SonoVue Microbubbles Research Articles

Optimal Control of SonoVue Microbubbles to Estimate Hydrostatic Pressure.

The measurement of cardiac and aortic pressures enables diagnostic insight into cardiac contractility and stiffness. However, these pressures are currently assessed invasively using pressure catheters. It may be possible to estimate these pressures less invasively by applying microbubble ultrasound contrast agents as pressure sensors. The aim of this study was to investigate the subharmonic response of the microbubble ultrasound contrast agent SonoVue (Bracco Spa, Milan, Italy) at physiological pressures using a static pressure phantom. A commercially available cell culture cassette with Luer connections was used as a static pressure chamber. SonoVue was added to the phantom, and radio frequency data were recorded on the ULtrasound Advanced Open Platform (ULA-OP). The mean subharmonic amplitude over a 40% bandwidth was extracted at 0–200-mmHg hydrostatic pressures, across 1.7–7.0-MHz transmit frequencies and 3.5%–100% maximum scanner acoustic output. The Rayleigh–Plesset equation for single-bubble oscillations and additional hysteresis experiments were used to provide insight into the mechanisms underlying the subharmonic pressure response of SonoVue. The subharmonic amplitude of SonoVue increased with hydrostatic pressure up to 50 mmHg across all transmit frequencies and decreased thereafter. A decreasing microbubble surface tension may drive the initial increase in the subharmonic amplitude of SonoVue with hydrostatic pressure, while shell buckling and microbubble destruction may contribute to the subsequent decrease above 125-mmHg pressure. In conclusion, a practical operating regime that may be applied to estimate cardiac and aortic blood pressures from the subharmonic signal of SonoVue has been identified.

Super-Resolution Contrast-Enhanced Ultrasound Methodology for the Identification of In Vivo Vascular Dynamics in 2D.

ObjectivesThe aim of this study was to provide an ultrasound-based super-resolution methodology that can be implemented using clinical 2-dimensional ultrasound equipment and standard contrast-enhanced ultrasound modes. In addition, the aim is to achieve this for true-to-life patient imaging conditions, including realistic examination times of a few minutes and adequate image penetration depths that can be used to scan entire organs without sacrificing current super-resolution ultrasound imaging performance.MethodsStandard contrast-enhanced ultrasound was used along with bolus or infusion injections of SonoVue (Bracco, Geneva, Switzerland) microbubble (MB) suspensions. An image analysis methodology, translated from light microscopy algorithms, was developed for use with ultrasound contrast imaging video data. New features that are tailored for ultrasound contrast image data were developed for MB detection and segmentation, so that the algorithm can deal with single and overlapping MBs. The method was tested initially on synthetic data, then with a simple microvessel phantom, and then with in vivo ultrasound contrast video loops from sheep ovaries. Tracks detailing the vascular structure and corresponding velocity map of the sheep ovary were reconstructed. Images acquired from light microscopy, optical projection tomography, and optical coherence tomography were compared with the vasculature network that was revealed in the ultrasound contrast data. The final method was applied to clinical prostate data as a proof of principle.ResultsFeatures of the ovary identified in optical modalities mentioned previously were also identified in the ultrasound super-resolution density maps. Follicular areas, follicle wall, vessel diameter, and tissue dimensions were very similar. An approximately 8.5-fold resolution gain was demonstrated in vessel width, as vessels of width down to 60 μm were detected and verified (λ = 514 μm). Best agreement was found between ultrasound measurements and optical coherence tomography with 10% difference in the measured vessel widths, whereas ex vivo microscopy measurements were significantly lower by 43% on average. The results were mostly achieved using video loops of under 2-minute duration that included respiratory motion. A feasibility study on a human prostate showed good agreement between density and velocity ultrasound maps with the histological evaluation of the location of a tumor.ConclusionsThe feasibility of a 2-dimensional contrast-enhanced ultrasound-based super-resolution method was demonstrated using in vitro, synthetic and in vivo animal data. The method reduces the examination times to a few minutes using state-of-the-art ultrasound equipment and can provide super-resolution maps for an entire prostate with similar resolution to that achieved in other studies.

