Nanobody-coupled microbubbles as novel molecular tracer

Camelid-derived single-domain antibody-fragments (similar to 15 kDa), 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 (mu Bs) and aimed to develop and characterize eGFP- and VCAM-1-targeted mu 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 mu Bs via streptavidin-biotin bridging. The ability of mu B-cAbGFP4 to recognize eGFP was tested as proof-of-principle by fluorescent microscopy and confirmed the specific binding of eGFP to mu B-cAbGFP4. Dynamic flow chamber studies demonstrated the ability of mu 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). mu B-cAbVCAM1-5 or control mu B-cAbGFP4 were injected intravenously and imaged using a contrast-specific ultrasound imaging mode. The echo intensity in the tumor was measured 10 min post-injection. mu B-cAbVCAM1-5 showed an enhanced signal compared to control mu Bs (p < 0.05). Using metabolic and site-specific biotinylation of nanobodies, a method to develop nanobody-coupled mu Bs was described. The application of VCAM-1-targeted mu Bs as novel molecular ultrasound contrast agent was demonstrated both in vitro and in vivo. (C) 2011 Elsevier B.V. All rights reserved.