Enzyme activating group type aggregation-induced emission photosensitizer as well as preparation method and application thereof

Abstract

The preparation method comprises the following steps: taking 4, 9-dibromobenzothiadiazole and a triphenylamine boric acid derivative as raw materials, and carrying out Suzuki coupling reaction to obtain a corresponding unilateral substitution product; further carrying out Suzuki coupling reaction with amino phenylboronic acid to obtain a corresponding amino-containing compound; and then in the presence of a condensation reagent, carrying out amidation reaction with carboxyl and amino protected glutamic acid, and further removing a protecting group through trifluoroacetic acid to obtain the AIE type photosensitizer capable of being activated by gamma-glutamyltransferase. The enzyme activated group type aggregation-induced emission photosensitizer obtained by the invention has efficient fluorescence quantum efficiency and ROS (reactive oxygen species) generation capability, and has a good application prospect in fluorescence imaging mediated photodynamic tumor treatment; meanwhile, the enzyme-activated group type aggregation-induced emission photosensitizer can be activated by gamma-glutamyltransferase, and has high targeting and killing effects on tumor cells, so that the problems of poor molecular tumor selectivity and poor tumor treatment effect of the traditional photosensitizer can be solved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and particularly relates to an enzyme-activated base type aggregation-induced luminescence photosensitizer and a preparation method and application thereof. Background Art

[0002] Photodynamic therapy (PDT) is widely used in clinical disease treatment due to its advantages such as non-invasiveness and selectivity. The PDT process includes three basic elements: light, photosensitizer and oxygen. PDT is to irradiate the diseased tissue or cells containing photosensitizer with light of a specific wavelength, and activate the photosensitizer molecules to produce reactive oxygen species (ROS), thereby destroying the diseased tissue and achieving therapeutic effects. ROS can be finely divided into type I ROS mainly composed of free radicals (superoxide anion radicals, hydroxyl radicals, etc.) and type II ROS mainly composed of singlet oxygen. Due to its high reactivity, ROS can react non-selectively with various biological molecules in cells, which causes corresponding toxic side effects. The high activity of ROS also makes it have a shorter action life (≤10ns) and action radius (≤10nm), which also affects the in vivo therapeutic effect of photosensitizer molecules to some extent. Currently commercialized photosensitizers (porphyrin derivatives and metal phthalocyanine compounds) belong to type II photodynamic photosensitizers. Since the production of singlet oxygen in type II photodynamic therapy depends on the oxygen concentration around the photosensitive molecules, the therapeutic effect of type II photosensitizer molecules decreases during long-term in vivo tumor treatment due to the hypoxia effect in the tumor microenvironment. In addition, since its hydrophobic rigid structure is easy to aggregate in the physiological environment, it leads to fluorescence aggregation quenching and decreased efficiency of reactive oxygen production, which is extremely unfavorable for its development in fluorescence imaging-mediated in vivo tumor treatment.

[0003] An ideal photosensitizer should include several important characteristics: 1) controllable photodynamic activity that can be specifically activated at the tumor site and inactivated at the non-tumor site; 2) good biocompatibility and low toxic side effects; 3) precise tumor cell targeting and low oxygen dependence. Tumor-targeted photosensitizer molecules can further improve the therapeutic effect of photodynamic therapy and reduce damage to healthy tissues.

[0004] Aggregation-induced emission (AIE) refers to the phenomenon that fluorescent molecules do not emit light or emit light weakly in a uniformly dispersed low-concentration state, but when the solubility decreases or the concentration reaches a certain limit, they will become brighter. That is, in the single-molecule state, AIE molecules emit light weakly, but after aggregation, the luminescence of these molecules is significantly enhanced. Although AIE molecules have significant aggregation luminescence enhancement advantages, the photosensitizer material systems reported so far with AIE properties are still very scarce, especially AIE-type photosensitizers with near-infrared emission and the ability to overcome hypoxic PDT treatment are even fewer.

[0005] Based on this, a new solution is needed. Summary of the invention

[0006] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a class of enzyme-activated aggregation-induced luminescence photosensitizer materials and a preparation method and application thereof.

[0007] According to one aspect of the present invention, the present invention provides an enzyme-activated aggregation-induced luminescence photosensitizer, wherein the enzyme-activated aggregation-induced luminescence photosensitizer has the following structure:

[0008]

[0009] Among them, the "π" bridge is an independent aromatic ring; R 1 It is a molecular rotor structure and is selected from one of triphenylamine, substituted triphenylamine, tetraphenylene group or substituted tetraphenylene group, and the structures of substituted triphenylamine and substituted tetraphenylene group are as follows, R 2 is H or an alkoxy chain,

[0010]

[0011] In the enzyme-activated aggregation-induced luminescence photosensitizer provided by the present invention, the enzyme-activated aggregation-induced luminescence photosensitizer has the following structure:

[0012]

[0013] In the enzyme-activated aggregation-induced luminescence photosensitizer provided by the present invention, the enzyme-activated aggregation-induced luminescence photosensitizer has the following structure:

[0014]

[0015] According to another aspect of the present invention, there is also provided a method for preparing the enzyme-activated aggregation-induced luminescence photosensitizer as described above, comprising the following steps:

[0016] Step S1, subjecting 4,9-dibromobenzothiadiazole and a triphenylamine boronic acid derivative or a tetraphenylethylene boronic acid derivative to a Suzuki coupling reaction in the presence of a catalyst to obtain a first intermediate product;

[0017] Step S2, subjecting the first intermediate product to a Suzuki coupling reaction with aminophenylboronic acid in the presence of a catalyst to obtain a second intermediate product;

[0018] Step S3, subjecting the second intermediate product to amidation reaction with carboxyl- and amine-protected glutamic acid in the presence of a condensing agent to obtain a third intermediate product;

[0019] Step S4, removing the protecting group of the third intermediate product by trifluoroacetic acid to obtain the enzyme-activated aggregation-induced luminescence photosensitizer.

