Hypoxia activating peptide-photosensitizer-drug conjugate as well as preparation method and application thereof

Abstract

The invention discloses a hypoxia activating peptide-photosensitizer-drug conjugate and a preparation method and application thereof in the technical field of medicine, the conjugate takes indocyanine with a high molar extinction coefficient as a photosensitive core, distorted conjugate units are introduced to two sides of indole, the characteristic of'limited movement in molecules' is given to the conjugate, and radiation and non-radiation energy dissipation are balanced. Meanwhile, an azo bond sensitive to hypoxia is reasonably introduced between a light treatment module and a chemotherapy module to serve as a responsive linker, and is further coupled with two-molecule integrin targeting peptide cRGD, so that the azo bond is self-assembled into nanospheres in an aqueous solution, and a triple targeting mechanism, namely multivalent integrin receptor targeting, aggregation and tumor microenvironment hypoxia selective activation, is realized. In an in-situ osteosarcoma mouse model, the conjugate realizes real-time near-infrared two-region imaging, shows potent tumor inhibition, effectively inhibits lung metastasis, and does not detect obvious systemic toxicity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a hypoxia-activated peptide-photosensitizer-drug conjugate, a preparation method and application thereof. BACKGROUND

[0002] To improve the selectivity and safety of cancer treatment, targeted prodrug strategies have been gradually developed. Antibody-drug conjugates (ADCs) using monoclonal antibodies as carriers were the first to achieve selective delivery of cytotoxic drugs and achieved clinical success, such as trastuzumab-mertansine conjugate and brentuximab. However, ADCs mainly improve the targeting accuracy and loading delivery efficiency, and do not solve the problem of long-term separation of diagnosis and treatment functions. In addition, its large molecular size limits penetration into solid tumors, and low drug-antibody ratio (DAR≤8) allows only high cytotoxicity load to be carried, which may cause severe systemic toxicity when released off-target, and may also have problems such as immunogenicity and high production cost.

[0003] Peptide-drug conjugates (PDCs) were proposed as an alternative, which use small molecule peptides instead of antibodies, with the advantages of smaller size, faster tissue distribution, and better tumor penetration, but are still susceptible to proteolytic degradation, thereby compromising targeting accuracy and in vivo stability. Notably, ADCs and PDCs mainly deliver chemotherapy or radiotherapy drugs, which are limited to a single treatment mode and lack the integrated diagnostic and therapeutic functions required for modern precision medicine. SUMMARY

[0004] The present application aims to solve the above technical problems, and provides a hypoxia-activated peptide-photosensitizer-drug conjugate, a preparation method and application thereof.

[0005] The present application achieves the above-mentioned purpose by the following technical solutions: As a first aspect of the present application, a hypoxia-activated peptide-photosensitizer-drug conjugate is provided, having the structure shown in Formula I: Formula I In Formula I, n is 1-5; Y - is an anion; R1 is an aromatic conjugated compound.

[0006] As a further optimization scheme of the present application, the anion is any one of Cl - , Br - , I - , BF4 - , PF6 - , ClO4 - , CH3COO - , p-toluenesulfonate or tetraphenylborate.

[0007] As a further optimization scheme of the present application, the structure of the aromatic conjugated compound is any one of formula R 1-1 , R 1-2 , R 1-3 , R 1-4 or R 1-5 . .

[0008] As a further optimization scheme of the present application, the structure of the cRGD is: .

[0009] As a second aspect of the present application, a preparation method of the hypoxia-activated peptide-photosensitizer-drug conjugate is also provided, comprising the following steps: (1) subjecting an indole boronic acid pinacol ester compound and R1Br containing an R1 group to Suzuki coupling reaction to obtain an intermediate I; (2) subjecting the intermediate I to nucleophilic substitution reaction with 5-chloropent-1-yn and MY to obtain an intermediate II; wherein M in MY is Na or K; (3) subjecting the intermediate II to Knoevenagel reaction with a chloro cyclohexadiene compound substituted with a diimine group to obtain an intermediate III; (4) subjecting the intermediate III to nucleophilic substitution reaction with a substituted benzene thiol compound to obtain an intermediate IV; (5) subjecting the intermediate IV to Click reaction with an azido-modified polyethylene glycol linked cRGD to obtain the hypoxia-activated peptide-photosensitizer-drug conjugate.

[0010] As a further optimization scheme of the present application, in step (1), the indole boronic acid pinacol ester compound and R1Br are subjected to reflux reaction in a solvent at 100°C for 24h in the presence of a catalyst Pd(PPh3)4 and a carbonate; wherein the molar ratio of the indole boronic acid pinacol ester compound, R1Br, Pd(PPh3)4 and the carbonate is 1:1.2:1.0:2.5; the carbonate is any one of potassium carbonate, sodium carbonate or cesium carbonate; the solvent is composed of deoxygenated 1,4-dioxane and deoxygenated water, and the volume ratio of the two is 5:1; After the reaction in step (1) is completed, dichloromethane is further added to the reaction product for extraction, the organic layer is collected, washed with saturated NaCl solution, and then dried, concentrated, and separated by silica gel column chromatography to obtain the intermediate I.

[0011] As a further optimization of the present application, in step (1), the structure of the indole boronic acid pinacol ester compound is: ; The structure of the intermediate I is: .

