Experimental Study On The Synergistic Effect Of Bifidobacterium And Polyethylenimine Modified Sodium Bicarbonate Loaded PLGA Nanoparticles On HIFU

BackgroundFocused Ultrasound Ablation Surgery（FUAS）is a non-invasive surgical approach,which uses High Intensity Focused Ultrasound（HIFU）to ablate solid tumors.Due to the propagation characteristics of ultrasound,the energy of HIFU will decrease with the increase of HIFU ablation depth.The gas-containing nanoparticles can increase the energy deposition in the HIFU target region by increasing the cavitation nuclei there,thus improves the efficiency of HIFU ablation.However,the tumor-targeting ability of gas-containing nanoparticles is not strong,which may cause damage to the non-HIFU ablation target region and reduce the safety of HIFU ablation.How to improve the efficiency and safety of HIFU ablation at the same time has become an urgent problem to be solved.In this study,based on the hypoxic and acidic microenvironment of solid tumors,a novel HIFU bio-targeted synergist was prepared,which not only had the targeting ability to the hypoxic region of solid tumors,but also could utilize the acidic microenvironment of solid tumors to produce gas to enhance HIFU ablation,so as to improve the efficiency and safety of HIFU ablation.ObjectivePoly（lactic-co-glycolic）acid（PLGA）Nanoparticles（NPs）modified with Polyethyleneimine（PEI）containing Sodium bicarbonate（Na HCO₃）（PEI-PLGA-Na HCO₃-NPs）with positive surface potential were prepared,which could enter the acidic tumor microenvironment of solid tumors and release Carbon dioxide（CO₂）to improve the efficiency and safety of HIFU ablation by the electrostatic adsorption with Bifidobacterium bifidum（B.bifidum）with negative surface potential and the targeting ability for the tumor hypoxic region.Methods1.Preparation and characterization of PEI-PLGA-Na HCO₃-NPs:PLGA as shell material,Na HCO₃ as core material,PEI as shell modification material,PEI-PLGA-Na HCO₃-NPs were prepared by the ultrasonic double emulsification method.The Transmission Electron Microscope（TEM）was used to observe the structure form of PEI-PLGA-Na HCO₃-NPs.The size and the surface potential of the nanoparticles were measured by laser particle size analyzer.The in vitro size and the surface potential stability of the nanoparticles were also measured by laser particle size analyzer.The encapsulation rate,drug loading rate and drug release characteristics in vitro of the nanoparticles were detected by the freeze-drying and acid-base titration methods.The biocompatibility of the nanoparticles was tested by the hemolysis test.2.Electrostatic adsorption and tumor targeting ability tests of B.bifidum combined with PEI-PLGA-Na HCO₃-NPs:the surface potential of B.bifidum was detected by laser particle size analyzer.Laser Scanning Confocal Microscope（LSCM）and Flow Cytometry（FCM）were used to detect the in vitro electrostatic adsorption rate between B.bifidum and PEI-PLGA-Na HCO₃-NPs.The subcutaneous breast cancer tumor transplantation model of BALB/c female mice were established.The distribution of B.bifidum in tumor-bearing mice was detected by anaerobic culture of tissue homogenate method.LSCM and in vivo fluorescence imaging of small animals were used to observe the electrostatic adsorption between B.bifidum and PEI-PLGA-Na HCO₃-NPs in tumor-bearing mice and the tumor targeting ability of PEI-PLGA-Na HCO₃-NPs combined with B.bifidum in tumor-bearing mice.3.Synergistic experiment of PEI-PLGA-Na HCO₃-NPs for HIFU ablation in vitro:in order to verify the synergistic effect of PEI-PLGA-Na HCO₃-NPs on HIFU ablation in vitro,the fresh bovine liver was used as the ablation medium,nine HIFU ablation sites were preset,and the ablation points were separated more than 3 cm,and the nine