[0020] In the preparation method provided by the present invention, in step S1, the triphenylamine boronic acid derivative is selected from one of 4-triphenylamine boric acid pinacol ester, 4-triphenylamine boric acid or 4-(diphenylamino)phenyl boric acid pinacol ester, the tetraphenylethylene boronic acid derivative is selected from one of (4-(1,2,2-triphenylvinyl)phenyl)boric acid or (4-(1,2,2-triphenylvinyl)phenyl)boric acid pinacol ester, and the mass ratio of 4,9-dibromobenzothiadiazole to the triphenylamine boronic acid derivative is 1:1 to 1:2; in step S2, the mass ratio of the first intermediate product to aminophenylboronic acid is 1:1 to 1:2; in step S3, the mass ratio of the second intermediate product to carboxyl and amino protected glutamic acid is 1:1 to 1:3.

[0021] In the preparation method provided by the present invention, the base for Suzuki reaction can be selected from sodium carbonate, sodium hydroxide, potassium hydroxide, potassium carbonate, the catalyst can be selected from tetrakistriphenylphosphine palladium, palladium chloride, and the condensation agent is HATU, HBTU and EDCI.

[0022] According to yet another aspect of the present invention, there is provided a use of the enzyme-activated aggregation-induced emission photosensitizer as described above in the preparation of a preparation for fluorescence imaging-mediated photodynamic tumor therapy.

[0023] In the application provided by the present invention, the enzyme-activated aggregation-induced luminescence photosensitizer is activated by γ-glutamyl transferase in tumor cells.

[0024] In the application provided by the present invention, the photodynamic activity of the enzyme-activated aggregation-induced luminescence photosensitizer is positively correlated with the content of γ-glutamyl transferase in tumor cells.

[0025] The implementation of the present invention can achieve the following beneficial effects:

[0026] The present invention introduces a kinetic (rotational / vibrational) electron donor (molecular rotor) on one side of 4,9-dibromonaphthothiadiazole to construct a strong charge transfer (CT) state while suppressing the intermolecular π-π stacking effect of the molecules in the aggregated state, thereby giving the material significant near-infrared emission and AIE characteristics; in addition, due to the strong degree of distortion between the electron donor and the acceptor, it is beneficial to the separation of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), thereby achieving a smaller singlet-triplet energy level difference (ΔE st), promoting the intergap crossing (ISC) of the excited singlet energy to the triplet state to achieve efficient ROS generation; at the same time, due to the restricted movement of the molecular aggregation state, the non-radiative excitation energy is effectively suppressed, which promotes the energy dissipation in the form of radiation transition, and the fluorescence efficiency is increased; therefore, the enzyme-activated base type aggregation-induced luminescence photosensitizer obtained by the present invention has high fluorescence quantum efficiency and ROS generation ability, and has good application prospects in fluorescence imaging-mediated photodynamic tumor therapy; the enzyme-activated base type aggregation-induced luminescence photosensitizer obtained by the present invention can be activated by γ-glutamyl transferase, and has high targeting and killing effects on tumor cells, thereby overcoming the problems of poor tumor selectivity and poor tumor treatment effect of traditional photosensitizer molecules; at the same time, its synthesis method is simple, the raw materials are easily available, the yield is high, and the obtained material structure is stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on the provided drawings without creative work:

[0028] Figure 1 :(a) shows the fluorescence spectra (fw) of TBmA in DMF / water mixture (10 μM); (b) shows the fluorescence spectra of TBmA, TBpA, TBmA-Glu and TBpA-Glu powders and DMF (fw w =0%) and DMF / water mixture (f w =99%) solution under 365 nm UV light; (c) shows DCFH (10 μM) as an indicator in PBS / DMSO (v / v=99:1) under white light (20 mW cm -2 ) after 15 min of irradiation, the total ROS generated by the photosensitizer (5 μM), DCF:λ ex = 488 nm; (d) shows the DCFH (10 μM) in the presence of TBmA (5 μM) with different water contents (f w )DMF / water solution, the relative fluorescence emission intensity (I / I 0 ) and illumination time (20 mW cm -2 ) relationship diagram, where I 0 = water content (f w ) In DMF / water solution, the fluorescence intensity of DCFH before illumination, λ ex =488nm; (e) shows the AIE curve of TBmA (α AIE =I / I 0 , where I0 = fluorescence intensity in DMF) and DCFH in DMF / water solution containing TBmA (5 μM) (f w ) and irradiated with light for 15 (20 mW cm -2 ) after the ROS changes; (f) shows the particle size of TBmA aggregates and the water content of the solution (f w ), the inset shows the particle size distribution of aggregated particles formed by TBmA at 65% (left) and 99% (right) water contents.

