Preparation,Characterization And Antiangiogenic Effects Of Recombinant Human Endostatin Loaded Lipid Microbubbles

ObjectiveTo prepare recombinant human endostatin loaded lipid microbubbles(RHEM) byBiotin-Avidin system,and investigate its characteristics and antiangiogenic effects.Methods1. The lipid microbubbles were prepared by the method of mechanical oscillation, andrh-ES was linked to the bubbles with Biotin-Avidin system. The form, particle sizeand distribution were observed by light microscope, and the drug-loading efficiencywas measured by ELISA.2. Ten H22liver cancer-bearing mice were randomly enrolled into two groups, RHEMgroup and SonoVue group, to perform contrast-enhanced ultrasonography, andanalyze the differences of parameters AT, TTP and PBD.3. Primary human umbilical vein endothelial celles (HUVEC) were cultured usingperfused-digestion method, and identified by immunohistochemistry examination onⅧ factor.4. The growth inhibitory effects of rh-ES on HUVEC were detected by MTT assay.5. HUVEC were divided into three groups, rh-ES, rh-ES+US and RHEM+US group.After administration for one or two hours, the cells were lysed to detect theintra-cellular concentration of rh-ES by ELISA.6. Chick embryos were randomly divided into7groups, rh-ES low, medium and highdose group, RHEM group, rh-ES+US group, RHEM+US group and saline controlgroup, respectively. After adimintration for48h, the growth of new blood vesselswere calculated and analyzed by the Image-pro plus6.0software.Results1. The prepared RHEM were smooth, with a density of （5.2±0.1）×10~8/mL. The meanparticle size was detected as (3.0±0.4) μm, and no less than82.4%microbubbles were in2～7μm range.The drug-loading efficiency was（800±70.53）μg every10~8microbubbles.2. After bolus injection of RHEM,the tumor echo enhanced firstly from the peripheral,then the whole tumor enhanced remarkably, and disappeared quickly, Thecontrast-enhanced parameters AT,TTP and PBD were similar to that of SonoVue.3. The primary cultured HUVEC were polygons or short spindle in shape, and most ofthe cytoplasm expressed Ⅷ factor.4. For the same rh-ES concentration, with prolonging of treatment time, the cellproliferation inhibition was more remarkable.In400ng/mL doseperiod,presented adose-time dependent manner.5. The intra-cellular drug contents of RHEM+US treatment for1h,2h were detected as(5.58±0.16) and (6.45±0.11) ng/mg total protein,which were significantly higherthan that of rh-ES and rh-ES+US groups（P <0.05）.6. In saline control group, the CAM showed abundant new vessles in radial uniformdistribution.However, in all the treatment groups, the new blood vessels reduced,especially the RHEM+US group,which had a new blood vessles area of (6.73±3.25)cm~2and a ratio of new blood vessles area/CAM area of (13.12±5.87)%,and thedifference was of significant when compared with any other group(P <0.05).Conclusion: RHEMs were successfully prepared by Biotin-Avidin system with optimalparticle size and high drug-loading efficiency. RHEM combined with ultrasonic irradiationcan increase the intra-cellular drug content, and significantly improve the antiangiogeniceffects in vitro.