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Assessment Of Targeting Capability Of Microbubbles Carrying Both Sialyl Lewis~x And Anti-p-selectin Monoclonal Antibody In High-shear Flow

Posted on:2012-02-05Degree:MasterType:Thesis
Country:ChinaCandidate:M Y LiFull Text:PDF
GTID:2214330368975679Subject:Internal Medicine
Abstract/Summary:PDF Full Text Request
Background and ObjectiveInflammation is a fundamental component of numerous cardiovascular disease processes, including atherosclerosis, myocardial ischemia reperfusion-injury, hypertension and so on. A technique that offers direct, real-time, non-invasive assessment of the inflammatory status of the endothelium would be clinically useful, potentially offering early diagnosis prior to overt clinical manifestations, as well as therapeutic monitoring of disease activity. Therefore, a wide array of noninvasive molecular imaging techniques for evaluating inflammation have been developed, such as ultrasound, MRI, CT and SPECT.Ultrasound molecular imaging is a recent development of noninvasive molecular imaging modality. Targeted ultrasound imaging involves the design and synthesis of microbubbles that adhere to endothelium under disease-specific conditions, so that the disease can be ultrasonically detected as a prolonged contrast effect that persists in tissue beyond the normal washout time of nontargeted microbubbles. As compared with traditional imaging approaches characterization of diseases by the detection of changes in physics or physiology, targeted contrast imaging could provide real-time, conveniencent, high spatial resolution and cheap capabilities for in vivo ultrsonic detection of phenotypic features of endothelium that predate clinical disease Inflammatory endothelium is characterized by the upregulation of a variety of leukocyte adhesion molecules, including P-, L-, E-selectin, intercellular adhesion molecule-1(ICAM-1) and vascular adhesion molecule-1 (VCAM-1) on the luminal surface. By binding to specific molecular markers expressed on the endothelial surface of inflammatory blood vessels, targeted ultrasound contrast agents permit ultrasonic detection of these cardiovascular disease.Achieving a high signal-to-noise ratio of targeted ultrasound contrast requires the ecruitment and retention of a sufficient fraction of the injected microbubbles on the intended target surface. Microbubbles are often targeted to specific molecular markers of disease using monoclonal antibodies, and ultrasound contrast agents targeting to specific molecular markers expressed on the endothelial surface of inflammatory blood vessels have been hired to detect and evaluate intravascular pathology, including myocardial ischemia-reperfusion injury and thrombus. Most antibodies attach to their respective antigens firmly (i.e. complex dissociation rate is slow), but the association is not always fast (i.e. antigen-antibody complex formation on-rate kinetics tends to be slow). If a microbubble bearing antibodies is rapidly moving through the blood vessel and has a limited time to interact with the vessel wall components, the rate of attachment of ligand to receptor becomes critical, leading to limited binding. Therefore, for the microcirculation targeted ultrasound contrast agents do improve visualization of specific structures, while molecular imaging of activated endotheliumin large- and middle-sized arteries by site-specific accumulation of contrast material is still difficult to achieve due to wall shear stress conditions in these vessels.Successful UCA retention requires that the molecular on-rate be rapid and off-rate be slow enough for bond formation to occur in the limited time and bind firmly. Microbubbles employ two distinct adhesion liglands, one of which mediates rapid catch and the other one mediates firm binding, could be able to improve the effect for molecular imaging in high-shear flow.Sialyl Lewisx as a clustered polymeric form of the fast-binding selectin ligand, is known to associate selectively and rapidly with selectins. Therefore, we sought to devise targeted microbubbles bearing both sialyl lewisx and anti-P-selectin antibodies, then its adhesive behavior and adhesive capability were evaluated by using a radial flow perfusion chamber in vitro and in vivo for molecular imaging in mice model of abdominal aortic inflammation, aiming at laying the groundwork for the development of microbubbles with stronger binding capability for molecular imaging in high-shear flow.Methods1. Biotinylated microbubbles