CN115433219B - Polar fluorescent probe with photodynamic property and preparation method and application thereof - Google Patents

Abstract

The invention relates to a polar fluorescent probe with photodynamic property, a preparation method and application thereof, wherein the structural formula of the polar fluorescent probe is as follows, so that not only can the fluorescent imaging of tumor cells and living body level tumor tissues be realized, but also healthy cells and tumor cells can be distinguished by utilizing the fluorescence signal difference caused by the polarity, and the specific imaging of living body level tumor tissues is realized; the compound can also generate singlet oxygen, has photodynamic therapy property, can effectively inhibit tumor growth, promotes apoptosis of tumor cells, and plays a good role in resisting tumor in nude mice transplanted tumor model.

Description

Polar fluorescent probe with photodynamic property and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a polar fluorescent probe with photodynamic properties, and a preparation method and application thereof.

Background

Cell polarity is an important parameter in the cellular microenvironment and is represented by the ordered arrangement of polar protein complexes and other polar biomolecules in the cell. Stabilization of cell polarity is an important guarantee of cell vital activities, such as cell division, differentiation, proliferation, etc. Studies have shown that cancer develops with reduced cell polarity, e.g., mutation of gene Par6 expressing polar molecules results in disruption of epithelial cell polarity; LKB1 kinase deficiency involved in cell polarity regulation can destroy the polarity of mammary epithelial cells, exacerbate tumor metastasis and invasion; atypical extracellular factor/planar cell polarity (Wnt/PCP) pathway signaling protein VANGL2 is overexpressed in basal breast cancer tissue, and the like. These studies reveal that reduced cell polarity is an important feature and marker of tumor cells. A fluorescence imaging guided precision photodynamic therapy (photodynamic therapy, PDT) strategy can accurately illuminate a tumor region by guiding a light source with a specific fluorescence response signal of tumor cells, thereby accurately treating the tumor. At present, various fluorescent probes based on fluorescence imaging of tumor microenvironments (weak acid, over-expressed enzyme, GSH and the like) and combined with PDT have been developed, but polar fluorescent probes with PDT function have not been reported yet.

Disclosure of Invention

The invention aims to provide a polar fluorescent probe HBT-CUR with photodynamic property on the basis of the prior art, which not only can realize fluorescent imaging of tumor cells and tumor tissues at living level, but also can distinguish healthy cells from tumor cells by utilizing fluorescence signal difference caused by polarity, thereby realizing specific imaging of tumor tissues at living level; the compound can also generate singlet oxygen, has photodynamic therapy property, can effectively inhibit tumor growth, promotes apoptosis of tumor cells, and plays a good role in resisting tumor in nude mice transplanted tumor model.

It is another object of the present invention to provide a method for preparing the polar fluorescent probe.

The invention also provides medical application of the polar fluorescent probe in fluorescent imaging diagnosis and photodynamic treatment effect, in particular to fluorescent imaging diagnosis and/or treatment of tumor cells and tissues in vitro and in vivo.

The technical scheme of the invention is as follows:

a polar fluorescent probe (HBT-CUR) with photodynamic property has the following structural formula:

the invention provides a polar fluorescent probe (HBT-CUR) concept, which mainly comprises the following points: (1) construction of an excellent fluorophore framework: 2- (2' -Hydroxyphenyl) Benzothiazole (HBT) is taken as an ESIPT fluorophore framework and an electron Donor group (Donor), and a stronger electron-deficient group difluoro borate structure is taken as an electron Acceptor group (accepter), so as to construct a molecule with a D-pi-A conjugated structure; (2) a polar sensitive group: introducing a difluoro borate structure as a polar sensitive group; (3) design of photosensitive Properties of the probes: the molecular structure has a strong Intramolecular Charge Transfer (ICT) effect, and the strong ICT effect can improve the efficiency of the excited state electron transfer, so that the yield of singlet oxygen is enhanced.

In the present invention, a method for preparing a polar fluorescent probe having photodynamic properties, comprising the steps of:

further, a method for preparing a polar fluorescent probe having photodynamic properties, comprising the following more detailed steps:

(1) In the presence of sodium hydroxide, performing a chemical reaction between a compound a and 2-amino thiophenol to prepare an intermediate HBT-1;

(2) In the presence of trifluoroacetic acid, performing a chemical reaction on the intermediate HBT-1 and hexamethylenetetramine to prepare an intermediate HBT-2;

(3) Under the protection of nitrogen, the compound b and boron trifluoride diethyl etherate are subjected to chemical reaction to prepare an intermediate C-1;

(4) In the presence of piperidine, the intermediate HBT-2 and the intermediate C-1 are subjected to chemical reaction to prepare the polar fluorescent probe HBT-CUR.

For the purposes of the present invention, in step (1), the molar ratio of compound a to 2-aminophenylsulfnol is 1:1 to 3, and may be, but not limited to, 1:1, 1:1.3, 1:1.5, 1:1.8, 1:2, 1:2.3, 1:2.5, 1:2.8 or 1:3, and the molar ratio of compound a to 2-aminophenylsulfnol is 1:2 for better effect.

In the step (1), the molar ratio of the compound a to the sodium hydroxide is 1:5-10, which may be, but not limited to, 1:5, 1:5.5, 1:6, 1:16.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9 or 1:10, and the molar ratio of the compound a to the sodium hydroxide is 1:8 for obtaining better effects.

In the step (1), the reaction temperature is 80 to 100℃and may be, but not limited to, 80℃85℃90℃95℃or 100℃and, for obtaining a better effect, 90 ℃.

Further, the reaction time is 1 to 3 hours, preferably 1 hour.

In the step (2), the molar ratio of the intermediate HBT-1 to the hexamethylenetetramine is 1:0.8-1.5, which may be, but is not limited to, 1:0.8, 1:0.9, 1:0.95, 1:1, 1:1.05, 1:1.2, 1:1.3 or 1:1.5, and the molar ratio of the intermediate HBT-1 to the hexamethylenetetramine is 1:1 for obtaining better effects.

In step (2), the mass-to-volume ratio of the intermediate HBT-1 to the trifluoroacetic acid is 40-50:1 mg/ml, which may be, but is not limited to, 40:1mg/ml, 43:1mg/ml, 45:1mg/ml, 45.3:1mg/ml, 45.4:1mg/ml, 45.6:1mg/ml, 48:1mg/ml or 50:1mg/ml, and the mass-to-volume ratio of the intermediate HBT-1 to the trifluoroacetic acid is 45.4:1mg/ml for obtaining a better effect.

In the step (3), the molar ratio of the compound b to the boron trifluoride diethyl ether is 1:1-3, which may be, but not limited to, 1:1, 1:1.3, 1:1.5, 1:1.8, 1:2, 1:2.3, 1:2.5, 1:2.8 or 1:3, and the molar ratio of the compound b to the boron trifluoride diethyl ether is 1:2 for obtaining better effect.

In step (4), the molar ratio of intermediate HBT-2 to intermediate C-1 is 1:0.8-1.5, which may be, but is not limited to, 1:0.8, 1:0.9, 1:0.95, 1:1, 1:1.05, 1:1.2, 1:1.3 or 1:1.5, and the molar ratio of intermediate HBT-2 to intermediate C-1 is 1:1 for better effect.

In step (4), the molar ratio of intermediate HBT-2 to piperidine is 1:0.8-1.5, which may be, but is not limited to, 1:0.8, 1:0.9, 1:0.95, 1:1, 1:1.05, 1:1.2, 1:1.3 or 1:1.5, and the molar ratio of intermediate HBT-2 to piperidine is 1:1 for better effect.

The fluorescent probe provided by the invention can be used as a photosensitizer for detecting cell polarity, can realize fluorescent imaging of tumor cells and tumor tissues at the living body level, can distinguish healthy cells from tumor cells by utilizing fluorescence signal difference caused by the polarity, and realizes specific imaging of the tumor tissues at the living body level.

The fluorescent probe provided by the invention can be used as a photosensitizer in photodynamic tumor treatment of tumors, has photodynamic treatment property, generates singlet oxygen, can effectively inhibit tumor growth, promotes tumor cell apoptosis, and plays a good role in resisting tumor in nude mice transplanted tumor models.

By adopting the technical scheme of the invention, the advantages are as follows:

the polar fluorescent probe (HBT-CUR) with photodynamic property can realize fluorescent imaging of tumor cells and tumor tissues at living body level, distinguish healthy cells from tumor cells by utilizing fluorescence signal difference caused by polarity, and realize specific imaging of tumor tissues at living body level; the compound can also generate singlet oxygen, has photodynamic therapy property, can effectively inhibit tumor growth, promotes apoptosis of tumor cells, and plays a good role in resisting tumor in nude mice transplanted tumor model.

The fluorescence quantum yield and maximum emission wavelength of the polar fluorescent probe (HBT-CUR) are obviously changed in solvents with different polarities (phi in toluene solution) _f ＝0.189，λ _em =600 nm; Φ in DMSO solution _f ＝0.035，λ _em 666 nm), can detect polarity change with high selectivity and high sensitivity, and has good photobleaching resistance, structural stability and visible light stability; meanwhile, the probe also has excellent photosensitive performance, and the singlet oxygen yield phi _Δ Can reachTo 0.70.

The probe HBT-CUR can generate stronger fluorescent signals in low-cell polarity environments of tumor cells (4T 1, MCF-7, hepG2, MDA-MB-231, 786-o), and only weak fluorescent signals in healthy cells (HK-2, MCF-10A), so that the difference of polarities can be used for distinguishing the tumor cells from the healthy cells.

The probe may also be used to induce apoptosis of tumor cells by generating Reactive Oxygen Species (ROS) by PDT. The probe can specifically image tumor tissues at the living body level, and the fluorescent signal is stable, so that the probe is suitable for long-time imaging (6 h); photodynamic therapy of tumor tissue at living level shows that after the probe is combined with phototherapy, the tumor volume of the MCF-7 nude mice transplanted tumor model is obviously reduced, which indicates that the probe has good anti-tumor activity.

