Photosensitive carrier, method for preparing the same and use thereof

Abstract

The application discloses a photosensitive carrier and a preparation method and application thereof, and belongs to the field of nanomaterials.The application relates to a glutathione-activated nano-immunopreparation compound with targeting property, and a structural general formula of the compound is shown as formula I.The photosensitive carrier is prepared by wrapping a TLR7 agonist in the nano-immunopreparation compound, so that the photosensitizer and the immunodrug activity are inhibited, the tumor-related antigens released after the photodynamic killing are combined with the activated immune checkpoint inhibitors to jointly induce a synergistic antitumor immune response, so that the growth of in-situ / distant tumors is inhibited and tumor metastasis is prevented; the activated photodynamic immunopreparation molecules not only realize synergistic treatment and obtain excellent tumor treatment efficiency, but also can avoid serious side effects in the tumor treatment process.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nanomaterials, and particularly relates to a photosensitive carrier and a preparation method and application thereof. BACKGROUND

[0002] Photodynamic therapy (PDT) has attracted considerable attention due to its minimally invasive and high spatiotemporal precision. Photosensitizers, as the key part of PDT, transfer the absorbed energy to the surrounding oxygen and produce reactive oxygen species (ROS), which then further react with nearby biological macromolecules (such as lipids, proteins, DNA) to kill cancer cells and complete PDT. Therefore, the effective enrichment of photosensitizers at the tumor site is a prerequisite for cancer patients to undergo PDT, and the tumor retention effect directly determines whether the treatment is accurate and effective. However, during in vivo PDT, most small organic molecule photosensitizers are rapidly cleared from the bloodstream, usually within a few hours or even tens of minutes, making it impossible for photosensitizers to effectively enrich and retain at the tumor site, resulting in low PDT efficiency. Therefore, it is urgent to improve the retention time of photosensitizers at the tumor site, which is of great significance to ensure accurate and efficient PDT in vivo.

[0003] In order to improve the treatment effect of cancer, a combined treatment method combining different treatment methods can usually be used. Among them, photodynamic therapy (PDT) is a phototherapy treatment method based on the interaction of photosensitizers, light sources and oxygen. When a laser of a specific wavelength is irradiated to a photosensitizer, strong oxidative singlet oxygen 1O2 is generated, which further destroys the biological active substances such as DNA and protein in tumor cells, and induces tumor cell apoptosis. Among various combined treatment methods, the strategy of PDT combined with immunotherapy shows its unique advantages. PDT can directly destroy tumor cells and tissues, damage the membrane structure of tumor cells, and cause a large increase in tumor-specific antigens. Such substances are called damage-associated molecular patterns (DAMP), mainly including Calreticulin exposed on the cell surface, High Mobility Group Box 1 (HMGB1) secreted by tumor cells to the outside world, and ATP molecules released by cells, making cancer cells sensitive to cancer immunity and making cold tumors into hot tumors.

[0004] Under the influence of tumor microenvironment cytokines, macrophages differentiate into different types of TAMs, mainly M1 and M2 types, and the process of macrophages differentiating into M1 and M2 types is called polarization. M1 macrophages are generally considered to be tumor-killing macrophages, mainly anti-tumor and immune promotion. While M2 macrophages exhibit immune suppression, promote tissue repair and tumor development. M1 and M2 TAMs exist in all stages of tumors; M1 is mainly in the early stage, and M2 is mainly in the middle and late stages. With the progression of the tumor, M1 gradually polarizes to M2, and the increase in the number of M2 TAMs also indicates poor prognosis. The two types of macrophages have different markers, metabolic characteristics and gene expression profiles. M1 macrophages secrete pro-inflammatory cytokines such as IL-12, tumor necrosis factor (TNF)-α, CXCL-10 and interferon (IFN)-γ, and produce high levels of iNOS, while M2 macrophages secrete anti-inflammatory cytokines such as IL-10, IL-13 and IL-4, and express abundant arginase-1, CD206, etc. Immune stimulation therapy aims to reshape the TME and reverse immune suppression, with the goal of activating anti-tumor immune responses. One of the treatment methods is imiquimod (IMQ), which is a hydrophobic small molecule drug that can repolarize innate immunity by activating the Toll-like receptor 7 (TLR-7) pathway. TLR7 agonists repolarize M2 macrophages to M1 macrophages, promoting phagocytosis, inflammation and antigen presentation.

[0005] As mentioned earlier, although the combined treatment mode of PDT and immunity has shown good prospects for clinical application, due to the short half-life of small molecule chemotherapy drugs and small molecule photosensitizers in the blood, they will be quickly cleared from the blood, thus not fully exerting their therapeutic effect. In addition, due to the lack of targeting, small molecule drugs often have high toxic side effects on healthy tissues. While nanomedicines exhibit high penetration and retention effects and higher drug delivery efficiency in vivo due to their special nanoscale size, they have attracted extensive research by researchers in recent years. Therefore, it is of great significance to develop activated PDT-Immunity combined therapy nanomedicines based on nanoscience. SUMMARY

[0006] In order to solve the problems existing in the prior art, the present application provides a photosensitive carrier and its preparation method and application, which is a targeted GSH-responsive nanomedicine for tumor photodynamic and immune combined therapy, for improving the therapeutic effect of cancer.

