Construction Of ROS-responsive Aggregation Nanoparticle Delivery Systems And Their Therapeutic Effects For Inflammatory Diseases

Reactive oxygen species-mediated oxidative damage at the site of inflammation is closely associated with the pathogenesis of many diseases associated with acute and chronic inflammation,such as acute liver injury,acute lung injury,neurodegenerative disease,cardiovascular disease,and inflammatory bowel disease（IBD）.The pathogenesis factors of inflammatory bowel disease are complex.Clinical basic drugs include aminosalicylic acid,NSAIDs,hormones,anti-TNF-α,small molecule drugs,etc.However,due to their untargeted distribution and drug characteristics in clinical treatment,drug side effects are inevitable.Drug-induced liver injury can develop into acute liver failure（ALF）,and severe patients even require liver transplantation.Recent studies have shown that oxidative stress,as a result of abnormally elevated levels of reactive oxygen species,is closely related to the pathogenesis of drug-induced tissue damage.Consequently,antioxidant therapy represents a potentially plausible approach for treating drug-induced tissue injury.Unfortunately,owing to their non-specific distribution,rapid metabolism,and low delivery efficiency,treatment with small-molecule antioxidants frequently affords limited efficacy and various adverse effects.Therefore,it is of great significance to develop specific,safe,and effective therapeutic drugs.According to the results of previous studies,the synthetic anti-oxidative stress material TPCD is effective in the prevention and treatment of various acute and chronic inflammatory diseases and plays an anti-inflammatory role.Thus,this project uses the oxidative polymerization effect of polyphenols to design a nano-delivery system that can respond to the targeted aggregation of active oxygen in the local area of inflammation.The targeting and biological therapeutic effect of reactive oxygen species（ROS）responsive aggregation nano-delivery system in an inflammatory disease model were investigated.The results showed that increasing the targeted aggregation of nanomedicines,increasing the concentration of nanomedicines in the inflammatory lesions,increasing the retention time in the inflammatory area,improving the therapeutic effect,reducing the non-specific distribution,and reducing the toxic and side effects.Methods1.Synthesis and characterization of reactive oxygen species amphiphilic polymers modified with polyphenols.Polyphenols reactive oxygen species responsive amphiphilic polymers were synthesized by the one-step reaction of the phenolic active monomer with amino group and amide of the NHS-active group at the end of the amphiphilic polymer.It was characterized by FT-IR and¹H NMR.Dopamine modifies DSPE-PEG to obtain DPD.DPS was obtained by modifying DSPE-PEG with serotonin hydrochloride and tyramine modified DSPE-PEG to get DPT.2.Preparation and performance evaluation of reactive oxygen species（ROS）responsive aggregation PLGA nanoparticles.PLGA is used as the hydrophobic core,and the amphiphilic polymer modified by polyphenol is used as the outer shell.The Nano-precipitation method is used to assemble nanoparticles with a core-shell structure.Nanoparticle DPN prepared by nano-precipitation assembly with DPD as the shell,Nanoparticle SPN prepared by nano-precipitation assembly with DPS as the shell,Nanoparticle TPN prepared by nano-precipitation assembly with DPT as the shell.To evaluate the performance of reactive oxygen species responsive aggregation PLGA nanoparticles,the particle size of the nanoparticles was detected by dynamic light scattering method after co-incubation with the local inflammatory microenvironment simulated and the colonic inflammatory tissue homogenate,and the cross-linked aggregation state was observed by transmission electron microscopy and scanning electron microscopy.After oral gavage,ROS-responsive aggregated PLGA nanoparticles were observed by in vivo imaging and laser confocal microscopy to targeting the distribution of inflammatory lesions in the colitis model in vivo.3.Synthesis and characterization of antioxidative stress material TPCDAccording to the foundation of the previous research work,the two functionally active groups PBAP and Tpl are covalently bonded to the framework material cyclodextrin,and characterized by infrared spectroscopy（FT-IR）and proton nuclear magnetic resonance spectroscopy（1H NMR）By detecting DPPH·radicals and hydrogen peroxide scavenging detecting its essential character.4.Preparation and characterization of TPCD nanoparticles and pharmacodynamics evaluation in an acute colitis model.The core-shell structure of TN nanoparticles was prepared by the nano-precipitation method from hydrophobic core TPCD and amphiphilic polymer shell DSPE-PEG.Then the particle size distribution,shape,and size of the nanoparticles were observed by particle size analysis and transmission electron microscopy In addition,the efficacy of TN in mice with acute colitis and its dose-effect relationship were