Improvement of Detection Sensitivity of Microbubbles as Sensors to Detect Ambient Pressure.

Microbubbles are considered a promising tool for noninvasive estimation of local blood pressure. It is reported that the subharmonic scattering amplitude of microbubbles decreases by 9 to 12 dB when immersed in the media under an ambient pressure variation from 0 to 180 mmHg. However, the pressure sensitivity still needs to be improved to satisfy clinical diagnostic requirements. Here, we investigated the effects of acoustic parameters on the pressure sensitivity of microbubbles through measuring the acoustic attenuation and scattering properties of commercially available SonoVue microbubbles. Our results showed that the first harmonic, subharmonic, and ultraharmonic amplitudes of microbubbles were reduced by 6.6 dB, 10.9 dB, and 9.3 dB at 0.225 mechanical index (MI), 4.6 dB, 19.8 dB, and 12.3 dB at 0.25 MI, and 18.5 dB, 17.6 dB, and 12.6 dB at 0.3 MI, respectively, when the ambient pressure increased from 0 to 180 mmHg. Our finding revealed that a moderate MI (0.25–0.4) exciting microbubbles could significantly improve their sensitivities to detect ambient pressure.

Potential causes of insufficient bladder contrast opacification and premature microbubble destruction during contrast-enhanced voiding urosonography in children.

Contrast-enhanced voiding urosonography (ceVUS) has been recognized as a child-friendly examination with high diagnostic accuracy for vesicoureteric reflux detection. A single bolus and the infusion techniques of ceVUS are described. Insufficient bladder contrast opacification during the filling phase and premature destruction of SonoVue microbubbles might occur. Data regarding SonoVue's features, doses, bladder contrast opacification, US bladder parameters, urine catheter, antibiotic prophylaxis, and childrens behaviors were collected to discover the possible causes of the contrast vanishing observed during bladder filling in 10% of examinations and in the later phase of ceVUS in 5% of examinations. An updated ceVUS examination protocol is suggested.

Effect of RNA Interference Targeting STAT3 Gene Combined with Ultrasonic Irradiation and SonoVue Microbubbles on Proliferation and Apoptosis in Keratinocytes of Psoriatic Lesions.

Background:Signal transducer and activator of transcription 3 (STAT3) was strongly expressed and activated in psoriatic keratinocytes (KCs) and correlated with the severity of psoriasis. The study aimed to investigate the effects of STAT3 small interfering RNA (siRNA) combined with ultrasonic irradiation and SonoVue microbubbles on the proliferation and apoptosis in KCs of psoriatic lesions and the relative mechanisms.Methods:Psoriatic KCs were transfected under four experimental conditions: (1) STAT3 siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (LUS group); (2) STAT3 siRNA only carried by Lipofectamine 3000 (L group); (3) the negative control of siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (siRNA-NC); (4) not treated as Blank. Cell Counting Kit-8 assay was used to evaluate the cell proliferation. Cell cycle analysis was detected with cycle test Plus DNA reagent kit associated with flow cytometer. FITC Annexin V apoptosis detection kit associated with flow cytometer was applied for apoptosis analysis. Fluo calcium indicator associated with flow cytometer was used to analyze intracellular free calcium concentration ([Ca2+]i). The expressions of cyclin D1 and Bcl-xL were detected both at the mRNA level by real-time reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by Western blotting. The obtained data were statistically evaluated by two-way analysis of variance.Results:STAT3 siRNA inhibited the growth of KCs in a time-dependent manner showing the highest proliferation inhibition in LUS group with proliferation ratio of 45.38% ± 5.85% at 72h (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced an altered cell cycle distribution of KCs showing the highest increases in G2/M-phase population up to 18.06% ± 0.36% in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced late apoptosis of KCs with the highest late apoptosis percentage of 22.87% ± 1.28% in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced the elevation of [Ca2+]i of KCs with the highest calcium fluorescence intensity mean of 1213.67 ± 60.51 in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced the downregulation of cyclin D1 and Bcl-xL expressions of KCs at mRNA and protein levels with the lowest expressions in LUS group with cyclin D1 expression of 51.81% ± 9.58% and 70.17% ± 4.22% at mRNA level and at protein level, respectively, and with Bcl-xL expression of 37.58% ± 4.92% and 64.06% ± 7.78% at mRNA level and at protein level, respectively (P < 0.05 vs. L group, siRNA-NC, or Blank).Conclusions:STAT3 siRNA inhibited the growth and induced the apoptosis in psoriatic KCs likely partly through altering cell cycle distribution, elevating [Ca2+]i, and downregulating cyclin D1 and Bcl-xL expressions. Silencing the target gene STAT3 in psoriatic KCs with siRNA combined with ultrasonic irradiation and microbubbles would contribute to a significant innovation as a new clinical therapy for psoriasis.

