A kind of active oxygen responsive photosensitizer and its preparation method and application

Abstract

The present invention relates to an active oxygen responsive photosensitizer and a preparation method and application thereof, and belongs to the field of chemical technology. The present invention provides an active oxygen responsive photosensitizer, which is obtained by connecting tetraphenylporphyrin (photosensitizer) and camptothecin (chemotherapeutic drug) through an active oxygen responsive thioketal; under the activation of the Cerenkov radiation released by the radionuclide labeled tumor targeting drug, the porphyrin part of the active oxygen responsive photosensitizer can generate active oxygen for photodynamic therapy, and at the same time, the generated active oxygen can cleave the thioketal linker to release the chemotherapeutic drug camptothecin for chemotherapy, therefore, the active oxygen responsive photosensitizer has the synergistic effect of photodynamic therapy and chemotherapy, has higher cytotoxicity to tumor cells, and thus exhibits an enhanced anti-tumor effect.

Description

Technical Field

[0001] The invention relates to an active oxygen responsive photosensitizer and a preparation method and application thereof, belonging to the technical field of chemistry. Background Art

[0002] Photodynamic therapy (PDT) is a non-invasive tumor treatment method that combines photosensitizers and corresponding light sources to selectively destroy target tissues through photodynamic reactions. It has the advantages of strong tumor targeting, low dark toxicity, short treatment time, and high repeatability. Traditional photodynamic therapy usually uses an external light source as the excitation light source. However, the external light source has poor radiation penetration depth, which leads to the long-term problem of poor tissue penetration in traditional photodynamic therapy.

[0003] Cherenkov radiation (CR) produced by radionuclides is a type of electromagnetic radiation with short wavelengths produced when charged particles move faster than the speed of light in a dielectric medium. Many medical β-decay radionuclides (such as 18 F. 68 Ga, 64 Cu, 90 Y and 131 I) It can generate Cherenkov radiation. Cherenkov radiation can be used as an internal light source to replace the external light source to activate the photosensitizer in the diseased tissue to produce reactive oxygen species and then induce photodynamic therapy to solve the problem of poor tissue penetration of traditional photodynamic therapy. However, compared with the external light source, the light intensity of Cherenkov radiation is weak, and the reactive oxygen species generated by the photosensitizer are also low. Therefore, the photodynamic therapy induced by Cherenkov radiation as the excitation light source has a poor therapeutic effect on tumors, which seriously limits the clinical application of Cherenkov radiation in photodynamic therapy.

[0004] It has been reported that photodynamic therapy can be combined with other tumor treatment methods (such as chemotherapy, photothermal therapy, and radiotherapy) to improve the therapeutic effect of tumors. Among them, due to the complementarity between photodynamic therapy and chemotherapy, the combination of these two treatment methods has received widespread attention. Chemotherapy makes cancer cells more sensitive to reactive oxygen species. Photodynamic therapy can downregulate the expression of P-glycoprotein, thereby inhibiting drug efflux activity and ultimately enhancing the therapeutic effect on multidrug-resistant (MDR) cancer. So far, researchers have constructed thousands of small molecules and nanomaterials to achieve synergistic therapy with photodynamic therapy and other tumor treatment methods. In order to reduce treatment side effects and enhance tumor selectivity, these types of small molecules and nanomaterials are usually designed to respond to the tumor microenvironment (acidity, redox, and enzymes) or exogenous stimuli (light, magnetic fields, and ultrasound). Considering the characteristics of photodynamic therapy, the reactive oxygen response strategy shows significant advantages in the combination of photodynamic therapy and chemotherapy. Under light irradiation, the reactive oxygen species produced by photosensitizers not only directly kill tumor cells, but also induce the release of chemotherapeutic drugs, providing a synergistic anti-tumor effect. However, at present, the research on photochemotherapy based on reactive oxygen species response strategy is limited to external light source excitation, and its therapeutic effect is still limited by the depth of tissue penetration. There is no report on photochemotherapy based on reactive oxygen species response strategy using Cherenkov radiation as internal light source excitation. Summary of the invention

[0005] In order to solve the above problems, the present invention provides an active oxygen responsive photosensitizer, wherein the active oxygen responsive photosensitizer has the following structure:

[0006]

[0007] In one embodiment of the present invention, the R is 2,2'-(propane-2,2-diylbis(sulfanediyl))diethanol; the 2,2'-(propane-2,2-diylbis(sulfanediyl))diethanol has the following structure:

[0008]

[0009] In one embodiment of the present invention, the active oxygen responsive photosensitizer has the following structure:

[0010]

[0011] The present invention also provides a method for preparing the above-mentioned active oxygen responsive photosensitizer, the method comprising: mixing compound I and compound II and performing an esterification reaction to obtain an active oxygen responsive photosensitizer; the compound I has the following structure:

[0012]

[0013] The compound II has the structure shown below:

[0014]

[0015] In one embodiment of the present invention, the method is: compound I, compound II, EDCI (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride), DMAP (4-dimethylaminopyridine) and dichloromethane are mixed and reacted to obtain a reaction solution; the reaction solution is purified to obtain an active oxygen responsive photosensitizer.

[0016] In one embodiment of the present invention, the mixing molar ratio of compound I, compound II, DMAP and EDCI is 1:1:1.2:1.2.

