| Focused ultrasound ablation surgery(FUAS)uses low-intensity ultrasound waves in vitro to focus on the lesion in vivo,and achieves the treatment purpose by ablating the lesion through thermal effect and cavitation effect with continuous output of acoustic energy.In the treatment of deep or large tumors,the shortcomings of FUAS are exposed: for example,decreased ablation efficiency,prolonged treatment time,and increased risk of thermal damage complications.Therefore,to address the above limitations,some researchers focus on synergistic substances that synergize FUAS therapy.The current research hotspot of synergistic substances is none other than lipid nanoparticles(NPs).Although lipid NPs improve the ablation efficiency of FUAS,their active targeting and safety were not well solved.The research group used anaerobic Bifidobacterium as the carrier of NPs in the early stage to improve the ability of synergistic substances to target tumors,which was confirmed to significantly enhance the efficiency of FUAS ablation,and opened a new chapter in the bio-targeted synergistic FUAS ablation of tumors.Pulse-focused ultrasound(PFUS)outputs energy intermittently,adjusting the duty cycle can make non-thermal effects such as cavitation effect dominate the mechanism,resulting in rapid disintegration and liquefaction of the focal tissues,without being affected by heat deposition,reducing the occurrence of thermal complications.In view of this,our study used Bifidobacterium bifidum(B.bifidum),a highly active bifidogenic bacterium,as a "target-seeking carrier" to couple PFH/DOX cationic lipid nanoparticles with imaging/potentiation effects(hereafter referred to as CL-PFH-DOX-NPs),to constitute a novel bio-targeted potentiator(herein after referred to as B.bifidum@CL-PFH-DOX-NPs),evaluate the efficacy,and safety of this potentiator to potentiate PFUS for the treatment of breast cancer,and preliminarily explore the mechanism of the treatment,providing clinical references to focused ultrasound for the treatment of tumors.ObjectiveTo research and explore the effect of B.bifidum@CL-PFH-DOX-NPs targeted synergistic PFUS in the treatment of breast cancer and to preliminarily investigate its treatment mechanism.Method1.Preparation and characterization of CL-PFH-DOX-NPs:CL-PFH-DOX-NPs were prepared by film hydration-ultrasonic double emulsification method,the structure and morphology were observed by TEM and FM.The DOX encapsulation rate,drug loading rate and in vitro release properties were measured by UV spectrophotometer.Cytotoxicity was evaluated by CCK-8.Ultrasound was used to evaluate the imaging ability of PFUS(10 % 3 s)before and after irradiation in vitro.Hemolysis assay to detect biocompatibility in vitro.2.Evaluation the coupling between B.bifidum and CL-PFH-DOX-NPs:LSCM and FCM were used to characterize and quantify the coupling between B.bifidum and CL-PFH-DOX-NPs,respectively.The potentials of B.bifidum,CL-PFH-NPs,CL-PFH-DOX-NPs and B.bifidum@CL-PFH-DOX-NPs and the particle diameters of B.bifidum,CL-PFH-DOX-NPs and B.bifidum@CL-PFH-DOX-NPs were detected by particle size analyzer.3.Safety test of B.bifidum@CL-PFH-DOX-NPs:12 BALB/C mice were randomly divided into 4 groups:(1)control group(without any treatment);(2)3 days after the injection of B.bifidum@CL-PFH-DOX-NPs;(3)7 days after the injection of B.bifidum@CL-PFH-DOX-NPs;(4)14 days after the injection of B.bifidum@CL-PFH-DOX-NPs.After the injection,follow-up blood tests were performed to evaluate the biocompatibility in vivo.Simultaneously,important organs of group(1)and(4)were taked,and were stained with H&E.4.Validation of B.bifidum@CL-PFH-DOX-NPs targeting:distribution of B.bifidum in vivo: tissue homogenization method was used to observe the growth of colonies in various organs and tumors.Effect of B.bifidum on ROS and ultrastructure of tumor: 24 tumor-bearing mice were randomly divided into 2 groups:(1)injection of PBS;(2)injection of B.bifidum;ROS were analyzed by LSCM on days 1,3,5 and 7 after injection of the corresponding solutions;ultrastructure was observed on day 7.CL-PFH-DOX-NPs targeting B.bifidum: 6 tumor-bearing mice were randomly divided into 2 groups:(1)injected with CL-PFH-DOX-NPs;(2)injected with B.bifidum and CL-PFH-DOX-NPs;the intensity and the trend of fluorescence in tumors were analyzed by FL for the 12 th,18th,32 