Non-linear acoustic emissions from therapeutically driven microbubbles

Acoustic detection of contrast agent microbubbles, infused to the vasculature for exposure to focused ultrasound, is now routinely undertaken to evaluate therapy and avoid irreversible tissue damage. Harmonic, subharmonic (to the frequency of the focused ultrasound, f0) and broadband emissions are often used to distinguish between stable and inertial cavitation activity, and associated bioeffects. The driven microbubble dynamic responsible for the generation of non-linear emissions, however, may not be well understood. Results from an investigation of single SonoVue microbubbles flowing through a capillary, for exposure to focused ultrasound at f0 = 692 kHz, will be presented. Dual high-speed imaging from orthogonal perspectives at circa 2 × 105, and shadowgraphically at 10 million frames per second, capture microbubble activity and shock wave generation. Acoustic emissions are simultaneously collected with a calibrated broadband needle hydrophone, and high frequency imaging array on passive receive, for cavitation mapping. The results indicate that non-linear emissions are mediated by periodic bubble collapse shock waves, with subharmonic emission occurring above a threshold driving pressure amplitude. Implications for detection and quantification of driven microbubble cavitation activity, as well as conventional classification as stable or inertial, will be discussed. [This work was supported by ERC Starting Grant TheraCav, 336189.]

Six-Transmembrane Epithelial Antigen of the Prostate-1 (STEAP-1)-Targeted Ultrasound Imaging Microbubble Improves Detection of Prostate Cancer In Vivo.

To investigate the feasibility of the 6-transmembrane epithelial antigen of the prostate-1 (STEAP-1)-targeted microbubbles for enhancing ultrasound imaging of prostate tumors in the nude mouse xenograft models. Contrast agents were established by conjugating biotinylated STEAP-1 monoclonal antibodies with streptavidin coated SonoVue microbubbles. Then, ordinary SonoVue (Bracco, Milan, Italy) microbubble and STEAP-1-targeted SonoVue microbubble were used, respectively, for contrast-enhanced sonography to detect prostate tumors in the nude mouse xenograft models. The characteristics, including peak intensity, time to peak, area under the curve, and mean transit time, were measured. The biological characteristics of STEAP-1-targeted SonoVue microbubbles were stable. STEAP-1-targeted SonoVue microbubbles can successfully conjugate to prostate cancer cell lines in vitro. Enhancement of ultrasound signal intensity was determined after injection of STEAP-1-targeted SonoVue microbubble, compared with ordinary SonoVue microbubble. Higher intensities of ultrasound signals in xenograft tumor of prostate cancer were associated with increased levels of STEAP-1 expression. Our results suggest that SonoVue microbubble carrying STEAP-1 monoclonal antibody could improve the ultrasound visualization of prostate cancer and identify the tumor more effectively in vivo. A prospective study is required to validate our finding in patients with prostate cancer.

Combining Acoustic Trapping With Plane Wave Imaging for Localized Microbubble Accumulation in Large Vessels.