[0017] In one embodiment of the present invention, the preparation method of the compound I is: pyrrole, benzaldehyde, methyl p-formylbenzoate and propionic acid are mixed and reacted to obtain a reaction solution; the reaction solution is purified to obtain an intermediate 1; the methyl ester of the intermediate 1 is removed to obtain compound I;

[0018] The intermediate 1 has the following structure:

[0019]

[0020] In one embodiment of the present invention, the preparation method of the compound II is as follows: dissolving methyl thioglycolate and acetone in methanol, dimerizing under the catalysis of sulfuric acid to obtain intermediate 2; purifying intermediate 2 after reduction with lithium aluminum hydride to obtain intermediate 3; dissolving triphosgene in dichloromethane to obtain a dissolving solution; adding camptothecin and DMAP (4-dimethylaminopyridine) to dichloromethane to obtain a suspension solution; dropping the dissolving solution into the suspension solution to obtain a mixed solution; reacting the mixed solution and intermediate 3 after mixing to obtain a reaction solution; purifying the reaction solution to obtain compound II;

[0021] The intermediate 2 has the following structure:

[0022]

[0023] The intermediate 3 has the following structure:

[0024]

[0025] The present invention also provides the use of the above active oxygen responsive photosensitizer in the preparation of tumor therapeutic drugs.

[0026] The present invention also provides a tumor therapeutic drug, which contains the above-mentioned active oxygen responsive photosensitizer.

[0027] In one embodiment of the present invention, the tumor therapeutic drug further contains a tumor targeting drug labeled with a radionuclide.

[0028] In one embodiment of the present invention, the radioactive nuclide is a beta decay nuclide; the beta decay nuclide is fluorine-18 ( 18 F), Gallium-68 ( 68 Ga), copper-64( 64 Cu), Yttrium-90( 90 Y) or iodine-131( 131 I).

[0029] In one embodiment of the present invention, the tumor targeting drug labeled with β-decay nuclide is [ 68 Ga]Ga-NOTA-Nb109, [ 68 Ga]Ga-BSA, [ 68 Ga]Ga-NOTA-FAPI-4,[ 18 F]F-FDG, [ 64 Cu]Cu-DOTA-VEGF, SIR-Spheres Y-90 Resin microspheres and / or [ 131 I]-MIBG.

[0030] The technical solution of the present invention has the following advantages:

[0031] The invention provides an active oxygen responsive photosensitizer, which is obtained by connecting tetraphenylporphyrin (photosensitizer) and camptothecin (chemotherapeutic drug) through an active oxygen responsive thioketal. Under the activation of Cerenkov radiation released by a radionuclide labeled tumor targeting drug, the porphyrin part of the active oxygen responsive photosensitizer can generate active oxygen for photodynamic therapy, and at the same time, the generated active oxygen can cleave the thioketal linker to release the chemotherapeutic drug camptothecin for chemotherapy. Therefore, the active oxygen responsive photosensitizer has a synergistic effect of photodynamic therapy and chemotherapy, has higher cytotoxicity to tumor cells, and thus exhibits an enhanced anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : ESI-MS characterization of compound Ⅰ.

[0033] Figure 2 : ESI-MS characterization of intermediate 2.

[0034] Figure 3 : ESI-MS characterization of intermediate 3.

[0035] Figure 4: ESI-MS characterization of compound Ⅱ.

[0036] Figure 5 : ESI-MS characterization of photosensitizer T1.

[0037] Figure 6 : H NMR spectrum characterization of photosensitizer T1.

[0038] Figure 7 : C NMR spectrum characterization of photosensitizer T1.

[0039] Figure 8 : ESI-MS characterization of compound III.

[0040] Fig. 9 : ESI-MS characterization of photosensitizer T2.

[0041] Fig.10 : H NMR spectrum characterization of photosensitizer T2.

[0042] Fig.11 : C NMR spectrum characterization of photosensitizer T2.

[0043] Fig.12 : Characterization results of compound Ⅰ, photosensitizer T1 and photosensitizer T2. Fig.12 In the figure, A, C and D are the UV absorption spectra in DMSO, water and BSA, respectively, and B is the fluorescence emission spectrum in DMSO.

[0044] Fig.13 : Detection diagram of reactive oxygen species generation by photosensitizer T1 and photosensitizer T2 in DMSO. Fig.13 In the figure, A is the dose relationship diagram, and B is the photosensitizer concentration relationship diagram.

[0045] Fig.14 : Detection diagram of reactive oxygen species generation by photosensitizer T1 and photosensitizer T2 in BSA. Fig.14 In the figure, A is the dose relationship diagram, and B is the photosensitizer concentration relationship diagram.

[0046] Fig.15 : Cerenkov imaging results of photosensitizer T1 and photosensitizer T2. Fig.15 In the figure, A is the Cerenkov imaging result in DMSO, and B is the Cerenkov imaging result in BSA.

[0047] Fig.16 : Quantification of fluorescence emission of photosensitizer T1 and photosensitizer T2 induced by radionuclides. Fig.16 In the figure, A is the radiation value in DMSO and B is the radiation value in BSA.

[0048] Fig.17: HPLC phase diagram of photosensitizer T1 and photosensitizer T2 under the action of radionuclide in DMSO.

[0049] Fig.18 : HPLC graphs of photosensitizer T1 and photosensitizer T2 under the action of radionuclides in BSA.

[0050] Fig.19 : Liquid chromatography-mass spectrometry analysis of the decomposition of photosensitizer T1 after exposure to radionuclides.

[0051] Fig. 20 : Results of cellular uptake analysis of photosensitizer T1 and photosensitizer T2 in A375-hPD-L1 cells. Fig. 20 In the figure, A is the bright field and fluorescence images, and B is the fluorescence quantitative analysis.

