The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies.

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ultrasound pulsing schemes, or novel transducers for contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery. The reasons for this are the accessibility of the embryo and vessel network of the CAM as well as the low costs of the model. An important step to get access to the embryo and CAM vessels is to take the egg content out of the eggshell. In this protocol, three methods for taking the content out of the eggshell between day 5 and 8 of incubation are described thus allowing the embryos to develop inside the eggshell up to these days. The described methods only require simple tools and equipment and yield a higher survival success rate of 90% for 5-day, 75% for 6-day, 50% for 7-day, and 60% for 8-day old incubated eggs in comparison to ex ovo cultured embryos (~50%). The protocol also describes how to inject cavitation nuclei, such as microbubbles, into the CAM vascular system, how to separate the membrane containing the embryo and CAM from the rest of the egg content for optically transparent studies, and how to use the chicken embryo and CAM in a variety of short-term ultrasound experiments. The in vivo chicken embryo and CAM model is extremely relevant to investigate novel imaging protocols, ultrasound contrast agents, and ultrasound pulsing schemes for contrast-enhanced ultrasound imaging, and to unravel the mechanisms of ultrasound-mediated drug delivery.

Corrections to "Targeted Microbubble Mediated Sonoporation of Endothelial Cells In Vivo".

In the above article [1], the authors regret that there was a mistake in calculating the mol% of the microbubble coating composition. For all experiments, the unit in mg/mL was utilized and the conversion mistake only came when converting to mol% in order to define the ratio between the coating formulation components. The correct molecular weight of PEG-40 stearate is 2046.54 g/mol [2], [3], not 328.53 g/mol. On page 1661, paragraph II-A, it should read "The coating was composed of DSPC (84.8 mol%; P 6517; Sigma-Aldrich, Zwijndrecht, The Netherlands);PEG-40 stearate (8.2 mol%; P 3440; Sigma-Aldrich); DSPE-PEG(2000) (5.9 mol%; 880125 P; Avanti Polar Lipids, Alabaster, AL, USA); and DSPE-PEG(2000)-biotin (1.1 mol%; 880129 C; Avanti Polar Lipids)".

Corrections to "Microbubble Composition and Preparation for High-Frequency Contrast-Enhanced Ultrasound Imaging: In Vitro and In Vivo Evaluation".

In the above article [1], the authors regret that there was a mistake in calculating the mol% of the microbubble coating composition used. For all experiments, the unit in mg/mL was utilized and the conversion mistake only came when converting to mol% in order to define the ratio between the coating formulation components. The correct molecular weight of PEG-40 stearate is 2046.54 g/mol [2], [3], not 328.53 g/mol. On page 556, Table I should read as shown here.

SPIO labeling of endothelial cells using ultrasound and targeted microbubbles at diagnostic pressures.

In vivo cell tracking of therapeutic, tumor, and endothelial cells is an emerging field and a promising technique for imaging cardiovascular disease and cancer development. Site-specific labeling of endothelial cells with the MRI contrast agent superparamagnetic iron oxide (SPIO) in the absence of toxic agents is challenging. Therefore, the aim of this in vitro study was to find optimal parameters for efficient and safe SPIO-labeling of endothelial cells using ultrasound-activated CD31-targeted microbubbles for future MRI tracking. Ultrasound at a frequency of 1 MHz (10,000 cycles, repetition rate of 20 Hz) was used for varying applied peak negative pressures (10-160 kPa, i.e. low mechanical index (MI) of 0.01-0.16), treatment durations (0-30 s), time of SPIO addition (-5 min- 15 min with respect to the start of the ultrasound), and incubation time after SPIO addition (5 min- 3 h). Iron specific Prussian Blue staining in combination with calcein-AM based cell viability assays were applied to define the most efficient and safe conditions for SPIO-labeling. Optimal SPIO labeling was observed when the ultrasound parameters were 40 kPa peak negative pressure (MI 0.04), applied for 30 s just before SPIO addition (0 min). Compared to the control, this resulted in an approximate 12 times increase of SPIO uptake in endothelial cells in vitro with 85% cell viability. Therefore, ultrasound-activated targeted ultrasound contrast agents show great potential for effective and safe labeling of endothelial cells with SPIO.

