Targeted Contrast-Enhanced Ultrasound Imaging of Angiogenesis in an Orthotopic Mouse Tumor Model of Renal Carcinoma

Previous studies have reported that microbubbles bearing targeting ligands to molecular markers of angiogenesis can be successfully detected by ultrasound imaging in various animal models of solid cancer. In the present study, we sought to investigate the activity of microbubbles targeted to vascular endothelial growth factor receptor 2 (VEGFR2) in an orthotopic model of renal cell carcinoma (RCC). Microbubbles conjugated to an anti-VEGFR2 antibody (MB_V) were compared with microbubbles conjugated to an isotype control antibody (MB_C) or naked microbubbles (MB_N). An orthotopic mouse model of human RCC was established by surgically implanting an established tumor within the renal capsule in mice. Tumor growth and blood flow were verified by B-mode and color Doppler ultrasound imaging. VEGFR2 expression within the tumor and renal parenchyma was detected by immunohistochemistry. The duration of contrast enhancement of MB_V was much longer than those of MB_N and MB_C when assessed over 10 min. The baseline-subtracted contrast intensity within the tumor was higher for MB_V than for MB_C and MB_N (p < 0.01). Additionally, the contrast intensity for MB_V was significantly higher in the tumor region than in normal parenchyma (p < 0.01). Microbubbles targeting VEGFR2 exhibit suitable properties for imaging angiogenesis in orthotopic models of renal cell carcinoma, with potential applications in life science research and clinical medicine.     

Targeting an Ultrasound Contrast Agent to Folate Receptors on Ovarian Cancer Cells

Objective. The purpose of the study was to synthesize and characterize folate‐targeted microbubbles (MB_F) as an ultrasound contrast agent and to evaluate their affinity to the folate receptor (FR) in vitro. Methods. Folate‐targeted microbubbles were prepared by incorporating 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[amino(polyethylene glycol)‐2000]‐folate into the lipid membrane of microbubbles. The diameter and concentration of the MB_F were determined by a cell counter and sizer. The MB_F, control microbubbles (MB_C), and MB_F with a free folic acid block‐ade were tested for binding specificity to human ovarian carcinoma SKOV3 cells, which overexpress the FR, by microscopy and confocal imaging, respectively. Results. The basic physical characteristics of MB_F were similar to those of MB_C. In the cell binding test, the adherence efficiency of MB_F to the SKOV3 cells (mean ± SD, 16 ± 5 microbubbles per cell; P < .01) was significantly higher than that of MB_C (0.7 ± 0.4 microbubbles per cell) or MB_F with the free folic acid blockade (0.7 ± 0.6 microbubbles per cell). Conclusions. Folate‐targeted microbubbles showed high affinity to SKOV3 cells with FR overexpression. They are potentially useful for ultrasonic molecular imaging and treatment of FR‐positive tumors and warrant further investigation.     

自制纳米级E-选择素靶向超声微泡对缺血心肌分子成像

目的 采用自制的携E-选择素抗体的纳米级靶向超声造影剂对大鼠缺血再灌注损伤后的缺血心肌进行超声分子成像,评估其检测缺血心肌的可行性。方法 采用生物素-链亲和素方法制备纳米级靶向微泡,应用荧光显微镜及流式细胞仪检测微泡形态及抗体连接效率;将26只SD大鼠随机分为E-选择素靶向微泡组(MB_E组,n=10)、IGg抗体微泡组(MB_IGg组,n=10)、普通微泡组(MB_C组,n=6),制备心肌缺血再灌注损伤模型,将冠状动脉左前降支阻断15 min后恢复灌注,于再灌注后6 h经尾静脉分别注射3种微泡行CEUS。后行病理学检查。结果 MB_E组缺血区域声学强度(VI)显著高于非缺血区(P<0.05),也显著高于MB_C组、MB_IGg组缺血区域的VI(P<0.05);MB_C组、MB_IGg组缺血区域与非缺血区的VI差异无统计学意义(P>0.05)。结论 纳米级E-选择素靶向超声微泡可以早期检测到缺血心肌,有望实现缺血心肌的"记忆"成像。     

Comparison of Magnetic Microbubbles and Dual-modified Microbubbles Targeted to P-selectin for Imaging of Acute Endothelial Inflammation in the Abdominal Aorta

Purpose

Ultrasound molecular imaging (UMI) has potential to evaluate an inflammatory profile of endothelium. However, it is less successful in large arteries. This study compared magnetic microbubbles (MBs) selectively targeted to endothelial P-selectin and dual-targeting MBs in vitro and in vivo.

