Responsive Microbubbles Research Articles

Explaining the correlation between subharmonic amplitude and ambient pressure for subharmonic-aided pressure estimation (SHAPE)

BackgroundSubharmonic aided pressure estimation (SHAPE) is an innovative non-invasive technique that leverages ultrasound subharmonic imaging to estimate pressure. This method exploits the “negative correlation between subharmonic amplitude and ambient pressure” observed in experimental settings. Despite extensive experimental validation and some promising results in clinical studies, the underlying mechanism of SHAPE remains incompletely understood. Although some studies have attempted to provide theoretical explanations, definitive conclusions have yet to be reached. In addition, theoretical investigations have mainly focused on the steady oscillation of bubbles under long pulse excitation, which contrasts with the short pulse excitation required for clinical SHAPE applications. An understanding of the SHAPE principle under short pulse excitation is needed. MethodsThe exponential elasticity model (EEM) was used to simulate Sonazoid bubbles, and a probe-to-probe acoustic propagation model was introduced to mimic a practical SHAPE scenario. The simulated acoustic signals in response to three-cycle sinusoidal pulse excitations were analyzed for spectral composition. The relationship between microbubble oscillation patterns and subharmonic characteristics was identified through detailed investigation. ResultsFor the excitation pulse of 2.5 MHz frequency and 350 kPa magnitude, bubbles larger than the resonance radius (2.29 μm) exhibited significant subharmonics in the magnitude spectrum, while bubbles smaller than the subharmonic resonance radius (3.85 μm) showed the activity of scattering subharmonic energy and the sensitivity to ambient pressure. The emergence of subharmonics when increasing excitation power was related to the increasing amplification of the bubble self-oscillation and the period-doubling features in the acoustically forced oscillation. The negative correlation between subharmonic amplitude and ambient pressure was attributed to the reduced self-oscillation caused by increasing ambient pressure and hence bubble size reduction. Microbubbles falling between 2 and 3 μm showed the desired subharmonic sensitivity to ambient pressure under the specified excitation conditions. ConclusionThe transient oscillatory behavior of microbubbles in response to short pulse excitation, characterized by a ringing down self-oscillation after the acoustic forcing effect has ceased, is crucial for understanding the subharmonic emergence and the observed negative correlation between subharmonic amplitude and ambient pressure. The proposed concepts of subharmonic resonance radius, subharmonic-significant bubbles, and subharmonic-active bubbles provide valuable insights into the diverse subharmonic behavior of microbubbles. The theoretical explanation of this negative correlation highlights the importance of using subharmonic-significant-and-active bubbles for SHAPE applications.

High-Speed Optical Characterization of Protein-and-Nanoparticle–Stabilized Microbubbles for Ultrasound-Triggered Drug Release

BackgroundUltrasound-triggered bubble-mediated local drug delivery has shown potential to increase therapeutic efficacy and reduce systemic side effects, by loading drugs into the microbubble shell and triggering delivery of the payload on demand using ultrasound. Understanding the behavior of the microbubbles in response to ultrasound is crucial for efficient and controlled release. MethodsIn this work, we characterize the response of microbubbles with a coating consisting of poly(2-ethyl-butyl cyanoacrylate) (PEBCA) nanoparticles and denatured casein. High-speed recordings are taken of single microbubbles, both in bright-field and fluorescence. ResultsThe nanoparticle-loaded microbubbles show resonance behavior, but with a large variation in response, demonstrating a substantial inter-bubble variation in mechanical shell properties. The probability of shell rupture and the probability of nanoparticle release were found to strongly depend on the microbubble size, and the most effective size was inversely proportional to the driving frequency. Both the rupture and release probabilities increased with increasing driving pressure amplitude. Rupture of the microbubble shell occurred after fewer cycles of ultrasound as the driving pressure amplitude or driving frequency was increased. ConclusionsThe results highlight the importance of careful selection of the driving frequency, driving pressure amplitude, and duration of the ultrasound to achieve the most efficient ultrasound-triggered shell rupture and nanoparticle release of protein-and-nanoparticle-stabilized microbubbles.

