A. van Wamel

Ultrasound and microbubble-targeted delivery of therapeutic compounds: ICIN Report Project 49: Drug and gene delivery through ultrasound and microbubbles

The molecular understanding of diseases has been accelerated in recent years, producing many new potential therapeutic targets. A noninvasive delivery system that can target specific anatomical sites would be a great boost for many therapies, particularly those based on manipulation of gene expression. The use of microbubbles controlled by ultrasound as a method for delivery of drugs or genes to specific tissues is promising. It has been shown by our group and others that ultrasound increases cell membrane permeability and enhances uptake of drugs and genes. One of the important mechanisms is that microbubbles act to focus ultrasound energy by lowering the threshold for ultrasound bioeffects. Therefore, clear understanding of the bioeffects and mechanisms underlying the membrane permeability in the presence of microbubbles and ultrasound is of paramount importance. (Neth Heart J 2009;17:82-6.)

Effects of diagnostic ultrasound parameters on molecular uptake and cell viability

The success of drug or gene delivery is limited by the inability of those components to cross biological barriers like the cell membrane. Ultrasound (US) has shown to increase cell membrane permeability in a process known as sonoporation. So far most investigations have used acoustic settings such as continuous wave or high intensity focused ultrasound, which are well far from the diagnostic range. Since a few years now, other studies used US waves with diagnostic conditions but in combination with contrast bubbles. The purpose of our study is to determine the effect of US alone on cell uptake using diagnostic parameters, and to correlate the sonoporation mechanism on the different diagnostic conditions. Monolayers of CHO cells, fixed on a membrane, are used whereby Texas-red labeled dextran (10 nm) is used as a marker molecule. The experimental parameters were: acoustic pressure 0.2-1.4 MPa P_, pulse length (10/spl mu/s-15/spl mu/s), duty cycle (0-0.75%), total exposure time (0-6 minutes) and addition of contrast microbubbles. Transmitted frequency was 1 MHz and the temperature was kept constant at 37/spl deg/C. The control cells showed no molecular uptake. For exposure times <30 s, sonoporation increased with increased MI. For exposure times > 30, sonoporation increased transiently with increasing MI reaching a maximum at MI 0.7. Using MI between 0.2-0.7, maximum sonoporation was reached after 2 minutes of exposure. Using ultrasound with MI 1.4 during 30 sec gave the highest molecular uptake (33%) but also very high cell lysis (40%). Under stronger diagnostic conditions (MI 1.4 and total exposure time above 2 min), lysis occurred up to 65% and almost no cells were sonoporated. Increasing repetition rate resulted in a higher cell lysis and very low sonoporation. Addition of contrast microbubbles resulted in a higher cell lysis but not in a higher molecular uptake. Significant molecular uptake can be induced by diagnostic pulsed US without using contrast bubbles. For pressures >1.4 MPa and exposure time >30s, (repetition rate 0.01 sec and 0.1% duty cycle) the US treatment results in cell-lysis. Both molecular uptake and cell viability strongly depend on total exposure time, applied MI, repetition rate, duty cycle, and addition of contrast microbubbles. Addition of contrast microbubbles enhanced the ultrasound effects.

Nonlinear imaging of targeted microbubbles with intravascular ultrasound

The nonlinear detection of targeted microbubbles at high ultrasound frequencies was investigated. A prototype nonlinear intravascular ultrasound (IVUS) system was employed using a 20 MHz fundamental frequency (F20) to examine 40 MHz second harmonic (H40) signals and a 40 MHz fundamental frequency (F40) to examine 20 MHz subharmonic (SH20) signals. An experimental biotinated micron to submicron lipid encapsulated agent was targeted to avidin coated agar-based tissue mimicking phantoms. An examination of bound bubble acoustic signatures demonstrated the feasibility of initiating H40 and SH20 signals. Imaging experiments showed improvements in contrast-to-tissue ratios (CTR) using both H40 and SH20 relative to fundamental frequency imaging. These results indicate the potential of high frequency nonlinear imaging as a means of improving the detection of targeted microbubbles.

Duration of ultrasound bubbles enhanced cell membrane permeability

Purpose: Ultrasound (US) has shown the ability to modulate the cell membrane permeability in a process known as sonoporation. In addition, the sonoporation process has been proven to be amplified when US is associated with contrast microbubbles. The purpose of this study is to quantify the duration of the sonoporation process for external molecules with different sizes. Method: monolayers of Chinese Hamster Ovary (CHO) cells, fixed on a membrane, were used and 3 fluorescent-labeled dextran molecules (10, 40 and 70 KDa) were used as markers. The US settings consisted of a burst of 10 cycles and 1 MHz at acoustic pressures between 0.2-1.0 MPa with a pulse repetition rate of 20 Hz. CHO cells were irradiated at 37/spl deg/C for 2 minutes after addition of microbubbles in a ratio of 1:1 cell. The cells were incubated with the 3 markers at t=0 sec, 10 sec, 30 sec, and 60 sec after US was applied and the respective uptake levels were measured. Conclusion: a negative correlation between maximum uptake and time after turning off US is demonstrated. Moreover, a higher maximum uptake level at the moment of ultrasound turn off results in a faster decay in uptake. In conclusion the duration of enhanced membrane permeability is limited with a maximal duration less than 60 sec. This depends on the size of the molecules but not on MI.

