Nanobody-coupled microbubbles as novel molecular tracer.

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 (?Bs) and aimed to develop and characterize eGFP- and VCAM-1-targeted ?Bs. 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 ?Bs via streptavidin-biotin bridging. The ability of ?B-cAbGFP4 to recognize eGFP was tested as proof-of-principle by fluorescent microscopy and confirmed the specific binding of eGFP to ?B-cAbGFP4. Dynamic flow chamber studies demonstrated the ability of ?B-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). ?B-cAbVCAM1-5 or control ?B-cAbGFP4 were injected intravenously and imaged using a contrast-specific ultrasound imaging mode. The echo intensity in the tumor was measured 10min post-injection. ?B-cAbVCAM1-5 showed an enhanced signal compared to control ?Bs (p