Laser-Activated Polymeric Microcapsules for Ultrasound Imaging and Therapy: In Vitro Feasibility.

Polymeric microcapsules with a light-absorbing dye incorporated in their shell can generate vapor microbubbles that can be spatiotemporally controlled by pulsed laser irradiation. These contrast agents of 6-8 µm in diameter can circulate through the vasculature, offering possibilities for ultrasound (molecular) imaging and targeted therapies. Here, we study the impact of such vapor bubbles on human endothelial cells in terms of cell poration and cell viability to establish the imaging and therapeutic windows. Two capsule formulations were used: the first one consisted of a high boiling point oil (hexadecane), whereas the second was loaded with a low boiling point oil (perfluoropentane). Poration probability was already 40% for the smallest bubbles that were formed (<7.5 µm diameter), and reached 100% for the larger bubbles. The hexadecane-loaded capsules also produced bubbles while their shell remained intact. These encapsulated bubbles could therefore be used for noninvasive ultrasound imaging after laser activation without inducing any cell damage. The controlled and localized cell destruction achieved by activation of both capsule formulations may provide an innovative approach for specifically inducing cell death in vivo, e.g., for cancer therapy.

Microbubble Composition and Preparation for High-Frequency Contrast-Enhanced Ultrasound Imaging: In Vitro and In Vivo Evaluation.

Although high-frequency ultrasound imaging is gaining attention in various applications, hardly any ultrasound contrast agents (UCAs) dedicated to such frequencies (>15 MHz) are available for contrast-enhanced ultrasound (CEUS) imaging. Moreover, the composition of the limited commercially available UCAs for high-frequency CEUS (hfCEUS) is largely unknown, while shell properties have been shown to be an important factor for their performance. The aim of our study was to produce UCAs in-house for hfCEUS. Twelve different UCA formulations A-L were made by either sonication or mechanical agitation. The gas core consisted of C4F10 and the main coating lipid was either 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; A-F formulation) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC; G-L formulation). Mechanical agitation resulted in UCAs with smaller microbubbles (number weighted mean diameter ~1 [Formula: see text]) than sonication (number weighted mean diameter ~2 [Formula: see text]). UCA formulations with similar size distributions but different main lipid components showed that the DPPC-based UCA formulations had higher nonlinear responses at both the fundamental and subharmonic frequencies in vitro for hfCEUS using the Vevo2100 high-frequency preclinical scanner (FUJIFILM VisualSonics, Inc.). In addition, UCA formulations F (DSPC-based) and L (DPPC-based) that were made by mechanical agitation performed similar in vitro to the commercially available Target-Ready MicroMarker (FUJIFILM VisualSonics, Inc.). UCA formulation F also performed similar to Target-Ready MicroMarker in vivo in pigs with similar mean contrast intensity within the kidney ( n = 7 ), but formulation L did not. This is likely due to the lower stability of formulation L in vivo. Our study shows that DSPC-based microbubbles produced by mechanical agitation resulted in small microbubbles with high nonlinear responses suitable for hfCEUS imaging.

Viability of endothelial cells after ultrasound-mediated sonoporation: Influence of targeting, oscillation, and displacement of microbubbles.

Microbubbles (MBs) have been shown to create transient or lethal pores in cell membranes under the influence of ultrasound, known as ultrasound-mediated sonoporation. Several studies have reported enhanced drug delivery or local cell death induced by MBs that are either targeted to a specific biomarker (targeted microbubbles, tMBs) or that are not targeted (non-targeted microbubbles, ntMBs). However, both the exact mechanism and the optimal acoustic settings for sonoporation are still unknown. In this study we used real-time uptake patterns of propidium iodide, a fluorescent cell impermeable model drug, as a measure for sonoporation. Combined with high-speed optical recordings of MB displacement and ultra-high-speed recordings of MB oscillation, we aimed to identify differences in MB behavior responsible for either viable sonoporation or cell death. We compared ntMBs and tMBs with identical shell compositions exposed to long acoustic pulses (500-50,000cycles) at various pressures (150-500kPa). Propidium iodide uptake highly correlated with cell viability; when the fluorescence intensity still increased 120s after opening of the pore, this resulted in cell death. Higher acoustic pressures and longer cycles resulted in more displacing MBs and enhanced sonoporation. Non-displacing MBs were found to be the main contributor to cell death, while displacement of tMBs enhanced reversible sonoporation and preserved cell viability. Consequently, each therapeutic application requires different settings: non-displacing ntMBs or tMBs are advantageous for therapies requiring cell death, especially at 500kPa and 50,000cycles, whereas short acoustic pulses causing limited displacement should be used for drug delivery.

