Study On The Treatment Of Multifunctional Biomimetic Nanomaterials For Ulcerative Colitis And Bladder Cancer As Well As The Underlying Mechanisms

Ulcerative colitis（UC）and bladder cancer are diseases that seriously threaten human health.Currently,although treatments for UC can alleviate symptoms,they are fraught with many drawbacks,such as significant side effects from the medications,low bioavailability,poor patient compliance,and high recurrence rate.The primary clinical treatments for bladder cancer include surgical resection,chemotherapy,radiotherapy,and immunotherapy.Despite each method having its characteristics,due to high metastasis and recurrence rates,multiple drug resistances of bladder cancer,and poor patient prognosis,the efficacy of a single treatment method often fails to meet clinical needs.Therefore,there is an urgent need to develop new therapeutic technologies to further enhance the treatment effects of UC and bladder cancer,effectively improving patients’quality of life.With the continuous development of nanotechnology,biomimetic nanomaterials have shown great potential in the field of disease treatment.Through rational design and preparation of biomimetic nanomaterials with specific functions,precise targeted drug delivery and controlled release can be achieved,allowing the drug to act directly on the lesion,reducing damage to healthy tissues,and lowering toxicity and side effects of the medication,thereby enhancing the treatment effects of the disease.Additionally,different types of biomimetic nanomaterials can be prepared based on the patient’s condition and individual differences,realizing personalized treatment and providing more accurate and effective treatment strategies.Based on this,UC and bladder cancer were selected as research subjects in the study.According to the characteristics of these two diseases,a series of biomimetic nanomaterials with different functions have been developed,and their performance has been explored.On this basis,the effect of the prepared biomimetic nanomaterials in treating UC and bladder cancer has been systematically studied at the cellular and animal levels,and the related mechanisms of action have been explored,achieving good results.The main research findings were as follows:1.Firstly,two cluster enzymes,Au₂₄Cu₁ and Au₂₄Cd₁（collectively named AU）,were synthesized using a single-atom substitution method,exhibiting highly efficienty enzyme-like activities（superoxide dismutase/catalase）.Various modern analytical techniques were used to characterize the prepared Au₂₄Cu₁ and Au₂₄Cd,confirming their successful synthesis and validating the enzyme-like activity of AU.Subsequently,AU was loaded into liposomes using hydration induction,sonication,and extrusion methods to prepare AU-LIP,which was then further characterized for analyses.The obtained AU-LIP was then disguised using a fusion membrane（denoted as CM）of neutrophil and erythrocyte membranes through a series of sonication and extrusion processes,leading to the creation of the biomimetic nanomaterial with inflammation targeting function,AU-LIP-CM.which was characterized by various analytical techniques to confirm its successful preparation.To mimic the state of cells during inflammation,RAW264.7 and Caco-2 cells were stimulated with lipopolysaccharide（LPS）and H₂O₂,respectively,to construct a cell oxidative damage model.The reactive oxygen species（ROS）scavenging ability of AU-LIP-CM was analyzed at the cellular level.Fluorescence microscopy imaging of ROS revealed that cells stimulated by inflammation showed a higher ROS fluorescence intensity,and it significantly decreased after AU-LIP-CM treatment,indicating the strong ROS scavenging capability of this biomimetic nanomaterial AU-LIP-CM.Additionally,LPS and Cp G oligodeoxynucleotides were used to induce inflammatory damage in RAW264.7 cells,and the anti-inflammatory activity of AU-LIP-CM was evaluated based on the expression levels of the inflammatory cytokine TNF-α.The results showed that cells treated with AU-LIP-CM significantly inhibited the secretion of the pro-inflammatory cytokine TNF-α,demonstrating the good anti-inflammatory effects of AU-LIP-CM and providing a basis for further studies in vivo UC treatment in animals.2.C57BL/6N mice were selected as experimental animals to construct a mouse model of UC.Combined with near infrared second window imaging technology,the dynamic distribution and therapeutic effect of AU-LIP-CM on UC mice were investigated.The results demonstrated that the biomimetic nanomaterial with inflammation targeting and immune evasion properties achieved efficient ROS scavenging in the UC mouse model.It effectively inhibited weight loss and shortening of the colon in mice,significantly reduced the disease activity index and H₂O₂ levels,repaired the intestinal barrier,increased the number of goblet cells and mucosal layer levels,and upregulated the expression of tight junction proteins,thereby realizing efficient treatment of UC,surpassing the therapeutic effect of5-aminosalicylic acid,and was a widely used first-line drug for UC treatment.Furthermore,to reveal the potential mechanism of AU-LIP-CM for the efficient treatment of UC,ROS immunofluorescence staining and fluorescence quantitative PCR detection of cytokine m RNA level were conducted on mouse colon