Drawings

FIG. 1 is a schematic diagram of the design principle of a probe HBT-CUR; the chinese corresponding to english in fig. 1 is as follows: donor represents a donor; the receptor represents a receptor; low polarity represents low polarity; fluorescence iamging represents fluorescence imaging; normal represents normal cells; cancer cells represent tumor cells; tomor represents a tumor; light represents light irradiation; PDT stands for photodynamic therapy; ICT stands for intramolecular charge transfer; the Borate ester represents a difluoroborate; HBT represents 2- (2' -hydroxyphenyl) benzothiazole;

FIG. 2 is a compound HBT-1 ¹ H NMR；

FIG. 3 is a compound HBT-1 ¹ H NMR；

FIG. 4 is a compound C-1 ¹ H NMR；

FIG. 5 is a probe HBT-CUR ¹ HNMR profile;

FIG. 6 is a probe HBT-CUR ¹³ CNMR profile;

FIG. 7 is a probe HBT-CUR ¹⁹ FNMR spectrum;

FIG. 8 is a high resolution mass spectrum of probe HBT-CUR: [ M-H ]] ^- calculated for C ₁₉ H ₁₄ BF ₂ NO ₃ ,384.0677；found, 384.0671.

FIG. 9 is an absorption spectrum and an emission spectrum of the probe HBT-CUR; wherein (A) in FIG. 9 is an ultraviolet absorption spectrum of a probe in Toluene (tolene), ethanol (Ethanol), 1, 4-Dioxane (Dioxane), acetonitrile (Acetonitrile), methanol (Methanol), methylene chloride (dichlormethane), acetone (acetate), dimethyl sulfoxide (DMSO), ethyl acetate (Ethylacetate), PBS (10 mM), and at 380nm of the abscissa, the curves represent PBS (10 mM), ethanol (Ethanol), dimethyl sulfoxide (DMSO), methanol (Methanol), 1, 4-Dioxane (Dioxane), acetonitrile (acetate), ethyl acetate (Ethylacetate), acetone (acetate), methylene chloride (dichlormethane) and Toluene (tolene) in order from low to high; FIG. 9 (B) shows fluorescence emission spectra of a probe (10. Mu.M) in the above solvent, and at 630nm on the abscissa, the curves represent, in order from low to high, PBS (10 mM), methanol (Methanol), ethanol (Ethanol), dimethyl sulfoxide (DMSO), acetonitrile (Acetonitrile), 1, 4-Dioxane (Dioxane), methylene chloride (dichlormethane), toluene (tolene), ethyl acetate (ethyl acetate) and Acetone (acetate);

FIG. 10 is a detection performance of the probe HBT-CUR; wherein (A) in FIG. 10 is the absorption spectrum of HBT-CUR (10. Mu.M) in PBS (0% -60%) solvent, and at 380nm on the abscissa, the curves represent 50% PBS, 60% PBS, 40% PBS, 30% PBS, 20% PBS and 50% PBS, in order from low to high; FIG. 10 (B) is a fluorescence spectrum of HBT-CUR (10. Mu.M) in PBS (0% -60%) solvent, with curves representing 60%, 50%, 40%, 30%, 20% and 50% PBS, in order from low to high, at 600nm on the abscissa; FIG. 10 (C) is a fluorescence plot of probe (10. Mu.M) at 365nm excitation in a solvent system with PBS content of 0% -60%; FIG. 10 (D) shows the correlation R between fluorescence intensity (10% -60% PBS system) and polarity parameter Δf ² ＝0.991(Ex＝405nm，slit：5nm)；

FIG. 11 is the stability of the probe HBT-CUR; wherein, in fig. 11 (a), HBT-CUR (10 μm) has a photobleaching resistance (ex=405 nm, slit:5 nm) obtained by continuously irradiating an excitation light source for 10min in 50% PBS and a Dioxane solution without PBS; FIG. 11 (B) shows the stability of HBT-CUR (10. Mu.M) when placed in 50% PBS with a PBS-free Dioxane solution at 37deg.C for 32 h; FIG. 11 (C) is a HBT-CUR (10. Mu.M) LED lamp (100 mW/cm in 50% PBS with a Dioxane solution without PBS ² ) Photostability upon irradiation for 30min (ex=405 nm, slit：5nm)；

FIG. 12 is a selectivity of probe HBT-CUR over polarity; wherein, in FIG. 12 (A), the fluorescence spectrum of the probe HBT-CUR (10. Mu.M) in PBS solution with the glycerol content increased from 10% to 50%, represents curves with glycerol concentrations of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% and 50%, which are not greatly different from each other and are substantially coincident with each other; FIG. 12 (B) shows the change in the maximum fluorescence intensity value of the probe HBT-CUR (10. Mu.M) in PBS solution from 10% to 50% in glycerol content;

FIG. 13 is a selectivity of probe HBT-CUR over polarity; wherein (A) in FIG. 13 is a fluorescence spectrum of the probe HBT-CUR (10. Mu.M) in PBS solution with pH increased from 4.0 to 9.0, representing curves with pH values of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0, respectively, which are not greatly different from each other and substantially coincide with each other; FIG. 13 (B) shows the change in the maximum fluorescence intensity value of the probe HBT-CUR (10. Mu.M) in PBS solution with pH increased from 4.0 to 9.0;

FIG. 14 is a selectivity of probe HBT-CUR over polarity; in FIG. 14, (A) is a probe (10. Mu.M) and active oxygen (O) ₂ ^·- ；H ₂ O ₂ ； HClO； ¹ O ₂ The method comprises the steps of carrying out a first treatment on the surface of the t-BuOOH; OH) (100. Mu.M), active Nitrogen (NO) ₂ ^- ；NO ₃ ^- ；ONOO ^- The method comprises the steps of carrying out a first treatment on the surface of the NO) (100 μm) fluorescence spectrum after 1min incubation at 37 ℃, all the enumerated curves representing active oxygen and active nitrogen substantially coincide; FIG. 14 (B) shows the maximum fluorescence intensity value of each group of solutions, 1.O ₂ ^·- (100μM)；2.H ₂ O ₂ (100μM)；3.HClO(100μM)；4. ¹ O ₂ (100μM)； 5.t-BuOOH(100μM)；6.·OH(100μM)；7.NO ₂ ^- (100μM)；8.NO ₃ ^- (100μM)；9.ONOO ^- (100μM)； 10.NO(100μM)；11.Dioxane；

FIG. 15 is a selectivity of probe HBT-CUR over polarity; in FIG. 15, (A) is a probe (10. Mu.M) and active sulfur (Na) ₂ S； NaHS；Na ₂ SO ₃ ；NaHSO ₃ ；CH ₃ SSSCH ₃ ；CysSSCys；GSSG；S ₈ The method comprises the steps of carrying out a first treatment on the surface of the GSH) fluorescence spectrum after incubation for 1min at 37 deg.cAll the listed curves representing active sulfur substantially coincide; FIG. 15 (B) shows the maximum fluorescence intensity value of each group of solutions, 1.Na ₂ S (100μM)；2.NaHS(100μM)；3.Na ₂ SO ₃ (500μM)；4.NaHSO ₃ (500μM)；5.CH ₃ SSSCH ₃ (100μM)；6.CysSSCys(1mM)；7.GSSG(1mM)；8.S ₈ (500μM)；9.GSH(10mM)；10.Dioxane；

FIG. 16 is a selectivity of probe HBT-CUR over polarity; in FIG. 16, (A) is a probe (10. Mu.M) obtained by mixing a metal ion (Sn) ²⁺ ； Cd ²⁺ ；Mn ²⁺ ；Co ²⁺ ；Cu ²⁺ ；Hg ²⁺ ；Zn ²⁺ ；Fe ²⁺ ；Ca ²⁺ ；Fe ²⁺ ；Al ³⁺ ；Ni ²⁺ ；Mg ²⁺ ；Li ⁺ ；Na ⁺ ；K ⁺ ；Ag ⁺ ) Fluorescence spectra after incubation for 1min at 37 ℃, all the enumerated curves representing different metal ions substantially coincide; FIG. 16 (B) shows the maximum fluorescence intensity value of each group of solutions, 1.Sn ²⁺ (1mM)；2.Cd ²⁺ (1mM)；3.Mn ²⁺ (1mM)；4.Co ²⁺ (1mM)；5.Cu ^{2 +} (1mM)；6.Hg ²⁺ (1mM)；7.Zn ²⁺ (1mM)；8.Fe ²⁺ (1mM)；9.Ca ²⁺ (1mM)；10.Fe ³⁺ (1mM)；11.Al ³⁺ (1mM)；12.Ni ²⁺ (1mM)；13.Mg ²⁺ (1mM)；14.Li ⁺ (1mM)；15.Na ⁺ (1mM)；16.K ⁺ (1mM)；17.Ag ⁺ (1mM)；18.Dioxane；

FIG. 17 is a selectivity of probe HBT-CUR over polarity; wherein (A) in FIG. 17 is a fluorescence spectrum of a probe (10. Mu.M) incubated for 1min at 37℃with amino acids (Gly; D-Ala; val; ile; L-Cys; L-Thr; asp; L-Glu; L-Arg; L-Lys; L-His; hcy; C), all of the enumerated curves representing different amino acids substantially coincide; FIG. 17 (B) shows the maximum fluorescence intensity value of each group of solutions, 1.Gly (1 mM); 2.D-Ala (1 mM); val (1 mM); ile (1 mM); 5.L-Cys (1 mM); 6.L-Thr (1 mM); asp (1 mM); 8.L-Glu (1 mM); 9.L-Arg (1 mM); L-Lys (1 mM); L-His (1 mM); hcy (1 mM); dioxane;

FIG. 18 shows the production of DCFH-DA in vitro detection probe HBT-CURGenerating active oxygen; in FIG. 18, (A) is a PBS solution of a probe (10. Mu.M) and DCFH-DA (40. Mu.M), and the solution is prepared by mixing the solution with a light source (LED, 100 mW/cm) ² ) Under irradiation, the fluorescence signal at 525nm changes; (ex=488nm, silt: 5); wherein the upper curve represents HBT-CUR+DCF-DA and the lower curve represents HBT-CUR+DCF-DA+light; FIG. 18 (B) shows that the solution exhibits a clear green fluorescence at 365nm excitation light;

