Preparation Of Fe₃O₄ Loaded Chitosan Nanobubbles For US/MR Dual-Mode Imaging

BackgroundUS/MR dual-mode imaging can overcome the shortcomings of a single imaging technology,obtain richer,more complete and more accurate image information,improve the detection rate of deseases.Therefore,US/MR dual-mode imaging has become a major development trend in modern medical imaging,the design and preparation of hybrid contrast agents that can enhance both ultrasound and magnetic resonance imaging have attracted much attention among domestic and foreign scholors.MIicrobubbles loaded with SPIONs can be used as US/MR dual-mode contrast agents.However,limited by the size of the microbubbles,most of the dual-mode contrast agents prepared currently are in micro size,which can only achieve intravascular imaging,but can't pass through the neovascular endothelial space of tumors?380-780nm?and concentrate in the diseased tissue through EPR effect,thus failing to achieve extravascular tissue imaging.Therefore,the new nano-scaled US/MR dual-mode contrast agent has become the focus of current molecular imaging research.The High-molecular polymer chitosan?CS?not only has high stability and good biocompatibility,but also can be degraded in vivo naturally.It is a kind of excellent biomedical material;Fe₃O₄ nanoparticles which can be prepared by coprecipitation method have high magnetization,therefore the preparation of nano-scaled US/MR dual-mode contrast agents can be realized by loading Fe₃O₄ nanoparticles on the CS nanobubbles which use CS as the raw material.ObjectiveNano-scaled US/MR dual-mode contrast agent-Fe₃O₄ loaded chitosan nanobubbles?Fe₃O₄-CS NBs?were prepared.Besides,basic properties of the nanobubbles were detected,their US/MR dual-mode imaging capabilities were explored.Methods1.Citric acid-modified Fe₃O₄ nanoparticles were prepared by coprecipitation method,and the surface morphology,size distribution,crystal structure,chemical structure and magnetic property of the modified Fe₃O₄ nanoparticles were characterized by a variety of detection tools.2.Using CS as the raw material,Fe₃O₄-CS nanobubbles were prepared by chemical crosslinking method and vacuum freeze drying technology.The internal structure,surface morphology,size distribution,crystal structure,chemical structure,magnetic property and iron content of the nanobubbles were characterized by a variety of detection tools.3.In vitro US/MR dual-mode imaging capability exploration:Ultrasonic and magnetic resonance scanning were performed on Fe₃O₄-CS nanobubbles with different concentrations of iron to obtain their in vitro T2WI images and ultrasonic images4.In vivo US/MR dual-mode imaging effect exploration:the liver of BALB/C mice was scanned by ultrasound and magnetic resonance before and after the injection of Fe₃O₄-CS nanobubbless?120.53±0.15?g Fe/ml?to observe the US/MR imaging effect in vivo.Results1.After being modified with citric acid,Fe₃O₄ nanoparticles with a hydrodynamic average size of 47.47±1.29 nm were round in shape,uniform in size,and well dispersed.The structure was consistent with the anti-spinel structure of Fe₃O₄ crystals and had good superparamagnetism.2.TEM showed that Fe₃O₄ nanoparticles were wrapped in the CS nanobubbles.The Fe₃O₄-CS nanobubbles with a hydrodynamic average size of 112.13±1.70 nm were regular in shape and uniform in size,showed good dispersion stability and superparamagnetism.After adding Fe₃O₄ with different content,the concentration of iron in Fe₃O₄-CS nanobubbles measured by ICP-OES was:11.69±0.04,22.46±0.07,43.39±0.07,74.31±0.10,120.53±0.15?g/ml.3.In vitro MR imaging experiments showed that the nanobubbles had a negative imaging ability,and the negative imaging ability would be enhanced with the increase of iron concentration;in vitro US imaging experiments showed that Fe₃O₄-CS nanobubbles had a certain ultrasound contrast ability,and could produce a strong ultrasound echo signal.4.In vivo MR imaging,after the injection of the contrast agent,the T2signal intensity in mouse liver significantly decreased,showing a negative imaging effect;in vivo US imaging,after the injection of the contrast agent,the echo signal in mouse liver was enhanced.ConclusionThe nano-scaled US/MR dual-mode contrast agent Fe₃O₄-CS nanobubbles are successfully prepared.The nanobubbles have good US/MR imaging capability,and is expected to play a role in clinical US/MR dual mode imaging.