[0012] As a further optimization of the present application, in step (2), the intermediate I, 5-chloropent-1-yn and MY are refluxed at 80°C under nitrogen protection for 20-25h in toluene to obtain the intermediate II; the molar ratio of the intermediate I, 5-chloropent-1-yn and MY is 1:1.5-3:3-5; After the reaction in step (2) is completed, the reaction product is diluted with DCM, washed with water, dried, concentrated, added with ether, and then precipitated, and then filtered, and the filter cake is washed with ether to obtain the intermediate II; The structure of the intermediate II is: .

[0013] As a further optimization of the present application, in step (3), the intermediate II and the di-imino-substituted chlorocyclohexadiene compound are added to a mixed solution of n-butanol and toluene in a volume ratio of 3:1, and reacted at 100°C for 5-8h, and then the reaction product is concentrated and separated by column chromatography to obtain the intermediate III; The molar ratio of the intermediate II and the di-imino-substituted chlorocyclohexadiene compound is 2-2.5:1; The structure of the di-imino-substituted chlorocyclohexadiene compound is: ; The structure of the intermediate III is: .

[0014] As a further optimization of the present application, in step (4), the intermediate III, the substituted benzene thiol compound and triethylamine are added to anhydrous DMF, and stirred at room temperature for 4-6h, and then the reaction product is concentrated and separated by column chromatography to obtain the intermediate IV; the molar ratio of the intermediate III, the substituted benzene thiol compound and triethylamine is 1:1-3:1-3; The structure of the substituted benzene thiol compound is: ; The structure of the intermediate IV is: ; The structure of the R2 group in the structure of the substituted benzene thiol compound or the intermediate IV is: .

[0015] As a further optimization scheme of the present application, in step (5), the intermediate IV, azido-modified polyethylene glycol linked cRGD, copper sulfate pentahydrate and sodium ascorbate are added to anhydrous DMF, stirred at room temperature for 10-12 h, after the reaction is completed, the reaction is concentrated, and then column chromatography is used for separation to obtain the hypoxia-activated peptide-photosensitizer-drug conjugate; The molar ratio of the intermediate IV, azido-modified polyethylene glycol linked cRGD, copper sulfate pentahydrate and sodium ascorbate is 1:2-3:0.3-0.5:0.4-0.6; The structure of the azido-modified polyethylene glycol linked cRGD is: .

[0016] As a third aspect of the present application, there is also provided a use of the hypoxia-activated peptide-photosensitizer-drug conjugate as described above in the preparation of a drug for the combined chemotherapy-photothermal-photodynamic treatment of osteosarcoma.

[0017] The present application has the following beneficial effects: The present application provides a hypoxia-activated peptide-photosensitizer-drug conjugate, which uses indocyanine with a high molar extinction coefficient (>10 5 The present application has the following beneficial effects: The present application provides a hypoxia-activated peptide-photosensitizer-drug conjugate, which uses indocyanine with a high molar extinction coefficient (>10 BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 A synthesis route map of the hypoxia-activated peptide-photosensitizer-drug conjugate provided for Example 1 is provided; Figure 2 A transmission electron microscope (TEM) picture of the IT-azo-RGD obtained in Example 1 dispersed in PBS is provided for Example 3; Figure 3High performance liquid chromatogram of the release of IT-azo-RGD (yellow peak) obtained in Example 1 at different time points under hypoxia (oxygen concentration of 1%) for Example 4; Figure 4 Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) diagram of the intracellular residue after incubation for 24 h under 1% O2 for Example 4; Figure 5 Intracellular hydroxyl radical (·OH) detection diagram of 143B cells treated under different conditions for Example 5; Figure 6 Cell activity diagram of IT-azo-RGD obtained in Example 1 on 143B, MG63 and Saos-2 under hypoxia for Example 6; Figure 7 Tumor fluorescence diagram of IT-azo-RGD obtained in Example 1 injected intravenously into orthotopic 143B osteosarcoma tumor-bearing mice over time for Example 8; Figure 8 Infrared thermal image diagram of the tumor region under 808 nm laser irradiation after model mice were injected with IT-azo-RGD obtained in Example 1 into the tail vein for Example 9; Figure 9 Tumor volume change test diagram of mice after treatment in four groups for Example 9; Figure 10 Tumor weight change test diagram of mice after treatment in four groups for Example 9; Figure 11 Body weight change test diagram of mice after treatment in four groups for Example 9; Figure 12 Tumor tissue section histopathological staining test diagram of mice after treatment in four groups for Example 9; Figure 13 Representative diagram of lung metastasis and corresponding H&E staining after treatment in four groups for Example 9; Figure 14 H&E staining diagram of main organs after treatment in four groups for Example 9; Figure 15 Mechanism of action of hypoxia-activated peptide-photo sensitizer-drug conjugate IT-azo-RGD provided in the present application. DETAILED DESCRIPTION

[0019] It is necessary to point out here that the following detailed description is only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application according to the above application content.

[0020] Example 1, Synthesis of Hypoxia Activated Peptide-Photosensitizer-Drug Conjugate (hereinafter referred to as IT-azo-RGD) The synthetic route is shown in Figure 1 .