[0007] To achieve the above object, the technical scheme adopted by the present application comprises:

[0008] In a first aspect, the present application provides a glutathione-activated nano-immunoprodrug compound with targeting property, the structural general formula of which is shown as formula I:

[0009]

[0010] In formula I, R is S.

[0011] The skeleton of the glutathione-activated nano-immunoprodrug compound with targeting property provided by the present application is composed of hydrophilic polyethylene glycol (PEG) and hydrophobic AIE photosensitizer through hydrophobic self-assembly. In order to improve its targeting property, RGD tumor targeting polypeptide is covalently combined at the end of polyethylene glycol; in order to respond to the high glutathione environment in cells, disulfide bond which can respond to the high expression of glutathione in tumor sites is connected between the photosensitizer and polyethylene glycol.

[0012] In a second aspect, the present application provides a preparation method of a glutathione-activated nano-immunoprodrug compound with targeting property, comprising the following steps:

[0013]

[0014] S1, under an inert atmosphere, refluxing 5'-bromo-[2,2'-bithiophene]-5-carbaldehyde compound A, (4-(diphenylamino)phenyl)boronic acid compound B, tetrakis(triphenylphosphine)palladium and potassium hydroxide in an organic solvent for 12-14 h to obtain product C;

[0015] 4-methylquinoline compound D and iodoethane are refluxed in acetonitrile for 18-20 h to obtain product E;

[0016] Under an inert atmosphere, product C and product E are refluxed in ethanol under catalysis of piperidine for 12-14 h to obtain product F;

[0017]

[0018] S2, under an inert atmosphere, refluxing product C, 4-methylquinoline compound D and methane sulfonic acid in dry N,N-dimethylformamide at 150-160℃ for 4-6 h to obtain product H;

[0019]

[0020] S3, under an inert atmosphere, refluxing product H and 9-bromononanoic acid in 1,2-dichlorobenzene at 150-160℃ for 20-24 h to obtain product I;

[0021]

[0022] S4, under inert atmosphere, the product I, N-hydroxysuccinimide, 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in dichloromethane at room temperature for 12-14h to obtain product J;

[0023]

[0024] S5, under inert atmosphere, the product J, tert-butyl (2-aminoethyl)carbamate and triethylamine in dichloromethane at room temperature for 12-14h to obtain product K after purification;

[0025]

[0026] S6, under inert atmosphere, the product K and trifluoroacetic acid in dichloromethane at room temperature for 12-14h to obtain product L;

[0027]

[0028] S7, under inert atmosphere, the product L and cRGD-PEG5000-S-S-NPC in pyridine catalysis at room temperature for 12-14h to obtain the product after purification.

[0029] Further, in the above technical solution, in step S1, the molar ratio of 5'-bromo-[2,2'-bithiophene]-5-carbaldehyde compound A to (4-(diphenylamino)phenyl)boronic acid compound B is 1:1.2-1.5;

[0030] The molar ratio of 4-methylquinoline compound D to iodoethane is 1:1.3-1.5;

[0031] The molar ratio of the product C to the product E is 1:1.2-1.5;

[0032] In step S2, the molar ratio of the product C, 4-methylquinoline compound D and methane sulfonic acid is 1:1.2-1.4-1.2-1.4;

[0033] In step S3, the molar ratio of the product H to 9-bromononanoic acid is 1:1.5-2;

[0034] In step S4, the molar ratio of the product I, N-hydroxysuccinimide, 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:10-13:2-2.3:2-2.3;

[0035] In step S5, the molar ratio of the product J to tert-butyl (2-aminoethyl)carbamate is 1:2-2.5;

[0036] The molar ratio of the product L to cRGD-PEG5000-S-S-NPC in step S7 is 1:1-1.2.

[0037] Further, in step S1 of the above technical solution, the added amount of tetrakis triphenylphosphine palladium is 20-30 mg.

[0038] The added amount of piperidine is 10-20 μL.

[0039] Further, in step S5 of the above technical solution, the added amount of triethylamine is 15-20 μL.

[0040] Further, in step S7 of the above technical solution, the added amount of pyridine is 10-20 μL.

[0041] In a third aspect, the present application provides a photosensitive carrier, which is the prodrug compound of claim 1 wrapping a TLR7 agonist.

[0042] Further, in the above technical solution, the TLR7 agonist wraps imiquimod.

[0043] In a fourth aspect, the present application provides a preparation method of a photosensitive carrier, wherein a TLR7 agonist and the nano-immunoprodrug compound are dissolved in an organic solvent to obtain a mixture; and the mixture is dialyzed to obtain the photosensitive carrier.

[0044] Further, in the above technical solution, the dialysis bag used in the dialysis has a molecular weight cut-off of 3500 Da.

[0045] In a fifth aspect, the present application provides an application of the photosensitive carrier or the photosensitive carrier prepared by the preparation method in the preparation of a nano-drug for tumor photodynamic and immune combined therapy.

[0046] In summary, the present application has the following beneficial effects:

[0047] Firstly, the present application realizes the dual inhibition of the activity of photosensitizer and immunological drug by covalently connecting AIE dye and RGD-PEG5000-S-S-NPC through tumor marker response to obtain a photodynamic immunoprodrug, and then loading an immune agonist.

