Supramolecular macrocyclic engineered protein regulator as well as preparation method and application thereof

Abstract

The invention discloses a supramolecular macrocyclic engineered protein regulator with an azo reductase response characteristic as well as a preparation method and application of the supramolecular macrocyclic engineered protein regulator. The preparation method comprises the following steps: synthesizing a catalase derivative CAT-SAC4A through a ring-opening reaction of an epoxy group, and loading HCQ through host-guest interaction of a supramolecular macrocyclic SAC4A to obtain the CAT-SAC4A-coated HCQ. The supramolecular macrocyclic engineered protein regulator CAT-SAC4A (at) HCQ can improve the problem that CAT is easily degraded by protease in vivo, obviously improves the intracellular level of CAT, and meanwhile, specifically responds to tumor high-expression azo reductase to release HCQ. Besides, the CAT-SAC4A-coated HCQ can obviously improve the hypoxia state of tumors and effectively block the autophagy process, so that strong anti-tumor immune response is stimulated, and the purposes of enhancing the tumor killing ability and relieving toxic and side effects are achieved.

Description

Technical Field

[0001] The present invention belongs to the field of nano biomedical materials, and relates to a preparation method of a supramolecular macrocyclic engineered protein regulator for regulating the tumor hypoxia-autophagy microenvironment and its application as a supramolecular radiotherapy sensitizer in improving the effect of tumor radioimmunotherapy. Background Art

[0002] In recent years, significant progress has been made in the field of cancer treatment, and the combined strategy of radiotherapy (RT) and immunotherapy has attracted much attention. However, tumor heterogeneity and the complex immunosuppressive tumor microenvironment (Tumor Microenvironment, TME) are still the main obstacles to treatment success.

[0003] Studies have shown that 50 - 60% of solid tumors exhibit significant hypoxia characteristics due to rapid proliferation and abnormal angiogenesis. This hypoxic state not only leads to radiation tolerance, but also promotes autophagy of tumor cells through pathways such as activation of hypoxia-inducible factor-1α (Hypoxia inducible factor-1α, HIF-1α), thereby weakening the immune killing effect mediated by T cells. In addition, the vicious cycle between hypoxia and autophagy further exacerbates the radioresistance of tumors: autophagy provides the energy and nutrients required for the survival of tumor cells, promotes their proliferation, and further aggravates hypoxia; while the ionizing radiation and hypoxic stress generated during radiotherapy will further induce autophagy, forming a vicious cycle of treatment resistance. This complex TME not only limits the effective delivery of drugs, but also reduces the effect of aerobic therapy, ultimately leading to an increased risk of tumor invasion and metastasis. Therefore, it is urgent to develop innovative strategies that can reprogram the tumor hypoxia-autophagy TME and synergistically enhance the effect of radio-immunotherapy.

[0004] Regarding the problem of tumor hypoxia, the existing technologies mainly focus on direct oxygen supply, in-situ oxygen production, and improvement of blood circulation. Among them, catalase (Catalase, CAT) shows great potential in improving tumor hypoxia because it can catalyze the decomposition of hydrogen peroxide into oxygen. However, simply relieving hypoxia cannot completely inhibit the survival of tumor cells mediated by autophagy. Hydroxychloroquine (Hydroxychloroquine, HCQ), as a clinically approved autophagy inhibitor, can effectively inhibit the autophagy process by alkalizing lysosomes to block autophagosome-lysosome fusion. However, the clinical application of CAT and HCQ faces many challenges: CAT is easily degraded by proteases in vivo, and there are problems such as immunogenicity and short half-life; while HCQ has poor solubility and lack of targeting, which limits its efficacy. Therefore, it is of great significance to design a nanocarrier that can simultaneously deliver CAT and HCQ and overcome the above limitations. Summary of the Invention

[0005] In recent years, supramolecular nano-platforms based on azocalix[4]arene (AC4A) have received extensive attention due to their unique azoreductase response characteristics in the tumor microenvironment (TME). Compared with traditional azoreductase-responsive materials, modified AC4A derivatives (such as sulfonated azocalix[4]arene, SAC4A) have high binding affinity and precise drug-loading ability.

