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/cm2). 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 10 minutes 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.

Synthesis, Functionalization, and Design of Magnetic Nanoparticles for Theranostic Applications

“…[517] Specifically, nanbobodies have been used as antagonistic drugs [518] and targeting agents of effector domains [519] in cancer immunotherapy. On the other hand, several molecular imaging modalities of SPECT [520] and PET, [521] optical imaging, [522] and ultrasound imaging [523] have been actively directed toward the target by means of nanobodies conjugated to them. Additionally, grafting nanobodies on nanoparticles have shown a considerable potential for increasing their affinity toward the target.…”

Crucial factors and emerging concepts in ultrasound-triggered drug delivery

“…Such microbubbles should be able to interact with molecules that become expressed in specific pathologies. In this case antibodies [41] or even nanobodies [42] are coupled to the surface of the bubbles, typically through avidin-biotin coupling [43].…”

Novel lactoferrin-conjugated amphiphilic poly(aminoethyl ethylene phosphate)/poly(L-lactide) copolymer nanobubbles for tumor-targeting ultrasonic imaging

“…11,15,16 NBs usually contain a gaseous perfluorocarbon or sulfur hexafluoride as cores, and their shells are composed of albumin, lipids, nonionic surfactants, and polymers. [16][17][18][19] Polymer-based hard-shell NBs are superior to phospholipid/surfactant-based soft-shell NBs because the hard shell makes those NBs more stable and thus have longer circulation life. 20,21 However, the mechanical properties, degradation time, and hydrophilic/lipophilic feature of the reported copolymers, such as polylactic acid, poly(lacticco-glycolic acid), and polycaprolactone, are difficult to adjust to meet the requirements of clinical ultrasound imaging.…”