DNA-loaded albumin microbubbles enhance ultrasound-mediated transfection in vitro

Ultrasound (US), with or without microbubbles, enhances gene transfer in cultured cells, but the effect is modest. We tested if attaching DNA to albumin microbubbles during bubble synthesis could enhance gene expression. Plasmid DNA was loaded on the albumin shell over a range of concentrations (500 to 10,000 microg/mL). Optimal gene expression occurred with loading doses of 4000 microg DNA/mL (4k-loaded bubbles). These microbubbles had diameters of 2.4 +/- 0.7 microm and carried 40 pg DNA/microbubble. DNA-loaded microbubbles had optimal transfection at higher delivered doses of DNA than unloaded bubbles mixed with plasmid. The 4k-loaded bubbles demonstrated a fivefold (p = 0.0003) increase in luciferase reporter expression over that with unloaded bubbles. Similarly, transfection efficiency was better for 4k-loaded microbubbles than unloaded microbubbles (41 +/- 3% vs. 9 +/- 3%, p < 0.0001). DNA loading of microbubbles enhances gene expression and transfection efficiency in US-targeted transfection in vitro and may represent an improved avenue for therapeutic gene delivery in vivo.     

载重组腺病毒Ad-EGFP/HIF-1α的超声造影剂的制备及其特性检测

目的 制备一种载重组腺病毒的脂质超声微泡造影剂,对其物理特性、载病毒的能力及在兔肝脏的显影效果进行研究.方法 采用机械振荡法及分层吸附法制备一种载重组腺病毒Ad-EGFP/HIF-1α的脂质超声微泡造影剂,用DFY-Ⅱ型超声图像定量分析诊断仪检测微泡粒径和浓度;检测其结合腺病毒的能力及观察对正常兔肝脏的显影效果.结果 载重组腺病毒Ad-EGFP/HIF-1α的超声微泡分布均匀,平均粒径为(1.17±0.82)μm,浓度为(2.50±0.12)×10~9/mL;该造影剂的最大腺病毒结合的效率为30%;体内造影显示该造影剂能够有效增强兔肝脏实质回声.结论 自制载重组腺病毒的微泡造影剂符合理想超声造影剂的要求,性质稳定,制备简易,载腺病毒效率高,为超声靶向微泡破裂基因释放技术的发展开辟新思路.     

载血卟啉的乳酸/羟基乙酸共聚物超声微泡造影剂制备及工艺优化

目的 探讨载声敏剂血卟啉(hematoporphyrin,HP)的高分子材料乳酸/羟基乙酸共聚物[Poly(lactic-co-glycolic acid),PLGA]超声微泡造影剂的优化制备工艺.方法 采用双乳化法制备包裹HP的PLGA超声微泡造影剂,通过正交设计筛选出比较理想的制备工艺,并对所制备的造影剂进行药物体外释放评估及体外超声造影观察.结果 载HP的PLGA造影剂(HP-PLGA)平均粒径602.3 nm,平均包封率63.5%,平均载药量2.15%,电位-(17.1±1.6)mV,体外14 d缓释约86.5%,体外超声显影良好.结论 通过采用双乳化法制备的HP-PLGA造影剂,具备缓释长效的特性,体外显像效果好,符合理想药物载体的基本特性,为实时监控下体内声动力治疗肿瘤提供了一种新型的药物剂型.     

载基因及穿膜肽脂质超声造影剂制备的实验研究

目的 研究自制的脂质超声造影剂载基因及穿膜肽的能力,评价其物理性质及显影效果.方法 采用机械振荡法制备载基因及穿膜肽的脂质超声造影剂,检测微泡形态、分布、浓度、粒径、表面电位及载基因和穿膜肽的能力,并观察其对兔心脏的显影效果.结果 自制载基因及穿膜肽脂质超声微泡造影剂的粒径为(2.27±0.38)μm,浓度为(3.07±0.42)×109个/ml,表面电位为(1.95±0.13)mV.该造影剂中基因的包封率为32%,穿膜肽的包封率为35%.体内造影显示该造影剂能有效增强心肌显影.结论 自制载基因及穿膜肽脂质超声造影剂符合理想超声造影剂的要求,载基因及穿膜肽的效率高,可作为携带基因等生物活性物质的载体材料.     

