Effect And Mechanism Of Ultrasmall Copper Nanoenzymes For The Treatment Of Inflammatory Diseases Via Scavenging Reactive Oxygen Species

BackgroundVarious internal and external injury factors,such as severe burn,trauma,infection and shock,will trigger a rapid and strong inflammatory reaction of the body immune system,which could result in the excessive uncontrolled systemic inflammatory response syndrome and cause a sharp decline in the function of multiple organs.The excessive and uncontrolled inflammation will lead to a variety of acute and chronic inflammatory diseases.Reactive oxygen species have been reported to be closely related to many acute and chronic inflammatory diseases.The excessive production of reactive oxygen species in the inflammatory sites is one of the most important causes of local tissue damage and aggravating inflammatory response.Removal of excessive reactive oxygen species is a feasible method for the treatment of inflammatory diseases.However,there are currently limited clinical methods available for the removal of excessive reactive oxygen species in the inflammatory diseases.Broad-spectrum reactive oxygen species scavengers such as N-acetyl cysteine and acetyl-L-carnitine are faced with problems of poor bioavailability,low stability and low reactive oxygen species scavenging efficiency,which limit their potential applications in the treatment of inflammatory diseases.The particle sizes of the vast majority of nanomaterials with reactive oxygen species scavenging properties are generally larger than the threshold of glomerular filtration barrier,which means that they could not be cleared through the kidney and thereby have potential biotoxicity.Nanomaterials that can be cleared through the kidney have the drawbacks of complex synthesis process and low reactive oxygen species scavenging efficiency,which limit their translation to the clinical practice.The development of ultrasmall nanomaterials with simple synthesis process,excellent reactive oxygen species scavenging performance,and renal clearance property is a safe and effective method for the treatment of inflammatory diseases.In this study,we reported the simple and efficient one-step development of ultrasmall Cu_5.4O nanoenzymes(Cu_5.4O USNPs)with multiple enzyme-mimicking properties and broad-spectrum reactive oxygen species scavenging abilities,and investigated the enzyme-mimicking properties,reactive oxygen species scavenging abilities and reactive oxygen species scavenging mechanisms of Cu_5.4O USNPs.In addition,the effect of Cu_5.4O USNPs on protecting cells from reactive oxygen species damage in vitro and the therapeutic effect of Cu_5.4O USNPs on the inflammatory diseases in vivo were further investigated.Simultaneously,the biosafety of Cu_5.4O USNPs both in vitro and in vivo was also investigated.Objective:This study devotes to the development of ultrasmall Cu_5.4O USNPs with broad spectrum reactive oxygen species scavenging ability and renal-clearable property to achieve therapeutic effect on various inflammatory diseases.Methods:1.Development and characterization of Cu5.4O USNPsThe Cu_5.4O USNPs were prepared by the redox reaction between copper chloride and ascorbic acid.Ascorbic acid served as both reducing agent and encapsulation agent.The ratio of copper chloride to ascorbic acid,the reaction time and the reaction temperature were tuned to determine the optimal conditions for the preparation of Cu_5.4O USNPs with both ultrasmall particle size and excellent catalytic activity.Transmission electron microscopy was used to characterize the morphology of Cu_5.4O USNPs,and Malvern particle size analyzer was used to characterize the hydrated particle sizes of Cu_5.4O USNPs.The valence states of copper elements in Cu_5.4O USNPs were characterized by the X-ray diffractometer and X-ray Auger electron spectroscopy.The functional groups of Cu_5.4O USNPs were characterized by the Fourier infrared spectroscopy.The reactive oxygen species scavenging capability of Cu_5.4O USNPs against H₂O₂,O₂·^-,·OH and ABTS free radicals,reactive oxygen species scavenging mechanisms