含生物素化脂膜超声造影剂的制备与初步评价

目的 制备一种含生物素化脂膜的超声造影剂,并对其功能进行初步评价.方法 以冷冻干燥法在自制＂脂氟显＂（对照微泡）的基础上制备含生物素化脂膜超声造影剂微泡,检测其理化性质和造影功能,同时应用激光共聚焦显微镜和平行板流动腔法观察含生物素化脂膜微泡与链亲和素的黏附性.结果 ①含生物素化脂膜微泡造影剂在理化性质与小鼠肝脏造影显像方面能力与对照微泡比较差异无统计学意义（P〉0.05）;②与异硫氰酸荧光素（FITC）标记的链亲和素孵育后,含生物素化脂膜微泡荧光检测呈阳性,对照微泡为阴性;③含生物素化脂膜微泡可结合于链亲和素包被的培养皿上,且随着链亲和素包被浓度的增加其结合稳定性也相应提高.结论 应用冷冻干燥法在制备＂脂氟显＂微泡造影剂的基础上,可成功制备含生物素化脂膜超声微泡造影剂,为今后制备靶向显影及载药（基因）超声造影剂奠定了基础.     

在生理血流条件下靶向超声微泡对P-选择素的靶向黏附效能

目的平行板流动腔评价在生理血流条件下携抗小鼠P-选择素单抗靶向超声微泡（MBp）的靶向黏附效能。方法采用＂亲和素-生物素＂桥接法构建MBp;在3种浓度（10、100和1000ng/ml）小鼠P-选择素Fc段（PSFc）包被的平行板流动腔和固定剪切应力下,以及最大包被浓度和不同剪切应力（0.2-1.7dyn/cm^2）下分别检测MBp的每分钟结合数量（结合率）,以抗小鼠P-选择素单抗封闭组和空白组为对照。在3种包被浓度下检测MBp达半数解离的剪切应力。所有分组样本数均为3。结果两对照组均未见有明显的MBp结合。实验组MBp结合率随包被浓度的增高而增加（P〈0.05）,然而与剪切应力呈现双向性（P〈0.05）。MBp达半数解离的剪切应力随包被浓度的增加而增大（P〈0.05）。结论MBp在生理条件下可与PSFc特异有效地结合,体外对靶向超声微泡的靶向黏附效能评价将有助于判定超声分子成像的效果和靶向微泡的在体应用环境条件。     

模拟动脉间歇脉搏血流评价携血管细胞黏附分子-1单抗靶向微泡黏附效能

目的 应用平行板流动腔模拟间歇脉动的动脉血流评价携血管细胞黏附分子-1单抗靶向微泡(MBv)进行高剪切应力下靶向的可行性.方法 采用"亲和素-生物素"桥接法构建MBv在1000ng/ml小鼠血管细胞黏附分子-1 Fc段(VFc)包被的平行板流动腔中,分为连续输注组及间歇输注组,在不同剪切应力(0.5～16 dyn/cm2)下,分别检测不同结合时间点(1～6 min)靶向微泡的结合数量.并用解离实验检测MBv达半数解离的剪切应力.所有实验均重复3次取平均值.结果 两组在0.5～2 dyn/cm2时均见有明显的MBv结合.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),且只有间歇输注组MBv能在4～8 dyn/cm2条件下实现靶向结合.解离实验显示靶向微泡达半数解离的剪切应力为(20.7±3.1)dyn/cm2.结论 在间歇脉动的液体流动状态下,MBv可在更高的血流剪切应力条件下与VCAM-1特异有效结合,有望用于进行动脉系统的超声分子成像.     

携IgG单抗靶向超声造影剂的构建及其靶向效能的体外评价

目的 构建携羊抗小鼠IgG单抗靶向超声造影剂(UCA-IgG)及体外评价其靶向黏附效能.方法 采用"亲和素-生物素"桥接法构建UCA-IgG,体外荧光法鉴定IgG单抗与生物素化造影剂特异结合.利用平行板流动腔在不同时间点、不同浓度小鼠IgG包被和不同剪切应力下,测定相应的UCA-IgG的结合数目及达到半数解离时的剪切应力.设不同浓度小鼠包被的平行板流动腔为实验组,羊抗小鼠IgG封闭组和空白组为对照组.结果 UCA-IgG发出明亮的绿色荧光,而普通脂质造影剂无荧光显示.实验组UCA-IgG结合数量随包被浓度的增高而增加(P<0.05),并与结合时间呈明显正相关(P<0.05),然而与剪切应力呈现双向性(P<0.05).而两对照组均未见明显的UCA-IgG结合.UCA-IgG达半数解离的剪切应力随包被浓度的增加而增大(P<0.05).结论 在不同血流动力学条件下UCA-IgG可与小鼠IgG特异有效结合,携羊抗小鼠IgG单抗造影剂可为其他特异靶向超声造影剂的体外研究提供有力的支持.     