FIG. 19 shows singlet oxygen yields in DPBF detection probes HBT-CUR in ethanol; wherein (A) in FIG. 19 is the probe (10. Mu.M) measured in the LED light source (100 mW/cm ² ) Irradiating for 1min, and changing absorption spectrum, wherein curves representing times of 60s, 50s, 40s, 30s, 20s, 10s and 0s are not greatly different and are basically overlapped; in FIG. 19 (B), DPBF (100. Mu.M) was irradiated with a light source for 1min, and the absorption spectrum was changed, and curves representing the times of 60s, 50s, 40s, 30s, 20s, 10s and 0s were not greatly different from each other and were substantially overlapped with each other; in FIG. 19 (C), the probe (10. Mu.M) and DPBF (100. Mu.M) were irradiated with a light source for 1min, and the change in absorbance spectrum, at 400nm on the abscissa, was represented by 60s, 50s, 40s, 30s, 20s, 10s and 0s in this order from low to high; in FIG. 19 (D), rose bengal (10. Mu.M) and DPBF (100. Mu.M) were irradiated with light for 1min, and the change in absorption spectrum, at 400nm on the abscissa, the curves represent 60s, 50s, 40s, 30s, 20s, 10s and 0s in this order from low to high; FIG. 19 (E) shows a linear relationship (R) between the absorbance at 410nm of the ethanol solution system of probe (10. Mu.M) and DPBF (100. Mu.M) ² =0.991); FIG. 19 (F) is a linear relationship (R) between Rose bengal (10. Mu.M) and DPBF (100. Mu.M) in terms of absorbance at 410nm ² ＝0.999)；

FIG. 20 is a graph showing cytotoxicity of HBT-CUR by MTT assay; wherein, FIG. 20 (A) shows cell viability of 4T1, 786-o, hepG2, MCF-7, MDA-MB-231, HK-2, MCF-10A cells incubated with different concentrations of probe HBT-CUR (0,2.5,5, 10, 20. Mu.M) for 24h, and in 5 sets of bar graphs in the figure, each set of bar graphs represents 4T1, 786-o, hepG2, MCF-7, MDA-MB-231, HK-2, MCF-10A cells in order from left to right; FIG. 20 (B) shows cell viability of 4T1, 786-o, hepG2, MCF-7, MDA-MB-231, HK-2, MCF-10A cells incubated with probe HBT-CUR (5. Mu.M) for various times (0, 6, 12, 24, 48 h), in 5 sets of bar graphs each representing 4T1, 786-o, hepG2, MCF-7, MDA-MB-231, HK-2, MCF-10A cells in sequence from left to right;

FIG. 21 is a biocompatible fluorescence imaging of probe HBT-CUR: the probe (5. Mu.M) was incubated with MCF-7 cells for various times (0, 5, 10, 15, 20 min), followed by staining of the nuclei with 4, 6-diamidino-2-phenylindole (DAPI) for 5min and confocal imaging; the probe is red fluorescent, and the DAPI is blue fluorescent; (Channel 1: lambda) _ex ＝405nm，λ _em ＝405-500nm；Channel2：λ _ex ＝405nm，λ _em ＝600-700 nm；Scale bar：40μm)；

FIG. 22 is a subcellular organelle co-localization fluorescence imaging of probe HBT-CUR: fluorescence imaging after incubation of the probe (5. Mu.M) with MCF-7 cells for 20 min; then stained with Lyso-Tracker Green DND-26 (66.7 nM), ER-Tracker Green (1. Mu.M), golgi-Tracker Green (NBD C6-cepamide, 3. Mu.M) and Mito-Tracker Green FM (200 nM), respectively; FIG. 4 is a graph showing the correlation between dye and probe intensity; column 5 is fluorescence intensity analysis of fixed cross section, in the first row of example 5, at an abscissa value of 0.5, the upper curve represents Lyso Green, the lower curve represents HBT-CUR, and in the second row of example 5, at an abscissa value of 1, the upper curve represents ER-Tracker, the lower curve represents HBT-CUR; in the third row of example 5, at an abscissa value of 0.5, the upper curve represents Gitracker, and the lower curve represents HBT-CUR; in the fourth row of example 5, at an abscissa value of 1.4, the upper curve represents Mito Green and the lower curve represents HBT-CUR; (Red channel:. Lambda.) _ex ＝405nm,λ _em ＝650-750 nm；green channel:λ _ex ＝488nm,λ _em ＝490-550nm Scale bar：40μm)；

FIG. 23 is a polarity test of probe HBT-CUR for different cell lines; after incubating the probe (5 μm) with different cell lines at 37 ℃ for 20min, confocal laser imaging was performed, wherein (a) in fig. 23 is 4T1; FIG. 23 (B) is MCF-7; FIG. 23 (C) is HepG2; FIG. 23 (D) is MDA-MB-231; FIG. 23 (E) is 786-o; FIG. 23 (F) is HK-2; FIG. 23 (G) is MCF-10A; FIG. 23 (H) is the flow chart (A) of FIG. 23 (G) Relative fluorescence intensities in total 7 groups of cells, data expressed as mean+ -SD (n=3), and experiments performed on groups of cells (786-o vs HK-2; MCF-7, MDA-MB-231 and 4T 1vs MCF-10A) using t-test for statistical difference analysis between groups, p is expressed as<0.001；(λ _ex ＝405nm，λ _em ＝600-700nm，Scale bar：40μm)；

FIG. 24 is the mean fluorescence intensity of flow cytometry assay probe (5. Mu.M) in tumor cells (4T 1, MCF-7, hepG2, MDA-MB-231, 786-o) versus normal cells (HK-2, MCF-10A); FIG. 24 (A) shows the average fluorescence intensity of cells; in fig. 24 (B) is the relative fluorescence intensity (n=3) in the 7 groups of cells, the Mean ± SD of the experimental data, the experimental groups of cells (786-o VS HK-2; 4T1, MCF-7 and MDA-MB-231VS MCF-10A) were subjected to an inter-group statistical difference analysis using T-test, p <0.001;

FIG. 25 is Calcein (AM)/Propidium Iodide (PI) apoptosis staining (time to PDT promote apoptosis); after incubation of the probe (5. Mu.M) with the cells for 20min, the probe was incubated with the cells in an LED (100 mW/cm ² ) Irradiating for 25min, and performing fluorescence imaging every 5 min; (Calcein AM: lambda) _ex ＝488nm，λ _em ＝500-550nm；PI：λ _ex ＝561nm，λ _em ＝590-640nm，Scale bar：100μm)；

FIG. 26 is a fluorescence imaging of Calcein (Calcein AM)/Propidium Iodide (PI) apoptosis staining; control: MCF-7 cells; light: irradiating with MCF-7 cell LED light source; HBT-CUR: incubating the probe (5 mu M) with the cells for 20min, and performing light-shielding treatment; HBT-cur+light: after incubating the probe (5 mu M) with the cells for 20min, irradiating by an LED light source; (Calcein AM: lambda) _ex ＝488nm，λ _em ＝500-550nm；PI：λ _ex ＝561nm，λ _em ＝590-640nm，Scale bar：100μm)；

FIG. 27 shows reactive oxygen species generated in DCFH-detecting cells; HBT-cur+light: after incubation of the probe (5. Mu.M) with MCF-7 cells for 20min, the probe was incubated with LED (100 mW/cm ² ) Illuminating for 25min; control: MCF-7 cells were not treated; HBT-CUR: incubating the probe (5 mu M) with MCF-7 cells for 20min, and treating in a dark place; light: MCF-7 cell LED (100 mW/cm) ² ) Illumination; after the above-mentioned respective cells were treated differently, DCFH (10μM) confocal fluorescence imaging; (lambda) _ex ＝488nm，λ _em ＝490-550nm，Scale bar： 40μm)；

FIG. 28 is a polarity detection of probe HBT-CUR in tumor tissue at the in vivo level; wherein, in FIG. 28 (A), a fluorescence image of 100. Mu.L of probe (final concentration 1mM, physiological saline: DMSO=9:1 v/v), (0 min,1min,5min,10min,20min,40min,60min,80min,120min,6h,12 h,24h,48 h) was injected intratumorally into tumor-bearing mice; fluorescence imaging of (B) tumor-bearing mice with intratumoral injection of 100 μl of solvent (physiological saline: dmso=9:1 v/v), (0 min,1min,5min,10min,20min,40min,60min,80min,120min,6h,12 h,24h,48 h); following intratumoral injection of the probe into the tumor-bearing mice in fig. 28 (C), the fluorescence intensity values over 120min, data mean±sd, are shown (n=6); in fig. 28 (D), after intratumoral injection of the probe into tumor-bearing mice, the fluorescence intensity values at time points (6 h,12h,24h,48 h) are shown as data mean±sd (n=6);

FIG. 29 is a in vivo horizontal fluorescence imaging of probe HBT-CUR; control: 100. Mu.L of blank solvent (physiological saline: DMSO=9:1 solution, v/v) was injected into right-side axillary normal tissue and left-side axillary tumor tissue; wherein, (a) in fig. 29 is a right normal tissue image of the mouse; wherein, (B) in fig. 29 is imaging of tumor tissue on the left side of the mouse; HBT-CUR: 100. Mu.L of probe (1 mM, physiological saline: DMSO=9:1, v/v) was injected into right-side underarm normal tissue and left-side underarm tumor tissue; fig. 29 (C) is a right normal tissue image of the mouse; fig. 29 (D) is a left tumor tissue image of the mouse; fig. 29 (E) shows the fluorescence signal intensity of tumor tissue of tumor-bearing mice injected with the probe in tumor and the fluorescence signal intensity of normal tissue alone (n=6), wherein p <0.001;

FIG. 30 is a study of different light source treatment regimens at the living body level, wherein 100. Mu.L of probe (1 mM, physiological saline: DMSO=9:1, v/v) was injected intratumorally, control was not given with exogenous light, and each of the other was given 300mW/cm, respectively ² Power light source 500 mW/cm ² Power light source, 1W/cm ² Irradiating by a power LED lamp for 30min each day; wherein (a) in fig. 30 is the change in body weight in the tumor-bearing mice over 14 days; wherein (B) in FIG. 30 is the tumor of tumor-bearing mice within 14 days Tumor volume change; wherein, in fig. 30 (C), mice were sacrificed after 14 days, and tumor tissue comparison plots were dissected;

