The Study On The Antimicrobial And Antitumor Effects Of Cu-Cy NPs With Potassium Iodide Mediated Photodynamic Therapy And Mechanism

Infection threats to human health,the incidence of drug resistant to multiple antibiotics is also increased year by year,bring certain difficulties and challenges in the clinical treatment.bacteria resistant to antibiotics is currently facing the worldwide difficult problem.Liver cancer,mainly hepatocellular carcinoma,is the fifth most common cancer in men and the eighth most common cancer in women.About 700,000 deaths are reported every year,making it the second and sixth leading cause of death among men and women respectively.the main treatment of primary liver cancer,whether surgical treatment or liver transplantation,due to the existence of some special factors,the treatment options are very limited.Photodynamic treatment(PDT)is an alternative therapy with anti-cancer therapeutic potential.It has become an important method for the treatment of a variety of cancers because of its characteristics such as small toxicity and adverse reactions,small trauma,accurate efficacy,no drug resistance,and the combination application with a variety of therapies.At present,the light source and photoconductive technology of PDT have been well solved,making photosensitizer has become the most critical problem in the field of PDT.It is very important to actively develop and research new photosensitive materials with excellent photosensitive properties.Cu-Cy NPs is a new type of nanomaterial which developed by Prof.Chen’s research team in 2016.It has unique characteristics:it can generate and release ROS by ultraviolet light,X-ray,microwave excitation,or ultrasound.It has low toxicity,easy synthesis,high cost performance,strong luminescence,small particles with good water solubility and absorbability and other characteristics.Potassium iodide(KI)is non-toxic,FDA-approved and commonly used as a health supplement to avoid iodine deficiency.KI is a compound made of 76%halogen iodine and 23%potassium,an alkali metal.Potassium iodide has been reported to enhance the antimicrobial APDT efficacy of the photosensitizers methylene blue(MB)and neomethylene blue(NMB)for the treatment of dental infections and the antimicrobial treatment of sinusitis.In summary,the clinical application of Cu-Cy NPs is still in the early stages of research.this project based on the principle of PDT in bacterial therapy as the basis of experimental research,to explore the effect of Cu-Cy NPs in antibacterial treatment;Based on the fact that other photo-sensitizers can be combined with KI to enhance the antimicrobial effect,we will further investigate whether KI also enhances the antimicrobial effect of Cu-Cy NPs to further explore the mechanism;Given that Cu-Cy NPs itself is known to have PDT antitumor effect,we will further explore whether KI can also enhance the PDT antitumor effect of this photosensitizer;To find a new solution for clinical bacterial multi-drug resistance,avoid the risk of tumor surgical treatment,improve the cure rate and reduce adverse reactions to explore a new feasible combination treatment measures.Part Ⅰ:The effect and mechanism of Cu-Cy NPs combined with KI mediated photodynamic therapy on Gram-positive and Gram-negative bacteriumPurposeTo evaluate the effect of KI on the photodynamic antimicrobial activity mediated by Cu-Cy NPs,and to explore its mechanism.Methods1.Cu-Cy NPs nanomaterials were synthesized by chemical method.The physical characteristics of Cu-Cy NPs were characterized by ultraviolet light excitation luminescence method,transmission electron microscopy,fluorescence and absorption spectrometers.2.The changes of singlet oxygen(1O2)produced by Cu-Cy NPs and KI were detected by RNO decolorization test.3.Colony formation assay was used to detect the killing effect of Cu-Cy NPs and KI on MRSA and E.coli.4.The effect