[0048] Secondly, after the prodrug molecule responds in the tumor cell, the photodynamic immunoprodrug can be activated under white light (100 mW / cm 2The activated photodynamic immunoprodrug molecule can efficiently kill tumor cells under light irradiation (within 3 min), realize photodynamic therapy, and cause immunogenic death of cells, thereby enhancing the immunogenicity of tumor cells, and further in in vivo experiments, the tumor-associated antigens released after photodynamic killing and the activated immune checkpoint inhibitors can induce synergistic anti-tumor immune responses, thereby inhibiting the growth of in situ / distant tumors and preventing tumor metastasis. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 A high-resolution mass spectrum of product C in the embodiment of the application.

[0050] Figure 2 A nuclear magnetic hydrogen spectrum of product C in the embodiment of the application.

[0051] Figure 3 A high-resolution mass spectrum of product F in the embodiment of the application.

[0052] Figure 4 A nuclear magnetic hydrogen spectrum of product F in the embodiment of the application.

[0053] Figure 5 A high-resolution mass spectrum of product H in the embodiment of the application.

[0054] Figure 6 A nuclear magnetic hydrogen spectrum of product H in the embodiment of the application.

[0055] Figure 7 A high-resolution mass spectrum of product I in the embodiment of the application.

[0056] Figure 8 A nuclear magnetic hydrogen spectrum of product I in the embodiment of the application.

[0057] Figure 9 A high-resolution mass spectrum of product J in the embodiment of the application.

[0058] Figure 10 A nuclear magnetic hydrogen spectrum of product J in the embodiment of the application.

[0059] Figure 11 A high-resolution mass spectrum of product K in the embodiment of the application.

[0060] Figure 12 A nuclear magnetic hydrogen spectrum of product K in the embodiment of the application.

[0061] Figure 13 A hydration particle size distribution diagram of the nanomedicine of the application.

[0062] Figure 14 A TEM diagram of the nanomedicine of the application.

[0063] Figure 15 Absorption spectrum of the photosensitizer, agonist and nanodrug of the present application.

[0064] Figure 16 GSH response curve of the nanodrug of the present application in PBS buffer solution.

[0065] Figure 17 Total reactive oxygen species detection of the photosensitizer and nanodrug in the examples of the present application.

[0066] Figure 18 Singlet oxygen detection of the photosensitizer and nanodrug in the examples of the present application.

[0067] Figure 19 Nanodrug incubation Co-localization coefficient change of nanodrug fluorescence and lysosome commercial dye Lyso-Tracker fluorescence after incubation of the nanodrug of the present application with RM-1 cells for different time.

[0068] Figure 20 Nanodrug of the present application under white light irradiation (100 mW / cm 2 , 3 min) condition, total reactive oxygen species DCFH-DA fluorescence at 525 nm in RM-1 cells.

[0069] Figure 21 Toxicity of the nanodrug of the present application and the photosensitizer alone to RM-1 cells and survival rate of RM-1 cells under light irradiation condition (white light, different light dose).

[0070] Figure 22 Fluorescence imaging of the nanodrug of the present application in the tumor site of mice at different time after intravenous injection.

[0071] Figure 23 Tumor volume detection for 7 days after intravenous injection of the nanodrug of the present application.

[0072] Figure 24 Body weight detection for 7 days after intravenous injection of the nanodrug of the present application.

[0073] Figure 25 HE staining of the tumor site and immunofluorescence detection of tumor-associated macrophages in the tumor site of mice after 7 days of treatment by intravenous injection of the nanodrug of the present application.

[0074] Figure 26 Immunofluorescence detection of T lymphocytes in the tumor site after 7 days of treatment by intravenous injection of the nanodrug of the present application.

[0075] Figure 27After the treatment of the nano-drug of the present application by intravenous injection for 7 days, the immune fluorescence detection of T lymphocytes in the spleen was performed. DETAILED DESCRIPTION

[0076] In order to better explain the present application, so as to be understood, the present application is described in detail by specific embodiments in combination with the drawings.

[0077] In order to better understand the above technical solutions, the exemplary embodiments of the present application will be described in more detail. It should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided so that the present application can be more clearly, thoroughly understood and the scope of the present application can be completely conveyed to those skilled in the art.

[0078] Example 1

[0079] A preparation method of a glutathione-activated nano-prodrug compound with targeting property comprises the following steps:

[0080]

[0081] S1: 5'-Bromo-[2,2'-bithiophene]-5-carbaldehyde compound A (273 mg, 1 mmol), (4-(diphenylamino)phenyl)boronic acid compound B (347-433 mg, 1.2-1.5 mmol), tetrakis(triphenylphosphine)palladium (20-30 mg) and potassium hydroxide (1.38 g, 10 mmol) in 35 mL of tetrahydrofuran / water (6:1, v / v) were refluxed at 60°C for 12 h under nitrogen. After cooling to room temperature and removing THF, the mixture was extracted with DCM three times. The organic phase was collected and dried with anhydrous sodium sulfate. After evaporation of the solvent, the mixture was purified by silica gel column chromatography using dichloromethane / petroleum ether (V:V=2:1) as eluent to obtain yellow solid product C (253 mg, 75%).

[0082] The mass spectrum and nuclear magnetic resonance characterization of product C are as follows: HRMS (ESI): m / z C 27 H 19 NOS2 calculated value [M+H] + 438.0908, test value 438.0973, its high resolution mass spectrum is shown in Figure 1 The nuclear magnetic resonance characterization is shown in Figure 2

[0083] 1 ​H NMR (500 MHz, Chloroform-d) δ 9.85 (s, 1H), 7.66 (d, J = 3.9 Hz, 1H), 7.50 - 7.42 (m, 2H), 7.31 (d, J = 3.8 Hz, 1H), 7.26 (d, J = 4.7 Hz, 2H), 7.23 (d, J = 3.9 Hz, 1H), 7.17 (d, J = 3.9 Hz, 1H), 7.13 (s, 2H), 7.12 (s, 2H), 7.06 (dt, J = 7.2, 3.2 Hz, 4H).