The capability of accumulating microbubbles using ultrasound could be beneficial for enhancing targeted drug delivery. When microbubbles are used to deliver a therapeutic payload, there is a need to track them, for a localized release of the payload. In this paper, a method for localizing microbubble accumulation with fast image guidance is presented. A linear array transducer performed trapping of microbubble populations interleaved with plane wave imaging, through the use of a composite pulse sequence. The acoustic trap in the pressure field was created parallel with the direction of flow in a model of a vessel section. The acoustic trapping force resultant from the large gradients in the acoustic field was engendered to directly oppose the flowing microbubbles. This was demonstrated numerically with field simulations, and experimentally using an Ultrasound Array Research Platform II. SonoVue microbubbles at clinically relevant concentrations were pumped through a tissue-mimicking flow phantom and exposed to either the acoustic trap or a control ultrasonic field composed of a single-peak acoustic radiation force beam. Under the flow condition at a shear rate of 433 s-1, the use of the acoustic trap led to lower speed estimations ( ) in the center of the acoustic field, and an enhancement of 71% ± 28%( ) in microbubble image brightness.

Stable cavitation using acoustic phase-change dodecafluoropentane nanoparticles for coronary micro-circulation thrombolysis

BackgroundThe thrombolysis in micro-circulation after acute myocardial infarction has been an unsolved issue, as elimination effect of acute thrombolysis and primary intervention were unsatisfied. Stable cavitation using acoustic phase-change nanoparticles may have potential for thrombolysis. Therefore, we sought to investigate a novel treatment method with dodecafluoropentane (DDFP) nanoparticles for rapid and effective thrombolysis in an in-vitro artificial vascular system, as a mimicking preparation of coronary circulation. MethodsTo simulate thrombus embolism in coronary circulation, an in-vitro artificial vascular system was established with cavitation effect using DDFP nanoparticles. For PBS blank control (group A), SonoVue microbubbles (group B) and DDFP nanoparticles (group C), the durations for cavitation effect were recorded and the thrombolysis efficiency with low intensity focused ultrasound irradiation in the in-vitro vascular system were analyzed with weight loss and pathological changes of thrombus before and after thrombolysis. ResultsThe optimal conditions for acoustic cavitation effect were power of 6 W for 20 min by ultrasound irradiation at 37 °C. The weight loss and weight loss rates of thrombus in group C (189.4 ± 30.2 mg and 34.2 ± 5.7%) were higher than those in group A (30.2 ± 16.0 mg and 5.2 ± 2.1%) and group B (84.0 ± 20.4 mg and 14.6 ± 1.5%) (P < 0.01, all). The duration for cavitation effect in group C (32.8 ± 3.9 min) was also longer than those in group A (0.0 ± 0.0 min) and group B (5.3 ± 0.3 min) (P < 0.01, all). ConclusionsBy stable and sustaining cavitation in targeted area, DDFP nanoparticles with ultrasound irradiation have significantly increased the thrombolysis efficiency, which has provided a powerful experimental foundation for potential coronary thrombolysis.

Acoustic Characterization and Enhanced Ultrasound Imaging of Long-Circulating Lipid-Coated Microbubbles.

A long-circulating lipid-coated ultrasound (US) contrast agent was fabricated to achieve a longer wash-out time and gain more resistance against higher-mechanical index sonication. Systemic physical, acoustic, and in vivo imaging experiments were performed to better understand the underlying mechanism enabling the improvement of contrast agent performance by adjusting the physical and acoustic properties of contrast agent microbubbles. By simply altering the gas core, a kind of US contrast agent microbubble was synthesized with a similar lipid-coating shell as SonoVue microbubbles (Bracco SpA, Milan, Italy) to achieve a longer wash-out time and higher inertial cavitation threshold. To bridge the structure-performance relationship of the synthesized microbubbles, the imaging performance of the microbubbles was assessed in vivo with SonoVue as a control group. The size distribution and inertial cavitation threshold of the synthesized microbubbles were characterized, and the shell parameters of the microbubbles were determined by acoustic attenuation measurements. All of the measurements were compared with SonoVue microbubbles. The synthesized microbubbles had a spherical shape, a smooth, consistent membrane, and a uniform distribution, with an average diameter of 1.484 μm. According to the measured attenuation curve, the synthesized microbubbles resonated at around 2.8 MHz. Although the bubble's shell elasticity (0.2 ± 0.09 N/m) was comparable with SonoVue, it had relatively greater viscosity and inertial cavitation because of the different gas core. Imaging studies showed that the synthesized microbubbles had a longer circulation time and a better chance of fighting against rapid collapse than SonoVue. Nano/micrometer long-circulating lipid-coated microbubbles could be fabricated by simply altering the core composition of SonoVue microbubbles with a higher-molecular weight gas. The smaller diameter and higher inertial cavitation threshold of the synthesized microbubbles might make it easier to access deep-seated organs and give prolonged imaging enhancement in the liver.