Procedures

MBs were modified with P-selectin antibody (MB_PM) or isotype control antibody (MB_CM) via a magnetic streptavidin bridge, and MBs were conjugated to P-selectin antibody (MB_P) or both P-selectin antibody and PAA-sialyl Lewis^x (MB_D) via regular streptavidin linker. Adherence of MBs was determined by using a parallel plate flow chamber at variable shear stress (0.5–24 dyn/cm²). Adhesive and magnetic behaviors of MBs were analyzed at 4.0 dyn/cm² or at a flow rate of 50 mm/s. Attachment of MBs to P-selectin was determined with contrast-enhanced ultrasound (CEU) imaging of murine abdominal aorta inflammation. The expression of P-selectin was assessed by immunohistochemistry.

Results

The adhesive efficacy of MB_D was greater than MB_P and MB_CM, but lower than MB_PM under all shear stress conditions (P?<?0.05). The behaviors of fast-binding and rolling slow down were noted in MB_D and MB_PM; meanwhile, magnetic shifting of MBs centerline was presented in MB_PM. Contrast video intensity (VI) from adhered MB_PM to P-selectin of the inflammatory aorta was significantly higher than those from MB_D and MB_P (P?<?0.05).

Conclusions

MB_PM may be a better molecular probe than MB_D for detection of P-selectin on aorta with CEU, likely due to the shifting of axial distribution. Thus, it may improve the detection of the inflammatory profile on large arteries by UMI.

     

对比液态氟碳脂质纳米粒与全氟丙烷脂质微泡体外显影及耐声压性

目的 与全氟丙烷（C₃F₈）脂质微泡造影剂比较,分析自制液态氟碳（PFOB）脂质纳米粒体外显影及耐声压性的优劣。方法 分别制备生物素化PFOB脂质纳米粒及生物素化C₃F₈脂质微泡,评估其稳定性,并观察其加入亲和素前后的体外显影效果。对2种造影剂在低声压（MI=0.28）及高声压（MI=0.56）环境下进行超声辐照,于辐照前及辐照10、20、30 s后观察显影情况及其差异。结果 2种造影剂加入亲和素后均发生聚集现象,粒径均较加入亲和素前明显增大（P均<0.05）;且加入亲和素前（t=16.225,P<0.001）、后2种造影剂间粒径差异均有统计学意义（t=-5.046,P<0.001）。稳定性观察期间PFOB脂质微粒内浓度无明显改变,而C₃F₈脂质微泡随放置时间延长浓度呈减低趋势。加入亲和素后,PFOB脂质纳米粒回声明显增强;C₃F₈脂质微泡加入亲和素前后显影效果均较好。低声压（MI=0.28）及高声压（MI=0.56）环境下,PFOB脂质纳米粒造影剂显影强度无明显改变,而C₃F₈脂质微泡显影强度随辐照时间延长呈减低趋势。结论 相较于C₃F₈脂质微泡,PFOB纳米脂质纳米粒造影剂粒径小、耐声压性好,更符合靶向超声造影剂的要求。     

一种新型靶向微泡-干细胞复合体的制备

[摘 要] 目的 制备一种新型靶向微泡-干细胞复合体。方法 采用“生物素-亲和素”桥连法构建携抗ICAM-1抗体的靶向微泡（MBICAM-1）。用带正电荷的多聚赖氨酸（PLL）对MBICAM-1进行包覆和修饰,使靶向微泡表面带正电荷。采用静电吸附法将PLL修饰的MBICAM-1与大鼠骨髓间充质干细胞（MSC）共同孵育,制备MBICAM-1-MSC复合体,流式细胞仪检测靶向微泡与MSC的结合率。结果 MBICAM-1经PLL修饰后,微泡表面带正电荷,但微泡形态、粒径以及与损伤内皮细胞（HUVEC）的结合率与未经PLL修饰的MBICAM-1无显著性差异（P＞0.05）。PLL修饰的MBICAM-1与MSC结合形成MBICAM-1-MSC复合体,且MSC与MBICAM-1之间的比例为1:40时,二者之间结合率达（29.45±2.88）%。结论 成功制备MBICAM-1-MSC复合体,当MSC与MBICAM-1之间的比例为1:40时,其结合率最大。 [关键词] 靶向微泡;骨髓间充质干细胞;复合体     

