Construction Of An Inflammatory Microenvironment-regulating Peptide Nanotherapy And Its Efficacies And Mechanisms For The Treatment Of Inflammatory Bowel Disease

Inflammatory bowel disease?IBD?is a group of chronic disorders that cause prolonged inflammation of the gastrointestinal tract.The main types of IBD include crohn's disease and ulcerative colitis.Although the etiology of IBD remains to be fully elucidated,corticosteroids,non-steroidal anti-inflammatory drugs and newly emerged biological therapies,such as antitumor necrosis factor and anti-adhesion therapies have been used for clinical treatment.These therapies,however,generally cause significant side effects for nonspecific distribution.Therefore,it is of high importance in developing specific,effective,and safe IBD therapies.The inflammatory molecules,immune cells,and microbial community lead to the proinflammatory microenvironment in the intestine that causes IBD.Consequently,restoring the abnormal inflammatory environment of IBD represents an intriguing strategy toward effective therapy.Recently,the resolution of inflammation has been considered as an active and tightly regulated process dominated by specific proresolving mediators,enabling suppression of inflammatory cytokine expression and clearance of apoptotic cells and microorganisms.Among different proresolving mediators,an endogenous protein annexin A1can effectively treat inflammatory diseases.Ac2-26 is an annexin A1 N-terminal derived peptide with pharmacological activities of the whole protein.This peptide can inhibit various aspects of the inflammatory response,such as cell adhesion and transmigration,thereby reducing the infiltration of neutrophils and monocytes/macrophages.As well documented,oxidative stress,characterized by excessive production of reactive oxygen species?ROS?,which can lead to the imbalance of oxidative stress or induce localized tissue injury,is closely related to the pathogenesis of IBD.Consequently,our hypothesis is that ROS-responsive NPs can serve as triggerable vehicles for oral delivery of peptide therapeutic Ac2-26.Also,we assume that nanotherapy-mediated simultaneous normalization of the proinflammatory microenvironment and dysbiosis in the intestine is an effective approach for the treatment of IBD.Herein,ROS responsive group,phenylboronic acid pinacol ester?PBAP?,was used as functional unit and was respectively bonded to?-cyclodextrin??-CD?to obtained OxbCD,and then developed an Ac2-26 peptide-containing nanotherapy for site-specific delivery to the inflamed colon in IBD.Both in vitro and in vivo studies were conducted to probe the mechanisms underlying therapeutic effects of this Ac2-26-derived proresolving nanotherapy.Methods1.Fabrication and characterization of Ac2-26-loaded OxbCD nanoparticlesA ROS-responsive material?OxbCD?was synthesized by functionalizing?-cyclodextrin??-CD?with an oxidation-labile compound PBAP.The obtained OxbCD was characterized by¹H NMR and Fourier transform infrared?FT-IR?spectroscopy.Ac2-26-loaded OxbCD nanoparticles?Ac2-26/OxbCD NP,defined as AON?were prepared by a modified nanoprecipitation/self-assembly method.Briefly,OxbCD was dissolved in methanol,into which Ac2-26 dissolved in DMSO was added.The obtained solution was added dropwise into deionized water containing DSPE-PEG and lecithin.After 2 h of incubation,solidified Ac2-26-loaded NPs were harvested.Following similar procedures,OxbCD NP?ON?and Ac2-26-containing PLGA NP?APN?were fabricated.Particle size and zeta-potential measurements were conducted on a Malvern Zetasizer Nano ZS instrument.Transmission electron microscopy?TEM?observation was carried out on a TECNAI-10 microscope.Scanning electron microscopy?SEM?was conducted on a FIB-SEM microscope.The loading content of FITC-Ac2-26 in different NPs was determined by fluorescence measurement of FITC-Ac2-26.2.In vitro hydrolysis,release study and stability of AONIn vitro hydrolysis of AON was performed at 37°C in PBS buffers with or without hydrogen peroxide.The release profiles of AON in PBS with hydrogen peroxide was was quantified by fluorescence spectroscopy.Through similar procedures,drug release in fluids simulating the alimentary tract environment was also examined.The stability of Ac2-26 and AON in various fluids including simulated gastric fluid,simulated intestinal fluid,murine gastric fluid,and colonic fluid,as well as stomachic and colonic homogenates was investigated