DNA-loaded microbubbles with crosslinked bovine serum albumin shells for ultrasound-promoted gene delivery and transfection

The microbubble is a kind of clinically applied ultrasound contrast agent in disease diagnosis that can also rupture under sonication to increase membrane permeability and promote gene entry into targeted cells. However, the development of ultrasound-mediated gene delivery might be restricted because genes and microbubbles were separated and would not reach the targeted cells simultaneously. Herein, a kind of crosslinked positive microbubbles (CPMBs) were prepared to load DNA as gene vectors to promote gene delivery efficiency. The BSA shell of the CPMBs was crosslinked with disulfide bonds, which obviously enhanced the stability of the CPMBs. Furthermore, the CPMBs revealed sonoporation effects comparable to those of clinically applied SonoVue microbubbles. As DNA and CPMBs were electrostatically linked as an entirety, they would reach cells simultaneously. Thus, with the aid of ultrasound, these DNA-loaded microbubbles promoted DNA entry into cytoplasm more effectively and obtained higher cellular uptake efficiency and better transfection efficiency than DNA-mixed microbubbles. Confocal microscopy results showed that rupturing of the CPMBs/DNA entire microbubbles under sonication could carry DNA directly into the cytoplasm or nucleus. All results indicated that the cytocompatible DNA-loaded microbubbles have promising prospects in ultrasound-mediated gene delivery.

Treatment of coronary micro-circulation thrombus by dodecafluoropentane acoustic vaporizable nano-droplet with ultrasound irradiation: an in vitro experimental study

Objective To explore the clinical value of a new tactic with novel dodecafluoropentane (C5F12, DDFP) acoustic vaporizable nano-droplets for the therapy of coronary micro-circulation thrombolysis. Methods Group A was poly butylene succinate buffer solution as blank control; group B was SonoVue microbubbles; group C was DDFP acoustic vaporizable nano-droplets, which were produced with emulsion process by ultrasonic oscillation instrument and rotary evaporator. The size of the nano-droplet was tested by Malvern laser particle size detector and observed with fluorescence microscope. The stability and efficiency of cavitation effect of group C for thrombolysis were tested in an in-vitro artificial vascular system with arterial thrombus model, comparing with group A and B. Weights and pathological sections of the inserted thrombus before and after the thrombolysis in these groups were compared for the thrombolytic efficiency. Results The average size of DDFP nano-droplets were (424.7±30.2)nm. More than 75% of the nano-droplets were ranged from 300-750 nm. The fluorescence microscope showed the DDFP nano-droplets were uniformed round-shaped and stable-structured with favorable dispersity; the nano-droplets had gradually turned into over 3 μm microbubbles under ultrasound irradiation. The thrombus weight loss after thrombolysis in group C [(199.0±35.8)mg, (32.1±4.4)%] was significantly larger than those in group A [(30.2±17.8)mg, (5.0±2.4)%] and B [ (72.6±20.7)mg, (12.7±2.8)%] (all P<0.01). Conclusions DDFP vaporizable nano-droplets with ultrasound irradiation is effective for thrombus elimination in coronary micro-circulation through lasting and stable cavitation effect. Key words: Sonication; Vaporizable nano-droplet; Thromboembolism; Thrombolytic therapy

High-speed photographic observation of the sonication of a rabbit carotid artery filled with microbubbles by 20-kHz low frequency ultrasound

