A triblock copolymer, preparation method and application thereof

Abstract

The present invention discloses a triblock copolymer and also discloses its corresponding preparation method, specifically including a method for preparing a triblock copolymer, which comprises the following steps: (1) A compound having the structure of formula (II) or formula (III) reacts with cyano-4-(phenylcarbonothioylthio) pentanoate in an organic solvent, followed by purification and drying to obtain an inducer PEG-CPPA; the organic solvent is dichloromethane or 1,4-dioxane; (2) The inducer PEG-CPPA obtained in step (1) reacts with monomers having the groups of formula (IV) and formula (V), followed by purification and drying to obtain a triblock copolymer. The triblock copolymer of the present invention contains a crown ether structure that can release intracellular iron ions, activate ferroptosis, and the polymer structure has hypoxia responsiveness, enabling specific activation of ferroptosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of polymer materials, and particularly relates to a triblock copolymer, a preparation method and an application thereof. Background Art

[0002] Ferroptosis is an iron-dependent, novel programmed cell death mode that is morphologically distinct from apoptosis, necrosis, and autophagy in cells. Mechanistically, ferroptosis is the cell death induced by lipid peroxidation of highly expressed unsaturated fatty acids that are iron-dependent on the cell membrane. The essence of ferroptosis is the depletion of glutathione and the decline in the activity of glutathione peroxidase (GPX4), resulting in the inability of lipid oxides to be metabolized through the glutathione reductase reaction catalyzed by GPX4. Subsequently, divalent iron ions oxidize lipids to generate reactive oxygen species, thus promoting the occurrence of ferroptosis. In addition, the uptake and metabolism of iron ions, as well as the supply and synthesis of fatty acids, are also important factors in ferroptosis.

[0003] As an iron-dependent cell death caused by lipid peroxidation, ferroptosis has a dual role in tumor biology and treatment. On the one hand, ferroptosis can eliminate tumor cells, cut off the blood supply of tumors, stimulate the attack of the immune system, and improve the response of tumor cells to other treatments. On the other hand, ferroptosis can also release some pro-inflammatory factors, trigger inflammatory reactions, change the tumor microenvironment, and reduce the immune susceptibility of tumor cells. Therefore, ferroptosis can be used as a new cancer treatment target, and by synergistically acting with other cell death pathways, it can increase the mortality rate of tumor cells and overcome the drug resistance of tumor cells.

[0004] Compared with normal cells, tumor cells have a higher demand for iron, which is called the "iron addiction" of tumor cells. This makes tumor cells more likely to undergo ferroptosis when the iron level is increased. For example, the iron-rich tumor microenvironment in renal cancer promotes the occurrence of ferroptosis in tumor cells.

[0005] Common ferroptosis inducers are mainly iron-based materials, such as iron oxide, magnetite, ferrocene, zinc-iron alloy, etc. They activate ferroptosis by delivering exogenous iron into cells, but the cytotoxicity of these iron-based nanomaterials cannot be ignored. Since cells themselves store a large amount of iron, activating ferroptosis using endogenous iron ions can solve the above problems and is the research focus in the current field of ferroptosis technology. Summary of the Invention

[0006] The object of the present invention is to provide a triblock copolymer and a preparation method thereof. Another object of the present invention is to provide its application.

[0007] Based on the above object, the present invention adopts the following technical solutions:

[0008] A triblock copolymer, the polymer structure is as shown in the general formula (I),

[0009]

[0010] In formula (I), R 1 is selected from hydrogen, alkyl or substituted alkyl;

[0011] R 2 is selected from -NH- or -R 6 (CH 2 ) r NH-, where R 6 is -O-, -OCONH-, -OCO-, -NHCOO- or -NHCO-, 1 ≤ r ≤ 10;

[0012] R 3 is selected from hydrogen or the following group, the group structure is as shown in formula (IV),

[0013]

[0014] R 4 is selected from hydrogen or the following group, the group structure is as shown in formula (V),

[0015]

[0016] R 5 is selected from hydrogen or the following group, the group structure is as shown in formula (VI)

[0017]

[0018] 20 ≤ n ≤ 500; 5 ≤ x ≤ 200; 5 ≤ y ≤ 200.

[0019] The alkyl is C1 - C40 alkyl; in the substituted alkyl, the substituents are mercapto, sugar residue, aldehyde group, carboxyl group, vinyl group, alkynyl group, succinimide, maleimide, biotin, RGD - like short peptide, LHRH - like short peptide or folic acid.

[0020] R 4 When it is a group of formula (V), the proportion of the crown - ether - structured group in the triblock copolymer is not less than 20%

[0021] The proportion of the crown - ether - structured group in the triblock copolymer is 40% - 60%.

[0022] R 1 is hydrogen, R 2 is -NH-, R 3 is a group of formula (IV), R 5When it is a group of formula (VI), the structural formula of the polymer is as shown in formula (VII).

