摘要
目的：
动脉粥样硬化斑块(Atherosclerosis Plaque)破裂是引发心肌梗死、脑血栓等心脑血管急性事件的主要原因。然而,动脉粥样硬化斑块的发生发展是个无声无息的慢性过程,大部分心脑血管事件发作前都没有先兆表现。因此,早期诊断动脉粥样硬化斑块有着十分重要的临床意义。但是目前临床尚无早期识别和评价动脉粥样硬化斑块的有效手段。其中,血管炎症反应在动脉粥样硬化斑块的发生发展方面扮演着非常重要的角色。如能无创性的对“斑块炎症”/“血管炎症”进行靶向分子成像,无疑可为临床早期诊断动脉粥样硬化斑块并对其进行危险分层提供非常有价值的手段,进而指导早期的干预治疗,避免或延缓事件的发生。因此,现有的无创性影像技术,如：超声、MRI、CT、SPECT等均在对“斑块炎症”/“血管炎症”的靶向分子成像进行积极探索。
超声分子影像技术(Ultrasound Molecular Imaging)是对粘附在血管内皮细胞上的特异分子进行靶向性对比超声成像(Contrast-Enhanced Ultrasound Imaging)。该技术通常应用靶向性超声微泡(Targeted Microbubbles)作为示踪剂,这种靶向性超声微泡既具有与红细胞相似的流变学特征,可顺利的通过组织微循环,又可与特异性靶向分子有效的结合,从而达到特异性评价循环系统血管内皮细胞上分子学变化或实现检测循环系统内固定的靶分子的目的。临床上有望用于评价血管内皮炎症、检测血栓形成或早期发现肿瘤等领域。而实现该技术的核心就是构建有效、特异的靶向超声微泡。目前常用的靶向超声微泡主要是通过主动性靶向机制,采用具有脂质外壳含惰性气体的微泡在其表面上装配特异性配体来实现。抗体、多肽和多糖等多种分子物质均可作为配体与超声微泡连接构建成特异的靶向性超声微泡。
动脉粥样硬化斑块被认为是一种慢性的炎症过程,在斑块发生发展的早期有大量的慢性炎症因子VCAM-1的表达,若能以其为靶点进行构建靶向超声微泡,将有望实现超声分子影像技术对斑块早期炎症的早期诊断。然而,动脉粥样硬化斑块位于高剪切应力的血流状态下,靶向超声微泡如何与动脉斑块内皮细胞上的靶分子有效结合,进而对动脉粥样硬化斑块的慢性炎症过程进行靶向超声分子成像?这是目前国际上亟待解决的难题。
为了实现动脉系统的超声分子成像,人们通过“双配体”连接技术构建靶向超声微泡增加靶向微泡的快速黏附以及使用超声辐射力改变微泡在血管内的“轴流”等方式进行了相关尝试,但均存在一些问题使其并未能很好的进行推广。在本课题组前期靶向超声分子成像研究成果基础上,我们假设将磁性靶向导航技术引入靶向超声微泡的制备中,制备出携带磁性物质和配体的靶向性超声微泡,通过磁性导航可以改变靶向性超声微泡在大中动脉中的轴向分布特征,引导靶向性超声微泡向成像目标(动脉)的管壁贴近并停留,进而有助于实现动脉系统的靶向超声分子成像。
动脉粥样硬化斑块早期炎症的识别是心血管领域的前沿热点,目前临床缺乏有效的评价手段。由于现有的靶向性超声微泡难以高效的结合在高剪切应力的大中动脉炎症斑块上,阻碍了无创、简便的超声分子影像技术在动脉粥样硬化斑块早期炎症评价中发挥作用。因此,本研究目的在于：应用生物素-亲和素桥接法制备出携VCAM-1单抗磁性靶向超声微泡(Magnetic microbubbles targeted to VCAM-1,MBv),在体和体外实验评价其靶向粘附效果,及其用于评价动脉粥样硬化斑块早期炎症的可行性。从而可为靶向超声微泡的构建提供新的思路和方法,为实现从分子水平无创性的评价动脉斑块炎症、早期识别斑块提供简便的手段,以指导预后。
方法：
一、模拟动脉间歇脉搏血流评价携VCAM-1单抗靶向微泡黏附效能
1、携VCAM-1单抗靶向微泡(Microbubbles targeted to VCAM-1, MBv)的制备及理化性质鉴定
通过声振法制备生物素化脂质超声微泡(Biotinayted microbubbles, MBb),通过生物素-亲和素桥接法先后加入链亲和素及生物素化抗小鼠VCAM-1单克隆抗体或同型抗体制备MBv及同型对照微泡(Microbubbles with isotype antibodies, MBi)。库尔特粒子计数仪检测微泡的粒径分布及浓度。
2、平行板流动腔法评价MBv在间歇脉搏血流的靶向黏附
应用1000ng/ml小鼠VCAM-1 Fc段包被的聚苯乙烯培养皿作为平行板流动腔体外检测微泡粘附稳定性的平台。MBv (5×106/ml)经微量注射泵分两组(持续滴注组和间歇滴注组)以不同剪切应力(0.5-16 dyn/cm2)通过以1000ng/ml VFc包被的平行板流动腔,镜下观察有微泡出现后录像6 min,观察每分钟微泡的靶向结合情况。并采用解离实验,每30秒倍增剪切应力(0.2-51.2dyn/cm2),检测MBv微泡达半数解离时的剪切应力。