[0029] Figure 2 :(a) Schematic diagram of GGT activating TBmA-Glu; (b) shows the molecular docking results of TBmA-Glu and GGT (PDB: 4GG2), TBmA-Glu is green, the surface of GGT molecule is a colored transparent surface, and the enlarged image shows the hydrogen bond formed between TBmA-Glu and GGT; (c) shows the change of TBmA-Glu fluorescence with incubation time in the presence or absence of GGT, and the arrows indicate the light irradiation time points (12 J·cm 2 ); (d) shows the LC / UV-Vis analysis of the in vitro enzymatic reaction product of TBmA-Glu and GGT. A 10 μM TBmA-Glu solution was added to a 10 mM HEPES buffer, pH 7.4, containing 0.1% DMSO as a co-solvent, and 5 units of GGT were added. The mixture was incubated at 37°C for 2 hours, and the absorbance at 450 nm was monitored. (e) shows the activity of GGT in the presence of different concentrations of TBmA-Glu, N=3, and the data are expressed as mean ± standard error; P value, ns: 0.001<**P<0.01, calculated using Student's t test.

[0030] Figure 3 : (a) shows the confocal laser scanning microscopy (CLSM) images of luciferase-transfected HepG2 and normal (LO2) cells co-incubated with TBmA-Glu (5 μM, 12 h); ex =455nm,λ em =650±20nm, scale bar: 30μm; (b) shows the CLSM images of HepG2 cells treated with TBmA-Glu (5μM) or TBmA (5μM) for 12h, scale bar: 30μM; (c) shows the cellular phototoxicity (IC50, μM) of TBmA, TBpA, TBmA-Glu and TBmA-Glu under normoxic and hypoxic conditions; (d) shows the activity of GGT in the presence of different concentrations of TBmA-Glu, data are expressed as mean ± standard error, N=3.

[0031] Figure 4:(a) Shows the generation of ROS in HepG2 cells. HepG2 cells were incubated with TBmA-Glu (2 μM) or 1% DMSO (Ctrl) for 12 h and then illuminated with a white laser array (12 J·cm -2 ) irradiation, DCFH-DA (10 μM) was used as ROS indicator, DCF, λ ex =488nm,λ em =500±20nm; TBmA-Glu, λ ex =465nm,λ em =700±20nm; (b) shows the scavenging rate of ROS induced by TBmA-Glu PDT by different ROS scavengers. Cells were pre-incubated with ROS scavengers for 2h (Tiron: 10mM, NaN3: 5mM, mannitol: 50mm, Ebselen: 50μM), and then incubated with TBmA-Glu (2μM) for 12h; (c) shows the effect of TBmA-Glu (2μM) on the GSSG / GSH ratio of cells; (d) shows the expression levels of GGT and GPX4 after HepG2 cells were treated with different concentrations of TBmA-Glu. TBmA-Glu was used as the indicator concentration for incubation for 24h. After incubation for 12h in the light treatment group, a white laser array (12J·cm -2 ) and then incubated for 12 h; (e) shows the expression level of GGT1 in HepG2 cells after incubation with TBmA-Glu (2 μM) or 1% DMSO (Ctrl) for 12 h; (f) shows the transmission electron microscopy images of HepG2 cells treated with TBmA-Glu (2 μM) under illumination and non-illumination conditions.

[0032] Figure 5 : (a) Shown is the structure of 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), HR-MS results of DOPE (10 μM) and TBmA (10 μM) co-incubated without (b) and after (c) light irradiation.

[0033] Figure 6 :(a) shows the photos of tumors isolated from nude mice in different treatment groups at the end of treatment; (b) shows the tumor growth curve of tumor-bearing mice. N=5, data are expressed as mean ± standard error; (c) shows the fluorescence images of organs and tumor tissues isolated from different treatment groups at the end of treatment, TBpA-Glu, λ ex =500nm,λ em =700nm (long pass filter); (d) shows the fluorescence imaging images of mice in the TBmA-Glu and TBmA PDT treatment groups at the beginning and end of treatment, TBpA-Glu, λ ex =500nm,λ em=700nm (LP); (e) shows the immunohistochemical results of GPX4 in tumor sections of mice in the TBmA-Glu treatment group, scale bar: 50μm.

[0034] Figure 7 :(a) shows the schematic diagram of the modeling process of orthotopic liver tumor model mice; (b) shows the fluorescence images of orthotopic liver cancer mice in the TBmA-Glu (5 mg / kg) treatment group at the beginning, middle and end of treatment, bioluminescence, λ em =550±50nm(BP). TBpA-Glu,λ ex =500nm,λ em =700nm(LP).

[0035] Figure 8 Shown are the fluorescent images of the organs of mice in each treatment group with orthotopic liver tumors, TBpA-Glu, λ ex =500nm,λ em =700nm(LP).

[0036] Fig. 9 : (a) shows the hematoxylin-eosin staining image of the liver of mice with liver orthotopic tumor model after treatment with TBmA-Glu (5 mg / kg). The enlarged image shows that the tumor tissue is fibrotic after TBmA-Glu photodynamic therapy; (b) shows the TUNEL staining result of the liver of mice with liver orthotopic tumor model treated with TBmA-Glu (5 mg / kg). Scale bar, 100 μm.

[0037] Fig.10 The Ki67 immunohistochemistry images of the liver of mice with orthotopic liver tumor model after TBmA-Glu (5 mg / kg) treatment are shown. Scale bar, 100 μm. DETAILED DESCRIPTION

[0038] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific embodiments of the present invention are now described in detail with reference to the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments described in this specification, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The present invention provides an enzyme-activated aggregation-induced luminescence photosensitizer, wherein the enzyme-activated aggregation-induced luminescence photosensitizer has the following structure:

[0040]

[0041] Among them, the "π" bridge is an independent aromatic ring; R 1It is a molecular rotor structure and is selected from one of triphenylamine, substituted triphenylamine, tetraphenylene group or substituted tetraphenylene group, and the structures of substituted triphenylamine and substituted tetraphenylene group are as follows, R 2 is H or an alkoxy chain. The alkoxy group may be a straight chain or branched chain alkyl group, for example, methoxy, ethoxy, propoxy, butoxy, isobutoxy, tert-butoxy, cyclohexaneoxy, etc.

