Experimental Study On Multifunctional Core-crosslinked Nanosystem Based On Manganese Dioxide Combined With Ultrasound For Breast Cancer Therapy

Due to the limitations of traditional cancer therapy methods such as surgery,radiotherapy and chemotherapy,researchers are urgently looking for new and efficient cancer diagnosis and therapy methods to treat tumors that threaten human health and life.Ultrasound（US）,as a truly non-invasive technology,has been applied in the clinical tumor treatment.Among them,low intensity focused ultrasound can increase the permeability of tumor blood vessels,improve the concentration of anti-tumor drugs in the tumor site and activate some drugs to produce cytotoxicity,thereby enhancing the therapeutic effect.High intensity focused ultrasound can lead to irreversible coagulative necrosis through the mechanical effect,thermal effect and cavitation effect of ultrasound,thus killing tumor cells.It is highly safe,easy to operate and has low side effects.In addition,with the rapid development of nanotechnology,nanomaterials with composite functions have gradually shown great application potential in tumor diagnosis and therapy due to their excellent properties.On the one hand,nanomaterials can overcome the limitations of complex physiological barriers in vivo on the targeted drugs delivery efficiency.On the other hand,nanomaterials can realize tumor imaging,which is of great significance in the early tumor diagnosis and therapy.The combination of ultrasound and nanotechnology can achieve effective synergy,thereby enhancing the therapeutic effect of tumors.In order to achieve the tumor-specific drugs delivery and effective anti-tumor effects,and to address the limitations of tumor therapy,we designed a new kind of nanomaterial with stable structure and tumor microenvironment responsive capability for drug delivery（Ce6-MnO₂/CCNP-HE-CPP）,and then the application of this nanosystem combined with ultrasound in tumor therapy was studied.The main research contents and results are as follows:ObjectiveTo develop a tumor microenvironment-responsive nanosystem with stable structure and good biocompatibility and to explore its anti-tumor activity in vitro and in vivo combined with ultrasound,achieving a variety of combined therapy strategies for efficient tumor suppression,it is expected to provide new opportunities for the development of nanosystem and ultrasound.Methods1.Synthesis and characterization of Ce6-MnO₂/CCNP-HE-CPP.The ultrathin manganese dioxide nanosheet with layer structure was firstly prepared by redox method,and the sonosensitizer chlorin e6（Ce6）was loaded onto the manganese dioxide nanosheet（Ce6-MnO₂）by adsorption.Ce6-MnO₂ was encapsulated into polyethylene glycol（PEG）-diselenide（PEG-HBSe）for improving the circulation stability.The multifunctional core-crosslinked nanoparticle（Ce6-MnO₂/CCNP-HE-CPP）with microenvironment-responsiveness and active targeting ability was finally constructed by visible light induced diselenide bond crosslinking and surface modification with targeting peptide（HE-CPP）.The morphology,particle size,zeta potential,stability,drug release behavior and hemolysis rate were studied.2.Anti-tumor activity of Ce6-MnO₂/CCNP-HE-CPP in vitro.The nanoparticles cellular uptake and distribution were measured by confocal laser scanning and flow cytometry,and the ROS generation ability was verified.CCK-8 assay was used to verify the nanomaterials and nanoparticles toxicity on cells.Cell apoptosis was detected by Annexin-V FITC/PI experiment.3.In vivo study on pharmacokinetics and multimodal imaging of Ce6-MnO₂/CCNP-HE-CPP.Pharmacokinetics was evaluated by measuring the drug concentrations in the blood at different time points.In vivo fluorescence imaging was used to verify the nanoparticles targeting ability and distribution in mice.The effect of Ce6-MnO₂/CCNP-HE-CPP on photoacoustic and magnetic resonance imaging in vivo and in vitro was observed.4.Anti-tumor activity of Ce6-MnO₂/CCNP-HE-CPP in vivo.The ROS production level of nanoparticles in tumor sites was investigated in female nude mice,and the tumor inhibition effect and biological safety of nanoparticles were evaluated by