Annemieke van Wamel

Ultrasound and Microbubble-Targeted Delivery of Macromolecules Is Regulated by Induction of Endocytosis and Pore Formation

Contrast microbubbles in combination with ultrasound (US) are promising vehicles for local drug and gene delivery. However, the exact mechanisms behind intracellular delivery of therapeutic compounds remain to be resolved. We hypothesized that endocytosis and pore formation are involved during US and microbubble targeted delivery (UMTD) of therapeutic compounds. Therefore, primary endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 to 500 kDa) using 1 MHz pulsed US with 0.22-MPa peak-negative pressure, during 30 seconds. Fluorescence microscopy showed homogeneous distribution of 4.4- and 70-kDa dextrans through the cytosol, and localization of 155- and 500-kDa dextrans in distinct vesicles after UMTD. After ATP depletion, reduced uptake of 4.4-kDa dextran and no uptake of 500-kDa dextran was observed after UMTD. Independently inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis significantly decreased intracellular delivery of 4.4- to 500-kDa dextrans. Furthermore, 3D fluorescence microscopy demonstrated dextran vesicles (500 kDa) to colocalize with caveolin-1 and especially clathrin. Finally, after UMTD of dextran (500 kDa) into rat femoral artery endothelium in vivo, dextran molecules were again localized in vesicles that partially colocalized with caveolin-1 and clathrin. Together, these data indicated uptake of molecules via endocytosis after UMTD. In addition to triggering endocytosis, UMTD also evoked transient pore formation, as demonstrated by the influx of calcium ions and cellular release of preloaded dextrans after US and microbubble exposure. In conclusion, these data demonstrate that endocytosis is a key mechanism in UMTD besides transient pore formation, with the contribution of endocytosis being dependent on molecular size.

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.

Oil-filled polymer microcapsules for ultrasound-mediated delivery of lipophilic drugs

The use of ultrasound contrast agents as local drug delivery systems continues to grow. Current limitations are the amount of drug that can be incorporated as well as the efficiency of drug release upon insonification. This study focuses on the synthesis and characterisation of novel polymeric microcapsules for ultrasound-triggered delivery of lipophilic drugs. Microcapsules with a shell of fluorinated end-capped poly(L-lactic acid) were made through pre-mix membrane emulsification and contained, apart from a gaseous phase, different amounts of hexadecane oil as a drug-carrier reservoir. Mean number weighted diameters were between 1.22 microm and 1.31 microm. High-speed imaging at approximately 10 million fames per second showed that for low acoustic pressures (1 MHz, 0.24 MPa) microcapsules compressed but remained intact. At higher diagnostic pressures of 0.51 MPa, microcapsules cracked, thereby releasing the encapsulated gas and model lipophilic drug. Using conventional ultrasound B-mode imaging at a frequency of 2.5 MHz, a marked enhancement of scatter intensity over a tissue-mimicking phantom was observed for all differently loaded microcapsules. The partially oil-filled microcapsules with high drug loads and well-defined acoustic activation thresholds have great potential for ultrasound-triggered local delivery of lipophilic drugs under ultrasound image-guidance.

High-speed photography during ultrasound illustrates potential therapeutic applications of microbubbles.

Ultrasound contrast agents consist of microscopically small encapsulated bubbles that oscillate upon insonification. To enhance diagnostic ultrasound imaging techniques and to explore therapeutic applications, these medical microbubbles have been studied with the aid of high-speed photography. We filmed medical microbubbles at higher frame rates than the ultrasonic frequency transmitted. Microbubbles with thin lipid shells have been observed to act as microsyringes during one single ultrasonic cycle. This jetting phenomenon presumably causes sonoporation. Furthermore, we observed that the gas content can be forced out of albumin-encapsulated microbubbles. These free bubbles have been observed to jet, too. It is concluded that microbubbles might act as a vehicle to carry a drug in gas phase to a region of interest, where it has to be released by diagnostic ultrasound. This opens up a whole new area of potential applications of diagnostic ultrasound related to targeted imaging and therapeutic delivery of drugs such as nitric oxide.

Optimization of ultrasound and microbubbles targeted gene delivery to cultured primary endothelial cells

Ultrasound and microbubbles targeted gene delivery (UMTGD) is a promising technique for local gene delivery. As the endothelium is a primary target for systemic UMTGD, this study aimed at establishing the optimal parameters of UMTGD to primary endothelial cells. For this, an in vitro ultrasound (US) setup was employed in which individual UMTGD parameters were systematically optimized. The criteria for the final optimized protocol were: (1) relative high reporter gene expression levels, restricted to the US exposed area and (2) induction of not more than 5% cell death. US frequency and timing of medium replacement had a strong effect on UMTGD efficiency. Furthermore, US intensity, DNA concentration and total duration of US all affected UMTGD efficiency. Optimal targeted gene delivery to primary endothelial cells can be accomplished with Sonovue® microbubbles, using 20 μg/ml plasmid DNA, a 1 MHz US exposure of Ispta 0.10 W/cm2 for 30 s with immediate medium change after UMTGD. This optimized protocol resulted in both an increase in the number of transfected cells (more than three fold) and increased levels of transgene expression per cell (170%).

