Dynamic contrast-enhanced ultrasound with carbon dioxide microbubbles as adjunct to arteriography of liver tumors

Purpose The goal of the study is to evaluate utility of contrast enhanced ultrasound (US) with carbon dioxide microbubbles in evaluation of hepatic lesions.Methods Twenty eight patients with single or multiple t hepatic lesions (11 hepatocellular carcinoma, 8 hemangiomas, 5 metastases, 1 adenoma, 1 focal nodular hyperplasia, 2 regenerative nodules) were examined. US exam was performed during intraarterial injection of 10 ml of CO₂ through the same catheter employed for liver arteriography. The US exam was videotaped in its salient phases. Characteristics of enhancement were evaluated and correlated with histological findings or patient follow up.Results Sonographic angiography clearly demonstrated vascularization of the lesions. Hepatocellular carcinoma, hemangioma, metastases, focal nodular hyperplasia, and regenerative nodules had very characteristic patterns. The injection of CO₂ allowed detection of small additional nodules.Conclusion Sonographic angiography can improve characterization and staging of hepatic tumors. Low cost and the simplicity of the technique should encourage further experimentation.     

Generation of Reactive Oxygen Species in Heterogeneously Sonoporated Cells by Microbubbles with Single-Pulse Ultrasound

To develop and realize sonoporation-based macromolecule delivery, it is important to understand the underlying cellular bioeffects involved. It is known that an appropriate level of reactive oxygen species (ROS) is necessary to maintain normal physiologic function, but excessive ROS triggers adverse downstream bioeffects. However, it is still unclear whether a relationship exists between intracellular ROS levels and sonoporation. Using a customized platform for 1.5-MHz ultrasound exposure (13.33 µs duration and 0.70?MPa peak negative pressure) and imaging the dynamics of sonoporation and intracellular ROS at the single-cell level, we quantified the exogenous molecular uptake and the concentration of intracellular ROS indicator to evaluate the extent of sonoporation and ROS change, respectively. Our results revealed that the intracellular ROS level was correlated with the degree of the sonoporation. (i) Within ~120?s of the onset of ultrasound, during which membrane perforation and complete membrane resealing occurred, intracellular ROS rapidly decreased because of extracellular diffusion of dichlorofluorescein through the perforated membrane and positively correlated with the degree of the sonoporation. (ii) In the following 270?s (120–390?s post-exposure), ROS generation in reversibly sonoporated cells gradually increased and was positively correlated with the degree of the sonoporation. (iii) The ROS level in irreversibly sonoporated cells reduced to depletion during this time interval. It is possible that ROS generation in reversibly sonoporated cells can impact their long-term fate. These results thus provide new insight into the biological response to sonoporation.     

Persistent Enhancement on Contrast-Enhanced Ultrasound Studies of Severe Crohn's Disease: Stuck Bubbles?

A small population of patients with severe Crohn's disease (CD) exhibit atypical lack of intensity decline on intestinal contrast-enhanced ultrasound. From a retrospective CD cohort examined with contrast-enhanced ultrasound, 104 patients were identified. Twenty study patients with severe active disease exhibited high peak enhancement (>23 dB) and minimal decline. From the same cohort, 84 control patients also exhibited high peak enhancement >23dB, but with typical intensity decline. Patient outcomes were assessed. Time–intensity curve analysis revealed a significantly higher (p < 0.0001) area under the curve (44.7 ± 1.5 dB·s), washout time and intensities at 60s and 120s in the study population compared with controls (40.0 ± 1.1 dB·s). Study patients had a worse overall outcome with surgery in 30% versus 10% (p?=?0.027) during follow-up. Heightened enhancement with lack of decline on contrast-enhanced ultrasound suggests microbubbles are stuck within the inflamed bowel wall for an extended period. This observation occurs in patients with severe disease and a bad outcome.     

Impact of Filling Gas on Subharmonic Emissions of Phospholipid Ultrasound Contrast Agents

Subharmonic signals backscattered from gas-filled lipid-shelled microbubbles have generated significant research interest because they can improve the detection and sensitivity of contrast-enhanced ultrasound imaging. However, the emission of subharmonic signals is strongly characterized by a temporal dependence, the origins of which have not been sufficiently elucidated. The features that influence subharmonic emissions need to be identified not only to better develop next-generation microbubble contrast agents, but also to develop more efficient subharmonic imaging (SHI) modes and therapeutic strategies. We examined the effect of microbubble filling gas on subharmonic emissions. Phospholipid shelled-microbubbles with different gaseous compositions such as sulfur hexafluoride (SF₆), octafluoropropane (C₃F₈) or decafluorobutane (C₄F₁₀), nitrogen (N₂)/C₄F₁₀ or air were insonated using a driving frequency of 10 MHz and peak negative pressure of 450 kPa, and their acoustic responses were tracked by monitoring both second harmonic and subharmonic emissions. Microbubbles were first acoustically characterized with their original gas and then re-characterized after substitution of the original gas with air, SF₆ or C₄F₁₀. A measureable change in intensity of the subharmonic emissions with a 20- to 40-min delayed onset and increasing subharmonic emissions of the order 12–18 dB was recorded for microbubbles filled with C₄F₁₀. Substitution of C₄F₁₀ with air eliminated the earlier observed delay in subharmonic emissions. Significantly, substitution of SF₆ for C₄F₁₀ successfully triggered a delay in the subharmonic emissions of the resultant agents, whereas substitution of C₄F₁₀ for SF₆ eliminated the earlier observed suppression of subharmonic emissions, clearly suggesting that the type of filling gas contained in the microbubble agent influences subharmonic emissions in a time-dependent manner. Because our agents were dispersed in air-stabilized phosphate-buffered saline, these results suggest that the diffusivity of the gas from the agent to the surrounding medium is correlated with the time-dependent evolution of subharmonic emissions.     