[0006] The present invention aims to develop an innovative supramolecular macrocycle-engineered protein regulator (CAT-SAC4A@HCQ), which has the following advantages: 1) Simple synthesis and low cost. 2) Good biosafety and easy to achieve clinical translation. After healthy BALB / c mice were randomly grouped, CAT-SAC4A@HCQ was injected into the tail vein. Blood samples were taken on the second and seventh days to detect blood indexes. No obvious changes were found in blood biochemical and blood routine parameters, which were within the normal physiological range, proving that CAT-SAC4A@HCQ has good biosafety. 3) Hypoxia-relieving ability: Breast cancer 4T1 cells were selected. After co-incubation with CAT-SAC4A@HCQ, a significant decrease in the expression of hypoxia-inducible factor HIF1-α was observed by confocal fluorescence microscopy. 4) Autophagy-blocking ability: Breast cancer 4T1 cells were selected. After co-incubation with CAT-SAC4A@HCQ, the expression levels of autophagy-related proteins were detected by Western blot. The LC3-II / LC3-I ratio increased significantly, and at the same time, the expression of autophagy substrate p62 increased, proving that CAT-SAC4A@HCQ can effectively block the autophagy process. 5) Compared with free CAT, CAT-SAC4A@HCQ showed more excellent antitumor radiotherapy effect: Colony formation assay: Breast cancer 4T1 cells were used as a cell model. At the same irradiation dose, CAT-SAC4A@HCQ could significantly reduce the number of tumor cell colonies and inhibit the growth of tumor cells.

[0007] In the present invention, SAC4A was first modified onto CAT based on the ring-opening reaction of epoxy groups to obtain CAT-SAC4A, and then HCQ was loaded through host-guest interaction to obtain CAT-SAC4A@HCQ. CAT decomposes hydrogen peroxide in the TME to generate oxygen to relieve tumor hypoxia and enhance radiotherapy-induced DNA damage; HCQ is precisely released in response to azoreductase highly expressed in the tumor site and further enhances the immune effect by inhibiting autophagy; the modification of the supramolecular macrocycle improves the stability and targeting of the system, avoiding immune clearance and drug leakage.

[0008] To achieve the above object, the present invention discloses the following technical solutions: A supramolecular macrocycle-engineered protein regulator CAT-SAC4A@HCQ, the structure of which is as follows: 1) Enzyme catalytic core: Composed of catalase (CAT), retaining its natural catalytic activity, capable of decomposing hydrogen peroxide into oxygen; 2) Supramolecular macrocyclic carrier: Sulfonated azocalix[4]arene (SAC4A), which is covalently linked to the amino group on the surface of the catalase through its epoxy group to form a CAT-SAC4A complex; 3) Drug loading unit: Hydroxychloroquine (HCQ) is encapsulated in the cavity of the sulfonated azocalix[4]arene through host-guest interaction to form a CAT-SAC4A@HCQ complex; Among them, the azo group of the sulfonated azocalix[4]arene can be specifically reduced by azoreductase in the tumor microenvironment, resulting in the targeted release of the hydroxychloroquine. At the same time, the catalytic activity of the catalase is not affected by the covalent modification; Structural feature description: 1) Covalent connection: The epoxy group of SAC4A and the amino group of CAT form a stable covalent bond through a nucleophilic ring-opening reaction, ensuring the structural stability of the complex in the physiological environment; 2) Host-guest loading: The hydrophobic cavity of SAC4A efficiently encapsulates HCQ through π-π stacking and hydrophobic interactions, with a loading rate ≥85%, and the drug release depends on the enzyme responsiveness of the tumor microenvironment; 3) Functional synergy: CAT maintains the catalytic oxygen production function to relieve tumor hypoxia; SAC4A realizes the spatio-temporal controlled release of HCQ to block protective autophagy; the two cooperate to induce oxidative stress and immunogenic cell death (ICD), reversing the immunosuppressive microenvironment.

[0009] The present invention further discloses a preparation method of the supramolecular macrocyclic engineered protein regulator CAT-SAC4A@HCQ, which is characterized by the following steps: (1) Synthesis of SAC4A: SAC4A is synthesized from 25, 26, 27, 28-tetrahydroxycalix[4]arene (C4A) and p-aminobenzenesulfonic acid as raw materials through a diazo coupling reaction. The steps are as follows: 1) Dissolve p-aminobenzenesulfonic acid (1.73 g, 10 mmol) and concentrated hydrochloric acid (2 mL, 37%) in 15 mL of water in a round-bottom flask. After cooling the solution to 2°C in an ice-water bath, slowly add an aqueous solution of sodium nitrite (10 mL, 10 mmol), while controlling the temperature below 5°C.