Pressure-Dependent Attenuation and Scattering of Phospholipid-Coated Microbubbles at Low Acoustic Pressures

Previous optical studies have shown threshold behavior of single-contrast agent microbubbles. Below the acoustic pressure threshold, phospholipid-coated microbubbles with sizes <5.0 mum in diameter oscillate significantly less than above the threshold pressure. Previous studies also revealed an acoustic pressure-dependent attenuation of ultrasound by microbubble contrast agents. In this study, we investigated whether pressure-dependent acoustic behavior may be explained by threshold behavior. For this purpose, pressure-dependent attenuation and scattering of a phospholipid-coated contrast agent were measured. Transmit frequencies between 1.5 and 6.0 MHz and acoustic pressures between 5 and 200 kPa were applied. Unlike the galactose-based contrast agent Levovist, the phospholipid-coated contrast agent BR14 showed a pressure-dependent attenuation. In addition, it was found that filtered suspensions with only microbubbles <3.0 mum in diameter show more pressure-dependent attenuation behavior than native suspensions of phospholipid-coated microbubbles. For the scattering measurements conducted at 3.0 MHz, the native suspension did not show any pressure-dependent behavior. However, the filtered suspension responded highly nonlinearly. Between 30 and 150 kPa, 16 dB additional scattered power was obtained. We concluded that threshold behavior of phospholipid-coated microbubbles results in pressure-dependent attenuation and scattering.

Single versus triple daily activation of the distractor: No significant effects of frequency of distraction on bone regenerate quantity and architecture

OBJECTIVES To study the effect of two different frequencies of distraction on the quantity and architecture of bone regenerate using micro-computed tomography, and to determine whether radiographic and ultrasonographic bone-fill scores provide reliable predictive value for the amount of new bone in the distraction area. MATERIAL AND METHODS Twenty-six skeletally mature rabbits underwent three full days of latency, after which midface distraction was started. Low-frequency group (n=12): a distraction rate of 0.9 mm/d achieved by one daily activation for 11 days to create a 10mm distraction gap. High-frequency group (n=12): idem, but three daily activations were used instead of one. Control group (n=2) underwent no distraction. After 21 days of consolidation, bone-fill in the distraction area was assessed by means of ultrasonography and radiography. Micro-computed tomography was used to quantify new bone formation and bone architecture. RESULTS Relative bone volume (BV/TV) showed a tendency towards a difference (P=0.09) between the low and high-frequency groups. No significant differences were found for bone architecture. No significant correlation between BV/TV values and bone-fill scores was found. CONCLUSIONS An increase in rhythm from one to three activations daily does not create significantly more bone. Bone-fill score values provided no reliable predictive value for the amount of new bone formation.

Generation of a droplet inside a microbubble with the aid of an ultrasound contrast agent: First result

textabstractNew ultrasound contrast agents that incorporate a therapeutic compound have become of interest. Such an ultrasound contrast agent particle might act as the vehicle to carry a drug or gene load to a perfused region of interest. The load could be released with the assistance of ultrasound. Generally, an increase in shell thickness increases the acoustic amplitude needed to disrupt a bubble. High acoustic amplitudes, however, have been associated with unwanted effects on cells. It would be interesting to incorporate a droplet containing drugs or genes inside a microbubble carrier. A liquid core surrounded by a gas encapsulation has been referred to as antibubble. In this paper, the creation of an antibubble with the aid of ultrasound has been demonstrated with high-speed photography.

Evaluation of the binding force between a biotinylated microbubble and a streptavidin-coated surface by ultrasound radiation

For molecular imaging using ultrasound, targeted ultrasound contrast agents are used that consist of encapsulated gas microbubbles. For this application, the strength of adhesion between a microbubble and a cell surface is an important factor. This research focuses on the evaluation of the binding force between a streptavidin-coated surface, which mimics a cell surface, and biotinylated lipid coated microbubbles by detaching the microbubbles with ultrasound radiation force. Our results show that ultrasound radiation force can be used to determine the binding force between a targeted microbubble and a surface.

Drug Delivery to Extravascular Tissue by Ultrasound‐activated Microbubbles

Drugs will be delivered to tissue more efficiently if the vascular endothelial permeability is increased. Although recent studies have established 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. To investigate endothelial layer permeability, we treated monolayers of human umbilical vein endothelial cells with ultrasound and the contrast agent BR14. Endothelial layer permeability was assessed by measuring the transendothelial electrical resistance (TEER) and the transendothelial transport of fluorescein. Ultrasound in combination with BR14 significantly decreased TEER to 68.0 ± 3.1% of initial values and temporally increased endothelial permeability for fluorescein by 38.1 ± 16.4 %. After treatment, no cell loss or damage was observed. In conclusion, ultrasound in combination with BR14 increased the endothelial layer permeability. This feature m...