Study On The "Self-switch" And "Reversible" Near-infrared Ratio Fluorescence Probe Of Tumor Micro-acid Environment

The acidic tumor environment is closely related to many factors that control tumor growth and metastasis.The detection of pH at the tumor site and the monitoring of pH changes have attracted widespread attention in the field of nanomedicine.In this paper,pH-sensitive near-infrared(NIR)fluorescent probe SiRB was synthesized by introducing boric acid groups into silicon rhodamine structure.Indocyanine Green(ICG)and SiRB were loaded into polylactic acid-glycolic acid copolymer(PLGA)with good biocompatibility to form pH-responsive near-infrared ratio fluorescent nanoprobe PLGA-SiRB-ICG.Experiments showed that the nanoparticles can accumulate efficiently at the tumor site by enhanced permeability and retention effect.PLGA accelerated degradation in weak acid environment is conducive to the release of the probe,SiRB has a good pH response in the range of pH 5.8 ~ 7.6,and the fluorescence intensity of ICG is almost unchanged with the change of pH.The fluorescence ratio of the two probes can quantitatively measure the pH value and pH value of the tumor site,which has important reference value for occurrence,development and metastasis of tumor.The main research contents of this topic can be divided into four parts:1.The synthesis and properties of "self-switching" near-infrared fluorescent probe SiRBIn this paper,the "self-switching" near-infrared fluorescent probe SiRB was successfully synthesized by using four-step method which 3-bromo-N,N-dimethylaniline and 2-bromophenylboronic acid as intermediates;the intermediates and final products were purified by column chromatography.The molecular structure and excitation / emission wavelength of SiRB were characterized by 1H-NMR,UV-Vis and fluorescence spectroscopy.The results showed that SiRB molecular structure consistent with the expected structure,and the excitation / emission wavelength are in the near infrared region.SiRB probe in the pH range of 5.8 to 7.6 with a sensitive pH response,and the study found that the fluorescence probe pH response had a good reversibility,was conducive to monitoring the tumor site of the pH changes.2.The preparation and properties of pH-responsive near infrared fluorescence probes based on SiRBUsing SiRB as a fluorescent pH indicator;Indocyanine Green(ICG)was used as the fluorescence reference reagent(the fluorescence intensity was not affected by pH change in a certain acid base range).The polymer PLGA was used as the carrier,and PLGA-SiRB-ICG was successfully obtained by solvent emulsification method.The PLGA-SiRB and PLGA-ICG were prepared by the same method.The results of transmission electron microscopy and UV-visible spectroscopy confirmed that PLGA loaded SiRB and ICG to form a core-shell structure.The results showed that the average particle size and potential of PLGA-SiRB-ICG are 95.13 ± 0.21 nm and-8.14 ± 0.13 mV,respectively.The particle size is uniform and stable.By comparing the UV-Vis spectra at different pH,the fluorescence ratio of PLGA-SiRB-ICG varied with pH from pH 5.8 to 7.6.The results showed that PLGA-SiRB-ICG had a higher release rate in the micro-acidic environment,which was beneficial to the release in the microenvironment of the tumor.The results showed that PLGA-SiRB-ICG had a higher release rate in the micro-acidic environment.3.Cell experiment of pH-responsive near infrared ratio fluorescence probe PLGA-SiRB-ICGThe cytotoxicity of PLGA-SiRB-ICG was investigated by using human breast cancer cell MCF-7 as the model cell.SRB results showed that PLGA-SiRB-ICG had no significant toxicity to MCF-7 cells at low concentration.When the concentration was more than 80 μg / mL,the preparation had certain toxicity to MCF-7 cells and the toxicity of free SiRB and ICG,which was significantly higher than that of the preparation group,indicating that the encapsulation of PLGA reduced the toxicity of the probe to the cells.We could see that the probe SiRB could enter and had a special targeting to the lysosome the cell by laser confocal fluorescence microscopy experiment.This study is expected to be applied to organelles.4.Fluorescence imaging and pH detection of pH-response near infrared ratio fluorescent nanoprobe PLGA-SiRB-ICGFirst,the relationship between the fluorescence ratio of the PLGA-SiRB-ICG and the pH value was recorded by the multi-mode living imaging system(KODAK).The linear relationship between them was investigated by linear regression analysis and the linear regression equation was obtained.Then,KM mice were used as model mice and ascites tumor S180 cells were used as model cells to construct the tumor-bearing mice model.Through the multi-mode in vivo imaging system,the detection of pH value of tumor tissue and major organs in mice was successful performed.Through the experimental results we can find: PLGA-SiRB-ICG on tumor tissue has a certain passive target,and the tumor site pH was significantly lower than other organs.Through the observation of different growth days,the longer the tumor growth days,the larger the size,the lower the p H,the results of the development stage of the tumor and micro-environment research has important reference value.