载tPA携RGDS的超声造影剂制备及释药促溶机理研究
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摘要
背景和目的:
血管内血栓形成是引起心肌梗塞、缺血性卒中、系统性栓塞、肺栓塞以及静脉血栓等多种疾病的共同机制,也是目前导致病人发病与死亡的主要原因。治疗血栓、恢复血供至关重要。与介入性溶栓方法相比,以组织型纤溶酶原激活物(tPA)为代表的药物溶栓简便易行、创伤性低,是目前临床非常期待的治疗手段,但是其疗效尚不能令人满意,而且昂贵的价格和出血性并发症也制约着药物溶栓的临床应用。
超声波及超声造影剂不仅在诊断领域、而且在治疗领域都得到了巨大的发展。国内外大量实验证明超声及声学造影剂具有明显的溶栓和助溶作用,其可能机理为:超声空化效应一方面可拉长、切断或损坏血栓中的纤维,增加溶栓药与血栓的结合位点,另一方面气泡破裂时产生的冲击波、微射流作为一种驱动力量,促使溶栓药向血栓内渗透,加快其结合速度,进而加速溶解。超声造影剂微泡的引入能增加空化核的数目,降低超声的空化阈值,明显提高空化效应的强度,并能增强药物的渗透作用。靶向微泡的研究使微泡能通过靶向配体与靶组织特异性结合,于局部聚集形成较高浓度,达到靶向显影或治疗的目的。目前研究证明使用能与激活血小板表面的糖蛋白Ⅱb/Ⅲa受体特异结合的配体分子制备靶向微泡,可以和血栓特异性结合。本科室前期也已成功制备携带RGDS短肽配体的脂质体微泡造影剂,并在体外实验中证实其能与血栓特异性结合,而且在超声波辐照下能显著提高溶栓率。以超声激发击破携带药物或基因的造影剂微泡定点释放是一种新型无创的药物释放及基因转移的方法,该方法可将药物包载于造影剂微泡中防止药物在体循环中降解失活,在增加药物疗效的同时又可减小其副作用。本科实验室已成功制备出包载紫杉醇的脂质微泡造影剂。那么,如果能将微泡载药技术、靶向结合作用和空化效应促溶作用三者有机结合在一起,将为解决药物溶栓所面临的问题提供一条新的可行之路。
本研究拟制备一种包载组织型纤溶酶原激活物(tPA)并同时携带RGDS短肽靶向配体的脂质体微泡造影剂,检测其体内外溶栓能力,并对其溶栓机理进行初步探讨。
方法:
1.采用冷冻干燥法和桥连剂共价键结合法制备载tPA携RGDS的脂质体微泡造影剂;光镜和荧光显微镜下观察其形态和大小以及tPA和RGDS在微泡上的定位情况,分别以Coulter颗粒计数仪、Zetasizer3000和pH计检测微泡的粒度粒径、表面电位和pH值。应用酶联免疫吸附法(ELISA)检测tPA的包封率和载药量;应用流式细胞仪检测微泡上RGDS携带率;采用琼脂糖纤维蛋白平板法检测该微泡的促纤溶活性;以该微泡造影剂对兔肝脏实质造影并以时间-强度曲线(Time-intensity curve, TIC)评价其增强显影功能。
2.制备体外人全血血栓;以不同频率(0.5MHz、1MHz和2MHz)和不同强度(0.7W/cm2、1.4W/cm2和1.8W/cm2)的脉冲超声波辐照溶栓,比较其溶栓率,并检测超声溶栓效果与超声频率及强度之间的相关性;建立以蠕动泵为动力源的体外循环模型;以占空比95 %、声强1.8W/cm2、频率2MHz的脉冲超声波辐照不同凝龄的血栓(2、6、12、24、48和72h),并给予凝龄2h的血栓不同时间的超声辐照(10、20、30和60min),比较溶栓率,评价超声溶栓率与血栓凝龄和辐照时间之间的相关性,并确定本研究超声体外溶栓条件;以不同剂量tPA溶栓(0.5、1、2、3、4和5μg/ml),比较溶栓率,确定体外溶栓实验tPA用量;分组以不同方法溶栓(对照、单纯超声辐照、单纯tPA、单纯载tPA携RGDS微泡、tPA+超声、普通微泡+超声、载tPA非靶向微泡+超声、普通微泡+tPA+超声及载tPA携RGDS微泡+超声),比较其溶栓率;对溶栓处理后的血栓块进行HE染色后光镜下做病理检查;以5FAM标记tPA,冰冻切片后荧光显微镜检查探讨超声辐照载tPA携RGDS脂质微泡的溶栓机理。
3.以钳夹法制作兔股动脉血栓模型;比较声学造影、彩色多普勒血流显像(CDFI)、能量多普勒显像(PDI)和灰阶血流显像技术(B-flow)对兔股动脉血流的评价情况;以不同方法溶栓(对照、单纯治疗超声、单纯tPA、tPA+治疗超声、tPA+普通微泡+治疗超声、载tPA携RGDS微泡+治疗超声、载tPA携RGDS微泡+诊断超声),以B-flow观察并比较兔股动脉再通情况和再通出现时间;测量并比较溶栓前后股动脉局部体温;以ELISA法测量并比较溶栓前后兔血液D-二聚体(D-D)水平。
结果:
1.成功制备载tPA携RGDS脂质微泡造影剂,平均粒径为2.08μm(0.6~4.7μm),表面电位-51.3,pH值5.58,载药量为(0.59±0.02)mg/ml,包封率为(81.12±2.44)%,RGDS短肽配体的携带率为(94.49±6.19)%,超声辐照后的造影剂溶液具有一定的溶栓活性,该造影剂能明显持续增强肝脏回声,TIC参数中峰值强度(Peak intensity,PT)为86.54±5.09,达峰时间(Time to peak,PT)为(46±8.94)s,平均渡越时间(Mean transit time,MTT)为(690±61.64)s。