by high-performance liquid chromatography?HPLC?.3.Cytotoxicity and in vitro cellular uptake of various nanoparticlesThe cytotoxicity of gradient concentrations of blank and Ac2-26-containing NPs on macrophage RAW264.7 quantified by MTT assay.RAW264.7 macrophage cells were incubated with FITC-AON for various durations.In vitro cellular uptake was observed by confocal laser scanning microscopy?CLSM?.Similarly,the dose and time-dependent internalization profile was examined after cells were incubated with FITC-AON via fluorescence activated cell sorting?FACS?.4.Anti-apoptosis and antioxidant activity of AON in macrophagesRAW264.7 cells were incubated with various formulations for 4 h,and then treated with H₂O₂ for 12 h.Apoptosis analysis was conducted using Annexin V-APC and propidium iodide?PI?detection kit.The antioxidant effect of AON on intracellular ROS generation in PMA-stimulated macrophages was detected by fluorescence microscopy and flow cytometry.ELISA kit was used to investigate the anti-inflammatory effect of AON on the secretion of TNF-?from RAW264.7 cells stimulated by LPS.5.Effect of AON on inflammatory cells in vitroTo induce production of neutrophils,thioglycollate?3.0 wt%?was intraperitoneally administrated to BALB/c mice.A transwell assay was performed to study anti-migration activity of Ac2-26 NPs in neutrophils.Neutrophils were fluorescently labeled using DiO membrane dye,and aged for 24 h in order to undergo apoptosis.Effects of AON on the macrophage efferocytosis of neutrophils was assessed by quantifying fluorescence via flow cytometry.RAW264.7 macrophages were stimulated in medium containing LPS and IFN-?for 24 h.Ability of AON on macrophage polarization was analyzed by flow cytometry.6.Study on biodistribution of Ac2-26 NPs in mice with acute colitisAcute ulcerative colitis in mice was induced by addition of 3%?w/v?DSS to the drinking water for 7 days.Mice received a single oral administration of Cy7.5-AON.At each defined time point,mice were euthanized.The colon segments and major organs including liver,spleen,kidney,intestine,and mesenteric lymph node?MLN?were isolated.Blood was also collected for analysis.Ex vivo imaging was carried out with a living imaging system.Similarly,colitic mice received a single oral administration of Ac2-26 or AON.The concentration of Ac2-26 in the whole blood,the colon tissues,and major organs was quantified by HPLC.The localization of orally delivered FITC-AON in the colon of mice with DSS-induced colitis was also observed by CLSM.To further investigate cellular uptake of Ac2-26 NPs,the experiments were performed in both healthy and colitis mice.Colitic mice were orally administered with Cy5-AON.After 4 h,the colon was resected,the uptake of AON by macrophages and neutrophils was measured by flow cytometry.7.Evaluation of therapeutic effect of AON on the acute or chronic colitis in miceThree colitis models were made including DSS-induced acute colitis model?7 days?,DSS-induced chronic colitis model?30 days?and IL-10^-/-mice with spontaneous chronic colitis.Healthy mice in the Normal group were not treated,while colitis mice in the Colitis,ON,APN,AON groups were orally administered with saline,blank OxbCD NP,APN,and AON,respectively.The dose was 250?g/kg of Ac2-26 for all Ac2-26-containing formations.The body weight and Disease activity index?DAI?of each animal were monitored daily.After various treatments,mice were euthanized,and the entire colon was collected.The colon length was measured and 1 cm of the distal section was used for histological assessment.The remaining section was taken for measuring the levels of tumor necrosis factor-??TNF-??,interferon-??IFN-??,interleukin-1??IL-1??,malondialdehyde?MDA?,and hydrogen peroxide as well as myeloperoxidase?MPO?activity.Blood samples were collected to quantify the biochemical markers relevant to liver/kidney functions and hematological parameters.Major organs including heart,liver,spleen,lung,kidney,stomach,intestine,and MLN were harvested and weighed.Intestinal mucosal recovery in mice with acute colitis was observed by mini-endoscopy and immunofluorescence staining.8.Preliminary safety evaluation of AONAON in saline was orally administered at a dose of 5.0 g/kg on C57BL/6J mice.After administration,the body weight of mice and their behaviors were monitored each day.After two weeks,blood samples were collected for hematological analysis.Gastrointestinal tissues and