of KI on the bactericidal activity of Cu-Cy NPs mediated PDT was detected by colony formation assay.5.Divalent copper ions were detected by electron spin resonance wave spectrometer(ESR).6.The effect of KI on 1O2 produced by Cu-Cy NPS mediated PDT was detected by RNO decolorization experiment.7.Amplex red/HRP fluorescence assay,iodine molecular starch assay and nitroblue tetraazolium(NBT)assay were used to determine the effects of KI on the enhancement of Cu-Cy NPs-mediated PDT on H2O2,iodine and superoxide.8.The effects of KI on the absorption and emission spectra of Cu-Cy NPs-mediated PDT were detected by UV-spectrophotometer and fluorescence spectrometer.Results1.Cu-Cy NPs characterization:Cu-Cy NPs solution is colorless and transparent under natural light,and can produce pink fluorescence under ultraviolet light irradiation.2.The maximum excitation wavelength of Cu-Cy NPs was 360nm,and the strongest emission wavelength was 607nm.The average particle size of Cu-Cy NPs is about 93±41nm,and the spherical shape of Cu-Cy NPs is highly crystalline.The polycrystalline electron diffraction pattern proves that Cu-Cy NPs nanomaterials have single crystal structure.3.Cu-Cy NPs has a strong absorption value in the ultraviolet region,the peak value is about 365nm,Cu-Cy NPs can produce 1O2 under the excitation of ultraviolet light.4.The antimicrobial effect of Cu-Cy NPs-mediated PDT could be activated by ultraviolet light.The single Cu-CyNPs-mediated PDT had obvious bactericidal effect on MRSA,but no bactericidal effect on E.coli.5.KI enhanced the bactericidal effect of Cu-Cy NPS mediated PDT on MRS A and E.coli.6.ESR was negative for the detection of divalence copper ions,and RNO decolorization showed that KI had a quenching effect on 1O2 produced by Cu-Cy NPs-mediated PDT.7.KI has a quenching effect on 1O2 produced by Cu-Cy NPS mediated PDT.8.KI increased the effect of Cu-Cy NPS mediated PDT in the detection of H2O2,iodine triions,1O2,appeared a new peak at 280-290nm.Conclusion1.Cu-Cy NPs combined with potassium iodide is effective against MRSA and E.coli.This improved the efficacy of Cu-Cy NPs alone in PDT therapy against Gram-negative bacteria.2.There was no copper release in the mixed solution of Cu-Cy NPs and KI,denying the Feton reaction caused by copper ions.3.The main mechanism of Cu-Cy NPs combined with potassium iodide enhancing bactericidal capacity is the generation of hydrogen peroxide,iodine triion and singlet oxygen.Part Ⅱ:Potassium iodide enhances the photodynamic antitumor effect of Cu-Cy NPs by mediating p53 to regulate mitochondrial apoptosis pathwayPurposeTo investigate the effect of potassium iodide on the photodynamic antitumor activity mediated by Cu-Cy NPs,to further explore its mechanism.Methods1.The physical characteristics of Cu-Cy NPs were characterized by the chemical synthesis,through ultraviolet illumination excitation luminescence method,transmission electron microscopy measurement of polycrystalline electron diffractive color,fluorescence spectrograph and absorption spectrograph,which were the same as those in Part Ⅰ.2.The experimental parameters of Cu-Cy NPs,UV and KI were explored by MTT cytotoxicity assay to further detect the cell activity of each group.3.Cell cloning assay was used to evaluate the proliferation ability of each group.4.Cell migration ability was evaluated by cell scratch test.5.Calcein-AM and EthD-1 dual fluorescence staining were used for cell Live/Dead assessment.6.The cell apoptosis was preliminarily determined and observed by DAPI staining.7.Cell apoptosis rate was determined by Annexin V-FITC/PI flow cytometry.8.Reactve oxygen species(ROS)detection kit was used to detect intracellular ROS production level by flow cytometry.9.The effect of Cu-Cy NPS mediated PDT on mitochondrial respiratory function was detected by Seahorse assay.10.The effect of Cu-Cy NPS mediated PDT on the change of