[0023]

[0024] A method for preparing a triblock copolymer polymer, comprising the following steps:

[0025] (1) A compound having the structure of formula (II) or formula (III) reacts with cyano-4-(phenylcarbonothioylthio) pentanoate (0.1 - 10eq, that is, the amount of cyano-4-(phenylcarbonothioylthio) pentanoate is 0.1 - 10 times the molar amount of the substrate compound having the structure of formula (II) or formula (III)) in an organic solvent, followed by purification and drying to obtain an inducer PEG-CPPA; the organic solvent is dichloromethane or 1,4-dioxane;

[0026]

[0027] (2) The inducer PEG-CPPA obtained in step (1) reacts with monomers having a group of formula (IV) (0.1 - 200eq, the amount is 0.1 - 200 times the molar amount of the substrate PEG-CPPA, the same applies hereinafter) and a group of formula (V) (0.1 - 200eq), followed by purification and drying to obtain a triblock copolymer polymer.

[0028] In step (2), in step (1), the inducer PEG-CPPA first reacts with a monomer having a group of formula (IV) (0.1 - 200eq) and an initiator (0.01 - 2eq) in an organic solvent, followed by purification and drying to obtain a product PEG-PBQAEM; then the product PEG-PBQAEM reacts with a monomer having a group of formula (V) (0.1 - 200eq) and an initiator (0.01 - 2eq) in an organic solvent, followed by purification and drying to obtain a triblock copolymer polymer; the organic solvent is 1,4-dioxane.

[0029] In step (2), the initiator is azobisisobutyronitrile;

[0030] A method for preparing a monomer having a group of formula (IV): A compound having the structure of formula (VIII) reacts with 2-aminoethyl methacrylate (0.1 - 10eq), followed by purification to obtain it;

[0031]

[0032] The preparation method of the compound with the structure of formula (VIII) includes the following steps: First, 2,5-dimethylbenzoquinone, sodium bisulfite (1 - 20eq), 3,3-dimethylacrylic acid (0.1 - 10eq) and methanesulfonic acid (10 - 200 mL) are mixed to obtain lactone 1. Then lactone 1 is dissolved in a mixed solution of acetonitrile (1 - 50 ml), acetone (0.01 - 10 ml) and water (0.1 - 50 ml), and N-bromosuccinimide (0.8 - 2eq) is added to obtain the compound with the structure of formula (VIII).

[0033] The preparation method of the monomer with the group of formula (V): 1-Aza-18-crown-6 reacts with methacryloyl chloride (0.1 - 10eq), and after purification, it is obtained.

[0034] The preparation method of the monomer with the group of formula (IV): The compound with the structure of formula (VIII) and 1-hydroxybenzotriazole (HOBt) (0.1 - 10eq) are dissolved in dichloromethane, and then 1,3-dicyclohexylcarbodiimide (DCC) (0.1 - 10eq), triethylamine (0.1 - 10eq), 2-aminoethyl methacrylate (0.1 - 10eq) and 4-dimethylaminopyridine (DMAP) (0.1 - 10eq) are added for reaction, and after purification, it is obtained.

[0035] Application of the triblock copolymer in the preparation of anti-tumor drugs.

[0036] The anti-tumor drug includes the triblock copolymer as a hypoxia-responsive ferroptosis inducer.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1) The triblock copolymer of the present invention contains a hydrophilic PEG block, which can increase the blood circulation time of the nano-drug. The crown ether not only acts as a hydrophobic block to promote the self-assembly of the polymer, encapsulate the hydrophobic drug chlorin e6, but also acts on the cell organelles in tumor cells, releases the endogenous iron ions stored in the cells, activates ferroptosis, and the quinone ester in the middle segment of the polymer also has hypoxia responsiveness. By artificially regulating the hypoxic environment of the tumor, the specific activation of ferroptosis is realized. The proton nuclear magnetic resonance spectrum shows the successful preparation of the triblock polymer, and the block ratio of the polymer is adjusted by adjusting the ratio of the monomer and the initiator.

[0039] 2) The nanoparticles prepared by the present invention have a particle size of about 200 nm. The particle size and transmission electron microscopy show that they are regular spherical in shape, with uniform morphology and good dispersibility. Small animal in vivo imaging and fluorescence co-localization show that the nanoparticles can accumulate in the tumor site, enter tumor cells, and act on organelles. Western Blot and immunofluorescence show the destruction of organelles and the release of endogenous iron ions induced by the nanoparticles. Flow cytometry and confocal analysis are used to detect the activation of ferroptosis and the reversal of the tumor immunosuppressive microenvironment. The in vitro anti-tumor results show that the nanoparticles designed by the present invention can significantly inhibit the proliferation of melanoma cells, promote apoptosis and necrosis, and exert a strong anti-tumor effect. At the same time, the in vivo tumor inhibition experiment also shows that the nanoparticles designed by the present invention can not only inhibit the primary tumor, but also induce a strong immune response to inhibit the growth of distal tumors. HE analysis of the main organs of mice verifies the safety of the nanoparticles designed by the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions of the present invention, the drawings required for use in the examples will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0041] Figure 1 1H NMR spectrum of the inducer PEG-CPPA prepared in Example 1 using deuterated dimethyl sulfoxide as a solvent;

[0042] Figure 2 1H NMR spectrum of the block copolymer PEG-PBQAEM-PAA18C6 of formula (I) prepared in Example 3 using deuterated dimethyl sulfoxide;

[0043] Figure 3 1H NMR spectrum of the monomer BQAEM of formula (IV) prepared in Example 2 using deuterated dimethyl sulfoxide as a solvent;

