Utilizing Inorganic/Organic Nanomedicine-induced Reactive Oxygen Species To Enhance Oxidative Stress For Cancer Therapy

Research background:Cancer,a global health concern,poses a severe threat to human well-being.Traditional treatment methods such as surgery,chemotherapy,and radiotherapy,while partially controlling cancer progression,exhibit limitations.Emerging treatments like Chemodynamic therapy(CDT),Photodynamic therapy(PDT),immunotherapy,photothermal therapy,and sonodynamic therapy,offer new hope for cancer treatment.In recent years,CDT and PDT have garnered significant attention in cancer treatment,each possessing unique treatment principles and characteristics.In terms of material selection,CDT requires suitable nano-catalysts,while PDT necessitates photosensitizers.Currently,both therapies face challenges in their development.CDT confronts issues such as drug selectivity,drug resistance,and side effects,while PDT is limited by inadequate light penetration depth and poor water solubility of photosensitizers.Hence,the future direction lies in developing novel CDT/PDT agents,enhancing efficacy,minimizing side effects,and overcoming limitations in practical application.Research Objective:To improve the efficacy of tumor treatment,the development of novel CDT/PDT agents to enhance therapeutic efficacy and reduce side effects is an important direction for future development.This study focuses on CDT and PDT,designing and synthesizing novel inorganic and organic nanomaterials to explore their potential applications as CDT and PDT agents in cancer treatment.For CDT,we designed and synthesized hydrophobic cerium oxide(CeO2)nanoparticles as nanozymes to catalyze intracellular reactive oxygen species(ROS)generation.These nanoparticles were coated with amphiphilic copolymer 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)](DSPE-PEG)to obtain highly stable DSPEPEG-CeO2.These nanoparticles showed long-term stability under physiological conditions,exhibiting good colloidal stability and high catalytic activity,which is beneficial for storage and in vivo applications.DSPE-PEG-CeO2 were able to increase ROS levels and induce cell apoptosis in cancer cells,thereby enhancing the efficacy of CDT and achieving efficient tumor cell killing.For PDT,we designed and synthesized the asymmetric triphenylamine-based dyes TPCA and TPCT as novel organic photosensitizers.TPCA and TPCT,composed of triphenyl amine,cyano-substituted stilbene,and phenothiazine,exhibited strong dual-state emission,mechanofluorochromism,and low toxicity.Under light irradiation,they could generate a large amount of 1O2 efficiently killing cancer cells and treating tumors.These research findings provide new insights and directions for exploring novel treatment modalities,enhancing efficacy,reducing side effects,and overcoming challenges in cancer treatment.Experimental Methods:PartⅠ:1.Synthesis of nanoparticles:Oil-soluble CeO2 nanoparticles were first prepared using a one-pot thermal decomposition method to make their surface more modifiable,and then we used amphiphilic DSPE-PEG surfactants to encapsulate the nanoparticles to prepare DSPE-PEG-CeO2.2.Nanoparticle characterization:The nanoparticles were characterized using UV-vis spectroscopy,scanning electron microscopy,and dynamic light scattering to determine the particle size distribution,Zeta potential,and dispersion and stability in solution.3.Biological detection:After treating HeLa and HT1080 tumor cell lines with nanoparticles,the levels of ROS,cell apoptosis,cell viability,Fe2+levels,etc.,were detected using flow cytometry,CCK-8 assay,immunofluorescence staining,protein immunoblotting,and other methods.4.In vivo experiments:Tumor-bearing mouse experiments verified the therapeutic effects of nanoparticles on tumors and their effects on other organs.PartⅡ:1.Synthesis of organic photosensitizers:Triphenyl amine,cyanobiphenyl,and phenothiazine were selected as functional units to synthesize TPCA and TPCT as organic photosensitizers through nucleophilic substitution reactions,Suzuki-Miyaura coupling,and Knoevenagel reactions.2.Structural characterization:The structures of TPCA and TPCT were determined using 1H/13C NMR spectroscopy and high-resolution mass spectrometry to confirm the molecular structure and purity.3.Optical properties:UV-vis spectroscopy,fluorescence spectroscopy,and fluorescence quantum yield measurements were used to test the UV absorption,fluorescence emission,and fluorescence quantum yields of TPCA and TPCT in solution and solid states,characterizing their optical properties.4.Quantum calculations:Density functional theory(DFT)and time-dependent density functional theory(TDDFT)were used to optimize the geometric configurations of TPCA and TPCT and calculate the ground/excited state electron cloud distribution and energy gap,analyzing the electronic structure