Construction Of Nanomaterials With The Disruption Capacity Of Intracellular Metal Ion Homeostasis For Tumor Therapy

Nowadays,malignant tumors have become the greatest threat to human health with the increasing incidence.Therefore,it is important to develop novel tumor treatment strategies with good biosafety,low toxic and specificity.Based on the important role of metal ions in a variety of intracellular biological processes,researchers have developed several nanomedicines for tumor therapy.Among them,the delivery of transition metal elements with Fenton/Fenton-like catalytic activity has attracted extensive research,which catalyzes the generation of reactive oxygen species（ROS）from hydrogen peroxide（H₂O₂）at tumor sites,causes apoptotic damage and promotes the accumulation of lipid peroxides（LPO）to induce apoptosis/ferroptosis in tumor cells.In addition to the delivery of exogenous metal elements,the depletion of endogenous metal ions is also a potentially effective strategy to disrupt intracellular ion homeostasis.Based on this,this thesis constructs a series of composite nanomaterials through both deliveries of exogenous metal ions and consumption of endogenous metal ions to achieve efficient tumor therapy by disrupting intracellular metal ion homeostasis.The performance in tumor therapy also explored by physicochemical characterization,cellular experiments and animal experiments.The specific contents are as follows:Part Ⅰ:To achieve exogenous metal ion delivery,a copper-based composite nanoparticle was prepared for the treatment of tumor cells by delivering exogenous Cu²⁺with the assistance of chemotherapeutic drug.Firstly,polyacrylic acid（PAA）was used as the soft template to form PAA-Cu₃（PO₄）₂ nanoparticles.The PAA-Cu₃（PO₄）₂-DOX-CMCS composite nanoparticles were obtained by doxorubicin（DOX）loading and carboxymethyl chitosan（CMCS）coating.The loading content and loading efficiency of DOX were measured to be 13.25%and 51.37%.The prepared composite nanoparticles can be effectively uptaken by tumor cells and acid response to release Cu²⁺and DOX.The Cu²⁺can deplete GSH and convert to Cu⁺to act as Fenton-like agents for ROS generation,which can cause LPO accumulation to induce tumor cells ferroptosis.The DOX was observed to be enriched near the nucleus at 3 h to induce tumor cells apoptosis.According to the cytotoxicity assay,160 ppm of PAA-Cu₃（PO₄）₂-DOX-CMCS composite nanoparticles could kill tumor cells more than 90%,indicating exogenous that delivery of Cu²⁺and DOX can achieve efficient treatment of tumor cellsPart Ⅱ:For the delivery of exogenous metal ions,a novel copper-based composite nanosheet was prepared for tumor elimination by delivery of exogenous copper ions with the assistance of biological enzmy.Firstly,using the property that ammonium bicarbonate（NH₄HCO₃）is easily decomposed by heat,basic cupric carbonate（Cu₂（OH）₂CO₃）nanosheets were synthesized under the reaction conditions of 40℃.The Cu₂（OH）₂CO₃-PDA-GOx-PVP composite nanosheets were obtained by glucose oxidase（GOx）loading and polyvinyl pyrrolidone（PVP）coating,the loading content and loading efficiency of GOx were measured to be 28.1%and 78.2%.The composite nanosheets have good stability under physiological p H conditions and good acid-responsive degradation properties under slightly acidic conditions.After uptaken by tumor cells,they could be degraded to release Cu²⁺and GOx.The Cu²⁺can deplete GSH and convert to Cu⁺to act as Fenton-like agents for ROS generation,the depleted GSH down-regulated the expression of GPX4 protein,which causes LPO accumulation.The GOx consumed glucose for adenosine triphosphate（ATP）down-regulation and H₂O₂ up-regulation,enhancing the Fenton-like action of Cu²⁺.Ultimately induces apoptosis and ferroptosis in tumor cells.In vivo experiments have shown that Cu₂（OH）₂CO₃-PDA-GOx-PVP composite nanosheets have good biosafety and antitumor effects.Finally,this method can be flexibly extended for the preparation of mono-element and multi-element carbonate nanomaterials with other transition metal elements（Fe,Mn,Co...）,which is a green and versatile synthetic method that can provide important implications for the efficient preparation of carbonate nanomaterials.Part Ⅲ:To achieve endogenous metal ion depletion,a novel“depletion-blockage”composite nanoparticle was prepared to induce tumor cell apoptosis by depleting the labile iron pool（LIP）.Firstly,poly（lactic-co-glycolic acid）（PLGA）,ethylenediaminetetraacetic acid（EDTA）and ebselen（EBS）were synthesized into PLGA-EDTA-EBS composite nanoparticles by double emulsion method.The composite nanoparticles can be effectively uptaken by tumor cells and acid-responsive release of EDTA and EBS.EDTA can rapidly consume intracellular iron elements by chelate Fe²⁺to form EDTA-Fe;EBS prevents tumor cells obtained extracellular iron elements by disrupting divalent metal transporter protein 1.The depletion of LIP caused by this strategy can lead to the reduction of ribonucleotide reductase activity and inhibit DNA synthesis,which directly performance that cell cycle-related proteins（Cyclin A2,Cyclin E1 and CDK2）down-regulation,G1 and G2/M phases of the cell cycle are significantly reduced,and the S phase is significantly increased in the number of tumor cells.On the other hand,it leads to the down-regulation of iron-sulfur cluster content,resulting in abnormal mitochondrial function,which directly showed the reduction of mitochondrial membrane potential,intracellular ATP synthesis and lactate efflux,and finally induces apoptosis of tumor cells.In vivo experiments have shown that PLGA-EDTA-EBS composite nanoparticles have excellent biosafety and antitumor effects.This strategy of tumor elimination based on the depletion of iron elements in tumor cells could provide important implications for the preparation of efficient and safe nanomedicines in the future.