Ultrasound-induced Microbubble Research Articles

Ultrasound-Responsive Nanodroplet-Based Targeted Therapy via Conversion to Microbubbles.

Ultrasound-based therapy is appealing as it can be used via a wireless approach at remote parts of the body including the brain. Microbubbles are commonly used in such therapy due to their highly sound-responsive property. However, the larger size of microbubbles limits selective targeting in vitro/in vivo. Here, we report the design of nanodroplets of 70-130 nm in size that can be easily converted to microbubbles via ultrasound exposure. The advantage of this approach is that smaller nanodroplets can be used for cell/subcellular targeting, and next, they can be used for therapy by converting to microbubbles. More specifically, folate/dopamine-terminated perfluorohexane nanodroplets are designed that are loaded with a molecular drug. These nanodroplets are used for selective cell targeting, followed by ultrasound-induced microbubble conversion that is associated with drug release and intracellular reactive oxygen species generation. This approach has been used for selective cell therapy applications. The designed nanodroplet and approach can be used for the enhanced therapeutic performance of existing drugs.

Feasibility of in vitro calcification plaque disruption using ultrasound-induced microbubble inertial cavitation

Percutaneous transluminal coronary angioplasty (PTCA) is a clinical method in which plaque-narrowed arteries are widened by inflating an intravascular balloon catheter. However, PTCA remains challenging to apply in calcified plaques since the high pressure required for achieving a therapeutic outcome can result in balloon rupture, vessel rupture, and intimal dissection. To address the problem with PTCA, we hypothesized that a calcified plaque can be disrupted by microbubbles (MBs) inertial cavitation induced by ultrasound (US). This study proposed a columnar US transducer with a novel design to generate inertial cavitation at the lesion site. Experiments were carried out using tubular calcification phantom to mimic calcified plaques. After different parameters of US + MBs treatment (four types of MBs concentration, five types of cycle number, and three types of insonication duration; n = 4 in each group), inflation experiments were performed to examine the efficacy of cavitation for a clinically used balloon catheter. Finally, micro-CT was used to investigate changes in the internal structure of the tubular plaster phantoms. The inflation threshold of the untreated tubular plaster phantoms was > 11 atm, and this was significantly reduced to 7.4 ± 0.7 atm (p = 5.2E-08) using US-induced MBs inertial cavitation at a treatment duration of 20 min with an acoustic pressure of 214 kPa, an MBs concentration of 4.0 × 108 MBs/mL, a cycle number of 100 cycles, and a pulse repetition frequency of 100 Hz. Moreover, micro-CT revealed internal damage in the tubular calcification phantom, demonstrating that US-induced MBs inertial cavitation can effectively disrupt calcified plaques and reduce the inflation threshold of PTCA. The ex vivo histopathology results showed that the endothelium of pig blood vessels remained intact after the treatment. In summary, the results show that US-induced MBs inertial cavitation can markedly reduce the inflation threshold in PTCA without damaging blood vessel endothelia, indicating the potential of the proposed treatment method.

Ultrasound-Induced Microbubble Cavitation for Targeted Delivery of MiR-29b Mimic to Treat Cardiac Fibrosis.

Cardiac fibrosis contributes to adverse ventricular remodeling and is associated with loss of miR-29b. Overexpression of miR-29b via plasmid or intravenous injection of microRNA mimic has blunted fibrosis, but these are inefficient and non-targeted delivery strategies. In this study, we tested the hypothesis that delivery of microRNA-29b (miR-29b) using ultrasound-targeted microbubble cavitation (UTMC) of miR-29b-loaded microbubbles would attenuate cardiac fibrosis and preserve left ventricular (LV) function. Lipid microbubbles were loaded with miR-29b mimic (miR-29b-MB) or negative control (NC) mimic (NC-MB), placed with cardiac fibroblasts (CFs) and treated with pulsed ultrasound. Cells were harvested to measure downstream fibrotic mediators. Mice received angiotensin II (ANG II) infusion causing afterload increase and direct ANG II-induced cardiac fibrosis. UTMC of miRNA-loaded microbubbles was administered to the heart at days 0, 3 and 7. Serial echocardiography was performed, and hearts were harvested on day 10. UTMC treatment of CFs with miR-29b-MB increased miR-29b and decreased fibrotic transcripts compared with NC-MB treatment. In vivo UTMC+NC-MB led to increased LV mass, reduction in cardiac function and increase in fibrotic markers, demonstrating ANGI II-induced adverse cardiac remodeling. Mice treated with UTMC+miR-29b-MB had preservation of cardiac function, downregulation of cardiac fibrillin and trends of lower COL1A1, COL1A2 and COL3 mRNA and decreased cardiac α-smooth muscle protein. UTMC-mediated delivery of miR-29b mimic blunts expression of fibrosis markers and preserves LV function in ANG II-induced cardiac fibrosis.