and charge transfer process of dye molecules.5.Mechano-responsive behavior:Solid powders were treated by mechanical grinding,and fluorescence spectroscopy was used to characterize the mechano-responsive behavior of TPCA and TPCT.6.Cytotoxicity experiments:MTT assay was used to test the cytotoxicity of the materials,and the relationship between material concentration and cell toxicity was confirmed.7.Lipid droplet imaging:Laser confocal microscopy was used to test the targeting of TPCA and TPCT to intracellular lipid droplets,and their dynamic imaging capability for observing lipid droplet metabolism was validated through starvation experiments and oleic acid stimulation experiments.8.In vivo experiments:Tumor-bearing mouse experiments and Hematoxylin and Eosin(HE)staining experiments were used to verify the therapeutic effects of the materials on tumors in animals and their effects on other tissues and organs.Experimental Results:1.The results of Part I research show that:(1)The synthesized DSPE-PEG-CeO2 have a size of 62 nm,a polydispersity index of 0.263,and a surface zeta potential of 15.3 mV(2)The UV-vis spectrum of DSPE-PEG-CeO2 shows a characteristic absorption peak at 299 nm,indicating that the synthesized nanoparticle system is homogeneous and does not contain other impurities.(3)Colloidal stability tests show that DSPE-PEG-CeO2 is stable when stored at 4℃,with the size of the nanoparticle colloidal solution remaining unchanged for 10 days,indicating that the nanoparticles have good stability.(4)Cell toxicity and proliferation experiments show that DSPE-PEG-CeO2 can induce morphological damage to HT1080 cells,reduce cell viability,and induce significant cell apoptosis.(5)Mechanistic studies show that DSPE-PEG-CeO2 induce a large amount of ROS production in cells,damaging intracellular DNA,disrupting mitochondrial membrane potential,and interfering with mitochondrial function,thereby killing cells.(7)In vivo tumor treatment experiments show that DSPE-PEG-CeO2 can effectively treat tumors in vivo without damaging other tissues and organs.2.The results of the Part Ⅱ research show that:(1)TPCA and TPCT have correct structures and do not contain impurities.(2)TPCA and TPCT are excellent dual-state luminescent dyes,showing strong emission in solution and solid states,with absolute fluorescence quantum yields(Φ)exceeding 37%.TPCA exhibits maximum yellow emission at 577 nm,with Φ of 48.6%,while TPCT exhibits maximum red emission at 621 nm,with Φ of 37.5%.(3)TPCA shows mechano-responsive fluorescence color change in its solid state,with a significant spectral shift(Δλ)of 41 nm,transitioning from yellow fluorescence to red fluorescence.TPCT does not exhibit mechano-responsive fluorescence color change properties.(4)TPCA and TPCT can target intracellular lipid droplets,enabling dynamic imaging of lipid droplet consumption and accumulation.(5)TPCA and TPCT can generate large amounts of 1O2 in cells,killing tumor cells.In vivo tumor treatment results show that these two dyes can effectively treat tumors under light conditions without damaging other tissues and organs.Conclusion:1.In this study,hydrophobic CeO2 nanoparticles were coated with DSPE-PEG surfactants,and a simple mixing-drying-rehydration method was adopted to obtain water-soluble DSPE-PEG-CeO2.DSPE-PEG-CeO2 have a uniform structure and size,maintaining their size unchanged for 10 days when stored at 4℃,showing high colloidal stability.2.DSPE-PEG-CeO2 has high surface activity and can effectively induce an increase in intracellular ROS levels through catalytic reactions,stimulating oxidative stress in tumor cells,efficiently killing tumor cells,and eliminating tumors in the body.3.DSPE-PEG-CeO2 damages DNA,disrupts mitochondrial membrane potential,and interferes with mitochondrial function by increasing intracellular reactive oxygen species levels,thereby killing cancer cells.4.TPCA and TPCT are triphenylamine,cyanophenylethylene,and phenothiazine-based organic dyes with an asymmetric D-π-A-π-D’ electronic structure.They exhibit strong fluorescence emission in dilute solutions and solid states,with a quantum yield exceeding 37%,making them typical excited-state fluorescent dyes.5.TPCA and TPCT have good environmental stimulus responsiveness.They are sensitive to solvent polarity,showing a significant redshift in their spectra as the solvent polarity increases,and their luminescence quantum yields gradually decrease.TPCA is sensitive to mechanical force,exhibiting significant mechanical fluorescence color change under shear force,changing from yellow to red,with a spectral redshift of 42 nm.6.TPCA and TPCT are excellent bioluminescent dyes and photosensitizers.They can specifically stain intracellular lipid droplets and efficiently generate 1O2 under light,stimulating oxidative stress in tumor cells and efficiently killing tumor cells.