Investigation On Synergistic Enhancement Of Tumor Therapy By Free Radical-ion Generator Based On The Response To Tumor Microenvironment

In recent years,the incidence of breast cancer is increasing,which seriously threaten the health of women.Free radical therapy has attracted extensively attentions in the tumor treatment for its high efficiency and weak possibility of drug resistance.Traditional radical treatments,such as photodynamic therapy（PDT）,sonodynamic therapy（SDT）and radiotherapy（RT）,are limited by hypoxia and penetration depth of light.Chemodynamic therapy（CDT）is a novel radical therapeutic means to kill tumor cells by catalyzing hydrogen peroxide（H₂O₂）to produce highly cytotoxic hydroxyl radicals（·OH）through Fenton or Fenton like reactions.CDT treatment effect is not limited by oxygen content and penetration depth of light,making it a research hotspot.However,insufficient content of H₂O₂and low catalytic efficiency of Fe³⁺in tumor cells seriously limits the efficiency of·OH generation.So it is an important issue to develop a synergistic enhancement effects therapy treatment to cooperation chemodynamic therapy（such as photothermal therapy,etc.）.Based on the above issues,this project intends to construct a simple and effective free radical nano-ion generator（CaO₂@ZIF8@MPN）which could respond to tumor microenvironment and synergistic enhancement of breast cancer treatment effect by chemodynamic therapy,Ca²⁺interference therapy and photothermal therapy.Metal peroxides（MPs）are composed of metal ions and peroxide groups（such as calcium peroxide,CaO₂）.Peroxides can specifically degrade in acidic environment to produce H₂O₂this makes peroxides as excellent donors of H₂O₂.However,CaO₂is unstable in water so it can rapidly degrade before reaching the tumor site,thus limiting its application in the field of tumor CDT.In this work,in order to improve the stability of CaO₂,a novel composite nanomaterial（CaO₂@ZIF8@MPN）was constructed based on nano-CaO₂,then entrapped with pH sensitive material2-methylimidazolium zinc salt（ZIF8）and modified by metal ion/polyphenol network(TA-Fe³⁺,MPN).This nanomaterial possesses both pH responsive and photothermal conversion properties.Under the faintly acidic environment of tumor cells,the nanomaterials gradually disintegrate,leading to a significant increase of intracellular H₂O₂and Ca²⁺.The dissociated tannic acid（TA）reduces Fe³⁺to the more catalytically active Fe²⁺.Subsequently,Fe²⁺catalyzes H₂O₂to generate highly toxic hydroxyl radical（·OH）,forming a accelerate Fe³⁺/Fe²⁺conversion cycle,which guarantees the highly efficient and rapid progress of the Fenton reaction.Meanwhile,because of the excellent photothermal properties of MPNs,the rate of disintegration in tumor cells after CaO₂@ZIF8@MPN being internalized could be accelerated,the intracellular release of Ca²⁺,TA,H₂O₂,Fe³⁺were improved and the production of·OH was further elevated,which could significantly improve the therapeutic effect of CaO₂@ZIF8@MPN.As an intracellular signal molecule,calcium ions play an important role in regulating various physiological functions of cells.Our previous have studies showed that Ca²⁺overload in mitochondria causes mitochondrial dysfunction,mitochondrial damage and apoptosis.Once CaO₂@ZIF8@MPN reaches the tumor site through blood circulation,it disintegrates in response to the tumor’s slightly acidic environment and releases a large amount of Ca²⁺.In the previous literature,Ca²⁺overload can reshape mitochondrial signal transduction,resulting in the decrease of mitochondrial membrane potential and intracellular ATP content,which can achieve the synergistic effect of enhanced CDT induced apoptosis of tumor cells.On the other hand,because of the coating of MPN,the nano-delivery system also has good photo-thermal characteristics.Under the irradiation of 808nm laser,the heat generated can increase the disintegration rate of CaO₂,which is beneficial to promote the release of Ca²⁺.Finally,the mitochondrial calcium overload was realized.In this study,transmission electron microscopy（TEM）and X-ray diffraction（XRD）were used to verify the successful preparation of CaO₂@ZIF8@MPN.The particle size is about 90 nm and the potential are about-18 m V.X-ray photoelectron spectroscopy（XPS）and TEM-mapping characterized the distribution and valence states of Ca,Fe,and Zn elements in CaO₂@ZIF8@MPN,which further proves the successful preparation of CaO₂@ZIF8@MPN.In pH sensitivity experiment,CaO₂@ZIF8@MPN did not disintegrate under the condition of pH 7.4.However,under the condition of pH 4.5 and 808 nm laser irradiation,the nanoparticles almost disintegrated so that H₂O₂,·OH and Ca²⁺are produced in large quantities,indicating that CaO₂@ZIF8@MPN has good pH responsiveness and can be released well in the weakly acidic environment of the lysosome.The photothermal conversion experiment shows that CaO2@ZIF8@MPN has good photothermal characteristics,and the photothermal conversion efficiency is 39.3%.The heat generated under 808nm laser irradiations is sufficient to promote the efficient progress of the CDT reaction.At the same time,in vivo and in vitro experiments show that the multifunctional nano delivery system has the characteristics of good biocompatibility,low toxicity and high stability,which can be effectively transported into tumor cells and retained in tumor for a long time.The anti-tumor effect was evaluated by tumor weight,H&E and TUNEL staining.The tumor inhibition rate of CaO₂@ZIF8@MPN reached to 77.7%and the results of nude mice body weight and H&E staining showed that CaO₂@ZIF8@MPN has good biological safety.In conclusion,the tumor microenvironmentally-responsive free radical ion generator（CaO₂@ZIF8@MPN）was successfully constructed based on integrated chemodynamic therapy,ion interference therapy and photothermal therapy,synergistically enhances the treatment of breast cancer,and provides a new idea for the development of cancer treatment strategies.