FIG. 31 is tumor treatment with probes at the in vivo level, HBT-CUR+light: tumor-bearing mice were injected intratumorally with 100 μl of probe (1 mM, normal saline: dmso=9:1, v/v), once every two days, with LEDs (300 mW/cm ² ) Irradiating for 30min by a light source; light: light irradiation was given for 30min daily, and 100 μl of blank solvent was injected; HBT-CUR: 100. Mu.L of probe (1 mM) (once every two days) was injected and treated in the dark; control: tumor-bearing mice naturally grow. Each group was treated for a period of 14 days; wherein (a) in fig. 31 is the change in body weight of the tumor-bearing mice within 14 days; fig. 31 (B) shows the tumor volume change in the tumor-bearing mice over 14 days; in fig. 31 (C), mice were sacrificed 14 days later, the mass of the tumor was dissected, and significant differences were analyzed between groups using t-test, representing p<0.001; FIG. 31 (D) is a photograph of the right underarm tumor tissue of a tumor-bearing mouse after 14 days of treatment; control; light; HBT-CUR; hbt-cur+light; in fig. 31 (E), mice were sacrificed 14 days later and the dissected tumor camera was photographed; FIG. 31 (F) shows H in tumor tissue dissected from mice sacrificed after 14 days &E, dyeing; control; light; HBT-CUR; HBT-CUR (Scale bar:50 μm);

FIG. 32 is H & E staining of heart tissue, liver tissue, spleen tissue, lung tissue, kidney tissue (Scale bar:50 μm) 14 days after treatment of tumor bearing mice from different treatment groups Control, light, HBT-CUR, HBT-CUR+light.

Detailed Description

The polar fluorescent probes of the present invention are further illustrated by the following examples, which are not intended to limit the invention in any way.

1. Method of implementation

1. Solution preparation

Preparing a probe solution: HBT-CUR (3.85 mg,0.01 mmol) was dissolved in 1.0mL of dimethyl sulfoxide to prepare a 10.0mM probe solution.

Preparation of Glutathione (GSH) stock solution: GSH (30.70 mg,0.10 mmol) was dissolved in deionized water (10.0 mL) to prepare a stock solution at a concentration of 10.0mM, and the stock solution was diluted to 1.0mM and 10.0mM for use.

Preparation of L-cysteine (L-Cys) stock solution: cys (12.10 mg,0.10 mmol) was dissolved in deionized water (10.0 mL) to give a stock solution at a concentration of 10.0mM, and the stock solution was diluted to 1.0mM and 10.0. Mu.M for use.

Preparation of homocysteine (Hcy) stock solution: hcy (13.50 mg,0.10 mmol) was dissolved in deionized water (10.0 mL) to prepare a stock solution at a concentration of 10.0mM, and the stock solution was diluted to 1.0mM and 10.0. Mu.M for use.

The preparation method of stock solutions of other substances (Gly, D-Ala, val, ile, L-Thr, asp, L-Glu, L-Arg, L-Lys, L-His, etc.) is the same as that described above.

ONOO ^- ；O ₂ ^·- ；H ₂ O ₂ ；HClO； ¹ O ₂ ；t-BuOOH；·OH；Na ₂ S；NaHS；CH ₃ SSSCH ₃ ；CysSSCys； GSSG；S ₈ ；Na ₂ SO ₃ ；NaHSO ₃ ；NO ₂ ^- ；NO ₃ ^- ；NO；Sn ²⁺ ；Cd ²⁺ ；Mn ²⁺ ；Co ²⁺ ；Cu ²⁺ ；Hg ²⁺ ；Zn ²⁺ ；Fe ²⁺ ； Ca ²⁺ ；Fe ³⁺ ；Al ³⁺ ；Ni ²⁺ ；Mg ²⁺ ；Li ⁺ ；Na ⁺ ；K ⁺ ；Ag ⁺ Double distilled water is used as solvent. OH by FeII (EDTA) with H ₂ O ₂ The Fenton reaction between the two. NO is produced from 3- (aminopropyl) -1-3-hydroxy-3-isopropyl-2-oxo-1-triazene (NOC-5, 50. Mu. Mol/ml).

All the above solutions were ready for use.

2. Cells

Species strain: MCF-7 (human breast cancer cell line), MDA-MB-231 (human breast cancer cell line), hepG 2 (liver cancer cell line), 4T1 (mouse breast cancer cell line), 786-o (human renal clear cell line), HK-2 (human renal cortex proximal tubular epithelial cell), MCF-10A (mammary epithelial cell). The source is as follows: cell bank of China academy of sciences, shanghai Fuheng science and technology Co., ltd.

3. Experimental animal

Species strain: healthy female Balb/C nude mice, weight 20-25g. The source is as follows: calvens laboratory animal Co., ltd.

2. Experimental method

2.1 Probe HBT-CUR Synthesis

The compound HBT-1 is synthesized by reacting trifluoromethyl phenol (compound a) as a raw material with 2-amino thiophenol, and then HBT-2 is synthesized by Duff reaction. The acetylacetone (compound b) is used as a raw material to synthesize C-1. Under the condition of piperidine, the C-1 and the HBT-2 are condensed by Knoevenagel to generate the polar fluorescent probe HBT-CUR. The design principle of the probe HBT-CUR is shown in figure 1.

2.1.1 Synthesis of Compound HBT-1

A mixture of 2- (trifluoromethyl) phenol (162.1 mg,1.0 mmol), 2-aminophenylsulfiol (260.9 mg,2.0 mmol) and 1mol/L NaOH (8.0 mL) was heated to 90℃under nitrogen and reacted for 1h. After cooling to room temperature, the mixture was adjusted to pH 7 with 1mol/L HCl (8.0 mL) to give a yellow precipitate. Filtration, washing of the filter cake with water (3X 10.0 mL) followed by recrystallization of the filter cake from methanol (8.0 mL) (reflux at 65 ℃ C., cooling crystallization) gave a white solid (R) _f =0.6, pe/ea=20:1, v/v) 132.0g of product, 60.2% yield. ¹ HNMR(400MHz,CDCl ₃ ) δ8.00 (d, j=8.0 hz, 1H), 7.90 (d, j=8.0 hz, 1H), 7.70 (d, j=8.0 hz, 1H), 7.51 (t, j=8.0 hz, 1H), 7.37-7.43 (m, 2H), 7.11 (d, j=8.0 hz, 1H), 6.96 (t, j=8.0 hz, 1H). As shown in fig. 2. LC-MS Found [ M-H ]] ^- :226.2.

Synthesis of 2.1.2HBT-2

HBT-1 (454.0 mg,2.0 mmol) was dissolved in 10.0mL of trifluoroacetic acid, and hexamethylenetetramine (280.0 mg,2.0 mmol) was added thereto and the mixture was reacted under reflux for 5 hours. The reaction mixture was then cooled to room temperature and the ph=7 was adjusted with 1mol/L NaOH, a yellow precipitate formed. The filter cake was washed with saturated brine and dried, and the product was a yellow solid. TLC monitoring (R) _f =0.5, pe/ea=20:1, v/v). 210.0mg of product with 40.3% yield. ¹ HNMR(400MHz,CDCl ₃ )δ10.53(s,1H),8.05-8.13(m,2H),7.93(t,J＝8.0Hz,2H),7.56(t, J＝8.0Hz,1H),7.46(t,J＝8.0Hz,1H),7.10(t,J＝8.0Hz,1H).LC-MS Found[M-H] ^- 254.0. As shown in figure 3.

Synthesis of 2.1.3C-1

Under the protection of nitrogen, compound b (100.1 mg,1.0 mmol) and boron trifluoride diethyl etherate (284.1 mg,2.0 mmol) are mixed and reacted for 12h at room temperature, and the product is separated out into transparent needlepoint crystals, filtered by suction, washed with water and dried for 3 times. TLC (R) _f =0.6, pe/ea=5:1, v/v) 133.2mg of product, 90.4% yield. ¹ HNMR(400MHz,CDCl ₃ )δ5.97(s,1H),2.03(s,6H).LC-MS Found[M+Na] ⁺ 171.0. As shown in figure 4.

2.1.4 Synthesis of polar fluorescent Probe HBT-CUR

HBT-2 (255.1 mg,1.0 mmol) and C-1 (141.1 mg,1.0 mmol) were dissolved in 20.0mL absolute ethanol, piperidine (544.6 mg,1.0 mmol) was added, the reaction was refluxed for 6h, the product precipitated out as an orange yellow precipitate, filtered off with suction, and washed three times with absolute ethanol. TLC monitoring (R) _f =0.6, pe:ea=1:1, v/v). 115.0mg of product was obtained in 29.1% yield. ¹ HNMR(400MHz,DMSO-D ₆ )δ8.32(d,J ＝16.0Hz,1H),8.24(d,J＝8.0Hz,1H),8.14(d,J＝8.0Hz,1H),8.08(d,J＝8.0Hz,1H),8.02(d,J＝8.0Hz, 1H),7.62(dd,J＝8.0,4.0Hz,1H),7.54(dd,J＝8.0,4.0Hz,1H),7.34(d,J＝16.0Hz,1H),7.18(t,J＝8.0Hz, 1H),6.56(s,1H),2.41(s,3H). ¹³ CNMR(100MHz,DMSO-D ₆ )δ＝193.4,180.6,168.7,157.6,151.2,142.0, 133.8,133.0,132.9,127.8,126.8,123.1,123.0,122.6,122.2,121.0,118.1,102.5,24.7ppm. ¹⁹ FNMR(376 MHz,CDCl ₃ ) Delta-136.10 ppm the correlation spectra are shown in FIGS. 5-8.