3、利用SPSS 13.0软件经行数据处理,两组微泡在不同剪切应力下每分钟结合的微泡个数之间比较采用单因素方差分析,微泡解离实验仍结合微泡百分数间比较用重复测量的方差分析,P<0.05有统计学意义。
二、携VCAM-1单抗靶向微泡评价动脉粥样硬化斑块早期炎症
1、动物分组及模型准备
小鼠分为4组：APOE小鼠高胆固醇饲养组(APOE-HCD组)、APOE小鼠普通饮食组(APOE-RD组),C57小鼠高胆固醇饲养组(C57-HCD组)和C57小鼠普通饮食组(C57-RD组)。
2、小鼠腹主动脉CEU检查
所有实验小鼠麻醉后分别随机(间隔30分钟)经静脉弹丸注射法给予MBv和MBi, CEU检查采用CPS成像技术进行,探头发射和接收频率分别为7.0和15 MHz,用低机械指数(MI=0.18)进行连续CEU观察8分钟并取图后给予高机械指数(MI=1.9)连续脉冲破坏3秒后取本底图像,所有图像均测量声强度(Video intensity, VI)并进行彩色编码处理。
3、小鼠腹主动脉免疫组化检查
小鼠超声成像后处死行腹主动脉免疫组化检测VCAM-1在管腔内皮细胞的表达；采用EnVision两步法检测,按操作说明书行常规脱蜡水合、抗原修复、抗及二抗孵育、DAB染色及苏木素复染、最后脱水、透明、中性树胶封片,于显微镜下观察并拍照。
4、利用SPSS 13.0软件经行数据处理,小鼠腹主动脉超声参数比较用单因素方差分析,两种微泡在不同动物实验组之间的声强度比较用两因素方差分析,P<0.05有统计学意义。
三、携VCAM-1单抗磁性靶向微泡的制备及体外评价
1、MBvM的制备及理化性质鉴定
通过生物素-亲和素桥接法利用磁性链亲和素制备磁性靶向微泡MBvM及磁性同型对照微泡(Magnetic microbubbles with isotype antibodies, MBiM)。库尔特粒子计数仪检测微泡的粒径分布及浓度。
2、荧光显微镜观察微泡的磁响应性
用DiI荧光MBb进行制备MBvM、MBv及MBiM,将微泡悬液吸出少许并用蒸馏水稀释后滴在玻片上,加上盖片后静置3分钟行荧光显微镜观察并照相。并用外置永磁铁在玻片一侧作用,荧光显微镜继续观察并录像。
3、平行板流动腔法评价磁性微泡的磁响应性及靶向黏附
A、平行板流动腔模型判断微泡磁响应性
MBvM、MBv和MBiM经微量注射泵以不同剪切应力(1-24 dyn/cm2)通过以1000 ng/ml VFc包被的平行板流动腔,各组均分别分为磁场作用组及无磁场作用组；给予镜下观察有微泡出现后录像5min,观察每分钟微泡的靶向结合情况,取第5分钟点计数视野内黏附微泡的个数进行比较。
B、平行板流动腔模型判断磁性微泡黏附效能
MBvM、MBv和MBiM经微量注射泵以不同剪切应力(1-24 dyn/cm2)通过以1000 ng/ml VFc包被的平行板流动腔,各组均先给予磁场作用5分钟,之后取消磁场作用并继续冲刷5分钟；观察每分钟微泡的靶向结合情况,取第5分钟点计数视野内黏附微泡的个数及第10分钟点计数视野内黏附微泡个数进行比较。
4、利用SPSS13.0软件经行数据处理,每组微泡不同剪切应力下每分钟结合的微泡个数之间比较采用具有一个重复测量因素两因素方差分析,微泡解离实验仍结合微泡百分数间比较用重复测量的方差分析,P<0.05有统计学意义。
四、携VCAM-1单抗磁性靶向微泡评价动脉粥样硬化斑块早期炎症
1、动物分组及模型准备
小鼠分为4组：APOE小鼠高胆固醇饲养组(APOE-HCD组)、APOE小鼠普通饮食组(APOE-RD组),C57小鼠高胆固醇饲养组(C57-HCD组)和C57小鼠普通饮食组(C57-RD组)。
2、小鼠腹主动脉CEU检查
所有实验小鼠麻醉后分别随机(间隔30分钟)经静脉弹丸注射法给予MBvM、MBv和MBiM(均约4×106个),小鼠背部下方放置5000GS强磁铁,微泡注射5分钟后取出, CEU观察10分钟并取图后给予高机械指数(MI=1.9)连续脉冲破坏3秒后取本底图像,所有图像均测量声强度(video intensity,VI)并进行彩色编码处理。
3、利用SPSS13.0软件经行数据处理,动物实验分组中三种微泡视频强度比较采用重复测量数据方差分析,微泡在各实验组间的比较采用两因素方差分析,P<0.05有统计学意义。
结果：
一、模拟动脉间歇脉搏血流评价携VCAM-1单抗靶向微泡黏附效能
1、MBv的制备及理化性质鉴定
库尔特检查示制备出的MBv的平均粒径约为(2.55±0.75)μm,浓度约为2.9×108个/ml。粒径分布均匀,大部分微泡集中在2-4μm之间。
2、平行板流动腔法评价MBv在间歇脉搏血流的靶向黏附
