In Vitro Anti-cancer Study On Co-delivered Nanoparticles Of Photosensitizer And Chemotherapeutics Based On Self-oxygenation Strategy

BackgroundCancer（malignant tumor）currently poses a serious threat to human health and has become one of the leading causes of death.Traditional cancer treatment methods include chemotherapy,radiotherapy and surgery.Moreover,some small-molecule chemotherapy drugs have also achieved some relatively good results in the treatment of cancer,but they can cause serious adverse reactions to normal tissues.Therefore,in order to overcome this difficulty,the combination of two or more anti-cancer drugs with different pharmacological mechanisms or different toxic and side effects for cancer treatment,that is,combined chemotherapy,has attracted much attention.Among the various treatment methods in recent decades,photodynamic therapy（PDT）relies on tissue oxygen and photosensitizer（PS）to generate reactive oxygen species（ROS）（such as singlet oxygen（¹O₂）,etc.）under the light beam.Treatment methods are widely concerned.Due to its unique advantages,it has recently been considered as one of the promising treatments for effective cancer treatment.However,considering the complex tumor structure and unique tumor microenvironment（TME）,the combination of chemotherapy and treatment using different drugs and/or mechanisms was proposed by the researchers because it can synergistically improve tumor treatment effects while reducing Side effects of cancer chemotherapy.Therefore,based on the concept of combined chemotherapy and the proposed nano-drug combined delivery system,the combination of PDT and chemotherapy to optimize the treatment of cancer treatment has attracted much attention.Malignant tumors include cancer cells and stromal cells,stromal cells such as endothelial cells,fibroblasts,and many immune cells,as well as the extracellular matrix（ECM）they produce.The complex formed by these complex structures is called the tumor microenvironment.Therefore,due to the deterioration of the microenvironment,abnormal blood vessels and uncontrolled proliferation of cancer cells will cause insufficient oxygen supply at the solid tumor site.According to reports,when the oxygen pressure is lower than 40 mm Hg,the efficiency of PDT for cancer treatment will be significantly reduced.Therefore,the therapeutic efficiency of PDT for cancer is still limited,and a new PDT strategy is urgently needed.AimWe designed a binary mixed nanoparticle co-delivery system and named it TPD&PZM.The system is formed by the physical mixing of two nanoparticles,and the surface of one of the nanoparticles is coated with Mn O₂.This material can trigger the decomposition of H₂O₂in the tumor microenvironment to produce O₂,which is used to relieve tumor hypoxia.Expect to improve the treatment efficiency of PDT.And the system was applied to breast cancer cell MCF-7 to detect its anti-cancer effect in vitro.In this study,we performed physical and chemical characterization of the nanosystem,cell uptake,in vitro ROS detection,and in vitro cytotoxicity experiments,hoping that it could alleviate tumor hypoxia and achieve a synergistic anti-cancer therapeutic effect.Methods1.Physical and chemical characterization:We prepared TP NPs and TPD NPs by the emulsion solvent volatilization method,prepared PP NPs and PZ NPs by the film dispersion method,and coated Mn O₂by the MES redox method.We detected the particle size and potential of TP NPs,TPD NPs,PP NPs,PZ NPs,and PZM NPs;then we observed the surface morphology and dispersion of several nanoparticles and nanomicelles using transmission electron microscopy;using ultraviolet spectrophotometry The meter measured the encapsulation rate and drug loading rate of the drug in nanoparticles and micelles,as well as the p H-sensitive in vitro release test;the nanoparticles and gel were detected by Fourier transform infrared spectroscopy（FTIR）and X ray diffraction（XRD）The structure of the beam is used to judge whether the drug is successfully loaded into the nanoparticles;and using X-ray photoelectron spectroscopy（XPS）to analyze whether the Mn O₂is successfully coated on the surface of PZ NPs.2.Cell uptake experiment:We prepared TPD NPs,using fluorescent labeling technology,using laser confocal microscope to observe the uptake of free DOX and TPD NPs in MCF-7 cells.3.Production of ROS in vitro:We prepared PZ NPs and PZM NPs,placed free Zn Pc,PZ NPs and PZM NPs with or without near-infrared light irradiation（NIR）,using H₂DCFH-DA detection kit for detection,and observed by fluorescence microscope The emergence of green fluorescence to judge the generation of ROS.4.In vitro cytotoxicity experiment:We first evaluated the safety of nanomaterials,and then detected the cytotoxicity of DOX,Zn Pc,TPD NPs,PZ NPs,PZM NPs,TPD NPs+PZ NPs,and TPD&PZM on MCF-7 cells,and then calculated the half-inhibitory concentrations respectively(IC₅₀)and Synergy Index(CI₅₀),the anti-cancer efficiency of the co-delivery system and the effect of improving PDT were evaluated.Results1.Transmission electron microscopy inspection results show that multiple nanoparticles present a uniform spherical structure,making them suitable and having good dispersibility.The results of Fourier infrared spectroscopy and X-ray diffraction show that multiple nanoparticles can be successfully synthesized and the analysis results of two X-ray photoelectron spectrometers show that the Mn ions on the surface of PZM NPs have a+IV valence,indicating the successful synthesis of Mn O₂.The results of in vitro drug release experiments show that the nanoparticles can respond to their p H and achieve controlled release of the drug.2.The results of cell uptake experiments showed that compared with nanoparticles,free DOX was taken up by cells poorly,and it was not possible to achieve good tumor accumulation.The effective accumulation will eventually achieve a good anti-cancer effect.3.In vitro ROS test results show that in the absence of infrared light,free drugs and nanoparticles did not detect the generation of ROS,indicating that photosensitizers cannot exert antitumor effects in the dark.However,when near-infrared light was added,both free drugs and nanoparticles produced ROS,and nanoparticles coated with Mn O₂produced the most ROS.4.In vitro cytotoxicity test results show that the two nanomaterials have low cytotoxicity to MCF-7 cells,which proves that the materials have certain safety.The results of drug-loaded nanoparticles on MCF-7 cytotoxicity showed that the combined delivery of the anti-cancer drug DOX and the photosensitizer Zn Pc had the lowest cell survival rate,indicating that the combined anti-cancer treatment effect is more significant.At the same time,the dosage of chemotherapy drugs can be reduced,thereby reducing toxic and side effects.ConclusionThe strategy of combining chemotherapy with PDT to improve the efficiency of cancer treatment has attracted widespread attention.In order to reduce the side effects of small-molecule chemotherapy drugs and overcome hypoxia at the tumor site to improve the efficacy of PDT treatment,in this study,we synthesized two types of nanoparticles and formed a binary mixed nanoparticle co-delivery system for breast Cancer treatment.We chose polylactic acid-glycolic acid（PLGA）,alpha-tocopherol polyethylene glycol 1000 succinate（TPGS）,methoxy polyethylene glycol-block poly-beta-amino ester（m PEG-PAE）and di Manganese oxide（Mn O₂）as a nanomaterial,respectively containing doxorubicin（DOX）and the photosensitizer zinc phthalocyanine（Zn Pc）We have characterized their physical and chemical properties and studied their anti-cancer effects on MCF-7 cells in vitro The results show that the nano-co-delivery system has high stability.Mn O₂can promote the decomposition of H₂O₂to produce O₂.This makes the tumor hypoxia relieved to improve the effect of PDT.More importantly,this improved PDT effect combined with the chemotherapy effect of DOX not only reduces the toxic and side effects of DOX,but also plays a joint anti-cancer effect.Compared with a single treatment method,the treatment effect of combined with chemotherapy and photodynamic therapy to relieve tumor hypoxia by self-generated oxygen is more satisfactory.