tissues.The results showed that AU-LIP-CM could efficiently scavenge ROS in the inflamed colon tissues,thereby enhancing the secretion of anti-inflammatory cytokines（IL-10and TGF-β）,inhibiting the secretion of pro-inflammatory cytokines（TNF-α,IL-6,IL-1β,and IFN-γ）,and alleviating inflammation.Additionally,at the genetic level,the mechanism of action of AU-LIP-CM for the treatment of UC was explored.Transcriptomic analysis results showed that AU-LIP-CM could change the gene characteristics of UC mice in response to ROS,restoring them to the levels of normal mice.Moreover,combined with functional information,results of the Kyoto encyclopedia of genes and genomes（KEGG）pathway enrichment analysis were screened to identify signaling pathways highly related to the therapeutic mechanism of action of AU-LIP-CM.Finally,to study the regulatory effect of AU-LIP-CM on the intestinal flora,16S r RNA microbiome sequencing and bioinformatics analysis were conducted on the collected mouse feces.The results showed that AU-LIP-CM could to some extent restore the diversity,richness,and evenness of the intestinal flora in UC mice,improve the relative abundance ratio of beneficial bacteria（i.e.,Bacteroidetes）to harmful bacteria（i.e.,Proteobacteria and Escherichia-Shigella）,successfully improving the ecological structure of the intestinal flora,further protecting the intestines.3.The preparation conditions for the reaction of Fe Cl₃?6H₂O and tannic acid（TA）to form Fe-TA were firstly optimized and then the chemotherapy drug doxorubicin（DOX）and glucose oxidase（GOx）were loaded into the Fe-TA metal-phenolic network structure by a one-step synthesis method to prepare Fe-TA-DOX-GOx.A stable transfected MB49 cell line with PD-L1 knockdown was constructed using lentiviral transfection,and plasmid transfection was performed on HEK293T cells to obtain PD-1 overexpressing HEK293T cells.Subsequently,cell membranes were separately obtained from these two types of cells to prepare a fusion membrane（denoted as CM）.The fusion cell membrane CM was coated onto Fe-TA-DOX-GOx by an ultrasonic method to obtain the biomimetic nanomaterial Fe-TA-DOX-GOx-CM.This material was characterized by various analytical techniques and its functionality was evaluated.The results indicated that this biomimetic nanomaterial Fe-TA-DOX-GOx-CM possesses dual-responsive release properties of p H and glutathione and dual tumor targeting abilities（homologous targeting and PD-1/PD-L1 interaction-mediated targeting）.Additionally,it has the capacity for quintuple synergistic antitumor therapy based on ferroptosis/starvation therapy/chemotherapy/photothermal therapy/immune checkpoint blockade therapy.The superiority of this biomimetic nanomaterial in vitro antitumor effects and its ability to induce immune responses were also assessed.The results showed that under laser irradiation,Fe-TA-DOX-GOx-CM demonstrated an effective synergistic tumor treatment effect,which could maximize the maturation of dendritic cells,activate CD8⁺T cells,and induce a strong tumor-specific immune response,laying a preliminary research foundation for its application in the treatment of in vivo bladder cancer.4.C57BL/6N mice were selected as experimental animals,and a bladder cancer mouse model was established to investigate the targeting behavior and photothermal conversion effect of Fe-TA-DOX-GOx-CM by an infrared thermal imager.The results showed that the coating of the fusion cell membrane was helpful to improve the aggregation of materials within tumor cells and enhance the photothermal conversion effect.Subsequently,a bilateral subcutaneous bladder cancer model in mice was constructed to comprehensively evaluate the therapeutic effects of different materials on bladder cancer.Observing the bilateral tumor growth in mice,the Fe-TA-DOX-GOx-CM group exhibited the best antitumor effect.Within 12 days post-treatment,the primary tumors in this group completely disappeared,and no tumor recurrence was observed during the subsequent 19-day observation period.The size of the distal tumors was also the smallest among all treatment groups.Additional analyses of the primary tumor sections revealed that tumor cells in the mice treated with laser and Fe-TA-DOX-GOx-CM exhibited the most severe damage,most apparent apoptosis,and the strongest inhibition.These results demonstrated the powerful functionality of the biomimetic nanomaterial with dual tumor targeting and a quintuple synergistic treatment effect,effectively eliminating primary tumors and inhibiting the growth of distal tumors.Furthermore,the mechanism of the material’s antitumor action was explored.It was found that post-treatment with laser and Fe-TA-DOX-GOx-CM released damage-associated molecular patterns,promoted the maturation of dendritic cells,presented antigens to activate T cells,and triggered an adaptive antitumor immune response to attack tumor cells in the body.Moreover,it reprogrammed the immunosuppressive tumor microenvironment,enhancing the adaptive antitumor immune response against primary and distal tumors.Finally,the experiments on preventing lung metastasis and recurrence showed that the generated antitumor immune response effectively achieved immune memory effect,inhibiting tumor recurrence and metastasis to a certain extent,and having a certain protective effect.