Construction Of Photodynamic/Hypoxia-regulated Metal Nanomedicines And Its Application In Breast Cancer Synergistic Therapy

Background:The characteristics of hypoxia in breast tumor microenvironment play a crucial role in its occurrence,development and metastasis,and greatly limit the effect of radiotherapy and chemotherapy.However,if the hypoxic characteristics of the tumor tissue microenvironment can be turned into therapeutic targets,it may have important implications for breast cancer treatment.Research based on this direction is being carried out at home and abroad.Using catalase-like active materials and photosensitizers to make metal nanoparticles and load chemotherapeutic drugs,catalyze the tumor microenvironment to generate oxygen,relieve tumor tissue hypoxia,and synergistically promote photodynamic therapy（PDT）and photothermal therapy agents and Chemotherapy is one of the most commonly used strategies.Combining oxygen-consuming materials and materials that can be activated by extreme hypoxia into metal nanosystems,using hypoxia to activate the cytotoxic effect of the composite materials for breast cancer treatment,is another strategy based on the hypoxia-dimensional microenvironment.In this study,based on the above two strategies,we designed and synthesized metal nanomedicines based on photodynamic/hypoxia regulation and observed their synergistic therapeutic effects on breast cancer.Part 1 Construction of a hypoxia-relieving nanosystem with synergistic amplification of ROS signals and its photodynamic therapy for breast cancerObjective:Construction of a hypoxia-relieved metal nanocoordination polymer with synergistic amplification of ROS signaling and observation of its synergistic therapeutic effect on breast cancer.Methods:1.Metal-coordinated nanoparticles were prepared by solution method illumination and loaded with 10-hydroxycamptothecin（CPT）to construct a multifunctional metal nanocomposite（CPM）.The surface morphology was characterized,and its drug loading,oxygen production efficiency,photothermal conversion efficiency,stability and reactive oxygen species（ROS）generation capacity were tested.2.The 4T1 breast cancer tumor-bearing nude mouse animal model was established.After intravenous injection of CPM,the tumor volume was observed in vivo,the tumor body and main organs were histologically analyzed,the temperature change of the tumor body was detected by an infrared camera,and the time and distribution of the drug to the tumor were analyzed by in vivo imaging.3.Breast cancer cells were cultured in vitro,CCK8 cytotoxicity assay was used to detect the killing ability of the drug on cells,flow cytometry was used to detect CPM-induced apoptosis and ROS,fluorescent labeling was used to detect the oxygen produced by the drug and changes in mitochondrial membrane potential,and Western blotting was used to detect Hypoxia-inducible factor-1α（HIF-1α）and Caspase 3expression.Results:1.According to TEM analysis,CPM has a shuttle structure,the particle size is about 140 nm,and the Zeta potential is negative.Photothermal conversion efficiency（35.3%）,in vitro oxygen production efficiency（maximum oxygen production concentration is 13.5 mg/ml）and good stability and can produce high abundance singlet oxygen.2.Through the small animal fluorescence imaging system,CPM has a good fluorescence tracking ability.After intravenous injection for 12 hours,it can achieve the best enrichment effect of the tumor,and no fluorescence signal is seen in the main organs;imaging by an infrared camera shows that CPM has a good tumor concentration.In vivo photothermal conversion（up to 47.5°C within 10 minutes）can effectively inhibit the growth of breast tumors and has good biological safety（HE staining of main organs showed no obvious abnormality）.3.CPM can effectively kill breast cancer cells（IC50=2.8±0.4μg/m L）,generate intracellular oxygen,induce apoptosis and reduce mitochondrial membrane potential（almost all covered by green fluorescence）,induce down-regulation of HIF-1αand up-regulation of Caspase 3 in tumor cells.Conclusions:CPM can effectively relieve tumor hypoxia,downregulate HIF-1αexpression,promote ROS generation,reduce mitochondrial membrane potential,kill breast cancer cells through the cascade PDT/PTT action of intracellular