th,and48th hours after injection.24 tumor-bearing mice were randomly divided into 2 groups as above,and the distribution of CL-PFH-DOX-NPs in the tumor was observed by LSCM at the corresponding time points.5.Evaluation of PFUS potentiation by CL-PFH-DOX-NPs in vitro:CL-PFH-DOX-NPs potentiated PFUS to kill breast cancer cells:6-well plate cells were randomly aliquoted into 4 groups:(1)contain free DOX;(2)contain CL-PFH-DOX-NPs;(3)contain free DOX in combination with low-power PFUS(5 w 4 s:150 s 300 s);and(4)contain CL-PFH-DOX-NPs in combination with low-power PFUS(5 w 4 s:150 s300 s),SEM and LSCM were used to observe the cell shape and intracellular DOX,and the cell survival rate was counted by Taipan blue staining.CL-PFH-DOX-NPs potentiated PFUS to destroy the bovine liver:the experimental setups were as follows:(1)PBS group and(2)CL-PFH-DOX-NPs group,the PBS or CL-PFH-DOX-NPs solutions was injected into the degassed bovine livers,and the focused ultrasound was irradiated(2 % for 150 s,10 % for 30 s,60 % for 30 s and 60 % for 30 s).Thermocouples measure temperature,visually observe changes in the bovine liver,and calculate the necrotic volume.6.Evaluation of B.bifidum@CL-PFH-DOX-NPs potentiated PFUS in ablating breast tumor:20 tumor-bearing mice were randomly divided equally into 4 groups:(1)PFUS group,(2)B.bifidum+PFUS group,(3)CL-PFH-DOX-NPs+PFUS group and(4)B.bifidum@CL-PFH-DOX-NPs+PFUS group.Injection of PBS,B.bifidum solution,PBS,B.bifidum solution,through tail vein for 3 consecutive days,respectively,and again on the 7th day after the injection,PBS,PBS,CL-PFH-DOX-NPs solution,CL-PFH-DOX-NPs solution,were injected respectively.The tumors were irradiated by PFUS(10 % for 30s),and the cavitation signals were detected by the PCD system,and ultrasound observation of gray scale changes in the target area,calculation of necrosis volume and EEF.H&E and TUNEL were used to evaluate the tumor destruction in the target area,and PCNA was used to evaluate the tumor proliferation at the target area junction.H&E was used to observe whether there was any obvious pathological damage to various vital organs in the tumor-bearing mice in the B.bifidum@CL-PFH-DOX-NPs+PFUS group.7.Evaluation of B.bifidum@CL-PFH-DOX-NPs potentiated PFUS in inhibiting breast tumor:25 tumor-bearing mice were randomly divided equally into:(1)control group(without any processing),(2)PFUS group,(3)B.bifidum+PFUS group,(4)CL-PFH-DOX-NPs+PFUS group and(5)B.bifidum@CL-PFH-DOX-NPs+PFUS group,the body weight and tumor changes were recorded.45 mice were randomly divided into 5 groups and treated as above,the changes of TNF-α,IFN-γ,IL-4,and IL-10 indexes were analyzed on the 3th,7th,and 14 th days after PFUS(10 % for 30 s)in each group.Results1.The CL-PFH-DOX-NPs were prepared successfully.The DOX encapsulation rate and loading rate were 73.46 % and 9.02 %,respectively.DOX released more completely after PFUS.The CL-PFH-DOX-NPs after PFUS reduced the cell survival rate,and the extent of the decrease was positively correlated with that of the CL-PFH-DOX-NPs concentration.In the gray-scale mode and contrast-enhanced mode,the echo changes of CL-PFH-DOX-NPs were significant after PFUS;the differences of EI were significant when compared with other solutions(****P < 0.0001).The hemolysis rate due to different concentrations of CL-PFH-DOX-NPs was lower than 5 %.2.The coupling between B.bifidum and CL-PFH-DOX-NPs:compared with the CL-PFH-NPs not encapsulated with DOX,B.bifidum adsorbed more CL-PFH-DOX-NPs on the surface,and the coupling rate between the two was 95.90 %,while that between the former was only22.10 %,which indicated that the construction of B.bifidum@CL-PFH-DOX-NPs was successful in vitro.The average potentials of B.bifidum,CL-PFH-NPs,CL-PFH-DOX-NPs,and B.bifidum@CL-PFH-DOX-NPs were-20.59 ± 1.25 m V,20.60 ± 3.01 m V,36.43 ± 1.17 m V,and 3.21 ± 1.46 m V,respectively.The potential of CL-PFH-DOX-NPs was higher than that of CL-PFH-NPs(**P < 0.01).The particle size of CL-PFH-DOX-NPs,the diameters of B.bifidum and B.bifidum@CL-PFH-DOX-NPs were approximately 227.90 ± 3.40 nm,0.5-0.7 μm,and 0.6-0.9 μm,respectively.3.B.bifidum@CL-PFH-DOX-NPs safety in vivo: under the treatment dose,there was no statistically significant difference in the blood routine and blood biochemical indexes between the control BALB/C mice and