[0084] S2: To a solution of ethyl iodide compound (1.24 g, 8 mmol) in acetonitrile (50 ml) was added 4-methylquinoline compound D (0.86 g, 6 mmol). The mixture was refluxed at 80 °C for 19 hours. After filtration, the solid was washed with diethyl ether three times and dried under vacuum to obtain the dark green solid product E (1.44 g, 80%).

[0085] S3: A solution of product C (87.5 mg, 0.2 mmol), product E (56.4 mg, 0.24 mmol) and piperidine (10 μΐ-20 μΐ) in dry ethanol (15 mL) was refluxed under nitrogen for 12-14 hours. The mixture was extracted three times with DCM, the organic phase was collected and dried over anhydrous sodium sulfate. Purification by column chromatography on silica gel using a mixture of dichloromethane / ethyl acetate (5:1 v / v) as eluent afforded the purple solid product F (98.7 mg, 78%).

[0086] Mass spectrometric characterization of product F is as follows: HRMS (ESI): m / Z C 39 H 31 N2S2 + Calculated [M] + 591.1923, found 591.1923, its high resolution mass spectrum is shown in Figure 3 Figure 1, and its NMR characterization is shown in Figure 4 Figure 2.

[0087] 1H NMR (500 MHz, DMSO-d6) δ 9.42 (t, J = 4.6 Hz, 1H), 9.35 (d, J = 6.6 Hz, 1H), 8.60 (d, J = 9.0 Hz, 1H), 8.54 (dd, J = 12.2, 8.7 Hz, 1H), 8.47 - 8.38 (m, 1H), 8.31 - 8.21 (m, 2H), 8.05 (ddd, J = 20.8, 11.0, 5.4 Hz, 3H), 7.95 (d, J = 15.6 Hz, 1H), 7.74 (d, J = 4.0 Hz, 1H), 7.61 (d, J = 8.3 Hz, 1H), 7.51 (d, J = 4.1 Hz, 1H), 7.48 (t, J = 5.4 Hz, 1H), 7.42 - 7.24 (m, 3H), 7.08 (h, J = 7.9 Hz, 5H), 6.97 (dd, J = 19.4, 8.3 Hz, 2H), 5.05 (q, J = 7.1 Hz, 2H), 1.59 (t, J = 7.3 Hz, 3H).

[0088]

[0089] S4: A solution of product C (175 mg, 0.2 mmol), 4-methylquinoline compound D (105 mg, 0.24 mmol) and methanesulfonic acid (30 mg, 0.24 mmol) in dry N,N-dimethylformamide (15-20 mL) was reacted at 150 °C under nitrogen for 6 hours. The mixture was extracted three times with DCM, the organic phases were collected and dried over anhydrous sodium sulfate. Purification by column chromatography on silica gel using a DCM / MeOH mixture (100:1 v / v) as eluent afforded product H as a red solid (143 mg, 75%).

[0090] Mass and NMR characterization of product H are as follows: HRMS (ESI): m / Z C 37 H 26 N2S2 + Calculated [M+H] + 563.1537, found 563.1603, with a high resolution mass spectrum as shown in Figure 5 Figure 1. NMR characterization as shown in Figure 6 Figure 2.

[0091] 1H NMR (500 MHz, DMSO-d6) δ 8.88 (d, J = 4.7 Hz, 1H), 8.46 (d, J = 8.5 Hz, 1H), 8.04 (d, J = 8.6 Hz, 1H), 7.85 (d, J = 4.6 Hz, 1H), 7.81 (t, J = 3.8 Hz, 1H), 7.77 (d, J = 3.2 Hz, 1H), 7.68 (t, J = 7.7 Hz, 1H), 7.59 (dd, J = 8.9, 2.5 Hz, 2H), 7.45 (d, J = 3.9 Hz, 1H), 7.43 (d, J = 3.7 Hz, 1H), 7.40 (d, J = 3.9 Hz, 1H), 7.37 (d, J = 3.9 Hz, 1H), 7.36 (s, 1H), 7.34 (s, 2H), 7.33 (d, J = 2.1 Hz, 1H), 7.13 - 7.05 (m, 7H), 7.02 - 6.97 (m, 2H).

[0092]

[0093] S5: A solution of product H (437 mg, 0.1 mmol), 9-bromononanoic acid (256 mg, 0.2 mmol) in dry 1,2-dichlorobenzene (15 mL) was heated at 150 °C under nitrogen for 24 h. The organic phase was directly purified by column chromatography on silica gel using a DCM / MeOH mixture (100:7 v / v) as eluent to give product I as a purple solid (254 mg, 55%).

[0094] Mass and NMR characterization of product I are as follows: HRMS (ESI): m / Z C 46 H 43 N2O2S2 + Calculated [M] + 719.2760, found 719.2753, with a high resolution mass spectrum as shown in Figure 7 Figure 1. NMR characterization as shown in Figure 8 Figure 2.

