Encapsulated Microbubbles Research Articles

A multifunctional theranostic contrast agent for ultrasound/near infrared fluorescence imaging-based tumor diagnosis and ultrasound-triggered combined photothermal and gene therapy

PurposeEncapsulated microbubbles (MBs) have been reported as new theranostic carriers for simultaneous imaging and ultrasound (US)-triggered therapy. Here, we designed a dual-modality US/NIRF contrast agent and extended its applications from image contrast enhancement to combined diagnosis and therapy with US-directed and site-specific targeting. MethodsGold nanorods (AuNRs) resonant at 880 nm together with the NIR797 dye were first encapsulated in lipid-shelled MBs to construct fluorescent gold microbubbles (NIR797/AuMBs) via thin film hydration and mechanical shaking in the presence of sulfur hexafluoride (SF6) gas. Then, polyethylenimine (PEI)-DNA complexes were electrostatically conjugated onto the surface of the NIR797/AuMBs, forming theranostic encapsulated MBs (PEI-DNA/NIR797/AuMBs). The potential of the PEI-DNA/NIR797/AuMBs for use as a dual-modality contrast enhancement agent was evaluated in vitro and in vivo. The antitumor effect of US/NIR laser irradiation mediating double-fusion suicide gene and photothermal therapy was also investigated using Bel-7402 cells and xenografts. ResultsThe developed theranostic AuMB complexes could not only provide excellent US and NIRF imaging to detect tumors but also serve as an efficient US-triggered carrier for gene delivery and photothermal ablation of tumors in xenografted nude mice. And US + laser exposure group showed a much higher rate of cell inhibition, apoptosis and necrosis as well as a higher Bel-7402 xenograft inhibition rate than the single gene therapy or single exposure (US or laser) group. ConclusionsPEI-DNA/NIR797/AuMBs would be of great value for providing more comprehensive diagnostic information and to guide more accurate and effective synergistic cancer therapy. Statement of SignificanceThis is an original paper focusing on developing a dual-modality US/NIRF contrast agent and extended its applications from image contrast enhancement to combined diagnosis and therapy with US-directed and site-specific targeting. The developed theranostic AuMB complexes could not only provide excellent US and NIRF imaging to detect tumors but also serve as an efficient US-triggered carrier for gene delivery and photothermal ablation of tumors in xenografted nude mice. PEI-DNA/NIR797/AuMBs would be of great value for providing more comprehensive diagnostic information and to guide more accurate and effective synergistic cancer therapy.

Deep Unfolded Robust PCA With Application to Clutter Suppression in Ultrasound.

Contrast enhanced ultrasound is a radiation-free imaging modality which uses encapsulated gas microbubbles for improved visualization of the vascular bed deep within the tissue. It has recently been used to enable imaging with unprecedented subwavelength spatial resolution by relying on super-resolution techniques. A typical preprocessing step in super-resolution ultrasound is to separate the microbubble signal from the cluttering tissue signal. This step has a crucial impact on the final image quality. Here, we propose a new approach to clutter removal based on robust principle component analysis (PCA) and deep learning. We begin by modeling the acquired contrast enhanced ultrasound signal as a combination of low rank and sparse components. This model is used in robust PCA and was previously suggested in the context of ultrasound Doppler processing and dynamic magnetic resonance imaging. We then illustrate that an iterative algorithm based on this model exhibits improved separation of microbubble signal from the tissue signal over commonly practiced methods. Next, we apply the concept of deep unfolding to suggest a deep network architecture tailored to our clutter filtering problem which exhibits improved convergence speed and accuracy with respect to its iterative counterpart. We compare the performance of the suggested deep network on both simulations and in-vivo rat brain scans, with a commonly practiced deep-network architecture and with the fast iterative shrinkage algorithm. We show that our architecture exhibits better image quality and contrast.

