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Evaluation Of Arterial Thrombus With Cyclic RGD-modified Microbubbles And Contrast-enhanced Ultrasound: An Experimental Study

Posted on:2011-08-22Degree:MasterType:Thesis
Country:ChinaCandidate:G Q HuFull Text:PDF
GTID:2154360308970095Subject:Internal Medicine
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Background and ObjectiveThe formation of thrombus or thromboembolism were the lastest key of the emergency events of cardic, brain and peripheral vescular, which were direct reasons of death or disability. The mortality of thromboembolic diseases repersented by myocardial infarction and stroke, have been accounted for more than half of the total number of deaths, far more than that of cancer, infectious diseases, respiratory diseases, in great extent, part of reasons were that thrombosis could not be detected in early stage so as to delay in trearments. Ultrasound is the primary imaging modality which is a widely used in clinical imaging. It has great clinical significance to diagnose and the follow-up of limb arterial and venous thrombosis, however, ultrasound has limited accuracy for detecting deep venous thrombosis, it depends on the invasive contrast venography and expensive computed tomography or magnetic resonance imaging. Therefore, if we can develop a technology capable of early non-invasive, sensitive and specific diagnosis of thrombosis and thus early treatment of thrombosis, which is critical to reducing patient mortality and the incidence of complications. It will have great clinical value. At present, although there are some international laboratories have succeed in evaluation of deep venous and atrial thrombosis in animals by the applications of ultrasonic molecular imaging technology, due to higher requirements of imaging technology, bubble stability, bubble concentrations and targeted capability of bubbles, ultrasound molecular imaging of arterial thrombosis is still difficult, no substantial progress are achieved in this field and few successsful research was reported in evaluation of arterial thrombosis by use of targeted microbubbles in home and abroad.Formation of thrombus partly depended on the activation and aggregation of platelets, the platelet glycoprotein GPⅡb/Ⅲa receptors are highly expressed on activated platelet surface, which bind to the arginine-glycine-aspartic acid (RGD) binding site that included in infibrinogen and other substances, and then facilitating platelet aggregation and early thrombus formation.if we attach the highly efficient ligands of platelet GPⅡb/Ⅲa receptors to the surface of microbubbles, thus the microbubbles can take the active tends to bind to activated platelet specifically and firmly via interactions between ligands and receptors, Study found that the synthetic oligo-peptide sequence RGD loop which simulate the body's natural RGD binding site, has a strong capability to attach platelet glycoprotein GPⅡb/Ⅲa receptor, if the surface of general microbubbles were conjugated with oligo-peptide sequence RGD loop, Then the microbubbles will posess strong targeted efficacy and it is expected to achieve early targeted ultrasound molecular imaging for the artery thrombus in combination with contrast ultrasound.Therefore, in this study, base on the general lipid microbubbles, we constructed the microbubbles targeted to GPⅡb/Ⅲa receptor (MBRGD) by covalently combinding its specific ligands-cyclic Arg-Gly-Asp (RGD) peptide to the shell of general lipid microbubbles. The binding abilities of MBRGD to GPⅡb/Ⅲa receptors immobilized on a culture dish to different shear stresses were assessed in a parallel plate flow chamber. By the application of MBRGD and contrast enhanced ultrasound (CEU), the ex-vivo thrombus and rats abdominal aortic thrombus were evaluated in a in vitro agarose flow chamber model and in vivo rat model of abdominal Aortic thrombosis. our aim is to explore the feasibility of targeted microbubble to evaluate aterial thrombosis.Methods1. Preparation of microbubbles and determine their biological properties.Cyclic pentapeptides and isotype nonspecific peptides were covalently attached to the lipid shell of general lipid microbubbles to prepare the targeted microbubbles (MBRGD) and isotype microbubbles (MBCON). They were also washed (2×) to remove excess free unincorporated lipid. MBRGD and MBCON were storaged in refrigerator at 4℃. Their mean diameters and density were measured by coulter counter.2. Evaluation the efficacy of MBRGD targeting to platelet glycoprpteinⅡb/Ⅲa(GPⅡb/Ⅲa) by using a parallel plate flow chamber2.1,Attachment study:MBRGD and MBCON (5×106/ml) were divided into three groups (each n=3)respectively, MBRGD, MBCON were drawn through the parallel plate flow chamber that coated with platelet glycoprotein GPⅡb/Ⅲa (1000ng/ml) at the shear stress of 0.6 dyn/cm2,1.8dyn/cm2 and 3.6 dyn/cm2, respectively. Quantitative analysis of microbubble accumulation was performed by counting the number of microbubbles adhered in the observed area under the microscope when the emergence of microbubble for 6min; Control experiments on a plate that lack of GPⅡb/Ⅲa, or was blocked with excess RGDfvV against GPⅡb/Ⅲa as controls (each n=3).2.2,Detachment study:The parallel plate flow chamber were coated with GPⅡb/Ⅲa (1000ng/ml). MBRGD or MBCON were drawn into the flow chamber and allowed to interact with the target surface by flotation at zero flow for 5 min. After 5 min, PBS was drawn through the flow chamber at a shear stress of 0.2 dyn/cm2 to remove stationary but not adhered microbubbles. The total number of microbubbles adhered was recorded and microbubble detachment was then assessed by drawing PBS through the flow chamber with incremental shear stress increases every 30s until the microbubbles were completely detached. Recorded the full video and took photographys at a fixed field of vision (the objective lens was 20 times).3. Evaluation the efficacy of MBRGD targeted to thrombus using agarose flow chamber model in vitro studyAfter the agarose