The aim of this study is to assess the physical damage of cavitation effects induced by low frequency ultrasound and microbubbles (MBs) to an in vitro vessel. A rabbit carotid artery filled with SonoVue MBs and methylene blue was irradiated with 20-kHz ultrasound, and the results were recorded by high-speed photography at 3000 frames per second. The carotid artery filled with MBs experienced a slight tremor during ultrasonication. Six intermittent blue flow events occurred in two places on the artery wall during the 5-s process. The duration of each leakage event was 90–360ms with an average of 200ms. Hematoxylin-eosin (H-E) staining demonstrated the separation of the carotid artery elastic membrane, local blood vessel wall defects and hole formation, and the surface of the ruptured area was rough and irregular. Another carotid artery was filled with a 0.9% NaCl solution and methylene blue as a control and irradiated with 20-kHz ultrasound. No blue liquid flow was seen, and no holes in the vessel were observed. H-E staining revealed intact vascular endothelial cells and smooth muscles with no vascular wall defects. Low-frequency ultrasound combined with MBs can cause a vessel to rupture and holes to form in a short time. High-speed photography is useful for observing transient changes caused by the effects of ultrasound cavitation on an in vitro vessel.

The Combination of Glycolytic Inhibitor 2-Deoxyglucose and Microbubbles Increases the Effect of 5-Aminolevulinic Acid-Sonodynamic Therapy in Liver Cancer Cells

Sonodynamic therapy (SDT) overcomes the shortcoming of photodynamic therapy in the treatment of cancer. Previous studies indicated that the glycolysis inhibitor 2-deoxyglucose (2-DG) potentiated photodynamic therapy induced tumor cell death and microbubbles (MBs) improved the SDT performance. We hypothesized that the combination of 2-DG and MBs will increase the effect of 5-aminolevulinic acid (ALA)-SDT in HepG2 liver cancer cells. When cells were treated with 5-min ALA-SDT and 2-mmol/L 2-DG, the cell survival rate decreased to 73.0 ± 7.1% and 75.2 ± 7.9%, respectively. Furthermore, 2 mmol/L 2-DG increased 5-min ALA-SDT induced growth inhibition and augmented ALA-SDT induced cell apoptotic rate from 9.8 ± 0.7% to 17.4 ± 2.2%. In the combination group (2-DG and ALA-SDT group), HepG2 cells possessed typical apoptotic characters. 2-DG also increased ALA-SDT associated intracellular reactive oxygen species generation and loss of mitochondrial membrane potential. Moreover, SonoVue MBs had stimulatory function on cell viability inhibition, apoptosis, reactive oxygen species production and mitochondrial membrane potential loss for combination treatment. This study suggests a promising therapeutic strategy using a combination of 2-DG, MBs and ALA-SDT for treating liver cancer.

Effects of RNA interference combined with ultrasonic irradiation and SonoVue microbubbles on expression of STAT3 gene in keratinocytes of psoriatic lesions.

The most effective sequence of small interfering RNA (siRNA) silencing STAT3 of psoriatic keratinocytes (KCs) was screened out, and the effects of the most effective siRNA combined with ultrasonic irradiation and SonoVue microbubbles on the expression of STAT3 of KCs and the dose- and time-response were investigated. Three chemically-synthetic siRNAs targeting STAT3 carried by Lipofectamine 3000 were transfected into KCs, and the effects on STAT3 expression were detected, then the most effective siRNA was selected for the subsequent experiments. The negative controls of siRNA (siRNA-NC) labeled with Cy3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles were transfected into KCs, then the optimal parameters of ultrasonic irradiation were determined. The most effective siRNA carried by Li-pofectamine 3000 combined with ultrasonic irradiation at the optimal parameters and SonoVue microbubbles was transfected into KCs, and the dose- and time-response of RNA interference was determined. The effect of RNA interference by the most effective siRNA at the optimal time and dose carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (LUS group) was compared with that only carried by Li-pofectamine 3000 (L group). The results showed that siRNA-3 achieved the highest silencing efficacy. 0.5 W/cm2 and 30 s were selected as the parameters of ultrasonic irradiation. The siRNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles could effectively knock down the STAT3 expression at mRNA and protein levels in dose- and time-dependent manners determined at 100 nmol/L with maximum downregulation on mRNA at 48 h, and on protein at 72 h after transfection. The LUS group achieved the highest silencing efficacy. It was concluded that siRNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles could effectively knock down the STAT3 expression in psoriatic KCs, and the optimized transfection condition and the sequence of siRNA-3 could serve for further research on gene therapy of psoriasis.