Targeted chemo-sonodynamic therapy treatment of breast tumours using ultrasound responsive microbubbles loaded with paclitaxel, doxorubicin and Rose Bengal

Mastectomy is a common surgical treatment used in the management of breast cancer but has associated physical and psychological consequences for the patient. Breast conservation surgery (BCS) is an alternative to mastectomy but is only possible when the tumour is of an appropriate size. Neo-adjuvant chemotherapy has been successfully used to downstage tumours and increase the number of patients eligible for BCS. However, the chemotherapies used in this approach are non-targeted and often result in significant side effects to the patient. In this manuscript, we evaluate the potential of ultrasound targeted microbubble destruction (UTMD) to deliver Rose Bengal-mediated sonodynamic therapy (SDT) in combination with paclitaxel (PTX) and doxorubicin (Dox) chemotherapy as a potential treatment for breast cancer. Efficacy of the combined treatment was determined in a three-dimensional (3D) spheroid model of human breast cancer and in a murine model of the disease bearing subcutaneous MCF-7 tumours. The results demonstrated a significant reduction in both the cell viability of spheroids and tumour volume following treatment with the drug loaded microbubbles and ultrasound compared to targets treated with the drug loaded microbubbles alone or a Cremophor EL suspension of PTX and Dox. In addition, the weight of animals that received the microbubble treatment was unchanged throughout the study while a reduction of 12.1% was observed for animals treated with a Cremophor suspension of PTX/Dox. These results suggest that UTMD-mediated chemo-sonodynamic therapy is an efficacious and well tolerated approach for the treatment of breast cancer.

Fast, Low-Frequency Plane-Wave Imaging for Ultrasound Contrast Imaging

Plane-wave ultrasound contrast imaging offers a faster, less destructive means for imaging microbubbles compared with traditional ultrasound imaging. Even though many of the most acoustically responsive microbubbles have resonant frequencies in the lower-megahertz range, higher frequencies (>3 MHz) have typically been employed to achieve high spatial resolution. In this work we implement and optimize low-frequency (1.5-4 MHz) plane-wave pulse inversion imaging on a commercial, phased-array imaging transducer in vitro and illustrate its use in vivo by imaging a mouse xenograft model. We found that the 1.8-MHz contrast signal was about four times that acquired at 3.1 MHz on matched probes and nine times greater than echoes received on a higher-frequency probe. Low-frequency imaging was also much more resilient to motion. In vivo, we could identify sub-millimeter vasculature inside a xenograft tumor model and easily assess microbubble half-life. Our results indicate that low-frequency imaging can provide better signal-to-noise because it generates stronger non-linear responses. Combined with high-speed plane-wave imaging, this method could open the door to super-resolution imaging at depth, while high power pulses could be used for image-guided therapeutics.

Interfacial Nanoprecipitation toward Stable and Responsive Microbubbles and Their Use as a Resuscitative Fluid

AbstractA new approach has been developed to prepare stable microbubbles (MBs) by interfacial nanoprecipitation of bioabsorbable polymers at air/liquid interfaces. This facile method offers robust control over the morphology and chemophysical properties of MBs by simple chemical modifications. This approach is amenable to large‐scale manufacturing, and is useful to develop functional MBs for advanced biomedical applications. To demonstrate this, a MB‐based intravenous oxygen carrier was created that undergoes pH‐triggered self‐elimination. Intravenous injection of previous MBs increased the risk of pulmonary vascular obstruction. However, we show, for the first time, that our current design is superior, as they 1) yielded no evidence of acute risks in rodents, and 2) improved the survival in a disease model of asphyxial cardiac arrest (from 0 to 100 %), a condition that affects more than 100 000 in‐hospital patients, and carries a mortality of about 90 %.

Interfacial Nanoprecipitation toward Stable and Responsive Microbubbles and Their Use as a Resuscitative Fluid.

A new approach has been developed to prepare stable microbubbles (MBs) by interfacial nanoprecipitation of bioabsorbable polymers at air/liquid interfaces. This facile method offers robust control over the morphology and chemophysical properties of MBs by simple chemical modifications. This approach is amenable to large-scale manufacturing, and is useful to develop functional MBs for advanced biomedical applications. To demonstrate this, a MB-based intravenous oxygen carrier was created that undergoes pH-triggered self-elimination. Intravenous injection of previous MBs increased the risk of pulmonary vascular obstruction. However, we show, for the first time, that our current design is superior, as they 1) yielded no evidence of acute risks in rodents, and 2) improved the survival in a disease model of asphyxial cardiac arrest (from 0 to 100 %), a condition that affects more than 100 000 in-hospital patients, and carries a mortality of about 90 %.