[0052] Fig.21 : Photosensitizer T1 and photosensitizer T2 are respectively 68 Cell viability after co-incubation of A375-hPD-L1 cells with Ga]Ga-NOTA-Nb109.

[0053] Fig. 22 : Photosensitizer T1 and [ 68 Fluorescence microscopy images of A375-hPD-L1 cells stained with PI after co-incubation of Ga]Ga-NOTA-Nb109 and A375-hPD-L1 cells. DETAILED DESCRIPTION

[0054] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0055] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0056] Triphosgene, methylthioglycolate, benzaldehyde, pyrrole, 4-dimethylaminopyridine (DMAP), methyl 4-formylbenzoate, 1,3-diphenylisobenzofuran (DPBF), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI) and (S)-(+)-camptothecin (CPT) involved in the following examples were purchased from Aladdin Co., Ltd. (Shanghai, China); the electrospray ionization mass spectrometry (ESI-MS) involved in the following examples was analyzed on a quadrupole tandem mass spectrometer ZMD4000 LC or MS (Waters), and the high performance liquid chromatography (HPLC) was detected by a pump (waters) with a C18 chromatographic column (250×4.6 mm, 10 cm, Phenomenex) and equipped with a dual-wavelength absorbance UV detector, and the NMR spectrum was analyzed on a Bruker 400MHz NMR spectrometer (Bruker).

[0057] Example 1: An active oxygen responsive photosensitizer T1

[0058] This embodiment provides an active oxygen responsive photosensitizer T1, and the active oxygen responsive photosensitizer T1 has the following structure:

[0059]

[0060] Example 2: A method for preparing active oxygen responsive photosensitizer T1

[0061] This example provides a method for preparing the active oxygen responsive photosensitizer T1 described in Example 1, and the specific steps are as follows:

[0062] Step 1: dissolving pyrrole (9.2 mmol) in propionic acid (40 mL) to obtain a dissolving solution; dissolving methyl p-formylbenzoate (2.3 mmol) and benzaldehyde (6.9 mmol) in propionic acid (10 mL) to obtain a mixed solution; dropping the dissolving solution into the mixed solution, stirring at 100° C. and 150 rpm for 0.5 h to obtain a reaction solution; first evaporating the solvent from the reaction solution by a rotary evaporator, then dissolving it in dichloromethane (100 mL), and then washing it with a saturated sodium bicarbonate solution (3×100 mL) and saturated brine (100 mL), respectively; taking the organic phase, adding anhydrous sodium sulfate (35 mmol) and concentrating it by rotary evaporation to obtain a crude product; purifying the crude product by an alumina column to obtain an intermediate 1;

[0063] Step 2: Dissolve the intermediate 1 in tetrahydrofuran (20 mL) to obtain a solution; add an aqueous sodium hydroxide solution (20 mL, concentration 2 mol / L) to the solution, and stir at 70°C, 150 rpm for 16 h to obtain a reaction solution; after the reactant is cooled to room temperature (25°C), the reaction solution is first acidified to pH = 1 with an aqueous hydrochloric acid solution with a concentration of 2.88 mol / L, and then the organic solvent is removed by rotary evaporation, and then diluted with water (40 mL), and finally extracted with dichloromethane (4×60 mL); the organic phase is first washed with water (100 mL), then dried by adding anhydrous sodium sulfate, and then concentrated to a small volume (3 mL) by rotary evaporation, and finally cold (0°C) n-hexane (50 mL) is added for precipitation to obtain a purple compound I (80 mg, yield 62%, C 45 The ESI-MS calculated value of H30N4O2 is 658.23, and the actual value is [M+H] + 659.31);

[0064] Step 3: Acetone (0.02 mol) and methyl thioglycolate (0.04 mol) were dissolved in methanol (5 mL) to obtain a solution; 98% (g / 100 mL) sulfuric acid (1.84 mL) was added dropwise to the solution, and the mixture was stirred at room temperature (25° C.) and 150 rpm for 48 h to obtain a reaction solution; chloroform (70 mL) was added to the reaction solution, and the mixture was washed with a saturated sodium bicarbonate solution (3×60 mL) and saturated brine (60 mL); anhydrous sodium sulfate (35 mmol) was added to the organic phase, and the mixture was concentrated by rotary evaporation to obtain a crude product; the crude product was purified by a silica gel column to obtain a transparent liquid intermediate 2 (3.207 g, yield 63%, C9H 16 The ESI-MS calculated value of O4S2 is 252.04, and the actual value is [M+Na] + 275.17);

[0065] Step 4: Dissolve the intermediate 2 (0.012 mol) in anhydrous tetrahydrofuran (50 mL) under nitrogen protection to obtain a solution; add lithium aluminum hydride (0.039 mmol) to tetrahydrofuran (50 mL) to obtain a suspension solution; place the solution in an ice bath, add the suspension solution within half an hour under stirring at 150 rpm, and stir at room temperature (25° C.) and 150 rpm for 1 hour to obtain a reaction solution; quench the reaction solution with a 15% NaOH aqueous solution (10 mL), then remove tetrahydrofuran by rotary evaporation, dissolve in dichloromethane (50 mL), and finally wash with saturated brine (50 mL); take the organic phase, add anhydrous sodium sulfate (35 mmol), and evaporate and concentrate it by rotary evaporator to obtain a crude product; purify the crude product by silica gel column to obtain intermediate 3 (1.369 g, yield 55%, C7H 16The ESI-MS calculated value of O2S2 is 196.05, and the actual value is [M+Na] + 219.17);

