Synthesis And Application Of Off-on Fluorescent Probes For Tumor Micro-environment Imaging

In recent years,with the development of cancer treatment,the detection of tumor markers under biomedical imaging has become a new trend.Fluorescence imaging has a far-reaching impact on the diagnosis and monitoring of cancer due to its high sensitivity,low cost,easy operation and so on.Glutathione(GSH)acts as biological molecules that contain thiol groups in the organism,which protects proteins or other cellular components from the oxidation of reactive oxygen species(ROS),and maintains the redox steady state in cells and organisms.The GSH play an important role in cellular redox homeostasis and protein function in living cells.The change of intracellular GSH concentrations is closely related to the biological disorders,such as cancer,heart disease and acquired immune deficiency syndrome(AIDS).Compared to normal tissue cells,the GSH in cell is highly expressed in the tumor region.It is very important to prepare a fluorescent probe capable of specifically detecting a biothiol molecule in vivo.However,most of the probe molecules with biothiol responsive their molecular excitation and emission wavelengths are located in the UV-visible region.The probe molecules are susceptible to autofluorescence interference,exsit strong background signal,low sensitivity,poor selectivity and damage to the tissue.Those shortcomings severely limited its application in biological imaging.In this paper,we synthesized the "off-on" type fluorescent probe with long-wavelength,highly sensitive and highly selective property.The switch-machanism fluorescent probe was loaded into the biodegradable PLGA polymer to form probe@PLGA nanoparticals.This nano diagnostic reagent has the characteristics of good biocompatibility and good pharmacokinetic parameters in vivo.Due to the presence of unstable ester bonds in its structure,the nanoparticles are easily degraded under tumor microenvironment conditions to release the probe.The switch-type probe used to assess tumors by different intensities of fluorescence.The designed “off-on” probe due to internal structure of the whole molecule photo-induced electron transfer(PET)mechanism exhibiting non-fluorescent(“off” state).But,the “off-on” probe molecule can recognize glutathione(GSH)with high selectivity and ultra-sensitivity in cells and tissues making whole probe molecule fluorescence intensity significantly increased(“on” state).The nanoparticles have property such as good biocompatibility,high sensitivity and not easily cleared from the blood and so on.The fluorescent probe has advantage such as excitation and emission wavelength is longer,relatively high quantum yield,high photochemical stability,good selectivity and sensitivity,so it can be applied to medical imaging to monitor tumor and intraoperative imaging in vivo.The fluorescent probe was synthesized by four-step chemical synthesis.The probe with tumor micro-environment response was successfully characterized by IR,NMR and ultraviolet and visible spectrophotometer(UV-Vis).The fluorescence probe was successfully loaded in blank PLGAs by the method of emulsion-solvent evaporation methods.The optical properties of probe molecules and its responsiveness to GSH were investigated by fluorescence emission spectroscopy.The nano-size of tumor diagnostic reagents with good biocompatibility was successful preparation.The blank PLGAs nanoparticles and the probe@PLGA nanoparticles were characterized by dynamic light scattering(DLS)particle size analyzer.The size,morphology and distribution of the probe@PLGA nanoparticles were characterized by transmission electron microscopy(TEM).The study of in vitro release behavior at pH=5.3 and pH=7.4 was measured by dialysis bag method.We choose MCF-7 cell lines as the model cells,The effect of each experimental group on the cell viability of breast cancer cells(MCF-7)was investigated by SRB method.We use fluorescence microscopy and flow cytometry for the diagnosis the in vitro properties of the nanosize reagents.The location of probe at the level of tumor cells was analyzed by laser confocal microscopy.Based on the S180 ascites cell lines,we constructed the tumor burdened KM mice model to investigate fluorescence imaging of probe and distribution of tissue in vivo.The pharmacokinetics of the nanoparticles in rats was investigated by high performance liquid chromatography(HPLC).The fluorescence spectra of the probes show that the synthesized fluorescent probes have the advantages of high selectivity,ultra-sensitivity and good optical stability.The average particle size of probe@PLGA nanoparticles prepared by emulsified solvent evaporation method was 94.49 ± 2.03 nm,the zeta potential was-8.97 ± 1.01 mV.The TEM images show that the nanoparticles are spherical and uniformly distributed.When the nanoparticles were stored at 4 ?,the nanoparticles can be stable for a long time,and has no obvious particle size changes were observed.The experimental results showed that the nanoparticles had good biocompatibility.The release of probe@PLGA nanoparticles at different pH values showed that pH = 5.3 had a higher cumulative release in vitro than pH = 7.4,and this release mechanism may caused by the PLGA nanoparticles are susceptible to tumor micro-environment.The cell cytotoxicity experiments show that probe@PLGA nanoparticles can significantly enhance their uptake behavior compared to free fluorescent probes.The fluorescent probe molecules can specifically target mitochondria in the cytoplasm rather than in the nucleus.Animal experiments show that the nanoparticles can targeted to the tumor through the high permeability and retention(EPR)effect.It shows strong fluorescence at the tumor site,which can be used to the definition of tumor region.In vivo pharmacokinetic experiments have shown that probe@PLGA nanoparticles prolonged blood circulation time in vivo and reduce the blood clearance rate,therefore the nanoparticles can be applied in complex in vivo environments.In this paper,we constructed a switch-mechanism fluorescence probe nanoparticles with tumor microenvironment responsive,which can defined the tumor region through different form of fluorescence.It has the advantages such as good selectivity,long emission wavelength,good sensitivity and rapid response time.It has important clinical significance and broad application prospect,and is used in in vivo fluorescence labeling and fluorescence surgery.This study lays a solid foundation for the future research.