Ultrasound-Mediated Microbubble Enhancement of Radiation Therapy Studied Using Three-Dimensional High-Frequency Power Doppler Ultrasound

Tumor responses to high-dose (>8 Gy) radiation therapy are tightly connected to endothelial cell death. In the study described here, we investigated whether ultrasound-activated microbubbles can locally enhance tumor response to radiation treatments of 2 and 8 Gy by mechanically perturbing the endothelial lining of tumors. We evaluated vascular changes resulting from combined microbubble and radiation treatments using high-frequency 3-D power Doppler ultrasound in a breast cancer xenograft model. We compared treatment effects and monitored vasculature damage 3 hours, 24 hours and 7 days after treatment delivery. Mice treated with 2 Gy radiation and ultrasound-activated microbubbles exhibited a decrease in vascular index to 48 ± 10% at 24 hours, whereas vascular indices of mice treated with 2 Gy radiation alone or microbubbles alone were relatively unchanged at 95 ± 14% and 78 ± 14%, respectively. These results suggest that ultrasound-activated microbubbles enhance the effects of 2 Gy radiation through a synergistic mechanism, resulting in alterations of tumor blood flow. This novel therapy may potentiate lower radiation doses to preferentially target endothelial cells, thus reducing effects on neighboring normal tissue and increasing the efficacy of cancer treatments.     

Dynamic Filtering of Adherent and Non-adherent Microbubble Signals Using Singular Value Thresholding and Normalized Singular Spectrum Area Techniques

Ultrasound molecular imaging techniques rely on the separation and identification of three types of signals: static tissue, adherent microbubbles and non-adherent microbubbles. In this study, the image filtering techniques of singular value thresholding (SVT) and normalized singular spectrum area (NSSA) were combined to isolate and identify vascular endothelial growth factor receptor 2-targeted microbubbles in a mouse hindlimb tumor model (n = 24). By use of a Verasonics Vantage 256 imaging system with an L12-5 transducer, a custom-programmed pulse inversion sequence employing synthetic aperture virtual source element imaging was used to collect contrast images of mouse tumors perfused with microbubbles. SVT was used to suppress static tissue signals by 9.6 dB while retaining adherent and non-adherent microbubble signals. NSSA was used to classify microbubble signals as adherent or non-adherent with high accuracy (receiver operating characteristic area under the curve [ROC AUC] = 0.97), matching the classification performance of differential targeted enhancement. The combined SVT + NSSA filtering method also outperformed differential targeted enhancement in differentiating MB signals from all other signals (ROC AUC = 0.89) without necessitating destruction of the contrast agent. The results from this study indicate that SVT and NSSA can be used to automatically segment and classify contrast signals. This filtering method with potential real-time capability could be used in future diagnostic settings to improve workflow and speed the clinical uptake of ultrasound molecular imaging techniques.     

Ultrasound and Microbubble-Induced Intra- and Intercellular Bioeffects in Primary Endothelial Cells

