Design,Synthesis And Biological Imaging Of Lysosomal Viscosity And Polarity Fluorescent Probes

Viscosity is an important biological microenvironment parameter in biological system,which has an important influence on the diffusion speed of many species in biological system.Polarity is another important parameter in microenvironment.Some specific inorganic and organic reactions are inseparable from polarity.In biological systems,especially at the cellular level,it determines the relationship between most protein and enzymes,and reflects the permeability of cell and organelle membrane components.The changes of cell viscosity and polarity are closely related to the occurrence and development of some diseases.Lysosome is a kind of organelle existing in eukaryotic cells,which contains a large number of hydrolases and is specially used to decompose various internal and external macromolecular substances.In this thesis,due to the advantages of good biocompatibility and low toxicity of polymers,the polymer fluorescent probes with viscosity and polarity sensitivity are designed and synthesized,and their spectral characteristics and applications in biological imaging are deeply studied.Its main contents are as follows:（1）Based on indole fluorophore and fluorescent polymer 2-hydroxyethyl methacrylate（PHEM）,we proposed a new polymer fluorescent probe In-PHEM with double response sites to track the viscosity changes of cell lysosomes.In-PHEM has excellent fluorescence performance and high photostability.The polymer fluorescent probe was used to monitor the viscosity changes of lysosomes under different physiological conditions,including inducer stimulation,PBS starvation and apoptosis.When cells are hungry,the viscosity of lysosomes will gradually increase,while in apoptotic cells,its viscosity will first increase and then decrease.Therefore,this work provides a new platform for monitoring the viscosity change of lysosomes,which is helpful to explore the relationship between the viscosity of lysosomes and related diseases.（Ⅱ）In-PDMAEMA,a ratio fluorescent polymer viscosity probe,was constructed based on naphthalimide fluorescence platform and polymer dmaema.Naphthimide fluorophore was selected to modify to synthesize initiator,and then the monomer DMAEMA was polymerized by atom transfer radical polymerization（ATRP）.Finally,the polymer fluorescent probe with viscosity response was synthesized by indole salt modification.In non-viscous environment or low viscosity environment,the polymer chain shows blue fluorescence.In the high viscosity environment,the blue fluorescence on the polymer chain is enhanced,and the red fluorescence appears at 603nm.The results of cell co-localization experiments show that the probe can enter the cell and be enriched in lysosomes.The probe can be used to detect the viscosity changes in lysosomes of living cells stimulated by monensin,lipopolysaccharide and nystatin,and also to detect the viscosity changes in lysosomes under oxidative stress caused by H₂O₂.The ratio polymer fluorescent probe provides a new tool for monitoring the viscosity change of lysosomes and studying the physiological function of lysosomes.（Ⅲ）Molecules with electron donor-π-electron acceptor（D-π-A）structure have a good response to polarity.Based on this,a lysosomal-targeted fluorescent polymer polarity probe NP-PDMAEMA was constructed with naphthalimide-aniline borate fluorescence platform and polymer DMAEMA.The probe has short fluorescence emission wavelength and high fluorescence intensity in low polarity environment.In the high polarity environment,the fluorescence emission wavelength is red-shifted and the fluorescence intensity is obviously reduced.In the co-location experiment,the Pearson coefficient of the probe reached 0.95,which means that it can effectively detect the polarity change of lysosomes.The polarity changes of lysosomes under cell starvation and dysfunction were studied,and the polarity changes of zebrafish under different stimuli were also studied.This study not only provides a new tool for monitoring the change of lysosomal polarity at the cellular level,but also provides more important data for clarifying the relationship between lysosomal polarity and biological diseases.