and multiple enzyme-mimicking properties of Cu_5.4O USNPs were further detected using detection kits and electron paramagnetic resonance spectroscopy.Compared with natural antioxidant enzymes,the stability of reactive oxygen species scavenging ability of Cu_5.4O USNPs over temperature and p H changes were explored.In addition,the changes of reactive oxygen species scavenging performance of Cu_5.4O USNPs after repeated use were detected.2.In vitro cytoprotective effect of Cu5.4O USNPs against reactive oxygen speciesIn order to detect the in vitro cytoprotective effect of Cu_5.4O USNPs against reactive oxygen species,HEK293 cells were co-cultured with various concentrations of Cu_5.4O USNPs and H₂O₂ at 250μM concentration for 24 h in vitro.Afterwards,the intracellular reactive oxygen species staining was performed using the reactive oxygen species probe DCFH-DA,and the intracellular scavenging of reactive oxygen species by Cu_5.4O USNPs after H₂O₂stimulation was further detected by the laser confocal microscopy and flow cytometry.The CCK-8 assay and Annexin V-FITC/PI flow cytometry were used to detect the cytoprotective effect of Cu_5.4O USNPs against cell apoptosis and necrosis induced by hydrogen peroxide in vitro.Transmission electron microscopy was used to observe the distribution of Cu_5.4O USNPs in cells.3.Therapeutic effect of Cu5.4O USNPs on the treatment of inflammatory diseasesTo evaluate the in vivo therapeutic effect of Cu_5.4O USNPs on inflammatory diseases,the acute kidney injury mouse model was established.The in vivo therapeutic effect of Cu_5.4O USNPs on the acute kidney injury mice model was systematically evaluated by detecting the survival curve,body weight change,serum creatinine and urea nitrogen levels,kidney injury markers HO-1 and KIM-1 levels,renal tissue reactive oxygen species staining and renal tissue HE staining of acute kidney injury mice after Cu_5.4O USNPs treatment.The in vivo pharmacokinetics,organ distribution and excretion mode of Cu_5.4O USNPs in the acute kidney injury mice were explored by collecting the blood,organs,urine and feces from the acute kidney injury mice at different time points.Transmission electron microscopy was used to observe the distribution of Cu_5.4O USNPs in the kidney of acute kidney injury mice.Afterwards,another inflammatory disease model,the mouse model of acute liver injury was established,and the in vivo therapeutic effect of Cu_5.4O USNPs on acute liver injury mice model was systematically evaluated by detecting the serum levels of alanine aminotransferase and aspartate aminotransferase,and observing HE staining sections of liver tissues from acute liver injury mice after Cu_5.4O USNPs treatment.Furthermore,the diabetic mouse wound model was established,and the therapeutic effect of Cu_5.4O USNPs on diabetic mice wound healing was observed and evaluated by continuous wound photography and HE staining sections of skin tissues at different time points after Cu_5.4O USNPs treatment.4.Therapeutic mechanism of Cu5.4O USNPs in the treatment of inflammatory diseasesThe underlying mechanism of Cu_5.4O USNPs in the treatment of inflammatory diseases was explored by the transcriptomics sequencing combined with bioinformatics analysis.The kidney tissues of mice in the acute kidney injury control group and Cu_5.4O USNPs treatment group were collected for transcriptomic sequencing,and the differentially expressed genes were analyzed by cluster analysis,enrichment analysis of KEGG signaling pathway and protein-protein interaction network analysis.The results of transcriptomic analysis were further verified by real-time fluorescence quantitative PCR,Western blot and ELISA experiments.5.In vitro and in vivo biosafety evaluation of Cu5.4O USNPsIn order to evaluate the in vitro cytotoxicity of Cu_5.4O USNPs,HEK293 cells were co-cultured with Cu_5.4O USNPs at different concentrations,and the cytotoxicity of Cu_5.4O USNPs was evaluated by the CCK-8 assay and phalloidin cytoskeleton staining of HEK293cells.The hemocompatibility of Cu_5.4O USNPs was evaluated by in vitro hemolysis assay.The in vivo acute and