Target tumor therapy in human gastric cancer cells through the combination of docetaxel-loaded cationic lipid microbubbles and ultrasound-triggered microbubble destruction.

It is well accepted that ultrasound-induced microbubble (USMB) cavitation is a promising method for drug delivery. Ultrasound-targeted destruction of cytotoxic drug-loaded lipid microbubbles (LMs) is used to promote the treatment of cancer. This study aimed to investigate the antitumor effects from a combination of docetaxel-loaded cationic lipid microbubbles (DLLM+) and ultrasound (US)-triggered microbubble destruction (UTMD) on gastric cancer (GC). It was found that the functional dose of DOC in this study was 1 × 10-9mol/L. We found that DLLM combined with the UTMD group showed greater growth inhibition of the cultured human gastric cancer cells (HGCCs) when compared with the other five groups by arresting the G2/M phase in the cell cycle. However, DLLM+ combined with UTMD showed a higher inhibition rate of tumor growth than DLLM combined with UTMD and that of the RC/CMV-p16 combined with UTMD in vitro and in vivo experiments. DLLM+ combined with UTMD significantly suppressed proliferation and promoted the apoptosis of HGCCs with more cells arrested in the G2/M phase. In addition, DLLM+ combined with UTMD suppressed the proliferation and induced apoptosis by arresting cells in the G2/M phase, which led to a great inhibition of GC progression. Thus, our results indicated that the combination of DLLM+ and UTMD might represent a novel and promising approach to chemotherapy for GC.

Mechanisms of ultrasonic de-agglomeration of oxides through in-situ high-speed observations and acoustic measurements

Ultrasonic de-agglomeration and dispersion of oxides is important for a range of applications. In particular, in liquid metal, this is one of the ways to produce metal-matrix composites reinforced with micron and nano sized particles. The associated mechanism through which the de-agglomeration occurs has, however, only been conceptualized theoretically and not yet been validated with experimental observations. In this paper, the influence of ultrasonic cavitation on SiO2 and MgO agglomerates (commonly found in lightweight alloys as reinforcements) with individual particle sizes ranging between 0.5 and 10 μm was observed for the first time in-situ using high-speed imaging. Owing to the opacity of liquid metals, a de-agglomeration imaging experiment was carried out in de-ionised water with sequences captured at frame rates up to 50 kfps. In-situ observations were further accompanied by synchronised acoustic measurements using an advanced calibrated cavitometer, to reveal the effect of pressure amplitude arising from oscillating microbubbles on oxide de-agglomeration. Results showed that ultrasound-induced microbubble clusters pulsating chaotically, were predominantly responsible for the breakage and dispersion of oxide agglomerates. Such oscillating cavitation clusters were seen to capture the floating agglomerates resulting in their immediate disintegration. De-agglomeration of oxides occurred from both the surface and within the bulk of the aggregate. Microbubble clusters oscillating with associated emission frequencies at the subharmonic, 1st harmonic and low order ultra-harmonics of the driving frequency were deemed responsible for the breakage of the agglomerates.

Ultrasound-Induced Microbubble Cavitation Combined with Paclitaxel-Loaded Nanoparticles for the Elimination of PC-3 Cells in vitro

Castration-resistant prostate cancer (CRPC) and its metastases are the main reasons for the high mortality of prostate cancer. Currently, paclitaxel (PTX)-based chemotherapeutics are used as first-line drugs to treat CRPC, but this treatment does not show good effects and is accompanied by serious side effects, which may be because intravenously injected chemotherapeutic drugs have difficulties gathering at the tumor site. Therefore, a safe and effective drug delivery carrier is urgently needed to enhance the therapeutic effects of chemotherapeutic drugs against CRPC. Methoxy polyethylene glycol-polylacticco-glycolic acid-polylysine (mPEG-PLGA-PLL) nanoparticles (NPs) have shown high drug encapsulation efficiency and good therapeutic effects against ovarian cancer and pancreatic cancer, but there are few studies on their treatment against CRPC. To expand the applications of mPEG-PLGA-PLL NPs, in this study, mPEG-PLGA-PLL NPs loaded with PTX (PTX-NPs) were synthesized. The synthesized PTX-NPs had a uniform particle size and no obvious aggregation. PTX-NPs can be uptaked by PC-3 cells, which significantly promotes the inhibition of proliferation and apoptosis effects of PTX on cells and reduces the expression levels of CDK6, Cyclin D1 and Bcl-2 (cyclins and an apoptosis inhibitor), and these effects can be further enhanced by ultrasound-induced microbubble cavitation (UIMC). Our research provides a new nanocarrier for the treatment of CRPC, laying the foundation for further research in the future.

