Remote manipulation of cells with ultrasound and microbubbles

Ultrasound in combination with contrast microbubbles has been shown to alter the permeability of cell membranes. This permeabilization feature is used to design new drug delivery systems using ultrasound and contrast agents. Although the exact underlying mechanisms are still unknown, one hypothesis is that oscillating microbubbles cause cell deformation resulting in enhanced cell membrane permeability. In this paper we show the actions of oscillating microbubbles on cultured cells under a microscope recorded with a fast framing camera at 10 million frames per second. Optical observations of microbubbles and cultured cells is possible through the use of a microscope mounted in front of the fast framing camera Brandaris128. The Brandaris128 is capable of recording a sequence of 128 images with a frame rate up to 25 million frames per second. Pig aorta endothelial cells were grown on the inside of an Opticell/spl trade/ container. A diluted suspension of experimental agents BR14 (Bracco Research, Geneva, Switzerland) was added. Ultrasound exposure consisted of one burst of 6 cycles at a frequency of 1 MHz and a P/spl I.bar/ of 0.5 MPa. During ultrasound transmission, the interactions between BR14 microbubbles and cultured cells were recorded using a frame rate of 10 million frames per second. Cell deformation as a result of vibrating microbubbles is studied. Cell deformation is quantified through measuring the displacement of the cells. Microbubble vibration is quantified by measuring its initial, maximal, and minimal radii. We observed that upon ultrasound arrival and microbubble oscillations, the cell membrane deforms up to a few micrometers in length as a result of the oscillation of the microbubble. The membrane deformation rate changes with the oscillation strength of the microbubble. During the sonication, changes in the cross-sectional distance of the cultured cells were observed due to microbubble vibrations. Depending on the maximal vibrations of the microbubble and the distance between the microbubble and the cell, the displacement of the cells varied form 0 to 20% of the cell size. This study reveals the action of oscillating microbubbles on cells. It is known that living cells sense mechanical forces thus there is no doubt that perturbation of the oscillating microbubbles results in profound alterations in the cellular content. This study is the beginning of revealing the functional relationships that lie beyond the remote manipulation of cells and ultrasound microbubble induced permeabilization of the cell membrane

Ultra-high-speed imaging of bubbles interacting with cells and tissue

Ultrasound contrast microbubbles are exploited in molecular imaging, where bubbles are directed to target cells and where their high-scattering cross section to ultrasound allows for the detection of pathologies at a molecular level. In therapeutic applications vibrating bubbles close to cells may alter the permeability of cell membranes, and these systems are therefore highly interesting for drug and gene delivery applications using ultrasound. In a more extreme regime bubbles are driven through shock waves to sonoporate or kill cells through intense stresses or jets following inertial bubble collapse. Here, we elucidate some of the underlying mechanisms using the 25-Mfps camera Brandaris128, resolving the bubble dynamics and its interactions with cells. We quantify acoustic microstreaming around oscillating bubbles close to rigid walls and evaluate the shear stresses on nonadherent cells. In a study on the fluid dynamical interaction of cavitation bubbles with adherent cells, we find that the nonspherical collapse of bubbles is responsible for cell detachment. We also visualized the dynamics of vibrating microbubbles in contact with endothelial cells followed by fluorescent imaging of the transport of propidium iodide, used as a membrane integrity probe, into these cells showing a direct correlation between cell deformation and cell membrane permeability

Ultrafast Microscopy Imaging of Acoustic Cluster Therapy Bubbles: Activation and Oscillation

Acoustic Cluster Therapy (ACT®) is a platform for improving drug delivery and has had promising pre-clinical results. A clinical trial is ongoing. ACT® is based on microclusters of microbubbles–microdroplets that, when sonicated, form a large ACT® bubble. The aim of this study was to obtain new knowledge on the dynamic formation and oscillations of ACT® bubbles by ultrafast optical imaging in a microchannel. The high-speed recordings revealed the microbubble–microdroplet fusion, and the gas in the microbubble acted as a vaporization seed for the microdroplet. Subsequently, the bubble grew by gas diffusion from the surrounding medium and became a large ACT® bubble with a diameter of 5–50 μm. A second ultrasound exposure at lower frequency caused the ACT® bubble to oscillate. The recorded oscillations were compared with simulations using the modified Rayleigh–Plesset equation. A term accounting for the physical boundary imposed by the microchannel wall was included. The recorded oscillation amplitudes were approximately 1–2 µm, hence similar to oscillations of smaller contrast agent microbubbles. These findings, together with our previously reported promising pre-clinical therapeutic results, suggest that these oscillations covering a large part of the vessel wall because of the large bubble volume can substantially improve therapeutic outcome.publishedVersio