[0066] Step 5: Dissolve triphosgene (0.14 mmol) in dichloromethane (3 mL) to obtain a solution; add (S)-(+)-camptothecin (0.24 mmol) and DMAP (0.96 mmol) to dichloromethane (10 mL) to obtain a suspension solution; add the solution dropwise to the suspension solution, and stir at room temperature (25°C) and 150 rpm for 0.5 h under nitrogen protection to obtain a reaction solution A; add an excess of intermediate 3 (0.72 mmol) to the reaction solution A, and stir at room temperature (25°C) and 150 rpm for 16 h to obtain a reaction solution B; dilute the reaction solution B with dichloromethane (50 mL), and wash it twice with a saturated ammonium chloride solution (50 mL) and a saturated brine (50 mL), respectively; take the organic phase, add anhydrous sodium sulfate (35 mmol), and evaporate and concentrate it by a rotary evaporator to obtain a crude product; purify the crude product by a silica gel column to obtain a yellow compound II (0.1 g, yield 73%, C 28 H 30 The ESI-MS calculated value of N2O7S2 is 570.14, the actual value is [M+Na]+593.24);

[0067] Step 6: Compound I (0.044 mmol), compound II (0.044 mmol), EDCI (0.053 mmol) and DMAP (0.053 mmol) were dissolved in anhydrous dichloromethane (10 mL), and stirred at room temperature (25°C) and 150 rpm for 16 h to obtain a reaction solution; the reaction solution was diluted with dichloromethane (50 mL), and then washed with saturated ammonium chloride solution (60 mL), saturated sodium bicarbonate solution (60 mL) and water (60 mL), respectively; anhydrous sodium sulfate (35 mmol) was added to the organic phase, and then evaporated and concentrated by rotary evaporation to obtain a crude product; the crude product was purified by silica gel column to obtain a purple active oxygen responsive photosensitizer T1 (38 mg, yield 78%, C 73 H 58 The ESI-MS calculated value of N6O8S2 is 1210.37, the actual value is [M+H] + 1211.51).

[0068] Compound Ⅰ, intermediate 2, intermediate 3, compound Ⅱ and active oxygen responsive photosensitizer T1 were analyzed by ESI-MS using an electrospray ionization source, and active oxygen responsive photosensitizer T1 was detected by HPLC using Waters 1525. The analysis and detection results are shown in Figures 1 to 7 .

[0069] The data results of hydrogen spectrum and carbon spectrum of active oxygen responsive photosensitizer T1 are as follows:

[0070] Hydrogen spectrum analysis: 1 H NMR (400MHz, CDCl3) δ8.87(m,J=8.3Hz,6H),8.78(d,J=4.8Hz,2H),8.52–8.40(d,,J=8Hz,2H),8.37–8.28(d,J=8Hz,2H),8.24 (m,6H),8.18(d,J=8.5Hz,1H),8.09(s,1H),7.87–7.68(m,11H),7.55(m,1H),7.34(s,1H),5.72(d,J=17.2Hz,1H),5.39(d,J= 17.2Hz,1H),5.16(d,J=18.9Hz,1H),5.01(d,J=18.8Hz,1H),4.65(t,J=6.8Hz,2H),4.43–4.28(m,2H),3.23–3.10(t,J=6.8Hz ,2H),3.02(t,J=7.2Hz,2H),2.26–2.18(s,1H),2.13–2.04(s,1H),1.69(d,J=2.8Hz,6H),0.92(t,J=7.5Hz,3H),-2.80(s,2H).

[0071] Carbon spectrum analysis: 13 C NMR (151MHz, CDCl3) δ167.2,166.5,157.2,153.5,152.2,148.8,147.2,146.4,145.6,142.1,134.6,130.9,130.6,129.6,129.4,12 8.3,128.0,127.9,127.8,126.7,120.6,120.4,118.5,96.0,77.9,67.7,67.1,64.1,56.7,49.8,31.9,31.1,29.7,29.4,28.9,7.5.

[0072] Comparative Example 1: A photosensitizer T2 without active oxygen response

[0073] This comparative example provides a non-active oxygen responsive photosensitizer T2, wherein the active oxygen responsive photosensitizer T2 has the following structure:

[0074]

[0075] Comparative Example 2: A method for preparing a non-active oxygen responsive photosensitizer T2

[0076] This comparative example provides a method for preparing the non-active oxygen responsive photosensitizer T2 described in comparative example 1, and the specific steps are as follows:

[0077] Step 1: dissolving triphosgene (0.14 mmol) in dichloromethane (3 mL) to obtain a solution; adding (S)-(+)-camptothecin (0.24 mmol) and DMAP (0.96 mmol) to dichloromethane (10 mL) to obtain a suspension solution; dropping the solution into the suspension solution, stirring at room temperature (25° C.) and 150 rpm for 0.5 h under nitrogen protection to obtain a reaction solution A; adding an excess of hexylene glycol (0.72 mmol) to the reaction solution A, stirring at room temperature (25° C.) and 150 rpm for 16 h to obtain a reaction solution B; diluting the reaction solution B with dichloromethane (50 mL), respectively, and washing twice with a saturated ammonium chloride solution (50 mL) and a saturated brine (50 mL); taking the organic phase, adding anhydrous sodium sulfate (35 mmol) and concentrating it by rotary evaporation to obtain a crude product; the crude product was purified by a silica gel column to obtain a yellow compound III (95 mg, yield 81%, C 27 H 28 The ESI-MS calculated value of N2O7 is 492.18, and the actual value is [M+Na] + 515.39);