Recent developments in the field of ultrasound (US) contrast agents have demonstrated that these encapsulated microbubbles can not only be used for diagnostic imaging but may also be employed as therapeutic carriers for localized, targeted drug or gene delivery. The exact mechanisms behind increased uptake of therapeutic compounds by US-exposed microbubbles are still not fully understood. Therefore, we studied the effects of stably oscillating SonoVue microbubbles on relevant parameters of cellular and intercellular permeability, i.e., reactive oxygen species (ROS) homeostasis, calcium permeability, F-actin cytoskeleton, monolayer integrity and cell viability using live-cell fluorescence microscopy. US was applied at 1-MHz, 0.1 MPa peak-negative pressure, 0.2% duty cycle and 20 Hz pulse repetition frequency to primary endothelial cells. We demonstrated increased membrane permeability for calcium ions, with an important role for H₂O₂. Catalase, an extracellular H₂O₂ scavenger, significantly blocked the influx of calcium ions. Further changes in ROS homeostasis involved an increase in intracellular H₂O₂ levels, protein nitrosylation and a decrease in total endogenous glutathione levels. In addition, an increase in the number of F-actin stress fibers and F-actin cytoskeletal rearrangement were observed. Furthermore, US-exposed microbubbles significantly affected endothelial monolayer integrity, but importantly, disrupted cell-cell interactions were restored within 30 min. Finally, cell viability was not affected. In conclusion, these data provide more insight in the interactions between US, microbubbles and endothelial cells, which is important for understanding the mechanisms behind US and microbubble-enhanced uptake of drugs or genes. (E-mail: ljm.juffermans@vumc.nl)     

Characterization of Submicron Phase-change Perfluorocarbon Droplets for Extravascular Ultrasound Imaging of Cancer

Because many tumors possess blood vessels permeable to particles with diameters of 200 nm, it is possible that submicron perfluorocarbon droplets could constitute a novel extravascular ultrasound contrast agent capable of selectively enhancing tumors. Under exposure to bursts of ultrasound of sufficient rarefactional pressure, droplets can undergo vaporization to form echogenic microbubbles. In this study, phase-change thresholds of 220-nm–diameter droplets composed of perfluoropentane were studied in polyacrylamide gel phantoms maintained at temperatures of 21–37°C, exposed to high-pressure bursts of ultrasound with frequencies ranging from 5–15 MHz and durations of 1 μs to 1 ms. The thresholds were found to depend inversely and significantly (p < 0.001) on ultrasound frequency, pulse duration, and droplet temperature, ranging from 9.4 ± 0.8 MPa at 29°C for a 1-μs burst at 5 MHz to 3.2 ± 0.5 MPa at 37°C for a 1-ms burst at 15 MHz. The diameters of microbubbles formed from droplets decreased significantly as phantom stiffness increased (p < 0.0001), and were independent of pulse duration, although substantially more droplets were converted to microbubbles for 1-ms pulse durations compared with briefer exposures. In vivo experiments in a mouse tumor model demonstrated that intravenously injected droplets can be converted into highly echogenic microbubbles 1 h after administration.     

Microbubble contrast imaging of the cardiovascular system of the chick embyro

Ultrasound imaging of the chick embryo cardiovascular system is limited to B-scan and Doppler technologies. This study demonstrates microbubble contrast imaging of the embryonic cardiovascular anatomy and physiology. Day 8–19 (Hamburger &; Hamilton Stage 34–43) chick embryos are examined in ovo using high-frequency ultrasound imaging through an opening in the blunt end (air cell) of the egg. A chorioallantoic vein is cannulated, and small boluses of octofluoropropane lipid microspheres (Definity®) are injected to visualize the chick embryo cardiovascular system. The entire chick embryo cardiovascular system including the two embryologic arteriovenous (AV) shunts can be visualized. More accurate physiologic measurements of ejection fractions and cardiac output measurements can be obtained using this technology. Microbubble contrast ultrasound imaging in the chick embryo greatly expands the ability to study cardiovascular development. Also, the two natural embryonic A-V shunts provide an excellent model to study the bioeffects of microbubbles in the arterial system.     

Quantitative Guidelines for the Prediction of Ultrasound Contrast Agent Destruction During Injection

Experiments and theory were undertaken on the destruction of ultrasound contrast agent microbubbles on needle injection, with the aim of predicting agent loss during in vivo studies. Agents were expelled through a variety of syringe and needle combinations, subjecting the microbubbles to a range of pressure drops. Imaging of the bubbles identified cases where bubbles were destroyed and the extent of destruction. Fluid-dynamic calculations determined the pressure drop for each syringe and needle combination. It was found that agent destruction occurred at a critical pressure drop that depended only on the type of microbubble. Protein-shelled microbubbles (sonicated bovine serum albumin) were virtually all destroyed above their critical pressure drop of 109 ± 7 kPa Two types of lipid-shelled microbubbles were found to have a pressure drop threshold above which more than 50% of the microbubbles were destroyed. The commercial lipid-shelled agent Definity was found to have a critical pressure drop for destruction of 230 ± 10 kPa; for a previously published lipid-shelled agent, this value was 150 ± 40 kPa. It is recommended that attention to the predictions of a simple formula could preclude unnecessary destruction of microbubble contrast agent during in vivo injections. This approach may also preclude undesirable release of drug or gene payloads in targeted microbubble therapies. Example values of appropriate injection rates for various agents and conditions are given.     

