Spectroscopic Investigation On The Interaction With Proteins And Sonodynamic Activities Of Phenothiazine Drugs

Sonodynamic therapy (SDT) is a new approach for cancer treatment based on photodynamic therapy (PDT). It was firstly proposed by Japanese scholars Umemura et al. in1989, based on the synergistic effects on tumor cells damage by the combination of hematoporphyrin and ultrasound. The attractive feature of SDT lies in its ability to focus ultrasound energy on malignancy sites buried deeply in tissues and locally activate the cytotoxicity of the sonosensitizer that preferentially accumulates in tumor sites, with minimal damage to peripheral healthy tissues. Compared with electromagnetic modalities such as laser beams or microwaves, this is a unique advantage in the application to non-invasive treatment of non-superficial tumors, which suggests that SDT has potential value in the application for targeted therapy of tumor.In the latest years, SDT has been widely investigated by focusing on the antitumor effects in vivo and/or in vitro and the mechanisms of the synergism between ultrasound and drugs by using different ultrasound parameters and different sonosensitizers. Most of them regarded the tumor cells as assault target, and achieved the goal of treating tumors through inducing tumor cells apoptosis. However, the intracellular targets of SDT have seldom been studied until now. The damage to intracellular substances, especially proteins that are highly abundant in cells, might be a more effective method to kill the tumor cells. If the proteins in the rumor cells were damaged by sonosensitizers under ultrasonic irradiation, the whole cells would undergo apoptosis abnormally.Sonosensitizer is an important factor affecting the efficacy of SDT. Nowdays, many sonosensitizers used in the research of SDT stem from traditional photosensitizers in PDT. Methylene blue (MB) and toluidine blue (TB), two phenothiazine derivatives, investigated and used as photosensitizers of PDT against several types of tumors, have been confirmed that they possess sonodynamic activities. These results demonstrate that phenothiazine derivatives have potential to be used as sonosensitizers in SDT. In addition, the study on the interaction between drugs and serum albumin (SA) can provide important information about drugs’ pharmacodynamics and pharmacokinetics, because the distribution, metabolism and efficacy of many drugs are correlated with their affinities towards SA. Furthermore, the study may also provide information for the structural features that determine the therapeutic effectiveness of drugs, and become an important research field in life sciences, chemistry, and clinical medicine.In this work, the interactions of three phenothiazine drugs, promethazine hydrochloride (PMT), dioxopromethazine hydrochloride (DPZ), and MB, with SA and sonodynamic activities of these phenothiazine drugs were studied by spectroscopic approach. The obtained results are as follow:1. The interaction between PMT and bovine serum albumin (BSA) was investigated by fluorescence and absorption spectra. The fluorescence of BSA was quenched remarkably by PMT. The fluorescence quenching and absorption spectra indicated that the fluorescence quenching mechanism was static quenching by forming a complex. The association constants and the number of binding sites were calculated at different temperatures. The BSA-PMT binding distance was determined to be less than7nm, suggesting that energy transfer from BSA to PMT may occur, but its contribution to fluorescence quenching is smaller than static quenching. The thermodynamic parameters of the interaction between PMT and BSA were measured according to the van’t Hoff equation and thermodynamic formula. The binding process was a spontaneous process in which Gibbs free energy change (AG) was negative. The negative enthalpy change (△H) and negative entropy change (AS) values indicated that the interaction of PMT with BSA was driven mainly by electrostatic interactions, but the van der Waals forces and hydrogen bonds can not be neglected. In addition, the synchronous fluorescence and three-dimensional fluorescence spectra showed that binding of PMT with BSA induced conformational changes in BSA.2. The interaction between DPZ and human serum albumin (HSA) was investigated by fluorescence, absorption and circular dichroism (CD) spectra. The results obtained from the fluorescence spectra indicated that DPZ has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The HSA-DPZ binding distance was determined to be less than7nm, suggesting that energy transfer from HSA to DPZ may occur, but its contribution to fluorescence quenching is smaller than static quenching. The thermodynamic parameters of the interaction between DPZ and HSA were measured. The binding process was a spontaneous process in which the AG value was negative. The negative AH and positive AS values indicated that hydrophobic forces play a major role in the binding of DPZ to HSA, but the electrostatic interactions can not be neglected. The experimental results of competitive displacement suggested that DPZ was located in site I within sub-domain IIA of HSA. In addition, the absorption, synchronous fluorescence, three-dimensional fluorescence and CD spectra showed that binding of DPZ with HSA induced conformational changes in HSA.3. The binding site of MB on HSA and the effect of Cu2+and Fe3+on the binding affinity of MB with HSA were investigated by fluorescence and absorption spectra. The experimental results of competitive displacement using the site probes ketoprofen and ibuprofen as specific markers suggested that MB was located in site I within sub-domain IIA of HSA. The association constants of MB binding to HSA were enhanced but to different extent in the presence of Cu2+and Fe3+, which indicated that the binding affinity of MB to HSA were enhanced by Cu2+and Fe3+, while the effect of different metal ions was different. The increase of the binding affinity of MB to HSA will prolong retention time and reduce the maximum effect of MB. The binding distance between MB and HSA and the conformation of HSA had no change in the presence of Cu2+and Fe3+, which indicated that the higher stability of MB-HSA-metal ion complex than MB-HSA complex induced the increase of the binding affinity of MB to HSA.4. The sonodynamic damage to protein in the presence of MB, PMT and DPZ were studied by fluorescence and CD spectra, selecting BSA as a model of protein. The mechanisms of the synergistic effects of ultrasound and drugs were studied by oxidation-extraction photometry with several reactive oxygen species (ROS) scavengers. The results indicated that the degree of ultrasound-induced protein damage was enhanced by these drugs. The results of synchronous fluorescence, three-dimensional fluorescence and CD spectra confirmed that the damage of protein induced by the synergistic effects of ultrasound and drugs was more serious than that when ultrasound or drug alone was applied. The damage of protein could be mainly due to the generation of ROS. The damage degree of protein increased with the increase of ultrasonic irradiation time and drugs’ concentration because of the increased quantities of ROS generation. The experimental results of oxidation-extraction photometry with several ROS scavengers indicated that both1O2and·OH were the important ROS of the ultrasound-inducing protein damage in the presence of MB, PMT and DPZ. Besides, there are other kinds of ROS generated in the SDT process of DPZ. These results indicated that MB, PMT and DPZ possess sonodynamic activities, and may be potential sonosensitizers for SDT.