Effect of acoustic parameters on the cavitation behavior of SonoVue microbubbles induced by pulsed ultrasound

SonoVue microbubbles could serve as artificial nuclei for ultrasound-triggered stable and inertial cavitation, resulting in beneficial biological effects for future therapeutic applications. To optimize and control the use of the cavitation of SonoVue bubbles in therapy while ensuring safety, it is important to comprehensively understand the relationship between the acoustic parameters and the cavitation behavior of the SonoVue bubbles. An agarose-gel tissue phantom was fabricated to hold the SonoVue bubble suspension. 1-MHz transmitting transducer calibrated by a hydrophone was used to trigger the cavitation of SonoVue bubbles under different ultrasonic parameters (i.e., peak rarefactional pressure (PRP), pulse repetition frequency (PRF), and pulse duration (PD)). Another 7.5-MHz focused transducer was employed to passively receive acoustic signals from the exposed bubbles. The ultraharmonics and broadband intensities in the acoustic emission spectra were measured to quantify the extent of stable and inertial cavitation of SonoVue bubbles, respectively. We found that the onset of both stable and inertial cavitation exhibited a strong dependence on the PRP and PD and a relatively weak dependence on the PRF. Approximate 0.25MPa PRP with more than 20μs PD was considered to be necessary for ultraharmonics emission of SonoVue bubbles, and obvious broadband signals started to appear when the PRP exceeded 0.40MPa. Moreover, the doses of stable and inertial cavitation varied with the PRP. The stable cavitation dose initially increased with increasing PRP, and then decreased rapidly after 0.5MPa. By contrast, the inertial cavitation dose continuously increased with increasing PRP. Finally, the doses of both stable and inertial cavitation were positively correlated with PRF and PD. These results could provide instructive information for optimizing future therapeutic applications of SonoVue bubbles.

Primary Porcine Brain Endothelial Cells as In Vitro Model to Study Effects of Ultrasound and Microbubbles on Blood-Brain Barrier Function.

Focused ultrasound (FUS) in the presence of microbubbles transiently and reversibly opens the blood-brain barrier (BBB) in rodents and humans, thereby providing a time window for increased drug delivery into brain tissue. To get insight into the underlying mechanisms that govern ultrasound (US)-mediated opening of the BBB, in vitro models are a useful alternative. In this paper, we have utilized an in vitro BBB model that consists of primary porcine brain endothelial cells (PBECs). PBEC monolayers are grown on permeable membranes, which allow assessment of key features of BBB function as well as US treatment. This experimental model is characterized by low permeability for both small molecules and proteins, has a high transendothelial electrical resistance, and expresses tight junctions and efflux pumps. Here, we compare the effects of inertial and stable cavitation in the presence of SonoVue microbubbles on PBEC monolayers' electrical resistance and permeability properties. Our results point out the fragility of PBEC monolayers, which enhances results variability. In particular, we show that handling of the inserts, such as medium change and transfer to the US setup, modifies the cellular response, and immunostaining of the monolayers introduces damage and cell detachment within the US-exposed monolayers. Our results indicate that stable cavitation might have a more pronounced impact on cell permeability as compared with inertial cavitation in vitro. This paper might contribute to further development of experimental setups that are suitable to characterize the impact of FUS and microbubbles on BBB properties in vitro.

Liposome-mediated transfection of wild-type P53 DNA into human prostate cancer cells is improved by low-frequency ultrasound combined with microbubbles.

