携抗ICAM-1超声造影剂对兔腹主动脉损伤内膜靶向显影的实验研究

目的探讨携抗ICAM-1的超声造影剂微泡靶向显影对损伤血管内膜的诊断价值.方法使用自制含氟碳声振白蛋白造影剂,制备FITC标记的携抗ICAM-1的靶向超声造影剂.荧光素标记普通造影剂微泡以示对照.新西兰大白兔12只,随机分2组,通过高脂饮食建立腹主动脉内膜损伤模型.模型建立前后分别经兔耳缘静脉推注普通(第1组)、靶向(第2组)2种造影剂微泡,超声监测不同时期、2类微泡在内膜显影情况的差异并做比较分析.造影后取腹主动脉标本做病理对照.结果模型建立前,2组造影结果无明显差异.内膜损伤后,靶向微泡持续显影时间明显延长,有延迟排空现象,与普通微泡造影结果相差显著.光镜见第2组内皮黏附微泡明显多于第1组,内膜面有强绿色荧光带形成.结论携抗黏附分子-1造影剂微泡可靶向黏附于损伤血管内膜,超声造影可据此判定早期血管内膜损伤.     

携IL-8单抗靶向超声造影剂与血管内皮细胞相互作用的实验研究

目的 观察自制携带IL-8单抗靶向超声造影剂与损伤血管内皮细胞的相互作用,探讨IL-8在粥样斑块形成过程中的作用和评价血管内皮功能的新方法.方法 采用交联剂将抗人IL-8单克隆抗体偶联到SonoVue微泡表面,制备靶向性超声微泡;倒置显微镜下分别观察SonoVue微泡、靶向性微泡与正常内皮细胞、损伤内皮细胞的结合作用,高倍视野下计数内皮细胞及黏附的微泡数,通过计算微气泡与内皮细胞的比值对两者之间的结合作用进行定量分析.结果 仅有极少量对照组SonoVue微泡黏附于正常及损伤内皮细胞;而镜下可见携IL-8单抗的SonoVue微泡黏附于内皮细胞,黏附于损伤内皮细胞表面的微泡数显著高于正常内皮细胞.结论 携IL-8单抗的靶向性超声造影剂能够特异性结合在损伤细胞表面,拓展了超声成像技术检测血管内皮损伤、评价血管内皮功能的新领域.     

携IL-8单抗的靶向超声造影剂对损伤心肌细胞的体外寻靶实验研究

目的 通过体外寻靶实验评价携IL-8单克隆抗体(简称单抗)靶向超声造影剂的靶向粘附效能,探讨IL-8在心肌梗死早期的作用机制。方法 采用共价偶联法制备携IL-8单抗靶向超声造影剂,利用使细胞缺氧、缺糖的方法制备损伤心肌细胞,通过靶向超声造影剂与损伤心肌细胞的体外结合实验检测其寻靶能力。结果 携IL-8单抗靶向微泡与损伤及损伤初期心肌细胞的粘附作用明显高于SonoVue微泡(P<0.05),且随着损伤程度的加重,携IL-8单抗靶向微泡与心肌细胞的黏附作用逐渐加强(P<0.05)。结论 携IL-8单抗靶向超声造影剂对损伤心肌细胞具有较强的靶向性。     

携CD54单抗的靶向超声造影剂增强兔腹主动脉内膜及粥样斑块显影的实验研究

目的　探讨携CD54单抗的超声造影剂在动脉粥样硬化中的诊断价值。方法　整和CD54单克隆抗体及白蛋白微泡制备靶向超声造影剂。新西兰大白兔 26只,高脂饮食建立动脉粥样硬化模型并随机分 3组。第 1、2组分别使用普通、靶向造影剂行腹主动脉超声造影,第 3组同时应用两种造影剂造影。视频密度法评价两种造影剂对动脉内膜、粥样斑块的造影增强效应,免疫组化检测白蛋白微泡在靶组织中分布情况。结果　各组造影后内膜、粥样斑块峰值视频密度与造影前基值相比均有显著增加 (P<0. 01)。第 1、2组间血管内膜、斑块峰值视频密度的差异有显著性意义 (P<0. 01)。第 3组使用普通造影剂的内膜、斑块峰值视频与使用靶向造影剂的对应峰值比较差异有显著性意义 (P<0. 05 )。腹主动脉壁上携CD54单抗的微泡免疫组化染色呈强阳性,普通微泡为弱阳性。结论　携CD54单抗造影剂对粥样硬化动脉内膜及斑块有靶向显影价值,可提高超声诊断敏感性。     

