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Drug transport from blood to extravascular tissue can locally be achieved by increasing the vascular permeability through ultrasound-activated microbubbles. However, the mechanism remains unknown, including whether short and long cycles of ultrasound induce the same onset rate, spatial distribution, and amount of vascular permeability increase. Accurate models are necessary for insights into the mechanism so a microvessel-on-a-chip is developed with a membrane-free extravascular space. Using these microvessels-on-a-chip, distinct differences between 2 MHz ultrasound treatments are shown with 10 or 1000 cycles. The onset rate is slower for 10 than 1000 cycles, while both cycle lengths increase the permeability in spot-wise patterns without affecting microvessel viability. Significantly less vascular permeability increase and sonoporation are induced for 10 versus 1000 cycles at 750 kPa (i.e., the highest studied peak negative acoustic pressure (PNP)). The PNP threshold for vascular permeability increases is 750 versus 550 kPa for 10 versus 1000 cycles, while this is 750 versus 220 kPa for sonoporation. Vascular permeability increases do not correlate with α v β 3 -targeted microbubble behavior, while sonoporation correlates with α v β 3 -targeted microbubble clustering. In conclusion, the further mechanistic unraveling of vascular permeability increase by ultrasound-activated microbubbles in a developed microvessel-on-a-chip model aids the safe and efficient development of microbubble-mediated drug transport., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)

Locally opening up the endothelial barrier in a safe and controlled way is beneficial for drug delivery into the extravascular tissue. Although ultrasound-induced microbubble oscillations can affect the endothelial barrier integrity, the mechanism remains unknown. Here we uncover a new role for F-actin in microbubble-mediated endothelial gap formation. Unique simultaneous high-resolution confocal microscopy and ultra-high-speed camera imaging (10 million frames per second) reveal that single oscillating microbubbles (radius 1.3-3.8 μm; n = 48) induce sonoporation in all cells in which F-actin remodeling occurred. F-actin disruption only mainly resulted in tunnel formation (75 %), while F-actin stress fiber severing and recoil mainly resulted in cell-cell contact opening within 15 s upon treatment (54 %) and tunnel formation (15 %). F-actin stress fiber severing occurred when the fibers were within reach of the microbubble's maximum radius during oscillation, requiring normal forces of ≥230 nN. In the absence of F-actin stress fibers, oscillating microbubbles induced F-actin remodeling but no cell-cell contact opening. Together, these findings reveal a novel mechanism of microbubble-mediated transendothelial drug delivery, which associates with the underlying cytoskeletal F-actin organization., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: After myocardial infarction, the lost myocardium is replaced by fibrotic tissue, eventually progressively leading to myocardial dysfunction. Direct reprogramming of fibroblasts into cardiomyocytes via the forced overexpression of cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) offers a promising strategy for cardiac repair. The limited reprogramming efficiency of this approach, however, remains a significant challenge., Methods: We screened seven factors capable of improving direct cardiac reprogramming of both mice and human fibroblasts by evaluating small molecules known to be involved in cardiomyocyte differentiation or promoting human-induced pluripotent stem cell reprogramming., Results: We found that vitamin C (VitC) significantly increased cardiac reprogramming efficiency when added to GMT-overexpressing fibroblasts from human and mice in 2D and 3D model. We observed a significant increase in reactive oxygen species (ROS) generation in human and mice fibroblasts upon Doxy induction, and ROS generation was subsequently reduced upon VitC treatment, associated with increased reprogramming efficiency. However, upon treatment with dehydroascorbic acid, a structural analog of VitC but lacking antioxidant properties, no difference in reprogramming efficiency was observed, suggesting that the effect of VitC in enhancing cardiac reprogramming is partly dependent of its antioxidant properties., Conclusions: Our findings demonstrate that VitC supplementation significantly enhances the efficiency of cardiac reprogramming, partially by suppressing ROS production in the presence of GMT., (© 2024. The Author(s).)

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 α v β 3 -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., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Phospholipid-coated targeted microbubbles are used for ultrasound molecular imaging and locally enhanced drug delivery, with the binding efficacy being an important trait. The use of organic solvent in microbubble production makes the difference between a heterogeneous or homogeneous ligand distribution. This study demonstrates the effect of ligand distribution on the binding efficacy of phospholipid-coated α ν β 3 -targeted microbubbles in vitro using a monolayer of human umbilical-vein endothelial cells and in vivo using chicken embryos. Microbubbles with a homogeneous ligand distribution had a higher binding efficacy than those with a heterogeneous ligand distribution both in vitro and in vivo. In vitro, 1.55× more microbubbles with a homogeneous ligand distribution bound under static conditions, while this was 1.49× more under flow with 1.25 dyn/cm 2 , 1.56× more under flow with 2.22 dyn/cm 2 , and 1.25× more in vivo. The in vitro dissociation rate of bound microbubbles with homogeneous ligand distribution was lower at low shear stresses (1-5 dyn/cm 2 ). The internalized depth of bound microbubbles was influenced by microbubble size, not by ligand distribution. In conclusion, for optimal binding the use of organic solvent in targeted microbubble production is preferable over directly dispersing phospholipids in aqueous medium.