2.以三种频率、三种能量超声辐照体外血栓,溶栓率均高于对照组,差异有显著性意义(P<0.05);固定频率为2MHz条件下,溶栓率与超声强度成正相关(r1 = 1.000, P<0.01);固定声强1.8W/cm2条件下,溶栓率与超声频率成负相关(r2 = -1.000, P<0.01);以不同剂量tPA于体外溶栓,随着tPA浓度的升高,溶栓率也有升高的趋势,但组别之间差异无显著意义(P >0.05);成功建立以蠕动泵为动力源的体外循环模型;以占空比95%,声强1.8W/cm2,频率2MHz的脉冲超声波辐照不同凝龄的血栓,凝龄12h以内,超声辐照后的溶栓率明显高于对照组,差异有显著性意义(P<0.05),超声波溶栓率与血栓凝龄成反比(r1 = -1.000,P<0.01);不同辐照时间条件下,超声体外溶栓率都明显升高,差异有显著性意义(P<0.01),超声波溶栓率与辐照时间成正比(r2 = 1.000,P<0.01);我们选择频率为2MHz、声强为1.8W/cm2的脉冲超声为本实验体内外超声溶栓条件,凝龄为2h的血栓,辐照时间为10min,tPA用量为1μg/ml;体外溶栓实验结果为,除单纯tPA-tMB组外,其余各组溶栓率均高于对照组,差异有显著性意义(P<0.05),单纯超声组与单纯tPA组之间无显著差异(P>0.05),但均小于tPA+超声组、普通微泡+超声组和载tPA非靶向微泡+超声组(P<0.05),后三者之间无显著差异(P>0.05),普通微泡+tPA+超声组与载tPA携RGDS微泡+超声组溶栓率最高(P<0.05),而两者之间无显著差异(P>0.05);病理检查可见,辐照血栓内可见“空洞样”结构,以普通微泡+tPA+超声组和载tPA携RGDS微泡+超声组最为明显;冰冻切片后荧光显微镜检查可见,无超声辐照的载tPA携RGDS脂质微泡黏附于血栓边缘,形成明亮的红色荧光带,超声辐照载tPA携RGDS脂质微泡后,血栓内部可见大量荧光分布。
3.以钳夹法成功建立兔股动脉血栓模型;以ATL5000和LOGIQ7超声诊断仪及自制脂质微泡造影剂脂氟显对兔股动脉造影,难以客观评价兔股动脉血流,CDFI和PDI易产生充盈缺损和溢出伪像,B-flow技术能客观显示兔股动脉血流,分辩率高,无伪像,操作简便;各溶栓方法体内溶栓后再通率差异有显著性意义(χ2= 20.00, P<0.01),联合使用tPA、普通脂质体造影剂和超声辐照与联合使用载tPA携RGDS靶向脂质体造影剂和超声辐照所得到的再通率最高(80%和70%),但二者之间的差异无显著性意义(χ2= 0.27, P = 0.61),联合使用诊断超声辐照和载tPA携RGDS脂质微泡所得再通率与对照组比较,差异无显著意义(χ2= 0.39, P= 0.50);联合使用tPA、普通脂质体造影剂和超声辐照三者再通出现时间主要集中于前20min,联合使用载tPA携RGDS靶向脂质体造影剂和超声辐照再通出现时间主要集中于后20min,差异有显著性意义(χ2= 6.83, P<0.05);各溶栓方法治疗前后,股动脉局部体温升高低于2℃,最高体温低于40℃;各溶栓方法治疗前后D-D水平都有显著升高(P<0.05),单纯使用tPA、联合使用tPA和超声辐照、联合使用tPA、空白微泡和超声辐照以及联合使用载tPA携RGDS靶向脂质体造影剂和超声辐照显著高于对照组和单纯使用超声组(P<0.01),但这四种方法中,联合使用载tPA携RGDS靶向脂质体造影剂和超声辐照治疗后的D-D水平却明显低于前三者(P<0.01)。
结论:
1.成功制备载tPA携RGDS脂质微泡造影剂,粒径符合注射要求,包封率及配体携带率较高,并具有增强显影功能,超声辐照后具有一定的溶栓活性。
2.体外超声溶栓效果与超声频率、血栓凝龄成反比,与超声强度、辐照时间成正比。
3.超声辐照载tPA携RGDS脂质微泡造影剂在体外能显著提高溶栓率。其可能机理为:载tPA微泡通过RGDS靶向配体与血栓特异性结合后于局部聚集,超声辐照产生的空化效应使微泡破裂释放药物,并促进药物溶栓作用。
4.超声辐照载tPA携RGDS脂质微泡造影剂在体内可达到良好的溶栓效果,而且不会对局部组织产生热损伤,所产生的纤溶产物也低于系统性应用tPA或超声及微泡辅助tPA溶栓方法。
Backgrounds and objectives
Thrombosis in blood vessels is the common mechanism of multiple diseases such as myocardial infarction, ischemia stroke, systemic embolism, pulmonary embolism and vein thrombus, etc. It’s also the main cause of the morbidity and the mortality. So it’s of great importance to treat thrombosis and restore the blood supply. Compared with the interventional thrombolysis, the drug thrombolysis such as t)ssue plasminogen activator (tPA) has some superiority because of the easy and noninvasive operation, which is one of the most expected ways in the clinic. However, its curative effect is not so satisfactory,and the high price and the bleeding complication also limit its clinical application.