[0021] (1) Compound 1 (630 mg, 2.20 mmol), 4,4'-(2-(4-bromophenyl)-2-styryl-1,1- diyl)dibenzene (885 mg, 2.64 mmol), tetrakis(triphenylphosphine)palladium (254 mg, 0.22 mmol), potassium carbonate (760 mg, 5.5 mmol) were added to a flask, which was evacuated and flushed with argon three times to remove oxygen. Subsequently, deoxygenated 1,4-dioxane and deoxygenated water (5:1 by volume) were added to the flask sequentially. The resulting mixture was heated to 100 °C under reflux for 24 h. After cooling to room temperature, the mixture was extracted with dichloromethane (DCM) three times. The organic layer was collected, washed with saturated aqueous sodium chloride solution, then dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel column chromatography to obtain compound 2 as a light yellow solid (696 mg, yield 76.5%).

[0022] The structural characterization data of compound 2 as a light yellow solid are as follows: 1 H NMR (600 MHz, Chloroform-d) δ 7.31 (d, J=7.9 Hz, 1H), 7.12–7.07 (m, 8H), 7.05 (dt, J=5.3, 2.1 Hz, 5H), 7.00(dd, J=6.7, 3.0 Hz, 2H), 6.93 (dd, J=7.9, 1.7 Hz, 1H), 6.88 (d, J=1.6 Hz,1H), 2.23 (s, 3H), 1.03 (s, 6H). 13 C NMR (151 MHz, Chloroform-d) δ 143.80, 143.74, 140.96, 140.87, 131.45, 131.43, 130.86, 127.75, 127.74, 127.73, 126.60, 126.47, 126.37, 125.17, 118.98, 53.25, 22.94, 15.52. HRMS (ESI + ) calcd for C 31 H 28 N + [M] +414.2217, found: 414.22275.

[0023] (2) A mixture of 5-chloro-l-pentynyl (502 mg, 4.9 mmol) and potassium iodide (1.63 g, 9.8 mmol) was dissolved in acetonitrile and stirred vigorously at 80 °C. After 1 h, compound 2 (1.01 g, 2.45 mmol) was added and stirred at 80 °C for 24 h under argon protection. The resulting residue was diluted with dichloromethane and then washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to concentrate. The resulting residue was dissolved in 2 mL of dichloromethane, precipitated with excess ether, and washed with excess ether three times. Compound 3 (699 mg, yield 47.0%) was obtained as an off-white solid, which was used directly in the next step without purification; The structural characterization data of compound 3 as an off-white solid are as follows: 1 H NMR (600 MHz, Chloroform-d) δ 7.50 (d, J=8.4 Hz, 1H), 7.20 (dd, J=8.4, 1.5 Hz, 1H), 7.17–7.15 (m, 3H), 7.13 (td, J=5.7, 5.0, 2.7 Hz, 6H), 7.09 (d, J=1.5 Hz, 1H), 7.04 (ddt, J=6.8, 4.8, 2.0 Hz, 4H), 7.00–6.98 (m, 2H), 4.84–4.79 (m, 2H), 3.15 (s, 3H), 2.47 (s, 2H), 2.21 (s, 2H), 2.06 (d, J=2.4 Hz, 1H), 1.34 (s, 6H). 13 C NMR (151 MHz, CDCl3) δ 146.59, 143.89, 143.17, 142.75, 142.53, 140.79, 139.01, 132.53, 131.49, 131.26, 128.34, 128.29, 127.96, 127.36, 126.75, 114.75, 82.08, 71.23, 54.23, 48.97, 26.76, 23.28, 17.50, 16.32. HRMS (ESI + ) calcd for C 36 H 34 N + [M] + :480.2686, found: 480.26485.

[0024] (3) Compound 3 (607.6 mg, 1.0 mmol), N-((1E,3E,5E)-5-(phenylimino)pent-1,3- dien-1-yl)aniline hydrochloride (Compound 5, 162 mg, 0.45 mmol) were dissolved in n- butanol and toluene (3:1) and stirred vigorously at 100 °C for 6 h. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography to give dark green solid Compound 4 (238 mg, yield 43.2%); The structural characterization data of green solid Compound 4 are as follows: 1 H NMR (600 MHz, Chloroform-d) δ 8.20 (d, J=13.9 Hz, 2H), 7.15-7.13 (m, 6H), 7.12-7.09 (m, 12H), 7.07-7.06 (m, 4H), 7.04-7.00 (m, 12H), 6.94 (d, J=1.6 Hz, 2H), 6.30 (dd, J=14.0, 2.4 Hz, 2H), 4.28 (t, J=7.4 Hz, 4H), 2.74 (t, J=6.3 Hz, 4H), 2.43 (td, J=6.5, 2.6 Hz, 4H), 2.09 (t, J=2.6 Hz, 2H), 2.06-2.01 (m, 4H), 1.93 (p, J=6.2 Hz, 2H), 1.39 (s, 12H). 13 C NMR (151 MHz, CDCl3) δ 172.19, 150.17, 144.03, 143.93, 143.41, 143.25, 141.94, 141.29, 140.58, 140.23, 140.19, 131.99, 131.55, 131.47, 131.37, 128.28, 128.09, 128.00, 127.83, 126.98, 126.80, 126.01, 110.29, 102.08, 83.05, 70.33, 48.76, 43.69, 28.07, 27.13, 26.22, 20.78, 16.38. HRMS (ESI + ) calcd for C 80 H 72 ClN2 + [M] + : 1095.5379, found: 1095.52168.