[0044] Figure 4 1H NMR spectrum of the monomer AA18C6 of formula (V) prepared in Example 2 using deuterated dimethyl sulfoxide as a solvent;

[0045] Figure 5 1H NMR spectrum of the block copolymer PEG-PBQAEM prepared in Comparative Example 2 using deuterated dimethyl sulfoxide;

[0046] Figure 6 1H NMR spectrum of the block copolymer PEG-PAA18C6 prepared in Comparative Example 2 using deuterated dimethyl sulfoxide;

[0047] Figure 7Particle size distribution of hPPAA18C6 nanoparticles in Example 4;

[0048] Figure 8 Structural changes of PEG-PBQAEM-PAA18C6 after treatment with sodium thiosulfate in Example 4;

[0049] Figure 9 Particle size distribution of the polymer drug-loaded micelles of hPPAA18C6@Ce6 in Test Example 2;

[0050] Figure 10 Drug release curve of the drug-loaded micelles prepared in Test Example 2 under treatment with sodium thiosulfate;

[0051] Figure 11 Result diagram of the endocytosis of the drug-loaded micelles prepared in Test Example 2 by B16F10 cells;

[0052] Figure 12 Result diagram of the investigation on the release of endogenous iron ions in B16F10 cells induced by the nanoparticles prepared in Example 4;

[0053] Figure 13 Result diagram of the investigation on the accumulation of lipid peroxides in B16F10 cells induced by the nanoparticles prepared in Example 4;

[0054] Figure 14 Result diagram of the investigation on the ferroptosis of B16F10 cells induced by the nanoparticles prepared in Example 4;

[0055] Figure 15 Result diagram of the investigation on the cytotoxicity of the nanoparticles prepared in Example 4 to B16F10 cells;

[0056] Figure 16 Result diagram of the investigation on the immunogenic cell death of B16F10 cells induced by the nanoparticles prepared in Example 4.

[0057] Figure 17 Result diagram of the investigation on the maturation of DC cells induced by the nanoparticles prepared in Example 4.

[0058] Figure 18 Effective anti-cancer effect of the polymer drug-loaded micelles of hPPAA18C6@Ce6 on B16F10 tumors. 1-PBS, 2-hPPAA18C6, 3-free Ce6, 4-hPP@Ce6 or 5-hPPAA18C6@Ce6 NPs were all intravenously injected. Ce6 at 1 mg / kg was injected on days 1, 4, and 7. For free Ce6, hPP@Ce6 or hPPAA18C6@Ce6 NPs, mice were irradiated at the tumor site 12 hours after intravenous injection (666 nm, 5 mW / cm 2 , 10 min);

[0059] Figure 19 Body weights of mice with different treatments, 1-PBS, 2-hPPAA18C6, 3-Ce6-free, 4-hPP@Ce6, or 5-hPPAA18C6@Ce6NPs in Test Example 11. (n = 5);

[0060] Figure 20 H&E staining of major organs of B16F10 transplanted tumor mice on the 14th day of the efficacy study in Test Example 11 (Heart: H, Liver: LI, Spleen: SP, Lung: LU, Kidney: K). Scale bar = 100 μm.

[0061] Figure 21 Analysis of immune cells in mice with different treatments, 1-PBS, 2-hPPAA18C6, 3-Ce6-free, 4-hPP@Ce6, or 5-hPPAA18C6@Ce6NPs in Test Example 11. In Figures B, C, D, and E, PBS, hPPAA18C6, Ce6-free, hPP@Ce6, hPPAA18C6@Ce6NPs in each group are shown from left to right.

[0062] In this application, since the test data involves fluorescence images, etc., for the convenience of analyzing and explaining the test effects, color figures of the specification drawings are provided as other supporting documents. Detailed implementation manners

[0063] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. However, the following embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other implementation manners obtained by those skilled in the art based on the embodiments of the present invention without creative efforts fall within the scope protected by the present invention.

[0064] Materials and methods used in the embodiments of the present invention

[0065] Materials

[0066] Cyano-4-(phenylcarbonothioylthio) pentanoate (CPPA-NHS) was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). PEG-NH 2(Mn = 5000 Da) was purchased from Pengshuo Biotech (Shanghai, China). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-Hydroxybenzotriazole (HOBt), 4-Dimethylaminopyridine (DMAP), Methacryloyl chloride (>98.0%), and Bovine serum albumin (BSA), Triethylamine (>99.0%) and 1-Aza-18-crown-6 (>98.0%) were purchased from Energy Chemical (Shanghai, China). Chlorin e6 (Ce6), a photosensitizer, was purchased from Aladdin (Shanghai, China). Reactive oxygen species (ROS) detection kit (2′,7′-Dichlorodihydrofluorescein diacetate, DCFH-DA), mitochondrial membrane potential detection kit and total glutathione detection kit were purchased from Beyotime (Shanghai, China). FeRhoNox TM-1 (Fe 2+ indicator) and Ru(dpp)Cl 2 (RDPP)(O 2 probe) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). All other solvents and reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. and used as described.

[0067] Instruments

[0068] 1) 1H nuclear magnetic resonance (NMR) spectra were recorded on an Agilent 400 MHz. 1

[0069] 2) The size of the nanoparticles was measured on a Malvern particle size analyzer.