平行板流动腔实验显示MBv在连续输注组在0.5-2 dyn/cm2时能与VFc包被的平行板流动腔有明显的结合。逐渐增加剪切应力(2-16 dyn/cm2)可见MBv的结合明显下降,在4 dyn/cm2后就难以看到靶向微泡的有效结合;
而在间歇输注组,MBv在各个剪切应力的靶向结合效率均明显较连续输注组高(P<0.05),其中在2 dyn/cm2时微泡的靶向结合效率最高,在4 dyn/cm2和8 dyn/cm2时仍见靶向微泡有效的结合,至16 dyn/cm2时仍见少量的靶向微泡结合。
解离实验显示平行板流动腔包被分组与剪切应力间存在交互作用(F=529.695,P<0.001),并可见MBv在1000 ng/ml VFc包被组更能抵抗血流剪切应力(F=12.011,P<0.001),达半数解离的剪切应力为(22.1±2.6)dyn/cm2,而在极高剪切应力(51.2 dyn/cm2)时仍能有微泡的滞留；在封闭组和空白组,MBv均无法有效结合,极低剪切应力即可导致大部分MBv解离。
二、携VCAM-1单抗靶向微泡评价动脉粥样硬化斑块早期炎症
1、小鼠CEU检查前后生命体征比较各组小鼠腹主动脉峰值血流速度,主动脉内径,以及心率等无显著差异。
2、小鼠腹主动脉CEU检查
MBv的CEU图像显示APOE-HCD组在微泡注射后8分钟仍可见明显的腹主动脉超声显影,而其他小鼠腹主动脉超声显影情况不明显；MBi在各动物分组8分钟时均无明显腹主动脉的超声显影增强。
彩色编码代表图像显示,APOE-HCD组8分钟后扣除本底的MBv成像彩色编码后较其它各组及各种微泡成像均明显增强,APOE-RD和C57-HCD组也可见8分钟后扣除本底的MBv成像较对照微泡稍增强,C57-RD组MBv及MBi均不增强。
对VI进行定量分析后,微泡分组和动物分组间存在交互效应,其中APOE-HCD-MBv组(10.21±1.60)显著高于其他各组及应用微泡的组合(F=62.203,P<0.001)
3、小鼠腹主动脉免疫组化检查
免疫组化检测显示APOE-HCD小鼠腹主动脉内膜粗糙,表面大量表达VCAM-1,而APOE-RD组和C57-HCD组也有少量VCAM-1表达,C57-RD小鼠腹主动脉内膜光滑,未见有VCAM-1表达。
三、携VCAM-1单抗磁性靶向微泡的制备及体外评价
1、MBvM的制备及理化性质鉴定
库尔特计数仪检测显示无论加入磁性还是非磁性亲和素为桥接,制备的靶向磁性微泡(MBvM)及非磁性靶向微泡(MBv)及对照微泡均(MBiM)的粒径分布和浓度均与生物素化脂质微泡(MBb)无显著差异,证明加入磁性亲和素材料并不影响微泡的稳定性。
2、荧光显微镜观察微泡的磁响应性
用外置永磁铁在玻片一侧作用,荧光显微镜观察发现非磁性靶向微泡MBv在磁场作用下保持静止状态,而磁性靶向微泡及磁性对照微泡MBvM及MBiM则迅速在磁场作用下趋向磁铁方向并聚集。
3、平行板流动腔法评价磁性微泡的磁响应性及靶向黏附
3.1、在没有磁场作用下,平行板流动腔显示MBvM与MBv靶向小鼠VCAM-1 Fc段的能力无论在任何剪切应力条件下均无显著性差异(F=0.131,P=0.877),与MBiM对比,两者只能在低于8dyn/cm2切应力条件下与小鼠VCAM-1 Fc段结合。MBiM在任何剪切应力作用下均无法与小鼠VCAM-1 Fc段有效结合。
3.2、在磁场作用5分钟后,MBvM在各剪切应力下的靶向结合比MBv及MBiM有显著性差异(F=70.236,P<0.0001),且能在16-24 dyn/cm2高剪切应力的条件下靶向结合；
3.3、磁场作用5分钟后,去掉磁场再作用5分钟,发现去掉磁场后,在4dyn/cm2以下切应力条件下MBvM在10分钟点的靶向略有增加,而在高切应作用下,10分钟时靶向黏附微泡的数量呈下降趋势,但仍有微泡有效的靶向。
3.4、磁场作用5分钟后,去掉磁场再作用5分钟,只有MBvM能在高剪切应力(8-24 dyn/cm2)继续保持有效的靶向(F=60.398,P<0.0001),而MBiM因为液体的冲刷作用,去掉磁场后无法保持其靶向能力。
四、携VCAM-1单抗磁性靶向微泡评价动脉粥样硬化斑块早期炎症
1、彩色编码代表图像显示,MBvM磁性靶向微泡在各动物分组10分钟后扣除本底的彩色编码后均较MBv及MBiM成像明显增强。
2、对VI进行定量分析后,微泡分组和动物分组间存在交互效应,其中APOE-HCD-MBvM组(28.1±1.8)显著高于其他各组及应用微泡的组合(F=77.545,P<0.001)。
结论：
一、靶向VCAM-1的微泡在间歇血流条件下可与VFc在高剪切应力的环境中有效的靶向结合,从而具有在脉动高剪切应力状态下的动脉系统处实现分子靶向的能力。
二、应用携VCAM-1单抗靶向微泡可对小鼠动脉粥样硬化斑块的早期炎症进行超声分子成像,但仍有进一步提高靶向效能的空间。