Acoustic Radiation Force for Vascular Cell Therapy: In Vitro Validation

Cell-based therapeutic approaches are attractive for the restoration of the protective endothelial layer in arteries affected by atherosclerosis or following angioplasty and stenting. We have recently demonstrated a novel technique for the delivery of mesenchymal stem cells (MSCs) that are surface-coated with cationic lipid microbubbles (MBs) and displaced by acoustic radiation force (ARF) to a site of arterial injury. The objective of this study was to characterize ultrasound parameters for effective acoustic-based delivery of cell therapy. In vitro experiments were performed in a vascular flow phantom where MB-tagged MSCs were delivered toward the phantom wall using ARF generated with an intravascular ultrasound catheter. The translation motion velocity and adhesion of the MB-cell complexes were analyzed. Experimental data indicated that MSC radial velocity and adhesion to the vessel phantom increased with the time-averaged ultrasound intensity up to 1.65 W/cm², after which no further significant adhesion was observed. Temperature increase from baseline near the catheter was 5.5 ± 0.8°C with this setting. Using higher time-averaged ultrasound intensities may not significantly benefit the adhesion of MB-cell complexes to the target vessel wall (p = NS), but could cause undesirable biologic effects such as heating to the MB-cell complexes and surrounding tissue. For the highest time-averaged ultrasound intensity of 6.60 W/cm², the temperature increase was 11.6 ± 1.3°C.     

Subharmonic,Non-linear Fundamental and Ultraharmonic Imaging of Microbubble Contrast at High Frequencies

There is increasing use of ultrasound contrast agent in high-frequency ultrasound imaging. However, conventional contrast detection methods perform poorly at high frequencies. We performed systematic in vitro comparisons of subharmonic, non-linear fundamental and ultraharmonic imaging for different depths and ultrasound contrast agent concentrations (Vevo 2100 system with MS250 probe and MicroMarker ultrasound contrast agent, VisualSonics, Toronto, ON, Canada). We investigated 4-, 6- and 10-cycle bursts at three power levels with the following pulse sequences: B-mode, amplitude modulation, pulse inversion and combined pulse inversion/amplitude modulation. The contrast-to-tissue (CTR) and contrast-to-artifact (CAR) ratios were calculated. At a depth of 8 mm, subharmonic pulse-inversion imaging performed the best (CTR = 26 dB, CAR = 18 dB) and at 16 mm, non-linear amplitude modulation imaging was the best contrast imaging method (CTR = 10 dB). Ultraharmonic imaging did not result in acceptable CTRs and CARs. The best candidates from the in vitro study were tested in vivo in chicken embryo and mouse models, and the results were in a good agreement with the in vitro findings.     

Microbubble Type and Distribution Dependence of Focused Ultrasound-Induced Blood–Brain Barrier Opening

Focused ultrasound, in the presence of microbubbles, has been used non-invasively to induce reversible blood–brain barrier (BBB) opening in both rodents and non-human primates. This study was aimed at identifying the dependence of BBB opening properties on polydisperse microbubble (all clinically approved microbubbles are polydisperse) type and distribution by using a clinically approved ultrasound contrast agent (Definity microbubbles) and in-house prepared polydisperse (IHP) microbubbles in mice. A total of 18 C57 BL/6 mice (n = 3) were used in this study, and each mouse was injected with either Definity or IHP microbubbles via the tail vein. The concentration and size distribution of activated Definity and IHP microbubbles were measured, and the microbubbles were diluted to 6 × 10⁸/mL before injection. Immediately after microbubble administration, mice were subjected to focused ultrasound with the following parameters: frequency = 1.5 MHz, pulse repetition frequency = 10 Hz, 1000 cycles, in situ peak rarefactional acoustic pressures = 0.3, 0.45 and 0.6 MPa for a sonication duration of 60 s. Contrast-enhanced magnetic resonance imaging was used to confirm BBB opening and allowed for image-based analysis. Permeability of the treated region and volume of BBB opening did not significantly differ between the two types of microbubbles (p > 0.05) at peak rarefractional acoustic pressures of 0.45 and 0.6 MPa, whereas IHP microbubbles had significantly higher permeability and opening volume (p < 0.05) at the relatively lower pressure of 0.3 MPa. The results from this study indicate that microbubble type and distribution could have significant effects on focused ultrasound-induced BBB opening at lower pressures, but less important effects at higher pressures, possibly because of the stable cavitation that governs the former. This difference may have become less significant at higher pressures, where inertial cavitation typically occurs.     