[0070] 3) Transmission electron microscopy (TEM) images were obtained on a FEI Tecnai F20 TEM (Jeol, Japan) at an acceleration voltage of 200 kV.

[0071] 4) UV-visible (UV-vis) spectra were recorded using a microplate reader.

[0072] 5) Confocal laser scanning microscopy (CLSM) images were taken on a TCS SP5 (Leica, Germany) microscope system.

[0073] 6) Flow cytometry analysis was performed using a BD Beckman Coulter flow cytometer (Brea, USA).

[0074] 7) In vivo imaging was performed on a small animal in vivo imaging system (Cambridge, UK).

[0075] Example 1 Preparation of Inducer PEG-CPPA

[0076] PEN-NH 2 ​(100 mg, 0.02 mmol), cyanide-4-(phenylcarbonothioylthio) pentanoate (CPPA-NHS, 45 mg, 0.12 mmol) and triethylamine (0.9 mg, 0.9 mmol) were dissolved in dichloromethane (5 mL). After stirring for 24 h, the mixture was precipitated into cold ether, and the precipitate was collected and dried in a vacuum drying oven to obtain PEG-CPPA (95 mg, yield 93%). The reaction formula is shown in Formula (1).

[0077] When the inducer PEG-CPPA uses deuterated dimethyl sulfoxide as the solvent 1 The HNMR spectrum (nuclear magnetic resonance hydrogen spectrum) is as Figure 1 shown.

[0078]

[0079] Example 2

[0080] Preparation of monomer BQAEM with the group of formula (IV): 2,5-dimethylbenzoquinone (10 g, 73.5 mmol) was dissolved in ether, and an aqueous solution of sodium bisulfite (178 g, 150 mL of water, 85%, 870 mmol) was added. The mixture was shaken until the ether layer was colorless. Extraction was carried out with ether. The obtained ether was washed with brine, dried over MgSO 4 dried, filtered and rotary evaporated to obtain 1,4-benzenediol (10.03 g, 72.6 mmol). 1,4-Benzenediol was mixed with 3,3-dimethylacrylic acid (8.00 g, 80 mmol) and methanesulfonic acid (111 mL). After stirring for 3 h under a nitrogen atmosphere at 85 °C, it was cooled to room temperature. 300 g of ice was added and stirred. The precipitate was extracted with ethyl acetate (4 × 100 mL). The organic layer was washed with saturated sodium bicarbonate solution and dried over MgSO 4 dried. After filtration and evaporation, a residue was obtained and recrystallized with ethyl acetate and n-hexane (1:1, v / v) to obtain lactone 1 (13.02 g, 81%, intermediate 1 in the reaction formula).

[0081] Lactone 1 (300 mg, 1.364 mmol) was dissolved in a mixture of acetonitrile (9 ml), acetone (1 ml) and water (9 ml), then N-bromosuccinimide (243 mg, 1.364 mmol) was added. After stirring at room temperature for 30 min, it was rotary evaporated. The residue was dissolved in ether (1 × 30 mL), and extracted with NaHCO 3 (3 × 10 mL), and the combined aqueous phase was washed with ether (3 × 10 mL). The aqueous solution was acidified to pH 2-3 with HCl and then extracted with ether (2 × 20 mL). After drying over MgSO 4 dried, evaporation gave a yellow oil product 2 (197 mg, 61%, product 2 in the reaction formula). The reaction formula is shown in Formula (2).

[0082]

[0083] The above product 2 (424 mg, 1.17 mmol) and 1-hydroxybenzotriazole (158 mg, 1.17 mmol) were dissolved in dichloromethane (10 mL). 1,3-Dicyclohexylcarbodiimide (309.5 mg, 1.17 mmol) was added to the above mixture, and the mixture was stirred at 0 °C for 30 min. Triethylamine (78.9 mg, 0.78 mmol), 2-aminoethyl methacrylate (100.6 mg, 0.78 mmol), and 4-dimethylaminopyridine (28.1 mg, 0.23 mmol) were added, and the reaction was stirred for 24 hours. The solution was cooled in an ice bath, filtered and evaporated. The residue was dissolved in ethyl acetate (5 mL) and filtered. The filtrate was washed with citric acid (5 mL, 5%, w / v), dried over magnesium sulfate, and evaporated to obtain a crude product as a yellow oil. Column chromatography was performed on silica gel using ethyl acetate as the eluent to purify the crude product, and a yellow solid BQAEM (467.5 mg, yield 97%) was obtained. The reaction formula is shown in formula (3).

[0084] When BQAEM uses deuterated dimethyl sulfoxide as the solvent, 1 the 1H NMR spectrum is as Figure 3 shown.