三、应用生物素-亲和素桥接法构建携VCAM-1单抗磁性靶向微泡,体外磁场作用下验证了其磁响应性,平行板流动腔证实该微泡在磁场作用下有更高的靶向黏附效能,提示其将能更好的应用于动脉系统的超声分子成像。
四、靶向VCAM-1磁性靶向微泡在磁场作用下可更好实现动脉粥样硬化斑块早期炎症的超声分子成像。
Objective
Traditionally, the diagnosis of atherosclerosis is possible only at advanced stages of disease, either by direct detection of arterial luminal narrowing or by evaluating the effect of arterial stenosis on organ perfusion or function. Molecular imaging with contrast-enhanced ultrasound (CEU) and targeted microbubbles offers the possibility of real-time, noninvasive visualization of molecular markers of cardiovascular disease using clinical ultrasound systems. Pre-clinical studies have demonstrated great potential for detection of microvascular inflammation, such as occurs in ischemia-reperfusion and cardiac transplant rejection. While CEU molecular imaging of early inflammatory changes of atherosclerosis has been demonstrated, there remain issues over the technical difficulties of targeting microbubbles in the setting of high shear stress in larger arterial vessels.
Ultrasound contrast microbubbles are traditionally used as blood flow tracers that exhibit rheological behavior similar to erythrocytes in vivo, and thus tend to remain close to the axial center of blood vessels. This behavior may disadvantage targeted microbubble agents used for molecular imaging of atherosclerosis in larger vessels, where early inflammatory changes occur within the vascular endothelium. Additionally, hemodynamic factors are very different in arteries as compared to the microvasculature, where endothelial shear stress is higher. In this setting, high shear stress forces may limit the rapid formation of adhesive bonds and hamper microbubble targeting, leading to a high rate of dislodgement of adhered microbubbles and a loss of targeting. To overcome this limitation, we have developed a microbubble agent that can be manipulated by a magnetic field (MF) to alter the axial distribution of microbubbles, increasing the number of circulating microbubbles that contact with the vascular endothelium, and potentially resulting in higher microbubble attachment.