2.2 measurement of absorption Spectrum and emission Spectrum of Probe HBT-CUR

2.2.1 determination of the ultraviolet absorption spectra of the probes in different solvents

The UV spectrum was measured using a UV-2401PC ultraviolet visible spectrophotometer. The probe was placed in a quartz cuvette, diluted to 10. Mu.M with 10 different solvents, and UV absorbance was measured.

2.2.2 determination of fluorescence spectra of probes in different solvents

The probe HBT-CUR was dissolved in 10 different solvents to prepare a 10. Mu.M sample solution to be measured, and fluorescence spectroscopy was measured. Each set of data was measured at least in triplicate. Results are expressed as mean±sd. Fluorescent spectrophotometer test conditions: for the probe HBT-CUR, the excitation wavelength is 405nm, the excitation slit width is 5nm, the emission slit width is 5nm, the scanning speed is 1200nm/min, and the emission spectrum range is 405-800nm. The voltage of the photomultiplier was set to 600V.

2.3 determination of fluorescence Quantum yields of Probe HBT-CUR in different solvents

Experiment adopts relative method to measure fluorescence quantum yield of probe in different solvents ^[104-105] Quinine sulfate is selected as a reference substance, and the calculation formula is as follows:

Φ _x ＝Φ _s (A _s F _x /A _x F _s )(n _x /n _s ) ²

Φ: fluorescence quantum yield

A: absorbance at excitation wavelength

F: integration area under corrected fluorescence spectrum curve

n: refractive index of solvent

2.4 detection Performance of Probe HBT-CUR

The probe HBT-CUR mother solution is diluted to 10 mu M by a Dioxane-PBS solvent system containing PBS (0-60%), the ultraviolet absorption spectrum and the fluorescence emission spectrum are respectively measured, and the correlation diagram of the fluorescence intensity at the maximum emission wavelength and the solvent polarity parameter Deltaf is drawn. The different solvent polarity parameters Δf were calculated using the lipppert Mataga formula:

TABLE 1 polarity parameter Table for binary mixed solvent

ε _mix ＝f _a ε _a +f _b ε _b (1)

Epsilon: dielectric constant of solvent

f is the percentage of the total solvent system

Refractive index of solvent

2.5 stability detection of Probe HBT-CUR

2.5.1 stability of the Probe

The probe stock was diluted to 10. Mu.M with Dioxane and 50% Dioxane-PBS solvent, respectively, and the fluorescence spectra were measured for both groups of samples while maintaining at 37℃for 2,4,8,16,32h, respectively.

2.5.2 photobleaching resistance of probes

The probe stock was diluted to 10. Mu.M with 50% water and a Dioxane solvent without water, respectively, and the two samples were continuously scanned under the same conditions for 10min, and the fluorescence spectrum was measured at each minute time point.

2.5.3 visible light stability of probes

The probe stock was diluted with Dioxane and 50% Dioxane-PBS solvent, respectively10. Mu.M, both samples were at 100mW/cm simultaneously ² The white light source is irradiated for 30min, and the fluorescence spectrum is measured every 5 min.

2.6 active oxygen detection of Probe HBT-CUR

DCFH-DA was activated by weighing DCFH-DA and dissolving it in DMSO to prepare a 1mM stock solution. Adding 0.5mL of mother liquor into 2mL of 0.01M sodium hydroxide aqueous solution, uniformly mixing the solution, standing at room temperature for 30min to fully hydrolyze and activate the solution, adding 10mL of sodium phosphate buffer solution (10 mM) into the hydrolysate to adjust the pH to 7.4, and storing at minus 20 ℃ in a dark place.

1 μl of probe HBT-CUR (10 mM) was added to 2mL of activated DCFH solution, and the solution was purified using LED (100 mW/cm ² ) After 1min of irradiation with the light source, the emission spectrum was measured with a fluorescence spectrophotometer. The excitation wavelength was 488nm, the excitation slit width was 5nm, and the emission slit width was 5nm.

2.7 measurement of singlet oxygen yield of Probe HBT-CUR

With 1, 3-Diphenylisobenzofuran (DPBF) ¹ O ₂ Trapping agent, rose bengal as standard reference (phi) _Δ =0.75), the singlet oxygen quantum yield (Φ) of the probe HBT-CUR was determined at room temperature _Δ ). HBT-CUR was dissolved in 1.0mL of methanol containing 20. Mu.M DPBF. Each prepared solution was prepared using 100mW/cm ² The LED lamp of (2) is irradiated for 1min at a time interval of 10s, the ultraviolet absorption spectrum is measured, and when the HBT-CUR and rose bengal in the test solution have the same OD value, the singlet oxygen quantum yield is calculated according to the following formula:

Φ _Δ singlet oxygen yield

K absorbance degradation Rate of DPBF at 410nm

2.8 Probe HBT-CUR cytotoxicity test

The cytotoxicity of the probe HBT-CUR was determined by MTT method. MCF-7, hepG 2, MDA-MB-231,4T1, 786-o, HK-2, MCF-10A cells were seeded at a density of 10,000 cells/well in 96-well plates, placed in 5% CO ₂ CulturingThe cells were cultured in a box until the cells entered the logarithmic growth phase, the culture medium was removed, and the cells were incubated with HBT-CUR at different concentrations for 48h, respectively. As a control, cells without HBT-CUR were used. For 24h, 20. Mu.L of MTT dye (3- [4, 5-dimethylthiazol-2-yl) was added to each well]-2, 5-diphenyltetrazolium bromide, 5mg/mL in PBS buffer), the incubation at 37 ℃ was continued for 4 hours. The remaining MTT solution was then removed and 150 μl of DMSO was added to each well to dissolve formazan crystals. After shaking the shaker for 10min, absorbance at 570nm was measured with an enzyme-labeled instrument. Each sample was repeated three times and the entire experiment was repeated three times.

2.9 cell culture, fluorescent imaging of cells

MCF-7 (DMEM high sugar medium), hepG 2 (DMEM high sugar medium), MDA-MB-231 (DMEM high sugar medium), 4T1 (DMEM/F12 high sugar medium), 786-o (1640 high sugar medium), HK-2 (low sugar medium), MCF-10A (special medium) were inoculated into a cell culture solution containing 10% fetal bovine serum, and cultured at 37℃in 5% CO ₂ Culturing in an incubator. Grown to log phase, digested with pancreatin and inoculated into confocal dishes.

2.9.1 cell uptake imaging

The cell culture broth was removed and washed 2 times with PBS. The probe HBT-CUR (final concentration 5. Mu.M) was added to the confocal dish and incubated with the cells for 20min, the medium was removed and washed 2 times with PBS. 600. Mu.L of paraformaldehyde was added and the mixture was fixed for 15min, paraformaldehyde was removed, and PBS was used for washing 2 times. After adding 500. Mu.L of DAPI dye, staining for 10min, removing the DAPI dye, washing 2 times with PBS, and adding 1.0mL of fresh incomplete medium for confocal fluorescence imaging.

The excitation wavelength of the channel 1 (blue fluorescent channel) is 405nm, and the emission wavelength range is 420-470nm; the excitation wavelength of channel 2 (red fluorescent channel) was 405nm and the emission wavelength range was 600-700nm. Analysis was performed using the Leica software (Lecia). All data are expressed in mean±sd (n=3).

2.9.2 cell Co-location imaging

The cell culture broth was removed and washed 2 times with PBS. Prepared ER-Tracker Green/Lyso-Tracker Green/Mito-Tracker Green/Golgi-TThe racker Green working fluid and the cells were incubated at 37℃with 5% CO ₂ The medium is incubated for 15-30min, the working solution is removed, and PBS is used for washing for 2 times. The incomplete medium containing the probe HBT-CUR (final concentration 5. Mu.M) was incubated with cells in confocal dishes for 20min with 1.0mL, medium removed and washed 2 times with PBS. 1.0mL of fresh incomplete medium was added and confocal fluorescence imaging was performed.

The excitation wavelength of the channel 1 (green fluorescence channel) is 488nm, and the emission wavelength range is 500-550nm; the excitation wavelength of channel 2 (red fluorescent channel) was 405nm and the emission wavelength range was 600-700nm. Analysis was performed using the Leica software (Lecia). All data are expressed in mean±sd (n=3).

2.9.3 cell confocal imaging

The cell culture broth was removed and washed 2 times with PBS. The probe HBT-CUR (final concentration 5. Mu.M) was added to the confocal dish and incubated with the cells for 20min, the medium was removed and washed 2 times with PBS. 1.0mL of fresh incomplete medium was added. Confocal fluorescence imaging was performed.

The excitation wavelength of channel 1 (red fluorescence channel) was 405nm and the emission wavelength range was 600-700nm. Analysis was performed using the Leica software (Lecia). All data are expressed in mean±sd (n=3).

Quantitative detection of 2.9.4 cell level fluorescence intensity

Cells were seeded at 100,000 cells/well in six well plates at 37℃in 5% CO ₂ Culturing until the cells adhere to the wall. The probe HBT-CUR (final concentration 5. Mu.M) was added to the well plate and incubated with the cells for 20min, the medium was removed and washed 2 times with PBS. 1.0mL pancreatin was added for digestion, and after resuspension with PBS, 500. Mu.L was pipetted into a flow tube for detection.

The excitation wavelength of the channel (PerCP) was 488nm and the emission wavelength was 675nm. Analysis was performed using flowjo10.6.2. All data are expressed in mean±sd (n=3).

2.9.5 apoptosis staining

Cells were seeded at 100,000 cells/well in six well plates at 37℃in 5% CO ₂ Culturing until the cells adhere to the wall. The probe HBT-CUR (final concentration 5. Mu.M) and the formulated Calcein/propidium iodide (Calcein Am/PI) working solution (1.0 mL) were added to the well plate,incubating with the cells for 20min, and incubating with 100mW/cm ² The LED lamp of (C) was irradiated for 25min, and imaging was performed by a fluorescence microscope.

The excitation wavelength of channel 1 (green channel) was 405nm and the emission wavelength was 455nm. Channel 2 (red channel) has an excitation wavelength of 488nm and an emission wavelength of 590nm. All data are expressed in mean±sd (n=3).