[0085]

[0086] Preparation of monomer AA18C6 having the group of formula (V):

[0087] 1-Aza-18-crown-6 (3.80 mmol, 1.00 g) was dissolved in anhydrous tetrahydrofuran (THF, 40 mL), and triethylamine (3.80 mmol, 0.384 g) was added. The mixed solution was maintained in an inert atmosphere. Methacryloyl chloride (3.80 mmol, 0.344 g) was dissolved in anhydrous THF (15 mL) and added dropwise to the above mixed solution at room temperature. The reaction mixture was vigorously stirred and heated at 40 °C for 1 h. It was filtered to remove triethylamine hydrochloride, and the filtrate was obtained as a transparent yellow oily product by evaporation under reduced pressure. The oily product was redissolved in dichloromethane (100 mL) and washed with distilled water (100 mL). The dichloromethane solution was dried over magnesium sulfate, filtered, and evaporated under reduced pressure. The crude product was purified by silica gel chromatography (EA:Hex, 20:1) to obtain a transparent colorless oil AA18C6 (1.08 g, yield: 89.9%). The reaction formula is shown in formula (4).

[0088] When AA18C6 uses deuterated dimethyl sulfoxide as the solvent, 1 the 1H NMR spectrum is as Figure 4 shown.

[0089]

[0090] Example 3 Preparation of Triblock Copolymer PEG-PBQAEM-PAA18C6

[0091] PEG-CPPA (113 mg, 0.05 mmol) of Example 1, BQAEM (43 mg, 1.0 mmol) of Example 2, and azobisisobutyronitrile (AIBN) (1.64 mg, 0.01 mmol) were dissolved in 1,4-dioxane (1 mL) under a nitrogen atmosphere. After stirring at 70 °C for 12 h, it was cooled to room temperature and precipitated in cold ether. The precipitate was collected, dissolved in 1,4-dioxane, and reprecipitated in cold ether. The dissolution-precipitation method was repeated once to remove unreacted monomers and initiators. The obtained precipitate was dried in a vacuum oven to obtain a yellow block polymer product PEG-PBQAEM (72 mg, yield: 40%).

[0092] PEG-PBQAEM (113 mg, 0.04 mmol), AA18C6 (139 mg, 0.25 mmol) of Example 2, and AIBN (1.64 mg, 0.01 mmol) were dissolved in 1,4-dioxane (1 mL) under a nitrogen atmosphere. After stirring at 70 °C for 12 h, it was cooled to room temperature and precipitated in cold ether. The precipitate was collected, dissolved in 1,4-dioxane, and reprecipitated in cold ether. The dissolution-precipitation method was repeated once to remove unreacted monomers and initiators. The obtained precipitate was dried in a vacuum oven to obtain a PEG-PBQAEM-PAA18C6 polymer product (172 mg, yield: 63%), and the reaction formula is shown in Formula (5).

[0093] When using deuterated dimethyl sulfoxide as the solvent 1 The 1H NMR spectrum is as Figure 2 shown

[0094] By comparing the peak of the methylene protons (δ 3.70 ppm), the peak of the methylene protons of BQAEM (δ 5.2 ppm), and the peak of the crown ether (δ 3.5 ppm), the degrees of polymerization of BQAEM and AA18C6, PBQAEM are 13 and 45, respectively.

[0095]

[0096] Comparative Example 2

[0097] PEG-CPPA (113 mg, 0.05 mmol), BQAEM (43 mg, 1.0 mmol) and AIBN (1.64 mg, 0.01 mmol) were dissolved in 1,4-dioxane (1 mL) under a nitrogen atmosphere. After stirring at 70 °C for 12 h, it was cooled to room temperature, precipitated in cold diethyl ether, the precipitate was collected, dissolved in 1,4-dioxane, and reprecipitated in cold diethyl ether. The dissolution-precipitation process was repeated once to remove any unreacted monomers and initiators. The resulting precipitate was dried in a vacuum oven to obtain a yellow block polymer PEG-PBQAEM (72 mg, yield: 42%), and the reaction formula is shown in Formula (6).

[0098] The 1 HNMR spectrum of PEG-PBQAEM with deuterated dimethyl sulfoxide as the solvent is as Figure 5 shown.

[0099]

[0100] PEG-CPPA (113 mg, 0.05 mmol), AA18C6 (145 mg, 1.0 mmol) and AIBN (1.64 mg, 0.01 mmol) were dissolved in 1,4-dioxane (1 mL) under a nitrogen atmosphere. After stirring at 70 °C for 12 h, it was cooled to room temperature and precipitated in cold diethyl ether. The precipitate was collected, dissolved in 1,4-dioxane, and reprecipitated in cold diethyl ether. This dissolution-precipitation process was repeated once to remove any unreacted monomers and initiators. The resulting precipitate was dried in a vacuum oven to obtain a yellow block polymer PEG-PAA18C6 (72 mg, yield: 40%), and the reaction formula is shown in Formula (7).

[0101] The 1 HNMR spectrum of PEG-PAA18C6 with deuterated dimethyl sulfoxide as the solvent is as Figure 6 shown.

[0102]

[0103] Example 4 Preparation of hPPAA18C6 nanoparticles

[0104] hPPAA18C6 nanoparticles self-assembled from PEG-PBQAEM-PAA18C6 were prepared by the solvent exchange method. PEG-PBQAEM-PAA18C6 was dissolved in THF (10 mg / mL), and 50 μL was taken and added to PBS buffer (950 μL, 10 mM, pH 7.4). It was left standing overnight, and the diameter and morphology of hPPAA18C6 were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The particle size distribution and Tyndall image of the nanoparticles are asFigure 7 as shown

[0105] It is known from Figure 7 that the particle size of the nanoparticles is about 200 nm, and they are evenly dispersed, showing an obvious Tyndall phenomenon.

