Studies On Damages Of DNA By Multifunctional Sonosensitizers Under Ultrasonic Irradiation

The tumor is a genetic disease.Under the influence of carcinogenic factors,the lossing and mutation of gene have effects on cells'morphology,function and metabolic process,resulting in the lossing of normal control ability and forming malignant tumor-cancer.The high mortality and death toll of cancer have become a great threat to human health and have a great impact on the survival and reproduction of human beings.Seeking effective methods to kill or cure cancer is currently one of the goals of human medical research.Sonodynamic therapy?SDT?is a method of inhibiting or killing tumor cells through antitumor factors which produced by ultrasound activating the sonosensitizers.SDT is a new method of tumor therapy based on Photodynamic therapy?PDT?.The energy source of SDT is ultrasound,and its ability of penetration and directionality are stronger than light's,which is more suitable for the treatment of non-superficial tumors.In addition,the sensitizers are easy to assemble on tumor cells,however which content and toxic side effects in normal cells of tumor patients are very low.With the further study of SDT,it has been applied more and more on clinically treating tumors and shows good therapeutic effects.In recent years,the more and more research of SDT for antitumor has been emerging.Most of the research subjects focus on tumor cells,and little research on intracellular macromolecules.Deoxyribonucleic acid?DNA?is a biological macromolecule that records genetic information.It can guide and participate in the survival and development of organisms.The damage of tumor cell's DNA is one of the effective ways to kill tumors.SDT method can be used to change the DNA base sequence of tumor cells and the conformation of DNA configuration by ultrasound induced sonosensitizers.SDT destroy the function of DNA as template,and then inhibit or kill tumor cells,in order to achieve the purpose of antitumor.Screening and developing the perfect,low toxicity and strong recognition for tumor cells of sonosensitizers is the key point to researchers in recent years.Calcein?Fluorescein-DA?and Alizarine complexone?Alizarine-DA?are two kinds of tricyclic polycarboxylic acid compounds with sonodynamic activities,after combined with Fe³⁺forming the Fluorescein-DA-Fe and Alizarine-DA-Fe.The Fluorescein-DA-Fe and Alizarine-DA-Fe are multifunctional sonosensitizers with antitumor activity,sonodynamic activity and sonocatalytic activity.In this paper,the interaction between two multifunctional sonosensitizers and DNA,and the damage to DNA by two multifunctional sonosensitizers under ultrasonic irradiation are studied.The details are listed as follows:?1?Fluorescein-DA-Fe and Alizarine-DA-Fe were synthesized by combined calcein?Fluorescein-DA?and alizarin complexone?Alizarine-DA?with iron?III?nitrate,respectively.The interaction between the Fluorescein-DA-Fe?or Alizarine-DA-Fe?and hs-DNA were investigated by methods of ultraviolet visible spectroscopy,fluorescence spectroscopy,circular dichroism spectrometry,viscosity and gel electrophoresis.The research results show that the interaction model of Fluorescein-DA-Fe and hs-DNA was groove binding mode or electrostatic interaction form,and the interaction model between Alizarine-DA-Fe and hs-DNA was intercalation binding model.The two sonosensitizers both can change the conformation of DNA.The fluorescence quenching mechanism of Fluorescein-DA-Fe and Fluorescein-DA affected by DNA was investigated by fluorescence spectroscopy.The number of binding sites?n?,quenching rate constant?K_q?and the binding constant?K_A?were calculated according to the fluorescence intensity.The experiment results show that both Fluorescein-DA-Fe and Alizarine-DA-Fe have combined with DNA at a ratio of 1:1.DNA have strong quenching effect to Fluorescein-DA-Fe,and Alizarine-DA-Fe have a strong fluorescence quenching effect to DNA-EB system.Both of the quench processing were a static quenching.?2?UV-Vis spectroscopy,fluorescence spectroscopy,viscosity and gel electrophoresis methods were used to investigate the damage of hs-DNA by ultrasonic activating Fluorescein-DA-Fe,and Alizarine-DA-Fe.The effects of ultrasonic time and drug concentrations were also demonstrated.The experimental results show as following:Both of Fluorescein-DA-Fe and Alizarine-DA-Fe have sonodynamic properties.The ultrasonic activating Fluorescein-DA-Fe and Alizarine-DA-Fe show a strong damage to DNA than ultrasound or sonosensiters alone.Fluorescein-DA-Fe and Alizarine-DA-Fe also have sonocatalytic properties.Fluorescein-DA-Fe have a strong damage to DNA comparing with Fluorescein-DA.Alizarine-DA-Fe have a strong damage of DNA comparing with Alizarine-DA.Fluorescein-DA-Fe have the most powerful damage to DNA under sonic irridation.The degree of DNA damage is proportional to the ultrasonic time and concentration of drug.?3?The number and species of reactive oxygen species?ROS?in each system were detected by oxidation extraction spectrometry.The effects of ultrasonic time and drug's concentration on ROS were also investigated.The ROS in Fluorescein-DA-Fe and Alizarine-DA-Fe system were identified by ROS scavenger.The mechanism of DNA damage by the Fluorescein-DA-Fe and Alizarine-DA-Fe were investigated.The experimental results show that the number of ROS are proportional to ultrasonic time and drug's concentration.ROS produced by ultrasonic activating Fluorescein-DA-Fe and Alizarine-DA-Fe are mainly ¹O₂,and?OH.The cell toxicity of the sonosensitiers was investigated by 3-?4,5-dimethylthiazole-2?-2,5-diphenyltetrazolebromide?MTT?method.The results indicate that Fluorescein-DA-Fe and Alizarine-DA-Fe all show inhibitory effects to mouse pancreatic cancer cells?MIN6?,and the complexes show a stronger cell toxicity under ultrasound.The toxicity of Fluorescein-DA-Fe is stronger than Alizarine-DA-Fe.The fatality rate of Fluorescein-DA-Fe was 70%,and the fatality rate of Alizarine-DA-Fe was 55%.