Ultrasound-Induced Microbubble Cavitation Combined with miR-34a-Loaded Nanoparticles for the Treatment of Castration-Resistant Prostate Cancer.

Currently chemotherapy drugs are usually used as first-line treatments for castration-resistant prostate cancer (CRPC), but they are ineffective and accompanied by serious side effects. MicroRNA-34a (miR-34a) simultaneously targets multiple genes related to the cell apoptosis in CRPC cells without obvious side effects. It has shown great potential in the treatment of CRPC. Previous studies focused on miR-34a increasing the sensitivity of chemotherapy drugs to chemoresistant prostate cancer cells. There are few researches on miR-34a alone in the treatment of CRPC. But the macromolecular miR-34a is difficult to enter the cell and is easily degraded by nuclease. Therefore, we constructed methoxy polyethylene glycol-polylacticco-glycolic acid-polylysine (mPEG-PLGA-PLL) nanoparticles to encapsulate miR-34a (miR-34a/NP). The results showed that miR-34a/NP protects miR-34a from degradation by nucleases and can be phagocytized by PC-3 CRPC cells. Ultrasound induces microbubble cavitation (UIMC) improves cell membrane permeability and capillary gaps, and further promotes miR-34a/NP to enter cells PC-3 and prostate cancer xenografts. The miR-34a/NP that enters the cell and tumor tissue releases miR-34a, which suppressed CRPC cells PC-3 proliferation, promoted its apoptosis, and inhibited the growth of CRPC xenografts. Our research verified that miR-34a/NP, especially combined with UIMC, has a significant anti-tumor effect on CRPC.

Ultrasound-induced microbubble cavitation via a transcanal or transcranial approach facilitates inner ear drug delivery.

Ultrasound-induced microbubble (USMB) cavitation is widely used to promote drug delivery. Our previous study investigated USMB targeting the round window membrane by applying the ultrasound transducer to the tympanic bulla. In the present study, we further extended the use of this technology to enhance drug delivery to the inner ear by introducing the ultrasound transducer into the external auditory canal (EAC) or applying it to the skull. Using a 3-dimensional-printed diffusion apparatus mimicking the pathway for ultrasound passing through and reaching the middle ear cavity in vitro, the models simulating the transcanal and transcranial approach demonstrated 4.8-fold- and 3.7-fold-higher delivery efficiencies, respectively. In an in vivo model of guinea pigs, by filling tympanic bulla with microbubbles and biotin-FITC, USMB applied transcanally and transcranially induced 2.8-fold and 1.5-fold increases in biotin-FITC delivery efficiencies, respectively. In addition, the gentamicin uptake by cochlear and vestibular hair cells and gentamicin-induced hair cell loss were significantly enhanced following transcanal application of USMB. On the 28th day after transcanal USMB, safety assessment showed no significant changes in the hearing thresholds and the integrity of cochlea. These are the first results to our knowledge to demonstrate the feasibility and support the potential clinical application of applying USMB via EAC to facilitate drug delivery into the inner ear.

Classification and prediction of inertial cavitation activity induced by pulsed high-intensity focused ultrasound

Classification and prediction of ultrasound-induced microbubble inertial cavitation (IC) activity may play an important role in better design of ultrasound treatment strategy with improved efficiency and safety. Here, a new method was proposed by combining support vector machine (SVM) algorithm with passive cavitation detection (PCD) measurements to fulfill the tasks of IC event classification and IC dose prediction. By using the PCD system, IC thresholds and IC doses were firstly measured for various ultrasound contrast agent (UCA) solutions exposed to pulsed high-intensity focused ultrasound (pHIFU) at different driving pressures and pulse lengths. Then, after trained and tested by measured data, two SVM models (viz. C-SVC and ε-SVR) were established to classify the likelihood of IC event occurrence and predict IC dose, respectively, under different parameter conditions. The findings of this study indicate that the combination of SVM and PCD could be used as a useful tool to optimize the operation strategy of cavitation-facilitated pHIFU therapy.