oxygen production,and exert an inhibitory effect on breast cancer cells by inducing mitochondrial apoptosis.Part 2 Hypoxia-activated nanosystem construction and its photodynamic therapy for breast cancer（Chapter 3）Objective:In this chapter,the hypoxic activation and chemodynamic therapy of breast cancer were realized by preparing an intracellular oxygen depleting metal-organic coordination polymer（ACT）for intracellular oxygen depletion in breast cancer to sensitize tirapazamine（TPZ）for hypoxic activation and chemodynamic therapy of breast cancer（CDT）.Methods:1.Nano-metal-organic coordination polymers（ACT）were prepared by copper ion(Cu²⁺)precipitation,and their drug loading.The particle size and surface morphology were characterized by TEM and spectroscopic analysis.Drug release capacity,and Fenton-like reaction efficiency were tested.2.A breast cancer cell model was established,and the CCK8 cytotoxicity assay was used to detect the killing ability of drugs on cells under normoxia and hypoxia.Flow cytometry was used to detect ACT-induced apoptosis and reactive oxygen species（ROS）production.The level of entry and ROS production,and the expression of cytochrome C（Cytochrome C）and Caspase 3 was detected by western blotting.Results:1.ACT has a spherical morphology with a diameter of about 150 nm.ACT has a high drug loading rate（14.5%）and p H/glutathione（GSH）responsiveness,and has a good hydroxyl oxygen radical（·OH）generation efficiency.2.ACT can effectively kill breast cancer cells under the action of PDT,has better killing effect and apoptosis induction effect under hypoxia,can efficiently take up cells and produce high abundance of ROS,and upregulate the expression of Cytochrome C and Caspase 3.Conclusions:ACT acts through the PDT/CDT cascade of intracellular oxygen depletion,rapidly releases the drug through the p H/GSH response of the tumor microenvironment,and then sensitizes the hypoxia-activated therapeutic drug TPZ,and effectively induces breast cancer cell apoptosis by up-regulating Cytochrome C and Caspase3.Part 3 Construction of targeted biparental nanomedicine and its photodynamic therapy for breast cancer（Chapter 4,Supplementary Chapter）Objective:In this chapter,a ligand-targeting strategy was used to design carrier-free amphiphilic nanomedicines（CTM）that actively target breast cancer cells for breast cancer-targeted chemotherapy-PDT therapy.Methods:1.Methotrexate（MTX）and D-α-tocopheryl succinate（TOS）were linked with cystamine,and then co-assembled with chlorin e6（Ce6）to form CTM nanomedicines.The particle size and surface morphology were characterized by TEM and spectral analysis,and their photosensitizer drug loading was detected,drug release capacity,stability and in vitro singlet oxygen（1O2）generation capacity.2.An animal model of breast cancer was established,CTM was injected intravenously,the tumor volume was observed in vivo,the tumor body and main organs were histologically analyzed,and the proliferation inhibition of cancer cells was analyzed by Ki67 immunohistochemistry.3.A breast cancer cell model was established,and CCK8 cytotoxicity assay was used to detect the killing ability of drugs on cells,flow cytometry was used to detect CTM-induced apoptosis and reactive oxygen species（ROS）production,fluorescence microscopy was used to analyze drug-induced ROS production,and fluorescence analysis was used to analyze death/survival cell ratio.Results:1.CTM nanomedicines are 140 nm particle size spheres.CTM has good photosensitizer loading capacity（15%）,p H/GSH responsiveness and time/concentration-dependent singlet oxygen（~1O₂）generation capacity.2.CTM has shown to respond to the tumor’s slightly acid and high GSH environment,and achieve the effect of drug release on demand.Combined with PDT,CTM can effectively generate ~1O₂,kill breast tumor cells,and induce cell apoptosis.3.CTM can effectively inhibit the growth of breast tumors.HE staining of the tumor shows obvious damage and necrosis.CTM significantly reduces the positive rate of Ki67.Conclusions:CTM actively targets breast cancer cells through folate receptors to achieve on-demand drug release and p H/GSH response,further combined with PDT/chemotherapy to kill breast cancer cells,inhibit breast tumor growth and proliferation,and has high biosafety.