Experimental Study On The Synergistic Effect Of Bifidobacterium Combined With PLGA Nanoparticles Loaded Liquid Fluorocarbon On HIFU Treatment

High intensity focused ultrasound is to accuumulate and focus the low energy ultrasound emitted in vitro on the target tissue in vivo.The irreversible coagulative necrosis of the target tissue is caused by mechanical effect,thermal effect and cavitation effect,so as to achieve the purpose of treatment.In practical application,the inherent limitations of HIFU are gradually revealed,for example,for the treatment of large or deep tumors,the ablation efficiency of HIFU is low,the treatment time is long,which may lead to many complications.In order to improve this situation,scholars will focus on the synergistic agents of synergistic HIFU treatment,such as lipiodol,ultrasonic microbubbles,liquid fluorocarbon nanoparticles,hydroxyapatite,mesoporous silica and so on.Although these synergists can improve the therapeutic efficiency of HIFU,the problem of active targeting and the safety of treatment have always existed.Herein,we prepared a new type of tumor targeting vector based on anaerobic Bifidobacterium longum(B.longum),which connects liquid fluorocarbon loaded polymer nanoparticles with HIFU synergistic effect to make it has tumor specific targeting and biosafety.Staying in the tumor target area for a long time,but not in the surrounding normal tissue,and can change the acoustic environment and microenvironment of tumor tissue,significantly enhance the effect of HIFU ablation,and provide a new idea for the further study of HIFU biological targeting synergist.Objective1.Liquid fluorocarbon(PFH)-loaded polylactic acid-glycolic acid copolymer(PLGA)nanoparticles(PFH/PLGA NPs)were prepared by optimizing the reaction conditions,PFH/PLGA NPs were conjugated to the surface of B.longum(PFH/PLGA-B.longum)by carbodiimide method,and their basic properties were detected.2.To observe the targeting ability of PFH/PLGA-B.longum on tumor.To study its effect on the acoustic environment of tumor tissue,and explore the feasibility of it as a HIFU biological targeting synergist.3.To evaluate the therapeutic effect of HIFU ablation of nude mice with HIFU biological targeting synergist PFH/PLGA-B.longum.Methods1.Using PLGA as the shell and liquid fluorocarbon perfluorohexane(PFH)as the core,PFH were prepared by ultrasonic double emulsification in PLGA as HIFU synergist.The surface morphology,particle size,Zeta potential were detected.The carboxyl groups on the surface of PLGA NPs were activated by carbodiimide method.the changes of chemical groups on the surface of NPs were detected by Fourier transform infrared spectroscopy(FTIR)and nuclear magnetic resonance hydrogen spectroscopy(~1H-NMR),and then co-incubated under anaerobic conditions.Ligation of PFH/PLGA NPs with anaerobic probiotics B.longum.Confocal laser microscopy(CLSM)and atomic force microscopy(AFM)were used to observe the connection;flow cytometry(FCM)was used to analyze the binding efficiencies;anaerobic culture in vitro was used to detect the biological activity of B.longum.2.15 tumor-bearing mice were randomly divided into 3 groups with 5mice in each group.PBS group,PFH/PLGA group were set up to compare with the experimental group PFH/PLGA-B.longum.The targeting ability and retention time of PFH/PLGA-B.longum in tumor were observed by small animal fluorescence imaging in vivo.The proliferation of B.longum in tumor was detected by gram staining and homogenate culture,and the effect of B.longum proliferation on tumor microenvironment was analyzed by Masson staining and CD34 staining.Quantitative analysis of acoustic characteristics and tissue hardness of tumor by nonlinear high energy ultrasound and biomechanical compression test.3.Pure HIFU group,PBS+HIFU group,B.longum+HIFU group,PLGA+HIFU group,PFH/PLGA+HIFU group and PFH/PLGA-B.longum+HIFU group were set up.Three groups(90 W 3 s,120 W 3 s,150W 3 s)were treated with HIFU with different gradients,and the dosage of HIFU was 90 W 3 s,120 W 3 s and 150 W 3 s,respectively.The changes of gray value of the target area were observed immediately after HIFU ablation in each group.The coagulative necrosis volume was observed by TTC staining 24 h later.The energy efficiency factors(EEF)of each group were calculated and immunohistochemical staining was used to evaluate the therapeutic effect.Blood samples were collected for biochemical analysis 1 d,7 d and 14 d after intravenous injection of the new HIFU biological targeting synergist.Results1.Laser scanning confocal microscopy(CLSM)and scanning electron microscopy(SEM)showed that the prepared PLGA and PFH/PLGA NPs had smooth surface,regular morphology,uniform particle size and good dispersion.PLGA and PFH/PLGA NPs measured by Malvern laser particle size meter were 160.7±5.4 nm and 