Effect of Molecular Weight on Sonoporation-Mediated Uptake in Human Cells

Ultrasound-induced microbubble destruction can enhance drug delivery to cells. The molecular weight of therapeutic compounds varies significantly (from <1 kDa for small molecule drugs, to 7–15 kDa for siRNAs/miRNAs, to >1000 kDa for DNA plasmids). Therefore, the objective of this study was to determine the relationship between uptake efficiency and molecular weight using equal molar concentrations. Uptake efficiency of fluorescent compounds with different molecular weights (0.3, 10 and 2000 kDa) was explored in vitro using human cardiac mesenchymal cells and breast cancer cells exposed to microbubbles and 2.5-MHz ultrasound pulses. Uptake by viable cells was quantified using flow cytometry. After correction for the fluorescence yield of each compound, there was a significant size-dependent difference in fluorescence intensity, indicating an inverse relationship between size and uptake efficiency. These results suggest that diffusion of therapeutic compounds across permeabilized cell membranes may be an important mechanism for ultrasound-mediated drug delivery.

Ultrasound-Induced Microbubble Cavitation for the Treatment of Catheterization-Induced Vasospasm

Highlights •In a porcine model, we investigated the therapeutic effectiveness of microbubble enhanced sonothrombolysis for treatment of catheter-induced vasospasm and vascular injury, a common complication after femoral arterial catheterization, particularly in children. •Our results show that microbubble cavitation mediated by brief application of diagnostic high mechanical index impulses is an effective noninvasive method for relieving catheter-induced vasospasm. •This approach could have potential as an effective treatment for reversal of pulse loss after peripheral arterial injury and vasospasm.

Passive acoustic mapping in aberrating media with the angular spectrum approach

The ability to localize and characterize ultrasound-induced microbubble oscillations through the intact skull with high spatial and temporal resolution holds significant promise for the diagnosis and treatment of brain diseases and disorders. In this study, we investigated the ability of angular spectrum (AS) method, a fast planar projection method, to perform passive acoustic mapping of microbubbles through an intact skull. Finite-difference time-domain numerical simulations were used to model microbubble emissions’ propagation through homogeneous, stratified, and 2D inhomogeneous (skull) environments approximately 80 mm by 160 mm. Reconstructions with the AS approach were performed with constant and effective sound speeds, as well as with multi-step propagation, to evaluate their ability to correct for induced aberrations and localize the microbubbles. We also investigated the impact of the receiver position on the localization accuracy. Results for skull simulations indicated that the multi-step AS method reduced the error in axial localization of the microbubbles by on the order of 50% compared with the effective sound speed method, while incurring approximately a 25% increase in computation time for each doubling of the number of propagation steps. Both AS methods were several orders of magnitude faster than time-domain reconstruction. Further investigation of the potential of this approach to correct skull aberrations is warranted.

Insights into mechanisms of sonothrombolysis using high speed imaging

Thrombotic arterial occlusion is the major cause of acute cardiovascular syndromes such as stroke and myocardial infarction. Exposing the thrombus to microbubbles in the presence of ultrasound facilitates thrombus disruption, and is thus a potentially powerful therapeutic strategy for thromboembolic diseases. However, optimization of “sonothrombolysis” is constrained by a lack of understanding of underlying mechanisms for blood clot disruption in response to ultrasound-induced microbubble vibrations. We tested the hypothesis that inertial cavitation induces mechanical clot disruption by optically characterizing lipid microbubble interactions with thrombus + ultrasound using an ultra-high speed microscopy imaging system capable of imaging at MHz frame rates. A microscope/acoustic stage was used to hold an experimentally created thrombus and microbubbles, which were insonified (1 MHz) during synchronized high speed imaging. Large amplitude microbubble oscillations in response to an inertial cavitation regime caused thrombus deformation and pitting. These data implicate a direct mechanical effect of oscillating microbubbles on mediating clot disruption.

