Studies On The Photosensitive Compounds Induced Damage Of Protein Molecule Under Low-frequency Ultrasound

Malignant tumor is one of the most deadly diseases for human being. Scientists from both domestic and overseas have done many researches to conquer it. Sonodynamic therapy (SDT) is a new anti-tumor therapy proposed by Japanese scholars Umemura et al in1989. The antitumor effect of SDT is based on the synergistic effects induced by hematoporphyrin (HP) which can remain preferentially in tumor tissues much longer than in normal tissues being activated with ultrasound. Many scholars have paid close attention to SDT for its theoretical significance and clinical value. Due to the specific accumulation of HP in tumor cells and the selective irradiation of the ultrasound, SDT does not damage healthy tissue, and avoids the side-effect of chemotherapy or radiotherapy. Therefore, it is suitable for those patients who can not be cured with the operation or in need of chemotherapy through veins, and the application of SDT is a promising way on treatment of tumors in deep tissues.In the latest years, many scholars have made lots of researches on the ultrasound apparatus, therapeutic effect on tumors and reaction mechanism of SDT choosing tumor cells as assault target, and achieved the goal of treating tumors through damaging the cell membrane. However, it is well known that cell membrane possesses certain repair ability, it make us think that the reasons for SDT’s antitumor effect are not only the cell membrane but also the active ingredient (such as protein and nucleic acid, etc) by the sonosensitizers penetrated into tumor cells. The reason for targeting the serum albumin lies in the knowledge that this kind of protein plays a critical role in the biological metabolism. If some biological macromolecules such as DNA, RNA, protein and enzyme in cells are damaged, the cells will die in a short time. In this dissertation, bovine serum albumin (BSA) was selected as target molecule, then fluoroquinolones (FQs), acridines (ADs) and chlorophyll derivatives (CPD) were chosen as sonosensitizers. They were used to study the synergy effect of ultrasound combined with FQs, ADs and CPD on the damage of BSA molecular at40KHz,1W/cm2. In addition, the interreaction of propylenediaminetertaacetic acid (PDTA)-rare earth metal complexe with BSA was also studied.Presently, the obtained results are as follow:1. Investigation on the damage of BSA by FQs under ultrasonic irradiation:the results showed that the quenching mechanism of BSA by levofloxacin (LVFX) and ciprofloxacin (CPFX) belongs to static quenching, and the effects of four metal ions on the interaction between FQs and BSA were studied. The BSA damage experimental results by FQs under ultrasonic irradiation showed that the damage degree of BSA was intensified with the increases of the ultrasonic irradiation time, temperature and FQs concentration. The ROS test result indicated that the singlet oxygen (1O2) and hydroxyl radical (·OH) were generated in the ultrasonic activation of FQs. At last, a synergistic effect from FQs and ultrasound was observed, so that the bacterial viability was reduced when FQs and ultrasound were combined.2. Investigation on the damage of BSA by ADs under ultrasonic irradiation:it was observed that there are strong interaction between acridines red (AR), acridines orange (AO) and acridines yellow (AY) with BSA, in which the array order of complex stability is BSA-AY> BSA-AO> BSA-AR and the quenching mechanisms of BSA all belong to static quenching. The experimental results on the damage of BSA molecules by ADs under ultrasonic irradiation showed that the damage degree of BSA was intensified with the increases of the ultrasonic irradiation time and ADs concentration. Furthermore, the ROS test result indicated that the amount of-OH was significantly more than1O2under ultrasonic irradiation in the presence of AY. In addition, metronidazole (MTZ), as a sensitizer, was proved to intensify obviously the BSA damage by amsacrine (AMSA) under ultrasonic irradiation, and the injury area of BSA is mainly tryptophan.3. Investigation on the damage of BSA by CPD under ultrasonic irradiation:the results showed that chlorophyllin (CHL) and metal-chlorophyin (Chl-M, M=Cu, Fe, Zn) can quench obviously the fluorescence of BSA and the mechanism of quenching all belong to static quenching. The binding force between CHL and BSA was mainly hydrophobic bond. Furthermore, it was observed that CHL and Chl-M activated by ultrasound could damage significantly the structure of BSA and the array order was Chl-Fe> Chl-Zn> CHL> Chl-Cu. In addition, the CHL-MTZ was synthesized by esterifi cation reaction and the damage of BSA by CHL-MTZ under ultrasonic irradiation was stronger than CHL’s, meanwhile CHL and CHL-MTZ were also decomposed under ultrasonic irradiation. Therefore, CHL-MTZ and CHL, as novel sonosensitizers, have good application prospect in SDT because of their low sonosensitive toxicity.4. Investigation on the interaction between [YⅢ(pdta)(H2O)]22-(Y-pdta) and BSA:the results showed that Y-pdta can quench strongly the fluorescence of BSA by static quenching. In the same time, Y-pdta can enhance the microenvironmental hydrophobicity around Trp residu. In addition, the major binding force is considered to be hydrophobic bond due to the thermodynamics related parameters.