[0042]

[0043] Preferably, the "π" bridge is a benzene ring, and the molecular rotor R 1 is triphenylamine, wherein R 2 For hydrogen.

[0044] The present invention also provides a preparation method for preparing the enzyme-activated aggregation-induced luminescence photosensitizer, comprising the following steps:

[0045] Step S1, subjecting 4,9-dibromobenzothiadiazole and a triphenylamine boronic acid derivative or a tetraphenylethylene boronic acid derivative to a Suzuki coupling reaction in the presence of a catalyst to obtain a first intermediate product;

[0046] Specifically, in one embodiment of the present invention, 4,9-dibromobenzothiadiazole and triphenylamine boronic acid derivatives or tetraphenylethylene boronic acid derivatives are used as raw materials, and the corresponding unilateral substitution product is obtained by Suzuki coupling reaction. Wherein, the triphenylamine boronic acid derivative is selected from one of 4-boronic acid triphenylamine pinacol ester, 4-boronic acid triphenylamine or 4-(diphenylamino)phenyl boronic acid pinacol ester, and the tetraphenylethylene boronic acid derivative is selected from one of (4-(1,2,2-triphenylvinyl)phenyl)boric acid or (4-(1,2,2-triphenylvinyl)phenyl)boric acid pinacol ester; the mass ratio of 4,9-dibromobenzothiadiazole to triphenylamine boronic acid derivatives or tetraphenylethylene boronic acid derivatives is 1:1 to 1:2, preferably 1:1.2.

[0047] Further, in one embodiment of the present invention, the catalyst is one selected from sodium carbonate, sodium hydroxide, potassium hydroxide, potassium carbonate and one selected from tetrakistriphenylphosphine palladium, palladium chloride, tri-tert-butylphosphine. Preferably, the catalyst is potassium carbonate and tetrakistriphenylphosphine palladium.

[0048] Step S2, subjecting the first intermediate product to a Suzuki coupling reaction with aminophenylboronic acid in the presence of a catalyst to obtain a second intermediate product;

[0049] Specifically, in one embodiment of the present invention, the unilateral substitution product after the coupling reaction is further subjected to a Suzuki coupling reaction with aminophenylboronic acid to obtain a corresponding amino-containing compound, wherein the mass ratio of the first intermediate product to aminophenylboronic acid is 1:1 to 1:2. The catalyst is selected from one of sodium carbonate, sodium hydroxide, potassium hydroxide, potassium carbonate and one of tetrakistriphenylphosphine palladium, palladium chloride and tri-tert-butylphosphine. Preferably, the catalyst is potassium carbonate and tetrakistriphenylphosphine palladium.

[0050] Step S3, subjecting the second intermediate product to amidation reaction with carboxyl- and amine-protected glutamic acid in the presence of a condensing agent to obtain a third intermediate product;

[0051] Specifically, in one embodiment of the present invention, an amine-containing compound is subjected to an amidation reaction with carboxyl- and amino-protected glutamic acid in the presence of a condensation reagent, wherein the mass ratio of the second intermediate product to carboxyl- and amino-protected glutamic acid is 1:1 to 1:3, and the condensation agent is BOC-L-glutamic acid-1-tert-butyl ester, HATU and N,N-diisopropylethylamine.

[0052] Step S4: removing the protecting group of the third intermediate product by trifluoroacetic acid to obtain the enzyme-activated aggregation-induced luminescence photosensitizer

[0053] Specifically, in one embodiment of the present invention, the protecting group is further removed by trifluoroacetic acid to obtain an AIE type photosensitizer that can be activated by γ-glutamyl transferase (GGT).

[0054] The present invention introduces a kinetic (rotational / vibrational) electron donor (molecular rotor) on one side of 4,9-dibromonaphthothiadiazole to construct a strong charge transfer (CT) state while suppressing the intermolecular π-π stacking effect of the molecules in the aggregated state, thereby giving the material significant near-infrared emission and AIE properties. In addition, due to the strong degree of distortion between the electron donor and the acceptor, it is beneficial to separate the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), thereby achieving a smaller singlet-triplet energy level difference (ΔE st ), promoting the intergap crossing (ISC) of the excited singlet energy to the triplet state to achieve efficient ROS generation. At the same time, due to the limited movement of the molecular aggregate state, the non-radiative excitation energy is effectively suppressed, which promotes the energy to dissipate energy through the radiation transition pathway, and the fluorescence efficiency is increased. Therefore, the material of the present invention has a high fluorescence quantum efficiency and ROS generation ability, and has a good application prospect in fluorescence imaging-mediated photodynamic tumor therapy. Therefore, GGT highly expressed in tumors can be oxidatively damaged to cause oxidative stress in tumor cells, and at the same time, the photosensitizer molecules can cause the accumulation of lipid peroxides (LOPs) in tumor cells through the photodynamic effect, thereby inducing ferroptosis of tumor cells.

