Application Of Cu/DVDMS Sonosensitizer For Enhanced Sonodynamic Therapy And Tumor Imaging

Part I Synthesis and characterization of Cu/DVDMS nanoparticlesObjective:In this study,ion-induced self-assembly technology was used to modify the small molecule drug DVDMS,eliminate the shortcomings of DVDMS and develop a highly efficient sonosensitizers based on the self-assembly between metal and DVDMS which could enhance sonodynamic therapy and combine chemodynamic therapy to improve the therapeutic effect.Methods:Cu/DVDMS nanoparticles were formed by self-assembly of DVDMS and Cu Cl₂solution with the help of polyvinylpyrrolidone.Firstly,Cu/DVDMS nanoparticles were characterized at the material level.The formation of Cu/DVDMS nanoparticles was verified by TEM image,zeta potential,dynamic light scattering,UV-vis absorption spectrum and XPS.SDT and CDT properties of the nanoparticles were evaluated at the material level using DFCH-DA probe and methylene blue.Results:1.The results of transmission electron microscopy,zeta potential,dynamic light scattering,UV-vis absorption spectrum and XPS fully proved the formation of Cu/DVDMS nanoparticles mainly because of the interaction between the sulfonate radicals of DVDMS and copper ions.2.Through the fluorescence intensity of DCFH-DA and degradation of MB,it was found that Cu/DVDMS nanoparticles could produce more ROS and mediate Fenton-like reactions under ultrasound.Conclusion:1.The design and synthesis of a novel self-assembly Cu-porphyrin nanosonosensitizer based on DVDMS was reported.2.The results of material level experiments showed that the nanoparticles could have a synergetic combination of sonodynamic therapy and chemodynamic therapy.Part II The therapeutic and imaging abilities of Cu/DVDMS nanoparticles in vitroObjective: The purpose of this study was to evaluate the therapeutic and imaging ability of Cu/DVDMS nanoparticles by 4T1 cells and 293 T cells.Confocal laser scanning microscopy and ICP-MS were used to assess cell uptake.Methods: 4T1 cells were used to assess the uptake of Cu/DVDMS nanoparticles by TEM,ICP-MS and confocal laser scanning microscopy.DFCH-DA probe was used to detect ROS production in 4T1 cells incubated with Cu/DVDMS nanoparticles and the flow cytometry analysis was performed.Next,the MTT assay was used to detect the cell viability of 293 T cells and 4T1 cells and evaluate the biosafety and cell killing capacity of Cu/DVDMS nanoparticles.Results: 1.The results showed that Cu/DVDMS nanoparticles reached the highest concentration in 4T1 cells at 3 hours after incubation and the concentration gradually decreased,indicating that Cu/DVDMS nanoparticles had good cell uptake.However,compared with DVDMS,the fluorescence of Cu/DVDMS nanoparticles was very weak and the quench occured which was consistent with the literature report.2.In this study,ROS generation ability of Cu/DVDMS nanoparticles was evaluated at the cellular level using 4T1 cells.Through fluorescence microscopy and flow cytometry,it was found that Cu/DVDMS nanoparticles could produce the most ROS under ultrasound.Conclusion: 1.It was found that Cu/DVDMS nanoparticles enter rapidly the cytoplasm.2.The cell experiments further proved that Cu/DVDMS nanoparticles possessed excellent biosafety for normal human cells and good ability to kill cancer cells under ultrasound,which should be attributed to the fact that Cu/DVDMS nanoparticles could enhance sonodynamic therapy,mediate fenton-like reaction and produce more ROS.Part III The therapeutic and imaging abilities of Cu/DVDMS nanoparticles in vivoObjective: The efficacy of nanoparticles was evaluated by dynamically monitoring the changes of tumor volume and body weight in tumor-bearing mice.PET was used to assess the imaging capability in vivo.Methods: BALB/c female mice were used to construct subcutaneous tumor models and animal experiments were conducted when the tumor volume reached 100mm3.On the one hand,PET imaging was carried out with the nanoparticles labeled by nuclide.On the other hand,in vivo therapy experiments were carried out to evaluate the therapeutic effect of Cu/DVDMS nanoparticles by dynamically monitoring the changes of tumor volume and observing the results of H&E staining and TUNEL staining.The in vivo safety of Cu/DVDMS nanoparticles was evaluated by dynamically monitoring the changes in body weight of mice and observing the H&E staining results of heart,liver,spleen,lung and kidney.Results: 1.The results of transmission electron microscopy and PET imaging showed that Cu/DVDMS nanoparticles possessed good uptake of tumor in tumor-bearing mice.2.The results of H&E staining and TUNEL staining showed that Cu/DVDMS nanoparticles could combine sonodynamic therapy with chemodynamic therapy and have a good therapeutic effect.3.H&E staining of heart,liver,spleen,lung,and kidney further proved that Cu/DVDMS nanoparticles had no obvious biological toxicity,little side effects and high safety in experimental mice.Conclusion:In the tumor environment,Cu/DVDMS could be reduced by GSH and lead to the release of Cu+ after the nanoparticles entered the cytoplasm of cancer cells.Cascade bioreactions induced by copper reduced intracellular GSH levels and generated a cytotoxic Fenton-like reaction to destroy tumor cells.Furthermore,this nanostructure could enhance sonodynamic therapy.Based on the systematical in vivo studies,the high ROS efficiency,bio-safety and excellent therapeutic effect of Cu/DVDMS were demonstrated.It was revealed that the nanoparticles could be efficiently delivered into the cytoplasm.It was believed that this report can represent a simple approach to combine SDT and CDT.