[0095] 1H NMR (500 MHz, DMSO-d6) δ 11.99 (s, 1H), 9.34 (ddt, J = 24.9, 18.0, 9.4 Hz, 1H), 8.96 (qt, J = 17.7, 8.8 Hz, 1H), 8.61 - 8.32 (m, 3H), 8.23 (h, J = 8.4 Hz, 1H), 7.97 (ddt, J = 29.1, 15.7, 6.4 Hz, 2H), 7.72 (d, J = 11.9 Hz, 1H), 7.59 (tt, J = 18.4, 8.0 Hz, 2H), 7.54 - 7.42 (m, 3H), 7.35 (tq, J = 16.8, 8.8, 8.0 Hz, 4H), 7.10 (dtq, J = 28.9, 14.4, 6.8 Hz, 6H), 7.00 - 6.83 (m, 2H), 5.19 - 4.77 (m, 2H), 2.19 (ddq, J = 25.4, 17.9, 10.0, 8.6 Hz, 2H), 2.03 - 1.81 (m, 2H), 1.49 (d, J = 36.8 Hz, 4H), 1.34 - 1.20 (m, 6H).

[0096]

[0097] S6: A solution of product I (800 mg, 0.1 mmol), N-hydroxysuccinimide (1.15 g, 1 mmol), 4-dimethylaminopyridine (400 mg, 0.2 mmol) and l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (410 mg, 0.2 mol) in dry dichloromethane (15 mL) was reacted under nitrogen at room temperature for 12 hours. The mixture was extracted three times with DCM, the organic phases were collected and dried over anhydrous sodium sulfate. Purification by column chromatography on silica gel using a DCM / MeOH mixture (100:4 v / v) as eluent gave the purple product J (600 mg, 72%).

[0098] Mass and NMR characterization of product J are as follows: HRMS (ESI): m / Z C 50 H 46 N3O4S2 + Calculated [M] + 816.2924, found 816.2916, with a high resolution mass spectrum as shown in Figure 9 NMR characterization as shown in Figure 10

[0099] 1 ​H NMR (500 MHz, DMSO-d6) δ 9.33 (d, J = 13.5 Hz, 1H), 9.08 - 8.88 (m, 1H), 8.64 - 8.33 (m, 3H), 8.24 (dt, J = 20.3, 12.4 Hz, 1H), 8.10 - 7.87 (m, 2H), 7.86 - 7.68 (m, 1H), 7.61 (dq, J = 20.3, 9.7, 8.5 Hz, 2H), 7.54 - 7.43 (m, 3H), 7.35 (q, J = 10.0, 9.0 Hz, 4H), 7.10 (dp, J = 28.9, 10.5, 9.2 Hz, 6H), 6.98 (s, 2H), 4.96 (d, J = 37.9 Hz, 2H), 2.81 (q, J = 14.9, 8.9 Hz, 4H), 2.70 - 2.55 (m, 4H), 1.97 (d, J = 32.4 Hz, 2H), 1.75 - 1.53 (m, 2H), 1.39 - 1.27 (m, 6H).

[0100]

[0101] S7: A solution of product J (89.5 mg, 0.1 mmol), tert-butyl (2-aminoethyl)carbamate (32 mg, 0.2 mmol) and triethylamine (15 μΐ) in dry dichloromethane (5 mL) was reacted under nitrogen at room temperature for 12 hours. The mixture was extracted three times with DCM, the organic phases were collected and dried over anhydrous sodium sulfate. Purification by column chromatography on silica gel using a DCM / MeOH mixture (100:2 v / v) as eluent afforded the purple product K (55.4 mg, 57.9%).

[0102] Mass and NMR characterization of product K are as follows: HRMS (ESI): m / Z C 53 H 57 N4O3S2 + Calculated [M] + 861.3867, found 861.3866, with a high resolution mass spectrum as shown in Figure 11 Figure 1. NMR characterization as shown in Figure 12 Figure 2.

[0103] 1H NMR (500 MHz, DMSO-d6) δ 9.31 (d, J = 6.6 Hz, 1H), 8.98 (d, J = 8.6 Hz, 1H), 8.51 (d, J = 8.8 Hz, 1H), 8.45 - 8.35 (m, 2H), 8.24 (t, J = 7.9 Hz, 1H), 8.06 - 7.92 (m, 2H), 7.79 - 7.70 (m, 2H), 7.61 (d, J = 8.4 Hz, 2H), 7.55 - 7.49 (m, 2H), 7.47 (d, J = 3.9 Hz, 1H), 7.35 (t, J = 7.8 Hz, 4H), 7.15 - 7.04 (m, 6H), 7.00 (dd, J = 8.7, 2.5 Hz, 2H), 4.92 (t, J = 7.1 Hz, 2H), 3.04 (q, J = 6.4 Hz, 2H), 2.95 (q, J = 6.4 Hz, 2H), 2.02 (t, J = 7.4 Hz, 2H), 1.93 (q, J = 7.7 Hz, 2H), 1.47 (d, J = 7.8 Hz, 3H), 1.37 (d, J = 5.8 Hz, 12H), 1.24 (s, 6H).

[0104]

[0105] S8: A solution of product K (89.5 mg, 0.1 mmol), trifluoroacetic acid (1 ml) in anhydrous dichloromethane (4 mL) was reacted at room temperature under nitrogen for 12 hours. The mixture was extracted with DCM three times, the organic phase was collected and dried over anhydrous sodium sulfate. The next step was carried out directly without purification.

