Mechanism Of Polyethylene Glycol Phenolic Rosmarinic Acid Nanopaticles With Delivery Of Interleukin-4 For Inflammatory Bowel Disease Treatment

Inflammatory bowel disease（IBD）is a kind of non-specific intestinal inflammatory disease with unknown etiology.At present,IBD has become a major medical burden with high incidence worldwide and is recognized as a major pathogenic risk factor for colorectal cancer.It is closely linked to immune disorders and excessive activation of M1 macrophages which secret various kind of pro-inflammatory cytokines,resulting in the chronic and high-level inflammation in colon.At present,the strategy of exogenous delivery of anti-inflammatory cytokines has attracted more attention from researchers.However,most current delivery systems face the problem of enzymatic degradation and denaturation of protein molecules,which may lead to immunogenic side effects and loss of pharmacological activity after administration.In addition,easy degradation,short circulating half-life and ineffective release are the main obstacles to effective delivery of cytokine proteins.Therefore,in this study,PEGylated rosatinic acid nanoparticles were synthesized and loaded with IL-4 cytokines,which were delivered to the site of intestinal inflammation to efficiently remove excessive ROS in the tissue and release IL-4,promote the polarization of M2 macrophages,improve the intestinal inflammatory microenvironment,and promote the process of tissue repair.Method1 Synthesis and characterization of PEGylated rosatinic acid nanoparticles loaded with IL-4 cytokines.Firstly,the m PEG-CPPA was obtained by esterification of hydroxyl group of m PEG（Mn 5000 Da）and carboxyl group of CPPA.Then,The m PEG-b-HEMA polymer was synthesized by reversible addition-fragmentation chain transfer（RAFT）polymerization using 2,2’-Azobis（2-methylpropionitrile）（AIBN）as the initiator.To synthesis m PEG-b-P（HEMA-r-RA）diblock polymer,RA and m PEG-b-HEMA was reacted for 24 h under argon atmosphere at 25℃.Finally,IL-4@PEGRA NAs were synthesized by assembly of m PEG-b-P（HEMA-r-RA）and IL-4via a facile solvent-exchange method,and different mass ratio of m PEG-b-P（HEMA-r-RA）to IL-4 was prepared to confirm favorable encapsulation efficiency of IL-4.To test the released protein under different conditions,enzyme-linked immunosorbent assay（ELISA）was performed.Additionally,the secondary structure of the proteins was performed by a circular dichroism（CD）spectrometer.Moreover,the free-radical scavenging capacities of IL-4@PEGRA was detected.Furthermore,the biocompatibility of IL-4@PEGRA was evaluated by hemolysis test,cytotoxicity test and animal test in vivo.2 Evaluation of PEGylated rosatinic acid nanoparticles loaded with IL-4 cytokines in vitro.Firstly,after H₂O₂ incubation,cell viability of Caco-2 was evaluated by CCK-8 kits and the percentage of apoptosis cells was detected by Annexin V-FITC/PI kits to assess the capacity of cytoprotection of IL-4@PEGRA.Then,BMDM cells were firstly incubated with LPS for24h under different conditions,followed by staining with CD206（a biomarker of M2 macrophages）and CD86（a biomarker of M1macrophages）.The images were captured by laser confocal microscope and the level of protein expression was tested by western blot.Finally,DCFH-DA probe was introduced to measure the oxidative stress level of live cells under different treatment which reflecting the effect of scavenging ROS of IL-4@PEGRA in vitro.3 Machanism of PEGylated rosatinic acid nanoparticles loaded with IL-4 cytokines for IBD treatment.Mouse colitis disease model was established by giving mice water containing 3%（w/v）dextran sulfate sodium salt（DSS）from day 0 for 9days and mice had consumed roughly the same amount of DSS water.Body weights of mice were recorded and disease activity index（DAI）was assessed daily.To quantify IL-4@PEGRA biodistribution,Di R-encapsulated IL-4@PEGRA were introduced intravenously to healthy mice and DSS-treated colitis mice.Mice entire colon was collected,imaged and colon length of each group was measured without stretching using a ruler.The activity of myeloperoxidase（MPO）and superoxide dismutase（SOD）,and the