Tom van Rooij

Targeted ultrasound contrast agents for ultrasound molecular imaging and therapy

Abstract Ultrasound contrast agents (UCAs) are used routinely in the clinic to enhance contrast in ultrasonography. More recently, UCAs have been functionalised by conjugating ligands to their surface to target specific biomarkers of a disease or a disease process. These targeted UCAs (tUCAs) are used for a wide range of pre-clinical applications including diagnosis, monitoring of drug treatment, and therapy. In this review, recent achievements with tUCAs in the field of molecular imaging, evaluation of therapy, drug delivery, and therapeutic applications are discussed. We present the different coating materials and aspects that have to be considered when manufacturing tUCAs. Next to tUCA design and the choice of ligands for specific biomarkers, additional techniques are discussed that are applied to improve binding of the tUCAs to their target and to quantify the strength of this bond. As imaging techniques rely on the specific behaviour of tUCAs in an ultrasound field, it is crucial to understand the characteristics of both free and adhered tUCAs. To image and quantify the adhered tUCAs, the state-of-the-art techniques used for ultrasound molecular imaging and quantification are presented. This review concludes with the potential of tUCAs for drug delivery and therapeutic applications.

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.

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.

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.

Acoustical response of DSPC versus DPPC lipid-coated microbubbles

Ultrasound contrast agents are widely used in clinical practice. Commercial lipid-coated microbubbles mainly consist of either DSPC or DPPC. Previous research on homemade microbubbles based on these lipids showed different shell micro-structures. In this study the acoustical behavior and shell properties are characterized by microbubble spectroscopy using the Brandaris 128 high-speed camera. DPPC microbubbles were found to be acoustically less stable. Resonance frequencies of both types were similar and both did not show compression- or expansion-only behavior. DPPC showed more nonlinear behavior, particularly more subharmonics; 10 out of 14 versus 4 out of 15 for DSPC. Moreover, the emitted acoustic pressures at the subharmonic frequency were higher for DPPC and measurable up to 2 cm from the bubble. Second harmonic behavior was similar for both types and present in ~70% of the bubbles, with pressures ~1 Pa. Shell elasticities were 0.17 (± 0.06) N/m for DSPC and 0.06 (± 0.08) N/m for DPPC. Shell viscosities increased with diameter and showed no relation with dilatation rates. Mean shell viscosities were equal at 1.3 · 10-8 kg/s. The presence of subharmonics and the higher emitted pressures make DPPC microbubbles more useful for contrast-enhanced ultrasound imaging, e.g. carotid imaging.

Influence of binding on the vibrational responses of targeted lipid-coated microbubbles

Microbubbles for molecular ultrasound imaging are targeted to disease-specific biomarkers. One of the main challenges is to acoustically distinguish free microbubbles from those that are bound to their molecular target. In this study we used the Brandaris 128 high-speed camera to compare the responses to ultrasound of two types of lipid-coated microbubbles (DSPC and DPPC) in their free and bound state aiming to acoustically discriminate them. We found larger shrinkage for DPPC than for DSPC microbubbles in both their free and bound condition, indicating higher acoustical stability for DSPC-based microbubbles. Upon binding the stability of both types did not change significantly. Frequencies of maximum response were similar for DSPC and DPPC microbubbles, whether they were free or bound. In their free state, more DPPC than DSPC microbubbles responded at the subharmonic and the second harmonic frequency, which did not change upon binding. Interestingly, the maximum relative radial excursions at the second harmonic frequency were higher for DSPC microbubbles when they had bound. In conclusion, bound DSPC and DPPC microbubbles had lower maximum relative radial excursions at the fundamental frequency, and had higher maximum relative radial excursions at the second harmonic frequency than the free microbubbles. We found no differences between bound DSPC and bound DPPC microbubbles. The higher maximum relative radial excursions of particularly bound DSPC microbubbles at the second harmonic frequency may provide opportunities to acoustically discriminate them from free microbubbles.