[0106]

[0107]

[0108] S9: A solution of product L, cRGD-PEG5000-S-S-NPC or cRGD-PEG5000-C-C-NPC, pyridine in anhydrous dichloromethane (4 mL) was reacted at room temperature under nitrogen for 12 hours. The mixture was transferred into 10 mL of DMSO and dialyzed (MWCO 3500 Da) against distilled water three times, and finally lyophilized to obtain RPST or RPCT, the formation of which was verified by particle size and TEM. As shown in FIGS. 1 and 2, RPST or RPCT can self-assemble into nanoparticles with an average diameter of 190 nm and 164 nm, respectively, under phosphate buffered saline (PBS) conditions. Figure 13 and Figure 14 As shown in FIGS. 1 and 2, RPST or RPCT can self-assemble into nanoparticles with an average diameter of 190 nm and 164 nm, respectively, under phosphate buffered saline (PBS) conditions.

[0109] S10: The Toll-like receptor 7 (TLR-7) compound Imiquimod is dissolved in dimethyl sulfoxide (DMSO) with RPST or RPCT to obtain a mixture, which is transferred to a dialysis bag and dialyzed against distilled water (MWCO 3500 Da) for three times to obtain the nanodrug RPST@IMQ or RPCT@IMQ loaded with the Toll-like receptor 7 (TLR7) agonist (IMQ, Imiquimod) and having photosensitive properties. As shown in Figure 13 and Figure 14 The characterization of these prepared nanoparticle samples using TEM and nanoparticle size analyzer shows that their average diameters are 122 nm and 106 nm, respectively, which are smaller than the average diameters of RPST or RPCT alone.

[0110] Example 2 Study on the physicochemical properties of nanodrug NPs

[0111] As shown in Figure 15 The specific absorption peak (320 nm) of the IMQ-loaded nanoparticles was detected in the absorption spectrum, which confirmed the successful encapsulation. The analysis of the photoluminescence (PL) spectra of the prepared RPS(C)T and RPS(C)T@IMQ in PBS and free TPA-KLI in DMSO shows a peak at 550 nm, indicating that RPS(C)T and RPS(C)T@IMQ can have the properties of the photosensitizer TPA-KLI. To prove the ability of the nanoparticles to release IMQ under high glutathione (GSH) conditions, we tested the ability of RPST@IMQ and RPCT@IMQ to release IMQ in a 10 mM GSH buffer. The results are shown in Figure 16 Under the condition of 10 mM GSH, about 60% of IMQ was released from the RPST@IMQ nanoparticles, while RPCT@IMQ hardly released the IMQ loaded therein.

[0112] Example 3 Performance detection test of nanodrug NPs

[0113] The compound F ((E)-4-(2-(5'-(4-(diphenylamino)phenyl)-[2,2'-bithiophen]-5-yl)vinyl)-1- ethylquinolinium-1-iodide), named TPA-KLI and used as a control, was used to evaluate the ROS production efficiency of RPST, RPCT, RPST@IMQ and RPCT@IMQ, with the purpose of judging whether the active oxygen yield of the photosensitizer changes after it forms nanoparticles by self-assembly. The active oxygen production efficiency was initially determined using 2',7'dichlorofluorescein diacetate (DCFH) as an indicator, which can emit fluorescence through the "opening" process triggered by ROS. As shown in Figure 17 DCFH alone has no fluorescence, and under white light irradiation (~ 10 mW cm-2 ) remained almost unchanged during the period. In contrast, in the presence of TPA-KLI, RPS(C)T or RPS(C)T@IMQ, the emission intensity of DCFH gradually increased during the same period of exposure to white light irradiation and the production rates of reactive oxygen species between TPA-KLI and RPST@IMQ were almost the same. In addition, as Figure 18 As shown, using 9,10-anthracenebis(methylene)dimethacrylate (ABDA) as an indicator, the singlet oxygen yields generated by TPA-KLI, RPS(C)T, and RPS(C)T@IMQ were significantly higher than those of the commercial Ce6 photosensitizer, and the singlet oxygen yields between TPA-KLI and RPSRPST@IMQ were nearly identical. Under the action of glutathione, the singlet oxygen yields of RPST and RPST@IMQ did not show significant changes. In summary, the designed macromolecular structures self-assembled through hydrophilic and hydrophobic structures to form nanocarriers (RPST, RPCT) and nanoparticles (RPST@IMQ, RPCT@IMQ). Compared with the free small molecule TPA-KLI, their reactive oxygen species and singlet oxygen yields did not change significantly, which is attributed to their unique AIE structure, which avoids aggregation-induced quenching.