[0106] Test Example 1 Hypoxic reactivity of PEG-PBQAEM-PAA18C6

[0107] Sodium metabisulfite (Na 2 S 2 O 4 ) was used as the hypoxia inducer.

[0108] The prepared hPPAA18C6 nanoparticles (1 mL, 5 mg / mL) were added to Na 2 S 2 O 4 (1 mM), and after stirring at 37 °C for 12 hours. The structure of the polymer changed as Figure 8 shown. The particle size distribution and Tyndall image of the nanoparticles are as Figure 7 shown.

[0109] It is known from Figure 7 that after treatment with sodium thiosulfate, the particle size of the nanoparticles became heterogeneous and disordered, the morphology of the nanoparticles was no longer complete and uniform, with different sizes, and the Tyndall phenomenon disappeared, indicating that the triblock polymer responded to hypoxia and its structure changed. As Figure 8 shown, the middle hydrophobic block became a hydrophilic block, the hydrophilic-hydrophobic ratio of the triblock polymer changed, and the self-assembly behavior was disrupted, and the nanostructure was no longer complete.

[0110] Test Example 2 Preparation and characterization of hPPAA18C6@Ce6 micelles

[0111] PEG-PBQAEM-PAA18C6 was dissolved in THF (50 μL, 10 mg / mL) and Ce6 (10 μL, 10 mg / mL) was added to PBS buffer (950 μL, 10 mM, pH 7.4), and left standing for 24 h. Then it was dialyzed in pure water for 12 h, and the water was changed 5 times during the dialysis process. The diameter size and morphology of hPPAA18C6@Ce6 were characterized by DLS and TEM. The particle size distribution of the nanoparticles is as Figure 9 shown. The content of Ce6 in the drug-loaded nanoparticles was determined by fluorescence spectroscopy, and the drug loading was 10%.

[0112] It is known from Figure 9 that the particle size of the drug-loaded nanoparticles is still about 200 nm, without obvious increase. The nano-drug has a Tyndall phenomenon like cotton thread and can be evenly dispersed in the solution.

[0113] Test Example 3

[0114] The hPPAA18C6@Ce6 micelles (50 μg Ce6 / mL, 1 mL) of Test Example 2 were placed in a dialysis bag (MWCO = 100 kDa) and immersed in PBS buffer (pH 7.4, 25 mL), with or without Na 2 S 2 O 4 (1 mM), and incubated with shaking at 37 °C. At determined time intervals, 100 μL of the release medium was extracted and refreshed with the same volume of release medium. The content of Ce6 in the harvested release medium was measured using a microplate reader (λex = 635 nm, λem = 660 nm). The drug release curve is as Figure 10 shown.

[0115] It can be Figure 10 seen that Ce6 can be rapidly released in a hypoxic environment, and 80% of Ce6 is released within 24 h. This indicates that the nano-drug prepared by the present invention can respond to the hypoxic environment and specifically release the encapsulated drug.

[0116] Test Example 4 Investigation of the endocytosis of the prepared drug-loaded micelles by B16F10 cells

[0117] First, B16F10 cells were seeded into glass-bottom culture dishes (5 × 10 5 ) and cultured for 24 h. The medium was replaced with fresh medium containing free Ce6 or hPPAA18C6@Ce6 (5 μg / mL Ce6). After incubation for 2, 4, and 8 h, the cells were washed 3 times with PBS buffer, stained with DAPI (5 μg / mL, 10 min), fixed with paraformaldehyde (4%, 10 min), and observed using CLSM (λex = 635 nm, λem = 660 nm). The results are as Figure 11 shown.

[0118] It can be Figure 11 seen that the cellular uptake of free Ce6 and hPPAA18C6@Ce6 by B16F10 cells gradually increases with time. This indicates that the nano-drug prepared by the present invention can be well endocytosed into cells.

[0119] Test Example 5 Release of endogenous iron ions from B16F10 cells induced by hPPAA18C6 nanoparticles

[0120] After B16F10 cells were treated with PBS, hPP, PPAA18C6, or hPPAA18C6 under hypoxia for 24 h, they were stained with DAPI (blue) and FeRhonoxRM-1 (red), respectively. As Figure 12 shown.

[0121] It can be Figure 12It is known that polymers containing the PAA18C6 block can release intracellular endogenous iron ions, and hypoxia-responsive triblock polymers can better expose the PAA18C6 block to release endogenous iron ions.

[0122] Experimental Example 6: Accumulation of lipid peroxides in B16F10 cells induced by hPPAA18C6 nanoparticles

[0123] After B16F10 cells were treated with PBS, hPP(PEG-PBQAEM), PPAA18C6(PEG-PAA18C6), or hPPAA18C6 nanoparticles under hypoxia for 24 h, they were stained with DAPI (blue) and BODIPY581 / 591-C11 (green) respectively. As Figure 13 shown.

[0124] It is Figure 13 known that polymers containing the PAA18C6 block can release intracellular endogenous iron ions, undergo Fenton reaction to generate peroxides. Moreover, hypoxia-responsive triblock polymers can better expose the PAA18C6 block to release endogenous iron ions and generate more peroxides.