[0010] 2) The resulting solution was slowly added to a solution of 25,26,27,28 - tetrahydroxycalix[4]arene (C4A, 1.0 g, 2.36 mmol) and sodium acetate (2.46 g, 30 mmol) in MeOH - DMF (26 mL, 5:8, V:V) to obtain a red suspension.

[0011] 3) After stirring at room temperature for 2 h, hydrochloric acid solution (37%) was added until the solution was adjusted to pH = 1. After reacting at 60 °C for 30 min, the mixture was filtered and washed with water and MeOH to obtain a light red solid SAC4A in quantitative yield.

[0012] (2) SAC4A was reacted with 1 - bromo - 2,3 - epoxypropane to obtain SAC4A - epoxy; the steps were as follows: 1) 35 mg of SAC4A was dissolved in 4 mL of DMF, and 400 mg of Na 2 CO 3 (100 mg / mL) and 1 - bromo - 2,3 - epoxypropane (1 g, 7.3 mmol) were added in sequence, and the mixture was stirred in the dark at room temperature for 24 h; 2) After the reaction, the insoluble Na 2 CO 3； 3) The supernatant was taken and dropped into an excess of ether to precipitate and remove the unreacted 1 - bromo - 2,3 - epoxypropane. Then the precipitate was collected and dried in vacuo to obtain SAC4A - epoxy; (3) SAC4A - epoxy was conjugated to CAT through the reaction between the amino group of CAT and SAC4A - epoxy to prepare CAT - SAC4A; the steps were as follows: 1) 100 mg of CAT powder was accurately weighed and added to a round - bottom flask, and 25 mL of Na 2 CO 3 buffer solution with a concentration of 100 mM was added, and the mixture was stirred at low speed until CAT was completely dissolved; 2) Subsequently, 479 μL of 50 mg / mL SAC4A - epoxy was added dropwise, and the mixture was stirred in the dark at room temperature for 24 h; 3) The product was separated by dialysis against water using (MWCO 10000), then ultrafiltration using MWCO 30000 and desalting using a desalting column with MWCO 7000; The methanol solution of HCQ with the same concentration of SAC4A as in CAT - SAC4A was mixed together, and the mixture was gently inverted and mixed evenly to obtain CAT - SAC4A@HCQ.

[0013] The present invention also discloses the application of the supramolecular macrocyclic engineered protein regulator CAT-SAC4A@HC in the preparation of drugs for enhancing tumor killing ability and reducing radiation-mediated toxicity. Experimental results show that: the number of cell clone colonies after treatment with CAT-SAC4A@HCQ is significantly lower than that of the PBS group and the free CAT group. This strategy can significantly relieve the tumor hypoxia state and block the protective autophagy process of tumor cells, thereby achieving the purpose of enhancing tumor killing ability and reducing toxicity by regulating the TME.

[0014] The present invention is described in more detail as follows: The present invention further studied the hypoxia-relieving situation of the supramolecular macrocyclic engineered protein regulator CAT-SAC4A@HCQ in 4T1 cells, and the steps are as follows: 1) Inoculate 4T1 cells at a density of 3×10 5 in a confocal microscope culture dish and incubate for 24 h.

[0015] 2) After the cells are fully adhered, discard the original culture medium and re-add the culture medium containing CAT (4 μM), CAT-SAC4A (20 μM), and CAT-SAC4@HCQ (20 μM / 20 μM) and act for 24 h.

[0016] 3) Add PBS to wash the cell surface and fix it with 4% paraformaldehyde solution at room temperature for 30 min.

[0017] 4) Re-add PBS to wash the cell surface and permeabilize it with pre-cooled ice methanol solution at -20°C for 15 min.

[0018] 5) After washing the cells with PBS again, block the cells with 1% BSA solution at room temperature for 1 h.

[0019] 6) Discard the BSA solution, wash the cells with PBS, add the HIF1-α primary antibody solution diluted with 1% BSA, and incubate overnight at 4°C.

[0020] 7) Discard the primary antibody solution, wash 3 times with PBS, add the fluorescent secondary antibody solution diluted with 1% BSA, and incubate at room temperature in the dark for 1 h.