The purpose of our study was to evaluate the finding of microbubble targeted to vascular cell adhesion molecule-1 (VCAM-1) coupled with a magnetic-guidance system could improve the efficacy of CEU molecular imaging of atherosclerosis in the aorta.
Methods
1. Binding capability of microbubbles targeted to VCAM-1 under pulsatile high-shear flow conditions
1.1. Microbubbles preparation and determine their biological properties.
Biotinylated, lipid-shelled microbubbles were prepared by sonication of dipalmitoyl phosphatidylcholine, poly (ethylene glycol) 40-stearate and biotin-poly (ethylene glycol) 2000-distearoylphosphatidylethanolamine with sonicator 3000 at maximum power in an atmosphere of perfluoropropane.1×108 biotinylated microbubbles were conjugated to either rat anti-mouse VCAM-1 monoclonal antibody (MBv) or isotype control (MBi) via a streptavidin bridge. The size distributions and concentrations of MBv and MBi were used coulter counter to counte.
1.2. Assessment of targeted microbubbles targeted to VCAM-1 with Parallel plate flow chamber.
The binding and retention of targeted microbubblesb (MBv) to VCAM-1Fc immobilized on a culture dish were assessed in a flow chamber at variable shear stress (0.5-16.0 dyn/cm2). The pulsatile flow conditions were generated and compared to the continuous flow conditions. The retentive ability of MBv was evaluated by the detachment test.
2. Ultrasound molecular imaging of early stage artherosclerosis with microbubbles targeted to VCAM-1
2.1. Animals Preparation and Diet
We used 4 different mouse models:apolipoprotein E-deficient (APOE-/-) mice on a hypercholesterolemic diet, APOE-/-mice on a regular diet, wild-type mice (C57BL/6) on a hypercholesterolemic diet, and wild-type mice on a regular diet which served as normal controls. Animals were anaesthetized and a jugular vein was cannulated for intravenous administration of microbubbles.