Nowadays, ultrasound and contrast agents have been developed greatly not only in the diagnostic realm but also in the therapeutic. A large amount of researches have demonstrated that ultrasound and microbubble contrast agents can resolve thrombus directly or accelerate thrombolysis. The possible mechanisms include: on the one hand, ultrasonic cavitational effect can stretch, cut and damage the fibers in thrombus and thus the sites to combinate thrombolytics will be increased; on the other hand, the shock wave and microstreaming produced during the microbubbles’rupture caused by ultrasound cavitation, as a driving force, will improve the penetration of drugs into thrombus, accelerate the combining speed and facilitate the declotting process. The introduction of microbubbles will increase the numbers of the cavitation nuclei, lower the cavitaton threshold of ultrasound, improve the power of cavitation and enhance the penetration of drugs. The researches on the targeting microbubbles make it possible to specifically bind the microbubbles to the target tissue through the targeting ligands and elevate the microbubble concentration at the local site, by which the goals of targeting imaging and therapy can be reached. It has been proved that the microbubbles targeting to thrombus can be prepared by adhering the specific ligand on their surface, which can specifically bind with glucoproteinⅡb/Ⅲa receptors on the surface of the activated platelets. Our lab has succeeded in preparing the lipid microbubbles conjugating RGDS peptides and confirmed that they could bind with thrombus specifically and improve the thrombolysis in vitro. A new strategy of drug and gene delivery, carrying drug molecules by microbubbles and releasing them by ultrasound irradiation, is becoming more and more attractive at present. By this way, the drugs encapsulated in microbubbles can avoid the degradation in the blood circulation, and their curative effects can be enhanced while their side effects can be reduced. Our lab has succeeded in preparing the lipid microbubbles entrapping paclitaxel. Therefore, if the technique of encapsulating drugs into microbubbles, the targeted binding effect and the acceleration to thrombolysis of cavitation are integrated together, a new solution for the problems of drug thrombolysis faced in the clinic presently may be worked out. In this research, we try to prepare a lipid microbubble contrast agent encapsulating tPA and carrying RGDS peptide, detect its thrombolysis ability in vitro and in vivo, and search for the mechanism primarily.
Methods
1. The lipid microbubble contrast agent encapsulating tPA and carrying RGDS was prepared by lyopyilization and covalent linkage methods. Its modality and size were observed under light microscope and the locations of tPA and RGDS in the microbubbles were observed under fluorescence microscope. The particle size and diameter, the surface potential and the pH value of the microbubbles were detected by Coulter events-per-unit-time meter, Zetasizer 3000 and pH meter, respectively. The encapsulation efficiency and the amount of loaded drug were measured by enzyme linked immunosorbent assay (ELISA). The conjugation rate of RGDS on the microbubbles was measured by flow cytometer. The activity of the tPA released by ultrasonic radiation-induced microbubble disruption was detected by agarose fibrin plate process. The contrast imaging of rabbit liver was performed with the microbubbles and its echo-enhancing function was evaluated by time-intensity curve (TIC).
2. The thrombus of human whole blood was prepared. The thrombus was irradiated by ultrasound of various frequencies (0.5MHz, 1MHz and 2MHz) and various intensities (0.7W/cm2, 1.4W/cm2 and 1.8W/cm2), the thrombolysis rates were compared, and the correlation between the ultrasonic thrombolysis rate and its frequency or its intensity was investigated. The extracorporeal circulation model was established with the peristaltic pump as the power source. The thrombi of various clotting times (2, 6, 12, 24, 48 and 72h) were irradiated by the pulse ultrasound of 95% duty cycle, 1.8W/cm2 intensity and 2MHz frequency, the thrombi clotting for 2h were irradiated by ultrasound for various times (10, 20, 30 and 60min), the thrombolysis rates were compared, the correlation between the ultrasonic thrombolysis rate and the clotting time of thrombus or the exposure time of ultrasound, and then the conditions of ultrasound for the in vitro thrombolysis experiment were decided. The drug thrombolysis was performed with tPA of various concentrations (0.5, 1, 2, 3, 4 and 5μg/ml) and the dosage used in the experiment in vitro was decided. Thrombolysis with different ways was performed in vitro (Control, ultrasound only, tPA only, tPA-encapsulated RGDS-conjugated microbubbles only, tPA + ultrasound, common microbubble + ultrasound, microbubbles encapsulating tPA without RGDS + ultrasound, common microbubbles + tPA + ultrasound and tPA-encapsulated RGDS-conjugated microbubbles + ultrasound) and the thrombolysis rates between the groups were compared. The pathological examination was performed to the treated clots. TPA was labeled by 5FAM with red fluorescence and the mechanism of the accelerated thrombolysis of tPA-encapsulated RGDS-conjugated microbubbles combined with ultrasound was investigated primarily by fluorescence microscopy following frozen section.
3. The thrombus model of rabbit femoral artery was established by clamping method. Ultrasound contrast, color Doppler flow imaging, power Doppler imaging and B-flow were compared in the evaluation of the blood flow in rabbit femoral artery. Thrombolysis was performed with different ways in vivo (control, therapeutic ultrasound only, tPA only, tPA + therapeutic ultrasound, tPA + common microbubbles + therapeutic ultrasound, tPA-encapsulated RGDS-conjugated microbubbles + therapeutic ultrasound and tPA-encapsulated RGDS-conjugated microbubbles + diagnostic ultrasound) and the recanalizations and their appearing times were observed and compared between groups. The local temperatures of femoral arteries and the D-dimer levels of rabbit blood were detected and compared before and after thrombolysis.