Microbubble Destruction During Intravenous Administration: A Preliminary Study

The concentration and size distribution of microbubble suspensions are important parameters for both diagnostic and therapeutic applications. The aim of this preliminary study was to investigate the relationship between changes in the microbubble population and the administration process variables, specifically syringe inner diameter, needle inner diameter, volume flow rate and the liquid in which the microbubbles are suspended. It was found that reducing either the syringe or needle inner diameter produced large reductions in microbubble concentration during administration, as much as 99.9% for needle inner diameters <0.24 mm. Increasing the volume flow rate up to 3 mL/min and changing the suspending fluid from distilled water to glycerol, however, were both found to reduce the degree of microbubble destruction. Further work is needed to fully explain these observations. However, investigation of the response of microbubbles to changes in hydrostatic pressure only, indicated that this was unlikely to be the main mechanism of destruction and hence that shear stress was a more important factor. Comparison with findings from another recent study of microbubble stability indicated that microbubble size, concentration and composition were also important parameters and should be taken into account in designing administration procedures for microbubble agents. It was concluded that current procedures should be reviewed, particularly for therapeutic applications, and that the results should also be taken into account when assessing the accuracy of microbubble size distribution measurements obtained using automatic particle sizing equipment in which microbubbles are made to flow under pressure. (E-mail: e_stride@meng.ucl.ac.uk)     

载组织型纤溶酶原激活物及精-甘-天-丝氨酸四肽超声微泡造影剂的实验研究

目的 制备一种同时携带组织型纤溶酶原激活物(Tissue plasminogen activator,tPA)与精-甘-天-丝氨酸四肽(RGDS)靶向配体的新型脂质超声微泡造影剂.方法 以冷冻干燥法制备包载tPA的脂质超声微泡造影剂并应用共价键桥连剂法结合RGDS配体.光镜及荧光显微镜观察其形态;测量其粒径、表面电位、pH值;以ELISA法测定tPA包封率;流式细胞仪检测RGDS携带率;琼脂糖纤维蛋白平板法检测其纤溶活性;以该造影剂对正常家兔肝脏进行静脉声学造影,绘制时间-强度曲线,检测其成像功能.结果 微泡膜上同时携带tPA和RGDS,浓度(7～9)×109/ml,粒径(2.08±0.93) μm,表面电位-51.3,pH值5.58,tPA包封率(81.12±2.44)%,RGDS携带率(94.49±6.19)%.微泡在超声辐照条件下显示溶栓活性.声学造影后,该造影剂能明显增强实验兔肝实质回声.结论 以冷冻干燥法和共价键桥连剂结合法能成功制备同时包载tPA并携带RGDS的脂质超声微泡造影剂,将为血栓的靶向释药促溶治疗提供一条新的可行之路.     

载10-羟基喜树碱靶向脂质微泡的制备及体外寻靶能力实验研究

目的 制备携人肝癌单抗Hab18载10-羟基喜树碱的脂质微泡,并观察其体外寻靶能力.方法 制备生物素化抗人肝癌Hab18单克隆抗体,检测其生物素化程度;用机械振荡法制备载药脂质微泡,以生物素-亲和素桥连方式构建携人肝癌单抗Hab18载10-羟基喜树碱的脂质超声微泡,检测其一般特性、包封率和载药量,以免疫荧光法检测微泡与抗体的连接情况,在光镜下观察靶向载药微泡的寻靶能力,并与载药非靶向微泡进行比较.结果 每个单抗分子平均可与13个生物素分子结合.载药靶向脂质超声微泡分布均匀,平均粒径为1.52 μm,包封率为76.32%,载药量为21.81%.免疫荧光法显示微泡表面可见明亮的红色环状荧光,体外寻靶实验显示该载药靶向微泡可与人肝癌7721细胞牢固结合.结论 携人肝癌单抗Hab18载10-羟基喜树碱的脂质超声载药靶向微泡可成功制备,其包封率和载药量较高,具有较强的体外寻靶能力.     

超声击破微泡对重组腺病毒Ad-EGFP/HIF-1α活性的影响

目的 探讨超声破坏载重组腺病毒Ad-EGFP/HIF-1α的微泡对Ad-EGFP/HIF-1α生物学活性的影响.方法 采用机械振荡法及分层吸附法制备携带重组腺病毒的脂质超声微泡造影剂,用DFY-Ⅱ型超声图像定量分析诊断仪检测微泡粒径.计算其浓度.将脐静脉内皮细胞(HUVEC)接种在24孔板中,随机分为以下5组:①空白对照组;②重组腺病毒Ad-EGFP/HIF-1α组;③载霞组腺病毒Ad-EGFP/HIF-1α微泡组;④超声+重组腺病毒Ad-EGFP/HIF-1α组;⑤超声+载重组腺病毒Ad-EGFP/HIF-1α微泡组.荧光显微镜及流式细胞仪检测各组在细胞内的表达及转染效率,R-PCR法检测HIF-1α的mRNA的表达.结果 载重组腺病毒的超声微泡的大小、粒径及分布与普通超声微泡相似.与对照组相比,各组间绿色荧光强度及转染率无明显差别;各组间HIF-1α mRNA的表达也无明显差别.结论 利用超声破坏载重组腺病毒Ad-EGFP/HIF-1α的微泡造影剂对腺病毒活性无明显影响,为拓展超声微泡介导的基因治疗领域提供了新的思路和方法.     