preparation:Biotinylated, lipid shelled decafl-uorobutane microbubbles were prepared by shearing of a C3F8-saturated aqueous suspension of DSPE -PEG2000- Bioti,DPPC,PEG4000. Microbubbles were then washed by superpurified water three times to remove exeess free unincorporated lipid. The concentration and size distribution of microbubbles were analysed with a Coulter Multisizer counter.2. Targeted microbubbles preparation:Biotinylated sialyl lewisx or biotinylated anti-P-selectin antibody were attached to the biotinylated microbubbles surface via multi-step avidin biotin bridging chemistry. Microbubbles were washed three times, streptavidin was then added to the washed microbubble dispersion. Following incubated at 4℃with 1.5μg of biotinylated sialyl lewisx or 7.5μg of biotinylated anti-P-selectin antibody per 107 microbubbles, Selectin-targeted (via sialyl lewisx) microbubbles (MBs) and P-selectin-targeted (via P-selectin monoclonal antibodies) microbubbles (MBP) were prepared. Dual-ligands (via both sialyl lewisx and P-selectin monoclonal antibodies) microbubbles (MBD) were prepared by simultaneous incubation with 0.75μg of biotinylated sialyl lewis" and 3.75μg of biotinylated anti-P-selectin antibodies per 107 microbubbles. A control (via isotype control antibodies) microbubbles (MBc), was also constructed by conjuncting 7.5μg rabbit monoclonal IgGi per 107 microbubbles. Coulter counter was then used to evaluate the characters of the microbubbles.3. Targeted microbubbles valuation:Targeted microbubbles and control microbubbles were incubated with both fluorescein-conjugated affinipure goat anti-rat IgG and rhodamine-conjugated affinipure goat anti-mouse IgG, using fluorescence microscope to identify the linking antibodies on the microbubbles. Then the binding rate of ligands in microbubbles was measured by quantitative flow cytometry.4. Evaluation the performance of microbubbles targeting to P-selectin by using a parallel plate flow chamberThe flow chamber Petri dish was incubated with 200μl of P-selectin.Fc at concentration in of 1μg/ml overnight at 4℃.4.1. Attachment study:four types of microbubbles (5×106/ml) were drawn through the parallel plate flow chamber that coated with P-selectin at the shear stress of 0.5 dyn/cm2,2.0dyn/cm2 and 4.0 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 6 min and the rolling micobubbles per second.4.2. Detachment study:The parallel plate flow chamber was coated with same concentration P-selectin. MBC, MBS, MBP and MBd were drawn into the flow chamber respectively and allowed to interact with the target surface by flotation at zero flow for 5 min. Then 0.9% sodium chloride solution 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 were assessed by increasing the shear stress every 30 s until the microbubbles completely detached. Recorded the full video and took photographys at a fixed field of vision (the objective lens was 20 times).4.3. A parallel plate flow chamber combined with a novel automated tracking algorithm, were used to analyze the transient velocities, rolling and firmly adherent numbers of microbubbles at various shear stress.5. Prepartion of mice model of abdominal aortic inflammation:12 mice were equally randomized into control group and inflammatory group, mice of inflammatory group were deal with local injection of cytokines IL-1βand TNF-αinto the retroperitoneal space around the abdominal aorta, and control one only with PBS. 6. Ultrasound molecular imaging:ultrasound molecular imaging was performed after intravenous injection of MBC, MBS, MBP and MBD in random order with 30 min interval. Second harmonic CEU imaging was taken to measure the ultrasound signal (video intensity, VI) from microbubbles. Abdominal aortic video intensity (VI) was measured by average pixel VI with a low mechanical index (MI) of 0.18 at 6 minute after injection subtracted from background.7. HE staining and P-selectin immunofluorescent:After targeted imaging, abdominal aortic of all mice were harvested for HE staining and immunofluorescent analysis of endothelial P-selectin expression.Results1. Characterization of microbubbles:The concentration of targeted microbubbles measured by Coulter Counter was from 1.239×108 to 1.341×108 per milliliter and the mean sizes distribution was from 2.182 to 2.378μm. There were no significant difference between both the concentrations and mean sizes (P>0.05).2. Targeted microbubbles valuation:Observed with fluorescence microscopy, MBc were absence of fluorescence, MBs emitted red fluorescence, MBP