Mecanism and applications of the nonlinear dynamic response to ultrasound contrast agent microbubbles

Ultrasound contrast agent (UCA) reflers to the agent that has specific acoustic properties to enhance the contrast in ultrasound imaging by composition of gas-filled microbubbles with micrometer-diameters. In a diagnostic ultrasound field, microbubbles in fluid create an acoustic impedance mismatch between fluid and surrounding tissue to increase the reflection of sound and achieve a better contrast. Ongoing developments improve diagnostic possibilities of UCA remarkably, whereas their potential therapeutic applications have also been investigated for a couple of decades. The nonlinear response of UCA microbubbles has clinical reflevance from both diagnostic and therapeutic perspectives. The aim of this review is to introduce the latest reflearch progress of our group regarding the mechanism and applications of the nonlinear dynamic response to UCA, which include (1) an all-in-one solution characterizing coated bubble parameters with the help of the light scattering technique and flow cytometry, which makes it possible to quickly integrate the size distribution with dynamic motions of thousands of microbubbles and easily verify the validities of different shelled bubble dynamic models; (2) the development of a new bubble dynamics model that takes into account both nonlinear shell elasticity and viscosity, which can not only be capable of simulating the “compression-only” behavior of microbubbles excited by large amplitude ultrasound but also eliminate the dependence of bubble shell parameters on bubble size; (3) the estimation of UCA inertial cavitation thresholds of two types of commercial UCA microbubbles (viz., SonoVue microbubbles coated with lipid shells and KangRun microbubbles coated with albumin shells) and the evaluation of the relationship between microbubble inertial cavitation thresholds and their shell parameters; and (4) the reflearches of DNA transfection efficiency and the reduction of cytotoxicity in gene delivery facilitated by UCA excited by 1-MHz focused ultrasound pulses, and the results indicate that the measured DNA transfection efficiency and sonoporation pore size generally increase with the enhancement of inertial cavitation dose, while the cell viability decreases linearly with the increase of International Classification of Diseases (ICD). These studies are of significance for better understanding the mechanism of ultrasound-induced microbubble nonlinear dynamics and investigating the effective quantification technique for microbubble cavitation activity, which are important for further optimizing therapeutic ultrasound effects and avoiding the side-effects.

New Insights into Mechanisms of Sonothrombolysis Using Ultra-High-Speed Imaging

Thrombotic arterial occlusion is the principal etiology for acute cardiovascular syndromes such as stroke, myocardial infarction and unstable angina. Exposing the thrombus to ultrasound and microbubbles facilitates thrombus disruption, making “sonothrombolysis” a potentially powerful therapeutic strategy for thromboembolic diseases. However, optimization of such a strategy, and hence clinical translation, is constrained by an incomplete understanding of mechanisms by which ultrasound-induced microbubble vibrations disrupt blood clots. We posit that previously reported sonothrombolytic efficacy using inertial cavitation regimes was due, at least in part, to mechanical clot disruption by oscillating microbubbles. To test this hypothesis, we optically characterized lipid microbubble interactions with thrombus in the presence of ultrasound using a recently developed ultra-high-speed microscopy imaging system to visualize microbubble acoustic behaviors at megahertz frame rates. A microscope/acoustic stage designed for the system allowed an experimentally created thrombus and microbubbles to be insonified at a co-localized acoustic and optical focus during synchronized high-speed imaging. Under inertial cavitation conditions, large-amplitude microbubble oscillations caused thrombus deformation and pitting. Acoustic radiation forces (Bjerknes forces) further augmented microbubble-thrombus interaction. These observations suggest that a direct mechanical effect of oscillating lipid microbubbles on an adjacent thrombus may play a role in mediating clot disruption in the presence of specific ultrasound conditions.

1:54 PM Abstract No. 95 - ■ FEATURED ABSTRACT Biodegradable starch nanoparticles are a novel and potentially effective and safe adjuvant for sonothrombolysis especially for more fibrin rich, aged and rigid clots