Enhanced microbubble contrast agent oscillation following 250\u2009kHz insonation

Microbubble contrast agents are widely used in ultrasound imaging and therapy, typically with transmission center frequencies in the MHz range. Currently, an ultrasound center frequency near 250 kHz is proposed for clinical trials in which ultrasound combined with microbubble contrast agents is applied to open the blood brain barrier, since at this low frequency focusing through the human skull to a predetermined location can be performed with reduced distortion and attenuation compared to higher frequencies. However, the microbubble vibrational response has not yet been carefully evaluated at this low frequency (an order of magnitude below the resonance frequency of these contrast agents). In the past, it was assumed that encapsulated microbubble expansion is maximized near the resonance frequency and monotonically decreases with decreasing frequency. Our results indicated that microbubble expansion was enhanced for 250 kHz transmission as compared with the 1 MHz center frequency. Following 250 kHz insonation, microbubble expansion increased nonlinearly with increasing ultrasonic pressure, and was accurately predicted by either the modified Rayleigh–Plesset equation for a clean bubble or the Marmottant model of a lipid-shelled microbubble. The expansion ratio reached 30-fold with 250 kHz at a peak negative pressure of 400 kPa, as compared to a measured expansion ratio of 1.6 fold for 1 MHz transmission at a similar peak negative pressure. Further, the range of peak negative pressure yielding stable cavitation in vitro was narrow (~100 kPa) for the 250 kHz transmission frequency. Blood brain barrier opening using in vivo transcranial ultrasound in mice followed the same trend as the in vitro experiments, and the pressure range for safe and effective treatment was 75–150 kPa. For pressures above 150 kPa, inertial cavitation and hemorrhage occurred. Therefore, we conclude that (1) at this low frequency, and for the large oscillations, lipid-shelled microbubbles can be approximately modeled as clean gas microbubbles and (2) the development of safe and successful protocols for therapeutic delivery to the brain utilizing 250 kHz or a similar center frequency requires consideration of the narrow pressure window between stable and inertial cavitation.

Shape oscillation and stability of an encapsulated microbubble translating in an acoustic wave.

Encapsulated microbubbles (EMBs) are associated with a wide variety of important medical applications, including sonography, drug delivery, and sonoporation. The nonspherical oscillations, or shape modes, of EMBs strongly affect their stability and acoustic signature, and thus are an important factor to consider in the design and utilization of EMBs. Under acoustic forcing, EMBs often translate with significant velocity, which can excite shape modes, yet few studies have addressed the effect of translation on the shape stability of EMBs. In this work, the shape stability of an EMB subject to translation is investigated through development of an axisymmetric model for the case of small deformations. The potential flow in the bulk volume of the external flow is modeled using an asymptotic analysis. Viscous effects within the thin boundary layer at the interface are included, owing to the no-slip boundary condition, using Prosperetti's theory [Q. Appl. Math. 34, 339 (1977)]. In-plane stress and bending moment due to the encapsulation are incorporated into the model through the dynamic boundary condition at the interface. The evolution equations for radial oscillation, translation, and shape oscillation of an EMB are derived, which can be reduced to model an uncoated gas bubble by neglecting the encapsulation properties. These equations are solved numerically to analyze the shape mode stability of an EMB and a gas bubble subject to an acoustic, traveling plane wave. The findings demonstrate the counterintuitive result that translation has a more destabilizing effect on an EMB than on a gas bubble. The no-slip condition at the encapsulating membrane is the main factor responsible for mediating this interfacial instability due to translation.

Shape stability of an encapsulated microbubble undergoing translational motion

Encapsulated microbubbles (EMBs) are associated with a variety of important diagnostic and therapeutic medical applications, including sonography, drug delivery, and sonoporation. The nonspherical oscillations, or shape modes, of EMBs strongly affect their stability and acoustic signature, and thus are an important factor in the design and utilization of EMBs. Under acoustic forcing, EMBs often translate with significant velocity, which can excite shape modes, yet few studies have addressed the effect of translation on the shape stability of EMBs. To investigate this phenomenon, an axisymmetric model is developed for the case of small shape oscillations. The exterior fluid is modeled as potential flow using an asymptotic analysis. Viscous effects within the thin boundary layer at the interface are included, owing to the no-slip boundary condition. In-plane stress and bending moment due to the encapsulation are incorporated into the model through the dynamic boundary condition at the interface. The evolution equations for radial oscillation, translation, and shape oscillation of an EMB are derived. These equations are solved numerically to analyze the shape mode stability of an EMB and a gas bubble subject to an acoustic, traveling plane wave. The findings demonstrate the counterintuitive result that translation more readily destabilizes an EMB than an uncoated gas bubble. The main factor responsible for mediating this translation-induced interfacial instability is the no-slip condition at the encapsulating membrane.