[0055] Example 1 Preparation of a GGT (γ-glutamyl transferase) activated AIE photosensitizer molecule

[0056]

[0057] The synthetic route is as follows:

[0058]

[0059] (1) 4,7-dibromo-2,1,3-benzothiadiazole (587.9 mg, 2.0 mmol), 4-(diphenylamino)phenylboronic acid pinacol ester (891.1 mg, 2.4 mmol), potassium carbonate (829.3 mg, 6 mmol) and tetrakistriphenylphosphine palladium (69.3 mg, 0.06 mmol) were added to a 100 mL round-bottom flask and dissolved with 40 mL 1,4-dioxane. The mixture was refluxed for 12 h under argon protection. After the reaction was completed, the catalyst was filtered off and the solvent was evaporated. The concentrated solution was washed with dichloromethane, saturated brine, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (PE:EA=5:1) to obtain orange-red powder intermediate 2 with a yield of 73%.

[0060] (2) Add the product 2 (475.0 mg, 1 mmol) obtained in the previous step, 4-aminophenylboronic acid (178.0 mg, 1.3 mmol), potassium carbonate (414.7 mg, 3 mmol) and tetrakistriphenylphosphine palladium (34.7 mg, 0.03 mmol) to a 50 mL round-bottom flask, and dissolve them with 20 mL 1,4-dioxane. Reflux the reaction under argon protection overnight. After the reaction is completed, filter to remove the catalyst, and evaporate the solvent. The concentrated solution is washed with dichloromethane solvent, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (PE:EA=3:1) to obtain red powder intermediate 3 with a yield of 56%. The NMR data are: 1 H NMR (500 MHz, DMSO-d 6 )δ(ppm):7.94(d,J=8.3Hz,2H),7.82(dd,J=22.7,8.2Hz,4H),7.35(t,J=7.8Hz,4H),7.10(d,J=7.8Hz,8H),6.72(d,J=8.2Hz,2H),5.45(s,2H). 13 C NMR (126 MHz, DMSO-d 6)δ(ppm):153.63,153.58,149.32,147.18,147.03,132.45,130.97,130.05,12 9.97,129.74,127.81,125.92,124.43,123.50,122.62,113.77.HR-ESI-MS(CH 3 OH)m / z:calculated for[M+H] + (C 30 H 23 N 4 S + )471.1643, found 471.1664.

[0061] (3) Add the intermediate 3 (235.3 mg, 0.5 mmol), BOC-L-glutamic acid-1-tert-butyl ester (227.5 mg, 0.75 mmol), HATU (384.2 mg, 1 mmol), and DIPEA (193.9 mg, 1.5 mmol) obtained in the previous step to a 50 mL round-bottom flask, respectively, and dissolve with 10 mL of tetrahydrofuran, and react at room temperature overnight. After the reaction, wash with saturated brine, extract with dichloromethane, dry with anhydrous sodium sulfate, and purify with column chromatography (PE:EA=1:1) to obtain a red powder 4 with a yield of 92%. The NMR data are:

[0062] 1 H NMR (500 MHz, DMSO-d 6 )δ(ppm):10.11(s,1H),7.98(dd,J=15.1,8.3Hz,4H),7.91(s,2H),7.77(d,J=8.3Hz,2H),7.36(t,J=7.8Hz,4H),7.20(d,J=7.9Hz,1H),7.12(d,J= 7.8Hz,7H),3.87(q,J=7.8Hz,1H),2.46(t,J=7.7Hz,3H),2.07-2.00(m,1H),1.83(dd,J=14.6,8.1Hz,1H),1.42(t,J=11.3Hz,18H).HR-ESI-MS(CH 3 OH)m / z:calculated for [M+H] + (C 44 H 46 N 5 O 5 S + )756.3241,found756.3234.

[0063] 13C NMR (126 MHz, DMSO-d 6 )δ(ppm):171.62,170.52,155.54,153.40,147.38,146.88,139.36,131.35,131.24,130.50,130.10,129.67,12 9.40,127.66,127.46,124.46,123.52,122.33,118.89,80.34,78.07,53.88,32.73,28.20,27.94,27.67,26.17.

[0064] (4) The intermediate 4 (270 mg, 0.2 mmol) obtained in the previous step was added to a 25 mL round-bottom flask, and 5 mL of a mixture of trifluoroacetic acid / water / triisopropylsilane (V) was added dropwise under ice-cooling. TFA / V H2O / V TIS =95:2.5:2.5). After reacting for 10 min, the temperature was raised to room temperature and the reaction was continued for 5 h. After the reaction was completed, the solvent was evaporated and the obtained solid was recrystallized from ethyl acetate to obtain a red solid compound 1 with a yield of 98%. The NMR data are: 1 H NMR (500 MHz, DMSO-d 6 )δ(ppm):10.41(s,1H),7.97(dd,J=12.9,6.7Hz,4H),7.88(s,2H),7.78(d,J=6.5Hz,2H),7.36( t,J=6.3Hz,4H),7.11(dd,J=6.7,3.7Hz,8H),3.67-3.58(m,1H),2.63-2.55(m,2H),2.06(m,2H). 13 C NMR (126MHz, DMSO) δ170.33,153.29,146.77,130.01,129.58,129.31,127.58 ,127.34,124.36,123.45,122.21,118.89,59.64,20.66,13.98.HR-ESI-MS(CH 3 OH)m / z:calculated for[M+H] + (C 35 H 30 N 5 O 3 S + )600.2064, found 600.2072.

[0065] Example 2 Preparation of a GGT-activated AIE photosensitizer molecule

[0066]

[0067] The synthetic route is as follows:

[0068]

[0069] The synthesis method of the above compound is the same as that of Example 1, except that 3-aminophenylboronic acid is used instead of 4-aminophenylboronic acid.