[0078] Step 6: Compound I (0.032 mmol), compound III (0.032 mmol), EDCI (0.038 mmol) and DMAP (0.038 mmol) were dissolved in anhydrous dichloromethane (10 mL), and stirred at room temperature (25° C.) and 150 rpm for 16 h to obtain a reaction solution; the reaction solution was diluted with dichloromethane (50 mL), and then washed with saturated ammonium chloride solution (60 mL), saturated sodium bicarbonate solution (60 mL) and water (60 mL), respectively; anhydrous sodium sulfate (35 mmol) was added to the organic phase, and then evaporated and concentrated by rotary evaporation to obtain a crude product; the crude product was purified by silica gel column to obtain a purple non-active oxygen responsive photosensitizer T2 (29.7 mg, yield 81%, C 72 H 56 The ESI-MS calculated value of N6O6 is 1132.41, and the actual value is [M+H] + 1133.45).

[0079] Compound III and the non-reactive oxygen responsive photosensitizer T2 were analyzed by ESI-MS using an electrospray ionization source, and the non-reactive oxygen responsive photosensitizer T2 was detected by HPLC using Waters 1525. The analysis and detection results are shown in Figures 8 to 11 .

[0080] The data results of the hydrogen spectrum and carbon spectrum of the non-reactive oxygen responsive photosensitizer T2 are as follows:

[0081] Hydrogen spectrum analysis: 1 H NMR (400MHz, CDCl3) δ8.88(d,J=4.3Hz,6H),8.81(d,J=4.8Hz,2H),8.45–8.41(d,J=8Hz,2H),8.32–8.29(m,2H),8 .27–8.23(m,6H),8.22–8.18(d,J=9.2Hz,1H),8.16(s,1H),7.83–7.73(m,11H),7.59–7.52(m,1H),7.37(s,1H),5 .73(d,J=17.2Hz,1H),5.41(d,J=17.2Hz,1H),5.26–5.08(m,2H),4.47(t,J=6.6,1.4Hz,2H),4.22(m,2H),2.38–2 .25(m,1H),2.15(m,1H),1.97–1.88(m,2H),1.82(m,2H),1.31–1.27(m,4H),1.01(t,J=7.5Hz,3H),-2.76(s,2H).

[0082] Carbon spectrum analysis: 13 C NMR (100MHz, CDCl3) δ166.4,165.8,156.3,152.8,151.3,147.8,145.9,145.3,144.8,141.1,133.5,130.0,129.6,128.8,128.5,127.3 ,127.0,126.9,126.8,126.8,125.7,119.5,119.3,117.6,95.0,76.7,68.0,66.1,64.2,48.8,31.0,28.7,27.7,27.5,24.8,24.4,6.6.

[0083] Experimental Example 1: Spectral properties of photosensitizers

[0084] This experimental example provides an experiment on the spectral properties of photosensitizers. The specific process is as follows:

[0085] The active oxygen responsive photosensitizer T1 prepared in Example 2 and the non-active oxygen responsive photosensitizer T2 prepared in Comparative Example 2 were respectively prepared into a solution with a concentration of 2 mM using DMSO (dimethyl sulfoxide), water and water containing 10% (v / v) bovine serum albumin; the ultraviolet absorption spectrum of the solution was measured using a Lambda-25 spectrometer (PerkinElmer) at room temperature (25°C) and a scanning wavelength range of 300 to 800 nm, and the fluorescence spectrum of the solution was measured using a Hitachi F-4600 (HITACHI) under the conditions of a recording range of 600 to 800 nm and an excitation wavelength of 420 nm. The measurement results are shown in FIG. Fig.12 .

[0086] like Fig.12 As shown in Figure A, T1 and T2 show typical absorbance spectra of TPP in DMSO, with a strong and sharp band (Soret band) at about 415 nm and four Q bands (515, 550, 593 and 647 nm). A moderate absorption peak at about 370 nm was observed in the absorbance spectra of T1 and T2, which is the absorption peak of the chemotherapy drug camptothecin. When excited with a 415 nm light source, both compounds showed fluorescence emission at 660 nm in DMSO, which is a typical fluorescence emission of tetraphenylporphyrin ( Fig.12 B). In water, the UV-visible absorption peaks of T1 and T2 become weaker and broader ( Fig.12 C), and at the same time, their fluorescence emission was significantly reduced ( Fig.12 D), which is mainly due to aggregation in aqueous media. Aggregation of photosensitizers will limit their ability to generate reactive oxygen species, while proteins can reduce the aggregation tendency of photosensitizers in aqueous media and restore their ability to generate reactive oxygen species. The absorption peak at 420nm becomes obvious in 10% BSA ( Fig.12 C), and their fluorescence intensity also increased significantly ( Fig.12 D), demonstrating that the protein can dissociate aggregates.