Interaction between encapsulated microbubbles: A finite element modelling study**Projects supported by the National Natural Science Foundation of China (Grant Nos. 11474161, 11474001, 116741731, 1774166, 11774168, 81527803, 81627802, and 81420108018), the Fundamental Research Funds for the Central Universities, China (Grant No. 020414380109, and the Qing Lan Project, China.

Theoretical studies on the multi-bubble interaction are crucial for the in-depth understanding of the mechanism behind the applications of ultrasound contrast agents (UCAs) in clinics. A two-dimensional (2D) axisymmetric finite element model (FEM) is developed here to investigate the bubble–bubble interactions for UCAs in a fluidic environment. The effect of the driving frequency and the bubble size on the bubble interaction tendency (viz., bubbles’ attraction and repulsion), as well as the influences of bubble shell mechanical parameters (viz., surface tension coefficient and viscosity coefficient) are discussed. Based on FEM simulations, the temporal evolution of the bubbles’ radii, the bubble–bubble distance, and the distribution of the velocity field in the surrounding fluid are investigated in detail. The results suggest that for the interacting bubble–bubble couple, the overall translational tendency should be determined by the relationship between the driving frequency and their resonance frequencies. When the driving frequency falls between the resonance frequencies of two bubbles with different sizes, they will repel each other, otherwise they will attract each other. For constant acoustic driving parameters used in this paper, the changing rate of the bubble radius decreases as the viscosity coefficient increases, and increases first then decreases as the bubble shell surface tension coefficient increases, which means that the strength of bubble–bubble interaction could be adjusted by changing the bubble shell visco-elasticity coefficients. The current work should provide a powerful explanation for the accumulation observations in an experiment, and provide a fundamental theoretical support for the applications of UCAs in clinics.

Numerical analyses of nonlinear behavior of microbubble contrast agents in ultrasound field and effective parameters.

This research aims to investigate the effects of some parameters on the nonlinear dynamics of an encapsulated microbubble. In the present study, the radial behavior of an encapsulated microbubble with modified Rayleigh-Plesset equation presented by Marmottant, van der Meer, Emmer, Versluis, de Jong, Hilgenfeldt, and Lohse [J. Acoust. Soc. Am. 118(6), 3499-3505 (2005)] is solved with matlab programming software. Radial dynamics of an individual microbubble for a free and coated state and effects of the added shell are also investigated. Results show that dilatational interfacial viscosity ( ks) of the shell and changes of surface tension have significant effects on the amplitude of oscillations, bubble dynamics, and bubble surface velocity. Regarding the fact that the velocity of the bubble surface has a significant role in stability and collapse of the bubble, it can be noted that this velocity is reduced considerably by adding the shell. Because of extensive usage of nonlinear microbubble oscillations in medical imaging techniques, the nonlinear response of a lipid-coated contrast agent called "compression-only" behavior is studied and effective parameters on this behavior are investigated. The results of bubble radius excursion reveal the fact that excitation frequency, pressure, and dilatational interfacial viscosity of the bubble affect the compression-only behavior.

A new expression for the second-order acoustical nonlinearity parameter B/A for a suspension of free or encapsulated bubbles

The presence of bubbles in the liquid introduces dispersion, increased attenuation and nonlinearities in the medium (bubbly liquid). The second-order acoustical parameter B/A describes the nonlinearity of the bubbly liquid. The nonlinear parameter B/A is an important property for characterizing different media and materials, and also important for ultrasound imaging. It can be derived from the equation of state of a fluid using the Taylor expansion to the second order. B/A has been studied extensively in industrial, chemical and biological fluids since Beyer proposed this thermodynamic technique. However, certain important aspect for this criterion remains unexplored specifically for bubbly liquid. Here, we develop a new formula based on the thermodynamic method which correlates both attenuation and phase velocity of ultrasound waves in liquids containing free or encapsulated microbubbles. These quantities can be measured directly using a broadband technique. The formula offered here is relatively simple and can be used to directly measure B/A from experiments; the new formula avoids using second harmonics that requires higher excitation pressures, as well as the direct thermodynamic method that gives rise to inaccuracy.