[0070] Among them, the NMR data of the second intermediate product is: 1 H NMR (500 MHz, CDCl 3 )δ(ppm):7.87(d,J=8.2Hz,2H),7.74(s,2H),7.38-7.26(m,8H),7.20(t,J=9.3Hz,7H),7.07(t,J=7.4Hz,2H),6.83(d,J=6.8Hz,1H). 13 C NMR (500MHz, CDCl 3 )δ(ppm)154.36,148.38,147.80,138.96,133.12,131.24,130.27,129.90,12 9.70,128.54,127.61,125.25,123.65,123.22,120.45,116.59.HR-ESI-MS(CH 3 OH)m / z:calculated for[M+H] + (C 30 H 23 N 4 S + )471.1643,found471.1638.

[0071] Among them, the NMR data of the third intermediate product is: 1 H NMR (500 MHz, DMSO-d 6)δ(ppm):10.07(s,1H),8.24(s,1H),7.98(d,J=8.3Hz,2H),7.94(d,J=7.4H z,1H),7.88(d,J=7.4Hz,1H),7.67(dd,J=16.8,7.9Hz,2H),7.47(t,J=7.9Hz ,1H),7.37(t,J=7.7Hz,4H),7.15-7.05(m,8H),3.87(q,J=8.0Hz,1H),2.49- 2.41(m,3H),2.05-1.97(m,1H),1.83(dd,J=14.5,8.1Hz,1H),1.41(s,18H). 13 C NMR (500 MHz, DMSO-d 6 )δ(ppm):171.62,170.51,155.53,153.39,153.34,147.48,146.87,139.44,137.33,131.85,131.63,130.38,130.17,129.67,128.8 6,128.38,127.39,124.49,123.84,123.55,122.29,119.68,118.91,80.33,78.06,53.88,32.69,28.18,27.66,26.19.HR-ESI-MS(CH 3 OH)m / z:calculated for[M+Na] + (C 44 H 46 N 5 O 5 S + )778.3034, found 778.3057.

[0072] Among them, the NMR data of the final compound is: 1 H NMR (500 MHz, DMSO-d 6 )δ(ppm):10.55(s,1H),8.26(s,1H),7.93(dd,J=20.0,7.9Hz,5H),7.85(d,J=7.5Hz,1H),7.73(d,J=8.1Hz,1H),7.64(d,J=7.8 Hz,1H),7.45(t,J=8.0Hz,1H),7.36(t,J=7.8Hz,4H),7.14-7.07(m,8H),2.59(d,J=8.1Hz,2H),2.15-2.04(m,2H),2.02(s,1H). 13C(500MHz,DMSO-d 6 )δ(ppm):153.41,147.49,146.88,139.53,137.33,131.85,131.67,130.39,130.19,129.71,128.83, 128.40,127.40,124.51,123.59,122.29,119.75,119.02,59.78,53.39,32.81,14.11.HR-ESI-MS(CH 3 OH)m / z:calculated for[M+H] + (C 35 H 30 N 5 O 3 S + )600.2064,found600.2089.

[0073] Example 3 Characterization of the photophysical properties of benzothiadiazolyl photosensitizer molecules

[0074] Figure 1 is the aggregation-induced emission properties of the materials obtained in Example 1 and Example 2. Wherein, TBmA and TBpA are AIE molecules obtained after TBmA-Glu and TBpA-Glu are catalyzed by GGT. Figure 1 As can be seen from (a) and (b), all four molecular solids can emit light, while only TBmA and TBpA have significant luminescence ability in aqueous solution, and TBmA-Glu and TBpA-Glu hardly emit light in aqueous solution and DMF solution. Therefore, it can be considered that the introduction of water-soluble glutamic acid groups can improve the water solubility of AIE photosensitizer molecules and inhibit their AIE properties in aqueous solution. At the same time, we also found that the four compounds TBmA (ROS ability is TBmA>TBpA>>TBmA-Glu>TBpA-Glu) have photodynamic activity Figure 1 (c) At the same time, its ROS production capacity is positively correlated with its aggregation state. Figure 1 (d).

[0075] Its possible

[0076] Example 4 Study on the Activation Effect of GGT on GGT-Activated Photosensitizer Molecules

[0077] Taking TBmA-Glu as the object, we studied the relationship between GGT and its photodynamic activity. From the catalytic activity of GGT, it can be seen that it can catalyze the departure of the water-soluble glutamine group in the TBmA-Glu molecule, thereby causing the lipid-soluble TBmA molecules to aggregate and activate its AIE properties ( Figure 2a). First, molecular docking was used to study the targeting ability of TBmA-Glu to GGT. From the docking results, it can be seen that the introduction of the AIE molecular core enables it to form intermolecular hydrogen bonds with multiple amino acids in the GGT protein, further enhancing the targeting and binding ability of glutamine to GGT ( Figure 3 b). Further co-incubation experiments with GGT showed that the fluorescence of the mixture system increased over time ( Figure 3 c). LC-MS results confirmed that the fluorescence enhancement was caused by GGT catalyzing TBmA-Glu to produce TBmA ( Figure 3 d). At the same time, if the mixed system is illuminated with light after incubation for 1 hour, the fluorescence intensity of the system will no longer change significantly ( Figure 3 c), the results also suggest that light treatment may have caused damage to the enzyme activity of GGT. Further enzyme activity research experiments have confirmed this hypothesis. Low concentrations of TBmA-Glu incubated with GGT and then exposed to light can significantly inhibit the enzyme activity of GGT. Based on the above results, it can be seen that the co-incubation of TBmA-Glu and GGT can produce TBmA with photodynamic activity, so further light exposure will cause oxidative damage to GGT and lose its activity.