Inside Cover: Interfacial Nanoprecipitation toward Stable and Responsive Microbubbles and Their Use as a Resuscitative Fluid (Angew. Chem. Int. Ed. 5/2018)

Inside Cover: Interfacial Nanoprecipitation toward Stable and Responsive Microbubbles and Their Use as a Resuscitative Fluid (Angew. Chem. Int. Ed. 5/2018)

Magnetically responsive microbubbles as delivery vehicles for targeted sonodynamic and antimetabolite therapy of pancreatic cancer

Magnetically responsive microbubbles (MagMBs), consisting of an oxygen gas core and a phospholipid coating functionalised with Rose Bengal (RB) and/or 5-fluorouracil (5-FU), were assessed as a delivery vehicle for the targeted treatment of pancreatic cancer using combined antimetabolite and sonodynamic therapy (SDT). MagMBs delivering the combined 5-FU/SDT treatment produced a reduction in cell viability of over 50% when tested against a panel of four pancreatic cancer cell lines in vitro. Intravenous administration of the MagMBs to mice bearing orthotopic human xenograft BxPC-3 tumours yielded a 48.3% reduction in tumour volume relative to an untreated control group (p<0.05) when the tumour was exposed to both external magnetic and ultrasound fields during administration of the MagMBs. In contrast, application of an external ultrasound field alone resulted in a 27% reduction in tumour volume. In addition, activated caspase and BAX protein levels were both observed to be significantly elevated in tumours harvested from animals treated with the MagMBs in the presence of magnetic and ultrasonic fields when compared to expression of those proteins in tumours from either the control or ultrasound field only groups (p<0.05). These results suggest MagMBs have considerable potential as a platform to enable the targeted delivery of combined sonodynamic/antimetabolite therapy in pancreatic cancer.

Estimation of Viscoelastic Properties of Cells Using Acoustic Tweezing Cytometry.

Recently developed acoustic tweezing cytometry uses ultrasound-responsive targeted microbubbles for biomechanical stimulation of live cells at the subcellular level. The purpose of this research was to estimate the viscoelastic characteristics of cells from the displacements of cell-bound microbubbles in response to ultrasound pulses on acoustic tweezing cytometry. Microbubbles were bound to NIH/3T3 fibroblasts and ATDC5 cells through an integrin-cytoskeleton linkage. The evolution of microbubble behaviors under irradiation by ultrasound pulses was captured by a high-speed camera and tracked by a customized algorithm. The total damping constant, stiffness, and rigidity of the cells were estimated by fitting the measured temporal displacement profiles to a Kelvin-Voigt-based model. The mean maximum displacement of the microbubbles attached to NIH/3T3 fibroblasts was much greater than that for ATDC5 cells. The mean fitted damping constant and stiffness ± SD for ATDC5 cells were 28.16 ± 7.08 mg/s and 0.5041 ± 0.1381 mN/m, respectively, and the values for NIH/3T3 fibroblasts were 13.12 ± 4.23 mg/s and 0.2591 ± 0.0715 mN/m. The rigidity for ATDC5 cells was 331.46 ± 106.50 MPa, whereas that for NIH/3T3 fibroblasts was 117.92 ± 34.83 MPa. The Arg-Gly-Asp-integrin-cytoskeleton system of NIH/3T3 fibroblasts appears to be softer than that of ATDC5 cells. The rigidity of ATDC5 cells was significantly greater than that of NIH/3T3 fibroblasts at the 95% confidence level. This strategy provides a novel way to determine the viscoelastic properties of the live cells.

Understanding Acoustic Cavitation Initiation by Porous Nanoparticles: Toward Nanoscale Agents for Ultrasound Imaging and Therapy.

