Research On The Preparation Of The Microbubble Ultrasound Contrast Agent Encapsulating TPA And Conjugating RGDS And The Mechanisms Of Drug Releasing And Thrombolysis Acceleration

Backgrounds and objectivesThrombosis 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.Methods1. 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. Results1. 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).Conclusions1. 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.