[0078] Example 5 Evaluation of Cellular Phototoxicity of GGT-Activated Photosensitizer

[0079] Considering that the photodynamic activity of GGT-activated photosensitizer molecules may be related to the expression of GGT in cells, the selective ability of TBmA-Glu on HepG2 cells with high GGT expression and LO2 cells with low GGT expression was studied. The results showed that TBmA-Glu can selectively accumulate and be activated in HepG2 cells ( Figure 3 a and 3b). Furthermore, we used three types of cells with different GGT expression levels, namely two tumor cells HepG2 (GGT high expression), HeLa (GGT medium expression) and LO2 (GGT medium expression) as research objects to evaluate the photodynamic activity of GGT-activated photosensitizer molecules TBpA-Glu and TBmA-Glu. First, the phototoxicity under normal oxygen conditions was studied. Tumor cells were plated in 96-well plates and cultured for 24 hours. They were first treated with different concentrations of photosensitizer molecules for 12 hours. The 96-well plates of the light-treated group were then exposed to white light (20mWcm -2) was irradiated for 10 minutes and then cultured for 8 hours, and then MTT was added and cultured for 4 hours. As shown in Table 1, when the GGT-activated photosensitizer molecules TBpA-Glu and TBmA-Glu had higher anti-tumor IC50 values ​​than the non-targeted photosensitizer molecules. At the same time, its IC50 value was negatively correlated with the expression of GGT. This result also shows that the phototoxicity of the photosensitizer molecules TBpA-Glu and TBmA-Glu was positively correlated with the content of GGT in the cells. In addition, further studies on the photodynamic activity under hypoxic conditions also found that the photodynamic activity of the photosensitizer molecules TBpA-Glu and TBmA-Glu was almost unaffected by changes in oxygen content. The results showed that both molecules achieved a killing effect on tumor cells through a type of photodynamics. However, overall, the photosensitizer molecule TBmA-Glu had better photodynamic activity than TBmA-Glu. Therefore, we took TBmA-Glu as the research object to further study the possible anti-tumor mechanism of the GGT-activated photosensitizer molecules.

[0080] Example 6 Evaluation of the photodynamic therapy mechanism of GGT-activated photosensitizer (TBmA-Glu)

[0081] HepG2 human liver cancer cells were used as the research object to study the photodynamic anti-tumor mechanism of TBmA-Glu. Figure 4 As can be seen from a, TBmA-Glu treatment significantly increased the level of ROS in HepG2 cells under illumination. Further treatment with different types of ROS scavengers showed that TBmA-Glu photodynamic therapy mainly led to the production of lipid peroxides and hydroxyl radicals in tumor cells ( Figure 4 b). Combined with the targeting ability of TBmA-Glu to GGT, further studies have found that TBmA-Glu photodynamic therapy can lead to a decrease in intracellular GGT levels ( Figure 4 d and Figure 4 e). Because GGT is mainly involved in the intracellular glutathione cycle, GGT loss can further induce a decrease in intracellular reduced GSH and an increase in the content of oxidized GSSG ( Figure 4 c). At the same time, in the WB experiment, we found that TBmA-Glu photodynamic treatment can also lead to a decrease in the level of GPX4 in tumor cells ( Figure 4 d), cell electron microscopy results also showed that the mitochondrial volume in the tumor cells of the light treatment group was shrunk, the mitochondrial membrane density was deepened, and the cell nucleus was intact, and the above phenomena were typical characteristics of cell ferroptosis. From the above results, it can be seen that TBmA-Glu photodynamic therapy caused GGT damage in tumor cells, increased intracellular ROS levels, and lipid peroxide accumulation ( Figure 5 ), GPX4 level is downregulated, further inducing ferroptosis of tumor cells.

[0082] Example 7 Study on the effect of photodynamic therapy of GGT-activated photosensitizer (TBmA-Glu) on in vivo tumor model

[0083] The tumor animal model was established by implanting HepG2 human liver cancer cells subcutaneously into nude mice. 3 Afterwards, the tumor-bearing mice were randomly divided into 5 groups and injected with TBmA, TBmA-Glu or an equal volume of saline (control group) through the tail vein to evaluate the therapeutic effect of the compound on the tumor. The drug was administered once every seven days, with a concentration of 5 mg / mL each time, and the treatment lasted for a total of 14 days. White light treatment (20 mW cm -2 ) for 10 minutes. During the treatment, the tumor volume and body weight of the mice were recorded every two days. At the end of the treatment, it was found that the tumor volume of the TBmA-Glu light-treated group was significantly smaller than that of the other treatment groups ( Figure 6 a and Figure 6 b). The fluorescence imaging results of the isolated organs and tumors in each group at the end of treatment showed that TBmA-Glu had good tumor targeting ( Figure 6 c). It can also be seen from the in vivo fluorescence imaging results of TBmA and TBmA-Glu after 12 hours of administration ( Figure 6 d), TBmA-Glu has good tumor targeting, and its fluorescence signal can also realize tumor monitoring during the photodynamic therapy of tumors. At the same time, after H&E staining of the tumor tissues of each treatment group, it can be seen that the TBmA-Glu phototherapy group can significantly induce a significant decrease in tumor tissue density and a significant increase in the number of apoptotic cells ( Figure 7 Further immunohistochemical results also proved that the expression of GPX4 in tumor cells in the TBmA-Glu phototherapy group was significantly lower than that in the non-phototherapy group, which also shows that TBmA-Glu can also induce ferroptosis of tumor cells in the photodynamic therapy of living tumors ( Figure 6 e).