ablation sites were divided into three groups:injected with dilute Hydrochloric Acid（HCl）group,injected with dilute HCl+PEI-PLGA-NPs group and injected with dilute HCl+PEI-PLGA-Na HCO₃-NPs group,there were three ablation sites in each group,and the concentration of dilute HCl was 0.000001 mol/L.After the above nine preset ablation sites were ablated,the ablation volume and the ultrasound Energy Efficiency Factor（EEF）of each group were calculated.4.B.bifidum combined with PEI-PLGA-Na HCO₃-NPs for the synergism of HIFU ablation in vivo:in order to verify the synergism of B.bifidum combined with PEI-PLGA-Na HCO₃-NPs for HIFU ablation in vivo,twelve tumor-bearing mice were divided into four groups as the ablation medium:PBS group,B.bifidum group,PEI-PLGA-Na HCO₃-NPs group and B.bifidum+PEI-PLGA-Na HCO₃-NPs group,there were three tumor-bearing mice in each group.The volume of coagulation necrosis and the ultrasound EEF in the target region were calculated and the tumor tissue sections were observed with Optical Microscope（OM）.To verify the safety of this bio-targeted synergist for HIFU ablation in vivo,six tumor-bearing mice were divided into two groups:the control group and the B.bifidum+PEI-PLGA-Na HCO₃-NPs group,with three mice in each group.After HIFU ablation of tumors in mice of the latter group,the heart,liver,spleen,lung and kidney tissues of the above six mice were removed for paraffin sections stained with Hematoxylin-Eosin（HE）,and then observed and compared with OM.In order to verify the biosafety of PEI-PLGA-Na HCO₃-NPs,twelve normal BALB/c mice were divided into four groups:the control group,the3 days after the injection of B.bifidum and PEI-PLGA-Na HCO₃-NPs via tail vein group,the 7 days after the injection of B.bifidum and PEI-PLGA-Na HCO₃-NPs via tail vein group and the 14 days after the injection of B.bifidum and PEI-PLGA-Na HCO₃-NPs via tail vein group,with three mice of each group.The normal BALB/c mice of the control group were not injected with B.bifidum or PEI-PLGA-Na HCO₃-NPs,and the bloods of the above twelve mice were collected for the blood routine and blood biochemical tests.Results1.PEI-PLGA-Na HCO₃-NPs showed a uniform core-shell structure under TEM.The average particle size of PEI-PLGA-Na HCO₃-NPs was258.0±3.3 nm and the average surface potential was 24.6±0.5 m V by the laser particle size analyzer.The average surface potential of PLGA-Na HCO₃-NPs was-12.9±0.3 m V.The encapsulation rate of PEI-PLGA-Na HCO₃-NPs was 48.8%while the drug loading rate was 1.4%,the in vitro release of Na HCO₃ was more complete under the acidic environment.The hemolysis rate induced by PEI-PLGA-Na HCO₃-NPs of the therapeutic dose concentration was less than 5%,indicating PEI-PLGA-Na HCO₃-NPs had a good biocompatibility.2.The average potential of B.bifidum measured by the laser particle size analyzer was-20.0±0.3 m V.After B.bifidum mixed with PLGA-Na HCO₃-NPs or mixed with PEI-PLGA-Na HCO₃-NPs in vitro,a large amount of PEI-PLGA-Na HCO₃-NPs was adsorbed around B.bifidum under LSCM,and the adsorption rate between PEI-PLGA-Na HCO₃-NPs and B.bifidum was 96.32%measured by FCM.There was basically no adsorption of PLGA-Na HCO₃-NPs around B.bifidum,and the adsorption rate between them measured by FCM was only 5.11%.The tissue homogenate of the heart,liver,spleen,lung,kidney and tumor of tumor-bearing mice injected with B.bifidum via tail vein were anaerobic cultured.The results showed that B.bifidum mainly proliferated in tumor tissues after been injected into tumor-bearing mice,and the amount of B.bifidum proliferated in tumor tissues reached the peak on the 7^th day after the injection of B.bifidum.The tumor tissue sections of tumor-bearing mice injected with B.bifidum and PEI-PLGA-Na HCO₃-NPs or injected with B.bifidum