Construction Of Novel Inorganic Nanozyme And Its Application In Scavengering Activity Of Cellular Reactive Oxygen Species

Reactive oxygen species（ROS）is a type of intermediate substances generated by incomplete metabolism of oxygen molecules in living organisms,mainly including superoxide anion(O₂^·-),hydrogen peroxide（H₂O₂）,hydroxyl radical（·OH）.Abnormally elevated ROS will cause oxidative stress in cells,leading to the occurrence of a variety of diseases.Therefore,the removal of excess ROS from cells has great physiological significance for the treatment of oxidative damage-related diseases.Nanozymes,as a class of nanomaterials with biocatalytic activity,can simulate the catalytic process of various natural oxidoreductases,and thus have a wide range of applications in the regulation of reactive oxygen species.Compared with natural enzymes and artificial macromolecular synthases,although nanozymes have the advantages of low preparation cost,high stability,and tunable catalytic activity,there are still some challenges such as low catalytic efficiency,unclear catalytic mechanism,and inability to precisely regulate catalytic activity.Meanwhile,the problem of biosafety in nanomaterials greatly limit the transformation of nanozymes into clinical nanomedicine.In this paper,aiming at the above-mentioned problems in nanozymes for the scavenging of cellular ROS,we designed and constructed a series of novel inorganic nanozymes with high catalytic efficiency,adjustable multiple enzyme-like activities and active scavenging strategies based on the strippability of double metal hydroxides（LDHs）,the tunability of interlayer metal elements and the self-driving characteristics of nanomotors.The relationship between material structure,elemental composition,exercise efficiency and ROS scavenging performance was systematically studied,so as to achieve efficient cellular ROS scavenging ability and application for alleviating mitochondrial oxidative damage as well as the treatment of inflammation.The specific research contents are summarized as follows:1.Defect-rich monolayer cobalt-aluminum LDH nanosheets were prepared to simulate the high activity of superoxide dismutase and used for relieving mitochondrial oxidative damage.A single-layer cobalt-aluminum LDH nanosheet（m-Co Al-LDH）with a thickness of only about 1.0 nm was successfully synthesized by a one-step hydrothermal in situ growth method.The abundant vacancies of the nanosheets can significantly improve the catalytic efficiency of the enzyme activity.Compared with natural SOD enzymes and other common SOD-like nanozymes,the k_cat of an active site is increased by 1-2 orders of magnitude,reaching3.30×10¹¹ M^-1 s^-1,which breaks the limitation of catalytic conversion efficiency in SOD-like activity of nanozymes.By comparing the vacancy concentration and enzymatic catalytic efficiency of Co Al-LDH with different thicknesses,it is proved that the excellent SOD-like activity of m-Co Al-LDH originates from the increase of vacancies following with reducing the number of layers,which leads to the increase of the overall number of active sites of the material.The role of different vacancies in affecting the catalytic efficiency of scavengering O₂^·-of m-Co Al-LDH was further elucidated by Density Functional Thoery（DFT）:the existence of Al vacancies greatly reduced the adsorption energy towards O₂^·-,while the exist of three vacancies（O vacancies,Co vacancies,and Al vacancies）synergistically to minimize the adsorption energy.From the perspective of the catalytic mechanism,the reason for the significant improvement of the catalytic conversion efficiency per active site of m-Co Al-LDH is revealed.Based on the above structural advantages,m-Co Al-LDH was applied to remove excess O₂^·-in mitochondria to repair mitochondrial oxidative damage.2.Ultrathin nickel-manganese LDH nanosheets with adjustable valence state were prepared to simulate the activity of multiple antioxidant enzymes for cellular reactive oxygen species scavenging and inflammation treatment.Using a simple solvothermal method,ultrathin nickel-manganese hydrotalcite nanosheets（U-Ni Mn-LDH）with Ni and Mn ratios of 2:1,3:1 and4:1 were successfully synthesized by changing the feeding ratio of Ni and Mn,respectively.Based on the ultrathin characteristics of LDH nanosheets,more active sites are exposed.Meanwhile,the ROS adsorption capacity is increased by using the positive charge of the main layer,which improves the overall removal efficiency of H₂O₂,O₂^·-and·OH.By comparing the GPx-like activity,SOD-like activity and OH scavenging efficiency of U-Ni Mn-LDH with three Ni and Mn ratios,combined with the analysis of Mn mixed valence ratios,.The relationship between each ROS scavenging efficiency and the different mixed valence ratios in Mn-based nanozymes were revealed:with the ratios of Mn³⁺/Mn²⁺,Mn²⁺/Mn⁴⁺,Mn³⁺/Mn⁴⁺increasing,the performance of the GPx-like enzyme activity,SOD-like enzyme activity,and the scavenging efficiency has improved,respectively.Based on the above conclusions,U-Ni₃Mn₁-LDH with the largest ratios of Mn³⁺/Mn²⁺,Mn²⁺/Mn⁴⁺and Mn³⁺/Mn⁴⁺exhibited the highest antioxidant properties.The scavenging efficiency of cellular ROS can reach 85%with the concentration of 0.2 mg m L^-1.Further with the good biocompatibility of U-Ni Mn-LDH,U-Ni₃Mn₁-LDH was used to mimic the antioxidant defense system in vivo to achieve effective treatment of ear inflammation in mice.3.An engineered Pt-based Janus nanomotor modified with black phosphorus quantum dots was constructed to realize the active scavenging of ROS and therapy for inflammation.The Janus Pt/AFSNs nanomotor was prepared by physical vapor deposition（PVD）method,which semi-loaded Pt nanoparticles on the surface of amino-modified Si O₂ nanoparticles.Then,BPQDs was modified on Janus Pt/AFSNs surface by electrostatic adsorption to successfully construct the compound Janus BPQDs/Pt/AFSNs nanomotor.Using the H₂O₂ overproduced in cells as fuel,the self-propelled motion of the nanomotor can be achieved through a concentration gradient generated by the process of removing H₂O₂ by Pt nanoparticles with CAT-like activity.Thus,the contact probability with ROS will increase during the self-driven movement of the nanomotor and the efficient removal of ROS can be realized.The modification of BPQDs increases the oxygen abundance on the surface of Pt（111）,which improves the reaction rate of Pt catalytic decomposition of H₂O₂ and the movement speed and diffusion coefficient of Janus BPQDs/Pt/AFSNs composite nanomomotor were2 times and 2.6 times higher than Janus Pt/AFSNs,respectively.The limitation of low H₂O₂ concentration on self-driven motion efficiency of Pt-based nanomotor is overcome.In addition,the inherent O₂^·-and·OH scavenging ability of BPQDs,synergistically enhances the ROS scavenging effect of Janus BPQDs/Pt/AFSNs.Meanwhile,the modification of BPQDs improves the biocompatibility and cell penetration of the material,ensuring the therapeutic effect of in vivo inflammation.In this paper,based on defect engineering,element proportion regulation and clearance strategy innovation respectively,nanzoyme for ROS scavenging has realized the leap from single enzyme activity to multiple enzyme activity as well as from static scavenging to dynamic scavenging,providing a new strategy for the design of nanozyme and the treatment of oxidative stress-related diseases.