超声脂质微泡与聚乳酸-聚乙醇酸共聚物纳米微球复合体在小鼠肝脏的定位释放

目的探讨新型超声微泡[荧光标记的聚乳酸-聚乙醇酸共聚物(PLGA)纳米微球与超声脂质微泡共价结合复合体]在一定声强的超声定位辐照下,PLGA纳米微球在小鼠体内肝脏组织的定位释放情况。方法用机械振荡法制备超声脂质微泡,双乳化法制备荧光标记的PLGA纳米微球,碳二亚胺法制备新型超声微泡。将实验小鼠分为3组:PLGA纳米微球组(NP组)、超声脂质微泡混合PLGA组(MB+NP组)、新型超声微泡组(MB-NP组),均用一定声强的超声经体表定点辐照小鼠肝脏。以激光共聚焦扫描荧光显微镜定性观察小鼠肝组织中PLGA纳米微球的分布。以DFY微泡测量软件对肝组织中荧光标记的PLGA纳米微球进行计数分析。结果 MB-NP组中的超声微泡周围布满数量不等的纳米微球,呈花环状,且PLGA纳米微球数量明显高于其他组(P均<0.01),MB+NP组的数量高于NP组(P<0.05)。结论新型超声微泡能够明显提高靶组织区内的PLGA纳米微球浓度,从而使PLGA携带的药物更多释放于靶组织中。     

制备RGDS/rt-PA双负载靶向微泡并初步评价其靶向性溶栓效果

目的 制备RGDS/rt-PA双负载靶向微泡,分析其对富血小板血栓（PRT）的靶向性和溶栓效果。方法 制备不同剂量梯度的RGDS及rt-PA单负载靶向微泡,以其最佳结合剂量、按照不同加入顺序（先加入RGDS,再加入rt-PA;先加入rt-PA,再加入RGDS;将RGDS及rt-PA混匀后加入）分别制备RGDS/rt-PA双负载靶向微泡,检测其结合率。比较裸微泡、单负载及双负载微泡的物理特性（直径、浓度及pH值）,不同剂量梯度的同一配体与微泡的单负载结合率,同一剂量梯度的不同配体与微泡的单负载结合率,加入顺序不同的配体与微泡的双负载结合率。制备体外PRT模型,检测RGDS/rt-PA双负载靶向微泡的声学显像特征、靶向溶栓能力。结果 不同微泡间的物理特性差异均无统计学意义（P均>0.05）。不同剂量梯度的rt-PA、RGDS与微泡单负载结合率的差异均有统计学意义,同一剂量梯度的rt-PA与微泡单负载的结合率均低于RGDS （P均<0.05）。加入顺序不同的配体与微泡的双负载结合率的差异有统计学意义（F=16.090,P=0.004）。将RGDS/rt-PA双负载靶向微泡注入血栓模型管腔后,超声可见均匀分布的点状高回声,血栓边界回声明显增强;扫描电镜下可见纤维蛋白网状结构明显破坏、纤维束断裂成细沙状,并可见变形融合的血细胞。结论 RGDS/rt-PA双负载靶向微泡性质稳定,与配体的结合率高,声学显像特征好,具备一定的PRT靶向性及溶栓能力。     

六氟化硫脂质微泡的制备研究

目的 制备六氟化硫脂质微泡,为靶向微泡的研制奠定基础.方法 以二硬脂酰磷脂酰胆碱、聚乙二醇磷脂酰乙醇胺为膜成分,六氟化硫为气体核心,薄膜一超声法制备微泡.以SonoVue作对照,观测自制微泡的形态、粒径、浓度、pH值、渗透压等各项理化参数.在二次谐波模式下观察微泡显影体外水囊及兔肾实质,测量分析增强图像峰值灰度.结果 自制微泡和SonoVue均大小均匀,圆形,中空透亮,平均直径分别为2.25μm和2.50 μm,粒径分布分别为0.4～10 μm和0.2～10 μm,90%微泡直径分别控制在6 μm和8 μm以内.初始浓度分别为5 x 108～10 X 108/ml和1 x 108～5 x 108/ml,配置后的稳定性均为6 h.显影水囊灰度值分别为121.67±6.76和122.33±4.53(P>0.05),兔肾造影增强峰灰度分别为72.00±7.21和74.65±10.93(P>0.05).结论 脂质微泡制备成功,具有良好的理化特性及显影效果.     

