Experimental Study Of Tumor Therapy Mediated By Multimodal Imaging Based On A Biological Targeting Synergistic Agent

BackgroundFocused Ultrasound Ablation Surgery(FUAS)or High intensity focused ultrasound(HIFU),as a new micro noninvasive technology,has been widely used in tumor therapy.However,with the increase of the depth of treatment,the ultrasonic energy attenuates,so the treatment of HIFU is inseparable from synergists.Varies synergists achieve the purpose of synergism of HIFU by changing the acoustic environment of the target area.However,the existing synergist has the limitations of single development mode and weak targeting,which limits its wider application in clinical.Single imaging mode is hard to provide comprehensive tumor diagnosis information,and it is difficult to provide better image guidance for HIFU.The exiting synergist have the defect of poor targeting,so it is difficult to achieve good imaging and treatment effect.If the power of HIFU treatment is increased,there may be a potential safety hazard,causing damage to normal tissues.Therefore,how to solve the problem of single development mode and weak targeting of synergists is the focus of current research.ObjectiveTo prepare a new biological targeting synergistic agent,which can target multimodal imaging,mediate HIFU treatment and enhance the therapeutic effect of HIFU.Methods1.ICG and PFH coloaded cationic lipid nanoparticles(CL-ICG-PFH-NPs)were prepared and the basic physical and chemical properties were determined.Bifidobacterium longum(B.longum)was cultured,the basic morphology of B.longum with gram staining was observed by light microscope.Use CLSM and flow cytometry to observe the connection between B.longum and CL-ICG-PFH-NPs.2.In vitro and in vivo multimodal imaging experiment: in vitro experiment,CL-ICG-PFH-NPs was diluted according to ICG concentration,and fluorescence,photoacoustic and ultrasonic images were collected,and imaging ability was evaluated.In vivo,B.longum related group was injected with quantitative B.longum every day for three consecutive days seven days before the imaging experiment.Fluorescence imaging,The nude mice were divided into three groups: B.longum group,CL-ICG-PFH-NPs group,B.longum + CL-ICG-PFH-NPs group.Living fluorescence images were collected After 6 h,24 h,48 h and 72 h of tail vein injection of CL-ICG-PFH-NPs.Photoacoustic imaging and ultrasound imaging,the nude mice were divided into four groups: PBS group,B.longum group,CL-ICG-PFH-NPs group,B.longum + CL-ICG-PFH-NPs group.The photoacoustic images were collected after intravenous injection of CL-ICG-PFH-NPs for 0 h,6 h,24 h,48 h and 72 h.In the ultrasonic imaging experiment,B mode and CEUS mode images before and after laser irradiation were collected and signals of tumor areas in each group were analyzed quantitatively in each imaging system.Use tissue sections and CLSM observe and verify the the targeting of biological targeting synergistic agent of tumors.3.Experimental study of biological targeted synergist to enhance HIFU treatment: PBS group,B.longum group,CL-ICG-PFH-NPs group,B.longum + CL-ICG-PFH-NPs group.Point irradiation(150 W,3 min)was used to observe the gray value change of tumor target area.TTC staining was carried out 24 hours after irradiation.The volume of coagulative necrosis area and EEF were observed and calculated.According to the above analysis,the synergistic effect of each group on HIFU was verified.He,PCNA and TUNEL of tumor tissue were analyzed to evaluate the ablation effect.CCK-8 was used to detect the safty of CL-ICG-PFH-NPs.Blood samples were collected after the injection of biological targeting synergistic agent.The tissues of normal organs in the biological targeting synergistic agent group were collected and stained with HE to evaluate the safety of HIFU.Results1.The appearance of CL-ICG-PFH-NPs were distributed with good dispersion under the light microscope.It was observed that the CL-ICG-PFH-NPs were of shell nuclear structure under the TEM.The average particle size of CL-ICG-PFH-NPs was 318 ± 17.3 nm and the average potential was 28.4 ± 4.71 mV.The encapsulation efficiency of ICG was 74%,the drug loading was 9.09 wt%.Temperature had a good linear relationship with ICG concentration.Under the light microscope,B.longum was blue purple long rod,the average surface potential was-29.7 ± 3.31 mV.A large number of CL-ICG-PFH-NPs were enriched around B.longum in the experimental group,and the connection rate of the two can be detected as high as 98.67%,while only 3.73% in the control group.Only a large number of B.longum can be seen in the tumor target area.The red signal intensity of the tumor target area in the biological targeting synergistic agent group is stronger than other groups.2.In vitro FL,PA,US experiments,CL-ICG-PFH-NPs has a good multimodal imaging effect,and the signal strength has a good linear relationship with ICG concentration.In vivo experiments,the biological targeted synergist group had good targeting to tumor tissue,FL signal and PA signal reached the peak 48 hours after the injection of nanoparticles,and the signal intensity of the biological targeted synergist group was far stronger than other groups.In vivo US imaging,the tumor target area was irradiated by laser.Only the biological targeted synergist group achieved enhanced US signal after irradiation(P<0.0001).3.In the HIFU synergistic experiment,the synergistic effect of the biological target group is better than other groups(P<0.001)by calculating the gray changes of the tumor,the EEF,the coagulative necrosis volume of the tumor after irradiation.HE,PCNA,TUNEL results showed that the synergistic effect of biological targeted synergist group was better than the other groups.The results of CCK-8 showed that CL-ICG-PFH-NPs is safe.After HIFU treatment,He staining of the important organs in the biological targeting synergistic agent group showed no obvious damage.Conclusions1.A biological targeting synergistic agent was successfully constructed,which has good and stable physical and chemical properties and good targeting for tumor tissue.2.The biological targeting synergist has good in vivo and in vitro multimodal imaging performance and the ability of synergizing HIFU.It can not only be used in the diagnosis of tumor as multimodal imaging agent,but also can enhance the therapeutic effect of HIFU on tumor at the same time of HIFU therapy.It is expected to achieve accurate diagnosis and treatment of tumor,improving the effectiveness and safety of HIFU.