Application Of Thrombin Beacon In Vascular Targeted Photodynamic Therapy

Vascular targeted photodynamic therapy(V-PDT)has become the first choice for treatment of vascular diseases such as Port wine stains and Age-related macular degeneration in clinical.Singlet oxygen(¹O₂)is believed to be the major cytotoxic agent in V-PDT,which can damage vascular endothelial cells,vascular closure,causing local tissue hypoxia and insufficient supply,and indirectly damage target tissues to achieve the purpose of treatment.Real-time monitoring of the PDT response and adjustment of the photodynamic dose can effectively improve the efficacy of V-PDT according to individual differences.Target activated molecular beacon based on fluorescence resonance energy transfer has functions of therapeutic and monitoring.They accurately respond to PDT biomarkers through specific peptide chains while generating ¹O₂,achieving real-time monitoring and evaluation of the treatment process.Thrombin is an important protease that causes blood vessel closure during V-PDT.The photosensitizer pyropheophorbide-a(Pyro)and the fluorescent molecule 5-carboxy-X-rhodamine(5-Carboxy-X-Rhodamine,ROX)was linked together by thrombin cleavable peptide chains to synthesis thrombin-activated molecular beacon(Pyro-thrombin cleavable peptide ROX,PPR).PPR has dual functions of diagnosis and treatment,which can monitor the treatment process of V-PDT in real time and indirectly evaluate the efficacy of V-PDT.Accordingly,the research work carried out in this paper is as follows:Firstly,The luminescence properties of synthetic thrombin-activated molecular beacon in the solution were analyzed to obtain the relationship between PPR and incubation time and concentration of thrombin;Secondly,The multi-optical imaging system was constructed based on the optical properties of thrombin-activated molecular beacon in order to obtain the ¹O₂ luminescence images.The resolution,field of imaging and sensitivity of imaging system were evaluated.The system can realize the simultaneous imaging of ¹O₂ luminescence,photosensitizer fluorescence and diagnostic fluorescence(ROX fluorescence)during V-PDT;Thirdly,The ¹O₂ luminescence image and ROX fluorescence image in V-PDT were recorded using multi-light path imaging system,showing that the vascular area contraction rate is positively correlated with ¹O₂ luminescence and ROX fluorescence.The dose-response relationship of V-PDT was quantitatively researched by direct and indirect dosimetry methods,laying a foundation for revealing the dose-response relationship of V-PDT.