Optical investigation of ultrasound induced encapsulated microbubble oscillations: threshold and hysteresis effects

In order to get more insight in the contribution of the shell upon the microbubble oscillating behaviour, we recorded the dynamic response of individual microbubbles dependent on the acoustic pressure using high-speed imaging. We investigated threshold and hysteresis effects in soft-shelled phospholipid microbubbles. In contradiction to the predictions of current bubble models, we have shown that depending on size, microbubbles need an initial acoustic pressure, before they start oscillating. This threshold behaviour was also found in the hysteresis curves of microbubbles below 5 μm. The presence of an oscillation threshold may be used to improve imaging techniques.

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.

11A-4 Molecular Imaging with Targeted Contrast Agents and High Frequency Ultrasound

Targeted ultrasound contrast agents (USCA) can be used as molecular imaging agents to visualize the expression of endothelial molecules (biomarkers). In a murine tumor model, three biomarkers for tumor vasculature were imaged using targeted USCA and the high frequency ultrasound system Vevo 770. Three biomarkers were targeted: VCAM-1, selectins (P and E), and VEGF receptors. Targeting was determined by ultrasound contrast imaging of the adhered USCA in tumors ranging in size from 3 up to 40 mm2. Dependent on the tumor size, targeting of ultrasound contrast agents to the tumor vasculature resulted in significant signal enhancement when compared to control contrast agent. Each targeted contrast agent had it own accumulation level during tumor growth indicating differential expression pattern of the investigated target molecules during tumor growth. High frequency imaging with targeted ultrasound contrast can provide dynamic and quantitative information about the molecular profile of the tumor vasculature. This can open the possibility for patient-tailored therapy and can provide a tool to monitor whether the therapy is having the desired effect.

P5B-13 Improved Ultrasound Contrast Agent Detection in a Clinical Setting

Optical studies have shown threshold behaviour of phospholipid-coated contrast agent microbubbles. Below the acoustic pressure threshold, phospholipid-coated microbubbles oscillate significantly less than above the threshold. For microbubbles smaller than 3.0 mum diameter, pressure-dependent scattering was measured, which is believed to be the result of threshold behaviour. The aim of this study is to investigate if threshold behaviour is useful to enhance the contrast in power modulation images. For levovist and BR14 suspensions (filtered and native), a programmable ultrasound system recorded power modulation images at 2 MHz and acoustic pressures between 25 and 250 kPa. Results were compared to intensities recorded with a commercial ultrasound system. An inverse relationship between the pressure-dependency of the scattering and microbubble size was observed. Threshold behaviour enhances the contrast in power modulation images. Using a suspension with microbubbles smaller than 2.0 mum, at 2 MHz transmit frequency and an acoustic pressure of 250 kPa, the CTR value was 33 dB, which is 13 dB higher compared to a native BR14 suspension.

1F-6 Transiently Increased Endothelial Layer Permeability by Ultrasound-activated Microbubbles

To enhance drug delivery to the extravascular tissue, a controlled, temporal and local increase in endothelial permeability is needed. 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 layers 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 permeability for fluorescein. Ultrasound in combination with BR14 significantly decreased TEER to 68.0 plusmn 3.1 % of initial values and temporally increased endothelial permeability for fluorescein by 38.1 plusmn 16.4 %. After treatment, no cell loss or damage was observed. In conclusion, ultrasound-activated BR14 microbubbles transiently increased the endothelial layer permeability. This feature may be used for future ultrasound-guided drug delivery systems

Creating antibubbles with ultrasound

Ultrasound contrast agents have been investigated for their potential applications in local drug and gene delivery. A microbubble might act as the vehicle to carry a drug or gene load to a perfused region of interest. The load has to be released with the assistance of ultrasound. We investigate the suitability of antibubbles for ultrasound-assisted local delivery. As opposed to bubbles, antibubbles consist of a liquid core surrounded by a gas encapsulation. Incorporating a liquid drop containing drugs or genes inside an ultrasound contrast agent microbubble, however, is technically challenging. An ultrasound-insonified microbubble generates a pressure field that is inversely proportional to the distance from the mi- crobubble. Therefore, an oscillating contrast agent microbubble may create a surface instability with a relatively big bubble at a short distance. For big enough instabilities, a drop may be formed inside the big bubble. Three different contrast agents were subjected to 0.5 MHz ultrasound, with mechanical indices >0.6. The contrast agents were inserted through an artificial capillary which led through the acoustic focus of the transducer. High-speed photographs were captured at a speed of 3 million frames per second and higher. We observed that ultrasound contrast microbubbles below resonance size may create visible surface instabilities with bubbles above resonance size. With an albumin-shelled contrast agent, we induced a surface instability that was big enough to create an antibubble inside a free (unencapsulated) gas bubble with an 8 micron diameter. The surface instability has been attributed to the presence of a contrast microbubble with a 3 micron diameter. This instability has the form of a re-entrant jet protruding into the gas bubble. The inward protrusion grew and subsequently drained, leaving a droplet with a five micron diameter inside the bubble. In a subsequent recording after 100 ms, only the gas bubble could be detected. Thus, the life- time of the antibubble was less then 100 ms. The presence of a surfactant on the interfaces might lead to an improved stability of an antibubble.