emitted green fluorescence, while MBD gave off both red and green fluorescence. All of the ligand-binding rates in MBD, MBs and MBP were more than 80%, there was not a significant difference among them (F= 1.773, P=0.248). All of above indicated the sialyl lewisx and the P-selectin monoclonal antibodies have linked well to the surface of microbubbles.3. Evaluation the performance of microbubbles targeting to P-selectin using a parallel plate flow chamberThe parallel plate flow chamber attachment experiments:MBs, MBP and MBD possessed a good targeted abilities to P-selectin at a certain shear stress (0.5-4.0 dyn/cm2), while MBc could not attach on the parallel plate. At the same shear stress, adhesive numbers and rolling numbers of MBs, MBP and MBD were much more than those of MBc(P<0.01). There was no significant difference among the rolling numbers of MBc at different shear stress conditions(F=4.287, P=0.070), while the adhesive numbers decreased at 4.0 dyn/cm2 compared to those at 0.5 and 2.0 dyn/cm2(F=6.833, P=0.028). In all flow conditions, the adhesive numbers and rolling numbers of MBs, MBP and MBD to the targets decreased with the shear stress increased(P<0.05). At 0.5 dyn/cm2 shear stress, there was not a significant difference among the rolling and adhesive numbers of MBS, MBP and MBD (P>0.05), while at 2.0 dyn/cm2, the rolling numbers of MBD and MBs to the targets were obviously greater than those of MBP (P<0.05), and the adhesive numbers increased from MBP, MBs to MBD in turns(P<0.05). At 4.0 dyn/cm2 shear stress, the rolling numbers of MBD and MBs to the targets were also obviously greater than those of MBP (P<0.05), while the adhesive numbers of MBD were more than those both of MBP and MBs(P<0.05). Flow chamber experiments show that dual-ligands microbubbles perform significantly better than their single-targeted counterparts at high-shear stress flow.The parallel plate flow chamber detachment experiments:As expected, MBc could not attach on the parallel plate even at low shear stress of (14.20±2.82) dyn/cm2, and either the half-maximal or the complete detachment shear stress increased gradually in MBC, MBS, MBD and MBP by turns (P<0.05).4. Ultrasound molecular imaging:With ultrasound molecular imaging, there was minimal signal for MBC, MBS, MBP and MBD in control mice aortic and MBc in inflammatory mice aortic. In comparison with control group, there was stronger signal enhancement from MBs, MBp and MBD in the inflammatory mice aorta, and MBD showed more signal enhancement than both MBS and MBP. The quantitative data showed that there was no significant difference among theⅥfrom MBC, MBS, MBp and MBD in the control mice group (F=1.199, P=0.336). In the inflammatory mice group,Ⅵof the aortic from MBD was significantly higher than MBs, MBp and MBC(F=36.349, P=0.000). TheⅥfor MBS, MBP and MBD in inflammatory mice group was obviously higher than that of control mice group (MBS:t=9.363, p=0.000; MBp:t=6.841, p=0.000; MBD:t=8.246, p=0.000), while there was no significantly difference ofⅥfor MBc in the two groups (t=0.099, p=0.923) 5. Pathological and immunohistochemisty examination:HE staining showed that the aortic vascular cells in control mice group were normal, while in inflammatory group, they were edema and infiltrated with a large number of leukocytes. P-selectin expressed abundantly in the aortic vascular cell in inflammatory group, while in control mice group P-selectin was below the level of detection.Conclusions1. Dual-liglands microbubbles(MBD) and their single-targeted counterparts targeted to P-selectin can be successfully constructed by combinding anti-P-selectin monoclonal antibodies and sialyl Lewis" to lipid microbubbles via "avidin- biotin" bridging chemistry.2. MBs, MBP and MBD showed different ways of adhesive behavior and adhesive capacilities in vitro. MBs exhibites unstable or transient adhesion with high rolling efficiency, while MBP reveales primarily firm adhesion with low rolling efficiency, and MBD exhibites firm adhesion with high rolling efficiency. MBD showed significantly better adhesive capacility than their single-targeted counterparts in high-shear flow.3. Ultrasound molecular imaging with MBD targeted to P-selectin in mice inflammatory abdominal aorta performed better than those with MBs or MBP.
Keywords/Search Tags:Targeted ultrasound microbubbles, Sialyl Lewis~x, Anti-P-selectin monoclonal antibody, Parallel plate flow chamber, Inflammation, Ultrasound molecular imaging
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