Studies On Preparation Of Targeted Ultrasound Microbubbles And The Interactions Between The Targeted Microbubbles And Human Vascular Endothelial Cells

PART 1. Studies on Preparation of Targeted UltrasoundMicrobubbles(1) Preparation study of Perfluorocarbon-filled Sonicated Albumin Contrast AgentsObjective: To explore the method of preparing ultrasound contrast agent using albumin and perfluorocarbon gas. The size, concentration, stability and myocardiai contrast of the prepared microbubbles were analyzed, respectively.Methods: Mixed with proper volumes of CsFsgas, the 5% human serum albumin was sonicated in a certain condition. The prepared microbubbles were constituted by albumin shell which encapsulates C₃F₈ gases. Microscope, blood cell counter and animal experiment were then applied to decide some parameters and myocardiai contrast of the self-made microbubbles.Results: The prepared albumin ultrasound contrast agents were lacte suspension. The size of microbubbles was 2 to 5μm. The mean concentration was (2.07±0.23)×10～8/ml. This microbubble suspension could be stored in refrigerator at 4℃ for a long time. In the animal experiments, intense myocardiai contrast images were obtained after intravenous injection of 2ml self-made contrast agents. There were no hemodynamic changes during the whole experiments.Conclusions: This self-made perfluorocarbon-filled sonicated albumin contrast agent was effective, safe and stable, which established the base of further preparation study of targeted contrast agents in heart diseases.(2) Methodology of antibody conjugating albumin microbubblesObjective: This study was performed to prepare albumin microbubbles with antibody binding by N-succinimidyl 3-(2-pyridyIdithio) propionate (SPDP) conjugation technology and to detect the most proper reaction conditions.Methods: The human IgG was coupled to albumin microbubbles on different quality ratios by SPDP conjugation technology. Slide agglutination and immunofluorescent assay were used to detect whether antibody biding with albumin microbubbles and to determine the best ratio.Results: The prepared immunomicrobubbles were positive in slide agglutination and immunofluorescent assay. When the quality ratio was 1:5, there was brightest reaction in immunofluorescent assay.Conclusions: The antibody can be effectively attached to the surface of albumin microbubbles by SPDP conjugation technology, which promoted further studies on preparation of targeted microbubbles.(3) Studies on preparation and immunological characterization of albumin ultrasound contrast agents with monoclonal antibody bindingObjectives: This study was performed to prepare albumin microbubble with monoclonal antibody binding by SPDP conjugation technology. This kind of microbubbles' targeting function was further explored in vitro studies. Methods: The mouse anti-human vascular cell adhesion molecule-1 (VCAM-1) monoclonal antibodies were covalently coupled to the albumin microbubbles' shells by SPDP. Slide agglutination test was used to detect whether antibody biding with the microbubbles. The TNF-α activated human umbilical vein endothelial cells (HUVEC) which over-express VCAM-1 were cultured. To detect the microbubbles' targeting function, the specific binding of immunomicrobubbles to the activated endothelial cells was observed in vitro.Results: The prepared immunomicrobubbles were positive in slide agglutination test. The activated endothelial cells were surrounded by plenty of anti-VCAM-1 conjugated microbubbles. While there was limited binding of control bubbles to normal or activated endothelial cells.Conclusions: The antibody can be effectively attached to the surface of albumin microbubbles by SPDP conjugation technology. And the studies in vitro had proved that the prepared immunomicrobubble could specifically bind to the targeted cells.PART 2. Interactions between Targeted Ultrasound Microbubbles and Human Vascular Endothelial Cells: In Vitro Studies(1) Studies on Interactions between Targeted Ultrasound Contrast Agents and Activated Vascular Endothelial CellsObjectives: This study was performed to explore the interaction between the activated endothelial cells and the self-made targeted ultrasound agents, which were conjugated anti-human vascular cell adhesion molecule-1 (VCAM-1) monoclonal antibodies. Meanwhile, a new method of assessing endothelial function was further explored based on the above interactions.Methods: Monoclonal antibodies to VCAM-1 were bound to the shells of the perfluocarbon gas-filled albumin microbubbles by covalently conjugation technology. The specific interaction between the microbubbles and the normal endothelial cells or activated endothelial cells was observed using inverted microscope, respectively. The microbubbles adherence was quantified by calculating the ratio of the adherent microbubbles and endothelial cells. The results were compared with the control bubbles.Results: There was rarely control microbubbles adherence to normal or activated endothelial cells. However, compared with normal endothelial cells, there were significantly more targeted microbubbles binding to the activated endothelial cells.Conclusions: The ultrasound agents with anti-human VCAM-1 monoclonal antibody can specifically bind to the activated endothelial cells, which offered a new insight to the noninvasive detection of endothelial dysfunction using ultrasound imaging techniques.(2) Studies on Interactions between Targeted Ultrasound Contrast Agents and damaged Vascular Endothelial Cells on different degreesObjectives: This study was performed to detect the interactions between the graded damaged endothelial cells and the self-made targeted ultrasound agents, which were conjugated anti-human vascular cell adhesion molecule-1 (VCAM-1) monoclonal antibodies.Methods: Human umbilical vein vascular cells were stimulated with TNF-a at the indicated concentration(0-300U/ml) for the same time(24h). Therewere six groups of endothelial cells which were damaged differently in degree. Cell surface expressions of VCAM-1 were detect by immunocytochemistry Monoclonal antibodies to VCAM-1 were bound to the shells of the perfluocarbon gas-filled albumin microbubbles by SPDP conjugation technology. The specific interactions between these microbubbles and each group of endothelial cells were observed using inverted microscope. The microbubbles adherence was quantified by calculating the ratio of the adherent microbubbles and endothelial cells. The results were compared between every two groups. And correlation analysis was performed between the targeted microbubbles' adherence and the cell surface expression of VCAM-1.Results: After TNF-a stimulation, the expressions of VCAM-1 in groups of damaged endothelial cells were obviously increased. And the expressions were dose-dependent. With the increase of TNF-a stimulation concentration, there were more and more targeted microbubbles adherent to the corresponding endothelial cells. And the correlation analysis showed there was positive correlation between the targeted microbubbles' adherence and the cell surface expression of VCAM-1.Conclusions: The ultrasound agents with anti-human VCAM-1 monoclonal antibody can specifically bind to the activated endothelial cells. And these ultrasound agents' adherence provides a new way to detect the damaged degrees of endothelial cells.