Study On The Preparation,pH Sensing And Cell Imaging Of Polythiophenes-based Nanoparticles

Intracellular pH（pHi）plays a very important role in many physiological and pathological processes.The pH value of typical mammalian cells is in the range of 4.5-8.0,and the pH values in different organelles are different.For example,the pHi in the cytoplasmic matrix is usually 6.8-7.4,while the pH in lysosome and mitochondria is about for4.5 and 8.0.A slight change in pHi can reflect the state of the cell and the metabolic process.In addition,a large number of studies have shown that the high acidity of cells is often associated with many diseases.Therefore,accurate measurement and visual imaging of intracellular pH is of great significance for biological research and clinical diagnosis.Fluorescent probe technology has the advantages of high sensitivity,simple operation,high signal-to-noise ratio,and good space-time resolution.It has become one of the effective analytical methods for pH measurement in cells.A large number of pH fluorescent probes have been reported,which are mainly based on derivatives of small molecule fluorescent dyes such as fluorescein,cyanine and anthraquinone.These probes generally have the disadvantage of poor water solubility and poor light stability,and it is impossible to track and observe changes in intracellular pH for a long time.Therefore,it is necessary to develop other pH probe systems.Conjugated polymers have strong light-trapping ability and multiplicative optical responsiveness,providing a new material system for sensing and imaging various biologically active substances.In this thesis,a polythiophene derivative（PTNEt₂）was designed and synthesized,which is characterized in that the polythiophene skeleton is a fluorescent reporter group and the side chain diethylamino group is a pH responsive group.The polymer is practically insoluble in water,but its protonated product is readily soluble in water.Using this property,a novel pH-dependent conjugated polymer nanoparticle assembly-deassembly pH sensing strategy was proposed.The pH detection principle is as follows:PTNEt₂nanoparticles（PTNPs）dispersed in water due to aggregation induced fluorescence quenching,the solution hardly emits fluorescence.As the pH decreases,the diethylamino group on PTNEt₂ in PTNPs is protonated,and the nanoparticles are deassembled due to electrostatic repulsion between the polymer chains,and the fluorescence of the system is restored.Conversely,as the pH increases,the diethylamino group on PTNEt₂ deprotonates,becomes a water-insoluble polymer,and reassembles into nanoparticles,and the fluorescence quenches the system.The main work of this paper is as follows:（1）Preparation and stability of conjugated polymer nanoparticles PTNPs.The conjugated polymer PTNEt₂ was first synthesized,and its chemical structure was determined by NMR.Then,PTNEt₂ nanoparticles（PTNPs）were prepared by reprecipitation method.The morphology and hydration diameter of the nanoparticles were characterized by TEM and DLS.The effects of polymer concentration on the particle size and morphology of the nanoparticles were investigated.The results show that the polymer forms spherical nanoparticles,and as the concentration of the polymer increases,the particle size of the nanoparticles increases and the stability decreases.The polymer nanoparticles prepared with a polymer concentration of 1 mg/mL have good stability and a suitable concentration,and the hydrate diameter is about 193 nm.Finally,using dynamic light scattering（DLS）,the effects of resting time,pH value,and common biological active substances in cells on the stability of nanoparticles were systematically studied.（2）The responsiveness of nanoparticle PTNPs to pH and cell imaging.The responsiveness of nanoparticle PTNPs to pH was studied in detail by UV absorption spectroscopy and fluorescence spectroscopy.The nanoparticles were dispersed in an aqueous solution of pH=8.0,and 0.1 M acetic acid was gradually added to the solution to gradually lower the pH of the system to 4.0.The results of UV titration spectroscopy indicated that with the increase of acidity of the system,the absorption peaks of the particles at 515 nm,550 nm,595 nm in the ultraviolet spectrum gradually shifted to 438 nm,and the color of the solution gradually changed from purple to pale yellow.The fluorescence emission spectrum shows that the fluorescence intensity increases with the decrease of the pH value of the system.According to the relationship between the fluorescence intensity and the pH value,the pKa of the nanoparticles was about 5.71（R²=0.9979）.When 0.1 M Na₂CO₃ aqueous solution was gradually added in the system with pH=4.0,the pH value of the system was gradually restored to 8.0.The results of spectral titration were as follows:as the basicity of the system gradually increased,the fluorescence intensity of the solution gradually weakened,and at 438 nm the UV absorption peak gradually shifted to 515 nm,550 nm,595 nm.Compared with other metal ions,only the addition of H⁺caused a significant change in fluorescence intensity of PTNPs,demonstrating that PTNPs have good selectivity for pH.MTT assay results indicate that PTNPs have low cytotoxicity and show good biocompatibility.The pH-dependent cell imaging experiments showed that the fluorescence intensity of the nanoparticles showed a good linear relationship with the change of intracellular pH（Y=2.4506-0.3077X,R²=0.9966）.Therefore,PTNPs are expected to be used for quantitative detection of intracellular pH.