Effects And Mechanisms Of Inflammation-targeting Prussian Blue Nanozyme For Treatment Of Inflammatory Bowel Disease

Part 1.Synthesis,Characterization and Safety Evaluation of PPBsObjective:A new,simple,economical and efficient PPBs was synthesized using polyvinylpyrrolidone?PVP?modified by one-step method.The size,structure,composition and physiological stability off PPBs were observed.ROS scavenging ability of PPBs were evaluated by a variety of methods based on its artificial nano-enzyme of POD-like,CAT-like and SOD-like activity.In addition,the biological safety of PPBs was evaluated by the effects of PPBs on different cells and mice.Methods:PVP and K₃[Fe?CN?₆]were mixed at a certain ratio and then added in HCl solution stirring until to clarified solution.The surface features and physiological stability of PPBs can be observed by transmission electron microscopy?TEM?,scanning and transmission electron microscopy?STEM?,Malvin Zetasizer Nanosize laser,UV-3101PC.In vitro antioxidant capacity of PPBs was evaluated by Electron spin resonance?ESR?,DZS-708,Microplate Reader and Bruker EMX.CCK-8 kit was used to evaluate the effect of PPBs on the proliferation of L929 cells and HT29 cells.Calcein-AM/PI staining was used to evaluate the cell viability.Blood smears were used to observe the effect of PPBs on the morphology and function of human erythrocyte.Blood routine tests,biochemical tests,Elisa and HE staining were used to analyze the effect of PPBs on the blood and main organs of Kunming mice.Results:We successfully designed a new PPBs of⁶0 nm.Depending on its reduction characteristics and multiple enzymes-like?peroxidase,catalase,superoxide?activity,PPBs can effectively eliminate various ROS substances including·OH,H₂O₂,O₂·-and·OOH.PPBs not only had good physical stability in water,physiological saline,saline of pH=5.0,DMEM containing serum,but also in acid solution of pH=0.1 and pH=1.0.Further more,its stability has no change at different temperature.Conclusion:In conclusion,PPBs could effectively remove excessive ROS in colonic tissue of IBD mice in time by intravenous administration,inhibiting the cascade development of inflammation,and alleviating the colonic inflammation in mice.Part 2.Effect of Intravenous Administration of PPBs on DSS-induced IBD MiceObjective: The mouse model of inflammatory bowel disease?IBD?induced by DSS is one of the most commonly used IBD models.DSS can promote the generation of ROS and a variety of inflammatory factors in the process of inflammation,activating multiple inflammatory signaling pathways and accelerating the process of inflammation.In this part,we will study the effects of PPBs on ROS elimination and enteritis inhibition in IBD mice after intravenous administration.Methods: The distribution of PPBs in various organs of mice was studied by ICP-OES and nuclear fixation red staining.The severity of colonic inflammation in mice was evaluated by DAI score,HE staining,MPO,ROS,IL-1?,IL-6,IFN-? and TNF-?.Results: The content of PPBs in colon and colonic mucosa of IBD mice was higher than that of normal mice at the same dose of intravenous administration.The ROS level in colon of mice in 3% DSS + PPBs group was significantly lower than that in IBD group,indicating that PPBs could target and accumulate in inflammation sites,play a better role of artificial nano-enzymes,effectively,timely and quickly remove ROS from inflammation sites,and effectively inhibit ROS-related inflammation process.Further experimental results showed that the DAI score of IBD mice was significantly decreased after PPBs treatment.HE staining confirmed that the colonic pathological inflammation of IBD mice was significantly improved after PPBs treatment.At the same time,the levels of MPO,IL-1?,IL-6,IFN-? and TNF-? in colon tissue of PPBs treatment group were significantly lower than those of IBD group,which proved that PPBs could regulate the expression of inflammatory factors and the activity of MPO.Conclusion: Inflammation causes increase ROS production and excess ROS will aggravate the inflammation in the body.PPBs can timely and effectively eliminate excessive ROS by preventing the development of the inflammatory cascade to inhibit inflammation in mice.The anti-inflammatory effect of PPBs in this systemic inflammatory model is a good paving for the study of PPBs in the treatment of inflammatory bowel disease in mice.Part 3.Effects and mechanism of PPBs on DSS-induced acute IBD miceObjective: By introducing Mn2+ to optimize and improve PPBs,a new,efficient and economical manganese Prussian Blue Nanozymes?MPBZs?was constructed.Its characteristics,stability,ROS scavenging ability and biosafety were evaluated,and the therapeutic effect and mechanism of MPBZs on DSS-induced IBD mice were explored.Methods: MPBZs are synthesized by a simple method.The characteristics of MPBZs were determined by TEM,STEM,Zetasizer Nanosize?Nano ZS90?and UV-3101 PC Shimadzu spectrometer.The antioxidant activity of MPBZs in vitro was evaluated by ESR,enzyme labeling and Bruker EMX spectroscopy.CCK-8 kit was used to evaluate the effects of MPBZs on cell proliferation,and blood routine test,biochemical test,Elisa,HE staining were used to analyze the effects of MPBZs on blood and main organs of Kunming mice.After oral administration of DSS-induced MPBZs in IBD mice,ICP-OES was used to analyze the tissue distribution of MPBZs,IVIS small animal fluorescence imaging system was used to analyze the content of MPBZs in colon at different time points.Immunofluorescence was used to analyze the distribution of MPBZs in colon mucosa.The levels of colonic inflammation were evaluated by DAI score,HE staining,MPO,ROS,IL-1?,IL-6,IFN-?.Finally,the inflammation-related genes and antioxidant-related genes affected by MPBZs were investigated by PCR ARRAY and bioinformatics analysis.Finally,the protein level was measured by Western-blotting.Results: MPBZs have good physiological stability and biosafety.It could target the inflammation site of intestinal mucosa by size and charge advantages.At the same dose of oral administration,the content of MPBZs in colon and colonic mucosa of IBD mice was higher than that of normal mice.The levels of ROS and H2O2 in colon of mice in IBD + MPBZs group were significantly lower than those in IBD group,which indicated that MPBZs could play the role of artificial nano-enzymes after targeting and accumulating inflammation sites,effectively,timely and quickly remove excessive ROS,and inhibit the process of ROS-related inflammation.After MPBZs treatment,the DAI score of IBD mice was significantly decreased.HE staining results confirmed that MPBZs treatment significantly improved the pathological inflammation of IBD mice colon.At the same time,the levels of MPO,MDA,IL-1?,IL-6,IFN-? and TNF-? in IBD + MPBZs group were decreased,which further confirmed that MPBZs could effectively reduce the expression of inflammatory factors and the activity of MPO and MDA.PCR ARRAY showed that MPBZs affected many signaling molecules in inflammation-related signaling pathways and oxidation-related signaling pathways.Bioinformatics analysis and Western-blotting results further confirmed that MPBZs could regulate the expression of signal molecules such as NFKB-50,NFKB-105,RELA,TLR4 PTGS1,PTGS2,and participate in inflammation-related signaling pathways and oxidation-related signaling pathways.Conclusion: Through size/charge mediation,MPBZs could target colonic inflammation sites,effectively eliminate excessive ROS,participate in a variety of inflammatory and oxidative signaling pathways and affect the expression of key signaling molecules such as NFKB-50,NFKB-105,Rela,Tlr4,Ptgs1,Ptgs2.Ultimately,colonic inflammation in mice was inhibited.