[0087] Experimental Example 2: Determination of active oxygen species induced by photosensitizer under Cherenkov radiation

[0088] This experimental example provides an experiment for measuring active oxygen species induced by photosensitizer under Cherenkov radiation. The specific process is as follows:

[0089] The active oxygen responsive photosensitizer T1 prepared in Example 2 was prepared into solutions with concentrations of 0.625, 1.25, 2.5, 5 and 10 μM respectively using DMSO (dimethyl sulfoxide) and water containing 10% (v / v) bovine serum albumin; DPBF (14 μL, 10 mmol, active oxygen indicator) was added to the solutions to obtain mixed solutions; the mixed solution without adding active oxygen responsive photosensitizer T1 was used as a control, and different radiation doses of [ 68 After adding Ga]GaCl3 (0.462, 0.925, 1.85 and 3.7 MBq), the mixture was shaken at room temperature (25°C) for 16 h, and then the fluorescence of DPBF was measured on SpectraMax M5 (λex=410 nm, λem=480 nm). The measurement results are shown in Figures 13-14 The same method was used to detect the non-active oxygen responsive photosensitizer T2 prepared in Comparative Example 2. The test results are shown in Figures 13-14 .

[0090] Compared with the control, the 68 The DPBF fluorescence in the mixed solution of Ga]GaCl3 was significantly reduced ( Fig.13 A and Fig.14 A). In addition, the fluorescence intensity of DPBF in the mixed solution depends on [ 68 The radioactive dose of Ga]GaCl3. 68 In the presence of Ga]GaCl3 (3.7MBq), the generation of active oxygen species by T1 or T2 also showed concentration dependence ( Fig.13 B and Fig.14 B). These results verify that T1 or T2 can generate reactive oxygen species under Cherenkov radiation activation, which provides a basis for studying the release of camptothecin activated by Cherenkov radiation.

[0091] Experimental Example 3: Fluorescence measurement experiment of photosensitizer induced by Cherenkov radiation

[0092] This experimental example provides a fluorescence measurement experiment of a photosensitizer induced by Cherenkov radiation. The specific process is as follows:

[0093] The active oxygen responsive photosensitizer T1 prepared in Example 2 was prepared into a solution with a concentration of 2.5 μM using DMSO (dimethyl sulfoxide) and water containing 10% (v / v) bovine serum albumin; a solution without active oxygen responsive photosensitizer T1 was used as a control, and [ 68After adding Ga]GaCl3 (7.4MBq), the samples were imaged using Cerenkov mode on an IVIS spectral imaging system (PerkinElmer, USA). Images with different emission wavelengths were obtained using no filter or selected filters (640-720nm), and the radiance of each sample was calculated from the region of interest (ROI) using dynamic imaging software. The imaging results are shown in Figures 15-16 The same method was used to detect the non-active oxygen responsive photosensitizer T2 prepared in Comparative Example 2. The test results are shown in Figures 15-16 .

[0094] In DMSO, the three samples showed similar Cerenkov radiation intensities in the unfiltered mode ( Fig.15 A and Fig.16 A). When the filter (>640nm) was applied, the fluorescence intensity of all three samples decreased. However, the samples with T1 or T2 showed brighter fluorescence than the control, verifying that the Cherenkov radiation activated the TPP derivatives to emit fluorescence. The same phenomenon was also observed in 10% BSA ( Fig.15 B and Fig.16 B).

[0095] Experimental Example 4: Drug release experiment of photosensitizer induced by Cherenkov radiation

[0096] This experimental example provides a drug release experiment of photosensitizer induced by Cherenkov radiation. The specific process is as follows:

[0097] The active oxygen responsive photosensitizer T1 prepared in Example 2 was prepared into a solution with a concentration of 2.5 μM using DMSO (dimethyl sulfoxide) and water containing 10% (v / v) bovine serum albumin; a solution without active oxygen responsive photosensitizer T1 was used as a control, and [ 68 Ga]GaCl3 (3.7MBq) to obtain a mixed solution; a mixed solution without vitamin C was used as a control, vitamin C (VC, 2mg) was added to the mixed solution to remove active oxygen, and then shaken at room temperature (25°C) for 16h, and then analyzed by HPLC using Waters1525 (for the mixed solution using 10% BSA, 200μL of ethanol was first added to precipitate the protein, then vortexed for 5 minutes, centrifuged, and finally the supernatant was collected for HPLC analysis), and the eluate after 6.17 minutes was collected and analyzed using ESI-MS. The analysis results are shown in Figures 17 to 19 The same method was used to detect the non-active oxygen responsive photosensitizer T2 prepared in Comparative Example 2. The test results are shown in Figures 17 to 19 .

[0098] To evaluate whether T1 releases camptothecin upon Cerenkov irradiation, T1 (2.5 μM) was mixed with [68 Ga]GaCl3 (3.7 MBq) and the samples were monitored by HPLC. 68 After incubation with Ga]GaCl3, a peak appeared at 6.17 minutes, which was confirmed by ESI-MS to be camptothecin ( Fig.17 A and Fig.19 ). Under the same conditions, the non-ROS-responsive photosensitizer T2 did not release camptothecin ( Fig.17 B) To verify whether the release of camptothecin is induced by ROS, vitamin C, an ROS scavenger, was added. Fig.17 As shown in A, no obvious peak was detected at 6.17 min in the presence of VC. In fact, no decomposition products were observed after co-incubation with vitamin C. In 10% BSA, Cerenkov radiation could also induce T1 to release camptothecin, while T2 still could not release camptothecin ( Fig.18 ). These results indicate that T1 can generate reactive oxygen species under Cerenkov induction and then release camptothecin.