携抗ICAM-1单抗和抗CD34单抗双靶向微泡的制备及体外寻靶研究

目的 制备同时携抗ICAM-1单抗和抗CD34单抗的双靶向微泡,鉴定其基本性质,并观察体外寻靶能力.方法 采用“生物素-亲和素”桥连法分别构建携抗ICAM-1单抗和抗CD34单抗双配体(MBD)、携抗ICAM-1单抗(MBICAM-1)和携抗CD34单抗(MBCD34)3种靶向微泡,光学显微镜下观察靶向微泡的形状,并用马尔文激光粒度分析仪、DFY超声图像定量分析仪、激光共聚焦显微镜、流式细胞仪检测靶向微泡的基本特性和抗体结合率.倒置显微镜下观察双靶向微泡与内皮祖细胞(EPCs)和损伤人脐静脉内皮细胞(HUVECs)的结合情况.结果 3种靶向微泡的形状、粒径、表面电位及抗体结合率差异均无统计学意义(P＞0.05),MBD的回声强度高于MBICAM-1、MBCD34和普通生物素化微泡(MBBiotin)(P＜0.01).MBD与EPCs和损伤HUVECs的结合率明显高于MBBiotin(P＜0.01),MBD和MBCD34与EPCs的结合率、MBD和MBICAM-1与损伤HUVECs的结合率差异均无统计学意义(P＞0.05).结论 成功制备了携抗ICAM-1单抗和抗CD34单抗的双靶向微泡,体外实验证实此双靶向微泡与内皮祖细胞和损伤血管内皮细胞均能特异性结合.     

携GL-7靶向微泡造影剂对高脂饮食兔腹主动脉内膜的靶向作用

目的 探讨携GL-7靶向微泡造影剂对高脂饮食兔腹主动脉内膜体外特异性结合及体内增强显影效果.方法 14只新西兰兔用高脂饮食法建立腹主动脉内膜损伤模型,并随机分为两组,4周后分别使用对照微泡和靶向微泡造影剂进行腹主动脉超声造影,以视频密度法评价两种造影剂对动脉内膜的增强效果,荧光显微镜观察两种造影剂体内结合情况及与动脉内膜荧光染色的结合情况及荧光强度统计分析.结果 对照微泡、靶向微泡造影后血管内膜回声均较造影前增强,靶向微泡造影与对照微泡造影比较,血管内膜峰值视频密度差异有统计学意义(P<0.05).荧光显微镜观察,靶向微泡血管腔内呈现绿色荧光,而对照微泡血管腔内仅有微弱的绿色荧光.动脉血管冰冻切片结果显示,靶向微泡造影组动脉内膜有绿色荧光表达,而对照微泡造影组绿色荧光表达较弱,两组荧光强度差异有统计学意义(P<0.05).结论 GL-7靶向微泡造影剂与兔动脉血管内膜体内、体外特异性结合,可显著增强兔腹主动脉内膜靶向显影.     

携IL-8单抗靶向超声微泡对损伤心肌细胞的体外寻靶实验研究

目的制备携IL-8单克隆抗体(以下简称单抗)靶向超声微泡,检测其基本特性,并探讨其对损伤心肌细胞的体外黏附能力。方法采用共价偶联法制备携IL-8单抗靶向超声微泡,利用缺氧、缺糖方法制备损伤心肌细胞,通过靶向超声微泡与损伤心肌细胞的体外结合实验检测其寻靶能力,并与Sono Vue微泡进行比较。结果携IL-8单抗靶向微泡与轻度及重度损伤心肌细胞的黏附比分别为(61.9±18.9)%和(86.6±5.1)%,明显高于Sono Vue微泡与轻度损伤及重度损伤心肌细胞的黏附比[(12.0±0.6)%和(11.8±1.0)%],差异均有统计学意义(均P0.01);且随着损伤程度的加重,携IL-8单抗靶向超声微泡与心肌细胞的黏附作用逐渐加强(r=0.945,P0.01)。结论携IL-8单抗靶向超声微泡对损伤心肌细胞具有较强的靶向性。     

