In Situ Pegylation Of Copper Phosphate Nanozyme And Its Application In Tumor Therapy

Recently,Oxidative stress therapy（OST）has been developed as an effective method in cancer treatment.The reactive oxygen species（ROS）was generated via the Fenton reaction,which selectively reacts with tumor microenvironment（TME）,thereby achieving the killing effects of tumor cells.OST has attracted increasing scientific attention owing to its highly compartment specific.The therapeutic effects of OST are often limited by the high reductive substance（GSH）concentration in tumor cells and TME,rendering the therapeutic efficacy.Combining OST with other therapies,while inhibiting the reduction of ROS by GSH provides strategies to improve therapeutic effects of OST.Copper-based nanomaterials generally exhibit excellent properties of photothermal conversion and peroxidase mimetic enzyme.It can mediate OST and photothermal therapy（PTT）combined therapy while depleting GSH in tumor cells,thus significantly improving therapeutic efficacy of OST.Nevertheless,the biocompatibility problems caused by the introduction of exogenous copper ions,as well as the biosafety problems as a result of the non-degradability by nanomaterials,greatly limit the clinical application of copper-based nanomaterials.To this end,this article first prepared a degradable copper phosphate nanomaterial（Cu₃（PO₄）₂）,which exhibits excellent photothermal conversion and peroxidase（POD）,catalase（CAT）,glutathione oxidase multi-enzyme activity.Cu₃（PO₄）₂ can selectively increase the level of ROS in the micro-acid environment of tumors,and at the same time consume the high concentration of GSH in tumor cells.Subsequently,a new strategy of in-situ PEGylation was developed,and Cu₃（PO₄）₂@PEGDA core-shell structured nanomaterial was successfully prepared,which realized the uniform surface modification of Cu₃（PO₄）₂ by PEGDA.Compared with unmodified Cu₃（PO₄）₂,Cu₃（PO₄）₂@PEGDA retains the original photothermal conversion and mimetic enzyme activity,while the degradation rate is significantly reduced,and the biocompatibility is significantly improved.Therefore,the Cu₃（PO₄）₂@PEGDA-mediated in vitro OST-PTT combined anti-tumor therapy was further explored.The specific research contents are as follows:1.Cu₃（PO₄）₂ with a particle size of about 330 nm was prepared by hydrothermal reaction using phosphoric acid and copper sulfate pentahydrate（Cu SO₄·5H₂O）.The in-situ coating of PEGDA on Cu₃（PO₄）₂ was successfully realized by using the peroxidase and photothermal properties of Cu₃（PO₄）₂.Under the irradiation of 808 nm laser,Cu₃（PO₄）₂ is used as catalyst and internal heat source,which makes H₂O₂ tend to be catalysed on the surface of Cu₃（PO₄）₂ and generate free radicals.Therefore,Cu₃（PO₄）₂@PEGDA with core-shell structure was successfully prepared by in-situ radical polymerization of PEGDA monomer on Cu₃（PO₄）₂ surface.2.Two materials were soaked in buffer solution or culture medium to explore the degradation rate of the materials.Compared with Cu₃（PO₄）₂,the degradation rate of Cu₃（PO₄）₂@PEGDA was significantly reduced,and the biocompatibility as well as water dispersion were significantly improved.The reason was that the presence of PEGDA layer significantly inhibited the degradation of Cu₃（PO₄）₂.3.The POD properties,catalase（CAT）properties,GSH oxidase properties and photothermal conversion properties of Cu₃（PO₄）₂@PEGDA materials were explored,and Cu₃（PO₄）₂@PEGDA mediation was successfully realized.The combination of oxidative stress-photothermal treatment and high-efficiency POD properties can generate ROS（·OH）in the micro-acid environment of tumors.On the one hand,the photothermal properties can mediate PTT,and on the other hand,the temperature rise caused by it significantly promotes the rate of catalytic reaction and enhances the efficacy of OST.In addition,it is different from tumor cells,which are neutral in normal tissue cells.The expression of CAT enzyme properties of Cu₃（PO₄）₂@PEGDA was promoted,which can inhibit normal tissue damage caused by the diffusion of H₂O₂.The consumption of glutathione reduces the ability of tumor cells to resist oxidative damage,which further enhances OST.The three major functions of GSH exhaustion,POD,and photothermal have amplified the anti-tumor effect in vitro,and the tumor cell inhibition rate reached 56.6%.4.Through staining of cells and analysis of subcellular structure,the mechanism of cell death was explored.The three properties of GSH consumption,POD,and light-to-heat have a synergistic effect,resulting in a significantly higher level of intracellular ROS in the treatment group than other groups,damaging to mitochondria,and mediating cell death.Further staining of DNA showed the apoptotic characteristics of chromatin condensation.At the same time,the staining of DNA by propidium iodide（PI）fluorescent dye proved the damage of cell membrane,which is a typical feature of necrosis.The TEM image of the cell section intuitively shows the damage of the cell membrane and nuclear membrane.In addition,the results of trypan blue（TB）staining indicate that the actual cell damage ratio is significantly higher than the results of the MTT method,and the reason for this result is the chelation of Cu²⁺.In summary,it is proved that Cu₃（PO₄）₂@PEGDA-mediated OST-PTT tumor cell death mode,mechanism and high-efficiency therapeutic effect.5.The good anti-tumor effect in vitro inspired the further exploration of the anti-tumor effect in vivo.The hemolysis and hematological analysis showed that the Cu₃（PO₄）₂@PEGDA had good biocompatibility in mice.And the relative tumor volume results showed that the tumor was significantly suppressed,and the tumor inhibition rate was up to 79.29%.The tumor site sections also showed obvious necrosis or apoptosis of the tumor,while there was no damage to the main organs.Together,these results indicate that Cu₃（PO₄）₂@PEGDA mediated in vivo OST-PTT efficacy,which is expected to provide the possibility for the further application of copper based nanomaterials in biomedicine.