Dual Catalytic Reactions Of Engineering Cu-based Nanomedicine For Lung Cancer Therapy

As the main cause of cancer mortality,lung cancer includes non-small cell carcinoma（NSCLC）and small cell carcinoma（SCLC）based on histological classification.Furthermore,according to morphological and immunohistochemical（IHC）features,NSCLC is mainly classified into adenocarcinoma,squamous cell carcinoma,large-cell lung carcinoma,alveolar carcinoma and adenosquamous carcinoma.Despite advances in understanding of risk factors,occurrence and progression,immune control and treatment options for lung cancer,its mortality still remains high although incidence and death rate decline over past decades.Clinically,chemotherapy is the most widely used regimen for lung cancer,such as cisplatin and gemcitabine,but due to its large side effects and other reasons,the therapeutic effect cannot reach the expected.In recent years,nanomaterials are rapidly developing in the field of tumor diagnosis and treatment by virtue of unique advantages,such as controllable synthesis,easy surface modification,and simultaneous loading of multiple substances,providing us with a new vision.More emphasis is put on chemodynamic therapy,photodynamic therapy and photothermal therapy of nanomaterials on tumors,among which,chemical kinetic therapy is the most widely studied.Based on the principle of Fenton reaction,iron-based nanomaterials can dissociate into metal ions in response to weak acid tumor environment,then catalyze the decomposition of local hydrogen peroxide（H₂O₂）,generating hydroxyl radicals which will kill tumor cells.However,due to the limited amount of local hydrogen peroxide in tumor site,the lack of enough lethal free radicals is still a great challenge.To overcome this difficulty,we designed and synthesized one nanoliposome（designated as Lipo-ART@CPNs）,synchronously loading the hydrophilic copper peroxide quantum nanodots（designated as CPNs）in the cavity and the hydrophobicity of artemisinin in membrane layer.After injected though tail vein,Lipo-ART@CPNs could gather together at tumor site owing to the enhanced permeability and retention effect（EPR）,release drugs from the liposomes under ultrasonic control,and decompose in response to tumor microenvironment,playing a role in anti-tumor.To be specific,the CPNs component of nanosystem can release copper ions(Cu²⁺)and H₂O₂under the slightly acidic tumor environment acting as a H₂O₂"self-supporting"platform,and generation of hydroxyl radicals（·OH）through Fenton-like reactions will ensue.Additionally,artemisinin released from the system can also be catalyzed by Cu²⁺to generate harmful free radicals due to the existence of peroxide bridge bonds in the chemical structure.A large number of free radicals will be produced by this copper-based dual catalytic strategy,which can react with membrane phospholipids,resulting in lipid peroxidation and inducing ferroptosis of tumor cells.As a non-apoptotic cell death,ferroptosis is mediated by disastrous reactive oxygen species（ROS）,which featured with iron and lipid peroxidation（LPO）accumulation.In addition to being a substrate for catalytic reactions,artemisinin is also an autophagy inducer that can induce ferritin autophagy（known as ferritinophagy）,increase the unstable iron content in cells,and further enhance ferroptosis.In this study,copper peroxide quantum dots were firstly synthesized under alkaline conditions using copper chloride dihydrate and hydrogen peroxide as raw materials,while under acidic conditions the products would be reversibly dissociated into copper ions and hydrogen peroxide.Transmission electron microscopy（TEM）,high-resolution electron microscopy,energy spectrum scanning,X-ray photoelectron spectroscopy（XPS）,energy dispersive X-ray spectroscopy（EDS）and other methods were used to characterize the physical properties of the as-synthesized materials such as morphology and size.The existence of peroxo group was verified by ultraviolet-visible-near infrared spectrum（UV-vis-NIR）spectrophotometer.The property of CPNs to dissociate Cu²⁺in weak acid was measured by inductively coupled plasma mass spectrometry（ICP-MS）.The ability of CPNs to self-supply hydrogen peroxide and produce hydroxyl radical through Fenton-like reaction was detected through electron paramagnetic resonance（ESR）.The catalytic reaction and corresponding kinetics were further assayed by Michaelis-Menten enzymatic kinetics.Then,the drug delivery system of Lipo-ART@CPNs were synthesized by thin film dispersionmethod,takingcholesterol,methoxylpolyethylene1,2-distearoyl-Sn-glycero-3-phosphoethanolamine(m PEG_2k-DSPE),dipalmitoyl-sn-phosphocholine（DPPC）,artemisinin（ART）and CPNs as raw materials.The nanoliposomes were negatively stained and their morphology and size were observed on a biological transmission electron microscopy（Bio-TEM）.The hydrate particle size and zeta potential of nanosystem were characterized by Malvern laser particle size analyzer.The ultrasonic controlled release of Cu²⁺from Lipo-ART@CPNs was recorded by ICP-MS.The radical production performance of Cu-based dual catalytic reaction and the initial reaction rate was tested by UV-vis-NIR spectrophotometer and microplate analyzer.In addition,we used confocal fluorescence microscopy to observe the free radical generation properties of Lipo-ART@CPNs at the cellular level of Lewis mouse lung cancer（LLC）cells.Secondly,cell counting kit-8（CCK8）assay kit was used to evaluate the safety of as-synthesized CPNs and Lipo-ART@CPNs at cellular level.Bio-TEM,confocal laser scanning microscope（CLSM）and Western blot were used to characterize autophagy and ferroptosis induced by Lipo-ART@CPNs.High-throughput sequencing was performed to examine genomic changes of LLC cells in response to Lipo-ART@CPNs.Babl/C nude mice were selected to establish a subcutaneous tumor-bearing mouse model to evaluate the tumor inhibition effect of Lipo-ART@CPNs.The results showed that the synthesized CPNs are 3-5 nm nanocrystals,in which copper valent is+2.Compared with neutral environment,weak acidic melieu（p H=5.5）is more likely to trigger dissociation of CPNs and release Cu²⁺and H₂O₂,which react with each other through Fenton-like reactions to produce hydroxyl radicals.The prepared Lipo-ART@CPNs are uniformly about 100 nm,with a spherical structure of negative charge.Ultrasound can accelerate the release of CPNs and ART.Compared with CPNs alone,Lipo-ART@CPNs produce more abundant free radicals,proving the existence of dual catalytic reaction.Further,free CPNs had great toxicity on normal cells,while nanoliposomes endowed CPNs high biocompatibility.After uptaken by tumor cells,Lipo-ART@CPNs can produce a large number of free radicals,damage mitochondrial membrane potential,inducing LPO,all of which are manifestations of ferroptosis.In addition to participating in catalysis,ART can act as an autophagy inducer to enhance the ferroptosis-like response led by Cu-base dual catalytic reactions.The underlying mechanism of Lipo-ART@CPNs on LLC cells was ascertained by reference transcriptome analysis.Last,therapeutic efficacy of Lipo-ART@CPNs in vivo was also demonstrated for the fact that it can effectively inhibit the growth of subcutaneous tumor in tumor-bearing mice.