[0125] Experimental Example 7: Ferroptosis induction in B16F10 cells by hPPAA18C6 nanoparticles

[0126] After B16F10 cells were treated with PBS, hPP(PEG-PBQAEM), PPAA18C6(PEG-PAA18C6), or hPPAA18C6 nanoparticles under hypoxia for 24 h, the cells were collected, washed 3 times with PBS, then lysed, and proteins were extracted for WB analysis. The results are as Figure 14 shown, Figure 14 where I is PBS, II is hPP, III is PPAA18C6, and IV is hPPAA18C6.

[0127] It is Figure 14 known that polymers containing the PAA18C6 block can induce autophagy, damage organelles, release intracellular endogenous iron ions, and induce ferroptosis. Moreover, hypoxia-responsive triblock polymers can better expose the PAA18C6 block to release endogenous iron ions and induce ferroptosis.

[0128] Experimental Example 8: Cytotoxicity of hPPAA18C6 nanoparticles to B16F10 cells

[0129] The cytotoxicity of hPPAA18C6 nanoparticles was investigated by MTT cytotoxicity assay. The specific steps are as follows:

[0130] Collect B16F10 cells for several passages and seed them into a 96-well plate, with 100 μL (10,000 cells) in each well; incubate in a cell culture incubator at 37 °C, saturated humidity, and 5% CO 2 for 24 h; after 24 h, discard the culture medium, dilute the prepared hPPAA18C6@Ce6 nanoparticles to different concentrations with the medium, add them to the 96-well plate, 100 μL in each well, with 3 replicates for each concentration; incubate in a cell culture incubator at 37 °C, saturated humidity, and 5% CO 2 for 8 h; irradiate with a laser (660 nm, 5 mW / cm 2 , 10 min), after 24 or 48 h, add 20 μL of MTT solution with a concentration of 5 mg / mL to each well and continue to incubate for 4 h; terminate the culture, aspirate the culture medium in the wells, add 200 μL of dimethyl sulfoxide to each well, oscillate at low speed for 10 min, and measure the absorbance of each well at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader to calculate the cell survival rate. The results are as Figure 15 shown.

[0131] It is Figure 15 known that both the free photosensitizer and the hPP@Ce6 nanoparticles encapsulated with the photosensitizer can produce the effect of photodynamic therapy after light irradiation, inhibit the proliferation of tumor cells, and promote their apoptosis. Moreover, after combining with ferroptosis induced by PAA18C6, the ability of hPAA18C6@Ce6 to kill tumor cells is significantly enhanced, demonstrating the ability of the PAA18C6 block copolymer to induce ferroptosis and its synergistic effect with photodynamic therapy.

[0132] Experimental Example 9 hPPAA18C6@Ce6 induces immunogenic cell death in B16F10 cells

[0133] Immunogenic cell death (ICD) is a type of programmed cell death that elicits an immune response by releasing tumor-associated antigens (TAAs) and danger-associated molecular patterns (DAMPs), including calreticulin (CRT), high-mobility group box 1 (HMGB1), and adenosine triphosphate (ATP). First, the ICD effect induced by the combination of Ce6 photodynamic therapy and hPAA18C6 ferroptosis was investigated through the characteristic biomarkers of ICD in B16F10 cells.

[0134] Experimental method for detecting the ICD effect in vitro: Seed B16F10 cells at 30,000 cells / well into a 24-well plate and incubate for 24 h. Replace the culture medium with fresh medium containing free Ce6, hPP@Ce6, hPPAA18C6@Ce6 (5 μg / mL Ce6). After incubating for 8 h, irradiate the cells (660 nm, 5 mW / cm 2 , 10 min). After incubating for another 24 hours, the cells are 488 anti-calreticulin staining was used to detect the expression of calreticulin in B16F10 cells. The results are as Figure 16 shown in a. ELISA and ATP detection kits were used to detect the levels of HMGB1 and ATP in the supernatant. The results are as Figure 16 shown in c and 16b, respectively.

[0135] As Figure 16 shown in a, in the cells treated with hPPAA18C6@Ce6, the fluorescence of CRT was the most obvious in all groups, indicating that hPPAA18C6@Ce6 induced ICD to the best extent. As Figure 16 shown in c and 16b, it was determined by the ATP detection kit that free Ce6 or hPP@Ce6 released more ATP than PBS due to the PDT effect, while the cells treated with hPPAA18C6@Ce6 showed the most significant ATP release, up to 70%. Similarly, compared with the treatment with free Ce6 or hPP@Ce6, the release amount of HMGB1 induced by hPPAA18C6@Ce6 was also the highest. This is because hPPAA18C6@Ce6 not only has the ability of photodynamic therapy but also can induce ICD through ferroptosis. Among them, blue (label 1) is PBS, red (label 2) is free Ce6, green (label 3) is hPP@Ce6, and purple (label 4) is hPPAA18C6@Ce6.