Ultrasonic characterization of ultrasound contrast agents

The main constituent of an ultrasound contrast agent (UCA) is gas-filled microbubbles. An average UCA contains billions per ml. These microbubbles are excellent ultrasound scatterers due to their high compressibility. In an ultrasound field they act as resonant systems, resulting in harmonic energy in the backscattered ultrasound signal, such as energy at the subharmonic, ultraharmonic and higher harmonic frequencies. This harmonic energy is exploited for contrast enhanced imaging to discriminate the contrast agent from surrounding tissue. The amount of harmonic energy that the contrast agent bubbles generate depends on the bubble characteristics in combination with the ultrasound field applied. This paper summarizes different strategies to characterize the UCAs. These strategies can be divided into acoustic and optical methods, which focus on the linear or nonlinear responses of the contrast agent bubbles. In addition, the characteristics of individual bubbles can be determined or the bubbles can be examined when they are part of a population. Recently, especially optical methods have proven their value to study individual bubbles. This paper concludes by showing some examples of optically observed typical behavior of contrast bubbles in ultrasound fields

Micromanipulation of endothelial cells: Ultrasound-microbubble-cell interaction

Ultrasound (US) in combination with contrast microbubbles has been shown to alter the permeability of cell membranes without affecting cell viability. This permeabilisation feature is used to design new drug delivery systems using US and contrast agents. The underlying mechanisms are still unknown. One hypothesis is that oscillating microbubbles cause cell deformation resulting in enhanced cell membrane permeability. This technical note reveals the interaction between oscillating microbubbles and endothelial cells under a microscope recorded with a fast framing camera at 10 million frames per second. A microbubble expansion of 100% resulted a 2.3-m displacement of the cell membrane. During the insonification, changes of approximately 15% in the cross-sectional distance of the endothelial cells were observed due to microbubble vibrations. In conclusion, the use of such a camera makes it possible to reveal the mechanisms of interactions between ultrasound, microbubbles and cells

A coalescence model for SonoVue(TM)

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The onset of microbubble vibration

A linear relationship between the relative expansion of an off-resonance ultrasound contrast microbubble and low acoustic pressures is expected. In this study, high-speed optical recordings of individual phospholipid-coated microbubbles were used to investigate this relationship for microbubbles ranging from 2 to 11 μm and for acoustic pressures ranging from 20 to 250 kPa at a driving frequency of 1.7 MHz. For microbubbles larger than 5 μm, the relative expansion (ΔD/D0) increased linearly with applied acoustic pressure, starting at the origin. The response of smaller microbubbles (<5 μm) also increased linearly with the applied acoustic pressure. However, linearity started at an acoustic pressure threshold value of 30 to 120 kPa for the different individual microbubbles. Below these pressure values, little or no oscillation was observed. The results may be explained by size-dependent mechanical properties of the phospholipid shells. An imaging technique such as power modulation imaging could profit from the presence of an acoustic pressure threshold in the microbubble response

HIGH-RESOLUTION ULTRASONOGRAPHY OF THE CUTANEOUS NERVE BRANCHES IN THE HAND AND WRIST

Ultrasonography can be used in the diagnosis of various neuropathies, including nerve injury. Nerves often involved in traumatic and iatrogenic injury are small cutaneous branches in the hand and wrist, which cannot be seen in detail using current ultrasound probes. This study explored the potential of high-resolution ultrasonography in seeing these nerve branches in the human. The VisualSonics Vevo 770 system with a 15-82.5 MHz probe was compared to a commonly used 5-12 MHz probe and ultrasound machine. The accuracy was validated by ultrasound guided dye injection into cadaver nerves, with subsequent anatomical dissection and verification. Results were confirmed in two healthy volunteers. The Vevo 770 system was able to accurately identify the small cutaneous nerves. It could also depict the median nerve and its fascicles in greater detail. This may be useful for clinical diagnosis, localisation and follow-up of neuropathies and nerve injuries

Increasing the Endothelial Layer Permeability Through Ultrasound-Activated Microbubbles

Drug delivery to a diseased tissue will be more efficient if the vascular endothelial permeability is increased. Recent studies have shown that the permeability of single cell membranes is increased by ultrasound in combination with contrast agents. It is not known whether this combination can also increase the permeability of an endothelial layer in the absence of cell damage. To investigate the feasibility of controlled increased endothelial layer permeability, we treated monolayers of human umbilical vein endothelial cells with ultrasound and the contrast agent BR14. Barrier function was assessed by measuring transendothelial electrical resistance (TEER). Ultrasound-activated BR14 significantly decreased TEER by 40.3% ?? 3.7% (p < 0.01). After treatment, no cell detachment or damage was observed. In conclusion, ultrasound-activated BR14 microbubbles increased the endothelial layer permeability. This feature can be used for future ultrasound-guided drug delivery system