chronic toxicity of Cu_5.4O USNPs was evaluated by detecting the blood routine,liver and kidney function indicators,and HE staining of organ sections at 24 h and 30days after single intravenous administration of Cu_5.4O USNPs.The in vivo toxicity of repeated intravenous administration of Cu_5.4O USNPs was evaluated by detecting the blood routine,liver and kidney function indicators,as well as observation of organ sections by transmission electron microscopy and HE staining of organ sections.Results:1.Development and characterization of Cu5.4O USNPsThe ultrasmall copper-based nanomaterials Cu_5.4O USNPs with broad spectrum reactive oxygen species scavenging ability was prepared by adjusting the conditions of the redox reaction between copper chloride and ascorbic acid,namely the molar ratio of copper chloride to ascorbic acid,the reaction time and the reaction temperature.Transmission electron microscopy showed that the prepared Cu_5.4O USNPs were spherical nanoparticles with uniform morphology.Malvern particle size analyzer was used to characterize the hydrated particle sizes of Cu_5.4O USNPs.The results revealed that the average hydrated particle size of Cu_5.4O USNPs was about 4.5nm.The valence states of copper elements in Cu_5.4O USNPs were characterized by the X-ray diffractometer and X-ray Auger electron spectroscopy.The results showed that the valence states of copper elements in the Cu_5.4O USNPs included both Cu⁺and Cu⁰ states.The Cu_5.4O USNPs possessed broad spectrum and highly efficient reactive oxygen species scavenging ability against H₂O₂,O₂·^-,·OH and ABTS free radicals,which could mimic the characteristics of a variety of natural antioxidant enzymes,such as CAT,SOD,GPx.The reactive oxygen species scavenging ability of Cu_5.4O USNPs was mainly attributed to the changes in the valence states of Cu⁺and Cu⁰.Compared with natural antioxidant enzymes,Cu_5.4O USNPs possessed good stability of reactive oxygen species scavenging performance over p H and temperature changes.Furthermore,Cu_5.4O USNPs maintained good reactive oxygen species scavenging performance after repeated use.2.In vitro cytoprotective effect of Cu5.4O USNPs against reactive oxygen speciesTo detect the in vitro cytoprotective effect of Cu_5.4O USNPs against reactive oxygen species,HEK293 cells were co-cultured with various concentrations of Cu_5.4O USNPs and H₂O₂ at 250μM concentration for 24 h in vitro.After being co-cultured with H₂O₂ at 250μM concentration and various concentrations of Cu_5.4O USNPs for 24 h in vitro,the intracellular reactive oxygen species in HEK293 cells were stained with the reactive oxygen species probe DCFH-DA and the levels of intracellular reactive oxygen species in HEK293 cells were further detected by the laser confocal microscopy and flow cytometry.The results of laser confocal microscope observation and flow cytometry showed that Cu_5.4O USNPs could effectively scavenge the increased intracellular reactive oxygen species after hydrogen peroxide stimulation.The results of CCK-8 assay and Annexin V-FITC/PI flow cytometry showed that Cu_5.4O USNPs could alleviate intracellular oxidative stress damage,reduce the percentage of cell apoptosis and cell necrosis caused by hydrogen peroxide and protect cells from hydrogen peroxide-induced cell death.Transmission electron microscopy was used to observe the distribution of Cu_5.4O USNPs in HEK293 cells.The results showed that Cu_5.4O USNPs could be found in the mitochondria and endocytosomes of HEK293 cells,which indicated that Cu_5.4O USNPs could enter cells through endocytosis and play the role of scavenging reactive oxygen species in the mitochondria of cells.3.Therapeutic effect of Cu5.4O USNPs on the treatment of inflammatory diseasesThe acute kidney injury mouse model was established according to the literature report.The survival time of acute kidney injury mice treated with Cu_5.4O USNPs was significantly prolonged,and there was no significant weight loss in the acute kidney injury mice with Cu_5.4O USNPs treatment.Compared with the acute kidney injury mice in the control group,the levels of serum