Ultrasound is widely applied in medical diagnosis and therapy due to its safety, high penetration depth, and low cost. In order to improve the contrast of sonographs and efficiency of the ultrasound therapy, echogenic gas bodies or droplets (with diameters from 200 nm to 10 µm) are often used, which are not very stable in the bloodstream and unable to penetrate into target tissues. Recently, it was demonstrated that nanobubbles stabilized by nanoparticles can nucleate ultrasound responsive microbubbles under reduced acoustic pressures, which is very promising for the development of nanoscale (<100 nm) ultrasound agents. However, there is still very little understanding about the effects of nanoparticle properties on the stabilization of nanobubbles and nucleation of acoustic cavitation by these nanobubbles. Here, a series of mesoporous silica nanoparticles with sizes around 100 nm but with different morphologies were synthesized to understand the effects of nanoparticle porosity, surface roughness, hydrophobicity, and hydrophilic surface modification on acoustic cavitation inception by porous nanoparticles. The chemical analyses of the nanoparticles showed that, while the nanoparticles were prepared using the same silica precursor (TEOS) and surfactant (CTAB), they revealed varying amounts of carbon impurities, hydroxyl content, and degrees of silica crosslinking. Carbon impurities or hydrophobic modification with methyl groups is found to be essential for nanobubble stabilization by mesoporous silica nanoparticles. The acoustic cavitation experiments in the presence of ethanol and/or bovine serum albumin (BSA) demonstrated that acoustic cavitation is predominantly nucleated by the nanobubbles stabilized at the nanoparticle surface not inside the mesopores. Finally, acoustic cavitation experiments with rough and smooth nanoparticles were suggested that a rough nanoparticle surface is needed to largely preserve surface nanobubbles after coating the surface with hydrophilic macromolecules, which is required for in vivo applications of nanoparticles.

Acoustic Mediated Drug Delivery System

Ultrasound contrast agents are highly echogenic micro bubbles with many unique properties. Micro bubbles can basically improve the sensitivity of conventional ultrasound imaging to the microcirculation. The resonance of micro bubbles in response to an incident ultrasound pulse results in nonlinear harmonic emission that serves as the signature of micro bubbles in micro bubble-specific imaging. Inertial cavitations and destruction of micro bubbles can produce a strong mechanical stress enhancing the permeability of the surrounding tissues, and can further increase the extravasations of drugs from the blood into the cytoplasm or interstitium. Stable cavitations by high-frequency ultrasound can also mildly increase tissue permeability without causing any damage even at a high acoustic pressure. It is cheap, widely available and portable. Using Doppler methods, flow information can be obtained easily and non-invasively. It is arguably the most physiological modality, able to image structure and function with less sedation than other modalities. This means that function is minimally disturbed, and multiple repeat studies or the effect of interventions can easily be assessed. Ultrasound is also unique in being both an imaging and therapeutic tool and its value in gene therapy has received much recent interest. Ultrasound biomicroscopy has been used for in utero imaging and can guide injection of virus and cells. Ultra high frequency ultrasound can be used to determine cell mechanical properties. The development of micro bubble contrast agents has opened many new opportunities, including new functional imaging methods, the ability to image capillary flow and the possibility of molecular targeting using labeled micro bubbles.

Enhanced delivery of microRNA mimics to cardiomyocytes using ultrasound responsive microbubbles reverses hypertrophy in an in-vitro model

Cardiovascular diseases (CVD) account for 36% of deaths in Europe and the United States. Gene therapy can act as a therapeutic modality for the treatment of CVD. The use of microRNA mimetics may be advantageous as they regulate important processes in health and pathology. A major hurdle for using miRNA therapies relates to site specific delivery and sufficient cellular uptake of material to achieve efficacy To assess the feasibility of ultrasound responsive microbubble mediated delivery of miR mimics to cardiomyocytes. Liposome/microbubble formulations were added to HL-1 cardiomyocytes in the presence/absence of ultrasound (US). Transfection efficacy and functionality was assessed using epifluorescent microscopy, flow cytometry and qRT-PCR. DNA Quantification post-ultrasound mediated transfection of HL-1s using microbubbles was quantified. The capability of miR-133 microbubble formulations to suppress hypertrophy were measured by quantifying changes in cell size. Ultrasound mediated microbubble formulations enhanced intracellular delivery of miR mimics in cardiomyocytes. Both complexed/encapsulated miR-microbubble formulations delivered functional miR mimics and showed no adverse effect on cardiomyocyte viability. Furthermore, ultrasound mediated microbubble transfection of miR-133 mimics reversed cardiomyocyte hypertrophy in an in-vitro model. This novel delivery method has the potential for further development as a targeted delivery strategy for miR therapeutics to the heart.

