Construction Of Porphyrin-Based Novel Bifunctional Nanocomposites And Its Application In Tumor Photodynamic-Chemodynamic Synergistic Therapy

Reactive oxygen species(ROS)mainly includes the hydroxyl radicals(·OH)and superoxide free radical(O2^·-),singlet oxygen(¹O₂),etc.Under the normal level,ROS participate in a variety of signal transduction and regulation in cells,plays an important role in physiological regulation,However,when ROS level increases,important macromolecular substances such as nucleic acids,proteins and lipids in cells will be damaged,leading to cell apoptosis or canceration.With the increase of ROS level in cancer cells,the scavenging rate of ROS also increased to maintain the balance of redox in cells.Therefore,cancer cells are more sensitive to exogenous ROS.Photodynamic therapy(PDT)and chemodynamic therapy(CDT)are two common treatment methods that utilize exogenous ROS.Due to their high selectivity,minimally invasive and controllable properties,PDT and CDT have great development prospects in tumor treatment.However,the long-term uncontrolled growth of tumor tissue leads to insufficient oxygen supply to blood vessels,leading to the gradual growth of solid tumors into hypoxic microenvironment,which seriously limits the therapeutic effect of PDT.At the same time,factors such as the slightly acidic environment of tumor cells and insufficient H₂O₂ content also limit the efficacy of CDT treatment to a certain extent.Therefore,it is of great significance to increase intracellular O₂ and H₂O₂ content and overcome the therapeutic limitations caused by tumor microenvironment in the treatment of cancer.In this study,we designed and synthesized a nanocomposite material that can solve the limitation of tumor microenvironment for improving tumor hypoxia and increasing the content of H₂O₂ in tumor.After combining PDT with CDT,we conducted anti-tumor therapy to evaluate its anti-tumor effect in vivo and in vitro.The specific research content is as follows:(1)Preparation and properties of PCN-224@CaO₂-CuO₂@PEG bifunctional nanoparticlesFirstly,porphyrin nanoparticle PCN-224 with particle size of about 90 nm were synthesized by hydrothermal synthesis,using meso-Tetra-(4-carboxyphenyl)porphine,zirconium oxychloride octahydrate and benzoic acid as raw materials.On the surface of PCN-224 nanoparticles,Ca(OH)₂ and Cu(OH)₂ nanometer layers were grown successively in situ,and then enough H₂O₂ was added to form PCN-224@CaO₂-CuO₂nanocomposite particles.In order to slow down the decomposition of CaO₂ and increase the water-solubility of the nanocomposite,we continued to coat the surface of the nanoparticles with a layer of PEG to prepare PCN-224@CaO₂-CuO₂@PEG nanoparticles,using a similar synthesis route to prepare PCN-224@CuO₂@PEG.The synthesized nanomaterials were characterized by scanning electron microscopy(SEM),transmission electron microscopy(TEM),X-ray photoelectron spectroscopy(XPS),X-ray diffractometry(XRD),multi-angle particle size analysis and Zeta potentiometer,UV-vis.The results show that PCN-224,PCN-224@CuO₂@PEG and PCN-224@CaO₂-CuO₂@PEG nanoparticles have been successfully prepared.The nanocomposite has a uniform morphology and good dispersibility.The singlet oxygen(¹O₂)and hydroxyl radical(·OH)generation capacity of the synthesized nanomaterials were studied.The results show that compared with PCN-224,PCN-224@CuO₂@PEG,H₂O₂ and O₂released by CaO₂ in PCN-224@CaO₂-CuO₂@PEG nanocomposite can further increase the content of·OH under photodynamic action or chemodynamic action.Finally,using reactive oxygen species fluorescent probe(DCFH-DA),we studied the ROS production capacity of PCN-224@CuO₂@PEG and PCN-224@CaO₂-CuO₂@PEG under the synergistic effect of photodynamic and chemodynamic under hypoxia conditions.The results showed that the content of ROS produced by PCN-224@CaO₂-CuO₂@PEG nanoparticles was significantly higher than that of PCN-224@CuO₂@PEG under hypoxia conditions,indicating that the ROS production capacity of PCN-224@CaO₂-CuO₂@PEG nanoparticles was highly efficient.(2)The antitumor effect of PCN-224@CaO₂-CuO₂@PEG bifunctional nanoparticles in vitroHFF-1,A549 and MCF-7 cells were cultured in vitro.The cytotoxicity of PCN-224@CaO₂-CuO₂@PEG nanoparticles to HFF-1,A549 and MCF-7 was studied by MTT colorimetry.Then MCF-7 cells were used as experimental cells to study and compare the fluorescence intensity of PCN-224,PCN-224@CuO₂@PEG and PCN-224@CaO₂-CuO₂@PEG nanoparticles in MCF-7 cancer cells under hypoxia and normal oxygen environment by using reactive oxygen species fluorescence probe(DCFH-DA)The anti-tumor effects of the above nanoparticles were studied by MTT colorimetry.Finally,the apoptosis of MCF-7 cells was studied by flow cytometry..The results showed that when the concentration of PCN-224@CaO₂-CuO₂@PEG nanoparticles porphyrin ligand(TCPP)in cell culture medium was less than 8?g/m L,the normal cells showed higher tolerance to the PCN-224@CaO₂-CuO₂@PEG nanoparticles than the cancer cells.The fluorescence of PCN-224@CaO₂-CuO₂@PEG nanoparticles was strongest in MCF-7cancer cells under both normal and hypoxia conditions.The results showed that PCN-224@CaO₂-CuO₂@PEG still had high ROS production capacity in cells At the same time,after laser irradiation,the tumor inhibition rate of PCN-224@CaO₂-CuO₂@PEG nanoparticles was about 80%in both normal and hypoxia environments,indicating that the nanocomplex has a strong tumor inhibition effect in vitro.Finally,the results of the flow cytometer showed that after laser irradiation,the apoptosis rate of the PCN-224@CaO₂-CuO₂@PEG group was about 75%,which again proved that the nanoparticles had an high anti-tumor effect.(3)The antitumor effect of PCN-224@CaO₂-CuO₂@PEG bifunctional nanoparticles in vivoAfter the tumor model was established in BALB/c female nude mice using 4T1tumor cells,PCN-224,PCN-224@CuO₂@PEG and PCN-224@CaO₂-CuO₂@PEG were injected intratumoral.The body weight and tumor size of the nude mice were measured during the treatment,and the tumor inhibition effect and biosafety of the nanoparticles in vivo were studied.After 14 days of treatment,the nude mice were sacrificed and the tumor tissues and major organs were collected for H&E and TUNEL staining to study the toxicity of the above nanoparticles to normal tissues and organs and the ability of inducing apoptosis of tumor cells.The results showed that PCN-224@CaO₂-CuO₂@PEG nanoparticles had good histocompatibility and did not cause obvious histotoxicity to normal tissues and organs.At the same time,after laser irradiation,compared with PCN-224,PCN-224@CuO₂@PEG nanoparticles,PCN-224@CaO₂-CuO₂@PEG nanoparticles showed better therapeutic effect and could significantly inhibit tumor growth in mice in vivo.