creatinine and urea nitrogen and the levels of kidney injury markers KIM-1 and HO-1 were significantly reduced in the acute kidney injury mice with Cu_5.4O USNPs treatment.Besides,the number of pathological casts and the levels of reactive oxygen species in the kidney tissues sections were significantly reduced in the acute kidney injury mice with Cu_5.4O USNPs treatment.The overall results indicated that Cu_5.4O USNPs could effectively scavenge excessive reactive oxygen species in the renal tissue of acute kidney injury mice and exhibited a good therapeutic effect on the acute kidney injury mice in vivo.The in vivo pharmacokinetics experiment results of Cu_5.4O USNPs in the acute kidney injury mice showed that the terminal elimination half-lives of the central component and peripheral component were 0.77 and 71.2 h,respectively.The results of organ distribution of Cu_5.4O USNPs in the acute kidney injury mice after intravenous administration showed that the Cu_5.4O USNPs were mainly distributed in the kidney,which was then followed by the liver.The accumulation of Cu_5.4O USNPs in the feces and urine changed in a time-dependent manner,and the cumulative amount of Cu_5.4O USNPs in the urine was significantly higher than that in the feces.Furthermore,Cu_5.4O USNPs could be found in the glomerular basement membrane,podocyte foot process,urinary space and cilia of renal tubular epithelial cells in the kidney tissues of acute kidney injury mice by transmission electron microscope observation.The acute liver injury mouse model was established according to the literature report.Compared with the acute liver injury mice in the control group,the levels of serum alanine aminotransferase and aspartate aminotransferase were significantly reduced in the acute liver injury mice with Cu_5.4O USNPs treatment.Besides,the areas of hepatocyte necrosis in the liver tissues sections were significantly reduced in the acute liver injury mice with Cu_5.4O USNPs treatment.The overall results indicated that Cu_5.4O USNPs exhibited a good therapeutic effect on the acute liver injury mice in vivo.To evaluate the therapeutic effect of Cu_5.4O USNPs on diabetic mice wound healing,the diabetic mouse wound model was established according to the literature report.Compared with the diabetic mice wounds in the control group,the wound healing rate of diabetic mice with topical Cu_5.4O USNPs treatment was significantly accelerated.The length of newly regenerated epithelium and the thickness of granulation tissues in the diabetic mice wounds with Cu_5.4O USNPs treatment were significantly higher than those of the control group.The overall results indicated that Cu_5.4O USNPs exhibited a good therapeutic effect on the diabetic mice wound healing in vivo.4.Therapeutic mechanism of Cu5.4O USNPs in the treatment of inflammatory diseasesThe underlying mechanism of Cu_5.4O USNPs in the treatment of inflammatory diseases was explored by the transcriptomics sequencing combined with bioinformatics analysis.Compared with the control group,a total of 5819 differentially expressed genes were identified in the kidney tissues of acute kidney injury mice with Cu_5.4O USNPs treatment.The5819 differentially expressed genes consisted of 2813 up-regulated genes and 3006down-regulated genes.The results of KEGG signaling pathway enrichment analysis of the differentially expressed genes showed that the MAPK and TNF/NF-κB signaling pathways were significantly inhibited in the kidney of acute kidney injury mice with Cu_5.4O USNPs treatment.The results of cluster analysis of the differentially expressed genes which are related to oxidative stress showed that several important antioxidant enzyme-related genes such as SOD1,SOD2,SOD3,GPX1,GPX3,GPX4,GPX6,and CAT were significantly upregulated in the kidney of acute kidney injury mice after treatment with Cu_5.4O USNPs.In addition,the differentially expressed genes which are associated with tissue damage and inflammation caused by oxidative stress,such as TLR4,NF-κB1,MMP9,MMP14,and HMOX1,were significantly downregulated in the kidney of acute kidney injury mice after