Guillaume Lajoinie

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.

Ultrafast vapourization dynamics of laser-activated polymeric microcapsules

Precision control of vapourization, both in space and time, has many potential applications; however, the physical mechanisms underlying controlled boiling are not well understood. The reason is the combined microscopic length scales and ultrashort timescales associated with the initiation and subsequent dynamical behaviour of the vapour bubbles formed. Here we study the nanoseconds vapour bubble dynamics of laser-heated single oil-filled microcapsules using coupled optical and acoustic detection. Pulsed laser excitation leads to vapour formation and collapse, and a simple physical model captures the observed radial dynamics and resulting acoustic pressures. Continuous wave laser excitation leads to a sequence of vapourization/condensation cycles, the result of absorbing microcapsule fragments moving in and out of the laser beam. A model incorporating thermal diffusion from the capsule shell into the oil core and surrounding water reveals the mechanisms behind the onset of vapourization. Excellent agreement is observed between the modelled dynamics and experiment.

On the Acoustic Properties of Vaporized Submicron Perfluorocarbon Droplets

The acoustic characteristics of microbubbles created from vaporized submicron perfluorocarbon droplets with fluorosurfactant coating are examined. Utilizing ultra-high-speed optical imaging, the acoustic response of individual microbubbles to low-intensity diagnostic ultrasound was observed on clinically relevant time scales of hundreds of milliseconds after vaporization. It was found that the vaporized droplets oscillate non-linearly and exhibit a resonant bubble size shift and increased damping relative to uncoated gas bubbles due to the presence of coating material. Unlike the commercially available lipid-coated ultrasound contrast agents, which may exhibit compression-only behavior, vaporized droplets may exhibit expansion-dominated oscillations. It was further observed that the non-linearity of the acoustic response of the bubbles was comparable to that of SonoVue microbubbles. These results suggest that vaporized submicron perfluorocarbon droplets possess the acoustic characteristics necessary for their potential use as ultrasound contrast agents in clinical practice.

In vitro methods to study bubble-cell interactions: Fundamentals and therapeutic applications.

Besides their use as contrast agents for ultrasound imaging, microbubbles are increasingly studied for a wide range of therapeutic applications. In particular, their ability to enhance the uptake of drugs through the permeabilization of tissues and cell membranes shows great promise. In order to fully understand the numerous paths by which bubbles can interact with cells and the even larger number of possible biological responses from the cells, thorough and extensive work is necessary. In this review, we consider the range of experimental techniques implemented in in vitro studies with the aim of elucidating these microbubble-cell interactions. First of all, the variety of cell types and cell models available are discussed, emphasizing the need for more and more complex models replicating in vivo conditions together with experimental challenges associated with this increased complexity. Second, the different types of stabilized microbubbles and more recently developed droplets and particles are presented, followed by their acoustic or optical excitation methods. Finally, the techniques exploited to study the microbubble-cell interactions are reviewed. These techniques operate over a wide range of timescales, or even off-line, revealing particular aspects or subsequent effects of these interactions. Therefore, knowledge obtained from several techniques must be combined to elucidate the underlying processes.

Surface curvature in triply-periodic minimal surface architectures as a distinct design parameter in preparing advanced tissue engineering scaffolds

Reproduction of the anatomical structures and functions of tissues using cells and designed 3D scaffolds is an ongoing challenge. For this, scaffolds with appropriate biomorphic surfaces promoting cell attachment, proliferation and differentiation are needed. In this study, eight triply-periodic minimal surface (TPMS)-based scaffolds were designed using specific trigonometric equations, providing the same porosity and the same number of unit cells, while presenting different surface curvatures. The scaffolds were fabricated by stereolithography using a photocurable resin based on the biocompatible, biodegradable and rubber-like material, poly(trimethylene carbonate) (PTMC). A numerical approach was developed to calculate the surface curvature distributions of the TPMS architectures. Moreover, the scaffolds were characterized by scanning electron microscopy, micro-computed tomography and water permeability measurements. These original scaffold architectures will be helpful to decipher the biofunctional role of the surface curvature of scaffolds intended for tissue engineering applications.

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.

