Delivery of Drugs with Ultrasound

In this article we discuss the potential role of microbubbles, traditionally used as ultrasound contrast agents, for site-specific drug delivery. To reach this goal, microbubbles capable of carrying a drug payload are being developed. To ensure that these microbubbles reach sufficient local concentration at disease sites, specific targeting for diseased tissues can be accomplished using several strategies. These strategies rely on either the intrinsic properties of microbubble shells or conjugation of monoclonal antibodies or other ligands to these shells that recognize antigens expressed in regions of disease. Site-specific delivery of antiinflammatory, antineoplastic, and thrombolytic drugs with microbubbles can be further enhanced by the ability to locally destroy microbubbles within an acoustic field, thereby releasing drugs and improving drug efficacy without systemic adverse effects. In the case of thrombi, ultrasound-mediated microbubble destruction also may facilitate the process of clot lysis. This review also will consider current limitations and technological advances required for the development of this field.     

超声联合微泡辅助卡铂抑制A549细胞活性的适宜剂量研究

目的 探讨超声辅助卡铂抑制体外A549细胞活性的适宜卡铂剂量及超声能量参数。方法 96孔板接种A549细胞,加入浓度梯度0-400>μg/ml的卡铂培养24小时后,每孔加入 10μlCCK8 溶液,孵育 1.5 小时,酶标仪测定在450nm处的吸光度值,计算细胞抑制率,应用Graphpad Prism 5软件的“药物剂量-效应模型”计算卡铂24小时IC50。设置空白组、US组、USMB组、CBP组、CBP+US组、CBP+USMB组,卡铂浓度为50 μg/ml,声强梯度范围为0.2-2.2W/cm2,频率1MHz,占空比10%,辐照时间1分钟,培养24小时,CCK8法测细胞存活率,应用SPSS24.0分析存活率差异,筛选有效剂量。光学倒置显微镜观察各组细胞形态学变化。结果 卡铂24小时IC50为65.72μg/ml。抑制A549细胞活性的强弱排序为CBP+USMB组>CBP+US组>CBP组>USMB组>US组,CBP+USMB组与其余4组之间差异有统计学意义(P<0.001),CBP(50μg/ml)+USMB(0.2-1.0W/cm2)5组之间差异无统计学意义(P>0.05)。处理组均可见细胞形态不规则、贴壁性降低,以CBP+USMB组为著。结论 卡铂50μg/ml、声强0.6W/cm2为24小时联合疗法适宜剂量。超声联合微泡可以与卡铂协同抑制A549细胞活性,成为肿瘤联合疗法的新策略。     

携VEGFR2靶向超声微泡在肾癌裸鼠模型中的显像研究

目的评价携血管内皮生长因子受体2(VEGFR2)抗体的靶向超声微泡在原位移植裸鼠肾癌模型中的显像效果。方法建立人肾透明细胞癌原位移植裸鼠模型,制备携VEGFR2抗体的靶向微泡(MBV)及携同型抗体IgG的对照微泡(MBC),每只裸鼠分别注射MBV及MBC(顺序随机,注射时间间隔30min)后行超声造影检查,比较注射两种微泡后10min内肿瘤的增强强度,并对肿瘤组织行VEGFR2免疫组化检测。结果超声造影示注射微泡后10s时两种微泡增强强度上升百分比比较差异无统计学意义,注射微泡后1,2,4,6,8,10min时MBV增强强度上升百分比明显高于MBC(P<0.05)。免疫组化显示肾肿瘤血管内皮VEGFR2高表达。结论应用携VEGFR2抗体的靶向微泡与肾癌新生血管内皮靶点特异性结合,能持续增强显像,可作为评价肿瘤新生血管及监测抗血管治疗疗效的重要分子成像方法。     

The effects of the microbubble suspension SH U 454 (Echovist) on ultrasound-induced cell lysis in a rotating tube exposure system