concentration of TNF-α、IL-1β、IL-6、IL-10 and IFN-γ,were measured by corresponding ELISA kits.The histological structure of colon in each group was observed and histopathological scoring was performed.ROS levels in colon tissues were assessed by DHE fluorescent staining using frozen sections.Paraffin sections were stained for CD86,CD206,and CD11b to evaluate the number of M1 macrophages,M2macrophages,and CD11b-positive immune cells in mouse colitis tissues.Fecal samples from the DSS and IL-4@PEGRA groups were collected for gut microbiota sequencing analysis.Colon tissue samples were collected from the DSS and IL-4@PEGRA groups,and RNA was extracted and analyzed by transcriptome sequencing.Results1 Synthesis and characterization of PEGylated rosatinic acid nanoparticles loaded with IL-4 cytokines.Macromolecular chain transfer Polyethylene glycol-conjugated4-cyano-4-（phenylcarbonothioylthio）pentanoic acid（m PEG-CPPA）was constructed by conjugating CPPA to m PEG-OH.Subsequently,m PEG₁₀₅-b-HEMA₂₀₉copolymerwassynthesizedvia m PEG-CPPA-initiated reversible addition-fragmentation chain transfer（RAFT）copolymerization of HEMA.RA containing catechol groups were grafted into the pendant hydroxyl groups of PHA by esterification to obtain m PEG₁₀₅-b-P(HEMA₁₉₅-r-RA₁₄),termed as PEGRA.The IL-4encapsulation capabilities of PEGRA significantly improved with an increase in the ratio of PEGRA to IL-4,eventually reaching the maximal encapsulation efficiency（approximately 90%）at the ratio of 20:1.Images obtained by transmission electron microscopy（TEM）indicated that PEGRA and IL-4@PEGRA NAs exhibited well-defined morphology with diameters of 90.5±5.1 and 97.31±4.6 nm,respectively.The diameter of PEGRA NAs and IL-4@PEGRA NAs,as determined using dynamic light scattering were 102.4±3.4 and 109.3±5.7 nm,respectively.The surface zeta potential of PEGRA NAs and IL-4@PEGRA NAs was-16.5±1.2 and-13.6±0.8 m V,respectively.Importantly,the binding of PEGRA and IL-4did not change the secondary structure of the latter.At 3 h,a significant increase in IL-4 release at 34.3%and 66.7%was observed in the presence of 200μM and 1m M H₂O₂,respectively and at 6 h,IL-4@PEGRA NAs released 57.3%and 83.7%of IL-4 in the presence of 200μM and 1 m M H₂O₂,respectively.Furthermore,various kinds of free-radicals（DPPH,ABTS,H₂O₂,·OH and O₂·^-）could be efficiently scavenged by IL-4@PEGRA.The hemoglobin leakage rate of IL-4@PEGRA even at a high concentration of 20μg m L^-1was<5%,suggesting its blood compatibility.The results of CCK-8 assay confirmed the low cytotoxicity of IL-4@PEGRA.The results of blood chemistry and organ histopathology revealed no significant difference between IL-4@PEGRA and control groups（p>0.05）,further indicating the remarkable biocompatibility of IL-4@PEGRA.2 Evaluation of PEGylated rosatinic acid nanoparticles loaded with IL-4 cytokines in vitro.Firstly,Caco-2 cells stimulated with 200μM H₂O₂for 24 h showed a significant increase of cell viability in the PEGRA and IL-4@PEGRA treated groups compared with IL-4 and H₂O₂treated groups.The results of flow cytometry showed that compared with the H₂O₂treatment group,the apoptosis rate induced by H₂O₂in the PEGRA and IL-4@PEGRA treatment groups was significantly reduced（p<0.01）.The results of immunofluorescence staining and Western Blot assay showed that IL-4@PEGRA could reverse the M1 polarization process of bone marrow derived macrophages（BMDMs）induced by LPS and polarize to M2macrophages.In addition,intracellular ROS levels were significantly reduced after IL-4@PEGRA treatment by 2’,7’-dichlorofluorescein diacetate（DCFH-DA）probe staining.The quantitative analysis results of flow cytometry also showed that when the cells were treated with PEGRA and IL-4@PEGRA,the percentage of fluorescent positive cells decreased from 40.3%to 14.4%and 6.36%,respectively（p<0.01）.3 Machanism of PEGylated rosatinic acid nanoparticles loaded with IL-4 cytokines for IBD treatment.First,a mouse model of acute colitis was successfully induced by DSS.The fluorescence intensity of IL-4@PEGRA group in the inflamed colon increased significantly compared to the healthy colon,especially 6 h after