2.2. Contrast Enhanced Ultrasound (CEU) Molecular Imaging
Ultrasound imaging was performed with a high-frequency linear-array probe (17L5) held in place. The abdominal aorta was imaged with fundamental imaging at 15 MHz to optimize the imaging plane in the longitudinal axis and the aortic diameter was measured. The peak flow velocity and pulse rates at the aorta were measured by pulsed-wave Doppler with a gate size set at the minimum setting. CEU was performed with Contrast Pulse Sequencing, which detects the nonlinear fundamental signal component from microbubbles. Imaging was performed at a centerline frequency of 7 MHz and a mechanical index (MI) of 0.18. Real-time imaging at 0.18 MI was performed after intravenous bolus injection of 1×106 magnetic microbubble, After continuous imaging for 8 minutes. The mechanical index was transiently increased to 1.0 for 3 s, to destroy adhered microbubbles, and subsequent post-destruction images were acquired at 0.18 MI, to obtain background images. To determine signal from retained microbubbles alone,3 post-destruction contrast frames representing any freely circulating microbubbles were averaged and digitally subtracted from 3 averaged pre-destruction frames use the Yabko MCE2.7 software (University of Virginia, USA) and then color-coded. Background-subtracted signal intensity was measured from a region of interest placed over the abdominal aorta.
2.3. Immunohistochemistry
Immunostaining for VCAM-1 was performed on frozen sections of the abdominal aorta after drying for 2 hours and fixing with paraformaldehyde for 15 minutes at room temperature. Rat anti-mouse VCAM-1 monoclonal antibody was used as a primary antibody with a secondary anti-rat antibody. Staining was performed with HRP substrate solution. Slides were counterstained with hematoxylin. Slides were visualized under microscope and photographed with a CCD camera.
3. Preparation of magnetic microbubbles targeted to VCAM-1 and in-vitro assessment.
3.1. Microbubble Preparation
Biotinylated, lipid-shelled microbubbles were as previously described. MBb were conjugated to either rat anti-mouse VCAM-1 monoclonal antibody (MBvM) or isotype control antibody (MBiM) via a magnetic streptavidin bridge. For a positive control microbubble, microbubbles were conjugated to a rat anti-mouse VCAM-1 monoclonal antibody via regular non-magnetic streptavidin bridge (MBv). For in vitro flow-chamber studies, microbubbles were fluorescently labeled by the addition of dioctadecyltetramethylindocarbocyanine (DiI) perchlorate to the aqueous suspension prior to sonication. Microbubble size, concentration and distribution were measured by electrozone sensing.
3.2. Assessment of Microbubbles in a Magnetic Field (MF)
The behavior of microbubbles within a MF was determined using an optical microscope. The microbubble suspension was gently shaken before one drop was applied to the microscope slide, and a cover slip applied. The images were recorded digitally with a CCD camera under a 10×objective lens and a 4×magnification tube. A magnet (5000 GS) was placed beside the visual field to assess microbubble behavior under MF.
3.3. Parallel Plate Flow-Chamber Adhesion Studies
PBS droplets (200μl) containing 1000 ng of recombinant mouse VCAM-1 Fc chimera were placed and fixed in a 1 cm diameter circular area on culture dishes. The flow chamber was placed on a microscope in a custom-designed stage and the culture dish side was inverted, and the MF placed underneath. A suspension of MBvM, MBv or MBiM (5×106 ml-1) was drawn through the flow chamber with an adjustable withdrawal pump with or without MF-guidance at an initial shear stress of 1 dyn/cm2. Because the MF was used to manipulate microbubble behavior at different shear stress conditions (1-24 dyn/cm2), MF-guidance was implemented for the first 5 min of microbubble infusion, after which it was removed and followed by a 5 min "flush". The number of microbubbles attached to the plate was determined over 20 optical fields (total area,0.5 mm2) at the 5 min and 10 min time points using Image Pro-Plus (IPP, Media Cybernetics) software to auto track and count microbubbles.