Nonlinear oscillation and interfacial stability of an encapsulated microbubble under dual-frequency ultrasound

Encapsulated microbubbles (EMBs) are widely used in medical ultrasound imaging as contrast-enhanced agents. However, the potential damaging effects of violent collapsing EMBs to cells and tissues in clinical settings have remained a concern. Dual-frequency ultrasound is a promising technique for improving the efficacy and safety of sonography. The system modeled consists of the external liquid, membrane and internal gases of an EMB. The microbubble dynamics are simulated using a simple nonlinear interactive theory, considering the compressibility of the internal gas, viscosity of the liquid flow and viscoelasticity of the membrane. The radial oscillation and interfacial stability of an EMB under single- and dual-frequency excitations are compared. The simulation results show that the dual-frequency technique produces larger backscatter pressure at higher harmonics of the primary driving frequency—this enriched acoustic spectrum can enhance blood-tissue contrast and improve the quality of sonographic images. The results further show that the acoustic pressure threshold associated with the onset of shape instability is greater for dual-frequency driving. This suggests that the dual-frequency technique stabilizes the encapsulated bubble, thereby improving the efficacy and safety of contrast-enhanced agents.

Coating microbubbles with nanoparticles for medical imaging and drug delivery.

Coating microbubbles with nanoparticles for medical imaging and drug delivery.

Modeling and parametric investigation of thick encapsulated microbubble's nonspherical oscillations.

Numerous studies have been carried out in the past few decades to investigate the radial oscillations of encapsulated microbubbles (MBs). Nonspherical oscillations also have gained attention, being unavoidable in actual applications of these bubbles. The present paper is intended to describe the nature of resonance trends of such spherical and nonspherical modes of a thick encapsulated MB filled with air and suspended in water. The shell material is assumed to be linear viscoelastic and quasi-incompressible. The considered isotropic and spherically isotropic material parametric range is limited to thick polymer shelled MBs. For the case of an isotropic material, shell viscosity has a major influence on the fundamental modes with meridional wave number n = 0, 4, especially for thicker bubbles, unlike for the case of the spherically isotropic material case considered, where the viscosity has very little influence. For most of the parametric range, n = 2, 3 modes are underdamped and their frequency is found to be lower than the n = 0, 4 modes, for both material cases. An interesting case is found for a spherically isotropic quasi-incompressible material case, where the first few nonspherical mode resonances are very close to radial mode resonance frequency.

Stability and natural frequency of nonspherical mode of an encapsulated microbubble in a viscous liquid

The dynamics of encapsulated microbubbles (EMBs) subject to an ultrasound wave have wide and important medical applications, including sonography, drug delivery, and sonoporation. The nonspherical shape oscillation of an EMB, termed as shape modes, is one of the core mechanisms of these applications and therefore its natural frequency is a fundamentally important parameter. Based on the linear stability theory, we show that shape modes of an EMB in a viscous Newtonian liquid are stable. We derive an explicit expression for the natural frequency of shape modes, in terms of the equilibrium radius of an EMB, and the parameters of the external liquid, coating, and internal gases. The expression is validated by comparing to the numerical results obtained from the dynamic equations of shape modes of an EMB. The natural frequency of shape modes shifts appreciably due to the viscosity of the liquid, and this trend increases with the mode number. The significant viscous effects are due to the no-slip condition for the liquid flow at the surface of an EMB. Our results show that when subject to an acoustic wave, the shape instability for an EMB is prone to appear if 2ωk/ωd = n, where ωk is the natural frequency of shape modes, ωd is the driving frequency of the acoustic wave, and n is a natural number. The effects of viscosity on the natural frequency is thus critical in setting the driving frequency of ultrasound to avoid or activate shape modes of EMBs, which should be considered in the applications of medical ultrasound.

Individual lipid encapsulated microbubble radial oscillations: Effects of fluid viscosity.