Enhanced delivery of microRNA mimics to cardiomyocytes using ultrasound responsive microbubbles reverses hypertrophy in an in-vitro model

BACKGROUND: Cardiovascular diseases (CVD) account for 36% of deaths in Europe and the United States. Gene therapy can act as a therapeutic modality for the treatment of CVD. The use of microRNA mim...

Binding dynamics of targeted microbubbles in response to modulated acoustic radiation force

Detection of molecular targeted microbubbles plays a foundational role in ultrasound-based molecular imaging and targeted gene or drug delivery. In this paper, an empirical model describing the binding dynamics of targeted microbubbles in response to modulated acoustic radiation forces in large vessels is presented and experimentally verified using tissue-mimicking flow phantoms. Higher flow velocity and microbubble concentration led to faster detaching rates for specifically bound microbubbles (p < 0.001). Higher time-averaged acoustic radiation force intensity led to faster attaching rates and a higher saturation level of specifically bound microbubbles (p < 0.05). The level of residual microbubble signal in targeted experiments after cessation of radiation forces was the only response parameter that was reliably different between targeted and control experiments (p < 0.05). A related parameter, the ratio of residual-to-saturated microbubble signal (Rresid), is proposed as a measurement that is independent of absolute acoustic signal magnitude and therefore able to reliably detect targeted adhesion independently of control measurements (p < 0.01). These findings suggest the possibility of enhanced detection of specifically bound microbubbles in real-time, using relatively short imaging protocols (approximately 3 min), without waiting for free microbubble clearance.

Single microbubbles vibrations measured with an “acoustical camera"

Measuring and understanding the nonlinear vibrations of contrast agent microbubbles in response to an ultrasound wave is essential to optimize the technique employed by an ultrasound scanner to distinguish microbubbles from tissue. In this context, an acoustical method was developed to retrieve the radial response of single microbubbles to a pressure wave by means of a low-amplitude probing wave. If the frequency of the latter is much higher than the spherical resonance frequency of the microbubble (typically between 1 and 10 MHz), the relative amplitude modulation (induced by a pressure wave) in the signal scattered in response to the probing wave is quasi-equal to the radial strain (i.e., relative variation in radius) induced by the pressure wave. A reference response to the probing wave acquired before and after the transmission of the pressure wave allows us to reveal asymmetry in microbubble oscillations. Although efficient nonlinear wave interaction is well known in bubbly liquids, we demonstrated that such an “acoustical camera” can extract quantitative information (the radius as function of time) on single bubble vibrations by analyzing the nonlinear coupling between two ultrasound waves.

Microbubbles and their Applications in Pharmaceutical Targeting

Microbubbles are small spherical gas filled bubbles in the size range of 1-10 microns. Their external coat is made of polymers or phospholipids. In combination with ultrasound, they have been explored as contrast agents for ultrasound and also carriers for drug and gene delivery. In response to ultrasound of lower mechanical index, microbubbles oscillate and vibrate to give a distinct signal in ultrasound imaging. At a higher mechanical index, these microbubbles rupture and break to deliver the drugs or genes enclosed in them or attached to their surface. The behaviour of microbubbles in response to ultrasound is a characteristic of the properties of microbubbles like its shell composition, shell thickness and the density and compressibility of the enclosed gas. Microbubbles have various applications in diagnostic imaging like echocardiography, and imaging of cancer cells, inflammed cells etc. They are also used as a medium for drug and gene delivery. Microbubbles can be further modified by binding specific ligands to their surface which specifically attach to certain cells so that selective action of the microbubbles can be seen at that location of cells.