the BALB/C mice of each experimental group injected with B.bifidum@CL-PFH-DOX-NPs(n.s P>0.05).There was no significant difference in the organs morphology between the mice of control group and that of mice injected with B.bifidum@CL-PFH-DOX-NPs on day 14.4.B.bifidum@CL-PFH-DOX-NPs targeting: B.bifidum colonies in tumor continued to increase,and B.bifidum colonies in each organ gradually decreased to almost disappeared on day 7,indicating that B.bifidum actively targeted the tumor.Increased ROS was observed in tumors after injecting B.bifidum.Tumor cells around B.bifidum showed damaging changes.The fluorescence peaks were higher(*P < 0.05)and lasted longer in the tumors of mice injected with B.bifidum and CL-PFH-DOX-NPs compared with those of mice injected with CL-PFH-DOX-NPs only.A large number of CL-PFH-DOX-NPs were seen in the tumors at 32 h after the injection of CL-PFH-DOX-NPs,which were more homogeneously dispersed.It indicated that B.bifidum@CL-PFH-DOX-NPs was successful in targeting tumors in vivo,and 32 h was the highest targeting efficiency time point.5.PFUS in vitro: cell experiments: 4T1 cells of CL-PFH-DOX-NPs+PFUS group had obvious "holes" on the cell surface,the most DOX in the cytoplasm,and the cell survival rate was significantly lower than that of the other groups(*P<0.05,**P<0.01,***P<0.001).Bovine liver experiment: bovine liver necrosis gradually changed from liquefied state to coagulation.The difference in necrotic volume between10 % and 60 % was insignificant,but both were higher than the volume of2 % and 100 %(**P < 0.01).The temperature of 10 % was lower than that of 60 %(**P < 0.01).The ablation volume of CL-PFH-DOX-NPs group in10 % was larger than that of the PBS group with the same parameters(*P <0.05).It suggested that the 10 % duty cycle may be the optimal parameter for safe and efficient ablation under this experimental condition.6.PFUS in vivo: cavitation signals were more significant in the B.bifidum@ CL-PFH-DOX-NPs+PFUS group and the highest rise in RMS amplitude was measured.The B.bifidum@CL-PFH-DOX-NPs+PFUS group had higher tumor gray change and necrosis volume than the other groups,with statistically significant differences(****P < 0.0001)and the smallest EEF(****P < 0.0001).Liquefaction necrosis and apoptosis were most obvious in the target area of the B.bifidum@CL-PFH-DOX-NPs+PFUS group,with a clearer junction area and low cell proliferation activity.There were no obvious pathological alterations in various organs of the tumor-bearing mice in the B.bifidum@CL-PFH-DOX-NPs group.It showed that B.bifidum@CL-PFH-DOX-NPs potentiated PFUS ablation was characterized by high efficiency,precision and safety.7.Growth and metastasis of breast tumor: the tumor of only B.bifidum@CL-PFH-DOX-NPs+PFUS group gradually shrank,which was significantly smaller than the rest of the experimental groups(*** P<0.001,****P<0.0001).At the end of the observation,there was no significant difference in the body weights of tumor-bearing mice in all groups,and only the CL-PFH-DOX-NPs+PFUS group and the B.bifidum@CL-PFH-DOX-NPs+PFUS group had no obvious metastatic foci in various organs.The TNF-α,IFN-γ and IL-4 concentration index of B.bifidum@CL-PFH-DOX-NPs+PFUS group were consistently higher than the other four groups(****P < 0.0001).It was confirmed that B.bifidum@CL-PFH-DOX-NPs potentiated PFUS inhibiting the growth and metastasis of breast tumor.Conclusion1.CL-PFH-DOX-NPs are prepared with obvious cytotoxicity and ultrasound imaging ability,excellent stability and biocompatibility in vitro.2.B.bifidum@CL-PFH-DOX-NPs successfully constructed have good safety in vivo,can specifically target tumors,causes damage to tumors in the process,and increase the retention and duration of CL-PFH-DOX-NPs within the tumor..3.CL-PFH-DOX-NPs well potentiates PFUS in vitro.When the cavitation effect dominates PFUS mechanism,a same ablation volume can be achieved at a lower temperature.4.B.bifidum@CL-PFH-DOX-NPs better potentiates PFUS ablation in vivo,and can achieve the purpose of potentiating PFUS for breast tumor treatment.The synergy between the antitumor proliferation effect of B.bifidum,the precise chemotherapy of DOX and the enhanced non-thermal and thermal effects of PFH phase change may be the key mechanism of treatment. |
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