4. Ultrasound molecular imaging of early stage artherosclerosis with magnetic microbubbles targeted to VCAM-1
4.1. Animals Preparation and Diet
We used 4 different mouse models:apolipoprotein E-deficient (APOE-/-) mice on a hypercholesterolemic diet, APOE-/-mice on a regular diet, wild-type mice (C57BL/6) on a hypercholesterolemic diet, and wild-type mice on a regular diet which served as normal controls. Animals were anaesthetized and a jugular vein was cannulated for intravenous administration of microbubbles.
4.2. Contrast Enhanced Ultrasound (CEU) Molecular Imaging
Ultrasound imaging was performed with a high-frequency linear-array probe (17L5) held in place. A MF (5000 GS) was placed under the abdomen of anesthetized mice. CEU was performed with Contrast Pulse Sequencing. Real-time imaging at 0.18 MI was performed after intravenous bolus injection of 1×106 MBvM, MBv or MBiM performed in random order. After 5 min of imaging the MF was manually removed, and continuous imaging continued for 10 minutes. The mechanical index was transiently increased to 1.0 for 3 s, to destroy adhered microbubbles, and subsequent post-destruction images were acquired at 0.18 MI, to obtain background images. To determine signal from retained microbubbles alone,3 post-destruction contrast frames representing any freely circulating microbubbles were averaged and digitally subtracted from 3 averaged pre-destruction frames use the Yabko MCE2.7 software (University of Virginia, USA) and then color-coded. Background-subtracted signal intensity was measured from a region of interest placed over the abdominal aorta.
Results
1. Binding capability of microbubbles targeted to VCAM-1 under pulsatile high-shear flow conditions
1.1. Both MBv and MBi were prepared successfully, the concentration of MBv was about 2.9 x 108/ml, the mean sizes was 2.55±0.75μm respectively.
1.2. The marked binding of MBv were seen in pulsatile and continuous flow conditions at low-shear flow conditions of 0.5-2 dyn/cm2, but the binding rate in the pulsatile flow group was higher (P< 0.05) than that in the continuous flow conditions. Furthermore, the marked binding of MBv was still noted at the highest shear rates (4-8 dyn/cm2) under pulsatile flow conditions, while it was not observed under continuous flow conditions. Microbubble detachment was assessed by increasing the flow every 30 s and observing the number of microbubbles remaining bound. Better retention of microbubbles was found on the 1000 ng/ml VCAM-1 Fc surfaces (F= 12.011, P< 0.001). The half detachment rate of MBv was high up to 22.1±2.6 dyn/cm2.
2. Ultrasound molecular imaging of early stage artherosclerosis with microbubbles targeted to VCAM-1
2.1 There were no significant differences in aortic peak velocity, aortic diameter and heart rates between the different animal groups.
2.2. Contrast Enhanced Ultrasound (CEU) Molecular Imaging The obviously signal enhancement was only observed for MBv in APOE-HCD mice (10.21±1.60), and was greater than any microbubbles used in any other animal groups (F=62.203, P<0.001).
2.3. Immunohistochemistry
On immunostaining, VCAM-1 expression was detected on the regions of luminal endothelial surface of the aorta in wild-type mice on hypercholesterolemic diet. In APOE-/-mice, there were visually intimal thickening and large atherosclerotic plaques protruding into the lumen, particularly in animals on hypercholesterolemic diet. Immunohistochemistry in APOE-/-mice demonstrated VCAM-1 expression on the endothelium, which was higher in animals on hypercholesterolemic diet.
3. Preparation of magnetic microbubbles targeted to VCAM-1 and in-vitro assessment.
3.1. Microbubble size, concentration and distribution were not significantly different between groups. After addition of magnetic streptavidin and biotinylated antibody to the biotinylated microbubble, the size distribution profile remained unchanged, suggesting a lack of aggregation.
3.2. While the behavior of microbubbles bearing magnetic streptavidin, magnetic microbubble and inactive magnetic microbubble, were influenced by the presence of a magnetic field, non-magnetic microbubble, remained stationary.