Mechanistic Insight into Sonoporation with Ultrasound-Stimulated Polymer Microbubbles

Sonoporation is emerging as a feasible, non-viral gene delivery platform for the treatment of cardiovascular disease and cancer. Despite promising results, this approach remains less efficient than viral methods. The objective of this work is to help substantiate the merit of polymeric microbubble sonoporation as a non-viral, localized cell permeation and payload delivery strategy by taking a ground-up approach to elucidating the fundamental mechanisms at play. In this study, we apply simultaneous microscopy of polymeric microbubble sonoporation over its intrinsic biophysical timescales–with sub-microsecond resolution to examine microbubble cavitation and millisecond resolution over several minutes to examine local macromolecule uptake through enhanced endothelial cell membrane permeability–bridging over six orders of magnitude in time. We quantified microbubble behavior and resulting sonoporation thresholds at transmit frequencies of 0.5, 1 and 2 MHz, and determined that sonic cracking is a necessary but insufficient condition to induce sonoporation. Further, sonoporation propensity increases with the extent of sonic cracking, namely, from partial to complete gas escape from the polymeric encapsulation. For the subset that exhibited complete gas escape from sonic cracking, a proportional relationship between the maximum projected gas area and resulting macromolecule uptake was observed. These results have revealed one aspect of polymeric bubble activity on the microsecond time scale that is associated with eliciting sonoporation in adjacent endothelial cells, and contributes toward an understanding of the physical rationale for sonoporation with polymer-encapsulated microbubble contrast agents.     

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.     

超声微泡携基因治疗肝脏疾病应用进展

超声靶向微泡破坏技术通过空化效应有效促进外源基因在目的组织中的转染,可通过抑制细胞某些基因的表达或抑制其信号通路而进行基因治疗,使基因治疗各类肝脏疾病逐渐成为可能。本文主要就超声微泡携基因在肝脏疾病中的应用进展进行综述。     

载BDNF脂质体纳米微粒脂膜微泡超声造影剂的制备与初步评价

目的 探索制备一种新型的耦连包载BDNF脂质体纳米颗粒的脂膜微泡超声造影剂(BDNF-UMCA)。方法 以冷冻干燥法在“脂氟显”制备的基础上加入一定比例的含生物素化棕榈酰磷脂酰甘油钠-聚乙二醇2000-生物素[DPPG-PEG(2000)-Biotin]制备含生物素的脂质超声微泡造影剂,通过链亲和素来耦连含生物素化PEG载BDNF脂质体纳米微粒制备BDNF-UMCA,检测其理化性质、载药量、包封率、稳定性和体内声学特性进行检测。结果 BDNF-UMCA平均粒径4.2±0.79 μm,浓度为1.02×10₉/ml;总药物含量为1.18±1.96 mg/mL,包封率为71.6±2.6%;BDNF-UMCA在4℃条件下,平均粒径和包封率均无明显的变化;在24℃条件下,载BDNF脂质体纳米微粒的平均粒径随时间逐渐增大,第1、3、5、7天的粒径与初始粒径比较具有明显的统计学差异(均P<0.05),包封率在24℃下各个时间点无明显的统计学差异(均P>0.05);BDNF-UMCA能显著增强实验动物肝脏显影,平均峰值强度为21.4±0.9 dB,平均达峰时间为4.7±0.4 s。结论 应用生物素-亲和素偶联可成功制备载BDNF脂质体纳米微粒的脂膜超声微泡造影剂,为靶向显影及药物通过血脑屏障释放提供工具。     

超声微泡靶向递送aFGF对糖尿病心肌病的治疗作用及其机制研究

 目的：观察SonoVue超声微泡靶向递送酸性成纤维细胞生长因子（aFGF）对糖尿病心肌病（DCM）大鼠左室舒缩功能的保护作用并初步探讨其机制。方法：24只健康雄性SD大鼠通过腹腔注射链脲佐菌素建立DCM模型,再随机平均分成DCM组与aFGF治疗组。另选择正常对照组12只。aFGF治疗组经尾静脉注射SonoVue-aFGF溶液并同时给予心肌定点超声辐照。干预后4周对所有大鼠行心导管检查,测定左室收缩末压力(LVESP)、左室舒张末压力(LVEDP)和左室内压最大上升/下降速率(LV±dp/dtmax)。处死大鼠取心肌组织,免疫组织化学染色检测心肌微血管密度（MVD),改良Masson胶原染色法测定心肌胶原容积分数（CVF）,TUNEL法检测心肌组织凋亡指数（AI）。结果：干预后4周,aFGF治疗组大鼠LVESP和LV±dp/dtmax与DCM组比较明显增加（P<0.01）,LVEDP较DCM组明显减低（P<0.01）。aFGF治疗组MVD测值与DCM组比较明显增加（P<0.01）,而CVF及AI较DCM组明显减低（P<0.01）。结论: 超声微泡靶向递送aFGF可有效改善DCM大鼠的左心室功能,有望成为治疗DCM的新方法。