Ying Luan

Ultrasound and microbubble mediated drug delivery: acoustic pressure as determinant for uptake via membrane pores or endocytosis.

Although promising results are achieved in ultrasound mediated drug delivery, its underlying biophysical mechanisms remain to be elucidated. Pore formation as well as endocytosis has been reported during ultrasound application. Due to the plethora of ultrasound settings used in literature, it is extremely difficult to draw conclusions on which mechanism is actually involved. To our knowledge, we are the first to show that acoustic pressure influences which route of drug uptake is addressed, by inducing different microbubble-cell interactions. To investigate this, FITC-dextrans were used as model drugs and their uptake was analyzed by flow cytometry. In fluorescence intensity plots, two subpopulations arose in cells with FITC-dextran uptake after ultrasound application, corresponding to cells having either low or high uptake. Following separation of the subpopulations by FACS sorting, confocal images indicated that the low uptake population showed endocytic uptake. The high uptake population represented uptake via pores. Moreover, the distribution of the subpopulations shifted to the high uptake population with increasing acoustic pressure. Real-time confocal recordings during ultrasound revealed that membrane deformation by microbubbles may be the trigger for endocytosis via mechanostimulation of the cytoskeleton. Pore formation was shown to be caused by microbubbles propelled towards the cell. These results provide a better insight in the role of acoustic pressure in microbubble-cell interactions and the possible consequences for drug uptake. In addition, it pinpoints the need for a more rational, microbubble behavior based choice of acoustic parameters in ultrasound mediated drug delivery experiments.

Brandaris 128 ultra-high-speed imaging facility: 10 years of operation, updates, and enhanced features

The Brandaris 128 ultra-high-speed imaging facility has been updated over the last 10 years through modifications made to the cameras hardware and software. At its introduction the camera was able to record 6 sequences of 128 images (500 × 292 pixels) at a maximum frame rate of 25 Mfps. The segmented mode of the camera was revised to allow for subdivision of the 128 image sensors into arbitrary segments (1-128) with an inter-segment time of 17 μs. Furthermore, a region of interest can be selected to increase the number of recordings within a single run of the camera from 6 up to 125. By extending the imaging system with a laser-induced fluorescence setup, time-resolved ultra-high-speed fluorescence imaging of microscopic objects has been enabled. Minor updates to the system are also reported here.

Lipid Shedding from Single Oscillating Microbubbles

Lipid-coated microbubbles are used clinically as contrast agents for ultrasound imaging and are being developed for a variety of therapeutic applications. The lipid encapsulation and shedding of the lipids by acoustic driving of the microbubble has a crucial role in microbubble stability and in ultrasound-triggered drug delivery; however, little is known about the dynamics of lipid shedding under ultrasound excitation. Here we describe a study that optically characterized the lipid shedding behavior of individual microbubbles on a time scale of nanoseconds to microseconds. A single ultrasound burst of 20 to 1000 cycles, with a frequency of 1 MHz and an acoustic pressure varying from 50 to 425 kPa, was applied. In the first step, high-speed fluorescence imaging was performed at 150,000 frames per second to capture the instantaneous dynamics of lipid shedding. Lipid detachment was observed within the first few cycles of ultrasound. Subsequently, the detached lipids were transported by the surrounding flow field, either parallel to the focal plane (in-plane shedding) or in a trajectory perpendicular to the focal plane (out-of-plane shedding). In the second step, the onset of lipid shedding was studied as a function of the acoustic driving parameters, for example, pressure, number of cycles, bubble size and oscillation amplitude. The latter was recorded with an ultrafast framing camera running at 10 million frames per second. A threshold for lipid shedding under ultrasound excitation was found for a relative bubble oscillation amplitude >30%. Lipid shedding was found to be reproducible, indicating that the shedding event can be controlled.

Non-linear Response and Viscoelastic Properties of Lipid-Coated Microbubbles: DSPC versus DPPC

For successful in vivo contrast-enhanced ultrasound imaging (CEUS) and ultrasound molecular imaging, detailed knowledge of stability and acoustical properties of the microbubbles is essential. Here, we compare these aspects of lipid-coated microbubbles that have either 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as their main lipid; the other components were identical. The microbubbles were investigated in vitro over the frequency range 1-4 MHz at pressures between 10 and 100 kPa, and their response to the applied ultrasound was recorded using ultrahigh-speed imaging (15 Mfps). Relative to DPPC-coated microbubbles, DSPC-coated microbubbles had (i) higher acoustical stability; (ii) higher shell elasticity as derived using the Marmottant model (DSPC: 0.26 ± 0.13 N/m, DPPC: 0.06 ± 0.06 N/m); (iii) pressure amplitudes twice as high at the second harmonic frequency; and (iv) a smaller amount of microbubbles that responded at the subharmonic frequency. Because of their higher acoustical stability and higher non-linear response, DSPC-coated microbubbles may be more suitable for contrast-enhanced ultrasound.