[0099] Experimental Example 5: Cellular uptake experiment of photosensitizer

[0100] This experimental example provides a photosensitizer cell uptake experiment. The specific process is as follows:

[0101] Experiment 1: A375-hPD-L1 cells (provided by Suzhou Zhihe Biopharmaceutical Technology Co., Ltd.) were cultured at 1×10 4 The cells were inoculated into a 96-well plate (3603, Corning) with an inoculum of 100 μL of DMEM medium (purchased from BI) containing 10% (v / v) fetal bovine serum (FBS) and cultured in an incubator at 37°C and 5% (v / v) CO2 for 16 h; after 16 h, the reactive oxygen species responsive photosensitizer T1 prepared in Example 2 was added to the 96-well plate to a final concentration of 0.25 and 0.5 μM, respectively; the mixed system of A375-hPD-L1 cells and reactive oxygen species responsive photosensitizer T1 at different concentrations was incubated in an incubator at 37°C and 5% (v / v) CO2 for 24 h; after 24 h, the 96-well plate was first washed with PBS buffer (pH 7.4, 0.01 M), and then the fluorescence intensity of each well in the 96-well plate was measured on a SpectraMax M5 (λex=415 nm, λem=660 nm), and the measurement results are shown in Fig. 20 The same method was used to detect the non-active oxygen responsive photosensitizer T2 prepared in Comparative Example 2. The test results are shown in Fig. 20 .

[0102] Experiment 2: A375-hPD-L1 cells (provided by Suzhou Zhihe Biopharmaceutical Technology Co., Ltd.) were cultured at 2×10 5The cells were inoculated into a glass-bottomed cell culture dish (bottom diameter 2 cm) and cultured in a 37°C, 5% (v / v) CO2 incubator using 3 mL of DMEM medium (purchased from BI) containing 10% (v / v) fetal bovine serum (FBS) for 16 h; after 16 h, the reactive oxygen species responsive photosensitizer T1 prepared in Example 2 was added to the culture solution to a final concentration of 0.5 μM; the mixed system of A375-hPD-L1 cells and the reactive oxygen species responsive photosensitizer T1 was incubated in a 37°C, 5% (v / v) CO2 incubator for 24 h; after 24 h, the 96-well plate was first washed with PBS buffer (pH 7.4, 0.01 M), and then the 96-well plate was observed under Olympus xcellence (Japan), and the observation results are shown in FIG. Fig. 20 The same method was used to detect the non-active oxygen responsive photosensitizer T2 prepared in Comparative Example 2. The test results are shown in Fig. 20 .

[0103] Obvious red fluorescence was observed in cells after incubation with T1 or T2 (0.25 μM or 0.5 μM) for 24 h, indicating that both compounds could be internalized by cells ( Fig. 20 A). Intracellular fluorescence quantitative analysis showed that the cellular uptake of T1 and T2 was concentration-dependent, and the A375-hPD-L1 cell line had similar uptake of them ( Fig. 20 B).

[0104] Experimental Example 6: Cytotoxicity experiment of photosensitizer induced by Cherenkov radiation

[0105] This experimental example provides a cytotoxicity experiment of photosensitizer induced by Cherenkov radiation. The specific process is as follows:

[0106] A375-hPD-L1 cells (provided by Suzhou Zhihe Biopharmaceutical Technology Co., Ltd.) were inoculated into a 96-well plate (3603, Corning) at a seeding capacity of 6000 cells per well, and 100 μL of DMEM medium containing 10% (v / v) fetal bovine serum (FBS) (purchased from BI Company) was used to culture in an incubator at 37°C and 5% (v / v) CO2 for 24 hours, and the supernatant was discarded to obtain the cells to be incubated; using DMEM medium without active oxygen responsive photosensitizer T1 as a control, 100 μL of DMEM medium containing 0.5 μM active oxygen responsive photosensitizer T1 was first mixed with the cells to be incubated, and incubated in an incubator at 37°C and 5% (v / v) CO2 for 0.5 hours, and then 100 μL of DMEM medium containing [ 68 Ga]Ga-NOTA-Nb109 (5.55MBq and 11.1MBq) ([ 68The Ga]Ga-NOTA-Nb109 labeling method can be found in the literature: G.Lv, X.Sun, L.Qiu, Y.Sun, K.Li, Q.Liu, Q.Zhao, S.Qin, J.Lin, PET Imaging ofTumor PD-L1 Expression with a Highly Specific Nonblocking Single-DomainAntibody, J.Nucl.Med., 61(2020)117-122.) DMEM medium was mixed with the cells to be incubated, and the cells were incubated in an incubator at 37°C and 5% (v / v) CO2 for 24 hours to obtain an incubation solution; 10 μL of MTT (purchased from Shanghai Biotechnology Co., Ltd., concentration of 5 mg / mL) was cultured in an incubator at 37°C and 5% (v / v) CO2 for 4 h, then the supernatant was discarded, DMSO (150 μL) was added, and shaking was performed for 10 min. Finally, the absorbance at 490 nm was measured using a microplate counter to determine the absorbance value of each well, and the cell survival rate of each group was calculated based on the relative absorbance value with the control group. The calculation results are shown in Fig.21 The same method was used to detect the non-active oxygen responsive photosensitizer T2 prepared in Comparative Example 2. The test results are shown in Fig.21 .

[0107] The calculation formula of cell survival rate is: cell survival rate = [(absorption intensity of experimental well-absorption intensity of blank well) / (absorption intensity of control well-absorption intensity of blank well)] × 100%.

