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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 (?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<0.05). Using metabolic and site-specific biotinylation of nanobodies, a method to develop nanobody-coupled ?Bs was described. The application of VCAM-1-targeted ?Bs as novel molecular ultrasound contrast agent was demonstrated both in vitro and in vivo.

SUBMITTER: Hernot S

PROVIDER: S-EPMC3294017 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Nanobody-coupled microbubbles as novel molecular tracer.

Hernot Sophie S Unnikrishnan Sunil S Du Zhongmin Z Shevchenko Talent T Cosyns Bernard B Broisat Alexis A Toczek Jakub J Caveliers Vicky V Muyldermans Serge S Lahoutte Tony T Klibanov Alexander L AL Devoogdt Nick N

Journal of controlled release : official journal of the Controlled Release Society 20111216 2

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-spec ...[more]

PMID: 22197777

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Project description:BackgroundThere is growing interest in limb contrast-enhanced ultrasound (CEU) perfusion imaging for the evaluation of peripheral artery disease. Because of low resting microvascular blood flow in skeletal muscle, signal enhancement during limb CEU is prohibitively low for real-time imaging. The aim of this study was to test the hypothesis that this obstacle can be overcome by intermediate- rather than low-power CEU when performed with an acoustically resilient microbubble agent.MethodsViscoelastic properties of Definity and Sonazoid were assessed by measuring bulk modulus during incremental increases in ambient pressure to 200 mm Hg. Comparison of in vivo microbubble destruction and signal enhancement at a mechanical index (MI) of 0.1 to 0.4 was performed by sequential reduction in pulsing interval from 10 to 0.05 sec during limb CEU at 7 MHz in mice and 1.8 MHz in dogs. Destruction was also assessed by broadband signal generation during passive cavitation detection. Real-time CEU perfusion imaging with destruction-replenishment was then performed at 1.8 MHz in dogs using an MI of 0.1, 0.2, or 0.3.ResultsSonazoid had a higher bulk modulus than Definity (66 ± 12 vs 29 ± 2 kPa, P = .02) and exhibited less inertial cavitation (destruction) at MIs ≥ 0.2. On in vivo CEU, maximal signal intensity increased incrementally with MI for both agents and was equivalent between agents except at an MI of 0.1 (60% and 85% lower for Sonazoid at 7 and 1.8 MHz, respectively, P < .05). However, on progressive shortening of the pulsing interval, Definity was nearly completely destroyed at MIs ≥ 0.2 at 1.8 and 7 MHz, whereas Sonazoid was destroyed only at 1.8 MHz at MIs ≥ 0.3. As a result, real-time CEU perfusion imaging demonstrated approximately fourfold greater enhancement for Sonazoid at an MI of 0.3 to 0.4.ConclusionsRobust signal enhancement during real-time CEU perfusion imaging of the limb is possible when using intermediate-power imaging coupled with a durable microbubble contrast agent.

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Nanobody-coupled microbubbles as novel molecular tracer.

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Nanobody-coupled microbubbles as novel molecular tracer.

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