Application Of Surface Enhanced Raman Spectroscopy In Organelle Targeted Detection And Therapy

Cell is the basic structure and function unit of organism,and it is the basic component of all organisms except for viruses.In eukaryote cells,subcellular compartments including cytoplasm,cell membrane,nucleus,mitochondria,lysosome,endoplasm and Golgi and so on,are essential functional units.They play important roles in regulating cellular biological functions,including cell differentiation,growth,apoptosis and the cellular transportation.In view of the significance of each subcellular compartment in cellular functions,their structural and functional disorders will cause various diseases,such as cancer,lysosomal storage disease,and various neurodegenerative diseases,and so on.Therefore,achiving the precise and accurate detection of the structure,function and microenvironment of organelles is of great significance for deeply understanding their roles in cell life activities and the diagnosis and treatment of diseases.Surface-enhanced Raman spectroscopy?SERS?is a spectroscopic technology that can simutaneously obtain the molecular structure information of substances and possess high sensitivity.Owing to its distinctive advantages including the narrow spectral peaks,light stability,and less damage to biological systems,SERS technology has been widely used in biological system.Although SERS technology has been initially used in organelle-related detection and treatment research,it still faces some problems.First,most of the studies are concentrated on the nucleus due to the lack of effective detection methods,little research on other organelles,and the accuracy of detection needs to be verified.Moreover,the research on organelle microenvironment is very scarce and needs further development.In addition,designing and preparing organelle-targeting probes to efficiently and selectively kill cancer cells has gradually become an effective strategy for cancer treatment.Although various treatment platforms are emerging,their treatment mechanism has not yet been clarified.Therefore,there is an urgent need to develop a method that can detect the structure and microenvironment information of organelles and real-time tracking their dynamic events,which is helpful to understand the structure and function of organelles in cells and therapeutic mechanisms in depth.Moreover,it can also provide some valuable insights in developing and preparing more effective diagnosis and treatment platforms.Aiming at the problems of SERS technology in the research of organelles,based on the SERS labeling and label-free strategy,in this paper,we developed a series of organelle-targeting probes to profile the structure and microenvironment of organelle and therapeutic mechanism based on the organelle targeted phototherapy.The main contents are as following:?1?We designed and prepared a serious of organelle targeted pH SERS nanosensors including nucleus,mitochondria and lysosome targeted nanoprobes.The nanosensor is composed of an AuNRs-enhanced substrate,a pH-sensitive reporter?4-mercaptopyridine,MPy?,and an organelle-targeting peptide.By monitoring the changes of the SERS spectrum of MPy at different pH values,the pH of specific organelles can be determined.With the assistance of the pH SERS nanosensors,we achieved the pH detection of mucleus,mitochondria and lysosome in cancer cells?HepG2?and normal cells?BNL.CL2?.The results show that the pH of nucleus,mitochondria and lysosome in HepG2 cells is 7.9±0.2?7.5±0.1and 4.7±0.3,while8.2±0.3?7.8±0.2 and 6.5±0.2 in BNL.CL2 cells.It shows that the nucleus pH of the cells is nearly neutral,mitochondria are weakly alkaline,and lysosomes are weakly acidic.Moreover,the organelle pH of cancer cells is slightly lower than that of normal cells.?2?To further study the structure and function of the nucleus,mitochondria and lysosome,and evaluate the different efficacy among different organelle-targeting platforms in the phototherapy?PTT?toward cancer cells,we designed biocompatible nucleus,mitochondria and lysosome targeting nanoprobes.These organelle-targeting probes are composed of AuNRs and organelle-targeting peptides,which not only own the strong SERS-enhancing ability,but also process high photothermal conversion efficiency,and can be used for organelle molecular detection and photothermal therapy.On the basis of the nanoprobes,we obtained the SERS spectra of the three organelles.The main components of the organelles were analyzed and compared.The results show that the organelles are mainly composed of proteins,lipids and carbohydrates.Different from the other two organelles,the nucleus has a lot of genetic material including DNA bases and phosphate backbones.Moreover,the PTT treatment for three different cancer cells based on the different organelle targeting nanoprobes were evaluated,the results demonstrating that mitochondria-and nucleus-targeting probes have stronger cancer-killing effects,compared with the lysosome targeting nanoprobes,and laid the foundation for the further invesyigation of organelle targeted therapy mechanisms.?3?In order to achieve the ROS detection at subcellular level and the dynamic monitoring during photothermal treatment,we designed and prepared a background-free SERS active nanoprobe.This nanoprobe was designed as an Au core-Ag shell nanoparticles?Au@Ag NPs?with a SERS reporter?4-mercaptobenzonitrile?resided in the inner of the core-shell.By modifying the mitochondria targeting peptide,the nanosensors can be delivered into the mitochondria and achieve the ROS detection.The sensing mechanism is mainly based on the etching of Ag shell by ROS,which causes the decreased SERS signal of MBN,according to the changes of the SERS signal,realizing the detection of ROS in mitochondria.In this study,reporter has a strong and sharp Raman band in the cellular Raman-silent region(1800-2800 cm^-1),which greatly eliminates the interference of biological macromolecules in the cell,improving the detection accuracy.Based on this strategy,the detection of ROS at the organelle level was achieved.Moreover,the changes of ROS in mitochondria during photothermotherapy were monitored.The results showed that with the increase of the PTT time,the ROS in the mitochondria increased significantly,until the time reached 8 min.Which revealed that the production of ROS is also one of the mechanisms of PTT induces cell death.?4?Based on the advantages of SERS spectroscopy technology in cell systems,for instance,SERS can realize the dynamic monitoring of biological macromolecules,we studied the PTT mechanism of mitochondrial targeted supercarbon dots?SCDs?using AuNPs as the SERS substrate.First,we prepared a kind of SCDs with strong absorption in the Vis-NIR region.By modifying the cancer cell membrane penetrating peptides and mitochondrial targeting peptides on the surface of SCDs,the selective accumulation of SCDs in mitochondria of cancer cells can be achieved,and induce the selective death of cancer cells during PTT process.It is to be noted that the PTT did no damage to normal cells and the cell viability difference between cancer cell?HepG2?and normal cell?L02?is as high as 70%,indicating that the mitochondrial target SCDs are highly specific and highly selective.In addition,we also found that the mitochondrial membrane potential decreased and the function was destroyed during this process.The dynamic changes of cell molecules during this process were further studied by SERS.The results showed that PTT will cause the structural changes of lipids,proteins and DNA,and then induced cell death.Which are helpful for the preparation of new CDs-based photothermal materials and the development of more efficient photothermal therapeutic platforms.