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 μm) than sonication (number weighted mean diameter ∼2 μm} ). 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.@en

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.@en

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)'.@en

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)'.@en

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)'.@en

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 (500–50,000 cycles) at various pressures (150–500 kPa). Propidium iodide uptake highly correlated with cell viability; when the fluorescence intensity still increased 120 s 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 500 kPa and 50,000 cycles, whereas short acoustic pulses causing limited displacement should be used for drug delivery.@en

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 (500–50,000 cycles) at various pressures (150–500 kPa). Propidium iodide uptake highly correlated with cell viability; when the fluorescence intensity still increased 120 s 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 500 kPa and 50,000 cycles, whereas short acoustic pulses causing limited displacement should be used for drug delivery.@en

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 (500–50,000 cycles) at various pressures (150–500 kPa). Propidium iodide uptake highly correlated with cell viability; when the fluorescence intensity still increased 120 s 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 500 kPa and 50,000 cycles, whereas short acoustic pulses causing limited displacement should be used for drug delivery.@en

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 (500–50,000 cycles) at various pressures (150–500 kPa). Propidium iodide uptake highly correlated with cell viability; when the fluorescence intensity still increased 120 s 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 500 kPa and 50,000 cycles, whereas short acoustic pulses causing limited displacement should be used for drug delivery.@en

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 786, paragraph II-A, it should read 'The coating was composed of 84.8 mol% DSPC (P6517, Sigma-Aldrich, Zwijndrecht, The Netherlands) or DPPC (850355, Avanti Polar Lipids, Alabaster, AL, USA); 8.2 mol% polyoxyethylene-40-stearate (PEG-40 stearate, P3440, Sigma-Aldrich); 5.9 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG(2000), 880125, Avanti Polar Lipids); and 1.1 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG(2000)-biotin, 880129, Avanti Polar Lipids).@en

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 786, paragraph II-A, it should read 'The coating was composed of 84.8 mol% DSPC (P6517, Sigma-Aldrich, Zwijndrecht, The Netherlands) or DPPC (850355, Avanti Polar Lipids, Alabaster, AL, USA); 8.2 mol% polyoxyethylene-40-stearate (PEG-40 stearate, P3440, Sigma-Aldrich); 5.9 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG(2000), 880125, Avanti Polar Lipids); and 1.1 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG(2000)-biotin, 880129, Avanti Polar Lipids).@en

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 αvβ3 expression in vitro and in vivo using αvβ3-targeted ultrasound contrast agents (UCAs). In the in vitro study, αvβ3 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 αvβ3 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 αvβ3 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 αvβ3 integrin. In conclusion, αvβ3-targeted UCA allows non-invasive assessment of the expression levels of αvβ3 on the vascular endothelium and may provide potential insights into early atherosclerotic plaque detection and treatment monitoring.@en

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 αvβ3 expression in vitro and in vivo using αvβ3-targeted ultrasound contrast agents (UCAs). In the in vitro study, αvβ3 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 αvβ3 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 αvβ3 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 αvβ3 integrin. In conclusion, αvβ3-targeted UCA allows non-invasive assessment of the expression levels of αvβ3 on the vascular endothelium and may provide potential insights into early atherosclerotic plaque detection and treatment monitoring.@en

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 αvβ3 expression in vitro and in vivo using αvβ3-targeted ultrasound contrast agents (UCAs). In the in vitro study, αvβ3 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 αvβ3 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 αvβ3 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 αvβ3 integrin. In conclusion, αvβ3-targeted UCA allows non-invasive assessment of the expression levels of αvβ3 on the vascular endothelium and may provide potential insights into early atherosclerotic plaque detection and treatment monitoring.@en

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 αvβ3 expression in vitro and in vivo using αvβ3-targeted ultrasound contrast agents (UCAs). In the in vitro study, αvβ3 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 αvβ3 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 αvβ3 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 αvβ3 integrin. In conclusion, αvβ3-targeted UCA allows non-invasive assessment of the expression levels of αvβ3 on the vascular endothelium and may provide potential insights into early atherosclerotic plaque detection and treatment monitoring.@en