Human erythrocytes resuspended at different hematocrits in autologous plasma at 37 degrees C were exposed to the therapeutic intensities of continuous-wave 0.75-MHz ultrasound in vitro in a rotating tube exposure apparatus designed to maximize the destructive effects of cavitational activity. Provided that large numbers of additional gas bubbles had not been introduced during the various preparative and manipulatory procedures, the addition of Echovist at final concentrations comparable with those currently being used for clinical investigations resulted in a statistically significant increase in the amount of cell lysis in vitro in those samples having hematocrits less than 2%. The amount of cell lysis produced at any given ultrasound intensity decreased with increasing hematocrit in both the controls and the cell suspensions containing Echovist until it was virtually zero in both cases at hematocrits of 5.5% or greater. The addition of Echovist to samples that already contained large numbers of stabilized gas bubbles and/or had hematocrits greater than 5.5% produced no detectable cell lysis even at ultrasonic intensities as high as 3 W/cm 2 spatial average, temporal average (SATA). It is therefore unlikely that Echovist would cause appreciable amounts of cell lysis when the gas bubbles were being exposed to ultrasound under the conditions used for clinical investigations in vivo.     

Myocardial perfusion and contrast echocardiography: review and new perspectives

Myocardial perfusion can be assessed qualitatively and quantitatively with new ultrasound contrast techniques. This article reviews progress and problems in this area, discussing intracoronary and aortic root injections in animals and humans. The technique has great potential clinical application for the identification of coronary flow reserve, and the assessment of the need for and outcome of coronary revascularization procedures. It may allow direct measurements of regional myocardial perfusion.     

Effect of pressure on intracardiac backscatter from microbubbles

Echocardiographic contrast agent is a solutionwhich contains microbubbles or can produce mi-crobubbles rapidly after injection into blood.The in-vestigators are now trying to find a contrast agentthat can yield opacification of left heart system andmyocardium after intravenous injection to evaluatethe myocardial perfusion.A systolic decrease in leftventricularcontrastintensities wasobserved in there-search of intravenous contrast echocardiography withthe use of sonicated albumin in human[1] .T…     

Advances in ultrasound-targeted microbubble-mediated gene therapy for liver fibrosis

Hepatic fibrosis develops as a wound-healing scar in response to acute and chronic liver inflammation and can lead to cirrhosis in patients with chronic hepatitis B and C. The condition arises due to increased synthesis and reduced degradation of extracellular matrix (ECM) and is a common pathological sequela of chronic liver disease. Excessive deposition of ECM in the liver causes liver dysfunction, ascites, and eventually upper gastrointestinal bleeding as well as a series of complications. However, fibrosis can be reversed before developing into cirrhosis and has thus been the subject of extensive researches particularly at the gene level. Currently, therapeutic genes are imported into the damaged liver to delay or prevent the development of liver fibrosis by regulating the expression of exogenous genes. One technique of gene delivery uses ultrasound targeting of microbubbles combined with therapeutic genes where the time and intensity of the ultrasound can control the release process. Ultrasound irradiation of microbubbles in the vicinity of cells changes the permeability of the cell membrane by its cavitation effect and enhances gene transfection. In this paper, recent progress in the field is reviewed with emphasis on the following aspects: the types of ultrasound microbubbles, the construction of an ultrasound-mediated gene delivery system, the mechanism of ultrasound microbubble–mediated gene transfer and the application of ultrasound microbubbles in the treatment of liver fibrosis.     

超声联合藤黄酸纳米粒抗人神经胶质瘤的研究

目的:研究藤黄酸对人神经胶质瘤细胞U87的有效作用浓度,以及载藤黄酸（gambogic acid,GA）的多孔脂质/PLGA杂化微泡（GA/PLGA）在超声作用下对U87的杀伤效果。方法:马尔文粒度仪检测GA/PLGA粒径电位分布情况;CCK-8法检测细胞活力;Annexin V/PI双染法检测细胞凋亡率。结果:每50 mg PLGA中添加4 mg GA制备出的GA/PLGA具有最高的包封率和载药量,分别为（95.57±1.89）%和（7.65±0.15）%;GA对U87细胞的半数抑制浓度（IC50）为1.5 μmol/L;US+GA/PLGA处理组对应U87细胞活力最低为（3.85±0.67）%;US+GA/PLGA处理组U87细胞凋亡率最高,为（75.97±0.49）%。结论:GA/PLGA微泡在超声作用下可以增强GA对U87的杀伤效果。     

