Effects Of Microbubbles Containing Nordy Irradiated By Ultrasound On Endothelial Cells

Background and purpose: Tumor invasion and metastasis is one of the important reasons of cancer deaths, and tumor angiogenesis is the basis of tumor growth and metastasis. Therefore, the anti-angiogenic therapy becomes a hot spot in current cancer treatment and research field. Targeted drug delivery system can selectively distributes drugs to the treated areas, increasing the drug concentration at the treated areas, decreasing the drug dose, reducing side effects and improving safety and effectiveness. Under ultrasonic action, the lipid microbubbles containing gases are cavitated, the energy caused by micro-bubble destruction in turn causes sonoporation effects, macromolecules can go into cells and be captured by cells in a short time. This method which uses lipid microbubbles contrast agent as a delivery vector has advantages of high efficiency, noninvasive and no immunogenic properties, therefore, lipid microbubbles have become a new means of targeted drug delivery in recent years. Nordy is a chiral compound synthesized based on the chemical structure of natural lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA). Recent studies have confirmed that NDGA can inhibit tumor angiogenesis and a variety of human cancers both in vivo and in vitro. In this study, we prepared a kind of lipid microbubbles contrast agent containing Nordy and investigated the effect of ultrasond-irradiated lipid microbubbles containing Nordy on the proliferation of endothelial cell.Methods: (1) lipid microbubbles containing Nordy was prepared by mechanical vibration method ; its morphology and size was observed by the optical microscope , and its particle size and its surface potential were detected by Zetasizer 3000HS; and the encapsulation rate was detected by high performance liquid chromatography (RP- HPLC) ; lipid microbubbles containing Nordy enhanced ultrosound imaging was demonstrated by contrast paulse sequencing (CPS) technique in rabbit liver; (2) perfusion with trypsin is used to obtain umbilical vein endothelial cells; (3) MTT colorimetric assay and trypan blue exclusion test are used to select the related parameters that ultrasound-irradiated lipid microbubbles effect on HUVEC; (4) the three-dimensional culture of vascular endothelial cells in vitro is used to observe the effect of lipid microbubbles containing Nordy on the formation of tubular structures; (5) the method which using the supernatant of tumor cells to culture EA.Hy926 cell lines is used to observe the effect of lipid microbubbles containing Nordy on the cells after induction, and RT-PCR method is used to observe the expression of tumor endothelial marker8 (TEM8) in the cells.Results: (1) the concentration of lipid microbubbles containing Nordy is (2.53±0.52)×109 / ml, particle size is 2.61±0.32μm, Zeta potential is + (17.52±2.04 )mV; the encapsulation rate of lipid microbubble containing Nordy is 27.1±2.01%, the drug loading is 10.9±0.90%; after injected through vein, microbubbles showed a good, sustained and enhancment imaging in the rabbit liver. (2) primary HUVEC cultured by perfusion with trypsinin were identified as HUVEC by cell morphology and immunohistochemistry. (3) When the concentration of Nordy is 50μmol / L, 100μmol / L, 150μmol / L, 200μmol / L respectively, the inhibition of cell proliferation shows a dose-effect relationship in the 24h. Nordy microbubbles are destructed under the conditions, frequency 1.5MHz, mechanical index 1.9, time 60s, 90s, 120s, 150s; the cell membrane temporarily opens to and captures macromolecules trypan blue dye in 120s. (4) comparing the inhibition of the tubular structure formation in the control group, simply Nordy microbubbles group, simple Nordy group, Nordy combined with ultrasound group and Nordy microbubbles combined with ultrasound group. Compared with other groups, the inhibition is more significant in Nordy microbubbles combined with ultrasound group. (5) after induction by supernatant of tumor cells cultured endothelial cells, EA.Hy926 cells has the characteristics of tumor-derived vascular endothelial cell (Td-EC). Nordy microbubbles combined with ultrasound can inhibit Td-EC to express TEM8.Conclusions: (1) lipid microbubble containing Nordy prepared by mechanical vibration method can go through the pulmonary vascular bed to meet the requirements of peripheral intravenous injection and can be used as a vector to deliver Nordy in vivo. (2) lipid microbubbles containing Nordy can significantly inhibit the formation of tubular structures in vitro. (3) lipid microbubbles containing Nordy can inhibit Td-EC to express TEM8 and make Td-EC normalize.