[0025] (4) IT-5-alkynyl (50 mg, 0.041 mmol) and (E)-4-((4-(bis(2- chloroethyl)amino)phenyl)azomethyl)benzyl (3-mercaptophenyl) carboxylate (41 mg, 0.082 mmol) were dissolved in 2 mL of anhydrous dimethylformamide under argon atmosphere, then triethylamine (12 µL, 0.082 mmol) was added and stirred for 12 h. After the reaction was completed, the mixture was purified by silica gel column chromatography to obtain compound 6 (49 mg, yield 70.0%) as a brown-green solid; The structural characterization data of brown-green solid compound 6 are as follows: 1 H NMR (600 MHz, DMSO-d6) δ 9.94 (s,1H), 8.48 (d, J=13.9 Hz, 2H), 7.79–7.76 (m, 4H), 7.55 (d, J=8.1 Hz, 2H), 7.52(s, 1H), 7.21 (d, J=6.0Hz, 2H), 7.15–7.09 (m, 18H), 7.02–6.98 (m, 6H), 6.97–6.94 (m, 10H), 6.92 (d, J=9.1 Hz, 2H), 6.88 (dd, J=8.2, 1.5 Hz, 2H), 6.81 (d,J=7.8 Hz, 1H), 6.30 (d, J=14.0 Hz, 2H), 5.24 (s, 2H), 4.13 (t, J=7.4 Hz, 4H),3.86 (t, J=6.9 Hz, 4H), 3.80 (t, J=6.7 Hz, 4H), 2.95 (t, J=2.6 Hz, 2H), 2.70(t, J=5.9 Hz, 4H), 2.28 (dt, J=7.3, 3.6 Hz, 4H), 1.93–1.88 (m, 2H), 1.83 (p,J=7.1 Hz, 4H), 1.11 (s, 12H). 13C NMR (151 MHz, DMSO) δ 174.27, 171.74, 153.15,151.98, 149.51, 149.21, 144.76, 143.32, 143.26, 142.96, 142.79, 141.08, 140.40, 140.19, 140.05, 139.94, 138.24, 137.22, 133.64, 130.84, 130.66, 130.60, 129.64, 128.81, 127.93, 127.79, 126.77, 126.58, 126.54, 125.22, 124.98, 121.98, 111.88, 110.54, 101.90, 83.47, 72.27, 51.90, 48.11, 40.98, 27.01, 26.56, 25.93, 22.11, 15.31. HRMS (ESI + ) calcd for C 104 H 95 Cl2N6O2S + [M] + :1563.6580, found: 1563.66110.

[0026] (5) Compound 6 (25.4 mg, 0.015 mmol) and cRGD-PEG4-N3 (26.3 mg, 0.030 mmol) were dissolved in 1 mL of N,N-dimethylformamide (DMF), to which was added a solution of copper sulfate pentahydrate (1.9 mg, 0.0075 mmol), sodium ascorbate (1.5 mg, 0.0075 mmol) and THPTA (3.3 mg, 0.0075 mmol) dissolved in 200 μL of water. The reaction solution was stirred at room temperature for 12 h. After the reaction was completed, the mixture was purified by Sephadex LH-20 resin column chromatography with dichloromethane and methanol (1:1) as the eluent to obtain brown-green solid compound 7 (IT-azo-RGD, 38 mg, yield 73.5%); The structural characterization data of brown-green solid compound 7 (IT-azo-RGD) are as follows: 1H NMR (600 MHz, DMSO-d6) δ 9.93 (s, 1H), 8.45 (d, J=13.3 Hz, 2H), 8.33 (s, 2H), 8.21 (s, 4H), 7.88(s, 6H), 7.77 (d, J=8.3 Hz, 4H), 7.69 (d, J=24.4 Hz, 2H), 7.54 (d, J=8.0 Hz,2H), 7.51 (s, 1H), 7.25–7.20 (m, 6H), 7.14 (d, J=7.2 Hz, 10H), 7.09 (t, J=6.6Hz, 14H), 7.00 (d, J=6.8 Hz, 6H), 6.94 (d, J=7.3 Hz, 14H), 6.88 (dd, J=13.8,7.6 Hz, 4H), 6.81–6.74 (m, 2H), 6.26 (d, J=13.8 Hz, 2H), 5.23 (s, 2H), 4.48(s, 2H), 4.47 (s, 4H), 4.16 (s, 2H), 4.11 (s, 4H), 3.85 (d, J=6.0 Hz, 4H),3.79 (m, 6H), 3.56–3.48 (m, 12H), 3.46–3.41 (m, 24H), 3.16 (s, 14H), 3.08 (s,4H), 2.95 (s, 4H), 2.77–2.70 (m, 6H), 2.63 (d, J=19.0 Hz, 4H), 2.28 (s, 4H),1.99 (tt, J=12.9, 7.2 Hz, 6H), 1.89 (s, 2H), 1.71 (s, 2H), 1.65–1.50 (m, 6H),1.45–1.38 (m, 4H), 1.13–1.06 (m, 14H). MALDI-TOF MS calcd for C 180 H 215 Cl2N 30 O 26 S + [M] + : 3315.5550, found: 3315.5558.