A mechanistic investigation of subharmonic response from polymer-shelled microbubbles in response to high-frequency ultrasound

Polymer-shelled ultrasound contrast agents (UCAs) can undergo a “compression only” behavior leading to shell rupture and nonlinear response of the released gas bubbles when excited below 10 MHz. This study investigated if polymer-shelled UCAs exhibited a similar behavior when excited at frequencies above 10 MHz. Four varieties of polylactide-shelled UCAs, each with a distinct shell-thickness-to-radius ratio (STRR), were employed; the STRRs were 7.5, 40, 65, and 100 nm/μm. Two experiments were performed: one examined the compression-induced rupture of UCA shells by subjecting them to static overpressure, and the other investigated subharmonic components in the backscattered signal produced by individual UCAs sonicated with 20-MHz tone bursts. The four UCAs exhibited distinctly different compression-induced rupture thresholds that were linearly related to their STRR, but were uncorrelated with UCA size. The subharmonic response of the UCAs increased with increasing STRR. Thus, the UCAs with larger STRRs were more resilient to rupture, but they produced significantly greater subharmonic activity. The results of this two-part study indicated that the polymer-shelled UCAs may not adhere to the rupture-based mechanism of subharmonic generation when excited at 20 MHz.

How Do Microbubbles and Ultrasound Interact? Basic Physical, Dynamic and Engineering Principles

Ultrasound contrast agents consisting of gas microbubbles stabilised by a polymer or surfactant coating have been in clinical use for several decades. Research into the biomedical uses of microbubbles, however, remains a highly active and growing field. This is largely due to their considerable versatility and the wide range of applications for which they have demonstrated potential benefits. In addition to contrast enhancement, diagnostic applications include: perfusion mapping and quantification and molecular imaging. In drug and gene therapy microbubbles can be used as vehicles which are inherently traceable in vivo and can provide both targeted and controlled release. In addition, the dynamic behaviour of the microbubbles in response to ultrasound excitation contributes to the therapeutic process. At low intensities microbubbles have been shown to mediate reversible enhancement of cell and endothelial permeability, including temporary opening of the blood brain barrier. At higher intensities they have been used as means of increasing the efficiency of thrombolysis, high-intensity focused ultrasound (HIFU) surgery and lithotripsy. The aim of this review is to describe the key physical principles which determine how microbubbles and ultrasound interact and the implications for their design, preparation and exploitation in diagnostic and therapeutic applications.

Ultrasound microbubble contrast agents for diagnostic and therapeutic applications : current status and future design

Ultrasound contrast agents are highly echogenic microbubbles with many unique properties. Microbubbles can basically improve the sensitivity of conventional ultrasound imaging to the microcirculation. The resonance of microbubbles in response to an incident ultrasound pulse results in nonlinear harmonic emission that serves as the signature of microbubbles in microbubble-specific imaging. Inertial cavitation and destruction of microbubbles can produce a strong mechanical stress enhancing the permeability of the surrounding tissues, and can further increase the extravasation of drugs from the blood into the cytoplasm or interstitium. Stable cavitation by high-frequency ultrasound can also mildly increase tissue permeability without causing any damage even at a high acoustic pressure. Microbubbles can carry drugs, release them upon ultrasound-mediated microbubble destruction, and simultaneously enhance vascular permeability to increase drug deposition in tissues. Various targeting ligands can be conjugated to the surface of microbubbles to attain ligand-directed and site-specific accumulation for targeted imaging. In addition to current developments in microbubble technology, this review introduces our studies of the applications of microbubble- specific imaging, ultrasound-aided drug delivery, and targeted imaging. These applications are promising but may require further improvement for clinical use.

MODIFICATION OF THE SIZE DISTRIBUTION OF LYSOZYME MICROBUBBLES USING A POST-SONICATION TECHNIQUE

The control of the physical and functional properties of ultrasound responsive microbubbles is of interest because of their potential applications in therapeutic and diagnostic medicine. Crosslinked lysozyme microbubbles, synthesized using high intensity low frequency (20 kHz) ultrasound, possess long-term stability and retain bactericidal property. However, the relatively broader size distribution of these microbubbles may limit their use in some applications. In this article, we introduce a post-sonication technique for modifying the size distribution of lysozyme microbubbles. The post-sonication of these microbubbles at very high ultrasound frequencies (213, 355, 647, and 1056 kHz) led to the selective destruction of microbubbles of certain size range, leading to changes in the size distribution of microbubbles. An increase in acoustic power (10 W–60 W) at a fixed frequency (1056 kHz) resulted in a relatively narrow size distribution of the microbubbles. Although the enzymatic activity of microbubbles was reduced by the post-sonication treatment, significant activity was still preserved.