| Organelles(including nucleus,mitochondria,lysosome,endoplasmic reticulum and Golgi apparatus,etc.)with a certain structure and function in a cell are the most important components and play an important role in cell metabolism,transportation,proliferation,differentiation,and apoptosis.Therefore,various organelles are often used as target sites for disease treatment,such as chemotherapy,photothermal therapy,and photodynamic therapy.The photosensitizer is irradiated with light of a specific wavelength,and a photochemical reaction occurs with the participation of oxygen in biological tissues,producing singlet oxygen and free radicals,destroying various biological macromolecules in tissues and cells,and finally causing tumor cell death.This is PDT,which has a long history since its discovery.Compared with traditional surgery,chemotherapy and other methods,PDT has its unique advantages,such as no or only minor trauma.However,the mechanism of treatment at the organelle level is still unclear.When photosensitizers work,how the cells react.Therefore,the detection of organelle structure,function,and microenvironment is very important to understand its role in PDT and the diagnosis and treatment of diseases,and it is helpful to design and develop more effective treatment platforms and methods.The discovery of Raman scattering effect in 1928 provided a new molecular spectroscopy method,namely Raman spectroscopy.Later,enhanced Raman signals were found on rough precious metal surfaces,and surface-enhanced Raman spectroscopy(SERS)was proposed.SERS enables the further development and application of Raman technology.SERS technology can obtain molecular structure information with ultra-high sensitivity and has the advantages of multi-component detection,narrow spectral peaks,and real-time in-situ non-destructive detection.It is especially suitable for the research and treatment of biological systems,and it has been widely used in the biological field,but it still faces some problems.For example,the research of SERS in the biological field mainly focuses on the nucleus due to the lack of effective organelle detection strategies.The research objects are relatively limited,and most of them are static targets.In addition,the research on the microenvironment within the organelles and their relationships is relatively scarce,and further development is needed.Although various treatment strategies and methods that target organelles are emerging in an endless stream,the treatment mechanism at the organelle level has not yet been fully clarified.Therefore,the development of a method that can track changes in the structure of organelles and microenvironmental information and its dynamic changes in real time during the treatment process is urgently needed,and can help us to understand the mechanism of targeted organelle therapy through the structure and function of the organelles,and provides the foundation for the development of more effective diagnosis and treatment platform.In view of the above,this thesis focuses on the two organelles,nucleus and mitochondria,based on the direct and indirect detection strategies of SERS,and explores the therapeutic mechanism of PDT from the structure of the organelle and the microenvironment.The main research contents are as follows:(1)A nuclear-targeted SERS nanoprobe was constructed for in-depth study of the structure and function of the nucleus,and to detect the dynamic changes of the nuclear structure during the PDT process.The nuclear targeting probe is mainly composed of gold nanorods(Au NRs)substrate and nuclear targeting peptides(NLS,CGGGPKKKRKVGG).Based on this strategy,we obtained the SERS spectrum of the melanoma cell B16 cell nucleus,and analyzed the main components in the cell nucleus through peak assignment,indicating that there mainly are DNA,protein and carbohydrates in the cell nucleus.This strengthens the understanding of the function and the fine structure of the nucleus,and obtained the dynamic changes of the main components in the nucleus during the PDT process.The effect of PDT was evaluated from the perspective of the nucleus.The dissolution of the tertiary structure of the protein(ie,the break of the disulfide bond)and DNA fragmentation mainly occurred in the nucleus,which laid the foundation for further research on the mechanism of targeted organelle PDT therapy.(2)The design proposed a new method to track the dynamics of the complete functional mitochondria during the photodynamic therapy process through the SERS method.Functional mitochondria were isolated and extracted from human breast cancer cells.Under the enhancement of Au NRs,SERS technology was used to detect the dynamic changes of mitochondria during the PDT process.Based on this method,we obtained the dynamic SERS spectrum of the mitochondria,and analyzed the main components of the mitochondria through the peak position assignment,deepened the understanding of the structure and function of the mitochondria,and obtained the dynamic changes of the main components,namely protein Denaturation,degradation,DNA double-strand break,base exposure,etc.,which caused the destruction of mitochondrial structure.The mechanism of PDT was evaluated from the changes in mitochondrial structure.(3)Designed and constructed the mitochondrial targeted p H nano-sensing probe to realize the detection of dynamic changes in the mitochondrial microenvironment during the PDT process.The nanoprobe is mainly composed of Au NRs,the p Hsensitive Raman reporter 4-mercaptopyridine(4-mercaptopyridine,4-MPy)and the mitochondrial targeting peptide(MLS,MLALLGWWWFFSRKKC).At the same time,combined with fluorescence imaging,the dynamic changes of two kinds of cancer cells(Hep G2 and MCF-7 cells)and one normal cell(LO2 cells)in the mitochondrial microenvironment during the PDT process were monitored in situ.In this work,the microenvironment in organelles and the relationship between them were explored,which has achieved an in-depth understanding of the mechanism of mitochondrial targeted photodynamic therapy. |
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