[0084] Example 8 Study on the effect of photodynamic therapy of liver in situ tumors using GGT-activated photosensitizer (TBmA-Glu)

[0085] To further verify the anti-tumor effect of TBmA-Glu, we established an animal model of orthotopic liver tumors. First, we constructed HepG2 cells that can express luciferase through lentiviral infection. The cells were directly injected into the liver to construct HepG2 orthotopic liver tumors. During the modeling process, the tumor growth was monitored by intraperitoneal injection of potassium luciferin (10 mg / kg) ( Figure 8a). After successful tumor modeling, TBmA-Glu (5 mg / kg) was injected into the tail vein. 12 hours after administration, a small incision (about 2 mm) was made in the mouse liver, and the optical fiber was inserted into the mouse abdominal cavity and photodynamic therapy (10 mW cm -2 ), 10 min. The good co-localization results of bioluminescence and TBmA-Glu fluorescence also indicate that TBmA-Glu can achieve tumor targeting well ( Figure 8 b). At the same time, the changes in tumor bioluminescence and TBmA-Glu fluorescence intensity during the treatment process and the fluorescence imaging results of various organs at the end of treatment were compared. Fig. 9 ) It can be seen that TBmA-Glu phototherapy can significantly inhibit the proliferation of liver in situ tumors. Further H&E and TUNEL staining pathological analysis of liver in situ tumor tissues in each treatment group showed that TBmA-Glu phototherapy group can significantly induce apoptosis of tumor tissue in the liver, and show obvious tumor necrosis and fibrosis ( Fig.10 a and Fig.10 b).

[0086] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific embodiments. The above-mentioned specific embodiments are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can make many modifications without departing from the scope of protection of the present invention and the claims, all of which belong to the scope of protection of the present invention.

Claims

1. An enzyme-activated aggregation-induced emission photosensitizer, characterized in that, the enzyme-activated aggregation-induced emission photosensitizer has the following structure: Among them, the "π" bridge is an independent aromatic ring; R 1 is a molecular rotor structure and is selected from one of triphenylamine, substituted triphenylamine, tetraphenylethylene or substituted tetraphenylethylene. The structures of substituted triphenyl and substituted tetraphenyl are as follows respectively, R 2 is H or an alkoxy chain 2. The enzyme-activated aggregation-induced emission photosensitizer according to claim 1, characterized in that, the enzyme-activated aggregation-induced emission photosensitizer has the following structure:

3. The enzyme-activated aggregation-induced emission photosensitizer according to claim 1, characterized in that, the enzyme-activated aggregation-induced emission photosensitizer has the following structure:

4. A method for preparing the enzyme-activated aggregation-induced emission photosensitizer according to any one of claims 1 to 3, characterized in that, it comprises the following steps: Step S1: 4,9-dibromobenzothiadiazole and a triphenylamine boronic acid derivative or a tetraphenylethylene boronic acid derivative are subjected to a Suzuki coupling reaction under the action of a catalyst to obtain a first intermediate; Step S2: The first intermediate and aminophenylboronic acid are subjected to a Suzuki coupling reaction under the action of a catalyst to obtain a second intermediate; Step S3: The second intermediate and carboxyl- and amino-protected glutamic acid are subjected to an amidation reaction under the action of a condensing agent to obtain a third intermediate; Step S4: The third intermediate is deprotected by trifluoroacetic acid to obtain the enzyme-activated aggregation-induced emission photosensitizer.

5. The preparation method according to claim 4, characterized in that, in Step S1, the triphenylamine boronic acid derivative is selected from one of 4-boronic acid triphenylamine pinacol ester, 4-boronic acid triphenylamine or 4-(diphenylamino)phenylboronic acid pinacol ester, and the tetraphenylethylene boronic acid derivative is selected from one of (4-(1,2,2-triphenylethynyl)phenyl)boronic acid or (4-(1,2,2-triphenylethynyl)phenyl)boronic acid pinacol ester; the mass ratio of 4,9-dibromobenzothiadiazole to the triphenylamine boronic acid derivative or the tetraphenylethylene boronic acid derivative is 1:1 to 1:2; in Step S2, the mass ratio of the first intermediate to aminophenylboronic acid is 1:1 to 1:2; in Step S3, the mass ratio of the second intermediate to carboxyl- and amino-protected glutamic acid is 1:1 to 1:

3.

6. The preparation method according to claim 4, characterized in that, the base for the Suzuki reaction can be selected from one of sodium carbonate, sodium hydroxide, potassium hydroxide, potassium carbonate, and the catalyst can be selected from one of tetrakis(triphenylphosphine)palladium and palladium chloride, and the condensing agent is HATU, HBTU and EDCI.

7. An application of the enzyme-activated aggregation-induced emission photosensitizer according to any one of claims 1-3 in the preparation of a preparation for fluorescence imaging-mediated photodynamic tumor therapy.

8. The application according to claim 7, characterized in that, the enzyme-activated aggregation-induced emission photosensitizer is activated by γ-glutamyltransferase in tumor cells.

9. The application according to claim 8, characterized in that, the photodynamic activity of the enzyme-activated aggregation-induced emission photosensitizer is positively correlated with the content of γ-glutamyltransferase in tumor cells.

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