Study On Preparation Of PH-Responsive Conjugated Polymer-based Nanoparticles,Sensing Property And Cell Imaging

Intracellular pH(pHi)is one of the important parameters of cellular physiological state and is closely associated with cell proliferation,apoptosis,autophagy,ion transport,endocytosis and cell polarization.pHi abnormalities will lead to cellular dysfunction and serious human diseases such as cancer,Alzheimer's disease,Parkinson's disease,etc.The intracellular microenvironment has different pH values,e.g.in mammalian cells,cytoplasmic pH is usually between 6.8-7.4,while the lysosomal interior is slightly acidic(pH 4.5-6.0).Therefore,visual monitoring of intracellular pH fluctuations not only helps to understand the physiological functions of pHi,but also has important implications for the diagnosis and treatment of related diseases.Fluorescent probe technology has the advantages of simple operation,fast response speed,high sensitivity,and the ability to achieve spatial and temporal resolution imaging,which has attracted widespread attention.Most of the currently reported pH fluorescent probes are based on response mechanisms such as intramolecular charge transfer(ICT),fluorescence resonance energy transfer(FRET)and excited state intramolecular plasmon transfer(ESIPT).In recent years,pH-responsive aggregation-de-agglomeration strategies based on conjugated polymer nanoparticles have proposed new ideas for the development of highly sensitive and selective pH fluorescent probes.In this thesis,we designed and synthesized a polythiophene derivative(PTPA)with a piperazine group in the side chain,and formed nanoparticles(PTPA-NPs)by self-assembly.The aggregation state of PTPA-NPs responds to pH changes with high selectivity,which can be used for quantitative detection of pH in aqueous solutions and lysosomal-targeted pH-responsive cell imaging.The main studies are as follows.1.Preparation and characterization of conjugated polymer nanoparticles PTPA-NPs.Polythiophene derivatives PTPA with N-ethyl piperazine in the side chain were prepared by Fe Cl3 oxidative polymerization and post-modification,and nanoparticles PTPA-NPs of PTPA were prepared in a mixture of THF/H2O(1/25,v/v)using nanoprecipitation method.Dynamic light scattering(DLS)results showed that the PTPA-NPs were well dispersed in water with a hydration diameter of approximately179 nm and a zeta potential of-15 m V;SEM photographs demonstrated the spherical shape of the nanoparticles.The effects of pH and common intracellular anions,cations and bioactive substances on the size and potential of the nanoparticles PTPA-NPs were investigated using the DLS system,and the results showed that the nanoparticles exhibited a highly selective response to pH,and their size increased with decreasing pH in the range of pH 4.0-8.0(at pH 4.0,the hydrated diameter of nanoparticles was about 342 nm;at pH 8.0,the hydrated diameter of nanoparticles is about 179 nm);in the range of pH 7.4-8.0,the zeta potential-34.5?-16.3 m V,and the nanoparticles are negatively charged;in the range of pH 3.8-6.0,the zeta potential 9.13?42.1 m V,and the nanoparticles are positively charged.2.pH sensing properties of conjugated polymer nanoparticles PTPA-NPs and cell imagingThe UV maximum absorption peak of PTPA-NPs nanoparticles in water was located at about 500 nm with a molar extinction coefficient of 7.57×10~3 L·mol^-1·cm^-1;the fluorescence quantum efficiency of the aqueous dispersion of PTPA-NPs almost did not emit fluorescence due to the fluorescence quenching caused by aggregation,which was less than 0.58%.With the gradual decrease of pH from 8.0 to 4.0 and the increase of H⁺ion concentration in the solution,the nitrogen atom on the N-ethyl piperazine group on the side chain of the conjugated polymer PTPA was protonated,and through the electrostatic repulsion between the polymer chains,the nanoparticles PTPA-NPs partially disaggregated,and their absorption peaks were blue-shifted from500 nm to about 420 nm,and the color of the solution changed from purplish red to pale yellow.The fluorescence emission intensity increased about 26-folds,showing an obvious OFF-ON fluorescence response,and the change of fluorescence intensity showed a good linear relationship with pH,which can be used for the quantitative determination of pH in aqueous solution.MTT experiments showed that the nanoparticles PTPA-NPs have good biocompatibility;co-localization experiments with the lysosomal probe Lyso-Tracker Deep Red It was shown that PTPA-NPs were mainly distributed in lysosomes and had lysosomal targeting ability.Therefore,PTPA-NPs were successfully used for pH-responsive fluorescence imaging in He La cells.