major organs were isolated and weighed for calculation of the organ index.Histopathological sections were prepared and stained with H&E.9.Study on the mechanism of anti-inflammatory and anti-oxidation effects of AON in vitroNon-selective formyl peptide receptor?FPR?antagonist Boc2 and selective FPR2antagonist WRW4 were used to investigate the mechanism of anti-inflammatory,anti-oxidation,anti-neutrophil migration and and promoting macrophage phagocytosis of AON.10.Effect of AON on the recruitment of inflammatory cells and epithelial wound healingColitic mice received oral administration of saline,Ac2-26,blank OxbCD NP,APN,and AON,respectively.After 7 days,the entire colon was collected.The number of neutrophils and macrophages were assessed by flow cytometry.In wound healing study,after 7 days of treatment,each mouse received FITC-dextran by oral gavage.Blood was sampled,and the FITC fluorescence was measured.11.Effect of AON on the resolution of inflammationPeritonitis was induced by i.p.administered with zymosan A in Balb/c mice.Mice received i.p.injection of saline,free Ac2-26,ON,APN,or AON.At predetermined time points,peritoneal exudates were collected.The neutrophils were assessed via a flow cytometer.The levels of TNF-?,IL-1?,MPO,MDA,and hydrogen peroxide in peritoneal exudates were quantified using the corresponding assay kits.12.Regulation of the Gut Microbiota by AONAcute colitis was induced in mice as aforementioned,and then randomly assigned mice received AON or saline by daily oral administration.Fecal samples were collected after 7days.Gut microbiota was analyzed and the concentration of short-chain fatty acids in feces was determined by GC-MS.Results1.A ROS-responsive material?OxbCD?was synthesized by functionalizing?-CD with an oxidation-labile compound PBAP.¹HNMR spectroscopy and FT-IR demonstrated the successful synthesis of OxbCD.The ¹H NMR spectrum revealed approximately 7 PBAP moieties were linked to each?-CD molecule.Ac2-26 peptide was encapsulated into an OxbCD NP,using a nanoprecipitation/self-assembly method,and this Ac2-26-containing,OxbCD-derived nanotherapy was defined as AON.Observation via TEM and SEM indicated that AON displayed a spherical shape.The mean diameter was 202±4 nm,with a relatively narrow distribution of size.AON had a negative?-potential of-37.4±0.6 mV.The drug loading content of Ac2-26 was 0.86?g/mg.AON displayed a rapid hydrolysis and release profile profile in 0.01 M PBS?pH 7.4?containing 1.0 mM of H₂O₂.HPLC was used to test whether Ac2-26 in AON is stable in simulated gastric or intestinal fluids.As expected,Ac2-26encapsulated in AON was clearly detected.In contrast,free Ac2-26 peptide completely hydrolyzed in all six solutions.Collectively,the results demonstrated that our AON can effectively protect Ac2-26 from degradation under gastrointestinal conditions.2.We evaluated the cytotoxicity of different NPs using RAW264.7 macrophages.Relatively high cell viability was observed for ON,AON,and APN across a broad range of concentrations.Flow cytometric and CLSM analysis revealed that AON can be efficiently internalized by macrophages and showed dose-dependent and time-dependent endocytosis.AON can significantly inhibit oxidative stress-mediated cell apoptosis by effectively scavenging ROS,remarkably suppress PMA-induced ROS generation,effectively reduced the TNF-?levels in macrophages treated with LPS,which was more effective than Ac2-26,ON,and APN.3.AON can effectively inhibit neutrophil migration,increase phagocytosis of apoptotic neutrophils,promote the macrophage phenotypic switching from an inflammatory M1phenotype?F4/80⁺CD86⁺?to an alternatively activated M2 phenotype?F4/80⁺CD206⁺?.4.Ex vivo imaging showed significantly higher fluorescent signals of Cy7.5-AON in colons from colitis mice compared to those of healthy mice.Compared to free Cy7.5-labeled Ac2-26 peptide,Cy7.5-AON generally exhibited higher fluorescent signals and higher retention in colons,and displayed extremely low fluorescence distribution in the blood.Consistently,the Cy7.5-Ac2-26 group exhibited higher non-specific fluoroescence distribution in major organs relevant to absorption,metabolism,and excretion,respectively.Also,the enhanced targeting of AON to the inflamed colon was demonstrated by fluorescence imaging of cryosections of colonic tissues.The levels of Ac2-26 in the colon,blood,MLNs,liver,spleen,and kidney of colitis mice were also quantified by