and PLGA-Na HCO₃-NPs were observed under LSCM,a large number of PEI-PLGA-Na HCO₃-NPs were found in tumor tissues while PLGA-Na HCO₃-NPs only existed in a small amount in tumor tissues.The adsorption rate in vivo between B.bifidum and PEI-PLGA-Na HCO₃-NPs was much higher than that between B.bifidum and PLGA-Na HCO₃-NPs.In vivo fluorescence imaging of small animals showed that PEI-PLGA-Na HCO₃-NPs could reach the tumor tissues more quickly,stay there for a longer time and had a higher peak concentration in the tumor tissues after been injected into tumor-bearing mice with the injection of B.bifidum previously compared with tumor-bearing mice without the injection of B.bifidum previously,and the peak concentration of PEI-PLGA-Na HCO₃-NPs in the tumor tissues appeared at the 24^th h after the injection（***P<0.001）.3.The in vitro HIFU ablation experiment of the bovine liver showed that the HIFU ablation volume of the group injected with dilute HCl and PEI-PLGA-Na HCO₃-NPs was significantly higher than that of the other groups（****P<0.0001）,while the ultrasound EEF of the group injected with dilute HCl and PEI-PLGA-Na HCO₃-NPs was significantly lower than that of the other groups（***P<0.001）.4.The in vivo HIFU ablation experiment of tumors showed that the volume of coagulated necrosis induced by HIFU ablation of the tumor-bearing mice injected with B.bifidum and PEI-PLGA-Na HCO₃-NPs via tail vein was significantly higher than that of the other groups（****P<0.0001）while the ultrasound EEF in the target region of the tumor-bearing mice injected with B.bifidum and PEI-PLGA-Na HCO₃-NPs via tail vein was significantly lower than that of the other groups（**P<0.01）.The nuclear karyopyknosis,fragmentation and dissolution induced by HIFU ablation in tumor tissue sections of the tumor-bearing mice injected with B.bifidum and PEI-PLGA-Na HCO₃-NPs via tail vein were more than those in the other groups.The HIFU ablation efficiency of solid tumors in the tumor-bearing mice injected with B.bifidum and PEI-PLGA-Na HCO₃-NPs was higher than that in the other groups.There was no obvious difference in the tissue sections of the heart,liver,spleen,lung and kidney between the tumor-bearing mice underwent HIFU ablation which were injected with B.bifidum and PEI-PLGA-Na HCO₃-NPs previously and those of the tumor-bearing mice without HIFU ablation observed under OM.The bio-targeted synergist was safe for the non-HIFU ablation target region when the HIFU ablation target region underwent HIFU ablation.Blood samples of the normal BALB/c mice were collected before and 3 days,7 days and 14 days after the injection of B.bifidum and PEI-PLGA-Na HCO₃-NPs for the blood routine and the blood biochemical tests,and there was no significant difference in the blood routine and the blood biochemical test results at each time point（P>0.05）.This novel HIFU ablation bio-targeted synergist showed a good biosafety.ConclusionsIn this study,PEI-PLGA-Na HCO₃-NPs was successfully constructed as a new type of HIFU ablation synergist with good physicochemical properties and biocompatibility.There was a strong electrostatic adsorption rate between B.bifidum and PEI-PLGA-Na HCO₃-NPs in vitro and in vivo.B.bifidum injected into tumor-bearing mice had a strong targeting ability to the solid tumors.With the electrostatic adsorption of B.bifidum proliferated in the solid tumors,PEI-PLGA-Na HCO₃-NPs injected into tumor-bearing mice also showed a strong targeting ability to the tumor tissues.More importantly,this study successfully verified that the bio-targeted synergist of HIFU ablation formed by B.bifidum and PEI-PLGA-Na HCO₃-NPs could synergize the HIFU ablation with a good biosafety.This novel HIFU bio-targeted synergist improved the efficiency and safety of HIFU ablation.