Synthesis Of Ultrafine Ceria Nanowires And Targeted Treatment Of Mice Acute Lung Injury

Background:Acute lung injury（ALI）is a common disease caused by virus or bacterial infection,inhalation of smoke or toxic gas and other causes.Due to the lack of specific treatment methods,the mortality rate of this disease is as high as 40%.The progression of ALI is a series of biological cascade reaction characterized by processes such as alveolar epithelial cells injury,neutrophils infiltration,hyaline membranes formation,alveolar edema,inflammatory and eventually fibrosis.The essence of ALI is that excessive reactive oxygen species（ROS）produced in the body leads to uncontrolled inflammatory reaction and excessive infiltration of inflammatory cells in the lung.Therefore,eliminating excessive ROS is an effective strategy to alleviate the symptoms of ALI.Ceria nanoparticles are effective and self-renewable ROS scavengers due to the existence of Ce³⁺and Ce⁴⁺valence states on their surface and reversible conversion under certain conditions.At present,ceria nanoparticles have been used to treat ROS disorder-related diseases such as ischemia reperfusion injury,central nervous system injury,depression and wound healing.The ROS scavenging capability of ceria nanoparticles is closely related to its size,where the smaller-sized nanoparticles featured with higher Ce³⁺concentration and larger surface area usually exhibit higher ROS scavenging capability.However,smaller-sized ceria nanoparticles preferred to accumulate in spleen and liver rather than lung,resulting in poor therapeutic effect and large side effects.Therefore,targeted treatment of lung diseases is an urgent problem to be solved.Recent studies have shown that some industrial nanomaterials with high aspect ratio may accumulate in the lung causing related diseases.On the other hand,nanomaterials with high aspect ratio have larger specific surface area compared with nanoparticles,resulting better catalytic activity.Based on these principles,we believe that ceria nanowires（CNWs）with high aspect ratio can achieve lung targeted therapy and have high ROS scavenging capacity.Methods:1.Ultrafine CNWs were synthesized by high temperature thermal decomposition method.The effects of reaction temperature,reaction time and TOPO concentrations on the size of CWNs were investigated.The morphology of the synthesized CWNs was observed by transmission electron microscopy（TEM）.High-resolution transmission electron microscopy（HRTEM）and selected area electron diffraction（SAED）were used to observe crystallinity of CNWs.The PEG was modified on the surface of CNWs under the suitable modification conditions,and the surface modification ratio was characterized by thermogravimetry（TGA）.The surface valence content of CWNs was determined by X-ray photoelectron spectroscopy（XPS）.Finally,the ROS scavenging ability of CNWs was evaluated by hydroxyl radical detection kit and superoxide anion scavenging experiment.2.The cytotoxicity of CWNs on RLE-6TN cells was detected by cell proliferation test.The ROS scavenging ability of CWNs in RLE-6TN cells and the anti-apoptosis performance of CWNs on RLE-6TN cells induced by H₂O₂ was evaluated by flow cytometry.At the same time,the intracellular ROS was visualized by fluorescence microscope,and the uptake of CWNs with different sizes by RLE-6TN cells was detected by inductively coupled plasma atomic emission spectrometry（ICP-AES）.3.The relationship between the size of CWNs and the distribution of tissues in vivo was revealed by measuring the content of CWNs in tissues after the injection of different lengths of CWNs into the tail vein of mice.The acute lung injury model of mice was established by instilling lipopolysaccharide（LPS）into the trachea.The degree of edema in the lung tissue of mice was evaluated by the ratio of dry and wet weight of the lung,and the injury of lung tissue in different treatment groups was observed by hematoxylin-eosin（HE）staining.Inflammatory factor（TNF-α,IL-1β,IL-6）concentration in bronchoalveolar lavage fluid（BALF）were detected with enzyme linked immunosorbent assay（ELISA）kit.Additionally,the therapeutic effect of CNWs for ALI model was evaluated through the determination of oxidative stress indicators malonydialdehyde（MDA）and myeloperoxidase（MPO）.Results:1.Ultrafine nanowires with a diameter of about 1.5 nm and a length of 14-161 nm were successfully prepared by adjusting the reaction temperature,reaction time and TOPO concentrations of the reaction system.In order to maintain the morphology and size of CNWs in hydrophilic modification process,the modification conditions are T=40℃,t=20 min.XPS results shown that the concentration of surface Ce³⁺decreases with the length increases of CNWs.The shortest CNW-22 has the highest surface Ce³⁺concentration and the best ROS scavenging ability.2.Three different lengths CNWs（CNW-22,CNW-57,CNW-140）exhibited a negligible toxicity to RLE-6TN cells at the 200μg m L^-1 concentration.In addition,CWNs can protect cells from oxidative stress injury induced by H₂O₂ though to inhibit the production of ROS in RLE-6TN cells.Finally,the uptake of CWNs by RLE-6TN cells is CNW-22<CNW-57<CNW-140.3.The results of blood circulation time and distribution in mice body of various CNWs（CNW-22,CNW-39,CNW-57,CNW-79,CNW-104,CNW-140）indicated that the shorter CNWs have a longer blood circulation time and the accumulation of CNWs in the lung was positively correlated with the length.Most importantly,the accumulation of CNW-140 in the lung was about 6.25 times that of CNW-22.4.The ROS scavenging ability of three different lengths CNWs（CNW-22,CNW-57,CNW-140）was investigated by LPS-induced acute lung injury model in mice.The degree of pulmonary edema and HE staining results suggested that CNW-140 treatment group had a more effective therapy for ALI.ELISA and oxidative stress indicators shown that CNW-140treatment group reduced the pro-inflammatory factor expression significantly,and decreased the degree of lipid peroxidation and the infiltration of neutrophils.Conclusion:1.Ultrafine CNWs with diameter of about 1.5 nm and length of 14-161 nm were prepared successfully by adjusting reaction temperature,reaction time and TOPO concentration.2.CNWs with shorter size have better ROS scavenging ability.In addition,the difference in enzymatic activity is attributed to the higher Ce³⁺ratio on shorter CNWs surface due to CNWs with different length have similar specific surface area.3.CNWs have good biocompatibility with RLE-6TN cells.At the same time,the uptake results of CNWs by RLE-6TN cells shown positive correlation between the length of CNWs and uptake concentrations.Additionally,CNWs can effectively inhibit the production of ROS caused by H₂O₂in RLE-6TN cells,protecting cells from oxidative stress,reducing the apoptosis.4.The accumulation of CNWs in the lung is positive correlated with the size of CNWs.The larger size of CNWs,the higher the concentration of CNWs in the lung.Our vivo experiments have proved that CNW-140 has better anti-inflammatory and antioxidant activity on ALI model,indicating that heigh aspect ratio CNWs can be used as a new candidate strategy for ALI treatment.