超声微泡连接特异性抗体的制备方法及靶向化处理因素对超声微泡物理性质的影响

目的 探讨超声微泡连接特异性抗体的制备方法及靶向化处理因素对超声微泡物理性质的影响.方法 应用生物素-链霉亲和素连接系统,使注射用六氟化硫微泡(SonoVue)与特异性抗血管细胞黏附分子-1(VCAM-1)抗体相连接,用间接免疫荧光法检测抗体与微泡的连接,用马尔文激光粒径分析仪分别测定生物素化处理前后微泡的粒径,采用超声诊断仪评价微泡靶向化构建前后的显像效果.结果 SonoVue与特异性抗体成功连接,间接免疫荧光检测呈阳性.生物素修饰后的微泡粒径分布变窄,与普通微泡的超声显像效果略有差异.结论 通过生物素-链霉亲和素系统可以成功靶向化构建超声微泡,靶向化处理因素对微泡的粒径有一定影响,对微泡的超声显影效果略有影响.     

低免疫原性靶向微泡的制备研究

目的 制备低免疫原性靶向微泡.方法 采用不同膜磷脂成份制备表面包埋或暴露生物素的靶向微泡(MBb或MBe),荧光鉴定"生物素-链亲和素"桥连技术构建靶向微泡的可靠性.以普通微泡(MBc)为参照,酶联免疫吸附法体外检测三组微泡与人血清反应后的补体3a(C3a)水平;平行平板流动腔实验检测各组微泡与包被有链亲和素培养皿的黏附力.结果 MBe及MBb均发出明亮的环形绿色荧光,而MBc未见荧光.MBb组的C3a水平[(1.037±0.047)ng/m1]明显低于MBe组[(1.326±0.042)ng/m1](P＜0.05),与MBc组[(1.004±0.031)ng/m1]差异无统计学意义(P＞0.05).平行平板流动腔实验显示MBb组黏附的微泡数[(15.2±11.3)个/视野]少于MBe组[(103.2±28.3)个/视野](P＜0.05),与MBc组[(17.8±11.9)个/视野]差异无统计学意义(P＞0.05).结论 成功制备屏蔽表面免疫原性物质的靶向微泡,为进一步活体实验提供了基础数据.     

靶向超声微泡MB_(ICAM-1)的制备及靶向实验研究

目的制备载抗ICAM-1(细胞间黏附分子-1)的靶向超声微泡MBICAM-1,鉴定制备微泡的基本性质,研究其在体内及体外的靶向能力。方法通过"生物素-亲和素"桥接法将抗ICAM-1结合到生物素化脂质微泡,制成MBICAM-1;检测制备微泡的浓度、粒径,观察微泡的形态;检测抗ICAM-1与微泡的结合率;ICAM-1质粒转染Hela细胞,观察转染后的Hela细胞与MBICAM-1结合,验证MBICAM-1体外靶向功能;制备家兔左侧跟腱炎症模型,用制备的微泡对家兔双侧跟腱行造影检查,验证MBICAM-1体内靶向能力。结果三种微泡平均粒径1.00~2.4μm,浓度2.4×108~10/ml;微泡的形态完整、规则,分布较匀;微泡和抗ICAM-1单抗的平均结合率为(86.5±5.3)%。MBICAM-1环绕在转染后的Hela细胞周围。MBICAM-1对家兔左侧跟腱造影呈高增强。结论采用"生物素-亲和素"桥接法可制备MBICAM-1;MBICAM-1在体外、体内均具有靶向功能。     

以HER2为靶点的靶向纳米级脂质微泡超声造影剂的制备及实验研究

目的 制备以HER2受体为靶点的靶向纳米级脂质微泡超声造影剂,并观察其体外寻靶能力及体外超声显像效果.方法 制备生物素化Herceptin单抗,检测其生物素化程度及生物学活性;薄膜水化-声振法制备生物素化纳米微泡,以生物素-亲和素为桥梁制备HER2为靶点的靶向纳米级脂质微泡超声造影剂,观察其对SKOV3卵巢癌细胞的体外寻靶能力及靶向结合的体外超声显像.结果 平均每分子Herceptin单抗可与16个生物素分子结合;与游离单抗相比,生物素化抗体的活性未见明显降低(P＞0.05).体外寻靶实验观察:靶向纳米微泡组SKOV3细胞表面有明显的红染纳米微泡与其牢固结合,沿细胞表面排列较规则;非靶向组SKOV3细胞表面未结合红染的纳米微泡.靶向纳米微泡体外超声显像:细胞爬片与靶向纳米微泡孵育后可明显增强超声显像效果,对照组均未见明显超声显像.结论 应用生物素-亲和素系统成功构建了以HER2为靶点的纳米级超声造影剂,与SKOV3细胞结合后有明显超声显像效果.     

