Intelligently Amphiphilic Nanodrugs For Tumor Targeted Therapy And Tumor Immunomodulation

Background:Bacteria,fungi,and viruses widely exist in various physiological cavities that communicate with the outside world.Among them,the relationship between gut microbiome and the tumors occurrence and development are the most systematic.The imbalance of microbiome can affect the occurrence and development of tumors through various ways.The effects of tumor chemotherapy and immunotherapy can be affected by the following progress,such as increasing of bacterial toxins,the remodelation of the immunosuppressive microenvironment by inflammatory factors,bacterial metabolites or related enzymes.In addition,commensal bacteria also exist in breast cancer,prostate cancer,esophageal cancer,gastric cancer,bowel cancer,pancreatic cancer,lung cancer,head and neck tumors,etc.in clinical pathological detection.Bacteria infiltrating tumors can affect the occurrence and development of tumors through direct or indirect effects.For example,Fusobaterium nucleatum can promote the resistance of colorectal cancer to chemotherapeutic drugs by regulating autophagy.Porphyromonas gingivalis can enter the pancreatic tissue of mice by swallowing and promote the occurrence and development of pancreatic cancer by changing the immune microenvironment.At present,antibiotics are one of the effective ways to remove pathogenic bacteria,but systemic administration of antibiotics can act on microorganisms in the body,which can easily cause the imbalance of gut microbiome whose diversity and abundance are difficult to restore.Compared with normal tissue,solid tumor tissue is heterogeneous.On the one hand,the tumor has abundant blood vessels,poor structural integrity,lack of lymphatic return,increased permeability.Which makes macromolecules,nano-sized particles,liposomes,etc.retained selectively,called enhanced permeability and retention effect.On the other hand,tumor tissue is acidic,glutathione level up-regulated and oxygen content decreased.Objective:In view of the characteristics of bacterial-infiltrating colorectal tumors,this project designed the hydrophobic antibiotic metronidazole(MTI)and the hydrophilic antitumor drug 5-fluorouridine(FDU)to form nanoparticles.They are linked by disulfide bonds to form amphiphilic small molecules by chemical synthesis,which will self-assemble into MTI-FDU nanoparticles in water through hydrophilic and hydrophobic interactions.To study its therapeutic effect on bacterial-infiltrating tumors and its regulating effect on tumor immune microenvironment without disturbance of normal microbiome.Methods:In this study,35 samples of human colorectal cancer and para-tumor normal tissues were collected.FISH technology was used to detect the degree of Fn infiltration in both,and immunohistochemical technology was used to detect the bacterial metabolite lipopolysaccharide in cancer and para-tumor normal tissues.The synthesis of MTI-FDU amphiphilic molecule was confirmed by HPLC,1H NMR,MS and UV-Vis.The particle size and morphology were detected and observed by DLS and TEM.The microplate reader was used to detect the absorbance of bacteria treated with MTI-FDU nano-drugs,the plane and Z-axis structure of biofilms stained by SYTO after drug treatment were photographed by CLSM,and the drug-sensitive Disk diffusion drug sensitivity test was used to detect the inhibitory effect of nano-drugs on bacterial.The inhibitory effect of nanomedicine on tumor cells was detected by CCK8 method,the effect of nanomedicine on tumor cell apoptosis was determined by Annexin V/PI staining,and the effect of nanomedicine on tumor cell migration ability was detected by Transwell assay.Small animal imaging to observe the tumor-targeting ability of nanomedicines.Two mouse models were constructed:colorectal cancer spontaneous model(AD/Fn)and colorectal cancer axillary model to evaluate the bacteriostatic and tumor-inhibiting abilities of nanomedicines.Flow cytometry was used to detect the level of immune cells infiltrated in tumor of mice.Elisa technology detects the expression levels of immune-related factors after drug treatment by nanodrugs.The changes of TLR4 and β-catenin were detected by immunohistochemistry.Results:Compared with the normal tissues,the level of Fn infiltration in colorectal cancer tissues and LPS level was significantly increased,the decreased patient survival rate was bound up with Fn.This project successfully designed and synthesized MTI-FDU nanoparticles with a size of 105±38nm,which has excellent target ability to tumor tissue.The original drug can be successfully released in a reducing environment.In vitro,it has a good inhibitory effect on Fn biofilm and