intravenous injection,when the fluorescence intensity in the inflamed colon was almost three times higher than that in the healthy colon.Especially at12 hours and 24 hours,the retention of IL-4@PEGRA in the inflammatory colon of the DSS group was higher than that of the normal colon of the healthy group（p<0.01）.In healthy mice,IL-4@PEGRA accumulates in the liver and spleen.In contrast,IL-4@PEGRA was significantly restricted to the colon and less accumulated in the liver and spleen in mice with acute colitis.The mice in DSS group showed reduced body weight,obvious bloody stool and weakened activity.On day 9,the IL-4@PEGRA group caused a significant increase in body weight（21.52±2.1 g）（p<0.01）.The DAI score of DSS group increased gradually,indicating a severe colitis state;IL-4@PEGRA At the end of day 9,the DAI score decreased significantly with a mean value close to 1（p<0.01）.The colon length was significantly shorter in the DSS group（4.46±0.32cm）,while the colon length was significantly longer in the IL-4@PEGRA treatment group（8.30±0.30cm）（p<0.01）.The DSS group showed disordered colonic structure,disordered mucosal structure,destruction of surface epithelium,disappearance of crypt glands and goblet cells,thickening of submucosa,and extensive inflammatory cell infiltration in representative colonic histology.The IL-4@PEGRA group showed almost normal colonic architecture and showed abundant goblet cells and preservation of cryptons,which were similar to the healthy group.Dihydroethidium（DHE）staining results showed that the fluorescence intensity of IL-4@PEGRA group was significantly lower than that of DSS group（p<0.01）.Compared with DSS group,the activity of SOD in IL-4@PEGRA group remained at a higher level（p<0.01）,while the activity of MPO in DSS group was significantly increased（p<0.01）.Compared with the DSS group,the number of CD11b immune cells in the IL-4@PEGRA group was significantly decreased（p<0.01）.The proportion of CD206 M2 labeled positive cells in IL-4@PEGRA group was significantly increased（p<0.01）,while the distribution and infiltration of CD86 M1 labeled positive cells were decreased（p<0.01）,which was consistent with the trend of Western Blot results.ELISA results showed that IL-4@PEGRA treatment significantly reduced the levels of IL-1β,IL-6,TNF-αand IFN-γin tissues（p<0.01）,and increased the expression of IL-10 gene（p<0.01）.IL-4@PEGRA had a significant effect on the gut microbiota compared to the DSS group,showing the ability to beneficially modulate the gut microbiota in IBD mice by ameliorating gut microbiota dysregulation,including an increase in probiotics and a decrease in pathogens.The PI3K/Akt signaling pathway was significantly inhibited in the IL-4@PEGRA group.The expression of genes associated with inflammation（IL-1β,IL-6,CCL2,Myd88,Tnf）,M1 macrophage differentiation（CD86）,proteolysis（Mmp2,Mmp3,Mmp9,Mmp14）,and cell apoptosis（Casp12）were significantly downregulated.Conclusion1.PEGylated rosatinic acid nanoparticles encapsulated with IL-4（IL-4@PEGRA）were successfully synthesized,which could release IL-4under the condition of a certain concentration of H₂O₂in vitro.IL-4@PEGRA could efficiently scavenge a variety of free radicals.Moreover,IL-4@PEGRA was highly biocompatible both in vitro and in vivo.2.IL-4@PEGRA could play a good role in the protection of Caco-2cells in the simulated high ROS environment in vitro.In addition,IL-4@PEGRA reversed the LPS-induced polarization of M1-type macrophages,instead promoted the polarization of M2-type macrophages,and reduced the production of ROS caused by LPS in vitro.3.IL-4@PEGRA exhibited significantly greater accumulation in the inflammatory colon tissues,and could reduce weight loss,blood in the stool and other symptoms caused by IBD;IL-4@PEGRA could also improve the inflammatory environment caused by IBD through promoting the polarization of M2 macrophages and scavenging excessive ROS in colon tissues.Additionally,IL-4@PEGRA improved the imbalance of intestinal microbiota and beneficially regulate the ability of intestinal microbiota in IBD mice.Finally,it promoted intestinal tissue repair and treated acute colitis in mice.