PH-responsive Multifunctional Nanoplatform To Cascade Enhance Sonodynamic Therapy Of Breast Cancer By Remodeling The Tumor Microenvironment

Background: Breast cancer is the most common malignancy in women and,according to statistics,1 in 6 women die from breast cancer.In recent years,the age of breast cancer patients has been decreasing,the prevalence rate and mortality rate of breast cancer have been rising,which poses a serious threat to women’s life and health,and has become a major social health problem.Therefore,early diagnosis and treatment of breast cancer has become particularly important.The new nanoplatform for integrated diagnosis and treatment brings new treatment options to breast cancer patients.This nanoplatform can provide structural and functional information of the tumor,and can also realize the treatment of the tumor.Among them,sonodynamic therapy（SDT）,with its advantages of high tissue penetration and non-invasiveness,represents a new method for the treatment of solid tumors and has great potential in the field of tumor therapy.However,the efficacy of SDT is often affected by the tumor microenvironment（TME）.Therefore,in this study,a cascade nanoplatform（Ce6/Pt@PDA-HA,PPCH）was constructed to remodel TME and improve the antitumor efficacy of SDT.In addition,the nanoplatform has dual-modality imaging capabilities that can be used for real-time monitoring and dynamic evaluation during early diagnosis and treatment of breast cancer.Purpose: To remodel the characteristics of TME and improve the efficacy of SDT,a cascade nanoplatform PPCH（Ce6/Pt@PDA-HA）was constructed,and the basic properties of PPCH were detected,the dual-modality imaging function of PPCH in vivo and in vitro was evaluated,and the antitumor SDT efficacy of PPCH in vivo and in vitro was studied.Methods: Polydopamine（PDA）was formed by oxidative self-polymerization of dopamine hydrochloride as nanocore.The cascade nanoplatform PPCH（Ce6/Pt@PDA-HA）was successfully prepared by in-situ reduction of H₂PtCl₆·6H₂O as Pt nanozymes using polyol-assisted reduction strategy,loading Pt nanozymes by catechol coordination,loading of sonosensitizer Ce6 by π-π stacking and electrostatic interaction,and loading of immunostimulant HA by surface modification through metal coordination and Michael addition reaction.X-ray diffraction and X-ray photoelectron spectroscopy were used to verify the successful loading of Pt nanozymes,and ultraviolet-visible spectrophotometer and Fourier transform infrared were used to verify the successful loading of Ce6 and HA.The morphology,particle size,dispersion and element distribution of PPCH were determined by transmission electron microscopy and energy spectrum.The particle size,potential and stability of PPCH were measured by NanoZS90 and zeta potentiometer.The degradation of PPCH was detected by scanning electron microscope.The drug loading and release of PPCH were detected by ultraviolet-visible spectrophotometer.The catalase-like properties of PPCH were monitored with a dissolved oxygen analyzer.The ability of PPCH to produce reactive oxygen species（ROS）after LIFU irradiation was measured by ultraviolet-visible spectrophotometer and fluorescence spectrometer.In vitro experiments,confocal laser scanning microscope（CLSM）and flow cytometry were used to detect PPCH uptake and subcellular localization.The ability of PPCH to reverse immunosuppression was analyzed by flow cytometry and ELISA.The effects of PPCH on tumor hypoxia,intracellular reactive oxygen species and drug release in vitro were evaluated by CLSM.The biosafety and cytotoxicity of PPCH in vitro were evaluated using CCK-8 and CAM/PI assays.The ability of PPCH in vivo and in vitro dual-modality imaging was tested by a small animal 3D visible light imaging system and photoacoustic imaging system.The biological distribution of PPCH in vivo,tumor targeting ability and the optimal LIFU time window of sonodynamic therapy were analyzed.In vivo experiments,the biosafety of PPCH,reversal of immunosuppressive effect and improvement of TME hypoxia were studied.By monitoring and recording the tumor volume of tumor-bearing mice treated in different groups,the tumor inhibition rate of different groups was calculated,and related pathological tests were performed to comprehensively evaluate the SDT antitumor therapeutic effect of PPCH in vivo.Results: In this study,the cascade nanoplatform PPCH（Ce6/Pt@PDA-HA）was successfully prepared.it had a clear spherical shape,uniform particle size,good dispersion and negative potential.The element mapping showed that Pt,C,N,and O were evenly distributed across the PPCH surface.PPCH had good stability and pH response,and could be degraded to different degrees under acidic conditions.The encapsulation rate of Ce6 in PPCH was（64.33 ± 0.52）% and the drug loading was（42.90 ± 0.36）%.In vivo and in vitro experiments,PPCH demonstrated good tumor targeting and dual-modality imaging capabilities.In the cascade reaction,HA transformed M2 macrophages into M1 macrophages in situ,reversed TME immunosuppression,and simultaneously produced H₂O₂ to achieve endogenous H₂O₂ self-replenishment.Then,Pt nanozymes used as catalase-like catalysts to decompose endogenous H₂O₂ and generate O₂,alleviating oxygen deficiency in TME.O₂ produced 1O₂ under the action of Ce6 and LIFU,and ROS levels were amplified.In vivo experiments,the study found that PPCH showed good biosafety.The PPCH + LIFU treatment group showed the best antitumor effect,which was significantly better than other treatment groups,and the difference was statistically significant.Immunofluorescence and pathological analysis of tumor tissues and major organs in different treatment groups were performed,and the conclusion was consistent with the treatment results.Conclusion: In this study,cascade nanoplatform PPCH was successfully prepared,which has good biosafety,p H response,tumor targeting and dual-modality imaging capability.Through the cascade reaction,PPCH remodeled the characteristics of TME,reversed immunosuppression,and alleviated the hypoxia of TME,which was an effective ROS amplifier,improved the efficacy of antitumor SDT,and provided new ideas for the accurate diagnosis and treatment of breast cancer.