colorectal cancer cells,MTI-FDU promoted cancer cell apoptosis rate and inhibit their migration ability.In vivo,MTI-FDU nanomedicine has obvious inhibitory effect on bacterial-infiltrating spontaneous colorectal cancer and axillary colorectal tumors and remove the bacteria infiltrating the tumor tissue without affecting the balance of gut microbiome.It could promote the increase of CD8+T cells,the decrease of Tregs and MDSC.Meanwhile MTI-FDU down-regulated cytokines IL-6,IL-22 and TNF-α.At protein level,nanomedicine affects the TLR4/β-catenin signaling pathway by inhibiting Fn,which decreases the expression of TLR4 and β-catenin in tumor tissue.Conclusion:MTI-FDU nanoparticles were successfully designed and synthesized in this study,which have significant inhibitory effects on Fn and colorectal tumor cells.It has excellent tumor inhibition and intratumoral bacteria removal ability on 2 types of colorectal tumor mouse models and did not cause gut microbiome imbalance.At the same time,it is also proved that the nanomedicine affects the TLR4/β-catenin signaling pathway by inhibiting Fn.In addition,MTI-FDU nanomedicines can modulate the inhibitory tumor immune microenvironment and reverse the changes induced by Fn in immune cells and cytokine.It is expected to provide innovative therapeutic drugs for tumor patients with confirmed intratumoral bacterial infiltration in clinic and pathology.This research will open up a new window for tumor nanomedicine research.Background: In recent years,studies have proved that many traditional anticancer drugs or targeted drugs can fully expose tumor cell antigens,release damage-associated molecular patterns(DMAP),and activate DCs in the process of killing tumors.DC cells present antigens to cytotoxic T cells and natural killer cells to regulate the tumor microenvironment,including cell composition,cytokines release,and cell flinction in tumor microenvironment,thereby activating the anti-tumor immune process.This process of tumor death is called tumor immunogenic cell death(ICD).During ICD progress,the Meat-me" signal is released,and the calretictdin(CRT)contained in the endoplasmic reticulum of tumor cells migrates to the surface of the cell membrane,and the high mobility group protein B1(HMGB1)is released into the cytoplasm and extracellular,and adenosine triphosphate(ATP)is released outside the cells,etc.The release of these signals is first step and important sign for ICD to activate the bodyfs anti-tumor immunity.Lactose(Lac)and its derivatives can combine with asialoglycoprotein receptor(ASGPR)on the surface of hepatocytes to deliver drugs into cells.Considered with the EPR effect of tumors,hydrophilic lactose linked the hydrophobic antitumor drug doxorubicin(DOX)to form amphiphilic small molecule(Lac-DOX).After self-assembling and DOX loaded to obtain Lac-DOX/DOX nanoparticles,which can be targeted to liver cancer tissue,and responsive drug release in the acidic environment of liver cancer,with excellent tumor treatment effect.Objective: To study whether the Lac-DOX/DOX nanoparticles constructed by the team could induce ICD phenomenon,the release of DAMPs such as CRT migration exposure,HMGB1 protein release,and ATP release was evaluated.Methods: Transcriptome sequencing was used to analyze the differential genes between Lac-DOX/DOX nanoparticles and DOX,GO clustering was used to analyze the cellular functions related to the differential genes,flow cytometry was used to detect the exposure of CRT protein on the cell membrane surface,and confocal microscope was used to photograph HMGB1 protein release progress,and microplate reader and ATP detection kit were used to detect the ATP release of tumor cells.Results: 9494 differential genes were found by sequencing the transcriptomes of Huh-7 cells treated with DOX and Lac-DOX/DOX.GO functional clustering analysis indicated that the functional changes caused by Lac-DOX/DOX nanomedicines were closely related to endoplasmic reticulum stress.The CRT migration rate was same with that cause by positive control drug,and the red fluorescence signal of HMGB 1 protein increased,which had migrated from the nucleus to the cytoplasm,and the release of ATP was significantly increased.Conclusion: Combining the above three indicators of CRT protein exposure,ATP release and HMGB1 protein release,it can be shown that after Lac-DOX/DOX nanomedicine treatment,dying Huh-7 cells will release DAMPs and induce the occurrence of ICD.It is beneficial to present DAMPs to DC cells,which is expected to trigger the body’s anti-tumor immunity.The in-depth understanding of this phenomenon will help the combination of chemotherapy and immunotherapy to obtain excellent liver cancer treatment effects.