Results
1. The lipid microbubbles encapsulating tPA and conjugating RGDS were prepared successfully, with the mean diameter of 2.08μm (0.6~4.7μm), the surface potential of -51.3, pH 5.58, the amount of loaded drug of (0.59±0.02) mg/ml, the encapsulation efficiency of (81.12±2.44) % and the conjugation rate of (94.49±6.19) %. After ultrasound exposure, the microbubble contrast solution had still some thrombolytic activity. The contrast microbubbles enhanced the echo of rabbit liver obviously and continuously and among the TIC index, PI was 86.54±5.09, PT was (46±8.94) s and MTT was (690±61.64) s.
2. Irradiated with ultrasound of 3 frequencies and 3 intensities, the thrombolysis rate was improved significantly (P<0.05), it was positively correlated with the frequency (r1=1.000, P<0.01) while negatively correlated with the intensity (r2=-1.000, P<0.01). The thrombolysis rate rose with the rise of tPA dosage, but there was no significant difference between groups (P>0.05) The extracorporeal circulation model was established successfully. Ultrasound improved the thrombolysis of the thrombus with clotting time lower than 12h (P<0.05) and the thrombolysis rate correlated with the clotting time negatively (r1 = -1.000,P<0.01). Ultrasound irradiation for various times all improved the thrombolysis (P<0.01) and the thrombolysis rate correlated with the exposure time positively (r2 = 1.000,P<0.01). We chose the pulse ultrasound of 2MHz and 1.8 W/cm2, thrombus of 2h, exposure time of 10min and tPA of 1μg/ml for the in vitro experiment. The thrombolysis rates in all the treating groups except for tPA-encapsulated RGDS-conjugated microbubbles only were higher than that in control (P<0.05), among which, there was no difference between ultrasound only and tPA only (P>0.05), but they are both lower than tPA + ultrasound, common microbubbles + ultrasound and tPA-encapsulated microbubble without RGDS + ultrasound (P<0.05), there was no difference between the latter 3 groups (P > 0.05), and it was highest in common microbubbles + tPA + ultrasound and tPA-encapsulated RGDS-conjugated microbubbles + ultrasound (P<0.05), between which, there was no significant difference (P>0.05). Pathological examination revealed that there are cavity-like structures inside the treated thrombus and it was the most obvious in common microbubbles + tPA + ultrasound group and tPA-encapsulated RGDS-conjugated microbubbles + ultrasound group. The fluorescence microscopy following frozen section revealed that the microbubbles adhering the edge of clot without ultrasound irradiation,, along which, a red fluorescent band wad observed under fluorescence microscopy, while with 10 min’s ultrasound exposure, a great deal of fluorescence signals were observed deep inside the clots.
3. The rabbit thrombus model was established by clamping method successfully. It was hard to evaluate the blood flow of rabbit femoral artery by ultrasound contrast with ATL 5000, LOGIQ 7 and self-made lipid microbubbles. It was easy to be interfered by artifacts of filling defect and flooding as CDFI and PDI were applied. It was objective, of high resolution, with no artifacts and easy-operational for B-flow technique to present the blood flow. The differences of recanalization rates between groups were significant (χ2=20.00, P<0.01), among which, it was highest in the groups of tPA + common microbubbles + therapeutic ultrasound (80%) and tPA-encapsulated RGDS-conjugated microbubbles + therapeutic ultrasound (70%) and their difference was of no significance between the two group (χ2= 0.27, P = 0.61). There was no significant difference between the group of tPA-encapsulated RGDS-conjugated microbubbles + diagnostic ultrasound and the control (χ2= 0.39, P= 0.50). The thrombolysis in tPA + common microbubbles + therapeutic ultrasound, which appeared mainly in the earlier 20min, was earlier than that in tPA-encapsulated RGDS-conjugated microbubbles + therapeutic ultrasound (χ2= 6.83, P<0.05), which appeared mainly in the later 20min. The changes of local temperature before and after treatment were lower than 2℃and the highest temperature didn’t exceed 40℃. The D-D level in each therapeutic group was raised significantly (P<0.05), among which, it was higher in tPA only, tPA + therapeutic ultrasound, tPA + common microbubbles + therapeutic ultrasound and tPA-encapsulated RGDS-conjugated microbubbles + therapeutic ultrasound (P<0.01) and it was lower in last group than the former three groups (P<0.01).
Conclusions
1. The lipid microbubble contrast agent, with the size and diameter fit for intravascular injection, the high encapsulation efficiency and ligand conjugation rate, the echo-enhancing function and the fibrinolysis activity with ultrasonic exposure, can be prepared successfully.
2. The sonothrombolysis in vitro is negatively correlated with the ultrasound frequency and the clotting time while positively correlated with the ultrasound intensity and the exposure time.
3. The tPA-encapsulated RGDS-conjugated microbubbles combined with ultrasound irradiation can improve the thrombolysis greatly, which is equivalent with the effect of combined application of systemic tPA, common microbubbles and ultrasound irradiation. The mechanism may be: The tPA-encapsulated RGDS-conjugated congregates locally with the targeted combination with thrombus through RGDS ligand, and then with the cavitaion effect caused by ultrasound irradiation, the microbubbles are disrupted and the entrapped tPA was released, which can accelerate the drug thrombolysis.
4. The tPA-encapsulated RGDS-conjugated microbubbles combined with ultrasound irradiation also have good thrombolysis in vivo. Its thermal effect will not damage the local tissue and the level of its fibrinolysis product is lower than the systemic tPA or the drug thrombolysis assisted by ultrasound and microbubbles.
