Copper-based Nanoscale Coordination Polymers Amplify Oxidative Stress To Enhance Radiotherapy

Radiotherapy(RT)has become one of the common therapies to treat cancer.Radiotherapy relies on irradiation to produce reactive oxygen species(ROS),such as hydroxyl radical(·OH),which in turn damages the DNA molecule.Recent studies have shown that ROS include hydroxyl radical(·OH),hydrogen peroxide(H₂O₂),singlet oxygen(¹O₂),superoxide anion radical(O₂·^-)and so on.Among these ROS,·OH is the most oxidizing and H₂O₂ is the weakest.However,there are still some reasons contributing to inadequate ROS production when tumor undergoes radiotherapy.Firstly,tumor tissues are composed of low-Z elements(carbon,hydrogen,oxygen,and nitrogen),resulting in insufficient X-ray deposition and·OH generation.What's more,the produced·OH would collide in pairs to produce H₂O₂spontaneously.Secondly,the metabolic reprogramming within tumor tissues would substantially increase the level of antioxidants,including glutathione(GSH),to rapidly quench·OH.Therefore,radiotherapy alone couldn't produce enough·OH to cause effective damage to tumor tissues.Chemodynamic therapy(CDT)has been considered as a promising therapeutic strategy,which could induce oxidative stress in tumor.In CDT,Fenton reagents utilize H₂O₂ to produce·OH via Fenton or Fenton-like reaction.CDT decomposes overexpressed H₂O₂ which is produced by abnormal metabolism and radiotherapy,inducing the production of more·OH to remedy the insufficient ROS generation by radiation.Comparing with traditional iron-based Fenton reagents,copper-based Fenton-like reagents have higher catalytic rate in a broader p H range.However,the REDOX potential of Cu²⁺/Cu⁺(?0.16 V)is so low that Cu⁺is easier to be oxidized.It's still challenging to obtain stable and free Cu⁺in aqueous solution.In this study,we constructed Copper-based nanoscale coordination polymers(Cu-NCPs)via self-assembly of Cu²⁺and 5'-guanosine monophosphate(5'-GMP)with a molar ratio of 1:4.After coordination,interestingly,partial Cu²⁺was converted into stable Cu⁺.Cu-NCPs had mixed-valence(Cu²⁺/Cu⁺),which could simultaneously and independently induce the Cu⁺-triggered Fenton-like reaction and Cu²⁺-triggered GSH elimination.Based on above properties,we hypothesized that when combining RT and CDT,Cu-NCPs could eliminate GSH and utilize radiotherapy byproducts H₂O₂ to induce more·OH to improve oxidative stress to enhance radiotherapy.This paper will discuss from the following four parts:The first part was about the preparation and characterization of Cu-NCPs.We constructed Cu-NCPs by one-step coordination.We used Dynamic light scattering and Field emission scanning electron microscopy to represent morphology and size of Cu-NCPs.We used X-ray photoelectron spectroscopy and ESR spectroscopy to analyze the valence state.We used FT-IR and X-ray diffractometer to analyze chemical composition and crystal form respectively.The results showed that Cu-NCPs with good stability had mixed valence copper(Cu²⁺/Cu⁺).The second part was about the mechanism in which Cu-NCPs amplified oxidative stress in vitro.We performed methylene blue bleaching and GSH and GSSG assay experiments to evaluate the ability of Cu-NCPs mediating Fendon-like reaction and eliminating GSH respectively.We used neocuproine to detect the content of Cu⁺in Cu-NCPs and Cu-NCPs+GSH groups.We also combined Cu-NCPs with different doses of radiotherapy to study the amplified Fenton reaction.The results showed that Cu-NCPs with mixed-valence copper(Cu²⁺/Cu⁺)could potentially amplify RT-induced oxidative stress via the generation of·OH and elimination of GSH.The third prat was cellular characterization of Cu-NCPs.We first studied the uptake behavior,elimination of GSH,induction of ROS and double-stranded DNA damage of Cu-NCPs in CT26 colorectal cancer cells.Then we performed the cytotoxicity experiments of Cu-NCPs+RT in several tumor cell lines.We also performed cell viability detection to evaluate the biosafety of Cu-NCPs in several normal cell lines.The results indicated that Cu-NCPs sensitized cells to radiation,with the combination treatment collectively amplifying intracellular oxidative stress,which synergistic inhibited tumor cell proliferation.The fourth part was therapeutic efficacy of Cu-NCPs sensitizing RT on CT26-bearing mice.We intravenously injected Cu-NCPs and performed ratiotherapy(5 Gy)after 6 hours on CT26-bearing mice.The results showed that the combination therapy could significantly inhibit tumor growth(98.2%),without causing systemic toxic and major organic damage.In this study,aiming at insufficient·OH produced by radiotherapy alone and challenge to obtain stable Cu⁺in aqueous solution,we constructed Cu-NCPs with mixed-valence copper(Cu²⁺/Cu⁺),to realize the synergism of radiotherapy and chemodynamic therapy.Radiotherapy could provide more substrates H₂O₂ for Fenton reaction,Cu-NCPs could eliminate GSH and induce more·OH to improve RT-mediated oxidative stress,which could amplify the therapeutic effect synergistically.This method could provide new strategy for radiosensitivity.