measuring the changes in tumor volume,body weight,survival curve of tumor mice and tumor sections pathological examination.Blood samples were collected on day 1,7,14 and 28 after the injection of nanoparticles for routine blood and blood biochemical analysis.5.Ce6-MnO₂/CCNP-HE-CPP for enhanced HIFU therapy.The oxygen production ability and improved the tumor hypoxic state were verified by HIF-αand blood oxygen saturation in the tumor site.In vitro and in vivo experiments were conducted to evaluate the feasibility and biosafety of nanoparticles for HIFU synergies.Results1.Ce6-MnO₂/CCNP-HE-CPP were successfully prepared with high drug loading rate（8.90%）and suitable particle size（175.8±7.2 nm）.The nanoparticles showed good stability in the physiological environment.Meanwhile,the MnO₂ nanosheet had the pH/GSH responsive stimulation ability and could control drug release,with the cumulative release amount reaching 85.4%at 48 h.The nanoparticle does not cause hemolysis and has good biocompatibility.2.Cell experiments showed that Ce6-MnO₂/CCNP-HE-CPP could effectively enter into the cells and generate reactive oxygen species（ROS）under ultrasound.The cell inhibition rate of Ce6-MnO₂/CCNP-HE-CPP under ultrasound irradiation was as high as 93.48%compared to free Ce6,showing good dose dependence and excellent anti-tumor efficacy.Annexin-V FITC/PI and CAM/PI double staining results demonstrated that cell proliferation was inhibited by the nanoparticles inhibit through inducing apoptotic pathways.3.The core-crosslinked strategy provides Ce6-MnO₂/CCNP-HE-CPP nanoparticles with long-term blood circulation time,making the half-life2.02 times that of free Ce6,while preventing premature drug leakage.The in vivo live imaging experiments showed that the nanoparticles could efficiently target tumors and increase accumulation.The fluorescence signal intensity reached the peak at 8 h after injection and lasted for 48 h.The drug concentration at the tumor site is 7.5 times that of free Ce6 after24 h of injection.The nanoparticles also showed significantly enhanced photoacoustic and MRI signals,and had the potential for multimodal imaging.4.In vivo anti-tumor activity experiments,MnO₂ was proved to improve the sonosensitizer cytotoxicity effect in hypoxic environment.In MDA-MB-231 tumor-bearing mice models,the tumor inhibitory effect of the nanoparticles was significantly stronger than that of other treatment groups,the tumor proliferation rate was the slowest and the tumor volume change was the smallest.There was no significant change in body weight,blood routine and blood biochemical indexes.5.HIF-1αimmunofluorescence section and blood oxygen saturation test results showed that Ce6-MnO₂/CCNP-HE-CPP could degrade and generate oxygen in tumor situ to improve the hypoxic environment.In the bovine liver model,the HIFU irradiation power was 150 W and the irradiation time was 5 s to achieve the excellent ablation effect.In the tumor-bearing mice models,the gray value of Ce6-MnO₂/CCNP-HE-CPP group after HIFU ablation was significantly higher than that of the HIFU group,the coagulation necrosis was obvious,and the energy efficiency factor was lower than that of the HIFU group,the results were significant difference（P<0.05）.H&E staining showed that the coagulation necrosis area in the nanoparticle group after HIFU ablation was clearly separated from the normal tissue,and the cell morphology and structure in the ablation area were disappeared.ConclusionIn this paper,the tumor microenvironment-responsive Ce6-MnO₂/CCNP-HE-CPP nanoparticles with active targeting capability were successfully constructed for sonosensitizer delivery to achieve the synergistic effect of multiple therapy methods,and increase the bioavailability of drugs.The nanoparticles overcame the premature drug release due to the instable structure of drug nanocarriers by core-crosslinked method,realized the controlled drugs release in tumor microenvironment and increased the drug concentration.It provides new ideas for addressing the limitations of traditional anti-tumor methods,overcoming the lack of stability of current nanocarriers,and solving the current problems of HIFU synergists.