Nanobody-coupled microbubbles as novel molecular tracer.

TitleNanobody-coupled microbubbles as novel molecular tracer.
Publication TypeJournal Article
Year of Publication2012
AuthorsHernot, S., S. Unnikrishnan, Z. Du, T. Shevchenko, B. Cosyns, A. Broisat, J. Toczek, V. Caveliers, S. Muyldermans, T. Lahoutte, A. L. Klibanov, and N. Devoogdt
JournalJ Control Release
Volume158
Issue2
Pagination346-53
Date Published2012 Mar 10
ISSN1873-4995
KeywordsAnimals, Antibodies, Biotinylation, Cell Line, Cell Line, Tumor, Contrast Media, Female, Green Fluorescent Proteins, Mice, Mice, Inbred C57BL, Microbubbles, Neoplasms, Ultrasonics, Vascular Cell Adhesion Molecule-1
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.

DOI10.1016/j.jconrel.2011.12.007
Alternate JournalJ Control Release
PubMed ID22197777
PubMed Central IDPMC3294017
Grant ListR21 CA102880-01A2 / CA / NCI NIH HHS / United States
R21/33 CA102880 / CA / NCI NIH HHS / United States
R33 CA102880-02 / CA / NCI NIH HHS / United States