[0136] Experimental Example 10 Induction of DC cell maturation by hPPAA18C6@Ce6

[0137] The hind limbs of C57 mice (6 - 8 weeks old) were taken, and the bone marrow cavity was rinsed with RPMI1640 medium to prepare a bone marrow suspension, and then red blood cells were removed with a red blood cell lysis solution. Then the bone marrow cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, 1% penicillin / streptomycin, 10 ng / mL murine recombinant murine granulocyte / macrophage colony-stimulating factor (GM-CSF), and 50 ng / mL IL-4. Half of the medium was replaced every other day. After repeating 3 times, BMDCs were obtained and used for the following experiments.

[0138] B16F10 cells were seeded on a 6-well plate at a concentration of 5×10 4 cells / well and cultured for 24 h. The medium was replaced with fresh medium containing PBS, free Ce6, hPP@Ce6, hPPAA18C6@Ce6 (5 μg / mL Ce6). After incubation for 8 h, the cells were irradiated (660 nm, 5 mW / cm 2 , 10 min). After incubation for 24 h, the supernatant was collected and BMDCs (50% fresh medium) were incubated for 24 h. Staining was performed with CD45, CD11c, CD80, and CD86, and then analyzed by flow cytometry. The results are as Figure 17As shown

[0139] It can be seen from Figure 17 that free Ce6 and hPP@Ce6 can induce the maturation of dendritic cells through photodynamic therapy, and hPPAA18C6@Ce6 can induce the maturation of dendritic cells through ferroptosis and photodynamic therapy, with the highest degree of maturation.

[0140] Experimental Example 11 In Vivo Antitumor Efficacy

[0141] The specific experimental steps are as follows:

[0142] (1) Female C57 mice were subcutaneously injected with B16F10 cells on the right (10 6 ) and left (10 5 ) flanks to establish primary and distant tumor models, as Figure 18 shown in A. When the volume of the right tumor reached approximately 100 mm 3 , the mice were divided into 5 groups and injected intravenously. PBS, hPPAA18C6, free Ce6, hPP@Ce6, and hPPAA18C6@Ce6 were injected at an equivalent dose of Ce6 (1 mg / kg).

[0143] (2) 12 h after injection, the right tumor was irradiated with a 660 nm laser at 5 mW / cm 2 for 10 min. The size and weight of the tumor were measured every other day. The tumor volume (V, mm 3 ) was calculated according to the formula: V = length × width × width / 2. As Figure 18 shown in B - E. The body weights of the mice in each group were weighed during this period, and the results are as Figure 19 shown.

[0144] (3) On the 14th day, the mice were sacrificed, and the main organs and tumors were excised.

[0145] (4) The tumors were weighed and imaged, and then the tumors were fixed with 4% formalin, embedded in paraffin, sectioned at a depth of 7 μm, stained with hematoxylin and eosin (H&E) and TUNEL-(G), and then subjected to histological evaluation, as Figure 18 shown in F and 18G. At the same time, the main organs were stained with H&E (heart: H, liver: LI, spleen: SP, lung: LU, kidney: K), and the results are as Figure 20 shown.

[0146] (5) To detect the apoptosis of tumor cells, the tumors were embedded in OCT, cryosectioned, and detected using a OneStepTUNEL apoptosis detection kit. At the same time, the immune cells were stained with fluorescently labeled antibodies for 20 min and then analyzed by flow cytometry.

[0147] It can be seen from Figure 18As shown by B-G, the tumors of mice treated with PBS increased rapidly to approximately 1000 mm within 14 days. 3 . Compared with free Ce6 that slightly inhibited tumor growth, hPP@Ce6 exhibited a stronger tumor inhibition level, mainly due to the increased accumulation of Ce6 at the tumor site through the nanosystem. The results showed that the PDT method delayed tumor growth but did not produce an inhibitory effect. Due to siderosis, treatment with hPPAA18C6 also showed a slight inhibition of primary tumor growth. The hPPAA18C6@Ce6 group had the strongest inhibition of primary and abdominal tumor growth, with the most obvious tumor cell necrosis and apoptosis, while the other treatment groups had no obvious inhibition of abdominal tumor growth.

[0148] It can be seen from Figure 19 and 20 that there were no obvious changes in the body weights of mice and no damage to normal organs in each group, indicating that the prepared nanodrugs had good biosafety.

[0149] As shown by Figure 21 , the infiltration ratios of CD45+CD8+ T cells and CD45+CD4+ T cells in the primary and distant tumors of the hPPAA18C6@Ce6 group were significantly increased compared with other control groups. At the same time, the numbers of MDSC cells and NK cells were detected. The results showed that CD45+CD8+ and CD45+NK1.1+ cells in the tumors were significantly increased after treatment with hPPAA18C6@Ce6, while CD45+CD11b+Gr1+ cells were significantly decreased. All these results indicate that hPPAA18C6@Ce6 can effectively destroy the primary tumor and exhibit an anti-tumor immune response of an in-situ vaccine against distant tumors.

Claims

1. A triblock copolymer, characterized in that, the structural formula of the polymer is shown as formula (Ⅶ): 20 ≤ n ≤ 500; 5 ≤ x ≤ 200; 5 ≤ y ≤ 200.

2. Use of the triblock copolymer according to claim 1 in the preparation of an anti-tumor drug.

3. The use according to claim 2, characterized in that, the anti-tumor drug comprises the triblock copolymer as a hypoxia-responsive ferroptosis inducer.

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