Prostate cancer is a common type of cancer in elderly men. The aim of the present study was to evaluate the effects of ultrasound exposure in combination with SonoVue microbubbles on liposome-mediated transfection of wild-type P53 genes into human prostate cancer cells. PC-3 human prostate cancer cells were exposed to ultrasound; duty cycle was controlled at 20% (2 sec on, 8 sec off) for 5 min with and without SonoVue microbubble echo-contrast agent using a digital sonifier (frequency, 21 kHz; intensity, 46 mW/cm2). The cells were divided into eight groups, as follows: Group A (SonoVue + wild-type P53), group B (ultrasound + wild-type P53), group C (SonoVue + ultrasound + wild-type P53), group D (liposome + wild-type P53), group E (liposome + SonoVue + wild-type P53), group F (liposome + wild-type P53 + ultrasound), group G (liposome + wild-type P53 + ultrasound + SonoVue) and the control group (wild-type P53). Following treatment, a hemocytometer was used to measure cell lysis, reverse transcription-quantitative polymerase chain reaction and western blotting were performed to detect P53 gene transfection efficiency, Cell Counting Kit-8 was employed to reveal cell proliferation and Annexin V/propidium iodide staining was used to determine cell apoptosis. Cell lysis was minimal in each group. Wild-type P53 gene and protein expression were significantly increased in the PC-3 cells in group G compared with the control and all other groups (P<0.01). Cell proliferation was significantly suppressed in group G compared with the control group and all other groups (P<0.01). Cell apoptosis levels in group G were significantly improved compared with the control group and all other groups (P<0.01). Thus, the results of the present study indicate that the use of low-frequency and low-energy ultrasound in combination with SonoVue microbubbles may be a potent physical method for increasing liposome gene delivery efficiency.

Comparison of the synergistic effect of lipid nanobubbles and SonoVue microbubbles for high intensity focused ultrasound thermal ablation of tumors

Microbubbles (MBs) are considered as an important enhancer for high intensity focused ultrasound (HIFU) treatment of benign or malignant tumors. Recently, different sizes of gas-filled bubbles have been investigated to improve the therapeutic efficiency of HIFU thermal ablation and reduce side effects associated with ultrasound power and irradiation time. However, nanobubbles (NBs) as an ultrasound contrast agent for synergistic therapy of HIFU thermal ablation remain controversial due to their small nano-size in diameter. In this study, phospholipid-shell and gas-core NBs with a narrow size range of 500–600 nm were developed. The synergistic effect of NBs for HIFU thermal ablation was carefully studied both in excised bovine livers and in breast tumor models of rabbits, and made a critical comparison with that of commercial SonoVue microbubbles (SonoVue MBs). In addition, the pathological changes of the targeted area in tumor tissue after HIFU ablation were further investigated. Phosphate buffer saline (PBS) was used as the control. Under the same HIFU parameters, the quantitative echo intensity of B-mode ultrasound image and the volume of coagulative necrosis in lipid NBs groups were significantly higher and larger than that in PBS groups, but could not be demonstrated a difference to that in SonoVue MBs groups both ex vivo and in vivo. These results showed that the synergistic effect of lipid NBs for HIFU thermal ablation were similar with that of SonoVue MBs, and further indicate that lipid NBs could potentially become an enhancer for HIFU thermal ablation of tumors.

Precise spatial control of cavitation erosion in a vessel phantom by using an ultrasonic standing wave

In atherosclerotic inducement in animal models, the conventionally used balloon injury is invasive, produces excessive vessel injuries at unpredictable locations and is inconvenient in arterioles. Fortunately, cavitation erosion, which plays an important role in therapeutic ultrasound in blood vessels, has the potential to induce atherosclerosis noninvasively at predictable sites. In this study, precise spatial control of cavitation erosion for superficial lesions in a vessel phantom was realised by using an ultrasonic standing wave (USW) with the participation of cavitation nuclei and medium-intensity ultrasound pulses. The superficial vessel erosions were restricted between adjacent pressure nodes, which were 0.87mm apart in the USW field of 1MHz. The erosion positions could be shifted along the vessel by nodal modulation under a submillimetre-scale accuracy without moving the ultrasound transducers. Moreover, the cavitation erosion of the proximal or distal wall could be determined by the types of cavitation nuclei and their corresponding cavitation pulses, i.e., phase-change microbubbles with cavitation pulses of 5MHz and SonoVue microbubbles with cavitation pulses of 1MHz. Effects of acoustic parameters of the cavitation pulses on the cavitation erosions were investigated. The flow conditions in the experiments were considered and discussed. Compared to only using travelling waves, the proposed method in this paper improves the controllability of the cavitation erosion and reduces the erosion depth, providing a more suitable approach for vessel endothelial injury while avoiding haemorrhage.