Original ContributionDynamics of Coated Microbubbles Adherent to a Wall
Introduction
The use of contrast agents in medical imaging with ultrasound is well established. The contrast agent is injected intravenously and is designed to enhance the contrast of the blood pool. The most common ultrasound contrast agent (UCA) is composed of a suspension of microbubbles (radius 0.5–5 μm), which are coated with a phospholipid, albumin or polymer shell. The coating reduces the surface tension σ and, therefore, the capillary pressure . Moreover, the coating increases the diffusive timescales and the combined effect prevents the bubble from quickly dissolving in the blood.
A promising application is noninvasive molecular imaging for selective diagnosis with ultrasound using contrast agent microbubbles. Targeting ligands are incorporated in the coating of ultrasound contrast agent microbubbles to selectively bind to biomarkers on the membrane of endothelial cells, which constitute the blood vessel wall. A series of challenges are encountered in the development of targeted microbubbles for molecular imaging applications.
The first question, as was stated by Lindner (2004), is whether bubbles adhering to a target cell produce strong enough acoustic signals. It was found that the response of adherent microbubbles is comparable to that of phospholipid-coated microbubbles (Zhao et al., 2006, Lankford et al., 2006). However, it remains to be seen if the concentration of adherent microbubbles in vivo will be high enough to produce signals in the order of normal contrast-enhanced ultrasound in perfusion imaging.
Another challenge that has received significant attention is the adhesion of the bubbles to the vessel wall under shear flow. Primary radiation force has been used to effectively push the bubbles towards the vessel (Dayton et al., 1999, Dayton et al., 2002, Takalkar et al., 2004, Rychak et al., 2007, Zhang et al., 2007, Doinikov et al., 2009). Engineering of the ligands has led to a method to increase the number of adherent microbubbles. The use of two distinct antibody-receptor pairs has been proposed (Eniola et al. 2003), as well as the use of a polymeric version of the ligand to increase the ligand surface density (Klibanov et al., 2006, Klibanov, 2009) and the use of a longer spacer arm (Ham et al. 2009).
Finally, one should be able to distinguish adherent microbubbles from freely circulating ones (Lindner 2004). The simplest approach is to wash out all the freely circulating microbubbles and image the remaining bubbles. The disadvantage is that it takes 5 to 10 min before all freely circulating bubbles are cleared by the liver and spleen and that there is no new supply of bubbles. Therefore, it would be beneficial to distinguish acoustically between adherent and freely circulating microbubbles. Considerable changes between adherent and nonadherent microbubbles were found, such as a change in the spectral response (Zhao et al. 2006) and a decrease in the acoustic response of adherent microbubbles with respect to nonadherent ones (Lankford et al. 2006).
Garbin et al. (2007) showed for one and the same bubble that the close proximity of a wall decreased the radial amplitude of oscillation by 50%. In vivo the bubbles circulate freely in the blood vessel and their position with respect to the wall is unknown. A change in the dynamics of a bubble solely due to the proximity of the boundary is, therefore, not sufficient to differentiate between freely circulating bubbles and adherent bubbles. Consequently, it is important to understand the influence of adherence to a wall on the bubble dynamics. Furthermore, we would like to investigate under what conditions the response of adherent and freely circulating bubbles can be differentiated, to optimize them for pulse-echo techniques.
Here, we investigate the change in the dynamics of adherent microbubbles with respect to bubbles in the unbounded fluid. In the Methods section, we describe the set-up, experimental methods and the preparation of the bubbles. The influence of targeting ligands, the proximity of the wall and the adhesion to the wall on the frequency of maximum response and the amplitude of oscillation will be shown in the Results section. In the Discussion section, we discuss the results and conclude.
Section snippets
Set-up
An OptiCell chamber (Thermo Fisher Scientific, Waltham, MA, USA) containing the contrast agent was positioned in a water tank. The custom designed water tank held the illumination fiber and the ultrasound transducer (PA168; Precision Acoustics, Dorchester, United Kingdom). The acoustic driving pulse was generated by an arbitrary waveform generator (Tabor 8026; Tabor Electronics, Tel Hanan, Israel). The pulse consisted of a burst of 10 cycles where the first and last three cycles were tapered
Targeting ligands
Figure 4A shows the frequency of maximum response as a function of the maximum amplitude of oscillation . The results for a BG-6437 phospholipid-coated microbubble (blue triangles) are compared with a BG-6438 functionalized microbubble (red squares). The radius of both microbubbles is 2.0 μm and the applied pressure and frequency are scanned to cover the parameter space from to 45 kPa and from to 4 MHz. The bubbles are located away from the Opticell wall.
We observe a
Discussion
In all experiments, we operate the system in a regime of large amplitude of oscillation, A1. With , the frequency of maximum response is hardly affected by the initial properties of the coating (Overvelde 2010a) and only the influence of the neighboring wall and adherence to the wall is investigated. We observe no influence of the targeting ligands on the dynamics of bubbles away from the wall, hence, no influence of the preparation protocol is expected on the behavior of the bubbles.
Conclusions
In conclusion, we investigated the influence of adhesion of a functionalized bubble to a target membrane on its frequency of maximum response and amplitude of oscillation. The bubble dynamics in the unbounded fluid was found to be unchanged for bubbles containing targeting ligands compared with phospholipid-coated microbubbles alone. A comparison of the response of a functionalized bubble in the unbounded fluid with the response of an adherent bubble resulted in a decrease of over 50% of the
Acknowledgments
The authors thank Peter Frinking and Thierry Bettinger from Bracco Research S.A. (Geneva, Switzerland) for discussions and for supplying the contrast agents. The authors thank Nenad Gajovic-Eichelman of the Fraunhofer Institute for the anti-fluorescein antibody and FITC-labeled BSA and Klazina Kooiman for fruitful discussions, help and practical information on the targeting of microbubbles. This work was partly financed by TAMIRUT, a Specific Targeted Research (STReP) project supported by the 6
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2020, Ultrasound in Medicine and BiologyCitation Excerpt :It could be related to effects on the bubble because of the proximity of the more rigid boundary. When a bubble is in contact with a very soft boundary, the effect of the boundary on its acoustic response is relatively small (Doinikov et al. 2012; Helfield et al. 2014), compared with the very significant damping of oscillations and a reduction in natural frequency when a bubble is in contact with a rigid boundary (Garbin et al. 2007; Overvelde et al. 2011). However, the stiffness whereby bubble oscillations become significantly influenced is unclear.
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Present address: Department of Chemical and Biomolecular Engineering, University of Pennsylvania, 220 South 33rd Street, Philadelphia PA 19104, USA.