Fluorescent imaging for ROS detection in 2.9.6 cells

The cell culture broth was removed and washed 2 times with PBS. Adding probe HBT-CUR (final concentration 5 μm) and 1.0mL of prepared DCFH-DA working solution into a confocal dish to incubate with cells for 20min, and then using 100mW/cm ² The LED lamp of (2) is irradiated for 25min.

The excitation wavelength of channel 1 (green fluorescence channel) is 488nm, and the emission and reception wavelengths are 500-550nm. Analysis was performed using the Leica software (Lecia). All data are expressed in mean±sd (n=3).

2.10 animal feeding

Healthy female Balb/C nude mice weighing 20-25g were supplied by the Hemsl laboratory animal company. The animal protocol is approved by the animal protection and use committee of Xuzhou medical university, and the animal experiment is conducted in accordance with the rule of using experimental animals by Chinese law. Animals should be familiar with the environment a week in advance before the experiment, and are kept in cages and groups under the natural circadian rhythm illumination condition, the temperature is 22+/-2 ℃, and the humidity is 50+/-10%.

2.11 establishment of nude mice transplantation tumor model

MCF-7 cells were cultured to log phase, digested, resuspended in normal saline and counted at 3X 10 ⁷ The cell viability was greater than 90% per mL. mu.L (3X 10) ⁶ Individual) cell suspensions were inoculated subcutaneously in the left upper limb axilla of nude mice.

2.12 fluorescence imaging of nude mice transplantation tumor model

2.12.1 influence of imaging time

6 tumors in the same batch are selected to have good conditions, and the tumor volume exceeds 200mm ³ Is a nude mouse of (2). After anaesthetizing nude mice with isoflurane gas, 100 μl probe HBT-CUR (1 mM) was injected intratumorally at a concentration of (1 min, 5min, 10min, 20min, 40min, 60min, 80 min), 120min, 6h, 12h, 24 h) were imaged using a Night OWL II LB983 small animal in vivo imager. Imaging conditions: excitation wavelength 475nm and emission wavelength 600nm. Analysis of the images and data was performed using the Indigo software.

2.12.2 in vivo horizontal tumor specific imaging

The same batch of 12 tumors with the tumor volume exceeding 200mm is selected in good condition ³ Is a nude mouse of (2). Randomly dividing into 2 groups, namely 6 groups of Control group and probe group, after anaesthetizing nude mice with isoflurane gas, the probe group is injected with 100 mu L of probe HBT-CUR (1 mM) in the left armpit tumor and the right armpit tissue of the tumor-bearing mice at the same time, the Control group is injected with the same amount of blank solvent (normal saline: DMSO=9:1) in the left armpit tumor and the right armpit tissue of the tumor-bearing mice at the same time, and imaging is carried out by adopting a Night OWL II LB983 small animal living body imager after 20 min. Imaging conditions: excitation wavelength 475nm and emission wavelength 600nm. Analysis of the images and data was performed using the Indigo software.

2.13 photodynamic therapy experiments at the Living level

Establishment of 2.13.1 treatment regimen

Nude mice with good conditions are selected, and the tumor volume exceeds 100mm ³ Is randomly divided into four groups of six. Is divided into Control group, light+HBT-CUR group (300 mW/cm) ² ，500mW/cm ² ，1mW/cm ² ) The same feeding conditions and growth environment are controlled. (1) Control group: the transplanted tumor mouse model is not subjected to any treatment and is fed under normal conditions; (2) light+hbt-CUR group: 100. Mu.L of HBT-CUR solution (final concentration 1mM, physiological saline: dimethyl sulfoxide=9:1v/v) was injected intratumorally every 24 hours, and administered daily (300 mW/cm ² ，500mW/cm ² ，1mW/cm ² ) And (3) carrying out illumination for 30min at different powers, and feeding under the same conditions as the control group. After the start of the experiment, the body weight of the mice was measured daily, and the tumor volume was treated for 14 days.

2.13.2 in vivo tumor treatment

Nude mice with good conditions are selected, and the tumor volume exceeds 200mm ³ Is randomly divided into four groups of six. Is divided into a Control group, a light+normal saline group and an HBT-CThe UR probe set and the light+HBT-CUR set, and the feeding conditions and the growth environment are controlled to be the same. (1) Control group: the transplanted tumor mouse model is not subjected to any treatment and is fed under normal conditions; (2) light+saline group: 100 mu L of physiological saline is injected into the tumor every 24 hours, and 300mW/cm is given for 30min every day ² Illuminating with a light source, and breeding under the same condition as the control group; (3) HBT-CUR Probe set: 100. Mu.L of HBT-CUR solution (final concentration 1mM, physiological saline: dimethyl sulfoxide=9:1v/v) was injected intratumorally every 24h, and the same conditions as those of the control group were maintained; (4) light+hbt-CUR group: 100. Mu.L of HBT-CUR solution (final concentration 1mM, physiological saline: dimethyl sulfoxide=9:1v/v) was injected intratumorally every 24 hours, and 300mW/cm was administered daily for 30min ² Is fed under the same condition as the control group. After the start of the experiment, the body weight of the mice, along with tumor volume, was measured daily until the treatment was completed. Daily photographs were taken and the state changes of the mice were recorded. After the experiment is finished, taking out the isolated tumor for photographing. H is applied to heart, liver, spleen, lung, kidney and tumor of mice&E pathology staining.

2.14 data processing

Each set of data was measured at least 3 times in parallel and the results were expressed as Mean ± standard deviation (Mean ± SD) and statistically analyzed using SPSS 16.0 software. The t-test was used for the analysis of the statistical differences between groups, and p <0.05 indicates that the differences were statistically significant.

3. Effect verification

1. Research on polarity detection performance of probe HBT-CUR

1.1 absorption Spectrum and emission Spectrum of Probe HBT-CUR

The probe HBT-CUR (10. Mu.M) was detected by absorption and emission spectra of 10 solvents including Toluene (tolene), ethanol (Ethanol), 1, 4-Dioxane (Dioxane), acetonitrile (Acetonitrile), methanol (Methanol), methylene chloride (dichlormethane), acetone (Acetone), dimethyl sulfoxide (DMSO), ethyl acetate (Ethylacetate), and PBS. As shown in FIG. 9A, the main absorption peak of the probe HBT-CUR is 400nm, and new absorption peaks of 600nm and 590nm respectively appear in DMSO and acetonitrile, and the double peak phenomenon is probably caused by the interaction between solvent molecules and the probe, so that the ketone type proportion of the probe in the ground state is improved. Subsequently, the fluorescence spectra of the probe HBT-CUR in 10 solvents were continuously examined, and as shown in FIG. 9B, the maximum emission wavelength of the probe was blue-shifted to 600nm in toluene solution with less polar solvent, and the wavelength was red-shifted to 666nm in DMSO solvent with greater polarity, indicating that the fluorescence properties of the probe were affected by the polarity of the solvent.

1.2 fluorescence Quantum yield of Probe HBT-CUR

In order to characterize the fluorescence properties of the probe HBT-CUR molecules, quinine sulfate was selected as a reference, and the fluorescence quantum yield was measured by a relative method, the measurement results are shown in Table 2, and it can be seen that the fluorescence quantum yield Φ of the probe HBT-CUR (10. Mu.M) in toluene solution with a smaller solvent polarity _f Can reach 0.189, and the fluorescence quantum yield phi in PBS solution with larger polarity _f Only 0.001 further illustrates that the probe is significantly affected by the polarity of the solvent.

TABLE 2 fluorescence quantum yield of probe HBT-CUR (10. Mu.M)

F, fluorescence intensity; absorbance is shown in the specification; n is the refractive index; epsilon: dielectric constant; phi _f Fluorescent quantum yield

1.3 detection Performance of Probe HBT-CUR

To further investigate whether the probe HBT-CUR specifically recognizes polarity, the Dioxane-PBS system was chosen as solvent to investigate the change in its spectral properties (FIG. 10). As the proportion of PBS increases from 0% to 60%, the ultraviolet absorption peak at 390nm gradually decreases and the absorption peak at 460nm gradually increases. Meanwhile, as the proportion of PBS increases, the fluorescence intensity at 620nm gradually decreases and the wavelength is red shifted. Then, the fluorescence intensity and the polarity parameter delta f are subjected to function fitting in a related relation, and the result shows that the fluorescence intensity and the polarity have good correlation R ² =0.991. The above experimental results fully demonstrate that the probe can sensitively detect polarity.

1.4 stability of Probe HBT-CUR

The stability of the probe is an important index of the performance of the probe, and a Dioxane-PBS system is adopted as a solventThe agent detects the photobleaching resistance and stability of the probe HBT-CUR (10. Mu.M). FIG. 11A shows that the fluorescence intensity of the probe is hardly changed within 10 minutes of continuous excitation of the excitation light, and the probe has strong photo-bleaching resistance. As shown in FIG. 11B, the probe solution was left at 37℃for 32 hours without significant change in fluorescence intensity. FIG. 11C shows the probe in an LED lamp (100 mW/cm) ² ) After 30min of irradiation, the fluorescence intensity is not obviously changed. The results show that the probe has better stability and photobleaching resistance.