Microbubble Stability is a Major Determinant of the Efficiency of Ultrasound and Microbubble Mediated in vivo Gene Transfer

In the search for an efficient nonviral gene therapy approach for the treatment of genetic disorders of cardiac and skeletal muscle such as Duchenne muscular dystrophy, ultrasound in combination with contrast enhancing microbubbles has emerged as a promising tool for safe and site-specific enhancement of gene delivery. Indeed, microbubble-enhanced gene transfer (MBGT) has been investigated for a wide variety of target sites using both reporter and therapeutic genes. Although a range of different microbubbles have been used for MBGT studies, comparison of their efficiencies is difficult because microbubble concentration and the ultrasound settings used for the application vary considerably. Only two studies to date have attempted a direct comparison of commercially available microbubbles, and both concluded that not all microbubbles show the same efficiencies with MBGT. Thus far, the reason for this is unclear. Here, the efficiency of three commercially available microbubbles—Optison, SonoVue and Sonazoid—was analyzed to understand the microbubble properties that are important for their function as an effective enhancer for gene transfer in vivo. In this study, plasmid DNA or antisense oligonucleotides were delivered by systemic injection with MBGT, focused on the heart. Gene delivery to the heart with equalized concentrations of the three microbubbles showed that Optison and Sonazoid are more efficient in MBGT compared with SonoVue, which showed the weakest gene transfer to the myocardium. Investigations into the properties of these microbubbles showed that size and shell composition did not directly influence MBGT, whereas the microbubbles with increased stability in an ultrasound field showed better MBGT results than those degrading faster. Moreover, the microbubble concentration used for MBGT was also found to be an important factor influencing the efficiency of MBGT. In conclusion, the stability of a microbubble was shown to be a major influential factor for its performance in MBGT, as is the concentration of the microbubbles used. These findings emphasize the importance of detailed investigations into the properties of microbubbles to allow the production of a microbubble specifically designed for optimum performance with MBGT. (E-mail: d.wells@imperial.ac.uk)     

载诺帝脂质微泡的制备及特性的实验研究

目的 制备载诺帝(去甲二氢愈创木酸)脂质微泡并测定其包封率、载药量及观察其体内超声显像效果.方法 采用机械振荡法制备载诺帝脂质微泡,测定其粒径大小、分布和Zeta电位、包封率、载药量;超声造影观察其在兔肝内的显像效果.结果 载药脂质微泡的浓度为(2.53±0.52)×109个/ml,粒径为(2.61±0.32)μm,Zeta电位为+(17.52±2.04)mV;脂质微泡诺帝的包封率(27.1±2.01)%,载药量为(10.9±0.90)%;微泡经外周静脉注射后,兔肝可见良好、持续的增强显像效果.结论 采用机械振荡法可以成功制备携带诺帝的脂质微泡,微泡可以通过肺血管床,符合静脉注射要求,在家兔体内获得了良好的显像效果.     

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.     

Property and contrast-enhancement effects of lipid ultrasound contrast agent: a preliminary experimental study

This work investigated the influence of some factors on the property in vitro of a self-made lipid ultrasound (US) contrast agent (LCA) and evaluated the relationship of acoustic pressure and enhancement effect in normal rabbit kidney parenchyma. In the in vitro studies, filling gas, solvent and concentration of LCA solution were investigated. Morphologic characteristics, concentration and mean diameter of microbubbles were considered as indices. In the in vivo studies, contrast-tuned imaging (CnTi) technique was used to investigate the enhancement effects in kidney parenchyma under nine acoustic pressure levels. Among the samples saturated with different filling gases, perfluoropropane (C(3)F(8)) resulted in the highest concentration of microbubbles and air, the lowest. Microbubbles filled with C(3)F(8) or sulfur hexafluoride (SF(6)) were quite stable and remained at a high level of concentration (above 2 x 10(9) microbubbles per mL) much longer than did air-filled microbubbles. Among the four solutions tested, 5% glucose solution and 0.9% saline solution showed higher initial concentrations and greater longevity than dextran 40 glucose solution (6%) or distilled water. The concentration of LCA solution had a positive correlation with the microbubble concentration. All microbubble samples under different test conditions remained shape-complete and no aggregation or fusion was observed. The mean diameter of microbubble samples was about 3.4 microm. Contrast intensity and longevity of CnTi enhancement in vivo showed an acoustic-pressure-dependent decrease. At 1 kPa, contrast intensity increased 224-fold (4.47/0.02) and the longevity of CnTi enhancement in the kidney parenchyma remained longer.     

