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Ultrasound contrast agents (UCA) consist of gas‐filled coated microbubbles with diameters of 1–10 µm. Targeted UCA can bind to biomarkers associated with disease through coating‐incorporated ligands, making ultrasound molecular imaging possible. The aim of our research was to compare the ligand distribution, binding area, and bound microbubble shape of 1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine (DSPC) based and 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine (DPPC) based lipid‐coated microbubbles using super‐resolution microscopy. Ligand distribution was studied by conjugating the fluorescent streptavidin Oregon Green 488 to the biotinylated microbubbles. An inhomogeneous streptavidin distribution was found when DSPC was the main coating lipid. When DSPC was replaced by DPPC, a more homogeneous streptavidin distribution was observed. Binding area of targeted microbubbles was studied using biotinylated microbubbles bound to a streptavidin‐coated surface. DSPC microbubbles had a significantly smaller binding area than DPPC microbubbles. Whereas the bound DSPC microbubbles remained spherical, the DPPC microbubbles were dome‐shaped. This study reveals that lipid‐coated microbubbles differ in ligand distribution, binding area, and bound microbubble shape solely on the basis of their main lipid component. Practical applications: A homogeneous ligand distribution, larger binding area and domed shape upon binding could be advantageous for binding of targeted microbubbles, thereby favoring DPPC over DSPC as main lipid for UCA for ultrasound molecular imaging. The findings of the present study can be used for the design of targeted microbubbles with improved binding capabilities and for the ongoing research to acoustically distinguish bound from unbound microbubbles. Targeted biotinylated DSPC and DPPC‐based microbubbles bound to streptavidin‐coated surface. Left graph: binding area; right panels: microbubbles (red fluorescent) bound to streptavidin‐coated surface (green fluorescent).

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Ultrasound contrast agents (UCAs) are used routinely in the clinic to enhance contrast in ultrasonography. More recently, UCAs have been functionalised by conjugating ligands to their surface to target specific biomarkers of a disease or a disease process. These targeted UCAs (tUCAs) are used for a wide range of pre-clinical applications including diagnosis, monitoring of drug treatment, and therapy. In this review, recent achievements with tUCAs in the field of molecular imaging, evaluation of therapy, drug delivery, and therapeutic applications are discussed. We present the different coating materials and aspects that have to be considered when manufacturing tUCAs. Next to tUCA design and the choice of ligands for specific biomarkers, additional techniques are discussed that are applied to improve binding of the tUCAs to their target and to quantify the strength of this bond. As imaging techniques rely on the specific behaviour of tUCAs in an ultrasound field, it is crucial to understand the characteristics of both free and adhered tUCAs. To image and quantify the adhered tUCAs, the state-of-the-art techniques used for ultrasound molecular imaging and quantification are presented. This review concludes with the potential of tUCAs for drug delivery and therapeutic applications.

Ultrasound contrast agents as drug-delivery systems are an emerging field. Recently, we reported that targeted microbubbles are able to sonoporate endothelial cells in vitro. In this study, we investigated whether targeted microbubbles can also induce sonoporation of endothelial cells in vivo, thereby making it possible to combine molecular imaging and drug delivery. Live chicken embryos were chosen as the in vivo model. αvß3-targeted microbubbles attached to the vessel wall of the chicken embryo were insonified at 1 MHz at 150 kPa (1 × 10,000 cycles) and at 200 kPa (1 × 1000 cycles) peak negative acoustic pressure. Sonoporation was studied by intravital microscopy using the model drug propidium iodide (PI). Endothelial cell PI uptake was observed in 48% of microbubble-vessel-wall complexes at 150 kPa (n = 140) and in 33% at 200 kPa (n = 140). Efficiency of PI uptake depended on the local targeted microbubble concentration and increased up to 80% for clusters of 10 to 16 targeted microbubbles. Ultrasound or targeted microbubbles alone did not induce PI uptake. This intravital microscopy study reveals that sonoporation can be visualized and induced in vivo using targeted microbubbles.

The use of stem cells for the repair of damaged cardiac tissue after a myocardial infarction holds great promise. However, a common finding in experimental studies is the low number of cells delivered at the area at risk. To improve the delivery, we are currently investigating a novel delivery platform in which stem cells are conjugated with targeted microbubbles, creating echogenic complexes dubbed StemBells. These StemBells vibrate in response to incoming ultrasound waves making them susceptible to acoustic radiation force. The acoustic force can then be employed to propel circulating StemBells from the centerline of the vessel to the wall, facilitating localized stem cell delivery. In this study, we investigate the feasibility of manipulating StemBells acoustically in vivo after injection using a chicken embryo model. Bare stem cells or unsaturated stem cells (<5 bubbles/cell) do not respond to ultrasound application (1 MHz, peak negative acoustical pressure P_ = 200 kPa, 10% duty cycle). However, stem cells which are fully saturated with targeted microbubbles (>30 bubbles/cell) can be propelled toward and arrested at the vessel wall. The mean translational velocities measured are 61 and 177 μm/s for P- = 200 and 450 kPa, respectively. This technique therefore offers potential for enhanced and well-controlled stem cell delivery for improved cardiac repair after a myocardial infarction.

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