Preparation And Ultrasound Imaging Of Amphiphilic Chitosan-polylactic Acid Nanobubbles

Objective:As a means of diagnosis of diseases,ultrasound imaging is considered to be one of the safest imaging methods.The appearance of contrast agents can significantly enhance the ultrasound signal and help ultrasound imaging in the diagnosis and treatment of diseases.Currently,the size of microbubble contrast agents on the market is in the micron level,which belongs to the blood pool contrast agent,and can not cross the vascular endothelium into the tissue gap,and the action time is short.Therefore,nano contrast agents with strong stability and biocompatibility have a broad prospect.In this study,microbubble contrast agents with good biocompatibility and nano particle size were prepared,which could penetrate the inner wall of blood vessels and achieve ultrasound imaging of extravascular tumors.Methods:A novel amphiphilic graft copolymer of chitosan and poly(lactic acid)was synthesized by oxidative degradation of chitosan(CS)to water-soluble chitosan oligosaccharide,using chitosan oligosaccharide and L-lactide(L-LA)as raw materials and triethylamine as catalyst in dimethyl sulfoxide solution.The chemical structure and physical properties of the graft copolymer were characterized by FT-IR,~1H-NMR,XRD and TGA.Then,the amphiphilic chitosan polylactic acid graft copolymer was used as the matrix material,and the liquid perfluoropentane(PFP)encapsulated nanobubbles were prepared by double emulsion solvent evaporation method.The size and stability of nanobubbles were studied by laser particle size analyzer.The biocompatibility of nanobubbles was studied by cytotoxicity test,cell migration test,blood biochemical analysis and hemolysis test.Clinical ultrasound imaging system was used for in vitro and in vivo ultrasound imaging.Results:FT-IR and ~1H-NMR results confirmed the successful grafting reaction of polylactic acid(PLLA)with chitosan,and the grafting rate was 365%.The average diameter of nanobubbles is 101.1±7 nm,Zata potential is-31.8±5 m V,with good monodispersity and uniform particle size.Transmission electron microscopy(TEM)results show that the nanobubbles are highly dispersed and spherical,with unique saccular structure.The nanobubbles have good stability at 4?.The results showed that the nanobubble solution with different concentrations had no effect on cell growth,and showed good biocompatibility in cytotoxicity,cell migration,blood biochemistry and hemolysis.Under the irradiation of ultrasound,nanobubbles can form ultrasonic high signal,which can significantly enhance the ultrasound imaging of tumor tissue in vivo.Conclusion:The results showed that the amphiphilic chitosan polylactic acid nanobubbles had good stability and biocompatibility,and showed the ability to enhance the ultrasonic signal in vivo and in vitro.All in all,nanobubbles as nano contrast agents have great potential in ultrasound imaging.