Experimental Study On Dual-mode Imaging And Inhibitory Effect Of Doxorubicin Loaded Targeted Microbubbles Mediated By Ultrasound On SU-DHL-4 Cells

OBJECTIVE:Prepare Rituximab conjugated,DOX and OA-SIOP loaded Microbubbles(RMBs).To investigate the ability of the RMBs being used in US/MRdual-mode imaging and to investigate the inhibitory effect of the RMBs mediated by ultrasound on SU-DHL-4 cells.METHODS:1?DOX and OA-SIOP loaded Microbubbles(MBs)were prepared using mechanic vibration method;RMBs were prepared using the "avidin-biotin"connection between biotinylated MBs and the biotinylated Rituximab;Observe DOX and Rituximab of the RMBs under Confocal Microscopy to make sure the RMBswere prepared successfully;Observe the shape of the RMBs under the SEM and the TEM;Size distribution.Zeta potential and drug-loading efficiency were further detected;Observe the stability of the RMBs;Drawing the DOX release curve of the RMBs irradiated by Ultrasound.2.Evaluate the imaging enhancement ability of the RMBs used in US/MR imaging in vitro.3.Observe the RMBsdircctedly accumulate around SU-DHL-4 cells with Confocal Microscopy;Verify the safety of RMBs with CCK-8;The SU-DHL-4 cells were divided into several groups,the morphologic changes were detected by DAPI staining,the cytotoxic effect were detected by CCK-8 and the cell apoptosis was detected by flow cytometry.RESULUTS:1?Confocal Microscopy images showed the RMBs were prepared successfuLly;SEM and TEM showed that the RMBs were spherical and the structure of RMBs was clear.The size distr pot ibution of the RMBs were(679.91126.6)nm;The Zeta ential of the RMBs were(-31.5±11.9)mV.The drug-loading efficiency of the RMBs were(27.31±0.47)%.The RMBs had appropriate stability.The DOX release curve of the RMBs irradiated by US showed that US could control the release of DOX from the RMBs.2.From the US imaging in vitro we observed that the grey value increased gradually with the increase of RMBs' concentration.RMBs can be used as US contrast agent.From the MR imaging in vitro we observed that in the RMBs group containing SIOP,T2-weighted images increased with the increase of RMBs' concentration,the signal of the images reduced gradually and in the RMBs group without SIOP,T2-weighted images of RMBs of all concentrations had no obvious difference between the control group.RMBs containing SIOP could be used as negative contrast agents for MRI.3?The best cell inoculation concentration for SU-DHL-4 cell lines was 10×104/mL.The IC50 of DOX for SU-DHL-4 cells was 0.052ug/mL.RMBs specifically targeted to CD20 positive SU-DHL-4 cell.RMBs had good biological safety.Among the DAPI images,the RMBs+US group showed the lowest density of cells and the most obvious apoptosis morphology.The cell vitality in RMBs+US group was(32.2±2.13)%,which was significantly lower than in other groups(p<0.05).The apoptosis rate in RMBs+US group was(46.43+2.50)%,which was significantly higher than in other groups(p<0.05).CONCLUSIONS:We prepared RMBs successfully.RMBs had the enhancement ability in US/MR imaging in vitro,so RMBs could work as an ideal US/MR dual-mode contrast agent.RMBs can greatly inhibit the proliferation and induce the apoptosis of DLBCL with the irradiation of US.RMBs may be a new therapy for DLBCL.