Ultrasound Bio-microscopy for Measurement of Coronary Artery Flow and Estimation of Infarct Size in a Mouse Model of Acute Myocardial Infarction

Ultrasound bio-microscopy was used to measure hemodynamic changes in the left main coronary artery after myocardial infarction (MI), and its usefulness in estimating infarct size was evaluated. MI was induced by left anterior descending artery ligation. Diastolic peak velocity (V_d), mean flow velocity (V_mean) and the velocity-time integral (VTI) were measured 2 and 6 h after MI. Serum troponin I levels were assayed 2, 6 and 12 h after MI. At 2 h, V_mean and VTI significantly differed between mice that underwent low and high left anterior descending artery ligation; V_d, V_mean and VTI were correlated with infarct size (r = −0.557, −0.693 and −0.672, respectively; all p < 0.01). Infarct size was more strongly correlated with 2-h ultrasound bio-microscopy measurements than with 2-h serum troponin I level. Measurement of coronary artery blood flow by ultrasound bio-microscopy may be useful for early estimation of infarct size in mice.     

High-Throughput,High-Frequency 3-D Ultrasound for in Utero Analysis of Embryonic Mouse Brain Development

With the emergence of the mouse as the predominant model system for studying mammalian brain development, in utero imaging methods are urgently required to analyze the dynamics of brain growth and patterning in mouse embryos. To address this need, we combined synthetic focusing with a high-frequency (38-MHz) annular-array ultrasound imaging system for extended depth-of-field, coded excitation for improved penetration and respiratory-gated transmit/receive. This combination allowed non-invasive in utero acquisition of motion-free 3-D data from individual embryos in approximately 2 min, and data from four or more embryos in a pregnant mouse in less than 30 min. Data were acquired from 148 embryos spanning 5 d of early to mid-gestational stages of brain development. The results indicated that brain anatomy and cerebral vasculature can be imaged with this system and that quantitative analyses of segmented cerebral ventricles can be used to characterize volumetric changes associated with mouse brain development.     

Effect of Microbubble Size on Fundamental Mode High Frequency Ultrasound Imaging in Mice

High-frequency ultrasound imaging using microbubble (MB) contrast agents is becoming increasingly popular in pre-clinical and small animal studies of anatomy, flow and vascular expression of molecular epitopes. Currently, in vivo imaging studies rely on highly polydisperse microbubble suspensions, which may provide a complex and varied acoustic response. To study the effect of individual microbubble size populations, microbubbles of 1–2 μm, 4–5 μm and 6–8 μm diameter were isolated using the technique of differential centrifugation. Size-selected microbubbles were imaged in the mouse kidney over a range of concentrations using a Visualsonics Vevo 770 ultrasound imaging system (Visualsonics, Toronto, Ontario, Canada) with a 40-MHz probe in fundamental mode. Results demonstrate that contrast enhancement and circulation persistence are strongly dependent on microbubble size and concentration. Large microbubbles (4–5 and 6–8 μm) strongly enhanced the ultrasound image with positive contrast, while 1–2 μm microbubbles showed little enhancement. For example, the total integrated contrast enhancement, measured by the area under the time-intensity curve (AUC), increased 16-fold for 6–8 μm diameter microbubbles at 5 × 10⁷ MB/bolus compared with 4–5 μm microbubbles at the same concentration. Interestingly, 1–2 μm diameter microbubbles, at any concentration, did not measurably enhance the integrated ultrasound signal at tissue depth, but did noticeably attenuate the signal, indicating that they had a low scattering-to-attenuation ratio. When concentration matched, larger microbubbles were more persistent in circulation. However, when volume matched, all microbubble sizes had a similar circulation half-life. These results indicated that dissolution of the gas core plays a larger role in contrast elimination than filtering by the lungs and spleen. The results of this study show that microbubbles can be tailored for optimal contrast enhancement in fundamental mode imaging. (E-mail: mb2910@columbia.edu)     