Laser-driven resonance of dye-doped oil-coated microbubbles: A theoretical and numerical study

Microbubbles are used to enhance the contrast in ultrasound imaging. When coated with an optically absorbing material, these bubbles can also provide contrast in photoacoustic imaging. This multimodal aspect is of pronounced interest to the field of medical imaging. The aim of this paper is to provide a theoretical framework to describe the physical phenomena underlying the photoacoustic response. This article presents a model for a spherical gas microbubble suspended in an aqueous environment and coated with an oil layer containing an optically absorbing dye. The model includes heat transfer between the gas core and the surrounding liquids. This framework is suitable for the investigation of both continuous wave and pulsed laser excitation. This work utilizes a combination of finite difference simulations and numerical integration to determine the dependancy on the physical properties, including composition and thickness of the oil layer on the microbubble response. A normalization scheme for a linearized version of the model was derived to facilitate comparison with experimental measurements. The results show that viscosity and thickness of the oil layer determine whether or not microbubble resonance can be excited. This work also examines the use of non-sinusoidal excitation to promote harmonic imaging techniques to further improve the imaging sensitivity.

Monodisperse Versus Polydisperse Ultrasound Contrast Agents: Non-Linear Response, Sensitivity, and Deep Tissue Imaging Potential

It has been proposed that monodisperse microbubble ultrasound contrast agents further increase the signal-to-noise ratio of contrast-enhanced ultrasound imaging. Here, the sensitivity of a polydisperse pre-clinical agent was compared experimentally with that of its size- and acoustically sorted derivatives by using narrowband pressure- and frequency-dependent scattering and attenuation measurements. The sorted monodisperse agents had up to a two-orders-of-magnitude increase in sensitivity, that is, in the average scattering cross section per bubble. Moreover, we found, for the first time, that the highly non-linear response of acoustically sorted microbubbles can be exploited to confine scattering and attenuation to the focal region of ultrasound fields used in clinical imaging. This property is a result of minimal pre-focal scattering and attenuation and can be used to minimize shadowing effects in deep tissue imaging. Moreover, it potentially allows for more localized therapy using microbubbles through the spatial control of resonant microbubble oscillations.

Laser-driven resonance of dye-doped oil-coated microbubbles: Experimental study

Photoacoustic (PA) imaging offers several attractive features as a biomedical imaging modality, including excellent spatial resolution and functional information such as tissue oxygenation. A key limitation, however, is the contrast to noise ratio that can be obtained from tissue depths greater than 1-2 mm. Microbubbles coated with an optically absorbing shell have been proposed as a possible contrast agent for PA imaging, offering greater signal amplification and improved biocompatibility compared to metallic nanoparticles. A theoretical description of the dynamics of a coated microbubble subject to laser irradiation has been developed previously. The aim of this study was to test the predictions of the model. Two different types of oil-coated microbubbles were fabricated and then exposed to both pulsed and continuous wave (CW) laser irradiation. Their response was characterized using ultra high-speed imaging. Although there was considerable variability across the population, good agreement was found between the experimental results and theoretical predictions in terms of the frequency and amplitude of microbubble oscillation following pulsed excitation. Under CW irradiation, highly nonlinear behavior was observed which may be of considerable interest for developing different PA imaging techniques with greatly improved contrast enhancement.

Nonlinear dynamics of single freely-floating microbubbles under prolonged insonation

The acoustic nonlinear responses of ultrasound contrast agent microbubbles (MBs) are of great interest for both diagnostic and therapeutic applications. Previously, optical and acoustical methods were developed to characterize single bubbles floating against a rigid wall. However, there is a need to develop an efficient approach for statistical measurement of single freely-floating MBs. Here we combine simultaneous optical sizing and sensitive acoustical characterization measurement to study quantitatively the nonlinear dynamics of single freely-floating bubbles under prolonged ultrasound exposure. The nonlinearity (ε2f, ε3f) and asymmetry (i.e., compression-dominant behavior) of bubble vibrations were found to increase with increasing oscillation amplitude, and reach the maximum for bubbles at resonance. Moreover, with the same fundamental response (εf), the second harmonic response (ε2f) of bubbles smaller than the resonance size is ~150% stronger than bubbles bigger than the resonance size. The data showed agreement with numerical simulations based on the shell-buckling model by Marmottant et al. The new system shows its great potential for in vitro characterization of contrast agent MB populations.