flow chamber model was prepared, equivalent control microbubbles (MBCON) or targeted ultrasound microbubbles (MBRGD) were randomly added into the flow chamber. After 30 min incubation with the thrombus fixed in self-made agarose flow chamber model, thrombus was washed with the continuous flow of PBS solution (15cm/s). When the thrombus was washed separately at 2,4,6, 8,10 min, imaging thrombus at different time points by contrast enhanced ultrasound (CEU) then the video intensity (Ⅵ) values of the images was measured.4. prepartion of rat model of abdominal Aortic thrombosis and CEU examination8 rats with non-occlusive thrombus in the abdominal aorta were created by inserting thrombin-soaked cotton threads via an iliac artery catheter, jugular vein catheter for injection of microbubbles, about one hour later there were non-occlusive aterial thrombosis on the suface of cotton surface. Two-dimensional ultrasound were used to determine the location of thrombus. Eight rats with non-occlusive thrombus were performed with CEU respectively by using MBCON and MBRGD, the intravenous injection of 10×106 microbubbles were made in random order with 30 minutes interval. After ten minutes of intravenous injection, microbubbles in the circulation were nearly eliminated, the ultrasound signal (video intensity,Ⅵ) from MBCON and MBRGD, were measured by second harmonic CEU imaging with pulsing interval time (PI) of ten seconds and a mechanical index (MI) of 0.18, transmission frequency of 7.0 MHz.After the first picture of CEU imaging being taken, the microbubbles were destroyed by two to three seconds of continuous imaging with a high MI of 1.9 and the background subtractedⅥof thrombus were measured.5. Examination of pathology:After CEU examination, all abdominal aortic were harvested for the examination of pathology and immunohistochemisty.Results1.Results for microbubble preparation:The density of MBRGD and MBCON were about 3.18~5.26×108/ml and 3.02~5.18×108/ml separately, the mean sizes for MBRGD and MBCON were about 2.13±1.18μm,2.18+1.11μm respectively.2. The efficacy of MBRGD targeted to GPⅡb/Ⅲa in the parallel plate flow chamber study:The parallel plate flow chamber experiments showed that MBRGD possessed a good targeted abilities to GPⅡb/Ⅲa at a certain shear stress (0.6-3.6 dyn/cm2), however, it could not attach on the parallel plate in the group lack of GPIIb/IIIa or blocked with excess RGDfvV against GPⅡb/Ⅲa. As expected, untargeted microbubbles(MBcoN) could not attach on the parallel plate even at low shear stress of 0.6 dyn/cm2 in the test groups or control groups. detachment study showed that the MBRGD could resist a certain shear stress, half-maximal detachment was achieved at a shear stress of (25.54±1.27) dyn/cm2. when the MBRGD were completely detached, the shear stress was up to (95.63±1.78) dyn/cm2, while MBCON can not resist low shear stress, its half-maximal detachment and maximal detachment were only achieved at a shear stress of (4.46±0.08) dyn/cm2 and (27.29 +0.74) dyn/cm2.3. In MBRGD group remarkable ultrasound contrast were observed in the first CEU pictures after 2 minutes washed by PBS, while there were no significant ultrasound contrast after 2 minutes washed by PBS in MBCON group. And after washed by PBS wash 2,4,6,8,10min, MBRGD still could attach to thrombus and stronger ultrasound contrast can be visualized than control MBCON-Before washed by PBS there were no significant difference (P>0.05);however, after washed by PBS, The video intensity of thrombus in MBRGD group reduced larger than MBCON.Ⅵvalues of thrombus in the MBRGD group were bigger than that in the MBCON group at each time points. (P<0.05).4.8 thrombi in rats abdominal aortas were successfully developed and each could be located with high-resolution B-mode ultrasound. As expected, all the thrombi were clearly visualized after intravenous application of specific MBRGD-Obvious contrast enhancement of thrombi in comparison to the abdominal aortas lumen was showed in all thrombi, This enhancement was located primarily at the proximal portion of the thrombi. After complete MBRGD destruction by high ultrasound mechanical index (MI) for several seconds, a notable loss of echogenicity was resulted in all thrombi.However, no obvious contrast enhancement of all thrombi can be observed when utilizing the nonspecific MBCON-Unlike the targeted microbubbles, the nonspecific MBRGD in conbination with CEU failed to increase thrombus detectability after intravenous application. Mean VI of the abdominal aortic thrombi with MBRGD (18.84±3.06) was about 3.15 folds higher than that of the thrombi with MBCON (5.99±1.18)(P<0.05).5. Pathological and immunohistochemisty examination:Part of the abdominal aorta were opened, thrombi could be visible; pathological examination:abdominal aortic platelet thrombosis were confirmed, formation of a large number of platelets trabecular were observed, and GPⅡb/Ⅲa receptors are expressed abundantly in the thrombus.Conclusions1. Microbubbles targeted to thrombus can be successfully constructed by covalently combinding oligopeptide RGD sequence to shell of lipid microbubbles, and the targeted microbubbles possess strong abilities to attach targets actively.2. MBRGD have a good targeting performance to thrombus and can resist to a certain degree of shear stress after adhesion to thrombus. In vitro evaluation of binding thrombus capability of targeted microbubble (MBRGD) by simulating shear stress in vivo environment could be helpful for predicting the effects of ultrasonic molecular imaging when MBRGD were used in vivo environment.3. The "active targeted CEU imaging" created by cyclic-modified microbubbles and CEU can effectively evaluate the aterial thrombosis in rat abdominal aortic, which may be used to diagnose the thrombosis in early stage, study targeting thrombolysis targeted therapy and monitoring the efficacy.
Keywords/Search Tags:Targeted ultrasound microbubbles, Thrombosis, Contrast enhanced ultrasound, RGD peptide
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