Therapeutic Effect And Mechanism Of Double-responsive Targeted Nanoparticle Loaded SiERN1 In Autoimmune Inflammatory Disease

ObjectiveAutoimmune inflammatory diseases are a type of systemic diseases that have been gradually recognized in recent years and are mainly characterized by inflammatory responses caused by immune disorders,such as rheumatoid arthritis(RA)and inflammatory bowel disease(IBD,including ulcerative colitis and Crohn’s enteritis),etc..The disease often involves multiple organs and multiple systems,with persistent disease and poor prognosis.Drug therapy is currently the main method for clinical treatment of such diseases,including nonsteroidal anti-inflammatory drugs,glucocorticoids,and biological agents.Despite the great success of clinical treatment,side effects such as myelosuppression,gastrointestinal reactions,drug resistance and immunosuppression greatly limit its clinical application.New drug development is a necessary topic.The successful clinical application of small interfering RNA(si RNA)drugs has brought new opportunities for the development of therapeutic drugs for such diseases.This project screened,designed,and constructed novel si RNA drugs,and designed and synthesized nanoparticle-loaded si RNA drugs with dual-responsiveness and targeting for the treatment of collagen-induced arthritis(CIA)model mice and IBD model mice.The inflammatory control,immune regulation,and tissue protection effects of the si RNA-nanoprodrug in the above models were investigated,and its mechanism of action was discussed in depth to provide new strategies for the treatment of RA and IBD.Methods1.First,the role of endoplasmic reticulum to nucleus signaling 1(ERN1,encoding inositol-requiring enzyme 1α,IRE1α)gene in the occurrence and development of RA disease was verified.ERN1 si RNA(si ERN1)with an ideal inhibition rate was designed,constructed,synthesized,and screened.At the same time,the ERN1 overexpressing adenovirus(ad ERN1)was successfully packaged by adenovirus construction and packaging technology.The expression of pro-inflammatory cytokines such as IL-1β,IL-6,and TNF-αafter knockdown or overexpression of ERN1 was detected by RT-q PCR and Western blot methods.2.Based on polyethyleneimine(PEI)and poly(β-amino amine)(PBAA),p H-responsive and reduction-responsive macrophage-targeted bilayer nanocarriers were designed and synthesized.si ERN1 was loaded by electrostatic adsorption,and further targeted modification was carried out.During the synthesis process,the intermediate products were detected by a series of detection methods such as gel permeation chromatography,~1H Nuclear Magnetic Resonance Spectroscopy,etc.,to ensure the correct assembly of the final product.The physicochemical properties of the nanoprodrugs were characterized by means of transmission electron microscopy,dynamic light scattering,Zeta potential,and drug release behavior.The biocompatibility of the nanoprodrugs was verified by MTT and hemolysis experiments.3.CIA model was established in DBA1/J mice.The Cy5-labeled nanoprodrugs was injected into the tail vein,and the drug distribution of the nanoprodrugs in the CIA model mice was observed at a predetermined time point to verify the targeting of the si ERN1 nanoprodrugs.The therapeutic effect of si ERN1 nanoprodrugs was assessed by arthritis score and hindpaw thickness by tail vein injection of each si ERN1 formulation.The cartilage and subchondral bone protection effects of si ERN1 nanoprodrugs were evaluated by Micro-CT,MRI,and histopathology.In addition,flow cytometry,RT-q PCR,Western blot,and immunofluorescence were used to evaluate the immune regulation and inflammation inhibition effects of si ERN1 nanoprodrugs in CIA model mice.4.IBD model was established in C57/BL6 mice.The therapeutic effect of the si ERN1 nanoprodrugs in an IBD model was evaluated by the changes in body weight,and the integrity of colon morphology,structure,and function.At the same time,flow cytometry,RT-q PCR,Western blot,and immunofluorescence experiments were used to evaluate the immune regulation and inflammation inhibition effects of si ERN1 nanoprodrugs in IBD model mice.5.The regulatory effect of IRE1αon macrophage polarization was detected by VMD,co-immunoprecipitation,fluorescence detection,flow cytometry,and RT-q PCR.In addition,the role of IRE1αin My D88-dependent TLR signaling pathway was verified by means of label-free quantitative proteomics assay and Western blot.Results1.The results of clinical samples showed that ERN1 expression was increased in the affected tissues of RA patients.Cell experiments confirmed that ERN1 was positively correlated with the expression levels of pro-inflammatory cytokines.It is suggested that ERN1 is related to the occurrence and development of RA.2.The si ERN1 with ideal inhibition rate was synthesized and screened,and a dual-responsive macrophage-targeted nanomaterial was constructed as a si ERN1 drug carrier.The characterization data show that the si ERN1nanoprodrugs has suitable particle size and surface potential,responsive drug release characteristics,and excellent biocompatibility.3.CIA mice model was successfully established.In CIA model mice,it was observed that the si ERN1 nanoprodrugs could be transported to the affected joint area for targeted therapy.The treatment of CIA model mice showed that,compared with other control groups,FA-PEG-R-NPs@si ERN1effectively delayed the disease progression in CIA mice,maintained immune homeostasis,inhibited the level of inflammation,and protected cartilage from damage.4.The targeting of si ERN1 nanoprodrugs was again demonstrated in IBD model mice.The therapeutic effect shows that FA-PEG-R-NPs@si ERN1 can also effectively control the disease severity of IBD model mice,regulate the immune homeostasis,effectively inhibit the level of local and systemic inflammation,and protect the integrity of intestinal tissue structure and function.5.In terms of mechanism,the data show that IRE1αcan affect the calcium ion concentration in macrophages by interfering with IP3R1/3function,thereby regulating macrophage polarization and regulating body immune homeostasis.In addition,IRE1αcan regulate the inflammatory state of the body through the My D88-dependent TLR signaling pathway.ConclusionERN1 is involved in the occurrence and development of RA,and is a promising gene target.The dual-responsive macrophage-targeted nanocarriers were designed and constructed in this study were loaded with si ERN1,which effectively realized the targeted therapy of RA.It showed superior therapeutic effect,regulation of immune homeostasis and chondroprotection in CIA model mice.In addition,the universality of the si ERN1 nanoprodrugs was verified in IBD model mice,showing reliable disease control effects,effectively maintaining immune homeostasis,and protecting the structural and functional integrity of the gut.In conclusion,si ERN1 nanoprodrugs provide a novel strategy for the treatment of complex autoimmune inflammatory diseases.