血管内血栓形成是引起心肌梗塞、缺血性卒中、系统性栓塞、肺栓塞以及静脉血栓等多种疾病的共同机制,也是目前导致病人发病与死亡的主要原因。治疗血栓、恢复血供至关重要。与介入性溶栓方法相比,以组织型纤溶酶原激活物(tPA)为代表的药物溶栓简便易行、创伤性低,是目前临床非常期待的治疗手段,但是其疗效尚不能令人满意,而且昂贵的价格和出血性并发症也制约着药物溶栓的临床应用。
超声波及超声造影剂不仅在诊断领域、而且在治疗领域都得到了巨大的发展。国内外大量实验证明超声及声学造影剂具有明显的溶栓和助溶作用,其可能机理为:超声空化效应一方面可拉长、切断或损坏血栓中的纤维,增加溶栓药与血栓的结合位点,另一方面气泡破裂时产生的冲击波、微射流作为一种驱动力量,促使溶栓药向血栓内渗透,加快其结合速度,进而加速溶解。超声造影剂微泡的引入能增加空化核的数目,降低超声的空化阈值,明显提高空化效应的强度,并能增强药物的渗透作用。靶向微泡的研究使微泡能通过靶向配体与靶组织特异性结合,于局部聚集形成较高浓度,达到靶向显影或治疗的目的。目前研究证明使用能与激活血小板表面的糖蛋白Ⅱb/Ⅲa受体特异结合的配体分子制备靶向微泡,可以和血栓特异性结合。本科室前期也已成功制备携带RGDS短肽配体的脂质体微泡造影剂,并在体外实验中证实其能与血栓特异性结合,而且在超声波辐照下能显著提高溶栓率。以超声激发击破携带药物或基因的造影剂微泡定点释放是一种新型无创的药物释放及基因转移的方法,该方法可将药物包载于造影剂微泡中防止药物在体循环中降解失活,在增加药物疗效的同时又可减小其副作用。本科实验室已成功制备出包载紫杉醇的脂质微泡造影剂。那么,如果能将微泡载药技术、靶向结合作用和空化效应促溶作用三者有机结合在一起,将为解决药物溶栓所面临的问题提供一条新的可行之路。
本研究拟制备一种包载组织型纤溶酶原激活物(tPA)并同时携带RGDS短肽靶向配体的脂质体微泡造影剂,检测其体内外溶栓能力,并对其溶栓机理进行初步探讨。
方法:
1.采用冷冻干燥法和桥连剂共价键结合法制备载tPA携RGDS的脂质体微泡造影剂;光镜和荧光显微镜下观察其形态和大小以及tPA和RGDS在微泡上的定位情况,分别以Coulter颗粒计数仪、Zetasizer3000和pH计检测微泡的粒度粒径、表面电位和pH值。应用酶联免疫吸附法(ELISA)检测tPA的包封率和载药量;应用流式细胞仪检测微泡上RGDS携带率;采用琼脂糖纤维蛋白平板法检测该微泡的促纤溶活性;以该微泡造影剂对兔肝脏实质造影并以时间-强度曲线(Time-intensity curve, TIC)评价其增强显影功能。
2.制备体外人全血血栓;以不同频率(0.5MHz、1MHz和2MHz)和不同强度(0.7W/cm2、1.4W/cm2和1.8W/cm2)的脉冲超声波辐照溶栓,比较其溶栓率,并检测超声溶栓效果与超声频率及强度之间的相关性;建立以蠕动泵为动力源的体外循环模型;以占空比95 %、声强1.8W/cm2、频率2MHz的脉冲超声波辐照不同凝龄的血栓(2、6、12、24、48和72h),并给予凝龄2h的血栓不同时间的超声辐照(10、20、30和60min),比较溶栓率,评价超声溶栓率与血栓凝龄和辐照时间之间的相关性,并确定本研究超声体外溶栓条件;以不同剂量tPA溶栓(0.5、1、2、3、4和5μg/ml),比较溶栓率,确定体外溶栓实验tPA用量;分组以不同方法溶栓(对照、单纯超声辐照、单纯tPA、单纯载tPA携RGDS微泡、tPA+超声、普通微泡+超声、载tPA非靶向微泡+超声、普通微泡+tPA+超声及载tPA携RGDS微泡+超声),比较其溶栓率;对溶栓处理后的血栓块进行HE染色后光镜下做病理检查;以5FAM标记tPA,冰冻切片后荧光显微镜检查探讨超声辐照载tPA携RGDS脂质微泡的溶栓机理。
3.以钳夹法制作兔股动脉血栓模型;比较声学造影、彩色多普勒血流显像(CDFI)、能量多普勒显像(PDI)和灰阶血流显像技术(B-flow)对兔股动脉血流的评价情况;以不同方法溶栓(对照、单纯治疗超声、单纯tPA、tPA+治疗超声、tPA+普通微泡+治疗超声、载tPA携RGDS微泡+治疗超声、载tPA携RGDS微泡+诊断超声),以B-flow观察并比较兔股动脉再通情况和再通出现时间;测量并比较溶栓前后股动脉局部体温;以ELISA法测量并比较溶栓前后兔血液D-二聚体(D-D)水平。
结果:
1.成功制备载tPA携RGDS脂质微泡造影剂,平均粒径为2.08μm(0.6~4.7μm),表面电位-51.3,pH值5.58,载药量为(0.59±0.02)mg/ml,包封率为(81.12±2.44)%,RGDS短肽配体的携带率为(94.49±6.19)%,超声辐照后的造影剂溶液具有一定的溶栓活性,该造影剂能明显持续增强肝脏回声,TIC参数中峰值强度(Peak intensity,PT)为86.54±5.09,达峰时间(Time to peak,PT)为(46±8.94)s,平均渡越时间(Mean transit time,MTT)为(690±61.64)s。