Quantification of Endothelial αvß3 Expression with High-Frequency Ultrasound and Targeted Microbubbles: In Vitro and In Vivo Studies.

Angiogenesis is a critical feature of plaque development in atherosclerosis and might play a key role in both the initiation and later rupture of plaques. The precursory molecular or cellular pro-angiogenic events that initiate plaque growth and that ultimately contribute to plaque instability, however, cannot be detected directly with any current diagnostic modality. This study was designed to investigate the feasibility of ultrasound molecular imaging of endothelial αvß3 expression in vitro and in vivo using αvß3-targeted ultrasound contrast agents (UCAs). In the in vitro study, αvß3 expression was confirmed by immunofluorescence in a murine endothelial cell line and detected using the targeted UCA and ultrasound imaging at 18-MHz transmit frequency. In the in vivo study, expression of endothelial αvß3 integrin in murine carotid artery vessels and microvessels of the salivary gland was quantified using targeted UCA and high-frequency ultrasound in seven animals. Our results indicated that endothelial αvß3 expression was significantly higher in the carotid arterial wall containing atherosclerotic lesions than in arterial segments without any lesions. We also found that the salivary gland can be used as an internal positive control for successful binding of targeted UCA to αvß3 integrin. In conclusion, αvß3-targeted UCA allows non-invasive assessment of the expression levels of αvß3 on the vascular endothelium and may provide potential insights into early atherosclerotic plaque detection and treatment monitoring.

Quantification of bound microbubbles in ultrasound molecular imaging.

Molecular markers associated with diseases can be visualized and quantified noninvasively with targeted ultrasound contrast agent (t-UCA) consisting of microbubbles (MBs) that can bind to specific molecular targets. Techniques used for quantifying t-UCA assume that all unbound MBs are taken out of the blood pool few minutes after injection and only MBs bound to the molecular markers remain. However, differences in physiology, diseases, and experimental conditions can increase the longevity of unbound MBs. In such conditions, unbound MBs will falsely be quantified as bound MBs. We have developed a novel technique to distinguish and classify bound from unbound MBs. In the post-processing steps, first, tissue motion was compensated using block-matching (BM) techniques. To preserve only stationary contrast signals, a minimum intensity projection (MinIP) or 20th-percentile intensity projection (PerIP) was applied. The after-flash MinIP or PerIP was subtracted from the before-flash MinIP or PerIP. In this way, tissue artifacts in contrast images were suppressed. In the next step, bound MB candidates were detected. Finally, detected objects were tracked to classify the candidates as unbound or bound MBs based on their displacement. This technique was validated in vitro, followed by two in vivo experiments in mice. Tumors (n = 2) and salivary glands of hypercholesterolemic mice (n = 8) were imaged using a commercially available scanner. Boluses of 100 µL of a commercially available t-UCA targeted to angiogenesis markers and untargeted control UCA were injected separately. Our results show considerable reduction in misclassification of unbound MBs as bound ones. Using our method, the ratio of bound MBs in salivary gland for images with targeted UCA versus control UCA was improved by up to two times compared with unprocessed images.

Targeted ultrasound contrast agents for ultrasound molecular imaging and therapy.

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.

Targeted microbubble mediated sonoporation of endothelial cells in vivo.

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.

Lipid shedding from single oscillating microbubbles.

