Preparation Of Gelsolin-targeted And Paclitaxel-loaded Ultrasound Contrast Agent And In Vitro Experiment

Objective Toprepare a kind of Gelsolin-targeted ultrasound contrast agent(CS-P-TX-PLGA-GSN)with PLGA and to evaluate its targeting and imaging effects in vitro.Methods Paclitaxel-loaded high molecular PLGA-COOH ultrasound contrast agent was prepared by a modified double emulsion technique and then conjugated with Gelsolin monoclonal antibody by carbodiimide technique.The physicochemical properties,such as the dispersity,the particle size and the zeta potential,were observed by light microscope and Malvern laser detector.Theentrapment efficiency,the drug loading efficiency and the property of releasing behavior were measured by high performance liquid chromatography(HPLC).The connectivity condition of ultrasound contrast agent with Gelsolin monoclonal antibody and the binding rate with fluorescent antibody were measured by immunofluorescence staining and flow cytometry respectively.Hca-F and its syngeneic cell line Hca-P are mouse hepatocarcinoma ascites cell lines with different lymphatic metastasis potential.Four experimental groups were set up:Hca-F cells with ordinary PLGA,Hca-F cells with antibody blocking,Hca-P cells with GSN-PLGA,Hca-F cells with GSN-PLGA.Each group was treated with the corresponding UCAs which were marked with coumarin-6 and then laser scanning confocal microscope was used to assess the binding of ultrasound contrast agents with Hca-F cells,and to evulate its effect on enhancing ultrasound imaging in vitro.Results The average diameter,the electric potential,the drug entrapment efficiency and the loading amount of CS-PTX-PLGA-GSN was(328.59±3.82)nm,(-11.46 ± 1.19)mV,(83.1±2.12)%,(8.31±0.21)%,respectively.The binding rate with Gelsolin monoclonal antibody was as high as 98.31%.The drug releasing test showed that the paclitaxel releasing rate was about 90.63% of 30 d.In vitro experiments showed that the Hca-F cells can intake more GSN-PLGA.The fluorescence intensity of the Hca-F cells with GSN-PLGAwas higher than that of Hca-P cells with GSN-PLGA(189.09±1.14)vs(140.15±0.98)(P<0.05),Nevertheless the fluorescence intensity of Hca-F cells with ordinary PLGA and Hca-F cells with antibody blocking was(108.39±1.29)and(123.31±1.25).All the other three groups were lower than that of Hca-F cells with GSN-PLGA(P<0.05).It showed that ultrasound contrast agent could enhance the ultrasound imaging greatly in vitro experiments.Conclusions The CS-PTX-PLGA-GSN nanoparticle with higher entrapment efficiency and loading amount can also enhance the ultrasound imaging greatly.