and characterize eGFP- and VCAM-1-targeted μBs. Anti-eGFP (cAbGFP4) and anti-VCAM-1 (cAbVCAM1-5) nanobodies were site-specifically biotinylated in bacteria. This metabolic biotinylation method yielded functional nanobodies with one biotin located at a distant site of the antigen-binding region of the molecule. The biotinylated nanobodies were coupled to biotinylated lipid μBs via streptavidin-biotin bridging. The ability of μB-cAbGFP4 to recognize eGFP was tested as proof-of-principle by fluorescent microscopy and confirmed the specific binding of eGFP to μB-cAbGFP4. Dynamic flow chamber studies demonstrated the ability of μB-cAbVCAM1-5 to bind VCAM-1 in fast flow (up to 5 dynes/cm(2)). In vivo targeting studies were performed in MC38 tumor-bearing mice (n=4). μB-cAbVCAM1-5 or control μB-cAbGFP4 were injected intravenously and imaged using a contrast-specific ultrasound imaging mode. The echo intensity in the tumor was measured 10min post-injection. μB-cAbVCAM1-5 showed an enhanced signal compared to control μBs (p<0.05). Using metabolic and site-specific biotinylation of nanobodies, a method to develop nanobody-coupled μBs was described. The application of VCAM-1-targeted μBs as novel molecular ultrasound contrast agent was demonstrated both in vitro and in vivo.
authors.affiliation: In vivo Cellular and Molecular Imaging, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium. Sophie.Hernot@gmail.com
authors.forename: Sophie
authors.initials: S
authors.lastname: Hernot
authors.forename: Sunil
authors.initials: S
authors.lastname: Unnikrishnan
authors.forename: Zhongmin
authors.initials: Z
authors.lastname: Du
authors.forename: Talent
authors.initials: T
authors.lastname: Shevchenko
authors.forename: Bernard
authors.initials: B
authors.lastname: Cosyns
authors.forename: Alexis
authors.initials: A
authors.lastname: Broisat
authors.forename: Jakub
authors.initials: J
authors.lastname: Toczek
authors.forename: Vicky
authors.initials: V
authors.lastname: Caveliers
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.forename: Tony
authors.initials: T
authors.lastname: Lahoutte
authors.forename: Alexander L
authors.initials: AL
authors.lastname: Klibanov
authors.forename: Nick
authors.initials: N
authors.lastname: Devoogdt
mesh_terms: D000818:Animals; D000906:Antibodies / Q000008:administration & dosage* / Q000276:immunology / Q000378:metabolism; D019921:Biotinylation; D002460:Cell Line; D045744:Cell Line, Tumor; D003287:Contrast Media / Q000008:administration & dosage*; D005260:Female; D049452:Green Fluorescent Proteins / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D045423:Microbubbles; D009369:Neoplasms / Q000000981:diagnostic imaging; D014465:Ultrasonics; D014463:Ultrasonography; D019010:Vascular Cell Adhesion Molecule-1 / Q000276:immunology*
chemical_list: D000906:Antibodies; D003287:Contrast Media; D019010:Vascular Cell Adhesion Molecule-1; D049452:Green Fluorescent Proteins