treatment with Cu_5.4O USNPs.The quantitative real-time PCR results showed that the m RNA expression levels of antioxidant enzyme-related genes SOD1,SOD2,SOD3,GPX1,GPX3,GPX6 and CAT in the kidney tissues of acute kidney injury mice treated with Cu_5.4O USNPs were significantly higher than those in the control group.The Western blot results showed that the NF-κB/IκB signaling pathway was inhibited in the kidney tissues of acute kidney injury mice with Cu_5.4O USNPs treatment.The ELISA results showed that the levels of TNF-αand IL-1βin the serum and kidney tissues of acute kidney injury mice treated with Cu_5.4O USNPs were significantly decreased.The overall results indicated that Cu_5.4O USNPs could reduce the inflammatory response of the kidney by downregulating the NF-κB/IκB signaling pathway and inhibiting the production of downstream pro-inflammatory factors TNF-αand IL-1β.5.In vitro and in vivo biosafety evaluation of Cu5.4O USNPsTo evaluate the in vitro cytotoxicity of Cu_5.4O USNPs,HEK293 cells were co-cultured with Cu_5.4O USNPs at different concentrations,and the cytotoxicity of Cu_5.4O USNPs was evaluated by the CCK-8 assay and phalloidin cytoskeleton staining of HEK293 cells.The CCK-8 assay results showed that Cu_5.4O USNPs exhibited no significant cytotoxicity when incubated with HEK293 cells at the concentrations ranging from 10 to 200 ng/m L for 24 and48 h in vitro.The phalloidin cytoskeleton staining results showed that Cu_5.4O USNPs at the concentration of 200 ng/m L exhibited no significant effect on the cell morphology after being co-cultured with HEK293 cells for 48 h in vitro.The in vitro hemolysis assay results showed that the hemolysis rate of Cu_5.4O USNPs was less than 5%at the concentrations ranging from0 to 500 ng/m L,which indicated that Cu_5.4O USNPs possessed good hemocompatibility at the concentrations ranging from 0 to 500 ng/m L.The in vivo acute and chronic toxicity of single dose administration of Cu_5.4O USNPs was evaluated.There were no abnormal changes in the blood routine,liver function indicators and kidney function indicators at 24 h and 30 days after the single dose intravenous administration of Cu_5.4O USNPs.Furthermore,no pathological damage in the major organs（heart,liver,spleen,lung,kidney）was observed in the HE staining tissues sections of mice at 24 h and 30 days after the single dose intravenous administration of Cu_5.4O USNPs.The in vivo toxicity of repeated intravenous administration of Cu_5.4O USNPs was also evaluated.After repeated intravenous administration for one week,Cu_5.4O USNPs were mainly distributed in the liver and kidney.Transmission electron microscope observation results showed that the cell morphology and structure of major organs were generally normal.In addition,no pathological damage was observed in the major organs of the mice,and there were no abnormal changes in blood routine,liver function indicators,and kidney function indicators in mice with repeated intravenous administration of Cu_5.4O USNPs.Conclusion:This study reported a simple and efficient one-step development of ultrasmall copper-based nanoenzymes(Cu_5.4O USNPs)with multiple enzyme-mimicking properties and broad-spectrum reactive oxygen species scavenging abilities for the treatment of various inflammatory diseases.The Cu_5.4O USNPs simultaneously possessing catalase-,superoxide dismutase-,and glutathione peroxidase-mimicking enzyme properties exhibited excellent cytoprotective effect against reactive oxygen species-mediated cell damage at the extremely low dosage and significantly improve the treatment outcomes in the acute kidney injury mice,acute liver injury mice and diabetic wound healing.Meanwhile,the ultrasmall size of Cu_5.4O USNPs enabled the rapid renal clearance of the nanomaterials,guaranteeing good biocompatibility.The broad-spectrum reactive oxygen species scavenging ability and good biocompatibility of Cu_5.4O USNPs will provide new ideas and reference for the development of copper-based nanomaterials with reactive oxygen species scavenging ability for the treatment of inflammatory diseases in the future.