[0108] When the dose is lower than 11.1MBq, [ 68 The cytotoxicity of Ga]Ga-NOTA-Nb109 to A375-hPD-L1 cells (>90% cell viability) was negligible ( Fig.21 In addition, in the absence of Cerenkov radiation excitation, 0.25mM or 0.5mM of T1 and T2 were almost non-cytotoxic (>90% cell viability) ( Fig.21 ). In contrast, when cells were treated with T1 or T2 and [ 68 When co-incubated with Ga]Ga-Nb109, the cell viability decreased significantly ( Fig.21 ). At a radiation dose of 5.55 MBq, T1 at a concentration of 0.25 μM or 0.5 μM reduced cell viability by 18% and 39%, respectively. A higher radiation dose (11.1 MBq) had more significant cytotoxicity. T2 showed lower cytotoxicity compared to T1. For example, T2 only showed a negative correlation with the 11.1 MBq [ 68Ga]Ga-Nb109 killed 34% of the cells under co-incubation, while T1 killed 56% of the cells under the same conditions. Under the activation of Cherenkov radiation emission, T1 can spontaneously produce reactive oxygen species and then release camptothecin through reactive oxygen-induced thioketal cleavage, and kill tumor cells through photodynamic therapy and chemotherapy combined therapy, while T2 cannot release camptothecin due to the lack of reactive oxygen responsive linker. Therefore, the anti-tumor effect of T1 comes from Cherenkov-induced photodynamic therapy and chemotherapy, but T2 only exhibits Cherenkov-induced photodynamic therapy.

[0109] Experimental Example 7: Propidium iodide staining experiment of cells induced by photosensitizer under Cerenkov radiation

[0110] This experimental example provides a cell propidium iodide staining experiment induced by photosensitizer Cerenkov radiation. The specific process is as follows:

[0111] A375-hPD-L1 cells (provided by Suzhou Zhihe Biopharmaceutical Technology Co., Ltd.) were inoculated into a 96-well plate (3603, Corning) at a seeding capacity of 6000 cells per well, and cultured in a 37°C, 5% (v / v) CO2 incubator for 24 h using 100 μL of DMEM medium (purchased from BI) containing 10% (v / v) fetal bovine serum (FBS), and the supernatant was discarded to obtain the cells to be incubated; the cells were incubated without the active oxygen responsive photosensitizer T1 and [ 68 Ga]Ga-NOTA-Nb109([ 68 The labeling method of Ga]Ga-NOTA-Nb109 can be found in the literature: G.Lv, X.Sun, L.Qiu, Y.Sun, K.Li, Q.Liu, Q.Zhao, S.Qin, J.Lin, PET Imaging ofTumor PD-L1 Expression with a Highly Specific Nonblocking Single-DomainAntibody, J.Nucl.Med., 61(2020)117-122.) DMEM medium without active oxygen responsive photosensitizer T1 and DMEM medium without [ 68 The DMEM medium of Ga]Ga-Nb109 was used as the control, and 100 μL of the DMEM medium containing the active oxygen responsive photosensitizer T1 (0.5 μM) and [ 68The DMEM medium containing 11.1 MBq of Ga]Ga-NOTA-Nb109 was mixed with the cells to be incubated, and incubated in an incubator at 37°C and 5% (v / v) CO2 for 24 h to obtain an incubation solution. After adding PI (10 μL, purchased from Bio-Time Corporation) to the incubation solution, the solution was first incubated in an incubator at 37°C and 5% (v / v) CO2 for 0.5 h, and then the cells were rinsed with PBS buffer (pH 7.4, 0.01 M), and the fluorescence of PI was observed under a fluorescence microscope. The results are shown in FIG. Fig. 22 .

[0112] PI is a live cell impermeable dye that can only stain dead cells, not live cells. Fig. 22 As shown, when adding T1 or [ 68 No obvious red fluorescence of PI was detected in cells co-incubated with T1 and [Ga]Ga-Nb109. 68 Obvious red fluorescence was observed in cells co-incubated with Ga]Ga-Nb109, indicating that the cells were effectively killed under this condition. All the results showed that the anti-tumor effect of Cerenkov radiation-induced photodynamic therapy and chemotherapy was more effective than that of Cerenkov radiation-induced photodynamic therapy alone.

[0113] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. An active oxygen responsive photosensitizer, characterized in that: The active oxygen responsive photosensitizer has the following structure:

2. A method for preparing the active oxygen responsive photosensitizer according to claim 1, characterized in that: The method comprises: mixing compound I and compound II and performing an esterification reaction to obtain an active oxygen responsive photosensitizer; the compound I has the structure shown below: The compound II has the structure shown below:

3. The method according to claim 2, characterized in that The method comprises the following steps: mixing compound I, compound II, EDCI, DMAP and dichloromethane and reacting the mixture to obtain a reaction solution; and purifying the reaction solution to obtain an active oxygen responsive photosensitizer.

4. The method according to claim 3, characterized in that The mixing molar ratio of the compound I, compound II, DMAP and EDCI is 1:1:1.2:1.

2.

5. Use of the active oxygen responsive photosensitizer according to claim 1 in the preparation of tumor therapeutic drugs.

6. A tumor treatment drug, characterized in that: The tumor therapeutic drug contains the active oxygen responsive photosensitizer according to claim 1.

7. The tumor treatment drug according to claim 6, characterized in that: The tumor therapeutic drug also contains a tumor targeting drug labeled with a radionuclide.

8. The tumor treatment drug according to claim 7, characterized in that: The radioactive nuclide is fluorine-18, gallium-68, copper-64, yttrium-90 or iodine-131.

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