[0027] Example 2, Near-infrared absorption and fluorescence spectra of IT-azo-RGD The compound 7 obtained from Example 1 was formulated into a 1.0 mmol / L dimethyl sulfoxide (DMSO) stock solution, which was then diluted with various solvents (dichloromethane, ethanol, dimethyl sulfoxide, water, phosphate buffer) to a final concentration of 5 μmol / L for subsequent near-infrared absorption spectroscopy measurements, and 10 μmol / L for emission spectrum evaluation. The absorbance and emission spectra measurements were performed using a quartz cuvette with an optical path of 1 cm.

[0028] The results obtained are shown in Table 1, IT-azo-RGD exhibits a maximum absorption / emission wavelength of 852 / 891 nm in dimethyl sulfoxide (DMSO) with a fluorescence quantum yield (ΦF) of 3.12%, and its emission spectrum is extended to 14.4% in the near-infrared II region (>1000 nm), meeting the requirements of deep tissue imaging.

[0029] Table 1. Photophysical properties of IT-azo-RGD 4 Maximum absorption wavelength (nm) Molar extinction coefficient (cm -1 M -1 ) Maximum emission wavelength (nm) Fluorescence quantum yield (%) Luminance (cm -1 M -1 ) Near-infrared second region emission ratio a (%) a Near infrared two Zone brightness (cm -1 M -1 ) b Solvent IT-azo-RGD 850 2.29 x 10 5 893 2.856527 14.4940 DCM (dichloromethane) 854 1.35 x 10 5 893 2.853848 13.6523 EtOH (ethanol) 852 1.59 x 10 5 891 3.124961 14.4714 DMSO (dimethyl sulfoxide) 866 1.12 x 10 5 916 0.111232 3.529 H20 (water) 8695.73×109140.095218.410 PBS phosphate buffered saline Note: a: a = 1,000-1,350 nm between the emission / total emission x 100%; b: near-infrared two area brightness = brightness x (a / 100).

[0030] Example 3, Test of IT-azo-RGD Particle Size The IT-azo-RGD obtained in Example 1 was dispersed in PBS and subjected to transmission electron microscopy (TEM) detection, and the results are shown in FIG. 3. As shown in FIG. 3, the IT-azo-RGD is a spherical nanostructure with an average diameter of about 100 nm. Figure 2

[0031] Example 4, Intracellular Drug Release Kinetics The oxygen environment of tumor tissue (usually <2.5% O2) and normal tissue (usually >5% O2) was simulated, and three oxygen concentration gradients of 1%, 2.5%, and 5% were set to explore the drug release efficiency of the IT-azo-RGD obtained in Example 1. The specific steps are as follows: 143B cells (Wuhan Saiver Biological Technology Co., Ltd., Catalog No.: STCC00058P; Brand: Zishan Biological) were inoculated in a 6-well plate (5 x 10 5 cells / well), and 1 μM of the IT-azo-RGD obtained in Example 1 was added under different oxygen concentrations for 2-24 h; the cells after trypsin digestion were collected, washed with ice bath PBS (pH = 7.4) for 3 times, and -20°C methanol was added for lysis by a probe ultrasonic instrument (pulse mode for 30 minutes, amplitude 50%, work / pause cycle 10 seconds); centrifugation (13000 x g, 20 minutes, 4°C) was performed to remove cell debris, and the supernatant was vacuum freeze-dried; after the residue was redissolved with 100 μL of methanol, HPLC quantitative detection was performed on the released phenylhydrazine mustard and IT-m-NH2-RGD (or IT-m-NH2-cRGD).

[0032] The results are shown in FIG. 4. As shown in FIG. 4, after 12 h, high performance liquid chromatography results showed the presence of three expected products (IT-m-NH2-RGD, phenylhydrazine mustard, and p-aminobenzyl alcohol), and no IT-azo-RGD obtained in Example 1 was detected (the yellow peak type is IT-azo-RGD, the blue peak type is p-aminobenzyl alcohol, the red peak type is phenylhydrazine mustard, and the purple peak type is IT-m-NH2-RGD); among them, IT-m-NH2-RGD has the following structure (I): Figure 3 (I) Phenylhydrazine mustard has the following structure (II): (II) ​​Subsequently, the MALDI-TOF analysis chart of the intracellular residue after 24h incubation under 1% O2 condition is shown in Figure 2, which confirms the existence of the three expected products (IT-m-NH2-RGD, aniline mustards and p-aminobenzyl alcohol), and the mass spectrum does not detect IT-azo-RGD. Figure 4

[0033] Example 5, Detection of Intracellular Reactive Oxygen Species Generation Normoxic condition (21% O2): when the confluence of 143B cells reached 60%, the DMEM medium was discarded and the cells were washed with PBS for 3 times; 5μM IT-azo-RGD obtained in Example 1 was added into the DMEM medium and the cells were incubated under normoxic condition for 3h; the medium containing IT-azo-RGD obtained in Example 1 was discarded and fresh medium containing HPF (10μmol) was added and incubated for 30min; after irradiation with 808nm laser (300mW·cm -2 ) for 5min, the cells were trypsinized and subjected to flow cytometry detection.

