Formation of Microbubbles for Targeted Ultrasound Contrast Imaging: Practical Translation Considerations.

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 co-surfactant 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 co-surfactant propyleneglycol (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 αVβ3, and control cRADfK-PEG-DSPE, without such affinity. Microbubbles were prepared by 45s 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 non-target muscle tissue in a contralateral leg was performed with a clinical scanner. Amalgamation technique with PG co-surfactant produced microbubbles at concentrations exceeding 2.10^9 particles per 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 in the absence of PG. After intravenous injection, peptide-targeted bubbles selectively accumulated in the tumor vasculature, with negligible accumulation in non-tumor contralateral leg muscle, or with control non-targeted 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 co-surfactant amalgamation technique generated peptide-targeted microbubbles amenable for bedside preparation and clinical translation. Pathway to clinical translation is simplified by the fact that most of the materials used in this study are either on FDA GRAS list, or can be procured as pharmaceutical grade substances.