Basic Copper Carbonate Nanocrystals For Tumor-specific Copper Ions-activated Cancer Therapy

Malignant tumor is a great threat to human life,which prompts researchers to devote themselves into exploring highly efficient treatment modalities.Therapeutic strategies against malignant tumors have been extensively explored via regulation of sophisticated tumor redox homeostasis.Tumor redox homeostasis maintains dynamic balance of reductive substances such as glutathione(GSH)and oxidizing substances such as reactive oxygen species(ROS).It has become a dominant issue to combat malignant tumor via disrupting redox-homeostasis and further amplifying oxidative stress.With rapid development of nanotechnology,valence-variable metal-based nanodrugs,possessing fenton-like properties,were extensively applied into efficient cancer treatment via consumption of reducing species,GSH,thus,amplifying oxidative stress such as ROS generation,and subsequently resulting in considerable apoptosis of tumor cells.Copper element,an essential component of human body,plays an essential role in effective cancer therapy due to its active chemical properties and high catalytic capacity.Copper ions can activate the chemodynamic therapy through consuming endogenous GSH,eliciting specific fenton-like reaction and subsequent production of cytotoxic hydroxyl radicals from hydroperoxide,together with high efficiency and negligible side effects.Additionally,copper can activate selective cytotoxicity of various chemotherapy drugs,such as disulfiram,which firstly metabolizes into diethyl dithiocarbamate ester(DDC),and further coordinates with copper ions to form cytotoxic Cu(DDC)2 complex,thus inducing apoptosis of cancer cells.Furthermore,copper ions can trigger chemodynamic therapy and chemotherapy,and subsequently induce the immunogenic cell death followed by immune activation,resulting in improved anticancer effect.Efficient targeted delivery of copper ions into tumors is the foundation of copper ions activated therapies,thus improving delivery efficiency is the key issue.In this thesis,we successfully synthesized basic copper carbonate albumin nanocrystals(CuCH-NCs)in albumin nanocages via biomineralization.The nanocrystals can specifically release Cu2+in acidic tumor microenvironment such as lysosomes,and further react with glutathione,resulting in the formation of cuprous ions(Cu+),thereby catalyzing the decomposition of hydrogen peroxide into cytotoxic hydroxyl radicals,so as to achieve more accurate and effective tumor therapy.In addition,further studies revealed that cuprous ions released in TME could also activate the chemotherapeutic effect of disulfiram,resulting in the synergistic effect of chemodynamic therapy and chemotherapy.CuCH-NCs can also synergize with anti-PD-L1 antibodies to amplify the therapeutic effect of immunotherapy.And this thesis is divided into the following four chapters:Chapter 1:In this chapter,the current situation of cancer and various cancer treatment strategies are briefly introduced,and the main inorganic metal nanomaterials are classified and introduced,together with emphasis on the progress of antitumor therapy based on copper ions,including copper ions activated chemodynamic therapy,chemotherapy and immunotherapy.In addition,we also lay emphasize on the regulation strategy of redoxhomeostasis to enhance therapeutic efficacy,and then proposes the design idea and research content of this thesis.Chapter 2:Preparation and characterization of basic copper carbonate nanocrystals.Firstly,basic copper carbonate nanocrystals with appropriate particle size and uniform size were prepared by modifying pH,temperature,reaction time and other conditions.The crystal structure of basic copper carbonate was confirmed by X-ray diffraction(XRD).And the release experiments showed that CuCH-NCs could release copper ions in acidic microenvironment and further react with glutathione to reduce the concentration of GSH and promote the conversion of Cu2+to Cu+.Under acidic pH and GSH conditions,CuCH-NCs can effectively catalyze the decomposition of hydrogen peroxide into hydroxyl radicals.Chapter 3:Studies on biological effects of basic copper carbonate nanocrystals at cellular level.CuCH-NCs exhibited time-dependent endocytosis and were endocytosed into cancer cells via clathrin-mediated endocytosis pathway,resulting in the colocalization with lysosomes,thereby promoting the release of copper ions.In addition,CuCH-NCs could consume intracellular GSH content and boost the production of cytotoxic hydroxyl radicals compared to that of CuCl2 via upregulation of apoptosis-related proteins,and inhibiting cell proliferation(IC50～0.37 mM).Meanwhile,CuCH-NCs had a strong killing effect on tumor cells,and the cytotoxicity of CuCH-NCs with disulfiram increased 7-fold(IC50～0.05 mM)via synergistic chemodynamic therapy and chemotherapy.In addition,CuCH-NCs triggered chemdynamic therapy distinctly improved the expression of calprotectin as the hallmark of immunogenic cell death,indicating the potential of synergistic effect with immunotherapy.Chapter 4:Antitumor effect of basic copper carbonate nanocrystals on animal models.IVIS imaging and biodistribution results verify the effective targeting capacity and prolonged retention for subsequent production of cytotoxic hydroxyl radicals.Furthermore,orthotopic breast tumor models were established to investigate the antitumor effect,showing that CuCH-NCs could delay tumor growth via intravenous injection of 5.0 mg kg-1 Cu at 1 and 7 days.After combination with disulfiram,the progression of orthotopic and metastatic tumors were significantly inhibited.Furthermore,upon synergy with anti-PD-L1 antibody,this chmodynamic-chemo-immunotherapy improved the production of cytotoxic T cells,thereby achieving highly efficient synergistic cancer therapy.Meanwhile,tumor microenvironment activated chemodynamic therapy exhibited distinct safety demonstrated by H&E staining and serum biochemistry assay.In this thesis,CuCH-NCs can achieve efficient targeted delivery of copper ions,and further trigger fenton-like reaction in tumor area,causing selective tumor inhibition.And CuCH-NCs also activated the cytotoxicity of disulfiram in tumor site via responsive release of copper ions in tumor microenvironment,thus avoiding systematic toxicity.In conclusion,CuCH-NCs achieved the more accurate and efficient cancer therapy through copper ions activated chemodynamic therapy and chemotherapy,causing immunogenic cell death,thus amplifying the anticancer efficacy of immunotherapy.