靶向性声学造影剂与血管内皮细胞相互作用的实验研究

目的 制备携抗人VCAM-1单克隆抗体的白蛋白声学造影剂，观察其与损伤血管内皮细胞的相互作用，探讨评价血管内皮功能的新方法。 方法 采用交联法将抗人VCAM-1单克隆抗体共价偶联到自制氟碳气体为核心的白蛋白微气泡表面，制备靶向性声学微气泡；倒置显微镜下分别观察普通白蛋白微气泡、靶向性微气泡与正常内皮细胞、损伤内皮细胞的结合作用，高倍视野下计数内皮细胞及黏附的微气泡的数目，通过计算微气泡与内皮细胞的比值对两者之间的结合作用进行定量分析。 结果 无论是正常内皮细胞，或损伤内皮细胞，仅见少量的对照组微气泡的黏附作用；而镜下可见大量携VCAM-1单抗的白蛋白微气泡黏附在损伤内皮细胞表面，黏附数目显著高于黏附于正常内皮细胞表面的数目。 结论 携VCAM-1单抗的靶向性声学造影剂能够特异性结合在损伤内皮细胞表面，开拓了超声成像技术检测血管内皮损伤、评价血管内皮功能新的研究领域。     

携细胞间黏附分子1抗体的Sono Vue微泡造影剂靶向识别受损内皮的实验研究

目的 探讨静电吸附法制备的携细胞间黏附分子1抗体靶向微泡在体外和体内的寻靶能力.方法 采用静电吸附法制备携ICAM-1抗体的靶向SonoVue微泡.体外培养经IL-1β刺激大量表达ICAM-1的人血管内皮细胞ECV304,采用免疫荧光法检测靶向微泡与其结合能力.构建兔急性心肌梗死模型,进行靶向微泡的心肌造影,采用冷冻切片和免疫荧光法榆测靶向微泡与受损血管内膜结合能力.结果 在体外实验中,可见大量携ICAM-1抗体靶向微泡与刺激后ECV304细胞紧密结合,仅有少量靶向微泡与正常ECV304细胞结合.体内实验中,可见大量携ICAM-1抗体靶向微泡在兔梗死心肌受损血管内膜处黏附,仅可见少量靶向微泡在正常血管内膜处黏附.结论 携ICAM-1抗体靶向微泡在体内、体外均能与受损血管内皮特异性结合,不仅有利于靶向超声造影显像,更为微泡携带药物或基因在局部定向释放开辟良好前景.     

携抗P-选择素单抗靶向微泡在炎症模型微循环中黏附机制及行为方式的研究

目的 荧光显微镜直视下对比评价携抗P-选择素单抗靶向微泡与同型对照微泡在微循环中的黏附机制及行为方式.方法 构建携荧光FITC的抗P-选择素单抗靶向微泡(MBp)和同型对照微泡(MBiso),并随机经静脉注入小鼠提睾肌炎症模型.20倍荧光显微镜直视下观察并记录5min内两种微泡在提睾肌微循环中的黏附情况,并对不同黏附方式的微泡进行计数.应用image-pro-plus分析软件对微泡进行定点追踪,并对其黏附过程中速度的变化进行定量测定.结果 荧光显微镜下观察可见MBp组与内皮黏附数量高达(8.4±2.1)个/视野,MBiso组仅为(0.8±0.8)个/视野,两者间差异有统计学意义(P＜0.01);MBp和MBiso组白细胞黏附数量分别为(3.6±0.6)个/视野、(2.2±0.8)个/视野,两者间差异无统计学意义(P＞0.05).微泡黏附过程速度变化曲线显示MBp和MBiso分别以流动速度逐渐和迅速降低两种方式实现对靶组织的黏附.结论 MBp和MBiso具有不同的黏附方式,MBp能更高效、特异地黏附于炎症组织血管内皮上,为评价血管内皮炎症反应或其他组织损伤的应用提供了理论基础.     

