Research On Novel Ruthenium-containing Metallopolymer-Based Optical Oxygen Nanosensors For Tumor Hypoxia Imaging

Hypoxia is one of the most important characteristics of the microenvironment of all solid tumors,which is directly related to the tumor invasion,metastasis and resistance to radiotherapy and chemotherapy.Therefore,the measurement of oxygen content in tumor is of great significance for early diagnosis,mid-term treatment and prognosis evaluation.Optical oxygen sensing technology is very attractive because of its non-invasiveness and high sensitivity,which shows a good prospect in the aspect of detection of tumor hypoxia.Because of their good biocompatibility,the fluorescent nanosensors thus have a long-time circulation in vivo.Due to the small particle size,those nanosensors can be passively enriched into tumor tissues by EPR effect.Because of the easy surface modification,those nanosensors are especially suitable for the detection of oxygen content in tumors and hypoxia imaging.Because of the aggregation-induced quenching effect,the oxygen probes such as Pt,Pd porphyrin and Ir complex can only be doped only in a small proportion in the nanosensors,which leads to the compromise of the fluorescence properties.Although Pt,Pd porphyrin and Ir complexes are very sensitive to oxygen,their poor stability is not suitable for long-term measurement in practical applications,which limits their application in optical oxygen nanosensors.Ruthenium complexes are the most widely studied oxygen probes,which not only have excellent photophysical properties and oxygen sensing properties,but also have no aggregation-induced quenching effect in solution or polymer environment.Herein,a series of novel ruthenium?II?-containing metallopolymers have been synthesized and prepared as nanosensors by a one-step reprecipitation method.The potential of this kind of nanosensors based on ruthenium?II?-containing metallopolymers in oxygen sensing has been discussed,the main results of the study are as follows:?1?The ruthenium?II?-containing metallopolymer?Ru-1?was synthesized by the coordination reaction of Ru?bpy?₂Cl₂ with the hydrophobic polymer PS-PMA-bpy containing bipyridine group,and its structure was confirmed by ¹H NMR,GPC,and spectroscopy analysis.Subsequently,PS-PEG-COOH was introduced to prepare nanoparticles with a diameter of about 80 nm and a negative Zeta potential,which emitted a red light of 603 nm when excited at 460 nm.After functionalizing the surface of nanoparticles,the fluorescent oxygen nanosensors Mito-NPs which can accumulate within the mitochondria were obtained,and their oxygen sensing performance in solution was studied.The results showed that the oxygen quenching response of Mito-NPs in aqueous solution was 75%,which was similar to that of Ru?bpy?₃ in aqueous solution.The emission intensity ratio of Mito-NPs under nitrogen and oxygen atmosphere was 3.9,and the intensity quenching was obvious,fit well by a Stern-Volmer curve?R²>0.999?,indicating that Mito-NPs has excellent oxygen sensing performance.?2?Hep G2 cells were co-cultured with the commercial Mito-Tracker Green and the mitochondrial-targeting fluorescent nanosensor Mito-NPs for a period of time.When excited at 488 nm,MTG glows green while Mito-NPs glows red.From the confocal fluorescence images,almost all red glows overlap with green glows and thus appear yellow.The Pearson's coefficient of Mito-NPs and MTG is about 0.43,which indicates that Mito-NPs can selectively target mitochondria.Finally,Mtio-NPs was incubated with the cells,and the mitochondria within the Hep G2 cells were stimulated by FCCP and Rotenone to regulate the oxygen content,thus the oxygenation and deoxidation processes within the cells were observed in real time.The results show that Ru 1-NPs has high photostability and excellent oxygen sensing properties,and can be further functionalized,which shows great potential in the detection of dissolved oxygen content and hypoxia imaging at the cellular and organelle levels.?3?On the basis of the above work,two novel metallopolymers Ru-2 and Ru-3were synthesized by using phen and dpp instead of bpy,their structures and photophysical properties were studied by ¹H NMR,GPC and spectral characterization.The UV-Vis absorption spectra showed that the maximum emission wavelength of the metallopolymer redshifted with the increase of the?-conjugation degree of the main ligand,and the quantum yield of Ru-3 increased to 8.8%.Subsequently,nanosensors were prepared by reprecipitation method.Zeta potential and spectral analysis indicated that the ruthenium complexes were not normally encapsulated in the hydrophobic core of the polymer,instead,they tended to be distributed on the surface of the nanoparticles,thus this unique distribution and nanostructure provides a guarantee for their excellent oxygen sensing performance.The quenching response of Ru 3-NPs is 81.9%,the emission intensity ratio is 5.9 under nitrogen and oxygen atmosphere.?4?The three dimensional multicellular tumor spheroids?MCTSs?were cultured by ultra-low adsorption surface method,and the hypoxia imaging in MCTSs using Ru-2and Ru-3 nanosensors was studied in detail.The results showed that both nanosensors could be rapidly ingested by tumor spheres and remained in the spheres for a long time without being metabolized due to the protection of PEGylation and small particle size.The Z-axis scanning function of confocal microscope is used to image the three-dimensional MCTSs.Through the analysis of Z-stack photographs,it can be distinguished that the peripheral fluorescence signal of the MCTSs is very weak,while the red signal of the inner core of the MCTSs is strong.At the same time,the variation of oxygen gradient in the MCTSs with distance can be measured in a simple semiquantitative analysis by the fluorescence of the nanosensors,which lays a foundation for its application in vivo.In a word,in this thesis,a series of ruthenium?II?-containing metallopolymers were designed and synthesized.Taking advantage of those metallopolymer,luminescent oxygen nanosensors were prepared in combination with PS and PS-PEG-COOH by a modified reprecipitation method.Oxygen sensing performance and hypoxia imaging within multicellular tumor spheroids were studied.86 figures,16 tables and 113 references are included in this thesis.