preparation-and-characterization-of-targeted-microbubbles

Preparation and Characterization of Targeted Microbubbles

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of ...

University of Virginia School of Medicine

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.

Nanobody-coupled microbubbles as novel molecular tracer.

For preparation of ligand-decorated microbubbles for targeted ultrasound contrast imaging, it is important to maximize the amount of ligand associated with the bubble shell. We describe optimization of the use of a biocompatible cosurfactant in the microbubble formulation media to maximize the incorporation of targeting ligand-lipid conjugate into the microbubble shell, and thus reduce the fraction of ligand not associated with microbubbles, following amalgamation preparation. The influence of the concentration of a helper cosurfactant propylene glycol (PG) on the efficacy of microbubble preparation by amalgamation and on the degree of association of fluorescent PEG-lipid with the microbubble shell was tested. Three sets of targeted bubbles were then prepared: with VCAM-1-targeting peptide VHPKQHRGGSK(FITC)GC-PEG-DSPE, cyclic RGDfK-PEG-DSPE, selective for αβ, and control cRADfK-PEG-DSPE, without such affinity. Microbubbles were prepared by 45 s amalgamation, with DSPC and PEG stearate as the main components of the shell, with 15% PG in aqueous saline. In vitro microbubble targeting was assessed with a parallel plate flow chamber with a recombinant receptor coated surface. In vivo targeting was assessed in MC-38 tumor-bearing mice (subcutaneous tumor in hind leg), 10 min after intravenous bolus of microbubble contrast agent (20 million particles per injection). Ultrasound imaging of the tumor and control nontarget muscle tissue in a contralateral leg was performed with a clinical scanner. Amalgamation technique with PG cosurfactant produced microbubbles at concentrations exceeding 2 × 10 particles/mL, and ∼50-60% or more of the added fluorescein-PEG-DSPE or VCAM-1-targeted fluorescent peptide was associated with microbubbles, about 2 times higher than that in the absence of PG. After intravenous injection, peptide-targeted bubbles selectively accumulated in the tumor vasculature, with negligible accumulation in nontumor contralateral leg muscle, or with control nontargeted microbubbles (assessed by contrast ultrasound imaging). For comparison, administration of RGD-decorated microbubbles prepared by traditional sonication, and purified from free peptide-PEG-lipid by repeated centrifugation, resulted in the same accumulation pattern as for translatable amalgamated microbubbles. Following amalgamation in the presence of PG, efficient transfer of ligand-PEG-lipid to microbubble shell was achieved and quantified. Purification of microbubbles from free peptide-PEG-lipid was not necessary, as proven by in vitro and in vivo targeting studies, so PG cosurfactant amalgamation technique generated peptide-targeted microbubbles are amenable for bedside preparation and clinical translation. The pathway to clinical translation is simplified by the fact that most of the materials used in this study either are on the United States Food and Drug Administration GRAS list or can be procured as pharmaceutical grade substances.

Sunil Unnikrishnan

University of Virginia School of Medicine

Preparation and Characterization of Targeted Microbubbles