HPLC after oral administration of free Ac2-26 or AON.In this case,the amount of Ac2-26 in colonic tissues of the free Ac2-26 group was much lower than that of the AON group at 4 and 24 h.Notably,almost no Ac2-26 was detected in the colon of the free peptide group at 24 h.For both groups,no intact peptide was detected in the blood,MLNs,and kidney at 4 and 24 h after oral administration.Additionally,only very low contents of Ac2-26 were found in the spleen and liver.5.To evaluate the therapeutic effect of ROS-responsive AON on colitis,we established DSS induced mouse acute and chronic colitis models and IL-10^-/-mice with spontaneous chronic colitis model.The animals were treated with AON by oral gavage.AON-treated mice showed less weight loss,lower DAI,and longer colon length,significantly less inflammation in the colonic mucosa.AON significantly reduced the levels of important proinflammatory cytokines including TNF-?,IFN-?,and IL-1?and representative markers related to oxidative stress including MDA,MPO and H₂O₂,showed no detectable injuries in the major organs and gastrointestinal tissues.AON exhibited superior anti-colitis capacity than Ac2-26,ON,Ac2-26 and ON mixture,and APN.6.The potential side effects after oral administration of a high dose of AON?5.0 g/kg?were examined in vivo.No significant weight and organ indexes of major organs differences were found between control and AON-treated groups.Representative hematological parameters and biomarkers relevant to hepatic and kidney functions were in normal ranges.Examination on H&E sections exhibited AON showed no discernable injuries in major organs.7.AON exerted its anti-inflammatory and proresolving actions through FPR2,and the anti-apoptotic activity in macrophages was largely attributed to FPR1 activation.8.AON decreased the number of neutrophils and macrophages recruited to the inflamed colons.FITC-Dextran was treated for colitic mice to reflect the changes of intestinal permeability.AON treatment can effectively protect the colonic mucosa and promote intestinal epithelial wound repair.9.Using a peritonitis model,we examined the proresolving capacity of AON.Local AON treatment significantly reduced the maximal number of neutrophils(?_max)and a resolution interval?R_i?,remarkably decreased the levels of proinflammatory cytokines,relieved oxidative stress.These results indicated that AON can expedite the resolution of inflammation.10.AON treatment reduced the expansion of Escherichia-Shigella and increased Prevotellaceae levels,increased SCFAs production,indicated that AON treatment can shift the microbial community profile from a dysbiotic state toward homeostasis for mice with DSS-induced colitis.Conclusions1.We developed a ROS responsive material OxbCD based on functionalizing?-CD with an oxidation-labile compound PBAP.Ac2-26 was efficiently loaded into OxbCD nanoparticle,resulting in a ROS-triggerable nanotherapy AON.At pH 7.4 and 1.0 mM H₂O₂,AON rapidly hydrolyzed,concomitant with efficient Ac2-26 release.In addition,Ac2-26 in AON was stable against hydrolysis in different simulated gastric/intestinal fluids.2.AON can be efficiently internalized by macrophages,effectively inhibited macrophage apoptosis as well as reduced ROS generation and inflammatory response,reduced neutrophil transmigration,enhanced the clearance of apoptotic neutrophils by macrophages and effectively switched macrophages from a proinflammatory M1 phenotype toward an anti-inflammatory M2 phenotype.3.AON can effectively target and accumulate in the inflamed colon and inflammatory cells of mice with DSS-induced colitis.AON treatment of mice with acute or chronic colitis afforded desirable outcomes,as manifested by significantly decreased weight loss,reduced DAI,as well as notably improved colonic morphology and microstructure.Compared to free Ac2-26,ON,Ac2-26/ON mixture and APN,AON significantly alleviated the symptoms of colitis.AON displayed good safety profile for oral administration.4.Examination the anti-colitis mechanisms of AON in vitro and in vivo indicate that AON exerted its anti-inflammatory and proresolving actions through FPR2,and the anti-apoptotic activity in macrophages was largely attributed to FPR1 activation.AON can decrease the recruitment of inflammatory cells,expedite the resolution of inflammation,restore epithelial barrier function and improve bowel health by shaping the gut microbiota and increasing SCFAs production.