一种新型载药超声微泡的制备

目的 探讨共价结合与静电吸附法连接超声微泡与乳酸羟基乙酸共聚物(PLGA)纳米微球的效率,制备一种新型载药超声微泡。方法 采用机械振荡法制备正电荷氨基化超声微泡(MB-NH₂)。以双乳化法制备负电荷PLGA纳米微球(NP),并用碳二亚胺法活化NP表面的羧基,得到活性PLGA纳米微球(NP-COOH)。分2组连接超声微泡与纳米微球:静电组(MB-NH₂+NP)、共价静电协同组(MB-NH₂+NP-COOH)。在光镜下不同时间点观察连接效果。结果 48 h后光镜下静电组超声微泡表面光滑,周围未见纳米微球聚集;共价静电协同组超声微泡表面不光滑,周围布满数量不等的小圆形的纳米微球,呈花环状。结论 在共价结合与静电吸附的协同作用下,可成功制备出一种新型载药超声微泡,有望为肿瘤治疗提供一种新思路。     

Acoustical Properties of Individual Liposome-Loaded Microbubbles

A comparison between phospholipid-coated microbubbles with and without liposomes attached to the microbubble surface was performed using the ultra-high-speed imaging camera (Brandaris 128). We investigated 73 liposome-loaded microbubbles (loaded microbubbles) and 41 microbubbles without liposome loading (unloaded microbubbles) with a diameter ranging from 3–10 μm at frequencies ranging from 0.6–3.8 MHz and acoustic pressures ranging from 5–100 kPa. The experimental data showed nearly the same shell elasticity for the loaded and unloaded bubbles, but the shell viscosity was higher for loaded bubbles compared with unloaded bubbles. For loaded bubbles, a higher pressure threshold for the bubble vibrations was noticed. In addition, an “expansion-only” behavior was observed for up to 69% of the investigated loaded bubbles, which mostly occurred at low acoustic pressures (≤30 kPa). Finally, fluorescence imaging showed heterogeneity of liposome distributions of the loaded bubbles.     

Sonicated echocardiographic contrast agents: reproducibility studies

This article describes the production, analysis, and reproducibility of forming microbubbles for contrast ultrasound imaging. The sonication method used to generate microbubbles was tested by four independent observers, and a subsequent laser particle counter analysis of microbubble size and concentration determined the reproducibility of the method. The results indicated that the mean bubble size was 3.3 +/- 1.2 microns for the entire group, based on three trials of each of the four participants. The characteristics of the bubble size of the microbubbles between observers were assessed with a Poisson distribution with the reproducibility based on the sample mean for each observer's trials. Standardization and calibration of the laser particle counter was accomplished with commercially available latex spheres, sonicated albumin microspheres, and a Coulter counter analysis. Our results indicate that the sonication technique generates small microbubbles with a reproducible uniform size distribution. The method of microbubble production is reproducible and can be widely applied for use in contrast echocardiographic perfusion imaging of tissue in a variety of research and clinical studies.     

机械振荡法制备负电荷超声造影剂的探讨

目的机械振荡法制备并评价适用于超声空化栓塞肿瘤微循环的负电荷脂膜超声造影剂。方法采用机械振荡法在不改变脂质及辅料总量前提下,通过改变配方中脂质成分比例以及聚乙二醇(PEG)的用量,制备三种配方超声造影剂,分别观察并检测超声造影剂的浓度、表面电位、pH值、粒径及分布。结果三种配方对造影剂的浓度、稳定性几乎无影响,但对其的电荷特性有明显影响。三种配方所制备微泡的粒径均值分别为1066.6nm、1605.4nm、1552.5nm;微泡表面电位均值:在蒸馏水中分别为-33.8mV、-57.6mV、-66.7mV,在生理盐水中分别为0.1mV、-37.5mV、-37.8mV。与配方1比较,配方2、3所制备微泡大小适中、表面带有更多负电荷(P<0.01),配方2、3间比较无明显差异(P>0.05)。结论采用机械振荡法,增加脂质成分中负电荷磷脂的比例,可以制备出表面带有更多负电荷的超声造影剂,为超声空化栓塞肿瘤微循环提供了实验基础。     

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)