Purpose It was postulated that microstreaming produced by ultrasound-induced microbubble (MB) cavitation will propel nanoparticles with sufficient force to help erode clots by a sandblasting-like process. This study tested the plausibility of this hypothesis using biodegradable starch nanoparticles (StNPs) to promote sonothrombolysis (STBL). StNPs were mixed with MBs and/or tPA to determine if these agents interacted synergistically with StNPs to produce more effective lysis of STBL-resistant clots. Materials and Methods Fibrin-rich clots were formed from fresh rabbit blood supplemented with 60 U of thrombin, incubated 24 h at 37 o C, and then cured for 24 h at 4 o C. In vitro STBL of 7.5-12.0 mg pieces of fibrin-rich clot was performed in a flow chamber (37.0 o C) infused with bovine calf serum containing biodegradable starch nanoparticles (StNPs). STBL was performed with pulsed ultrasound (1 MHz or 3 MHz) and intensities of 0.05-2.0 W/cm 2 . STBL efficacy was quantified as % clot mass loss during a 15 min treatment. MBs (1 μm or 3 μm diameter) and/or tPA (0.01 mg/mL) were then combined with the StNPs to determine if they interacted synergistically to improve STBL efficacy. Results StNPs with a mean diameter of 250 nm were two-fold more effective than MBs for STBL of fibrin-rich, and rigid clots at both 1 MHz and 3 MHz. They interacted synergistically with MBs and tPA to provide an additional two- to four-fold more effective STBL. Conclusion Surprisingly, 250 nm StNPs were an effective adjuvant for STBL without MBs or tPA, but interacted synergistically with MBs and/or tPA to provide even more effective STBL, as postulated. StNPs are not destroyed by ultrasound and so provide longer-lasting STBL; plus their biocompatibility and biodegradability (within 2 h) eliminate potential toxicity concerns. Their nm size likely permits StNPs to penetrate deeper through the smaller pores within fibrin-rich clots than MBs. The current model is that as the deeper penetrating StNPs promote clot lysis so that MBs and tPA can then penetrate fibrin-rich clots more readily and interact with the ultrasound to enhance STBL efficacy further.

Enhancement Effect of Ultrasound-Induced Microbubble Cavitation on Branched Polyethylenimine-Mediated VEGF165 Transfection With Varied N/P Ratio

One isoform of the vascular endothelial growth factor, VEGF165, has been reported to be a dominant mediator and regulator of angiogenic process, which plays an important role in treating cardiovascular diseases and chronically ischemic wounds. Branched polyethylenimine (bPEI) has been widely used as a non-viral delivery vector for gene therapy. Although bPEI-mediated DNA transfection efficiency can be raised by increasing the PEI nitrogen:DNA phosphate (N/P) ratio, cytotoxicity increases as well. In this study, the enhancement effect of microbubble inertial cavitation (IC) on bPEI-mediated VEGF165 transfection was investigated, in an effort to optimize transfection efficiency using low N/P ratios. HEK 293T cells, mixed with bPEI:VEGF165 complexes, were exposed to 1-MHz ultrasound pulses. The results show that: (1) IC activity induced by microbubble destruction can be quantified as an IC “dose” (ICD) and will increase with increasing acoustic driving pressure; (2) larger sonoporation pores can be generated by increasing ICD; (3) the transfection efficiency can be enhanced by increasing ICD until reaching a saturation level; and (4) microbubble IC activity has less cytotoxicity than bPEI, although a combinatorial effect of microbubble IC activity and bPEI could be observed on cell viability. The results suggest that, with appropriate ultrasound parameters, it is possible to optimize bPEI-mediated VEGF transfection efficiency using relatively low N/P ratios by employing ultrasound-induced microbubble inertial cavitation.

Influence of lipid shell physicochemical properties on ultrasound-induced microbubble destruction

We present the first study of the effects of monolayer shell physicochemical properties on the destruction of lipid-coated microbubbles during insonification with single, one-cycle pulses at 2.25 MHz and low-duty cycles. Shell cohesiveness was changed by varying phospholipid and emulsifier composition, and shell microstructure was controlled by postproduction processing. Individual microbubbles with initial resting diameters between 1 and 10 microm were isolated and recorded during pulsing with bright-field and fluorescence video microscopy. Microbubble destruction occurred through two modes: acoustic dissolution at 400 and 600 kPa and fragmentation at 800 kPa peak negative pressure. Lipid composition significantly impacted the acoustic dissolution rate, fragmentation propensity, and mechanism of excess lipid shedding. Less cohesive shells resulted in micron-scale or smaller particles of excess lipid material that shed either spontaneously or on the next pulse. Conversely, more cohesive shells resulted in the buildup of shell-associated lipid strands and globular aggregates of several microns in size; the latter showed a significant increase in total shell surface area and lability. Lipid-coated microbubbles were observed to reach a stable size over many pulses at intermediate acoustic pressures. Observations of shell microstructure between pulses allowed interpretation of the state of the shell during oscillation. We briefly discuss the implications of these results for therapeutic and diagnostic applications involving lipid-coated microbubbles as ultrasound contrast agents and drug/gene delivery vehicles.