低频诊断超声联合脂氟显微泡开放小鼠血脑屏障的可逆性研究

目的探讨低频诊断超声联合脂氟显微泡开放小鼠血脑屏障(blood-brain barrier,BBB)的可逆性及伊文思蓝(Evans blue,EB)透过率。方法健康C57BL/6小鼠48只,随机分为对照组6只和观察组42只。观察组再随机分为0、0.5、1、2、3、8、10 h亚组各6只,低频诊断超声辐照同时经尾静脉注射脂氟显微泡1 mL/kg,并分别于超声辐照0、0.5、1、2、3、8、10 h经尾静脉注射质量分数2%EB溶液50 mg/kg;对照组不进行低频诊断超声辐照,经尾静脉注射1 mL/kg生理盐水后注射质量分数2%EB溶液50 mg/kg。注射1 h后处死小鼠取脑组织,观察脑组织蓝染深度及面积,采用紫外分光光度法定量分析脑组织EB含量。结果对照组及观察组8、10 h亚组未见脑实质蓝染,观察组0、0.5、1、2、3 h亚组辐照区脑实质均有不同程度蓝染,且1 h时蓝染深度最深、面积最大;观察组0、0.5、1、2、3 h亚组脑组织EB含量[(45.43±2.89)、(50.94±1.25)、(79.79±1.12)、(51.55±1.20)、(31.60±1.77)μg/g]均高于对照组[(8.32±0.12)μg/g](P<0.05),8、10 h亚组脑组织EB含量[(8.34±0.09)、(8.31±0.07)μg/g]与对照组比较差异无统计学意义(P>0.05);观察组0、0.5、1 h亚组脑组织EB含量依次升高(P<0.05),1、2、3 h亚组脑组织EB含量依次降低(P<0.05)。结论低频诊断超声联合脂氟显微泡开放小鼠BBB呈动态可逆的过程,BBB在超声辐照后1 h开放程度最大,于超声辐射8 h恢复正常。     

携载G250单克隆抗体的纳米微泡靶向于肾细胞癌的体内外实验研究

目的 本研究拟制备携载G250单克隆抗体的纳米微泡,在体内和体外研究其对肾细胞癌的靶向性.方法 机械振荡法制备空白脂质纳米微泡并观察其稳定性,生物素-亲和素连接技术将纳米微泡与G250单克隆抗体相连,采用免疫荧光进行验证.细胞免疫荧光实验鉴定所使用的两种肾细胞癌细胞(786-O和ACHN细胞)中G250的表达情况,靶向结合实验鉴别靶向纳米微泡对786-O和ACHN两种肾细胞癌细胞的体外寻靶能力.在肾细胞癌的裸鼠皮下移植瘤模型中,使用纳米微泡进行超声造影,并将采集到的动态图像在定量分析软件Qlab 8.1中进行数据分析.结果 成功制备了平均粒径为404.9 nm空白纳米微泡和平均粒径为611.4 nm靶向纳米微泡,稳定性能好,同时免疫荧光证实了靶向纳米微泡构建成功,能够与FITC标记的二抗结合,从而显示出绿色荧光;细胞免疫荧光证实G250抗原在786-O细胞中呈膜表达,而ACHN细胞中不表达.在细胞结合实验中发现靶向纳米微泡能够特异性的结合786-O细胞,但不能结合于ACHN细胞.体内超声造影显像从平均值和最大值分析,靶向纳米微泡和空白微泡在786-O肾细胞癌细胞的裸鼠模型的显影效果存在显著差异[平均值:(11.74±0.52) vs (16.34±0.40),P=0.001;最大值:(13.07±0.94) vs (18.09±0.82),P=0.003].结论 G250靶向的纳米微泡可在体外能够特异性的结合G250表达阳性的肾细胞癌细胞,在动物体内能够明显的增强移植瘤的超声显影效果.