| Objective:The aim of this study is to prepare an acoustic contrast agent by coupling an anti-interleukin-8monoclonal antibody (anti-IL-8mAb) with SonoVue microbubbles, as well as to discuss the methodology of such coupling. Based on these, by interacting the anti-IL-8mAb-bound targeted ultrasound agent with normal endothelial cells and injured endothelial cells respectively, in vitro targeting adhesion of the contrast agent was evaluated, for exploring the role of IL-8in formation of atherosclerotic plaques, and for suggesting a new approach for evaluation of the vascular endothelial function, and thus providing new imaging evidence for both the early clinical diagnosis and the treatment of atherosclerosis.Method:1. Through N-succinimidyI-3-(2-pyridyldithiol)propionate (SPDP) crosslinking, the anti-IL-8mAb was bounded to SonoVue microbubbles under the procedures as follow:the anti-IL-8mAb was dissolved in phosphate-buffered saline (PBS), before the addition of SPDP solution. The resultant solution was then centrifuged and washed, to obtain a pyridine dithiothymine-carrying monoclonal antibody. Afterwards, excessive dithiothreitol (DTT) acetic acid solution was added to carry out a chemical reaction. Centrifugation and washing was performed again, for obtaining a sulfydryl-containing antibody solution. SonoVue microbubble suspension was added into SPDP solution and washed, before being mixed and reacted with the sulfydryl-containing antibody solution. Hence, the anti-IL-8mAb-bound acoustic microbubble suspension was eventually prepared.2. The validity test on coupling anti-IL-8mAb with acoustic microbubbles:(1) The slide agglutination experiment:on clean glass slides, the coupled and uncoupled suspension were mixed with goat anti-mouse IgG serum and normal saline, respectively and stilled for several minutes. The microbubble status was examined later by using an optical microscope.(2) The immunofluorescence staining experiment:The goat anti-mouse IgG-fluorescein isothiocyanate (FITC) was mixed and reacted with both the anti-IL-8mAb coupled and uncoupled SonoVue microbubbles suspensions, respectively, and the results of florescence staining were then observed by an fluorescence microscope.3. The human umbilical vein endothelial cells were cultured in a human RPMI1640medium until sub-fusion state was reached. After being digested by0.25%trypsin, a portion of the cells was added into PBS buffer solution to establish the normal endothelial cell model group; while another portion of the cells was subjected to tumor necrosis factor-alpha(TNF-a) intervention, to establish the injured endothelial cell model group. Both model groups were continuously cultured for another24hours, before immunocytochemical staining test was employed to examine the expression of IL-8on cellular surface.4. The respective interactions between the uncoupled SonoVue microbubbles, the targeted microbubbles and the the normal endothelial cells, the injured endothelial cells were observed by an inverted microscope. The numbers of endothelial cells and the adhering microbubbles within a highly magnified view were counted, for calculating the ratio of microbubble to endothelial cell, which was able to suggest a quantitative analysis on interactions between microbubble and cell.Result:1. Uncoupled SonoVue microbubbles were homogeneous in the size (with a diameter ranging from2-4μm), and evenly distributed in a single and scattering manner; The concentration of SonoVue microbubbles in the anti-IL-8mAb coupled suspension was slightly lower than tat in the uncoupled suspension, but the size, shap and properties of microbubble in both suspensions were almost identical, i.e. without any significant difference. 2. During the slide agglutination experiment, when the anti-IL-8mAb-bound SonoVue microbubble suspension mixed with the secondary anti-(goat anti-mouse IgG serum) antibody solution, a clear agglutination was observed by an optical microscope; When uncoupled SonoVue microbubble suspension mixed with the secondary antibody solution, or when both the anti-IL-8mAb-bound SonoVue microbubble suspension and the uncoupled SonoVue microbubble suspension mixed normal saline respectively, no significant agglutination was observed by an optical microscope. In the immunofluorescence staining experiment:green fluorescence was only observed for the anti-IL-8mAb-bound SonoVue microbubbles, not any fluorescence was observed on the uncoupled SonoVue microbubbles.3. In the immunocytochemical staining test, nuclei of vascular endothelial cells in the normal group depicted a bluish purple color, and the cellular surface remained unstained; after induction of TNF-a, nuclei of vascular endothelial cells in the injured group also depicted a bluish purple color, but the cell surface was almost stained to brown.4. When the uncoupled microbubbles and the anti-IL-8mAb-bound microbubbles interacted with normal endothelial cells and injured endothelial cells, respectively, the quantitative analysis that conducted by using inverted microscope showed that the difference in the adhesion to the normal endothelial cells between the two types of microbubble was insignificant; however, the adhesion to injured endothelial cells of the anti-IL-8mAb-bound targeted microbubbles was significantly higher than that of uncoupled microbubbIes(P<0.01). For uncoupled SonoVue microbubbles, the adhesion to injured endothelial cells was slight higher than that to normal endothelial cells, but the difference was statistically insignificant; for anti-IL-8mAb bound targeted microbubbles, the adhesion to injured endothelial cells was significantly higher than that to normal endothelial cells (P<0.01).Conclusion:1. The result of slide agglutination experiment showed that an obvious agglutination took place when anti-IL-8mAb bound acoustic microbubbles suspension mixed and reacted with the secondary antibody. In addition, the result of immnuofluorescence experiment showed an obvious green fluorescence appeared on the surface of the anti-IL-8mAb-bound acoustic microbubble. Both results confirmed that anti-IL-8mAb has bounded to the surface of microbubble, i.e. the targeted contrast agent was successfully prepared.2. After SPDP crosslinking, although the concentration of prepared targeted microbubbles decreased, the size, shape and properties of the microbubble did not varied significantly, indicating that the coupling with SPDP did not change the physical and biological properties of the acoustic contrast agent, i.e. the acoustic contrast agent was still in vivo applicable and valid.3. After the normal endothelial cells get injured by TNF-a intervention, the nuclei of injured endothelial cells depicted a blueish purple color, and the cellular surface depicted a brown color in the immunocytochemical staining test. The staining result confirmed the presence of IL-8on the surface of endothelial cells, and proofed that the TNF-a successfully induced the endothelial cells to secret IL-8.4. The result of immunocytochemical staining test showed that after the induction by TNF-a, a high level expression of IL-8that secreted by the injured endothelial cells was observed, which suggested a possible new approach to accurately detect injured endothelial cells by using anti-IL-8mAb as targeted antibody and coupling it to the surface of SonoVue microbubbles.5. The result of the in vitro cell experiment show that abundant of anti-IL-8mAb bound targeted microbubbles adhered to the surface of injured endothelial cell, indicating the anti-IL-8mAb maintained its immunological competence after bounding to microbubble surface.6. From the in vitro cell experiment, although several uncoupled SonoVue microbubbles was observed being able to adhere to the surface of injured endothelial cells, but this number was significantly lower than that of anti-IL-8mAb-bound targeted microbubbles, suggesting that the interaction between the targeting cells and the anti-IL-8mAb-bound targeted microbubbles, which was prepared by SPDP crosslinking, was highly efficient and specific.7. The in intro cell experiment showed that by employing the interaction between anti-IL-8mAb-bound targeted microbubbles and the specific antigen-antibody on the surface of injured endothelial cell, the adhesion to injured endothelial cells was drastically improved, which proposed the possibility of applying targeted ultrasound contrast technique in the detection of endothelial injury and the assessment of endothelial functions. |
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