2.以三种频率、三种能量超声辐照体外血栓,溶栓率均高于对照组,差异有显著性意义(P<0.05);固定频率为2MHz条件下,溶栓率与超声强度成正相关(r1 = 1.000, P<0.01);固定声强1.8W/cm2条件下,溶栓率与超声频率成负相关(r2 = -1.000, P<0.01);以不同剂量tPA于体外溶栓,随着tPA浓度的升高,溶栓率也有升高的趋势,但组别之间差异无显著意义(P >0.05);成功建立以蠕动泵为动力源的体外循环模型;以占空比95%,声强1.8W/cm2,频率2MHz的脉冲超声波辐照不同凝龄的血栓,凝龄12h以内,超声辐照后的溶栓率明显高于对照组,差异有显著性意义(P<0.05),超声波溶栓率与血栓凝龄成反比(r1 = -1.000,P<0.01);不同辐照时间条件下,超声体外溶栓率都明显升高,差异有显著性意义(P<0.01),超声波溶栓率与辐照时间成正比(r2 = 1.000,P<0.01);我们选择频率为2MHz、声强为1.8W/cm2的脉冲超声为本实验体内外超声溶栓条件,凝龄为2h的血栓,辐照时间为10min,tPA用量为1μg/ml;体外溶栓实验结果为,除单纯tPA-tMB组外,其余各组溶栓率均高于对照组,差异有显著性意义(P<0.05),单纯超声组与单纯tPA组之间无显著差异(P>0.05),但均小于tPA+超声组、普通微泡+超声组和载tPA非靶向微泡+超声组(P<0.05),后三者之间无显著差异(P>0.05),普通微泡+tPA+超声组与载tPA携RGDS微泡+超声组溶栓率最高(P<0.05),而两者之间无显著差异(P>0.05);病理检查可见,辐照血栓内可见“空洞样”结构,以普通微泡+tPA+超声组和载tPA携RGDS微泡+超声组最为明显;冰冻切片后荧光显微镜检查可见,无超声辐照的载tPA携RGDS脂质微泡黏附于血栓边缘,形成明亮的红色荧光带,超声辐照载tPA携RGDS脂质微泡后,血栓内部可见大量荧光分布。
3.以钳夹法成功建立兔股动脉血栓模型;以ATL5000和LOGIQ7超声诊断仪及自制脂质微泡造影剂脂氟显对兔股动脉造影,难以客观评价兔股动脉血流,CDFI和PDI易产生充盈缺损和溢出伪像,B-flow技术能客观显示兔股动脉血流,分辩率高,无伪像,操作简便;各溶栓方法体内溶栓后再通率差异有显著性意义(χ2= 20.00, P<0.01),联合使用tPA、普通脂质体造影剂和超声辐照与联合使用载tPA携RGDS靶向脂质体造影剂和超声辐照所得到的再通率最高(80%和70%),但二者之间的差异无显著性意义(χ2= 0.27, P = 0.61),联合使用诊断超声辐照和载tPA携RGDS脂质微泡所得再通率与对照组比较,差异无显著意义(χ2= 0.39, P= 0.50);联合使用tPA、普通脂质体造影剂和超声辐照三者再通出现时间主要集中于前20min,联合使用载tPA携RGDS靶向脂质体造影剂和超声辐照再通出现时间主要集中于后20min,差异有显著性意义(χ2= 6.83, P<0.05);各溶栓方法治疗前后,股动脉局部体温升高低于2℃,最高体温低于40℃;各溶栓方法治疗前后D-D水平都有显著升高(P<0.05),单纯使用tPA、联合使用tPA和超声辐照、联合使用tPA、空白微泡和超声辐照以及联合使用载tPA携RGDS靶向脂质体造影剂和超声辐照显著高于对照组和单纯使用超声组(P<0.01),但这四种方法中,联合使用载tPA携RGDS靶向脂质体造影剂和超声辐照治疗后的D-D水平却明显低于前三者(P<0.01)。
结论:
1.成功制备载tPA携RGDS脂质微泡造影剂,粒径符合注射要求,包封率及配体携带率较高,并具有增强显影功能,超声辐照后具有一定的溶栓活性。
2.体外超声溶栓效果与超声频率、血栓凝龄成反比,与超声强度、辐照时间成正比。
3.超声辐照载tPA携RGDS脂质微泡造影剂在体外能显著提高溶栓率。其可能机理为:载tPA微泡通过RGDS靶向配体与血栓特异性结合后于局部聚集,超声辐照产生的空化效应使微泡破裂释放药物,并促进药物溶栓作用。
4.超声辐照载tPA携RGDS脂质微泡造影剂在体内可达到良好的溶栓效果,而且不会对局部组织产生热损伤,所产生的纤溶产物也低于系统性应用tPA或超声及微泡辅助tPA溶栓方法。
Backgrounds and objectives
Thrombosis in blood vessels is the common mechanism of multiple diseases such as myocardial infarction, ischemia stroke, systemic embolism, pulmonary embolism and vein thrombus, etc. It’s also the main cause of the morbidity and the mortality. So it’s of great importance to treat thrombosis and restore the blood supply. Compared with the interventional thrombolysis, the drug thrombolysis such as t)ssue plasminogen activator (tPA) has some superiority because of the easy and noninvasive operation, which is one of the most expected ways in the clinic. However, its curative effect is not so satisfactory,and the high price and the bleeding complication also limit its clinical application.