1.5 Probe HBT-CUR Selectivity

It is necessary to examine the effect of other species on the polarity of the probe detection in vitro. Therefore, first, the interference of viscosity on the detection polarity of the probe was examined, and as shown in FIG. 12, the fluorescence spectrum of the probe (10. Mu.M) was not significantly changed under the different viscosity (glychol 10% -50%) environments, and no fluorescence response was seen. Next, it was examined whether pH has an effect on the detection polarity of the probe, and as shown in FIG. 13, the fluorescence spectrum of the probe (10. Mu.M) did not change significantly under different pH conditions (4.0-9.0), and no fluorescence response was seen. Next, the interference of reactive oxygen species and reactive nitrogen species on the detection performance of the probe was investigated, as shown in FIG. 14, for the probe (10. Mu.M) and reactive oxygen species (ROS, including O ₂ ^·- ；H ₂ O ₂ ；HClO； ¹ O ₂ The method comprises the steps of carrying out a first treatment on the surface of the t-BuOOH; OH), reactive Nitrogen (RNS, including NO ₂ ^- ；NO ₃ ^- ；ONOO ^- The method comprises the steps of carrying out a first treatment on the surface of the NO) the species after incubation at 37 ℃ did not affect its fluorescence spectrum nor did it change the maximum fluorescence intensity value, only exhibited fluorescence on after solvent change to Dioxane. Then, the interference of active sulfur on the detection performance of the probe was investigated, and as shown in FIG. 15, the probe (10. Mu.M) and active sulfur (RSS, including Na ₂ S；NaHS； Na ₂ SO ₃ ；NaHSO ₃ ；CH ₃ SSSCH ₃ ；CysSSCys；GSSG；S ₈ The method comprises the steps of carrying out a first treatment on the surface of the GSH) did not cause a change in fluorescence signal. Subsequently, it was examined whether or not the metal ion activated the probe fluorescence signal, as shown in FIG. 16, the probe (10. Mu.M) was mixed with the metal ion (Sn ²⁺ ；Cd ²⁺ ； Mn ²⁺ ；Co ²⁺ ；Cu ²⁺ ；Hg ²⁺ ；Zn ²⁺ ；Fe ²⁺ ；Ca ²⁺ ；Fe ²⁺ ；Al ³⁺ ；Ni ^{2 +} ；Mg ²⁺ ；Li ⁺ ；Na ⁺ ；K ⁺ ；Ag ⁺ ) There is little fluorescence response. Finally, it was investigated whether the amino acid activated the probe fluorescence signal, and as shown in FIG. 17, the probe (10. Mu.M) and amino acid (Gly; D-Ala; val; ile; L-Cys; L-Thr; asp; L-Glu; L-Arg; L-Lys; L-His; hcy) probe fluorescence spectra did not change significantly. The results show that the probe can not generate fluorescent signals due to different pH values, different viscosities, active oxygen, active nitrogen, active sulfur, metal ions and amino acids, and the probe can generate fluorescent signals only in a solvent system (Dioxane) with lower polarity, so that the probe has good selectivity and is not interfered by environment and other substances.

1.6 photosensitive Properties of Probe HBT-CUR

The photosensitive properties of the probes were examined by measuring the Reactive Oxygen Species (ROS) and singlet oxygen yields produced by the probes. First, the active oxygen produced by the probe was detected by 2, 7-dichlorofluorescein (DCFH-DA) (FIG. 18). The probe (10. Mu.M) showed no fluorescence response signal in the absence of light source irradiation. Under the irradiation of a light source, the system generates a remarkable fluorescent signal at 525nm, and under the irradiation of a 365nm ultraviolet lamp, the solution emits bright green fluorescence. The above results indicate that the probe HBT-CUR can generate active oxygen by illumination.

Then, rose bengal (phi) _Δ =0.75) as a reference, the singlet oxygen yield of the probe (10 μm) in ethanol was detected by Diphenylisobenzofuran (DPBF) (100 μm) (fig. 19). As shown in FIGS. 19A and 19B, the probe (10. Mu.M) and DPBF (100. Mu.M) were irradiated with a light source in an ethanol solution for 1min with little change in the ultraviolet absorption spectrum, indicating that the method is suitable for detection of the singlet oxygen yield of the probe. FIG. 19C shows that the probe and DPBF were in ethanol solution, the light source was irradiated for 1min, and the absorbance of the system at 410nm was gradually decreased; FIG. 19E shows that absorbance at 410nm is linear with illumination time (R ² =0.991); detection of absorbance change at 410nm with ethanol solution of rose bengal (10. Mu.M) by the same methodThe correlation between (fig. 19D and 19F) is calculated by the formula: phi _Δ,sample ＝Φ _Δ,standard (K _sample /K _standard )，(Φ _Δ Singlet oxygen yield; k. the absorbance degradation rate of DPBF at 410nm was calculated to obtain the singlet oxygen yield (. Phi.) of the probe HBT-CUR in ethanol _Δ =0.52). Singlet oxygen yields of the probe in different solvents were determined and calculated as described above. As a result, as shown in Table 3, the probe had a higher singlet oxygen yield (. Phi.) in Dioxane _Δ =0.70). The above results indicate that HBT-CUR is a good performing type II photosensitizer.

TABLE 3 singlet oxygen yields (. Phi.) in different solvents _Δ )

Reference:Rose bengal

2. Probe HBT-CUR cell level fluorescence imaging

2.1 cytotoxicity test

Before performing the cell experiments, cytotoxicity assays of the probe HBT-CUR were first performed. A total of 7 cell lines of mouse breast cancer cells (4T 1), human kidney clear cell carcinoma cells (786-o), human breast cancer cells (MCF-7), human liver cancer cells (HepG 2), triple negative human breast cancer cells (MDA-MB-231), human renal cortex proximal tubular epithelial cells (HK-2) and human mammary epithelial cells (MCF-10A) were selected, and the cytotoxicity of the probe HBT-CUR was detected by the MTT method (FIG. 20). Different concentrations of probe HBT-CUR (0,2.5,5,10, 20. Mu.M) were incubated with these 10 cell lines. As a result, it was found that when the probe concentration was less than 5. Mu.M, the survival rate of the cells was 90% or more. The cell viability was still around 80% after 48h of subsequent incubation of HBT-CUR (5 μm) with these 10 lines of cells for 0,6, 12, 24. The above results demonstrate that the probe HBT-CUR (5. Mu.M) is less toxic and can be used for cell level imaging.

2.2 biocompatibility of probe HBT-CUR

The cytocompatibility of the probe is an important indicator of whether the probe can be used for cell imaging. Here, the probe (5. Mu.M) was incubated with MCF-7 cells for various times (0, 5, 10, 15, 20 min), followed by nuclear staining with 4, 6-diamidino-2-phenylindole (DAPI) for 5min and confocal imaging. As shown in FIG. 21, the probe incubated with the cells for about 5min showed red fluorescence when entering the cells, and blue fluorescence when DAPI was mainly accumulated in the nuclei. Indicating that the probe can enter cells with good biocompatibility.

2.3 distribution of probes in cells

In order for a cell to function more orderly, different regions in the cell have different cell polarities. Studies have shown that lysosomes undergo structural changes, increase in number and size, and exhibit reduced lysosomal activity during tumor formation, which may lead to changes in lysosomal polarity. In order to study the distribution of the probe HBT-CUR in tumor cells, a lysosome Green dye (Lyso-Tracker Green), an endoplasmic reticulum Green probe (ER-Tracker Green), a Golgi Green probe (Golgi-Tracker Green), and a mitochondrial Green probe (Mito-Tracker Green) were selected for subcellular organelle co-localization imaging with the probe HBT-CUR (5. Mu.M) respectively. As shown in FIG. 22, the co-localization imaging effect of the probe and the lysosome is good, and the pearson correlation coefficient is 0.96; the pearson correlation coefficients for endoplasmic reticulum, golgi apparatus and mitochondria co-localization were 0.78, 0.76 and 0.65, respectively. The analysis of the fluorescence intensity of the cross section of the cell imaging diagram shows that the coincidence of the fluorescence intensity of the lysosome and the peak type is better. The above results indicate that HBT-CUR has good lysosomal localization ability.

2.4 tumor cell specific imaging

To demonstrate that the probe HBT-CUR can specifically recognize tumor cells, 7 cell lines of mouse breast cancer cells (4T 1), human renal clear cell carcinoma cells (786-o), human breast cancer cells (MCF-7), human liver cancer cells (HepG 2), triple negative human breast cancer cells (MDA-MB-231), human renal cortex proximal tubular epithelial cells (HK-2), and human mammary epithelial cells (MCF-10A) were subjected to fluorescence imaging with the probe HBT-CUR (5. Mu.M). As shown in FIGS. 23A-E, bright red fluorescence was seen in the tumor cell lines described above, 4T1, 786-o, MCF-7, hepG 2, MDA-MB-231, which was due to the low polarity of the tumor. As shown in FIGS. 23F-G, HK-2 and MCF-10A showed little intracellular fluorescence signal. As shown in FIG. 23H, the cell groupings (786-oVSHK-2; MCF-7 and MDA-MB-231VS MCF-10A) were analyzed for statistical differences between groups using t-test, and the results showed that the fluorescence intensities in the tumor cell lines were significantly different from those in the normal cell lines. In summary, the probe specifically images tumor cells by detecting the difference in polarity in the cells, thereby distinguishing tumor cells from normal cells.

2.5 quantitative detection of HBT-CUR cell level imaging with Probe

To further demonstrate the ability of the probes to specifically image tumor cells, the average fluorescence intensity within the cells was quantitatively analyzed by flow cytometry after incubating the probes (5 μm) with 7 cell lines for 20 min, respectively (fig. 24). As can be seen from fig. 24A, the average fluorescence intensity of tumor cells was significantly higher than that of normal cells. FIG. 24B compares MCF-7, MDA-MB-231,4T1 and MCF-10A, respectively; 786-o and HK-2, and statistically analyzed, the results showed that there was a significant difference in the mean fluorescence intensity between tumor cells and normal cells. The results further demonstrate that the probes can specifically differentiate tumor cells.

2.6 apoptosis staining

In order to study whether the probe HBT-CUR can effectively induce apoptosis of tumor cells, the cells are stained by Calcein AM/PI, the living cells can be stained green by Calcein AM, and the apoptotic cells can be stained red by PI. As a result, as shown in FIG. 25, the cells incubated with the probe were irradiated with an external light source for 0 to 25min, the green fluorescent signal was gradually decreased, and the red fluorescent signal was gradually increased, indicating that the cells were rapidly apoptotic under the coexistence of the probe and the light source. To further demonstrate that apoptosis is the result of the combined action of Light source irradiation and probe, cells were grouped into Control, light, HBT-CUR, HBT-CUR+light, and fluorescence imaging results showed in FIG. 26, control, light, HBT-CUR showed very little red fluorescence signal, and HBT-CUR+light showed a large area of red fluorescence signal. The above results indicate that the probe HBT-CUR causes rapid apoptosis in the presence of an external light source.