3.3. Both MBvM and MBv demonstrated similar attachment to plates coated with VCAM-1 Fc within the flow chamber in the absence of MF-guidance (F=0.131, P=0.877), while MBiM had minimal bubble attachment at the initial shear stress of 1 dyn/cm2. For all microbubbles, attachment decreased exponentially as shear stress increased, becoming minimal at shear rates>8 dyn/cm2. Upon MF-guidance, attachment of MBvM was significantly higher than MBv (F= 70.236, P< 0.0001). When MF-guidance was applied for the initial 5 min of infusion, both magnetic microbubble and inactive magnetic microbubble demonstrated binding at higher shear flow (8-16 dyn/cm2), while the attachment of MBiM remained unchanged. After termination of MF-guidance and a 5 min "flush", minimal MBiM attachment remained at low shear flow; however MBvM remained firmly attached even at high shear flows of 12-16 dyn/cm2 and was significantly greater than MBv and MBiM at each level of shear stress (F= 60.398, P< 0.0001).
4. Ultrasound molecular imaging of early stage artherosclerosis with magnetic microbubbles targeted to VCAM-1
Background-subtracted color-coded CEU images showed the obviously signal enhancement was observed for MBvM in APOE-HCD mice. After termination of MF-guidance and 5 min "flush", in wild-type mice on regular diet, signal for MBvM was low and similar to MBiM and MBv. In the other three groups, background-subtracted signal intensity for MBvM was greater than MBv and MBiM.The interaction between targeted microbubble agent and animal group was highly significant (F= 77.545, P< 0.001), suggesting that the difference in signal for MBvM depended on the animal group (disease severity).
Conclusions
1. The targeted microbubbles binding to VCAM-1 specific and effective at high-shear stress under pulsatile flow conditions. The molecular ultrasound imaging could be potentially use in the high-shear conditions artery system.
2. MBv can use to ultrasound molecular imaging of early stage inflammation in artherosclerosis, but still need improvement.
3. MBvM can be manipulated by a magnetic field and have the better binding than MBv and MBiM, using this method could provide better ultrasound molecular imaging of artherosclerosis.
4. Magnetic field-guided molecular CEU imaging using magnetic microbubbles targeted to endothelial VCAM-1 improves the detection of the early stages of atherosclerosis in mice.
引文
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1. Klibanov AL, Rychak JJ, Yang WC, Alikhani S, Li B, Acton S, Lindner JR, Ley K, Kaul S. Targeted ultrasound contrast agent for molecular imaging of inflammation in high-shear flow. Contrast Media Mol Imaging.2006; 1:259-266
2. Takalkar AM, Klibanov AL, Rychak JJ, Lindner JR, Ley K. Binding and detachment dynamics of microbubbles targeted to p-selectin under controlled shear flow. J Control Release.2004;96:473-482
3. Kaufinann BA. Ultrasound molecular imaging of atherosclerosis. Cardiovascular Research.2009;83:617-625
4. Rychak JJ, Klibanov AL, Ley KF, Hossack JA. Enhanced targeting of ultrasound contrast agents using acoustic radiation force. Ultrasound Med Biol.2007;33:1132-1139
5. Rychak JJ, Li B, Acton ST, Leppanen A, Cummings RD, Ley K, Klibanov AL Selectin ligands promote ultrasound contrast agent adhesion under shear flow. Mol Pharm.2006;3:516-524
6. Rychak JJ, Lindner JR, Ley K, Klibanov AL. Deformable gas-filled microbubbles targeted to p-selectin. J Control Release.2006; 114:288-299
7. Kaufinann BA, Sanders JM, Davis C, Xie A, Aldred P, Sarembock IJ, Lindner JR. Molecular imaging of inflammation in atherosclerosis with targeted ultrasound detection of vascular cell adhesion molecule-1. Circulation. 2007;116:276-284
8. Cho Y-K, Yang W, Harry BL, Klibanov AL, Hossack JA, Ley K. Abstract 3559:Dual-targeted contrast enhanced ultrasound imaging of atherosclerosis in apolipoprotein e gene knockout mice. Circulation.2006;114:Ⅱ_759-c-
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