Lipid-coated microbubbles are used clinically as contrast agents for ultrasound imaging and are being developed for a variety of therapeutic applications. The lipid encapsulation and shedding of the lipids by acoustic driving of the microbubble has a crucial role in microbubble stability and in ultrasound-triggered drug delivery; however, little is known about the dynamics of lipid shedding under ultrasound excitation. Here we describe a study that optically characterized the lipid shedding behavior of individual microbubbles on a time scale of nanoseconds to microseconds. A single ultrasound burst of 20 to 1000 cycles, with a frequency of 1 MHz and an acoustic pressure varying from 50 to 425 kPa, was applied. In the first step, high-speed fluorescence imaging was performed at 150,000 frames per second to capture the instantaneous dynamics of lipid shedding. Lipid detachment was observed within the first few cycles of ultrasound. Subsequently, the detached lipids were transported by the surrounding flow field, either parallel to the focal plane (in-plane shedding) or in a trajectory perpendicular to the focal plane (out-of-plane shedding). In the second step, the onset of lipid shedding was studied as a function of the acoustic driving parameters, for example, pressure, number of cycles, bubble size and oscillation amplitude. The latter was recorded with an ultrafast framing camera running at 10 million frames per second. A threshold for lipid shedding under ultrasound excitation was found for a relative bubble oscillation amplitude >30%. Lipid shedding was found to be reproducible, indicating that the shedding event can be controlled.

Imaging microvasculature with contrast-enhanced ultraharmonic ultrasound.

Atherosclerotic plaque neovascularization was shown to be one of the strongest predictors of future cardiovascular events. Yet, the clinical tools for coronary wall microvasculature detection in vivo are lacking. Here we report an ultrasound pulse sequence capable of detecting microvasculature invisible in conventional intracoronary imaging. The method combines intravascular ultrasound with an ultrasound contrast agent, i.e., a suspension of microscopic vascular acoustic resonators that are small enough to penetrate the capillary bed after intravenous administration. The pulse sequence relies on brief chirp excitations to extract ultraharmonic echoes specific to the ultrasound contrast agent. We implemented the pulse sequence on an intravascular ultrasound probe and successfully imaged the microvasculature of a 6 days old chicken embryo respiratory organ. The feasibility of microvasculature imaging with intravascular ultrasound sets the stage for a translation of the method to studies of intra-plaque neovascularization detection in humans.

Acoustical properties of individual liposome-loaded microbubbles.

A comparison between phospholipid-coated microbubbles with and without liposomes attached to the microbubble surface was performed using the ultra-high-speed imaging camera (Brandaris 128). We investigated 73 liposome-loaded microbubbles (loaded microbubbles) and 41 microbubbles without liposome loading (unloaded microbubbles) with a diameter ranging from 3-10 µm at frequencies ranging from 0.6-3.8 MHz and acoustic pressures ranging from 5-100 kPa. The experimental data showed nearly the same shell elasticity for the loaded and unloaded bubbles, but the shell viscosity was higher for loaded bubbles compared with unloaded bubbles. For loaded bubbles, a higher pressure threshold for the bubble vibrations was noticed. In addition, an "expansion-only" behavior was observed for up to 69% of the investigated loaded bubbles, which mostly occurred at low acoustic pressures (≤30 kPa). Finally, fluorescence imaging showed heterogeneity of liposome distributions of the loaded bubbles.

In vivo characterization of ultrasound contrast agents: microbubble spectroscopy in a chicken embryo.

The dynamics of coated microbubbles was studied in an in vivo model. Biotinylated lipid-coated microbubbles were prepared in-house and were injected into a chick embryo chorioallantoic membrane (CAM) model on the fifth day of incubation. The microbubbles, ranging between 1.0 and 3.5 µm in diameter, were insonified in the frequency range of 4-7 MHz. Two amplitudes of acoustic pressure were applied: 300 kPa and 400 kPa. The fundamental and subharmonic responses were recorded optically with an ultra-fast camera (Brandaris 128) at 20 million frames per second. A subharmonic response was observed for 44% of the studied bubbles. From the data the frequency of the maximum fundamental and subharmonic response was derived for each individual bubble and resulted in the resonance curves of the microbubbles. All the bubbles showed shell (strain) hardening behavior for a higher acoustic pressure. We conclude that the subharmonic oscillations observed in this study belonged to the transmit at resonance (TR) regime.