Analysis Of Physicochemical Properties And Anti-tumor Mechanism Of A Novel Photosensitizer Sodium Porphin

Background and purposeBreast cancer is the leading cause of cancer-related deaths among women in the worldwide.Drug resistance and often severe side effects of surgical interventions,radiation,and chemotherapy have driven the emergence of new therapies.Duing to the temporal and spatial control of the treatment with minimal systemic toxicity,photodynamic therapy(PDT)is an effiective method for breast cancer.PDT is based on a photochemical reaction among photosensitizer,light,and molecular oxygen.Photosensitizer is the core for the successful of PDT.Ideal PS should have high stability,water-solubility,high phototoxicity,and low dark toxicity etc.The improvement and development of PDT is along with the discovery and development of photosensitizers.Photosensitizer is typically divided into three generations based on the time and technological progress.In recent years,a variety of second generation photosensitizers which have enriched the clinical application continuously appeared to overcome the limitations of first generation.Second generation photosensitizers generally included the following sections:Porphins,phthalocyanines,chlorins,and others.Chlorins with high quantum yield of singlet oxygen and intense absorption band at longer wavelengths are the key objects for researching at home and abroad.One of the most widely studied PS is HPPH which has good photodynamic activity and used for treatment oralcancer,esophageal cancer,gastric cancer and other solid tumors.Nevertheless,HPPH is poor hydrophilicity in aqueous media and need to be dissolved in toxic organic solvents in practical use.NPe6 and m-THPC are other novel photosensitizers,belonging to chlorin compounds,which show satisfactory antitumor effects as reported in clinical studies.But they also have shortcomings,such as skin phototoxicity,delay of neuralgia and high cost.The disadvantages above mentioned restrict the PDT clinical applications.S-PS,gained independent intellectual property in China,is a newly developed PS which pertains to chlorins and has a strong absorption peak at 600-700 nm.Because of its high purity(99.5%),researchers can reduce dose to achieve good therapeutic effect.S-PS and HPPH have same starting material and ring structure.And,preliminary studies showed that S-PS-PDT could significantly damage cholangio carcinoma cells and the destruction was more prominent when joined VEGF.These early findings constitute a valuable reference for future S-PS-PDT explorations.S-PS as a new type of photosensitizer,is still in the early study phase.The aim of this study is to investigate the physical and chemical properties of S-PS,evaluate the anti-tumor effect and BSA damage of S-PS-PDT.This work could be a valuable reference for further investigation in the synergy effect of S-PS and light activation.Methods and ResultsPart 1 The analysis of physical and chemical properties of S-PS.UV-vis spectra and fluorescence spectra were applied to analyse the physical and chemical properties of S-PS.The absorption spectra of S-PS indicated that the strongest absorption peak was around 398 nm.The S-PS absorption was gradually reduced along with the extension of illumination time.The fluorescence emission spectra indicated the position of the maximum emission located at 650 nm.Besides,the singlet oxygen yeild of S-PS was higher than Ce6?HMME?HP?PpIX?but lower than HPPH.Part 2 Cytotoxicity of S-PS-PDT to MDA-MB-231and 4T1 cells.Cell viability was analyzed in two types of cells using conventional MTT assay Cell viability followed S-PS-PDT was significantly decreased with the increasing of light dose and the concentration of S-PS,while S-PS alone showed no inhibitory effect on cells.And Annexin V-FITC and PI staining indicated that S-PS-PDT triggered cell apoptotic response.Simutaneously,cell membrane and DNA damages were detected.According to flow cytometry detections,ROS level for cells which subjected to S-PS-PDT significantly enhanced,indicating that the phototoxic effect was medicated by ROS.Further,the S-PS distribution results manifested that S-PS mainly located in the mitochondria of MDA-MB-231and 4T1 cells,we speculated that mitochondria may be the main lesion site.Part 3 Damage effect of S-PS-PDT on BSA.The absorption spectra and fluorescence spectra were executed to evaluate the damage of S-PS on BSA.The results demonstrated an interaction between BSA and S-PS.S-PS-PDT exhibited superior BSA hyperchromicity and fluorescence quenching compared with S-PS and light alone.Synchronous fluorescence intensities of BSA subjected to S-PS or S-PS-PDT were gradually decreased.The decline extent of AX=60 nm was more serious than the corresponding ones at ??=15 nm.The results suggested that S-PS-PDT could damage protein structure.The site of action between the S-PS and BSA was closer to the Trp residues than the Tyr residues.Part 4 Metabolic researches of S-PS in 4T1 tumor-bearing miceS-PS biodistribution in 4T1 tumor-bearing mice was analyzed by fluorescence spectra.The results indicated that S-PS in tumor increased and reached the highest level at 4 h after intravenous injection,and its metabolic activity was slow between 2?8 h which suggested a retention effect in tumor.Between 6 h(89.80%of the peak)and 8 h(74.51%of the peak),the S-PS concentration in the tumor was still much higher,meanwhile the S-PS concentration of other tissues held a lower level.In order to improve the effect of treatment and minimize side effects on healthy tissues,6 h after administration might be an appropriate time point for laser radiation.Part 5 The anti-tumor effect of S-PS-PDT in vivo.We used 4T1 tumor-bearing mice as a model to evaluate the effects of S-PS-PDT through tumor volume,histopathological and mice survival.The results revealed thatS-PS-PDT treatment markedly inhibited tumor growth and prolonged the survival of the tumor-bearing mice.The effect in repressing tumor growth on S-PS-PDT and HPPH-PDT was similar under the same condition of treatment.Histopathological,clinical signs and body weight were measured and no obvious changes were observed.In conclusion,this approach is a relatively safe and effective method.ConclusionIn summary,we have studied the physical and chemical properties of S-PS,performed in vitro and in vivo experiments,BSA damage experiment to assess the damage effects on breast cancer and the possible mechanism about S-PS-PDT.The results showed that S-PS could be effectively activated by light and displayed high phototoxicity on tumor which suggested that S-PS is a promising photosensitizer in clinical PDT.