[0114] Example 4 Intracellular Imaging of Nanodrug NPs

[0115] The small molecule TPA-KLI was used as a control to verify the intracellular ROS production efficiency of RPST, RPCT, RPST@IMQ and RPCT@IMQ. The purpose was to determine whether the intracellular ROS production rate changed after the photosensitizers self-assembled into nanoparticles. 5 cells / mL) were inoculated into a 35 mm diameter confocal culture dish. The cells were then exposed to the following different treatments, including G1: incubation with RPST@IMQ; G2: incubation with RPST; G3: incubation with RPCT@IMQ; G4: incubation with RPCT; G5: incubation with TPA-KLI. The drug incubation time was 24 h, and the concentration was 5 μmol / mL, which was measured based on the content of the photosensitizer in the dosage form. After the drug incubation was completed, the cells in the culture dish were incubated with 10 μM DCFH-DA for 90 min. Afterwards, a white light LED (50 mW / cm 2 Cells were irradiated for 3 min. Cells were imaged using an OLYMPUS FV-1000 inverted confocal fluorescence microscope with a 60× oil immersion lens. The excitation wavelength for DCFH-DA was 488 nm, and the acceptance wavelength was 510-550 nm. Figure 19The co-localization coefficient was about 0.9 after 24 hours of incubation with RPST@IMQ, indicating that RPST@IMQ mainly accumulated in lysosomes after entering cells. We then used CLSM to measure the ability of AIE-PS to generate ROS in cells. As shown in Figure 20 After light triggering, bright green fluorescence of ROS sensor DCFH-DA was observed in cells, indicating effective ROS generation. In summary, the formed nanocarriers (RPST, RPCT) and their nanoparticles (RPST@IMQ, RPCT@IMQ) can all generate reactive oxygen species in cells, and the yield of reactive oxygen species is almost unchanged compared with free small molecule TPA-KLI. According to previous reports, the therapeutic mechanism of ROS generated by light-triggered photosensitizers can rapidly damage the biological function of organelles, leading to apoptosis, necrosis and autophagy. Thus, the nanodrugs enhance the therapeutic effect of cancer.

[0116] Example 5 Cell survival rate test of nanodrugs

[0117] In this experiment, the MTT method was used to test the cell survival rate.

[0118] Operation steps: RM-1 cells were seeded in a 96-well plate (1 x 10 4 cells per well and incubated in 100 μL DMEM). The cells were grown to a density of 80%, and the nanodrugs (0 μmol / L, 5 μmol / L) were incubated for 24 hours. Then irradiated with a white light lamp (0, 1, 3, 5, 10, 30, 50, 100 mW / cm 2 ) for 5 minutes. After further incubation for 2 hours, 100 μL of MTT (0.5 mg / mL) solution was added to each well, and the cells were incubated at 37°C for 4 hours. Then, the culture medium was carefully removed, and 100 μL of DMSO was added to dissolve the formazan crystals. The absorbance value was measured at 570 nm and 630 nm (background) using a Bio-Rad microplate reader, and the cell survival rate was calculated:

[0119]

[0120] As shown in Figure 21 , when 0 μM of nanoparticles were used under the condition of 30 J / cm 2 light irradiation (white light) irradiation, almost no cell toxicity was observed, and the cell survival rate was more than 90%, indicating good biocompatibility under only light irradiation. In contrast, when the cells were irradiated with 30 J / cm 2The growth of tumor cells was inhibited by about 80% when incubated with 5 μΜ of RPST@IMQ, RPST, RPCT@IMQ, RPCT under light irradiation (white light). These results indicate that the nanodrugs exhibit high cytotoxicity at a concentration of 5 μΜ under irradiation.

[0121] Example 6 Mouse tumor killing test of nanodrugs

[0122] C57 mice of 4-6 weeks old were selected and injected subcutaneously with 2 x 10 6 RM-1 cells to establish C57 tumors. For in vivo imaging, when the primary tumor volume reached 150 mm 3 , tumor-bearing mice were randomly divided into 4 groups (RPCT, RPST, RPCT@IMQ, RPST@IMQ). The mice were first injected with the above drugs (10 mg Kg -1 ), and then the heart, liver, spleen, lung, kidney and tumor were collected at 4 h, 12 h and 24 h, and finally imaged using a NightOWL II LB983 small animal in vivo imaging system. The fluorescence was excited at 580 nm and collected at 650 ± 20 nm. For tumor killing test, when the primary tumor volume reached 50 mm 3 , tumor-bearing mice were randomly divided into 10 groups (PBS, RPCT, RPST, RPCT@IMQ, RPST@IMQ, PBS+Light, RPCT+Light, RPST+Light, RPCT@IMQ+Light and RPST@IMQ+Light, n = 5), the mice were first injected with the above drugs (10 mg Kg -1 ), and the light group was treated with white light at a light intensity of 100 mw cm -2 for 10 minutes at 24 hours and 36 hours, respectively. Then the body weight and tumor volume of the mice were recorded within 16 days. Finally, the tumors and spleens of the mice were removed and immunofluorescence section studies were performed. The tumor volume was calculated using the following formula:

[0123]

[0124] V represents the tumor volume of the mouse, a represents the longest diameter of the tumor area, and b represents the diameter in the vertical direction according to a of the tumor area. All animal experiments involved in this study were carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (8th edition, 2011) and were approved by the Local Research Ethics Review Committee of the Animal Ethics Committee of Dalian University of Technology.

