Ultrasound-triggered Phase Transition Nanodroplets As A Multimodal Imaging Contrast Agent For Liver Cancer

The liver cancer is an aggressive cancer that was diagnosed commonly through ultrasound (US) and magnetic resonance imaging (MRI). But they were difficult to detect small tumor. In order to improve the accuracy of the disease, the contrast agents and multimodal imaging often were used. Microbubbles could slightly cause the signals to change in magnetic field. However, as ultrasound contrast agents, microbubbles are still considered as blood-pool agents due to their large size and poor stability.In this study, the uniform-size ultrasound-triggered phase transition nanodroplets were prepared via the premix SPG membrane emulsification method. After encapsulating DiR and SPIO, the feasibility of nanodroplets as multimodal imaging contrast agents for MRI/US/fluorescence imaging were evaluated both in vitro and in vivo. Based on this study, the nanodroplets only encapsulating DiR were investigated. The main results are as follows:(1) The nanocapsules and nanodroplets were successfully prepared via SPG membrane emulsification method. The size of nanodroplets was348.7±2.3nm with PDI of0.072±0.010and zeta potential of-3.36±0.43mV, which was stable within one year. The TEM showed nanodroplets existed as core-shell spheres.(2) The phase transition property of nanodroplets was confirmed. Although the boiling point of PFP was lower than37℃, PFP was still maintained as liquid in the polymer shell at37℃. The phase transition from droplets to bubbles with evaporation didn’t happen until the nanodroplets were heated above50℃or activated by ultrasound.(3) The DiR-SPIO-NDs were successfully prepared via SPG membrane emulsification method. The size of DiR-SPIO-NDs was385.0±5.0nm with PDI of0.169±0.011, zeta potential of11.40±1.51mV, and concentration of2.8x109/mL. The TEM showed that DiR-SPIO-NDs existed as spherical core-shell structure, and DiR and SPIO dispersed in the shell or core. Under the ultrasound excitation, the size of DiR-SPIO-NDs were enlarged2.5times. Furthermore, with the increase of the nanodroplets concentration, the fluorescent signal intensity was enhanced. The r2relaxivity of127.9mM-1s-1suggested the MRI applicability of DiR-SPIO-NDs.(4) The biocompatibility and multimodal imaging capability of DiR-SPIO-NDs were studied. The MTT and hemolysis studies demonstrated that the nanodroplets were biocompatible and safe. After intravenous injection of DiR-SPIO-NDs, the T2-weighted images of MRI and fluorescence images both showed that the images contrast in liver of rats or mice model were enhanced. Moreover, the ultrasound imaging in mice tumor as well as MRI and fluorescence imaging in liver of rats or mice showed that the DiR-SPIO-NDs had long-lasting contrast ability in vivo. These in vitro and in vivo findings suggested that DiR-SPIO-NDs could potentially be a fluorescence/MRI/US tri-modal imaging contrast agent in the diagnosis of liver tissue disease.(5) The DiR-NDs were successfully prepared via SPG membrane emulsification method. The size of DiR-NDs was708.7±34.8nm with PDI of0.154±0.034, zeta potential of-0.75±0.24mV, and concentration of7.4x109/mL. The TEM showed that DiR-NDs existed as spherical structure. Under the ultrasound excitation, the size of DiR-NDs were enlarged1.6times. The R2*of DiR-NDs was measured for different volume fraction, and the r2relaxivity was about10.0(volume fraction)-1s-1for in vitro MRI studies.(6) The biocompatibility and multimodal imaging capability for liver cancer of DiR-NDs were studied. The MTT and hemolysis studies demonstrated that the nanodroplets were biocompatible and safe. After injection of DiR-NDs, though the fluorescence intensities in the liver and spleen were most strongest, a little fluorescence signal in tumor of animals bearing in situ VX2liver cancer model was observed, which suggested the DiR-NDs could partly accumulate in tumor due to passive targeting of nanoparticles. Furthermore, in vivo MRI showed that the tumor boundaries of rabbits bearing in situ VX2liver cancer were gradually clear at5min after the intravenous injection of DiR-NDs, which was consistent with the findings of US imaging. These in vitro and in vivo results suggested that DiR-NDs could potentially be a fluorescence/MRI/US tri-modal imaging contrast agent for detection of liver cancer.In summary, the uniform-sized stable nanodroplets were successfully prepared by SPG membrane emulsification method. Under ultrasound irradiation at37℃, the nanodroplets could undergo a phase transition, and transform to microbubbles for ultrasound/fluorescence/MRI multimodal imaging in the diagnosis and therapy of liver cancer.