A Comparison of Sonothrombolysis in Aged Clots between Low-Boiling-Point Phase-Change Nanodroplets and Microbubbles of the Same Composition

We present enhanced cavitation erosion of blood clots exposed to low-boiling-point (−2°C) perfluorocarbon phase-change nanodroplets and pulsed ultrasound, as well as microbubbles with the same formulation under the same conditions. Given prior success with microbubbles as a sonothrombolysis agent, we considered that perfluorocarbon phase-change nanodroplets could enhance clot disruption further beyond that achieved with microbubbles. It has been hypothesized that owing to their small size and ability to penetrate into a clot, nanodroplets could enhance cavitation inside a blood clot and increase sonothrombolysis efficacy. The thrombolytic effects of lipid-shell-decafluorobutane nanodroplets were evaluated and compared with those of microbubbles with the same formulation, in an aged bovine blood clot flow model. Seven different pulsing schemes, with an acoustic intensity (I_SPTA) range of 0.021–34.8 W/cm² were applied in three different therapy scenarios: ultrasound only, ultrasound with microbubbles and ultrasound with nanodroplets (n = 5). Data indicated that pulsing schemes with 0.35 W/cm² and 5.22 W/cm² produced a significant difference (p < 0.05) in nanodroplet sonothrombolysis performance compared with compositionally identical microbubbles. With these excitation conditions, nanodroplet-mediated treatment achieved a 140% average thrombolysis rate over the microbubble-mediated case. We observed distinctive internal erosion in the middle of bovine clot samples from nanodroplet-mediated ultrasound, whereas the microbubble-mediated case generated surface erosion. This erosion pattern was supported by ultrasound imaging during sonothrombolysis, which revealed that nanodroplets generated cavitation clouds throughout a clot, whereas microbubble cavitation formed larger cavitation clouds only outside a clot sample.     

Contributions of mechanical and sonochemical effects to cell membrane damage induced by single-shot pulsed ultrasound with adjacent microbubbles

Purpose  The objective was to investigate the contributions of mechanical effects due to kinetic force induced by the dynamic behavior of microbubbles and sonochemical effects due to free radicals produced by inertial cavitation to cell membrane damage under sonoporation conditions in which cells with adjacent microbubbles were irradiated with single-shot pulsed ultrasound. Methods  The free radical scavenger cysteamine was used to control the occurrence of sonochemical effects, and the ratios of cells with membrane damage to intact cells were compared in the presence and absence of cysteamine. To determine the optimal dose of cysteamine, free radical production on exposure to burst pulse ultrasound was investigated using KI-starch solutions with different concentrations (0–5 mM) of cysteamine. High-speed observation of the dynamic behavior of Levovist microbubbles during ultrasound exposure was also carried out in the presence and absence of cysteamine, and the difference in the ratios of the maximum bubble diameter to the initial diameter was evaluated. Next, human prostate cancer cells with adjacent Levovist microbubbles were exposed to single-shot pulsed ultrasound with a center frequency of 1 MHz, a peak negative pressure of 1.1 MPa, and a pulse width of 3 μs, and the percentages of cells with membrane damage were evaluated by fluorescent microscopy using propidium iodide in the presence and absence of cysteamine. Results  It was confirmed that cysteamine at a concentration of 5 mM completely suppressed sonochemical effects without causing a change in the dynamic response of microbubbles to pulsed ultrasound. The percentages of cells with membrane damage in the presence and absence of cysteamine (5 mM) were 10.3% ± 4.1% (n = 13) and 8.7% ± 3.9% (n = 9), respectively. No significant difference was found (P = 0.36). Conclusion  The results indicate that cell membrane damage induced by single-shot pulsed ultrasound with adjacent microbubbles was due mainly to mechanical effects, not to sonochemical effects.