Mitochondria membrane potential(MMP)was detected.11.The effect of Cu-Cy NPS-mediated PDT on the mRNA expression levels of p53,Bax,Bcl-2,Survivin,Caspase-3 and Caspase-9 in HepG2 cells was detected by Reverse transcription Polymerase chain reaction(PCR).12.Western blot(WB)was used to detect the effects of Cu-Cy NPS-mediated PDT on the expression levels of p53,Bax,Bcl-2,Survivin,Caspase-3 and Casepase-9 in HepG2 cells and tissues.13.Subcutaneous tumorigenesis test of nude mice,HE staining,immunohistochemical staining method was used to further verify the experimental effect by extracting tissue proteins.Results1.Cu-Cy NPs is relatively safe and has no toxic effect on cells when the concentration of Cu-Cy NPs is less than 50μg/ml.2.The experimental parameters were obtained by MTT experiment:Cu-Cy NPs 12.5μg/mL,KI 3.75mg/mL,UV irradiation for 5min.3.In the HepG2,Hep3B and Huh7 cell lines,Cu-Cy NPs reduced the cell activity after UV irradiation,and the effect was stronger after KI addition,with statistical difference between them.And the effect is stronger in HepG2.It had no effect on the activity of HL-02 normal cells.It was further demonstrated that PDT was selective and specific to tumor tissue.4.Cu-Cy NPs mediated PDT inhibited cell migration,and the inhibitory effect was significantly enhanced with KI addition,with statistical significance(P<0.05).5.Cu-Cy NPs mediated PDT decreased the clonal ability of cells,and the clonal ability decreased more obviously after KI addition.6.Cu-Cy NPs-mediated PDT can make HepG2 cell nuclei condensed and reduced,marginalized,nuclear membrane cleaved into blocks or apoptotic bodies and other typical cell morphological characteristics of apoptosis,and the apoptosis was more obvious with the addition of KI.7.Cu-Cy NPs mediated PDT induced mitochondrial respiratory dysfunction,resulting in a decrease in ATP,which was more pronounced with the addition of KI.8.Cu-Cy NPs-mediated PDT induced a decrease in mitochondrial membrane potential(MMP),and the decrease was more significant with the addition of KI.9.Cu-Cy NPs mediated PDT increased intracellular production of reactive oxides(ROS).KI can enhance this effect.10.Cu-Cy NPS-mediated PDT induced increased expression of p53,Casepase3 and 9,and decreased expression of Bcl-2/Bax and Survivin in HepG2 cells.11.WB results of cells and tissues showed that Cu-Cy NPs combined with KI-mediated PDT increased the expression of p53,Bax,Casepase-3 and Casepase-9 proteins,and decreased the expression of Bcl-2 and Survivin proteins.KI can enhance this effect.12.The tumor transplantation model in nude mice was successfully established,and Cu-Cy NPs mediated PDT to inhibit the growth of HepG2 tumor tissue.In the center of the tumor model,the liver,spleen,lung and kidney organs were not damaged.Immunohistochemistry indicated the expression of Pro-Casepase-3.The effect of KI is more obvious.Conclusion1.Cu-Cy NPs mediated PDT decreased the activity of HepG2 cells,inhibited cell proliferation,migration and cloning ability,and resulted in decreased cell survival rate,and enhanced the effect of KI.2.Cu-Cy NPs mediated PDT induced HepG2 cell apoptosis,and the apoptosis was enhanced with KI,with statistically significant difference(P<0.05).3.Cu-Cy NPs mediated PDT increased intracellular ROS,decreased mitochondrial membrane potential,decreased ATP production,and impaired respiratory function.The effect of adding KI is obvious.4.Cu-Cy NPS mediated PDT induced p53 upregulation in HepG2 cells and tissues,Survivin decline,and caspase-3,caspase-9,and Bcl-2/Bax activation.The effect of adding KI is obvious.5.HepG2 tumor model was successfully established.KI combined with Cu-Cy NPs mediated PDT to inhibit the growth of tumor cells,and there was no damage to animal organs.6.KI enhances the antitumor effect of Cu-Cy NPs mediated PDT and provides a new strategy for combined treatment of PDT.