[0125] The results showed that, as Figure 22As shown, the fluorescence signal in the tumor gradually increased, reaching a peak at 24 hours after injection, indicating that RPS(C)T@IMQ significantly accumulated in the tumor, which is beneficial for primary tumor treatment. As shown in Figure 23 The inhibition rates of tumor growth for RPST@IMQ+Light, RPST+Light, RPCT@IMQ+Light, RPCT+Light were 91.2%, 62.4%, 67.2%, 58.8%, respectively. No significant changes in body weight were observed throughout the treatment Figure 24 ), which indicated that RPS(C)T and RPS(C)T@IMQ did not cause severe systemic toxicity. As shown in Figure 25 H&E staining showed that RPST@IMQ had significant tumor necrosis in all light-treated groups. Further, immunofluorescence staining was used to evaluate the types of macrophages at the tumor site, and the results showed that RPST@IMQ+Light significantly led to a decrease in M2 tumor-associated macrophages and an increase in M1 tumor-associated macrophages, further confirming that RPST@IMQ+Light can repolarize M2 tumor-associated macrophages to M1 tumor-associated macrophages, with good therapeutic effect. These results excited us, and we further investigated the changes in T cells in the tumor microenvironment. As shown in Figure 26 Immunofluorescence staining of CD4+ and CD8+ T cells showed that fewer CD4+ CD8+ T cells were observed in the tumor tissue of mice treated with PBS compared to mice treated with PBS, and a large number of CD4+ CD8+ T cells were observed with RPST@IMQ+Light. As shown in Figure 27 We also observed similar trends in CD4+ and CD8+ T cell infiltration in the main immune organ, the spleen, further confirming the activation of anti-tumor immunity. Immunofluorescence staining also showed that there were more CD4+ CD8+ T cell infiltrations with RPST@IMQ+Light. In summary, RPST@IMQ+Light effectively increased the number and activity of CD8+ T cells within the tumor, and the combination of photosensitizer and IMQ showed the best efficacy. The mechanism of RPST@IMQ+Light includes TAM repolarization, normalization of tumor vasculature, and reduction of M2 macrophage inhibition of T cells, thereby enhancing T cell infiltration and cytotoxicity. These findings indicate that photoimmunotherapy activates specific T lymphocytes while attenuating immune suppression.

[0126] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A targeted glutathione-activated nanoimmune prodrug compound, characterized in that: The general structural formula of the nanoimmune prodrug compound is shown in Formula I: In formula I, R is S.

2. The method for preparing the targeted glutathione-activated nanoimmune prodrug compound according to claim 1, characterized in that: The steps include: S1. Under an inert atmosphere, reflux 5'-bromo-[2,2'-bithiophene]-5-carboxaldehyde compound A, (4-(diphenylamino)phenyl)boronic acid compound B, tetrakistriphenylphosphine palladium, and potassium hydroxide in an organic solvent for 12-14 hours to obtain product C; 4-Methylquinoline compound D is reacted with iodoethane in acetonitrile under reflux for 18-20 hours to obtain product E; Under an inert atmosphere, product C and product E are refluxed in ethanol under the catalysis of piperidine for 12-14 hours to obtain product F; S2. Under an inert atmosphere, product C, 4-methylquinoline compound D, and methanesulfonic acid are reacted in dry N,N-dimethylformamide at 150-160° C. for 4-6 hours to obtain product H; S3. Under an inert atmosphere, product H and 9-bromononanoic acid are reacted in 1,2-o-dichlorobenzene at 150-160° C. for 20-24 h to obtain product I; S4. Under an inert atmosphere, product I, N-hydroxysuccinimide, 4-dimethylaminopyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are reacted in dichloromethane at room temperature for 12-14 h to obtain product J; S5. Under an inert atmosphere, product J, tert-butyl (2-aminoethyl)carbamate, and triethylamine are reacted in dichloromethane at room temperature for 12-14 h, and product K is obtained after purification; S6. Under an inert atmosphere, product K and trifluoroacetic acid are reacted in dichloromethane at room temperature for 12-14 hours to obtain product L; S7. Under an inert atmosphere, the product L was reacted with cRGD-PEG5000-SS-NPC under pyridine catalysis at room temperature for 12-14 h, and the product was obtained after purification.

3. The preparation method according to claim 2, characterized in that In step S1, the molar ratio of 5'-bromo-[2,2'-bithiophene]-5-carboxaldehyde compound A to (4-(diphenylamino)phenyl)boronic acid compound B is 1:1.2-1.5; The molar ratio of 4-methylquinoline compound D to ethyl iodide is 1:1.3-1.5; The molar ratio of the product C to the product E is 1:1.2-1.5; In step S2, the molar ratio of the product C, 4-methylquinoline compound D and methanesulfonic acid is 1:1.2-1.4-1.2-1.4; In step S3, the molar ratio of the product H to 9-bromononanoic acid is 1:1.5-2; In step S4, the molar ratio of the product I, N-hydroxysuccinimide, 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:10-13:2-2.3:2-2.3; In step S5, the molar ratio of the product J to tert-butyl (2-aminoethyl)carbamate is 1:2-2.5; In step S7, the molar ratio of the product L to cRGD-PEG5000-SS-NPC is 1:1-1.

2.

4. A photosensitive carrier, characterized in that The photosensitive carrier is the prodrug compound according to claim 1 encapsulating the TLR7 agonist.

5. The photosensitive carrier according to claim 4, characterized in that The TLR7 agonist includes imiquimod.

6. The method for preparing a photosensitive carrier according to claim 4 or 5, characterized in that: The TLR7 agonist and the nanoimmune prodrug compound are dissolved in an organic solvent to obtain a mixture; and the mixture is dialyzed to obtain the product.

7. The preparation method according to claim 6, characterized in that The molecular weight cut-off of the dialysis bag in the dialysis is 3500Da.

8. Use of the photosensitive carrier according to claim 4 or 5, or the photosensitive carrier prepared by the preparation method according to claim 6 or 7, in the preparation of nanomedicines for combined photodynamic and immunotherapy of tumors.

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