Ultrasound-stimulated microbubble dynamics have been shown to be dependent on intrinsic bubble properties, including size and shell characteristics. The effect of the surrounding environment on microbubble response, however, has been less investigated. In particular, microbubble optimization studies are generally conducted in water/saline, characterized by a 1 cP viscosity, for application in the vasculature (i.e., 4 cP). In this study, ultra-high speed microscopy was employed to investigate fluid viscosity effects on phospholipid encapsulated microbubble oscillations at 1 MHz, using a single, eight-cycle pulse at peak negative pressures of 100 and 250 kPa. Microbubble oscillations were shown to be affected by fluid viscosity in a size- and pressure-dependent manner. In general, the oscillation amplitudes exhibited by microbubbles between 3 and 6 μm in 1 cP fluid were larger than in 4 cP fluid, reaching a maximum of 1.7-fold at 100 kPa for microbubbles 3.8 μm in diameter and 1.35-fold at 250 kPa for microbubbles 4.8 μm in diameter. Simulation results were in broad agreement at 250 kPa, however generally underestimated the effect of fluid viscosity at 100 kPa. This is the first experimental demonstration documenting the effects of surrounding fluid viscosity on microbubble oscillations, resulting in behavior not entirely predicted by current microbubble models.

Reparable Cell Sonoporation in Suspension: Theranostic Potential of Microbubble.

The conjunction of low intensity ultrasound and encapsulated microbubbles can alter the permeability of cell membrane, offering a promising theranostic technique for non-invasive gene/drug delivery. Despite its great potential, the biophysical mechanisms of the delivery at the cellular level remains poorly understood. Here, the first direct high-speed micro-photographic images of human lymphoma cell and microbubble interaction dynamics are provided in a completely free suspension environment without any boundary parameter defect. Our real-time images and theoretical analyses prove that the negative divergence side of the microbubble's dipole microstreaming locally pulls the cell membrane, causing transient local protrusion of 2.5 µm in the cell membrane. The linear oscillation of microbubble caused microstreaming well below the inertial cavitation threshold, and imposed 35.3 Pa shear stress on the membrane, promoting an area strain of 0.12%, less than the membrane critical areal strain to cause cell rupture. Positive transfected cells with pEGFP-N1 confirm that the interaction causes membrane poration without cell disruption. The results show that the overstretched cell membrane causes reparable submicron pore formation, providing primary evidence of low amplitude (0.12 MPa at 0.834 MHz) ultrasound sonoporation mechanism.

Low Intensity Ultrasound Mediated Liposomal Doxorubicin Delivery Using Polymer Microbubbles.

Cardiotoxicity is the major dose-limiting factor in the chemotherapeutic use of doxorubicin (Dox). A delivery vehicle that can be triggered to release its payload in the tumoral microvasculature but not in healthy tissue would help improve the therapeutic window of the drug. Delivery strategies combining liposomal encapsulated Dox (LDox), microbubbles (MBs), and ultrasound (US) have been shown to improve therapeutic efficacy of LDox, but much remains to be known about the mechanisms and the US conditions that maximize cytotoxicity using this approach. In this study, we compared different US pulses in terms of drug release and acute toxicity. Drug uptake and proliferation rates using low-intensity US were measured in squamous cell carcinoma cells exposed to LDox conjugated to or coinjected with polymer MBs. The aims of this study were: (1) to compare the effects of low- and high-pressure US on Dox release kinetics; (2) to evaluate whether conjugating the liposome to the MB surface (DoxLPX) is an important factor for drug release and cytotoxicity; and (3) to determine which US parameters most inhibit cell proliferation and whether this inhibition is mediated by drug release or the MB/US interaction with cells. Low-pressure US (170 kPa) at high duty cycle (stable cavitation) released up to ∼ 70% of the encapsulated Dox from the DoxLPX, thus improving Dox bioavailability and cellular uptake and leading to a significant reduction in cell proliferation at 48 h. Flow cytometry showed that US generating stable oscillations of DoxLPX significantly increased cellular Dox uptake at 4 h after US exposure compared to LDox. Drug uptake was correlated with cytotoxicity at 48 h. Our results demonstrate that Dox-containing liposomes conjugated to polymer MBs can be triggered to release ∼ 70% of their payload using noninertial US. Following release, Dox became bioavailable to the cells and induced significantly higher cytotoxicity compared to nonreleased encapsulated drug. Our findings show promise for targeted drug delivery using this theranostic delivery platform at low US intensities.

The role of encapsulated microbubbles in the diagnosis of stenosis in arteries

Numerical modelling of sound scattered by ultrasound contrast agent bubbles in arteries is presented. Nonlinear dynamics of an encapsulated microbubble coupled with transmission of sound through liquid-filled flexible tubing is developed. Based on the frequency response, a normal artery can be distinguished from that of a stenosed artery. Effects of parameters like incident pressure amplitude, driving frequency and degree of stenosis are studied. It is further found that the effect of variable pressure produced by blood flow does not have a significant effect on the observed sound pressure levels.