含氟烷人血白蛋白靶向超声造影剂的制备研究

目的为靶向超声造影剂的制备进行基础实验研究。方法将含氟烷人血白蛋白超声造影剂与ICAM-1抗体混合,加入0.1%戊二醛,4℃孵育2h,促使抗体与微泡外壳充分交联。普通光镜评估靶向微泡的大小、形态。免疫荧光法进行靶向微泡的鉴定。结果普通光镜下可见携ICAM-1抗体的靶向微泡大小、形态与普通白蛋白微泡对照无明显改变。荧光显微镜证实微泡与ICAM-1抗体整合成功。结论戊二醛交联法适用于白蛋白靶向超声造影剂的制备,方法简便,成本低,重复性好。     

Deformable gas-filled microbubbles targeted to P-selectin.

Ultrasound contrast microbubbles have been successfully targeted to a number of intravascular disease markers. We hypothesized that targeted delivery could be improved further, by making the microbubbles deformable, leading to increased microbubble-endothelium adhesion contact area and stabilized adhesion. Activated leukocytes utilize such strategy; they deform after binding to inflamed endothelium in the vasculature. Lipid-shell microbubbles were targeted to the endothelial inflammatory protein P-selectin with a monoclonal anti-P-selectin antibody attached to the microbubble shell. Deformable microbubbles were created by controlled pressurization with partial gas loss, which generated an average excess shell surface area of approximately 30% and the formation of outward-projected wrinkles and folds. Targeted microbubble adhesion and deformability were assessed in the parallel plate flow chamber under shear flow. Sustained adhesion of deformable microbubbles at wall shear stresses between 0.4 and 1.35 dyn/cm(2) was consistently better than adhesion of wrinkle-free microbubbles. Over this shear range, targeted wrinkled microbubbles were deformed by shear flow, unlike wrinkle-free microbubbles. In a murine cremaster inflammation model, a significant improvement of deformable microbubble targeting was observed by intravital microscopy. Overall, the mechanical aspects of adhesion, such as particle shape, deformability and surface microstructure, are important in engineering efficient site-targeted particle-based agents for medical imaging and therapy.     

An in vivo validation of the application of acoustic radiation force to enhance the diagnostic utility of molecular imaging using 3-d ultrasound

For more than a decade, the application of acoustic radiation force (ARF) has been proposed as a mechanism to increase ultrasonic molecular imaging (MI) sensitivity in vivo. Presented herein is the first noninvasive in vivo validation of ARF-enhanced MI with an unmodified clinical system. First, an in vitro optical-acoustical setup was used to optimize system parameters and ensure sufficient microbubble translation when exposed to ARF. 3-D ARF-enhanced MI was then performed on 7 rat fibrosarcoma tumors using microbubbles targeted to α_vβ₃ and nontargeted microbubbles. Low-amplitude (<25 kPa) 3-D ARF pulse sequences were tested and compared with passive targeting studies in the same animal. Our results demonstrate that a 78% increase in image intensity from targeted microbubbles can be achieved when using ARF relative to the passive targeting studies. Furthermore, ARF did not significantly increase image contrast when applied to nontargeted agents, suggesting that ARF did not increase nonspecific adhesion.     

Therapeutic effects of paclitaxel-containing ultrasound contrast agents

Drug delivery vehicles that combine ultrasonic and molecular targeting are shown to locally concentrate a drug in a region-of-interest. The drug delivery vehicles, referred to as acoustically active lipospheres (AALs), are microbubbles surrounded by a shell of oil and lipid. In a region limited to the focal area of ultrasound application, circulating AALs are deflected by radiation force to a vessel wall and can subsequently be fragmented. Ligands targeting the alphavbeta3 integrin are conjugated to the AAL shell and increase in vitro binding by 26.5-fold over nontargeted agents. Toxicity assays demonstrate that paclitaxel-containing AALs exert a greater antiproliferative effect after insonation than free paclitaxel at an equivalent concentration. Lastly, ultrasound and molecular targeting are combined to deliver a model drug to the endothelium and interstitium of chorioallantoic membrane vasculature in vivo.     

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.