[0034] Hypoxic condition (1% O2): 143B cells were first pretreated under hypoxic condition for 6h; the subsequent steps were consistent with the normoxic group, but the incubation of IT-azo-RGD obtained in Example 1 and the incubation of HPF (10μmol) were all carried out under hypoxic condition.

[0035] Figure 5 It is shown that IT-azo-RGD obtained in Example 1 releases IT-m-NH2-RGD in cells under hypoxic condition, and generates •OH and O2 ⁻ through type I electron transfer pathway, thereby enhancing the efficacy of hypoxia-selective PDT (photodynamic therapy).

[0036] Example 6, In vitro Cytotoxicity Test Human osteosarcoma cell lines 143B, Saos-2 (Wuhan Saver Biological Technology Co., Ltd.; Catalog No.: STCC00100P; Brand: Zishan Biological) and MG63 (Wuhan Saver Biological Technology Co., Ltd.; Catalog No.: STCC11401P; Brand: Zishan Biological) were seeded in 96-well plates at 5000 cells / well and pre-cultured at 37℃ for 24h: (1) normoxic light cytotoxicity evaluation: gradient concentrations of IT-azo-RGD or IT-m-NH2-RGD obtained in Example 1 were added for treatment; (2) hypoxic light cytotoxicity test: first pre-equilibrium under hypoxic condition (1% O2) for 6h, then add the compounds for treatment, and then incubate under 21% O2 condition for 12h. All groups were irradiated with 808nm laser (300mW·cm -2 ​, 10 minutes) irradiation, continue to incubate for 12h; use CCK-8 method to detect cell viability: add CCK-8 reagent to incubate for 2h, use enzyme-labeled instrument to detect absorbance at 450nm, calculate survival rate according to the following formula: Cell survival rate (%) = [(OD treatment group-OD blank group) / (OD control group-OD blank group)]x100%; At the same time, set up parallel experiments without laser irradiation to evaluate baseline cytotoxicity.

[0037] Figure 6 It is shown that IC 50 values of IT-azo-RGD obtained in Example 1 for 143B, MG63 and Saos-2 under hypoxic conditions are significantly reduced to about 1 / 10, 1 / 20 and 1 / 5 of their corresponding normoxic values, which is consistent with the results of selective cleavage of azo bond and efficient release of aniline nitrogen mustard under hypoxic environment.

[0038] Example 7, construction of osteosarcoma animal model Suspension of 143B-luc cells (3.0x10 7 / mL, prepared with PBS) is transplanted intramedullary to establish orthotopic osteosarcoma xenograft model: use a sterile 1.0mL syringe to puncture the tibial plateau to the medullary cavity, inject 30μL of cell suspension into the left tibial medullary cavity; incubate for 18 days, verify by vernier caliper measurement and bioluminescence, and wait until the tumor volume reaches the target value (about 350mm 3 ).

[0039] Example 8, in vivo near-infrared two-zone (NIR-II) blood circulation imaging and biodistribution study IT-m-NH2-RGD and IT-azo-RGD obtained in Example 1 (0.20mM, 100μL PBS) are injected into the tail vein of BALB / c nude mice (n=3) bearing 143B xenograft tumor to perform NIR-II fluorescence imaging test: under 808nm laser (100mW·cm -2 ) irradiation, real-time in vivo imaging data is collected by indium gallium arsenide (InGaAs) imaging system; after 60h of injection, main organs and tumors are removed to observe the in vivo distribution and tumor enrichment of IT dye; images are processed by Image-J software to analyze the fluorescence intensity at tumor site.

[0040] As shown in Figure 7 , although the two groups show comparable tumor fluorescence intensity within 12h after injection, IT-azo-RGD reaches peak tumor aggregation at 24h.

[0041] Example 9, evaluation of anti-tumor effect Mice bearing orthotopic osteosarcoma are randomly divided into 4 groups (n=5 for each group) for treatment evaluation.

[0042] The specific groupings are as follows: Group I (PBS): 100 μL of PBS was injected via the tail vein; Group II (PBS+L): PBS was injected via the tail vein, and 24 hours later, the tumor site was locally irradiated with an 808 nm laser (300 mW·cm). -2 Group III (IT-azo-RGD): 100 μL IT-azo-RGD solution (0.40 mM) was injected via tail vein; Group IV (IT-azo-RGD+L): 100 μL IT-azo-RGD (0.40 mM) was injected via tail vein, and 808 nm laser irradiation was performed on the tumor site 24 h later according to the parameters of Group II.

[0043] A periodic treatment regimen was adopted: the drug was administered once every 4 days, and corresponding treatment was performed 24 hours after administration; for 20 consecutive days, tumor progression and the overall physiological status of mice were assessed every 48 hours using bioluminescence imaging, tumor volume and weight measurement, and body weight monitoring; tumor volume was calculated by measuring the volume of the tumor-side limb minus the volume of the contralateral healthy limb; longitudinal bioluminescence imaging was used to supplement volume data and compare the treatment effects of each group; after 20 days of treatment, mice were sacrificed, and major organs (heart, liver, spleen, lung, and kidney) were harvested for histopathological evaluation.

[0044] The results are as follows: Figure 8 , Figure 9 , Figure 10 , Figure 11 , Figure 12 , Figure 13 , Figure 14 As shown.

