Chemical Modification Enhances Tumor-targeted Colonization Of Probiotic E. Coli Nissle 1917 And Synthesis And Application Of Novel Bisacridine Nucleic Acid Fluorescent Dyes

Cancer is the main cause of death at present,and it is also one of the main obstacles to the improvement of human life expectancy in the 21st century.Traditional anticancer therapy,such as surgery,chemotherapy and radiotherapy,is still not ideal in curing cancer.(Origin of bacterial Therapy for Cancer)as a new means of cancer treatment,tumor bacterial therapy(Bacteria cancer therapy,BCT)has made great progress in laboratory research and clinical trials.BCT is mainly based on the advantage that facultative or specific anaerobes can naturally target tumor tissues and proliferate in tumor tissues.Through genetic engineering,bacteria are constructed to kill tumors by using bacteria as drug delivery carriers or expressing target proteins(such as cytotoxins,prodrug invertase,immunomodulators,etc.).However,because of the pathogenicity of the bacteria used,the clinical application of live bacteria in BCT is limited.EscherichiacolistrainNissle1917(EcN)is a kind of non-pathogenic probiotics.Because it has no pathogenicity to human body and greatly improves the safety of the application of live bacteria,it has been paid more and more attention in BCT.However,as a probiotic bacteria,the lipopolysaccharide(LPS)on the surface of EcN can trigger the immune response of the body,which leads to side effects such as inflammatory reaction.Therefore,it is necessary to modify the surface of EcN so as to reduce the immune response induced by BCT.Based on this,the photoinitiated electron transfer-reversible addition chain transfer polymerization(photo induced electron transfer-reversible addition-fragmentation chain-transfer polymerization,PET-RAFT)method was used to initiate the polymerization on the living EcN surface with 2(2-ethoxyethoxy ethyl acrylate)as the monomer and BTPA-NHS as the chain transfer agent,so that the bacterial surface was modified by graft polymer to obtain polymer-coated bacteria(Polymer Coated Bacteria,PCB).In order to verify the occurrence of bacterial surface polymerization,through the synthesis of fluorescent monomer and participation in the polymerization reaction,the fluorescence of bacterial surface was observed under laser fluorescence confocal microscope,indicating that the fluorescent monomer was modified on the bacterial surface.In order to verify that the inflammatory response of PCB to the body is less than that of EcN,and the retention of PCB in blood is more than that of EcN,injected the same amount of PCB and EcN,into normal mice,the blood routine and inflammatory factors were determined.The results showed that the blood routine and inflammatory factors of mice injected with PCB were closer to those of normal mice,and the content of PCB in blood was 9.4 times higher than that of EcN,.In order to verify the higher efficiency and longer customization time of bacterial colonization to tumor tissue,the same amount of PCB and EcN,were injected into tumor-bearing mice.It was observed that the time of PCB colonization in tumor tissue was 12 days,the time of EcN colonization in tumor tissue was 7 days,and the ratio of the number of PCB colonization in tumor tissue to the number of EcN colonization in tumor tissue was up to 234 times.The above experimental results show that chemically modified probiotics E.coli Nissle1917 can enhance the efficiency of targeted tumor colonization.As one of the most important macromolecules in organisms,nucleic acid needs specific methods and specific instraments to observe its morphological structure and other properties,such as staining nucleic acid,so as to study its shape,molecular size and so on.At present,there are many nucleic acid fluorescent dyes commonly used in the laboratory,but dyes such as ethidium bromide(EB),acridine orange(AO),thiazole orange and other dyes have high cytotoxicity and environmental pollution,while SYBR series dyes have low cytotoxicity,but they are expensive.In order to solve this problem,we designed and synthesized a green nucleic acid fluorescent dye with low price and low toxicity to human body.In this paper,a new type of nucleic acid fluorescent dye-GEP-Green was designed and synthesized using acridine orange as raw material.In the experiment of linear range of 0-4000 ng/mL DNA concentration,the fluorescence intensity is linear when the concentration of DNA is in the range of 31.25 ng/mL-500 ng/mL,and the linear equation is y=0.34x+8.55(R2=0.9812).In the staining experiment of living cells,the living cells were stained with the same concentration(0.3 ?mol/L)of GEP-Green staining solution and acridine orange staining solution at the same time.The staining results showed that the cells stained with acridine orange were stained with acridine orange,while the cells stained with GEP-Green staining solution were not stained,which indicated that the dye molecule would not enter the living cells to dye the nucleus.In the cell activity experiment,MTT experiments were carried out on different concentrations of GEP-Green dye solution and acridine orange dye solution.The results showed that the cell activity was significantly affected when the concentration of GEP-Green dye solution was 12 ?mol/L,while when the concentration of acridine orange dye solution was 1.5 ?mol/L,it had a significant effect on cell activity.In the gel staining experiment,at the same concentration(1.5?mol/L),the band of GEP-Green was clearly visible when the content of DNA was 2.5 ng,and the band of acridine orange was clearly visible when the content of DNA was 10 ng.The above experimental results show that GEP-Green is a non-toxic and economical nucleic acid fluorescent dye.