investigated.5.Targeting and pharmacological evaluation of reactive oxygen species aggregated TPCD nanoparticles in acute colitis.According to the above-mentioned preparation method of nanoparticles,TPCD is used as the hydrophobic core and polyphenol responsive polymer is used as the shell to construct nanoparticles DTN（with DPD as the shell）,STN（with DPS as the shell）,and TTN（with DPT as the shell）.Subsequently,The size and shape of nanoparticles were characterized by particle size analyzer and transmission electron microscopy.Moreover,in vivo imager was used to investigate the targeted aggregation of inflammatory lesions in the model of acute colitis,and the pharmacodynamics of responsive aggregated TTN nanoparticles.6.Preparation,characterization,and physicochemical properties evaluation of AC2-26loaded ATTN nanoparticles.The Ac2-26 and TPCD were used as hydrophobic cores,which are dissolved in methanol,DPT and lecithin were dissolved in ultra-pure water,and the nanoparticles were assembled by the precipitation method.The encapsulation rate,drug loading,and drug release of the nanoparticles were detected by fluorescence spectrophotometer.After co-incubation with simulated gastrointestinal physiological environment fluid,HPLC detects the structural stability of peptide drugs.7.Evaluation of biological effects of AC2-26 loaded ATTN nanoparticles in vitro.LPS induced the inflammatory outbreak of RAW264.7 cells,and the anti-inflammatory ability of ATTN was investigated.The oxidative stress of RAW264.7 cells was stimulated with PMA and the anti-oxidative stress ability of ATTN was investigated by flow cytometry and confocal microscopy.The chemokine MCP-1 was used to induce RAW264.7 cell migration and activated neutrophil migration experiments to investigate the ability of ATTN to inhibit cell migration.8.Effectiveness evaluation of AC2-26 loaded ATTN nanoparticles in acute colitisAcute colitis model mice were induced by DSS,and ATTN was administered orally.Body weight,colon length,colon H&E staining,tight junction protein occludin immunofluorescence staining,PAS staining,oxidative stress and inflammatory indexes MPO,H₂O₂,TNF-α,IL-1βwere investigated.The colonic morphology of mice was observed by mouse endoscope,and the fluorescence intensity of blood was measured by fluorescence spectrophotometer after oral gavage of FITC-Dextran（3500-5000Da）for 4 h.9.Targeted aggregation of reactive oxygen species aggregation nanoparticles in an acute lung injury model after intravenous injection.BALB/C mice were treated with lipopolysaccharide（LPS）nasal drops to establish an acute lung injury model.In vivo imaging was performed to observe the targeting distribution of PLGA nanoparticles DPN,SPN,and TPN in the lungs by reactive oxygen species（ROS）injection through the tail vein.10.Targeted aggregation of ROS-aggregation nanoparticles in an intravenous model of acute liver injury.The acute liver injury model was established by intraperitoneal injection of APAP in C57BL/6J mice.ROS-responsive aggregated PLGA nanoparticles DPN,SPN and TPN were injected into the tail vein to observe the targeting distribution of PLGA nanoparticles in the liver by in vivo imaging.Through intravenous injection of TPCD nanoparticles TN and TTN,in vivo imaging observes the target distribution in the liver.11.Evaluation of the therapeutic effect of reactive oxygen species aggregation nanoparticles on acute liver injury model.The acute liver injury model was established by intraperitoneal injection of APAP,and TTN nanoparticles were injected intravenously.The liver H&E staining,TUNEL apoptosis staining,serum biochemical indexes including ALT,AST,TBIL,DBIL,oxidative stress and inflammation indexes such as MPO,H₂O₂,TNF-α,and IL-1βwere investigated.12.Safety evaluation of reactive oxygen species responsive aggregation nanoparticles TTN in vivo by oral administration.C57BL/6J mice were gavaged with TTN nanoparticles at 175mg/kg and 350mg/kg for 7days,and then observed for 7 days,the examination included blood routine:red blood cell,white blood cell,platelet,hemoglobin;liver and kidney function:ALT,AST,CREA,UREA;the main organs were fixed with paraformaldehyde,and the pathological sections were stained for observation.Results1.The active polyphenolic monomers dopamine,serum tryptophan,and tyramine were modified at the hydrophilic end of the amphiphilic polymer,and DPD,DPS,and DPT were characterized.ROS-responsive aggregated PLGA nanoparticles（DPN,SPN,TPN）were constructed with a polyphenolic ROS-responsive polymerization amphiphilic polymer as the shell and PLGA as the core.The average particle size is about 160nm～190nm,and the Zeta potential is about-20m V.DPN,SPN,and TPN were co-incubated with MPO/ROS or colonic inflammatory tissue homogenate,and then the nanoparticles were cross-linked and aggregated.The fluorescence of DPN,SPN,and TPN in the inflammatory lesions of the colitis model was significantly enhanced by in vivo imaging and laser confocal imaging.2.Based on PPAP,Tpl,and cyclodextrin,the anti-oxidative stress material