2.7 detection of reactive oxygen species in cells

The PDT process mainly promotes the photosensitizer to generate a large amount of active oxygen through illumination, so that cells are subjected to excessive oxidative stress and apoptosis occurs. To investigate the cause of apoptosis by the probe, after incubating the probe (5. Mu.M) with MCF-7 cells for 20min, the cells were incubated with LED (100 mW/cm ² ) The light source irradiates for 25min, DCFH (10 mu M) is added, and the DCFH reacts with active oxygen to emit a green fluorescent signal of 520 nm. Blank treatment, probe addition and light shielding treatment and irradiation treatment without probe light source are used as control. As shown in FIG. 27, the MCF-7 cells incubated with the probe and irradiated with the light source showed bright green fluorescence signals, and the control MCF-7 cells had only weak green fluorescence response signals, which indicated that the probe generated active oxygen under the irradiation of the external light source. The above results demonstrate that the probe induces apoptosis of tumor cells by PDT-generated ROS.

3. Probe HBT-CUR in vivo level imaging

The imaging ability of the probe HBT-CUR in tumor tissue was studied at the in vivo level as a result of the cell experiments. First, the retention time of the probe HBT-CUR in tumor tissue is examined, and the tumor volume is larger than 200mm ³ The tumor-bearing mice of (1) were randomly divided into two groups, 100. Mu.L of probe (1 mM, dimethyl sulfoxide: physiological saline=1:9, v/v) was injected into the tumor of the tumor-bearing mice of the experimental group, 20. Mu.L of blank solvent (dimethyl sulfoxide: physiological saline=1:9, v/v) was injected into the tumor of the tumor-bearing mice of the control group, and the living body level imaging was performed at 0min,1min,5min,10min,20min, 40min,60min,80min,120min,6h,12h,24h,48h after the injection. As shown in FIG. 28, the fluorescence intensity in the tumor reached the fluorescence intensity peak (10 times) at 10min, and a strong fluorescence signal (6 times) was still detected within 6 hours, and the fluorescence signal gradually decreased after 6 hours, but was less than 2 times after 48 hours. The probe is shown to be capable of realizing tumor tissue imaging, generating a stronger fluorescent signal in a shorter time (10 min) in tumor tissue, and being capable of imaging for a longer time, and is shown in fig. 29.

4. Photodynamic treatment of tumor tissue at the level of the probe HBT-CUR living body

The results of in vivo horizontal imaging indicate that the probe can be used for visual imaging of tumor tissues. The therapeutic effect of the probe on tumor tissue in a nude mouse transplanted tumor model is discussed. First, a suitable light source treatment regimen is sought. Tumor volume is larger than 100mm ³ Is randomly grouped, each group is 6 with the number of 300mW/cm ² A power light source; 500mW/cm ² A power light source; 1W/cm ² A power light source; control. Mice in each group were injected intratumorally with 100 μl of probe (1 mM, dimethyl sulfoxide: physiological saline=1:9, v/v), once every two days, with no light irradiation given to the control, and 300mW/cm for each of the other groups, respectively ² Power light source 500mW/cm ² Power light source, 1W/cm ² The mice were treated for 14 days with light from a power light source for 30min daily (once every 5min interval) and changes in body weight and tumor volume were recorded over 14 days (fig. 30). Figure 30A shows little change in mouse body weight during treatment. Fig. 30B shows that Control tumor volume increases dramatically within 14 days, while different power light sources treat tumor volume decreases, indicating that the probe can effectively inhibit tumor growth. As shown in FIG. 30C, the tumor tissue is taken out for photographing after 14 days of treatment of the mice, the tumor volume of the treatment group is obviously reduced compared with that of the Control group, and the light source power is 300mW/cm ² To 1W/cm ² The tumor inhibition was only slightly enhanced, indicating 300mW/cm ² Is sufficient to efficiently excite the probe to produce singlet oxygen. The experimental result shows that the probe HBT-CUR can effectively inhibit the growth of tumor tissues by PDT, and has good anti-tumor effect.

It was further confirmed that the inhibition of tumor growth by the probe was by PDT. First, tumor volumes of greater than 200mm are measured ³ Is randomly divided into four groups of 6 tumor-bearing mice, and the number is: control, light, HBT-CUR, HBT-CUR+light. HBT-CUR+light, with intratumoral injection of 100. Mu.L of probe (1 mM, dimethyl sulfoxide: physiological saline=1:9, v/v), was administered once every two days, 300mW/cm daily ² The LED light source of (1) irradiates for 30min (irradiates once every 5min and irradiates for 30min in total); the Control does not carry out any treatment, and the tumor-bearing mice naturally grow; light tumor-bearing mice were given intratumoral injections of 100 μl of blank solutionThe agent (dimethyl sulfoxide: physiological saline=1:9, v/v), was injected once every two days, and administered 300mW/cm daily ² The LED light source of (1) irradiates for 30min (irradiates once every 5min and irradiates for 30min in total); 100. Mu.L of probe (1 mM, dimethyl sulfoxide: physiological saline=1:9, v/v) in tumor of HBT-CUR tumor-bearing mice was administered once every two days and subjected to light-shielding treatment. Treatment was performed for 14 days. The body weight of each group of mice (fig. 31A) was recorded over 14 days as a function of tumor volume (fig. 31B). The results show that the body weight of the mice has almost no obvious change within 14 days, compared with the control group, the tumor volume of the HBT-CUR+light group is obviously reduced, which shows that the probe has obvious inhibition effect on tumors under the irradiation of an external Light source and has good biocompatibility. After 14 days, the left side underarm tumor tissue of the nude mice was photographed with a camera and compared (FIG. 31D), and the results showed that the tumor volume of the left side underarm of the tumor-bearing mice (1.36D-a) of the HBT-CUR+light group was significantly smaller than that of the other groups. Mice were then sacrificed and the internal organs (heart, liver, spleen, lung, kidney) and tumor tissues of the tumor-bearing mice were dissected, the isolated tumor tissues were weighed (fig. 31C) and photographed with a camera (fig. 31E), and the results showed that the tumor-bearing mice with the HBT-CUR intratumoral injection probes had significantly lower volume and weight of isolated tumor tissues than the other groups. And subjecting the isolated tumor tissue (FIG. 31F) to H with the organ (FIG. 32) &E pathology staining. The result shows that Control, light is dense with the mouse tumor cells of the HBT-CUR group, the cell nuclei are deeply stained and have obvious allotype, and the blood vessels in the tumor are relatively abundant and no necrotic foci are seen; most of tumor cells in the HBT-CUR+light group are completely necrotized, cell nuclei are also disappeared, peripheral areas are scattered on part of the cell nuclei, the cell nuclei are in an obvious shrinkage state, the nuclear membrane is not clearly identified, and the coloring level is not clear. The isolated organs of the tumor-bearing mice, such as heart tissue, liver tissue, spleen tissue, lung tissue and kidney tissue, are almost normal, and have no obvious pathological changes. In conclusion, the probe can inhibit tumor growth by PDT under the irradiation of an external light source.

The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.

Claims (13)

1. A polar fluorescent probe with photodynamic property has the following structural formula:

。

2. the method for preparing the polar fluorescent probe according to claim 1, which comprises the following steps:

(1) In the presence of sodium hydroxide, performing a chemical reaction between a compound a and 2-amino thiophenol to prepare an intermediate HBT-1;

(2) In the presence of trifluoroacetic acid, performing a chemical reaction on the intermediate HBT-1 and hexamethylenetetramine to prepare an intermediate HBT-2;

(3) Under the protection of nitrogen, the compound b and boron trifluoride diethyl etherate are subjected to chemical reaction to prepare an intermediate C-1;

(4) In the presence of piperidine, the intermediate HBT-2 and the intermediate C-1 are subjected to chemical reaction to prepare the polar fluorescent probe HBT-CUR, wherein the specific synthetic route is as follows:

。

3. the method for preparing a polar fluorescent probe according to claim 2, wherein in the step (1), the molar ratio of the compound a to 2-aminophenylsulfol is 1:1 to 3; the molar ratio of the compound a to the sodium hydroxide is 1:5-10.

4. The method for preparing a polar fluorescent probe according to claim 3, wherein in the step (1), the molar ratio of the compound a to 2-aminophenylsulfol is 1:2; the molar ratio of the compound a to the sodium hydroxide is 1:8.

5. The method for preparing a polar fluorescent probe according to claim 2, wherein in the step (1), the reaction temperature is 80 to 100 ℃; the reaction time is 1-3 h.

6. The method for preparing a polar fluorescent probe according to claim 5, wherein in the step (1), the reaction temperature is 90 ℃; the reaction time was 1 h.

7. The method for preparing a polar fluorescent probe according to claim 2, wherein in the step (2), the molar ratio of the intermediate HBT-1 to hexamethylenetetramine is 1:0.8-1.5; the mass volume ratio of the intermediate HBT-1 to the trifluoroacetic acid is 40-50:1 mg/ml.

8. The method for preparing a polar fluorescent probe according to claim 7, wherein in the step (2), a molar ratio of the intermediate HBT-1 to hexamethylenetetramine is 1:1; the mass-volume ratio of the intermediate HBT-1 to the trifluoroacetic acid is 45.4:1mg/ml.

9. The method for preparing a polar fluorescent probe according to claim 2, wherein in the step (3), the molar ratio of the compound b to boron trifluoride diethyl etherate is 1:1-3; in the step (4), the molar ratio of the intermediate HBT-2 to the intermediate C-1 is 1:0.8-1.5; the molar ratio of the intermediate HBT-2 to the piperidine is 1:0.8-1.5.

10. The method for preparing a polar fluorescent probe according to claim 9, wherein in the step (3), the molar ratio of the compound b to boron trifluoride etherate is 1:2; in step (4), the molar ratio of intermediate HBT-2 to intermediate C-1 is 1:1; the molar ratio of intermediate HBT-2 to piperidine was 1:1.

11. The use of the polar fluorescent probe according to claim 1 for preparing a medicament for detecting cell polarity.

12. Use of the polar fluorescent probe of claim 1 in the preparation of a medicament for photodynamic tumour therapy.

13. The use of claim 12, wherein the polar fluorescent probe produces singlet oxygen by PDT to kill tumor cells.