Targeted microbubble mediated sonoporation of endothelial cells in vivo

Ultrasound contrast agents as drug-delivery systems are an emerging field. Recently, we reported that targeted microbubbles are able to sonoporate endothelial cells in vitro. In this study, we investigated whether targeted microbubbles can also induce sonoporation of endothelial cells in vivo, thereby making it possible to combine molecular imaging and drug delivery. Live chicken embryos were chosen as the in vivo model. αvß3-targeted microbubbles attached to the vessel wall of the chicken embryo were insonified at 1 MHz at 150 kPa (1 × 10 000 cycles) and at 200 kPa (1 × 1000 cycles) peak negative acoustic pressure. Sonoporation was studied by intravital microscopy using the model drug propidium iodide (PI). Endothelial cell PI uptake was observed in 48% of microbubble-vessel-wall complexes at 150 kPa (n = 140) and in 33% at 200 kPa (n = 140). Efficiency of PI uptake depended on the local targeted microbubble concentration and increased up to 80% for clusters of 10 to 16 targeted microbubbles. Ultrasound or targeted microbubbles alone did not induce PI uptake. This intravital microscopy study reveals that sonoporation can be visualized and induced in vivo using targeted microbubbles.

Viability of endothelial cells after ultrasound-mediated sonoporation: Influence of targeting, oscillation, and displacement of microbubbles

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

Impulse response method for characterization of echogenic liposomes

An optical characterization method is presented based on the use of the impulse response to characterize the damping imparted by the shell of an air-filled ultrasound contrast agent (UCA). The interfacial shell viscosity was estimated based on the unforced decaying response of individual echogenic liposomes (ELIP) exposed to a broadband acoustic impulse excitation. Radius versus time response was measured optically based on recordings acquired using an ultra-high-speed camera. The method provided an efficient approach that enabled statistical measurements on 106 individual ELIP. A decrease in shell viscosity, from 2.1 × 10(-8) to 2.5 × 10(-9) kg/s, was observed with increasing dilatation rate, from 0.5 × 10(6) to 1 × 10(7) s(-1). This nonlinear behavior has been reported in other studies of lipid-shelled UCAs and is consistent with rheological shear-thinning. The measured shell viscosity for the ELIP formulation used in this study [κs = (2.1 ± 1.0) × 10(-8) kg/s] was in quantitative agreement with previously reported values on a population of ELIP and is consistent with other lipid-shelled UCAs. The acoustic response of ELIP therefore is similar to other lipid-shelled UCAs despite loading with air instead of perfluorocarbon gas. The methods described here can provide an accurate estimate of the shell viscosity and damping for individual UCA microbubbles.

Segmented high speed imaging of vibrating microbubbles during long ultrasound pulses

Detailed information about the response of microbubbles to long ultrasound pulses (>100 cycles) is hampered by the limited time span ultra fast-framing cameras (> 10 MHz) cover. We therefore developed a new imaging mode for the Brandaris 128 camera [1], facilitating high speed imaging during small time windows (segments), equally distributed over a relatively large time span.

Loss of gas from echogenic liposomes exposed to pulsed ultrasound.

The destruction of echogenic liposomes (ELIP) in response to pulsed ultrasound excitations has been studied acoustically previously. However, the mechanism underlying the loss of echogenicity due to cavitation nucleated by ELIP has not been fully clarified. In this study, an ultra-high speed imaging approach was employed to observe the destruction phenomena of single ELIP exposed to ultrasound bursts at a center frequency of 6 MHz. We observed a rapid size reduction during the ultrasound excitation in 139 out of 397 (35%) ultra- high-speed recordings. The shell dilation rate, which is defined as the microbubble wall velocity divided by the instantaneous radius, [Formula: see text] /R, was extracted from the radius versus time response of each ELIP, and was found to be correlated with the deflation. Fragmentation and surface mode vibrations were also observed and are shown to depend on the applied acoustic pressure and initial radius. Results from this study can be utilized to optimize the theranostic application of ELIP, e.g. by tuning the size distribution or the excitation frequency.

Liposome shedding from a vibrating microbubble on nanoseconds timescale

When ultrasound contrast agents microbubbles (MBs) are preloaded with liposomes, they can be applied as a potential drug delivery vehicle. The fate of the liposomes under ultrasound excitations is of prime interest for investigations, since it is an essential step in the application of drug delivery. Previous studies on regular lipid-shelled MBs have shown lipid shedding phenomena, accompanied by MB shrinkage under ultrasound excitations. Here we present a multi-modal study to optically characterize shedding behavior of liposome-loaded MBs (lps-MBs) based on high-speed fluorescence imaging. First, the dynamics of shedding were resolved by the Brandaris camera operating at up to 2 million frames per second (Mfps). Shedding of shell material was observed after few cycles of the excitation pulse. Second, a parametric study using a Photron camera running at 75 kfps indicates a significant influence of MB resonance on the shedding behavior. Third, the shedding behavior was investigated as a function of the MB oscillatory dynamics, facilitated by combination of the two fast cameras. We found a threshold of the relative amplitude of oscillations (35%) for the onset of lipids shedding. Overall, the shedding behavior from lps-MBs could well be controlled by the excitation pulse.