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.@en

The authors regret that there was a mistake in reporting the mol% of the microbubble coating composition used. For all experiments, the unit in mg/mL was used and the conversion mistake occurred only when converting to mol% to define the ratio between the coating formulation components. The correct molecular weight of PEG-40 stearate is 2046.54 g/mol (Shen et al. 2008; Kilic and Bolukcu 2018), not 328.53 g/mol. On page 1433, the sentence should read “The lipid coating was composed of 84.8 mol% DSPC (P6517, Sigma-Aldrich, Zwijndrecht, Netherlands) or DPPC (850355, Avanti Polar Lipids, Alabaster, AL, USA); 8.2 mol% polyoxyethylene-40-stearate (PEG40 stearate, P3440, Sigma-Aldrich); 5.9 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)2000 (DSPE-PEG2000, 880125, Avanti Polar Lipids); and 1.1 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG2000-biotin) (880129, Avanti Polar Lipids).” This correction does not change the conclusions published in this work. The authors apologize for any inconvenience caused.@en

Photoacoustic (PA) imaging can be used to monitor flowing blood inside the microvascular and capillary bed. Ultrasound speckle decorrelation based velocimetry imaging was previously shown to accurately estimate blood flow velocity in mouse brain (micro-)vasculature. Translating this method to photoacoustic imaging will allow simultaneous imaging of flow velocity and extracting functional parameters like blood oxygenation. In this study, we use a pulsed laser diode and a quantitative method based on normalized first order field autocorrelation function of PA field fluctuations to estimate flow velocities in an ink tube phantom and in the microvasculature of the chorioallantoic membrane of a chicken embryo. We demonstrate how the decorrelation time of signals acquired over frames are related to the flow speed and show that the PA flow analysis based on this approach is an angle independent flow velocity imaging method.@en

Ultrasound insonification of microbubbles can locally enhance drug delivery by increasing the cell membrane permeability. To aid development of a safe and effective therapeutic microbubble, more insight into the microbubble-cell interaction is needed. In this in vitro study we aimed to investigate the initial 3D morphology of the endothelial cell membrane adjacent to individual microbubbles (n = 301), determine whether this morphology was affected upon binding and by the type of ligand on the microbubble, and study its influence on microbubble oscillation and the drug delivery outcome. High-resolution 3D confocal microscopy revealed that targeted microbubbles were internalized by endothelial cells, while this was not the case for non-targeted or IgG1-κ control microbubbles. The extent of internalization was ligand-dependent, since αvβ3-targeted microbubbles were significantly more internalized than CD31-targeted microbubbles. Ultra-high-speed imaging (~17 Mfps) in combination with high-resolution confocal microscopy (n = 246) showed that microbubble internalization resulted in a damped microbubble oscillation upon ultrasound insonification (2 MHz, 200 kPa peak negative pressure, 10 cycles). Despite damped oscillation, the cell's susceptibility to sonoporation (as indicated by PI uptake) was increased for internalized microbubbles. Monitoring cell membrane integrity (n = 230) showed the formation of either a pore, for intracellular delivery, or a tunnel (i.e. transcellular perforation), for transcellular delivery. Internalized microbubbles caused fewer transcellular perforations and smaller pore areas than non-internalized microbubbles. In conclusion, studying microbubble-mediated drug delivery using a state-of-the-art imaging system revealed receptor-mediated microbubble internalization and its effect on microbubble oscillation and resulting membrane perforation by pores and tunnels.@en

Bacteria encased in a biofilm poses significant challenges to successful treatment, since both the immune system and antibiotics are ineffective. Sonobactericide, which uses ultrasound and microbubbles, is a potential new strategy for increasing antimicrobial effectiveness or directly killing bacteria. Several studies suggest that sonobactericide can lead to bacterial dispersion or sonoporation (i.e., cell membrane permeabilization); however, real-time observations distinguishing individual bacteria during and directly after insonification are missing. Therefore, in this study, we investigated, in real-time and at high-resolution, the effects of ultrasound-induced microbubble oscillation on Staphylococcus aureus biofilms, without or with an antibiotic (oxacillin, 1 µg/mL). Biofilms were exposed to ultrasound (2 MHz, 100–400 kPa, 100–1000 cycles, every second for 30 s) during time-lapse confocal microscopy recordings of 10 min. Bacterial responses were quantified using post hoc image analysis with particle counting. Bacterial dispersion was observed as the dominant effect over sonoporation, resulting from oscillating microbubbles. Increasing pressure and cycles both led to significantly more dispersion, with the highest pressure leading to the most biofilm removal (up to 83.7%). Antibiotic presence led to more variable treatment responses, yet did not significantly impact the therapeutic efficacy of sonobactericide, suggesting synergism is not an immediate effect. These findings elucidate the direct effects induced by sonobactericide to best utilize its potential as a biofilm treatment strategy.@en