Nowadays, ultrasound and contrast agents have been developed greatly not only in the diagnostic realm but also in the therapeutic. A large amount of researches have demonstrated that ultrasound and microbubble contrast agents can resolve thrombus directly or accelerate thrombolysis. The possible mechanisms include: on the one hand, ultrasonic cavitational effect can stretch, cut and damage the fibers in thrombus and thus the sites to combinate thrombolytics will be increased; on the other hand, the shock wave and microstreaming produced during the microbubbles’rupture caused by ultrasound cavitation, as a driving force, will improve the penetration of drugs into thrombus, accelerate the combining speed and facilitate the declotting process. The introduction of microbubbles will increase the numbers of the cavitation nuclei, lower the cavitaton threshold of ultrasound, improve the power of cavitation and enhance the penetration of drugs. The researches on the targeting microbubbles make it possible to specifically bind the microbubbles to the target tissue through the targeting ligands and elevate the microbubble concentration at the local site, by which the goals of targeting imaging and therapy can be reached. It has been proved that the microbubbles targeting to thrombus can be prepared by adhering the specific ligand on their surface, which can specifically bind with glucoproteinⅡb/Ⅲa receptors on the surface of the activated platelets. Our lab has succeeded in preparing the lipid microbubbles conjugating RGDS peptides and confirmed that they could bind with thrombus specifically and improve the thrombolysis in vitro. A new strategy of drug and gene delivery, carrying drug molecules by microbubbles and releasing them by ultrasound irradiation, is becoming more and more attractive at present. By this way, the drugs encapsulated in microbubbles can avoid the degradation in the blood circulation, and their curative effects can be enhanced while their side effects can be reduced. Our lab has succeeded in preparing the lipid microbubbles entrapping paclitaxel. Therefore, if the technique of encapsulating drugs into microbubbles, the targeted binding effect and the acceleration to thrombolysis of cavitation are integrated together, a new solution for the problems of drug thrombolysis faced in the clinic presently may be worked out. In this research, we try to prepare a lipid microbubble contrast agent encapsulating tPA and carrying RGDS peptide, detect its thrombolysis ability in vitro and in vivo, and search for the mechanism primarily.
Methods
1. The lipid microbubble contrast agent encapsulating tPA and carrying RGDS was prepared by lyopyilization and covalent linkage methods. Its modality and size were observed under light microscope and the locations of tPA and RGDS in the microbubbles were observed under fluorescence microscope. The particle size and diameter, the surface potential and the pH value of the microbubbles were detected by Coulter events-per-unit-time meter, Zetasizer 3000 and pH meter, respectively. The encapsulation efficiency and the amount of loaded drug were measured by enzyme linked immunosorbent assay (ELISA). The conjugation rate of RGDS on the microbubbles was measured by flow cytometer. The activity of the tPA released by ultrasonic radiation-induced microbubble disruption was detected by agarose fibrin plate process. The contrast imaging of rabbit liver was performed with the microbubbles and its echo-enhancing function was evaluated by time-intensity curve (TIC).
2. The thrombus of human whole blood was prepared. The thrombus was irradiated by ultrasound of various frequencies (0.5MHz, 1MHz and 2MHz) and various intensities (0.7W/cm2, 1.4W/cm2 and 1.8W/cm2), the thrombolysis rates were compared, and the correlation between the ultrasonic thrombolysis rate and its frequency or its intensity was investigated. The extracorporeal circulation model was established with the peristaltic pump as the power source. The thrombi of various clotting times (2, 6, 12, 24, 48 and 72h) were irradiated by the pulse ultrasound of 95% duty cycle, 1.8W/cm2 intensity and 2MHz frequency, the thrombi clotting for 2h were irradiated by ultrasound for various times (10, 20, 30 and 60min), the thrombolysis rates were compared, the correlation between the ultrasonic thrombolysis rate and the clotting time of thrombus or the exposure time of ultrasound, and then the conditions of ultrasound for the in vitro thrombolysis experiment were decided. The drug thrombolysis was performed with tPA of various concentrations (0.5, 1, 2, 3, 4 and 5μg/ml) and the dosage used in the experiment in vitro was decided. Thrombolysis with different ways was performed in vitro (Control, ultrasound only, tPA only, tPA-encapsulated RGDS-conjugated microbubbles only, tPA + ultrasound, common microbubble + ultrasound, microbubbles encapsulating tPA without RGDS + ultrasound, common microbubbles + tPA + ultrasound and tPA-encapsulated RGDS-conjugated microbubbles + ultrasound) and the thrombolysis rates between the groups were compared. The pathological examination was performed to the treated clots. TPA was labeled by 5FAM with red fluorescence and the mechanism of the accelerated thrombolysis of tPA-encapsulated RGDS-conjugated microbubbles combined with ultrasound was investigated primarily by fluorescence microscopy following frozen section.
3. The thrombus model of rabbit femoral artery was established by clamping method. Ultrasound contrast, color Doppler flow imaging, power Doppler imaging and B-flow were compared in the evaluation of the blood flow in rabbit femoral artery. Thrombolysis was performed with different ways in vivo (control, therapeutic ultrasound only, tPA only, tPA + therapeutic ultrasound, tPA + common microbubbles + therapeutic ultrasound, tPA-encapsulated RGDS-conjugated microbubbles + therapeutic ultrasound and tPA-encapsulated RGDS-conjugated microbubbles + diagnostic ultrasound) and the recanalizations and their appearing times were observed and compared between groups. The local temperatures of femoral arteries and the D-dimer levels of rabbit blood were detected and compared before and after thrombolysis.