[0045] Figure 8 The image shows an infrared thermogram of the tumor region under 808nm laser irradiation in Group IV. Infrared thermography confirms the efficient photothermal conversion in vivo achieved by the IT-azo-RGD obtained in Example 1. The tumor temperature in Group IV rose to 49.3°C within 10 minutes, while that in Group I was 35.1°C.

[0046] Figure 9 This is a graph showing the change in tumor volume after treatment in Example 9; Figure 10 This is a graph showing the changes in tumor weight in mice after treatment in four groups in Example 9; Figure 11 The image shows the weight changes of mice after treatment in four groups in Example 9; the IT-azo-RGD obtained in Example 1 showed a significant inhibitory effect on tumor growth. The treatment period was 20 days. During this period, the tumor volume in Group IV did not increase significantly, while the tumor volume in Group I and Group II increased the fastest. Figure 9 , Figure 10 ). In addition, the body weight of Group IV mice did not show obvious decrease during the treatment ( Figure 11 ), which indicated that the IT-azo-RGD used in Example 9 did not have obvious toxic side effects.

[0047] Figure 12 are the test images of H&E and TUNEL staining of tumor cells after treatment of the four groups in Example 9; Figure 13 are the representative images of lung metastasis and the corresponding test images of H&E staining after treatment of the four groups in Example 9; Figure 14 are the test results of H&E staining of mouse organs after treatment of the four groups in Example 9; through the histopathological staining experiment, there are extensive nuclear fragmentation, chromatin condensation and reduced Ki67 positive rate in the tumor of Group IV, especially in the hypoxic core area, which is better than that of Group III ( Figure 12 ). Lung metastasis analysis shows that ( Figure 13 ) there are multiple metastatic nodules in Group I and Group II, fewer in Group III, and no metastatic nodules are detected in Group IV. In addition, the results of H&E staining experiments on organs such as heart, liver, spleen, lung and kidney show that ( Figure 14 ) the IT-azo-RGD used in Example 9 does not show obvious toxic side effects on normal tissue organs, and shows good biological safety.

[0048] In summary, the conjugate IT-azo-RGD of the application introduces an azo bond sensitive to hypoxia as a responsive linker between the light treatment module and the chemotherapy module, and two integrin targeting peptides cRGD, which can self-assemble into nanoparticles in water and release at the tumor site, thereby realizing a triple targeting mechanism: multivalent integrin receptor targeting (through double cRGD), passive accumulation (through the EPR effect of nanospheres) and selective activation of release of nitrogen mustard chemotherapy drugs by the hypoxic tumor microenvironment ( Figure 15 ); The conjugate IT-azo-RGD of the application shows maximum absorption / emission wavelength of 852 / 891 nm in dimethyl sulfoxide (DMSO), and the fluorescence quantum yield (ΦF) is 3.12%, and 14.4% of its emission spectrum extends to the near-infrared two region (>1000 nm), which meets the requirements of deep tissue imaging; After the conjugate of the application is incubated under 1% O2 for 12 h, the corresponding chromatographic peak of the conjugate completely disappears, and the MALDI-TOF result of the intracellular residue further confirms the existence of the three expected products; The conjugate drug of the application shows enhanced therapeutic effect after drug release, the photo-thermal conversion efficiency reaches 59.7%, and hydroxyl radicals (•OH) are generated efficiently through type I photodynamic process, so that the conjugate can realize near-infrared two-zone imaging guided chemotherapy-photo-thermal-photo-dynamic combined treatment; The conjugate of the application shows obvious tumor enrichment within 24 hours after intravenous injection into orthotopic 143B osteosarcoma tumor-bearing mice, and the signal-to-noise ratio of normal tissues is as high as 5.1, and the subsequent fluorescence decay is consistent with the typical liver clearance mode, which confirms the metabolic safety. The conjugate has the dual ability of high sensitivity whole body vascular imaging and precise orthotopic osteosarcoma detection / monitoring; In the treatment of orthotopic osteosarcoma model, the conjugate realizes real-time near-infrared two-zone imaging, realizes synergistic strong tumor inhibition (inhibition rate 97.7%) through hypoxia-activated chemotherapy and enhanced photo-thermal-photo-dynamic effect, effectively inhibits lung metastasis, and no obvious systemic toxicity is detected.

[0049] These findings highlight the potential of IT-azo-RGD as a rationally designed peptide-photo-sensitizer-drug conjugate for integrated cancer diagnosis and treatment.

[0050] The above-described embodiments only express several embodiments of the application, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the patent of the application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the application, a number of modifications and improvements can be made, which are all within the protection scope of the application.

Claims

1. A hypoxia-activated peptide-photosensitizer-drug conjugate, characterized in that, It has the structure shown in Equation I: ； Formula I In Equation I, n is 1~5; Y - R1 is an anion; R1 is an aromatic conjugated compound.

2. The hypoxia-activating peptide-photosensitizer-drug conjugate according to claim 1, characterized in that, The anion is Cl. - ,Br - I - BF4 - PF6 - ClO4 - CH3COO - Any one of p-toluenesulfonate or tetraphenylborate.