TPCD was synthesized and characterized by hydrogen spectrum and infrared spectrum,and its DPPH·free radical and hydrogen peroxide scavenging ability showed a dose-dependent relationship.The average particle size of TN nanoparticles constructed based on TPCD was about 90nm,and through the establishment of an acute colitis model,it was evaluated that TN nanoparticles could improve the inflammatory response of colitis model mice.3.ROS-responsive aggregation TPCD nanoparticles:STN,DTN,TTN,the average particle size is about 85nm～95nm,In vivo imaging of the established acute colon model proved that the fluorescence intensity of STN,DTN,and TTN in the local colon was significantly enhanced than that of TN,and the therapeutic effect of TTN in IBD showed that TTN was better than TN.4.The ATTN nanoparticles loaded with the polypeptide Ac2-26 have a particle size of about 90nm,an encapsulation rate of 34%,a drug load of 2.72μg/mg,and maintain the stability of the peptide structure in the simulated gastrointestinal fluid.At the cellular level.ATTN can inhibit inflammatory cell migration,cellular inflammatory response（TNF-α）,and oxidative stress level expression at the cellular level.Besides,ATTN in the treatment of colitis model in mice,can reduce the local colon MPO,H₂O₂,TNF-α,and IL-1βexpression,improve the colon mucosal morphology,reduce ulcer formation,reduce colon lamina propria infiltration of neutrophils and macrophages,improve colon epithelial permeability change,protect the colon mucosa,improve the intestinal epithelial barrier protection function.5.In the acute lung injury model,in vivo imaging showed that the fluorescence intensity of DPN,SPN,and TPN in the lung was significantly increased compared with that in the PN group.In the acute liver injury model,in vivo imaging showed that the fluorescence intensity of DPN,SPN,and TPN in the liver was significantly increased compared with that in the PN group.6.In the acute liver injury model,in vivo imaging results proved that the nanoparticle concentration of TTN was higher than that of TN in the inflammatory liver tissue.In the efficacy evaluation experiment,TTN could significantly improve serum liver function indexes and inflammatory response indexes,and inhibit drug-induced apoptosis.7.In the acute toxicity test of oral TTN nanoparticles,it was proved that TTN nanoparticles had no pathological effects on the main organs,and there were no significant differences in blood routine and liver and kidney function tests between TTN nanoparticles and normal groups.Conclusion1.Polyphenols ROS-responsive polymerization amphiphilic polymers DPD,DPS,and DPT were successfully synthesized and characterized.In this study,phenolic compounds with amino groups were modified at the hydrophilic end of the biosafety amphiphilic polymer DSPE-PEG through an amide reaction.2.We successfully constructed and characterized the targeted aggregation of ROS-aggregated PLGA nanoparticles in the inflammatory disease model.In this study,DSS-induced acute colitis,LPS-induced acute lung injury,and APAP-induced acute liver injury inflammatory disease models were mainly used.Tyramine-modified PLGA nanoparticles（TPN）showed the strongest local fluorescence intensity and the most excellent targeted aggregation of local inflammatory tissues.As well as simulating the inflammatory microenvironment in vitro,TPN exhibits the best responsive cross-linking and aggregation effect.3.We successfully constructed and characterized the targeted aggregation of ROS-responsive aggregated TPCD nanoparticles in the inflammatory disease model and the biological evaluation in vivo.In this study,the anti-oxidative stress material TPCD was synthesized and characterized.In this study,DSS-induced acute colitis and drug APAP-induced acute liver injury inflammatory disease models were mainly used.Tyramine-modified TPCD nanoparticles（TTN）have the strongest local fluorescence intensity and the most outstanding targeted aggregation of local inflammatory tissues.In addition,TTN has a better therapeutic effect than TN in the inflammatory disease model.And oral TTN has good biological safety.4.Load the anti-inflammatory peptide Ac2-26 into ROS-responsive aggregated nanoparticles TTN to obtain ATTN,and evaluate its biological effects in vivo and in vitro.The physical and chemical properties of ATTN were characterized,and the structural stability of the peptide molecules in a simulated gastrointestinal physiological environment was proved.In vitro,through cell-level experiments,ATTN can interfere with the oxidative stress,inflammation outbreak,and cell migration of macrophages.ATTN can interfere with the migration of neutrophile granulocyte under the activated state,which proves that it can alleviate the inflammatory response of cells.When administered orally in vivo,ATTN can inhibit the expression of inflammatory factors in colitis,inhibit the increase of intestinal epithelial permeability,inhibit inflammatory cell infiltration,reduce colon bleeding and ulcer formation,and improve the therapeutic effect.