Present-day people from England and Wales have more ancestry derived from early European farmers (EEF) than did people of the Early Bronze Age 1 . To understand this, here we generated genome-wide data from 793 individuals, increasing data from the Middle to the Late Bronze Age and Iron Age in Britain by 12-fold, and western and central Europe by 3.5-fold. Between 1000 and 875 BC, EEF ancestry increased in southern Britain (England and Wales) but not northern Britain (Scotland) due to incorporation of migrants who arrived at this time and over previous centuries, and who were genetically most similar to ancient individuals from France. These migrants contributed about half the ancestry of people of England and Wales from the Iron Age, thereby creating a plausible vector for the spread of early Celtic languages into Britain. These patterns are part of a broader trend of EEF ancestry becoming more similar across central and western Europe in the Middle to the Late Bronze Age, coincident with archaeological evidence of intensified cultural exchange 2-6 . There was comparatively less gene flow from continental Europe during the Iron Age, and the independent genetic trajectory in Britain is also reflected in the rise of the allele conferring lactase persistence to approximately 50% by this time compared to approximately 7% in central Europe where it rose rapidly in frequency only a millennium later. This suggests that dairy products were used in qualitatively different ways in Britain and in central Europe over this period., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Volumetric ultrasound imaging of blood flow with microbubbles enables a more complete visualization of the microvasculature. Sparse arrays are ideal candidates to perform volumetric imaging at reduced manufacturing complexity and cable count. However, due to the small number of transducer elements, sparse arrays often come with high clutter levels, especially when wide beams are transmitted to increase the frame rate. In this study, we demonstrate with a prototype sparse array probe and a diverging wave transmission strategy, that a uniform transmission field can be achieved. With the implementation of a spatial coherence beamformer, the background clutter signal can be effectively suppressed, leading to a signal to background ratio improvement of 25 dB. With this approach, we demonstrate the volumetric visualization of single microbubbles in a tissue-mimicking phantom as well as vasculature mapping in a live chicken embryo chorioallantoic membrane.

Phospholipid-coated microbubbles are ultrasound contrast agents that, when functionalized, adhere to specific biomarkers on cells. In this concise review, we highlight recent developments in strategies for targeting the microbubbles and their use for ultrasound molecular imaging (UMI) and therapy. Recently developed novel targeting strategies include magnetic functionalization, triple targeting, and the use of several new ligands. UMI is a powerful technique for studying disease progression, diagnostic imaging, and monitoring of therapeutic responses. Targeted microbubbles (tMBs) have been used for the treatment of cardiovascular diseases and cancer, with therapeutics either coadministered or loaded onto the tMBs. Regardless of which disease was treated, the use of tMBs always resulted in a better therapeutic outcome than non-tMBs when compared in vitro or in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Spontaneous Ca2+ release (SCR) can cause triggered activity and initiate arrhythmias. Intrinsic transmural heterogeneities in Ca2+ handling and their propensity to disease remodeling may differentially modulate SCR throughout the left ventricular (LV) wall and cause transmural differences in arrhythmia susceptibility. Here, we aimed to dissect the effect of cardiac injury on SCR in different regions in the intact LV myocardium using cryoinjury on rat living myocardial slices (LMS). We studied SCR under proarrhythmic conditions using a fluorescent Ca2+ indicator and high-resolution imaging in LMS from the subendocardium (ENDO) and subepicardium (EPI). Cryoinjury caused structural remodeling, with loss in T-tubule density and an increased time of Ca2+ transients to peak after injury. In ENDO LMS, the Ca2+ transient amplitude and decay phase were reduced, while these were not affected in EPI LMS after cryoinjury. The frequency of spontaneous whole-slice contractions increased in ENDO LMS without affecting EPI LMS after injury. Cryoinjury caused an increase in foci that generates SCR in both ENDO and EPI LMS. In ENDO LMS, SCRs were more closely distributed and had reduced latencies after cryoinjury, whereas this was not affected in EPI LMS. Inhibition of CaMKII reduced the number, distribution, and latencies of SCR, as well as whole-slice contractions in ENDO LMS, but not in EPI LMS after cryoinjury. Furthermore, CaMKII inhibition did not affect the excitation-contraction coupling in cryoinjured ENDO or EPI LMS. In conclusion, we demonstrate increased arrhythmogenic susceptibility in the injured ENDO. Our findings show involvement of CaMKII and highlight the need for region-specific targeting in cardiac therapies., (© 2021 Dries et al.)

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., Competing Interests: The authors report no declarations of interest., (© 2021 The Author(s).)

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.