Acoustic Characterization of a Vessel-on-a-Chip Microfluidic System for Ultrasound-Mediated Drug Delivery

Ultrasound in the presence of gas-filled microbubbles can be used to enhance local uptake of drugs and genes. To study the drug delivery potential and its underlying physical and biological mechanisms, an in vitro vessel model should ideally include 3-D cell culture, perfusion flow, and membrane-free soft boundaries. Here, we propose an organ-on-a-chip microfluidic platform to study ultrasound-mediated drug delivery: the OrganoPlate. The acoustic propagation into the OrganoPlate was determined to assess the feasibility of controlled microbubble actuation, which is required to study the microbubble-cell interaction for drug delivery. The pressure field in the OrganoPlate was characterized non-invasively by studying experimentally the well-known response of microbubbles and by simulating the acoustic wave propagation in the system. Microbubble dynamics in the OrganoPlate were recorded with the Brandaris 128 ultrahigh-speed camera (17 million frames/s) and a control experiment was performed in an OptiCell, an in vitro monolayer cell culture chamber that is conventionally used to study ultrasound-mediated drug delivery. When insonified at frequencies between 1 and 2 MHz, microbubbles in the OrganoPlate experienced larger oscillation amplitudes resulting from higher local pressures. Microbubbles responded similarly in both systems when insonified at frequencies between 2 and 4 MHz. Numerical simulations performed with a 3-D finite-element model of ultrasound propagation into the OrganoPlate and the OptiCell showed the same frequency-dependent behavior. The predictable and homogeneous pressure field in the OrganoPlate demonstrates its potential to develop an in vitro 3-D cell culture model, well suited to study ultrasound-mediated drug delivery.

Focal areas of increased lipid concentration on the coating of microbubbles during short tone-burst ultrasound insonification

Acoustic behavior of lipid-coated microbubbles has been widely studied, which has led to several numerical microbubble dynamics models that incorporate lipid coating behavior, such as buckling and rupture. In this study we investigated the relationship between microbubble acoustic and lipid coating behavior on a nanosecond scale by using fluorescently labeled lipids. It is hypothesized that a local increased concentration of lipids, appearing as a focal area of increased fluorescence intensity (hot spot) in the fluorescence image, is related to buckling and folding of the lipid layer thereby highly influencing the microbubble acoustic behavior. To test this hypothesis, the lipid microbubble coating was fluorescently labeled. The vibration of the microbubble (n = 177; 2.3–10.3 μm in diameter) upon insonification at an ultrasound frequency of 0.5 or 1 MHz at 25 or 50 kPa acoustic pressure was recorded with the UPMC Cam, an ultra-high-speed fluorescence camera, operated at ~4–5 million frames per second. During short tone-burst excitation, hot spots on the microbubble coating occurred at relative vibration amplitudes > 0.3 irrespective of frequency and acoustic pressure. Around resonance, the majority of the microbubbles formed hot spots. When the microbubble also deflated acoustically, hot spot formation was likely irreversible. Although compression-only behavior (defined as substantially more microbubble compression than expansion) and subharmonic responses were observed in those microbubbles that formed hot spots, both phenomena were also found in microbubbles that did not form hot spots during insonification. In conclusion, this study reveals hot spot formation of the lipid monolayer in the microbubble’s compression phase. However, our experimental results show that there is no direct relationship between hot spot formation of the lipid coating and microbubble acoustic behaviors such as compression-only and the generation of a subharmonic response. Hence, our hypothesis that hot spots are related to acoustic buckling could not be verified.

Response to ultrasound of two types of lipid-coated microbubbles observed with a high-speed optical camera

Microbubbles (MBs) can be coated with different lipids, but exact influences on acoustical responses remain unclear. The distribution of lipids in the coating of homemade MBs is heterogeneous for DSPC and homogeneous for DPPC-based MBs, as observed with 4Pi confocal microscopy. In this study, we investigated whether DSPC and DPPC MBs show a different vibrational response to ultrasound. MBs composed of main lipid DSPC or DPPC (2 C-atoms less) with a C4F10 gas core, were made by sonication. Microbubble spectroscopy was performed by exciting single MBs with 10-cycle sine wave bursts having a frequency from 1 to 4 MHz and a peak negative pressure of 10, 20, and 50 kPa. The vibrational response to ultrasound was recorded with the Brandaris 128 high-speed camera at 15 Mfps. Larger acoustically induced deflation was observed for DPPC MBs. For a given resting diameter, the resonance frequency was higher for DSPC, resulting in higher shell elasticity of 0.26 N/m as compared to 0.06 N/m for DPPC MBs. Shell viscosity was similar (~10-8 kg/s) for both MB types. Non-linear behavior was characterized by the response at the subharmonic and second harmonic frequencies. More DPPC (71%) than DSPC MBs (27%) showed subharmonic response, while the behavior at the second harmonic frequency was comparable. The different acoustic responses of DSPC and DPPC MBs are likely due to the choice of the main lipid and the corresponding spatial distribution in the MB coating.