Microflow-induced shear stress on biomaterial wall by ultrasound-induced encapsulated microbubble oscillation**Projects supported by the National Natural Science Foundation of China (Grant Nos. 11174077 and 11474090), the Natural Science Foundation of Hunan Province, China (Grant No. 13JJ3076), the Science Research Program of Education Department of Hunan Province, China (Grant No. 14A127), and the Doctoral Fund of University of South China (Grant

A model of an ultrasound-driven encapsulated microbubble (EMB) oscillation near biomaterial wall is presented and used for describing the microflow-induced shear stress on the wall by means of a numerical method. The characteristic of the model lies in the explicit treatment of different types of wall for the EMB responses. The simulation results show that the radius-time change trends obtained by our model are consistent with the existing models and experimental results. In addition, the effect of the elastic wall on the acoustic EMB response is stronger than that of the rigid wall, and the shear stress on the elastic wall is larger than that of the rigid wall. The closer the EMB to the wall, the greater the shear stress on the wall. The substantial shear stress on the wall surface occurs inside a circular zone with a radius about two-thirds of the bubble radius. This paper may be of interest in the study of potential damage mechanisms to the microvessel for drug and gene delivery due to sonoporation.

Microstreaming velocity field and shear stress created by an oscillating encapsulated microbubble near a cell membrane

Sonoporation mediated by microbubbles is being extensively studied as a promising technology to facilitate gene/drug delivery to cells. However, the theoretical study regarding the mechanisms involved in sonoporation is still in its infancy. Microstreaming generated by pulsating microbubble near the cell membrane is regarded as one of the most important mechanisms in the sonoporation process. Here, based on an encapsulated microbubble dynamic model with considering nonlinear rheological effects of both shell elasticity and viscosity, the microstreaming velocity field and shear stress generated by an oscillating microbubble near the cell membrane are theoretically simulated. Some factors that might affect the behaviors of microstreaming are thoroughly investigated, including the distance between the bubble center and cell membrane (d), shell elasticity (χ), and shell viscosity (κ). The results show that (i) the presence of cell membrane can result in asymmetric microstreaming velocity field, while the constrained effect of the membrane wall decays with increasing the bubble-cell distance; (ii) the bubble resonance frequency increases with the increase in d and χ, and the decrease in κ, although it is more dominated by the variation of shell elasticity; and (iii) the maximal microstreaming shear stress on the cell membrane increases rapidly with reducing the d, χ, and κ. The results suggest that microbubbles with softer and less viscous shell materials might be preferred to achieve more efficient sonoporation outcomes, and it is better to have bubbles located in the immediate vicinity of the cell membrane.

Intelligent controlling microbubble radial oscillations by using Slave–Master Feedback control

Dynamics of acoustically driven microbubbles in ultrasonic fields are known to be complex and uncontrollable phenomena indicative of a highly active nonlinear as well as chaotic behavior. In this paper, a method based on Slave–Master Feedback (SMF) to suppress unstable radial oscillations of contrast agents is presented. In the proposed control process, the encapsulated microbubbles as the slave system is coupled with a dynamical system as the master, so that the output of the coupled system is able to produce a stable oscillation. A great virtue of this control technique is its flexibility. In comparison with existing techniques, the present dynamical chaos control method does not need to know more than one variable. The numerical results show its strong impact on reducing the chaotic oscillations to regular ones.

Localized high pressure near an aspheric encapsulated microbubble

Based on hydrodynamics, the pressure of the liquid outside an aspheric encapsulated bubble driven by ultrasound is studied, and its analytical expression is derived. Numerical simulation shows that 1) the aspheric shape of an encapsulated bubble makes little influence on the pressure of the liquid far away from the bubble; 2) the pressure is extremely high at some local places of the liquid near an aspheric encapsulated bubble, and the pressure values at these places are apparently larger than those for a spherical encapsulated bubble at the same conditions. This phenomenon is of significance in the applications such as high intensity ultrasound therapy, drug delivery, cell membrane perforation, etc. As the ultrasound frequency shifts to the resonance frequency of an encapsulated bubble, or bubble shape deviates from sphericity, the localized high pressure becomes even greater.