213.4±3.6 nm,respectively.The surface Zeta potentials of them were-9.55±3.82 mV and-16.3±5.46 mV,respectively.PFH encapsulation efficiencies was 44.45±1.27%.After adding EDC/NHS to activate the carboxyl groups modified on the surface of NPs,the results of FTIR and ~1H-NMR showed that NHS and PLGA were synthesized successfully.CLSM and AFM showed that PFH/PLGA NPs were conjugated to the surface of B.longum(PFH/PLGA-B.longum)by carbodiimide method,and the stability of B.longum and NPs was good.The binding rate of PFH/PLGA NPs to B.longum was 84.34±4.35%by FCM detection,which was significantly different from control group(P<0.05).The growth characteristics of B.longum cultured in anaerobic culture were similar to the B.longum without any special treatment.2.The prepared PFH/PLGA-B.longum had a strong tumor-specific targeting property.The fluorescence signal of the tumor was still strong in the small animal fluorescence imaging at 168 h,but there was no fluorescence signal in other tissues and organs.The results of Gram staining and homogenate culture showed that B.longum only colonized and proliferated in tumor tissues and not distributed in other organs.Masson staining and CD34 staining showed that the density of collagen fibers and microvessels in PFH/PLGA-B.longum group were 11.31±2.33%and 7.38±2.26%respectively,which were significantly different from those in other groups without B.longum injection(P<0.05).The mean sound velocity,acoustic attenuation and elastic modulus measured by nonlinear high energy ultrasound system and biomechanical compression experiment in PFH/PLGA-B.longum group were 1788.14±72.92 m/s,2.86±0.59dB/cm and 14.89±3.54 KPa,respectively.The efficient and stable connection between B.longum and PFH/PLGA NPs can enrich the NPs in the tumor site for longer time,but none in other tissues and organs,and the fluorescence intensity was significantly different from that of the control group(P<0.05).Masson and CD34 staining sections showed that the proliferation of B.longum in the tumor increased the synthesis of collagen fibers and inhibited the formation of neovascularization.The sound velocity,sound attenuation and elastic modulus of PFH/PLGA-B.longum group were significantly higher than those of uninjected B.longum group,which revealed the effect of B.longum on tumor acoustic environment.3.The results of HIFU ablation experiment showed that the gray value of PFH/PLGA-B.longum group was the highest when three groups of different gradient HIFU doses were used,which was significantly different from the other HIFU treatment groups(P<0.05).TTC staining showed that the coagulative necrosis volume in PFH/PLGA-B.longum group was the largest,which was significantly different from other HIFU treatment groups(P<0.05).In analysis of energy efficiency factors,the energy efficiency factor of PFH/PLGA-B.longum group was the lowest,which was significantly different from that of other HIFU treatment groups(P<0.05).Compared with 120 W 3 s and 150 W 3 s,the energy efficiency factor of 90 W 3 s group was the lowest among the three groups(P<0.05),and there was significant difference between 90 W 3 s group and the other two groups(P<0.05).The pathological sections of H&E,PCNA and TUNEL staining showed that in PFH/PLGA-B.longum group,the nuclear fragmentation and dissolution were more obvious after HIFU ablation,the proliferating cell nuclear antigen(PI)was the lowest,and the cell apoptosis rate(AI)was the highest.The percentages were 11.61±3.81%and 77.50±5.74%,respectively,which were significantly different from the other treatments(P<0.05).Creatinine(CREA),blood urea nitrogen(BUN),alanine transaminase(ALT),aspartate transaminase(AST)and total bilirubin(TBIL),there were no significant changes before and after injection(P>0.05).Conclusion1.PFH/PLGA NPs were successfully conjugated to the surface of B.longum by covalent coupling,and PFH/PLGA-B.longum was synthesized.After anaerobic culture in vitro,maintained no significant difference in biological activity compared with B.longum without special treatment.2.PFH/PLGA-B.longum was enriched in the tumor region for a long time,but not in other tissues and organs.Histopathological sections showed that the synthesis of collagen fibers increased and neovascularization was inhibited.In PFH/PLGA-B.longum group,the sound velocity,sound attenuation and elastic modulus increased significantly,which indicated that PFH/PLGA-B.longum could change the acoustic environment and microenvironment of tumor tissues.3.The prepared HIFU biological targeting synergist PFH/PLGA-B.longum has an effect on the acoustic environment of the tumor tissues and synergistic enhancement of HIFU ablation,it has good biocompatibility and safety in vivo,which provides a new strategy and method for the further research of HIFU biological targeting synergist.