3. The hypoxia-activating peptide-photosensitizer-drug conjugate according to claim 1, characterized in that, The aromatic conjugated compound has the structure of formula R. 1-1 R 1-2 R 1-3 R 1-4 Or R 1-5 Any one of them: 。 4. The hypoxia-activating peptide-photosensitizer-drug conjugate according to claim 1, characterized in that, The structure of the cRGD is as follows: 。 5. A method for preparing the hypoxia-activated peptide-photosensitizer-drug conjugate as described in any one of claims 1 to 4, characterized in that, Includes the following steps: (1) The indoleboronic acid pinacol ester compound and R1Br containing the R1 group were subjected to a Suzuki coupling reaction to obtain intermediate I; (2) The intermediate I is subjected to a nucleophilic substitution reaction with 5-chloropentyne and MY to obtain intermediate II; wherein, M in MY is Na or K; (3) Intermediate II and a bisimin-substituted chlorocyclohexadiene compound undergo a Knoevenagel reaction to obtain intermediate III; (4) The intermediate III is subjected to a nucleophilic substitution reaction with a substituted benzenethiol compound to obtain intermediate IV; (5) The intermediate IV is reacted with the azide-modified polyethylene glycol-linked cRGD compound by a Click reaction to obtain the hypoxia-activated peptide-photosensitizer-drug conjugate.

6. The method for preparing the hypoxia-activated peptide-photosensitizer-drug conjugate according to claim 5, characterized in that, In step (1), in the presence of catalyst Pd(PPh3)4 and carbonate, the indoleboronic acid pinacol ester compound and R1Br are refluxed in a solvent at 100°C for 24 h to obtain intermediate I; wherein, The molar ratio of the indoleboronic acid pinacol ester compound, R1Br, Pd(PPh3)4 and carbonate is 1:1.2:1.0:2.5; the carbonate is any one of potassium carbonate, sodium carbonate or cesium carbonate; the solvent is composed of deoxygenated 1,4-dioxane and deoxygenated water, with a volume ratio of 5:

1. After the reaction in step (1) is completed, dichloromethane is added to the reaction product for extraction, the organic layer is collected, washed with saturated NaCl solution, the organic layer is dried, concentrated, and separated by silica gel column chromatography to obtain intermediate I.

7. The method for preparing the hypoxia-activated peptide-photosensitizer-drug conjugate according to claim 5, characterized in that, In step (1), the structure of the indoleboronic acid pinacol ester compound is as follows: ； The structure of intermediate I is as follows: 。 8. The method for preparing the hypoxia-activated peptide-photosensitizer-drug conjugate according to claim 5, characterized in that, In step (2), intermediate I, 5-chloropentyne and MY are refluxed in toluene at 80°C for 20-25 h under nitrogen protection. The reactants are concentrated and then separated by column chromatography to obtain intermediate II. The molar ratio of intermediate I, 5-chloropentyne and MY is 1:1.5-3:3-5. After the reaction in step (2) is completed, the reaction product is diluted with DCM, washed with water, dried and concentrated, precipitated by adding diethyl ether, filtered, and the filter cake is washed with diethyl ether to obtain intermediate II. The intermediate II has the following structure: 。 9. The method for preparing the hypoxia-activated peptide-photosensitizer-drug conjugate according to claim 5, characterized in that, In step (3), intermediate II and the diimino-substituted chlorocyclohexadiene compound are added to a mixed solution of n-butanol and toluene in a volume ratio of 3:1, and reacted at 100°C for 5-8 hours. The reactants are concentrated and then separated by column chromatography to obtain intermediate III. The molar ratio of intermediate II to the diimino-substituted chlorocyclohexadiene compound is 2-2.5:

1. The diimine-substituted chlorocyclohexadiene compound has the following structure: ； The structure of intermediate III is as follows: 。 10. The method for preparing the hypoxia-activated peptide-photosensitizer-drug conjugate according to claim 5, characterized in that, In step (4), intermediate III, substituted benzenethiol compounds, and triethylamine are added to anhydrous DMF and stirred at room temperature for 4-6 hours. The reactants are concentrated and then separated by column chromatography to obtain intermediate IV. The molar ratio of intermediate III, substituted benzenethiol compound and triethylamine is 1:1~3:1~3; The structure of the substituted benzylthiol compound is as follows: ； The structure of intermediate IV is as follows: ； The structure of the R2 group in the substituted benzenethiol compound or intermediate IV is as follows: 。 11. The method for preparing the hypoxia-activated peptide-photosensitizer-drug conjugate according to claim 5, characterized in that, In step (5), intermediate IV, azido-modified polyethylene glycol linked cRGD, copper sulfate pentahydrate, and sodium ascorbate are added to anhydrous DMF and stirred at room temperature for 10-12 hours. The reactants are concentrated and then separated by column chromatography to obtain the hypoxia-activated peptide-photosensitizer-drug conjugate. The molar ratio of intermediate IV, azido-modified polyethylene glycol linked cRGD, copper sulfate pentahydrate, and sodium ascorbate is 1:2-3:0.3-0.5:0.4-0.

6. The azide-modified polyethylene glycol linked cRGD has the following structure: 。 12. The use of the hypoxia-activating peptide-photosensitizer-drug conjugate as described in any one of claims 1 to 4 in the preparation of a drug for the combined chemotherapy-photothermal-photodynamic therapy of osteosarcoma.

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