Results
1. The lipid microbubbles encapsulating tPA and conjugating RGDS were prepared successfully, with the mean diameter of 2.08μm (0.6~4.7μm), the surface potential of -51.3, pH 5.58, the amount of loaded drug of (0.59±0.02) mg/ml, the encapsulation efficiency of (81.12±2.44) % and the conjugation rate of (94.49±6.19) %. After ultrasound exposure, the microbubble contrast solution had still some thrombolytic activity. The contrast microbubbles enhanced the echo of rabbit liver obviously and continuously and among the TIC index, PI was 86.54±5.09, PT was (46±8.94) s and MTT was (690±61.64) s.
2. Irradiated with ultrasound of 3 frequencies and 3 intensities, the thrombolysis rate was improved significantly (P<0.05), it was positively correlated with the frequency (r1=1.000, P<0.01) while negatively correlated with the intensity (r2=-1.000, P<0.01). The thrombolysis rate rose with the rise of tPA dosage, but there was no significant difference between groups (P>0.05) The extracorporeal circulation model was established successfully. Ultrasound improved the thrombolysis of the thrombus with clotting time lower than 12h (P<0.05) and the thrombolysis rate correlated with the clotting time negatively (r1 = -1.000,P<0.01). Ultrasound irradiation for various times all improved the thrombolysis (P<0.01) and the thrombolysis rate correlated with the exposure time positively (r2 = 1.000,P<0.01). We chose the pulse ultrasound of 2MHz and 1.8 W/cm2, thrombus of 2h, exposure time of 10min and tPA of 1μg/ml for the in vitro experiment. The thrombolysis rates in all the treating groups except for tPA-encapsulated RGDS-conjugated microbubbles only were higher than that in control (P<0.05), among which, there was no difference between ultrasound only and tPA only (P>0.05), but they are both lower than tPA + ultrasound, common microbubbles + ultrasound and tPA-encapsulated microbubble without RGDS + ultrasound (P<0.05), there was no difference between the latter 3 groups (P > 0.05), and it was highest in common microbubbles + tPA + ultrasound and tPA-encapsulated RGDS-conjugated microbubbles + ultrasound (P<0.05), between which, there was no significant difference (P>0.05). Pathological examination revealed that there are cavity-like structures inside the treated thrombus and it was the most obvious in common microbubbles + tPA + ultrasound group and tPA-encapsulated RGDS-conjugated microbubbles + ultrasound group. The fluorescence microscopy following frozen section revealed that the microbubbles adhering the edge of clot without ultrasound irradiation,, along which, a red fluorescent band wad observed under fluorescence microscopy, while with 10 min’s ultrasound exposure, a great deal of fluorescence signals were observed deep inside the clots.
3. The rabbit thrombus model was established by clamping method successfully. It was hard to evaluate the blood flow of rabbit femoral artery by ultrasound contrast with ATL 5000, LOGIQ 7 and self-made lipid microbubbles. It was easy to be interfered by artifacts of filling defect and flooding as CDFI and PDI were applied. It was objective, of high resolution, with no artifacts and easy-operational for B-flow technique to present the blood flow. The differences of recanalization rates between groups were significant (χ2=20.00, P<0.01), among which, it was highest in the groups of tPA + common microbubbles + therapeutic ultrasound (80%) and tPA-encapsulated RGDS-conjugated microbubbles + therapeutic ultrasound (70%) and their difference was of no significance between the two group (χ2= 0.27, P = 0.61). There was no significant difference between the group of tPA-encapsulated RGDS-conjugated microbubbles + diagnostic ultrasound and the control (χ2= 0.39, P= 0.50). The thrombolysis in tPA + common microbubbles + therapeutic ultrasound, which appeared mainly in the earlier 20min, was earlier than that in tPA-encapsulated RGDS-conjugated microbubbles + therapeutic ultrasound (χ2= 6.83, P<0.05), which appeared mainly in the later 20min. The changes of local temperature before and after treatment were lower than 2℃and the highest temperature didn’t exceed 40℃. The D-D level in each therapeutic group was raised significantly (P<0.05), among which, it was higher in tPA only, tPA + therapeutic ultrasound, tPA + common microbubbles + therapeutic ultrasound and tPA-encapsulated RGDS-conjugated microbubbles + therapeutic ultrasound (P<0.01) and it was lower in last group than the former three groups (P<0.01).
Conclusions
1. The lipid microbubble contrast agent, with the size and diameter fit for intravascular injection, the high encapsulation efficiency and ligand conjugation rate, the echo-enhancing function and the fibrinolysis activity with ultrasonic exposure, can be prepared successfully.
2. The sonothrombolysis in vitro is negatively correlated with the ultrasound frequency and the clotting time while positively correlated with the ultrasound intensity and the exposure time.
3. The tPA-encapsulated RGDS-conjugated microbubbles combined with ultrasound irradiation can improve the thrombolysis greatly, which is equivalent with the effect of combined application of systemic tPA, common microbubbles and ultrasound irradiation. The mechanism may be: The tPA-encapsulated RGDS-conjugated congregates locally with the targeted combination with thrombus through RGDS ligand, and then with the cavitaion effect caused by ultrasound irradiation, the microbubbles are disrupted and the entrapped tPA was released, which can accelerate the drug thrombolysis.
4. The tPA-encapsulated RGDS-conjugated microbubbles combined with ultrasound irradiation also have good thrombolysis in vivo. Its thermal effect will not damage the local tissue and the level of its fibrinolysis product is lower than the systemic tPA or the drug thrombolysis assisted by ultrasound and microbubbles.
引文
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