Multifunctional PLGA Nanoparticles Based On Astragalus Polysaccharide For FUAS-mediated Tumor Immunotherapy

Studies have shown that Focused Ultrasound Ablation Surgery(FUAS)releases cell debris and exposes tumor antigens to form an "in situ tumor vaccine",inducing the body to produce a systemic anti-tumor immune response.However,the immune level activated by FUAS alone is not sufficient for clinical application.Combining the FUAS-induced "tumor vaccine" with an active immune adjuvant is expected to overcome immune tolerance and improve the body’s immune response to the "tumor vaccine".Multifunctional ultrasonic molecular probes have unique advantages in FUAS personalized tumor treatment.This experiment intends to prepare an ultrasonic-responsive multifunctional nano-immune adjuvant,which activates the whole body while locally ablating tissue Immunotherapy,preliminary verification and discussion of the tumor immunotherapy strategy mediated by nano-immune adjuvant combined with FUAS.PurposeA multifunctional nano-immune adjuvant APS/Au NR/PLGA was prepared to explore the effect and mechanism of enhancing FUAS tumor immunotherapy,FUAS ablation efficiency and PAI imaging function.MethodsNanoparticles APS/Au NR/PLGA were prepared by double emulsification method.Observe its morphology,measure the particle size and potential,detect the encapsulation rate of APS and Au NR,optimize the preparation parameters,and test the cytotoxicity in vitro and the safety in vivo.Analyze the photoacoustic imaging capabilities.Enhance the rate of apoptosis in vivo and reduce the rate of viable cells to verify the non-thermal synergistic effect on FUAS.In vivo live rabbit liver experiment,calculate the volume of liver necrosis and the energy efficiency factor EEF,and evaluate its in vivo thermal ablation efficiency.Verify the immune function that promotes DC maturation in vitro.detect the expression rates of DC surface antigens CD80,CD86,MHC-II,and the phagocytosis rate of FITC-dextran(1 mg/ml).ELISA was used to detect the interleukin IL-12 and interferon IFN-γ in the cell supernatant.concentration.To verify the effect and mechanism of enhancing immunity in vivo,ELISA detected cytokines TNF-α,IFN-γ,IL-4,IL-10,immunoglobulin Ig G1,Ig G2 a,H&E staining of tumors,heart,liver,spleen,lung and kidney.Tumor tissue TUNEL and VEGF staining.Immunohistochemistry was used to detect the positive rates of CD80 and CD86 antigens on the surface of DCs adjacent to the tumor,and the positive rates of CD3 and CD8 antigens on the surface of tumor T cells.Observe and record the body weight,tumor size and survival time of mice.ResultsThe nano immune adjuvant APS/Au NR/PLGA was successfully prepared.The nano particles have regular morphology,spherical shape,uniform size and good dispersibility.They can be prepared repeatedly with an average particle size of 255.00±0.1717 nm.The property is stable,the encapsulation rate Au NR is 56.09±4.33%,APS is 54.89±2.07%.It can be prepared repeatedly and has good biological safety,providing a technical basis for subsequent experiments.APS/Au NR/PLGA in the 680-900 nm excitation spectrum,700 nm is the optimal absorbance,with a good photoacoustic signal.The photoacoustic signal in vitro has a certain linear relationship with the concentration,but the intratumoral dispersion is poor after local intratumoral injection,and only a good photoacoustic signal can be seen in the injection channel.In vitro synergistic experiments,APS/Au NR/PLGA nanoparticles can significantly enhance the FUAS cavitation effect and enhance the efficiency of killing tumor cells;in vivo synergistic experiments,the nanoparticles can significantly enhance the ablation efficiency and reduce the need for ablation of tissue per unit volumeEnergy,and as the concentration of nanoparticles increases,the synergistic effect increases.APS/Au NR/PLGA nanoparticles can significantly promote DC maturation in vitro,APS/Au NR/PLGA can significantly enhance anti-tumor immune factors in vivo,and the concentration of anti-tumor immune factors increased significantly 3 days after surgery,which was 2-3higher than that of FUAS group alone Times,the immunization time is prolonged,so that Th2 type immunity gradually reverses to Th1 type immunity.The positive rate of immunohistochemical DC homing and intratumoral T cell infiltration was significantly increased.Nanoparticles could significantly promote DC homing and intratumoral T cell infiltration,inhibit tumor growth and prolong survival time.ConclusionIn this experiment,a multifunctional nano-immune adjuvant APS/Au NR/PLGA that can significantly enhance the anti-tumor immune response after FUAS and prolong the immunization time is prepared.Strategy,nano-immune adjuvant combined with FUAS-mediated immunotherapy has broad application prospects in malignant tumors.