LHRHa靶向微泡造影剂的制备及体外寻靶实验

目的 制备人卵巢癌靶向超声造影剂,观察其体外寻靶能力。方法 采用生物素-链霉亲和素法制备促黄体生成素释放激素类似物(LHRHa)靶向脂质微泡,以免疫荧光染色验证LHRHa与脂质微泡的结合情况,并以普通脂质微泡作为对照,在倒置显微镜下观察LHRHa靶向脂质微泡对人卵巢癌A2780/DDP的体外寻靶能力。结果 LHRHa靶向脂质微泡免疫荧光阳性;体外寻靶实验显示LHRHa靶向脂质微泡能够与表面存在LHRH受体的A2780/DDP细胞特异性结合,而普通脂质微泡则不能结合。结论 利用生物素-链霉亲和素方法可成功制备LHRHa靶向脂质微泡造影剂,该靶向造影剂具有体外寻靶能力。     

制备结合链酶亲和素超声造影剂的实验研究

目的：探索制备一种结合链酶亲和素的脂膜超声微泡，以适用于亲和素－生物素法制备靶向超声造影剂，并评价其理化性质。方法：分4组于机械或超声振荡之前或之后，加入链酶亲和素完成超声造影剂的制备，采用浮选法洗涤。观察并检测洗涤前后各组微泡大小、形态、浓度、荧光亮度、微泡与链酶亲和素的结合情况。结果：机械或超声振荡制备的各组结合链酶亲和素的超声造影剂其浓度、形态与普通微泡比较无明显差异，但易静置分层。超声振荡法制备的微泡粒径稍大、浓度偏低。荧光显微镜观察：洗涤前各种不同方法所制备的微泡均能激发出明亮的红色荧光；洗涤后微泡浓度由1010左右降到107左右，微泡荧光亮度仍为“3级”。微泡与链酶亲和素的结合率，各种制备方法间比较差异无显著性意义（P>0.05），其结合率高达98%以上。结论：机械和超声振荡均可制备结合链酶亲和素的负电荷超声造影剂，微泡与其结合率高，为完成亲和素－生物素法制备靶向超声造影剂提供了重要基础。     

Targeted ultrasound contrast agent for molecular imaging of inflammation in high‐shear flow

Targeted ultrasound contrast materials (gas‐filled microbubbles carrying ligands to endothelial selectins or integrins) have been investigated as potential molecular imaging agents. Such microbubbles normally exhibit good targeting capability at the slower flow conditions. However, in the conditions of vigorous flow, binding may be limited. Here, we describe a microbubble capable of efficient binding to targets both in slow and fast flow (exceeding 4 dyne/cm² wall shear stress) using a clustered polymeric form of the fast‐binding selectin ligand sialyl Lewis^X. Microbubbles were prepared from decafluorobutane gas and stabilized with a monolayer of phosphatidylcholine, PEG stearate and biotin‐PEG‐lipid. Biotinylated PSLe^x (sialyl Lewis^X polyacrylamide) or biotinylated anti‐P‐selectin antibody (RB40.34) was attached to microbubbles via a streptavidin bridge. In a parallel plate flow chamber targeted adhesion model, PSLe^x bubbles demonstrated specific adhesion, retention and slow rolling on P‐selectin‐coated plates. Efficiency of firm targeted adhesion to a P‐selectin surface (140 molecules/µm²) was comparable for antibody‐carrying bubbles and PSLe^x‐targeted bubbles at 0.68 dyne/cm² shear stress. At fast flow (4.45 dyne/cm²), PSLe^x‐targeted bubbles maintained their ability to bind, while antibody‐mediated targeting dropped more than 20‐fold. At lower surface density of P‐selectin (7 molecules/µm²), targeting via PSLe^x was more efficient than via antibody under all the flow conditions tested. Negative control casein‐coated plates did not retain bubbles in the range of flow conditions studied. To confirm echogenicity, targeted PSLe^x‐bubbles were visualized on P‐selectin‐coated polystyrene plates by ultrasound imaging with a clinical scanner operated in pulse inversion mode; control plates lacking targeted bubbles did not show significant acoustic backscatter. In vivo, in a murine model of inflammation in the femoral vein setting, targeting efficacy of intravenously administered PSLe^x‐microbubbles was comparable with targeting mediated by anti‐P‐selectin antibody, and significantly exceeded the accumulation of non‐targeted control bubbles. In the inflamed femoral artery setting, PSLe^x‐mediated microbubble targeting was superior to antibody‐mediated targeting. Copyright © 2006 John Wiley & Sons, Ltd.     

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.     

