Preparation And Characterization Of Highly Sensitive Nanothermometers For Segmented Indication And Real-Time Monitoring Of Hyperthermia Temperature

The safety and efficacy of hyperthermia mainly depends on the local hyperthermia temperature and the distribution of temperature field.Therefore,in the process of hyperthermia,how to realize the segmented indication of the mild hyperthermia temperature,so that the mild hyperthermia temperature of 39°C～43°C can be easily observed and identified,and its temperature field distribution can be monitored in real time,which is an urgent problem to be solved.In order to solve the above problems,a novel highly sensitive polymer nanothermometer was developed to provide segmented indication and real-time monitoring of hyperthermia temperature and temperature field distribution.In order to achieve the purpose of this study,two organic small molecule fluorescent dyes with different fluorescence activation mechanisms were prepared,combined with temperature-sensitive polymers with different phase transition temperatures,and then the temperature-sensitive fluorescent polymers with different phase transition temperatures were coupled by click chemistry to obtain a two-block dual-temperature fluorescent polymer nanothermometer.Through the characterization of the molecular structure,temperature sensitivity,spectral performance,in vitro temperature measurement performance and biological imaging of the product,the controllable performance of the polymer nanothermometer and its excellent temperature sensitivity,fluorescence performance and in vivo and in vivo temperature measurement ability are confirmed.The specific chapters and contents of this paper are as follows:Chapter 1:Firstly,it briefly describes the importance of temperature monitoring for understanding cell activity and early diagnosis and treatment of diseases.Then,the existing temperature measurement technology,the types of nanometer thermometers and the principle of temperature measurement are reviewed,and the necessity of segmental indication and real-time monitoring of the temperature and temperature field distribution in the process of hyperthermia is introduced in detail.Based on the current actual needs,our solution is finally proposed,that is,to design a two-block dual temperature-sensitive fluorescent polymer nanothermometer,and the corresponding structure,composition,specific preparation method and related applications are described.Chapter 2:Design,synthesis and performance research of polarity-responsive thermosensitive fluorescent polymers based on fluorescent donors.In this chapter,we first synthesized a polar-responsive fluorescent dye nitro benzofuran acrylate monomer（NBD-AA）as a donor fluorophore for fluorescence resonance energy transfer,and verified that its maximum ultraviolet absorption peak is at 460 nm,the maximum fluorescence emission peak is around 540nm.Then it was randomly copolymerized with the thermosensitive monomer N-isopropyl acrylamide（NIPAM）by reversible addition-fragmentation chain transfer（RAFT）polymerization to obtain a polar responsive temperature-sensitive fluorescent polymer P（NIPAM-co-NBDAA）with a phase transition temperature（LCST₁）of 32.5°C.In the temperature-variable fluorescence spectrum test,the fluorescence intensity of the temperature-sensitive fluorescent polymer shows obvious abrupt enhancement between 32°C and 34°C.In a wider temperature range（24°C～40°C）,as the temperature gradually increased,the maximum change of the fluorescence intensity of P（NIPAM-co-NBDAA）reached 4.9 times.In addition to fluorescence intensity,the quantum yield and fluorescence lifetime of P（NIPAM-co-NBDAA）also increased significantly with increasing temperature,and the quantum yield（11.6%）and fluorescence lifetime（4.14 ns）at 38°C were about 4 times that of it at 24°C（2.7%,1.05 ns）.The above research results prove that the polarity-responsive thermosensitive fluorescent polymer P（NIPAM-co-NBDAA）based on a fluorescent donor was successfully synthesized.Chapter 3:Design,synthesis and performance research of thermosensitive fluorescent polymers with high phase transition temperature based on fluorescent acceptors.In this chapter,we first synthesized a rhodamine B derivative（RhB-AA）as an acceptor fluorophore for fluorescence resonance energy transfer,and verified that its maximum ultraviolet absorption peak is around 558 nm,and its maximum fluorescence emission peak is around 580 nm.Then,using reversible addition-fragmentation chain transfer（RAFT）polymerization,we randomly copolymerized N-isopropylacrylamide（NIPAM）and N,N-dimethylacrylamide（DMAM）,and obtained the phase transition temperature（LCST）is the thermosensitive polymer P（NIPAM-co-DMAM）at 42.5℃.Finally,the acceptor fluorophore RhB-AA was modified to the end of the temperature-sensitive polymer P（NIPAM-co-DMAM）by thiol-ene click chemical reaction,and the temperature-sensitive fluorescent polymer P（NIPAM-co-DMAM）-RhBAA with a phase transition temperature（LCST₂）of 42.8°C was obtained.P（NIPAM-co-DMAM）-RhBAA has a characteristic ultraviolet absorption peak at 563 nm and a characteristic fluorescence emission peak at 585 nm,the grafting rate of RhB-AA reaches 90.9%,and the quantum yield and fluorescence lifetime are 28.5%and 29.7 ns respectively.In the temperature-variable fluorescence spectrum test,the fluorescence intensity,quantum yield and fluorescence lifetime of the temperature-sensitive fluorescent polymer have no obvious changes with the increase of temperature.It can be seen that the temperature-sensitive fluorescent polymer P（NIPAM-co-DMAM）-RhBAA was successfully synthesized and has good photophysical properties.Chapter 4:The design,synthesis and performance research of the nano-thermometer with segmented indication and real-time monitoring.In this chapter,we coupled P（NIPAM-co-NBDAA）and P（NIPAM-co-DMAM）-RhBAA prepared in Chapters 2 and 3 by click chemistry to obtain two thermosensitive and the block temperature-sensitive fluorescent polymer PNN-b-PND-RhBAA was successfully synthesized by nuclear magnetic（¹H-NMR）,gel chromatography（GPC）,ultraviolet absorption spectroscopy（UV-vis）and fluorescence emission spectroscopy（FL）.Since the emission spectrum of NBD-AA is connected in series with the excitation spectrum of RhB-AA,and the two phase transition temperatures of the two-block thermosensitive polymer can adjust the distance between NBD-AA and RhB-AA,the two-block thermosensitive fluorescent polymer can exhibit different fluorescence under different temperature ranges through the fluorescence resonance energy transfer（FRET）effect.In the temperature-variable fluorescence spectrum test,the nano-thermometer showed a good linear relationship between the ratio of the fluorescence intensities(I₅₈₅/I₅₄₀)of the two fluorophores and the temperature within the temperature range of 32°C to 45°C,and the relative thermal sensitivity could reach 13.18%℃^-1,the temperature resolution can reach 0.08℃,and the real-time monitoring of temperature can be realized by calculating the fluorescence intensity ratio.Furthermore,in vivo studies of zebrafish,we confirmed that the nanothermometer PNN-b-PND-RhBAA has high biocompatibility and in vivo temperature measurement ability,that is,it can complete the three-color conversion from no fluorescence to green fluorescence to red fluorescence with the increase of ambient temperature,so as to realize the segmented indication of temperature.Chapter 5:At the end of this paper,we summarize the work of the full text,point out the deficiencies of some work,and put forward the prospect of the research direction in the next stage.