血管生成靶向微泡的鉴定及黏附实验

目的 体外评价"亲和素-生物素"法制备靶向整合素αvβ3的微泡(MBαvβ3)及其黏附人脐静脉内皮细胞(HUVECs)的效能.方法 "亲和素-生物素"法桥连αvβ3抗体于磷脂微泡,体外荧光法鉴定其表面"生物素-亲和素-生物素"结构,应用平行平板流动腔(PPFC)技术评价MBαvβ3特异性黏附HUVECs的效能.结果 加入荧光标记的亲和素后,生物素化微泡(MBB)表面可见明亮荧光,普通微泡未见荧光;MBB加入荧光标记的蛋白A也未见荧光;MBB结合亲和素后,再加入荧光标记的生物素或蛋白A,前者表面可见明亮荧光,后者未见荧光.PPFC内,MBαvβ3组和普通微泡组结合数分别为(9.9±3.1)微泡/HUVEC和(0.8±0.3)微泡/HUVEC(P<0.05).结论 "生物素-亲和素"法能成功制备MBαvβ3,具有特异黏附血管内皮细胞的能力.

Abstract：

Objective To identify microbubbles targeted (MBt) to alpha(v)beta(3) (αvβ3) via biotin-avidin bridge and evaluate the adhesion to human umbilical vein endothelial cells (HUVECs) in vitro.Methods MBt produced via biotin-avidin bridge were validated using fluorescence in vitro.Adhesion of αvβ3-integrin targeted MBt (MBαvβ3) to HUVECs was tested using the parallel plate flow chamber (PPFC) test.Results Bright green fluorescence was observed on the biotinylated microbubbles(MBB) incubated with fluorescein isothiocyanate labeled streptavidin (FITC-SA) and on MBB-SA incubated with FITC labeled biotin.There was no fluorescence seen on non-targeted control microbubbles,MBB incubated with FITC labeled protein A and MBB-SA incubated with FITC labeled protein A. The adherent rate of MBαvβ3 was significantly higher than MBt with non-specific antibody (MBN) in PPFC test,with 9.9±3.1 of MBαvβ3 and 0.8±0.3 of MBN adhered to HUVECs,respectively(P<0.05).Conclusions Avβ3 targeted microbubbles using biotin-avidin bridging method is highly efficient and reliable for HUVECs.     

Laser Doppler anemometry measurements of the shear stresses on ultrasonic contrast agent microbubbles attached to agar

Ultrasonic contrast agents are currently being developed to target and bind to specific areas of interest such as atheromous plaque. A microbubble has been developed in-house which can be targeted to attach to specific cell-lines. To assess the feasibility of using the microbubble in vivo, the shear stresses which the bound microbubbles can withstand need to be known. A flow chamber was developed for use with intravascular ultrasound (IVUS) and laser Doppler anemometry (LDA). Biotin was incorporated into the microbubble shells and streptavidin was used to attach them to agar. IVUS at 40 MHz was then used to image the attached microbubbles under steady flow at a range of flow rates from 75 to 480 mL min(-1) through a flow area of 9 mm(2). LDA was employed to find high resolution velocity profiles of the flow in the chamber at a selection of these flow rates and the shear stresses on the bubbles were calculated. The bubbles were found to remain attached to the agar for shear stresses of up to 3.4 Pa. This compares with mean physiological arterial shear stresses of less than 1.5 Pa for pulsatile flow.     

Dynamics of Targeted Microbubble Adhesion Under Pulsatile Compared with Steady Flow

Hemodynamic flow variations at low fluid shear stress are thought to play a critical role in local atherosclerotic plaque initiation and development and to affect plaque instability. Targeted microbubbles are being developed as intravascular agents for identifying atherosclerotic lesions using ultrasound. How variations in local hydrodynamic flow influence the adhesiveness of targeted microbubbles is not well understood. We postulated that rates of targeted microbubble binding and accumulation differ when subjected to steady flow (SF) as compared with oscillatory or pulsatile flow (PF), because PF imposes non-uniform blood rheology and periodic acceleration and deceleration of blood velocity, when compared with SF. We assessed the binding rates of targeted microbubbles in seven randomly assigned PF and seven matched SF replicate runs at low (<1 Pa) and intermediate (≥1 and <2.5 Pa) wall shear stress (WSS) by drawing 4.8 × 10⁶ microbubbles mL⁻¹ over streptavidin-coated substrates, immobilized within a parallel plate flow chamber at a calculated density of 81 binding sites μm^-2. Selective binding and accumulation of targeted microbubbles was recorded in a single field of view using real-time video microscopy. Microbubble accumulation was modeled to obtain flow-mediated microbubble binding kinetics (amplitude, A, and rate constant, k). PF elicited higher microbubble accumulation rates, in comparison to SF. The rates of microbubble accumulation differed significantly between PF and SF (p < 0.05) at intermediate WSS but not at low WSS (p > 0.05). The rate of microbubble accumulation decreased as WSS increased.