[0021] 8) Discard the secondary antibody solution, wash 3 times with PBS, continue to add 500 μL of DAPI staining solution, and stain at room temperature in the dark for 10 min.

[0022] 9) Wash the cells with PBS, then add PBS buffer and infiltrate at 4°, and place on the sample stage of a confocal fluorescence microscope to take fluorescence photos.

[0023] The present invention also discloses the regulation of the expression of autophagy-related proteins by the supramolecular macrocyclic engineered protein regulator CAT-SAC4A@HCQ in 4T1 cells, and the steps are as follows: 1) Take 4T1 cells in the logarithmic growth phase and inoculate them in a 6-cm diameter culture dish, and culture them in an incubator for 24 h.

[0024] 2) Re-add the medium containing CAT (4 μM), CAT-SAC4A (20 μM), and CAT-SAC4@HCQ (20 μM / 20 μM) and incubate for 24 h. After replacing with fresh medium, irradiate with γ-rays (6 Gy), and continue to culture for 24 h.

[0025] 3) Digest the cells with trypsin and collect them, and lyse them in RIPA lysis buffer containing PMSF (1 mM) at 4 °C for 30 minutes. Centrifuge at 12,000 rpm for 10 minutes at 4 °C to obtain the supernatant containing total cellular proteins. The extracted proteins are further quantified using a BCA protein assay kit.

[0026] 4) Subsequently, separate different samples of equal amounts of protein (50 μg) using an SDS-PAGE gel and electrophoretically transfer them to a PVDF membrane.

[0027] 5) Block non-specific binding sites using a TBST solution containing 5% skim milk. Then, incubate the PVDF membrane with primary antibodies (such as anti-ATG5 antibody (Abmart), anti-Beclin-1 antibody (Abmart), anti-LC3 antibody (CST), anti-p62 antibody (Abmart)) overnight.

[0028] 6) After incubation with the primary antibody, incubate the membrane with a horseradish peroxidase-conjugated anti-rabbit antibody for 1 h, and detect the luminescence using an enhanced chemiluminescence (ECL) detection reagent.

[0029] GAPDH is detected as a housekeeping protein control.

[0030] The present invention also studied the evaluation of the radiotherapy sensitization ability of the supramolecular macrocyclic engineered protein regulator CAT-SAC4A@HCQ on 4T1 cells, and the steps are as follows: 1) Take 4T1 cells in the logarithmic growth phase and inoculate them into 12-well plates at a density of 500 cells per well, and culture them in an incubator for 24 h.

[0031] 2) Add CAT, CAT-SAC4A, and CAT-SAC4@HCQ with the same drug concentration to the 12-well plates respectively and continue to culture for 24 h.

[0032] 3) After replacing with fresh culture medium, ionizing radiation with irradiation doses of 0, 2, 4, and 6 Gy was carried out respectively, and then placed in an incubator for another 7 days to wait for the formation of cell clone clusters, and the cell clone clusters in each group were counted.

[0033] The present invention further studied the hematological indexes after injecting the supramolecular macrocyclic engineered protein regulator CAT-SAC4A@HCQ into BALB / c mice to evaluate the biosafety of this supramolecular drug. The steps are as follows: 1) Healthy BALB / c mice at 5-6 weeks of age were randomly divided into 5 groups, and PBS (200 μL), CAT (200 μL, 80 μM), CAT-SAC4A (200 μL, 400 μM), and CAT-SAC4A@HCQ (200 μL, 400 μM) were injected via the tail vein respectively.

[0034] 2) On the 2nd and 7th days after drug injection, 100 μL of blood was collected from the posterior orbital venous plexus of mice into an anticoagulant tube and gently shaken evenly, and the changes in blood routine indexes were detected using an automatic hematology analyzer.

[0035] 3) On the 7th day after drug injection, the whole blood of mice was put into a 1.5 mL centrifuge tube. After the plasma coagulated, it was centrifuged in a centrifuge (3000 rpm, 5 min), and the upper layer of serum was aspirated for the next detection of liver and kidney function indexes.

[0036] The present invention mainly solves the problems that CAT and HCQ have poor stability, cannot effectively relieve tumor hypoxia and block autophagy, resulting in poor radiosensitization effect. It focuses on investigating the physicochemical properties of supramolecular engineered nanoparticles and their effects on enhancing radiation-mediated inhibition of tumor proliferation. The main difficulty lies in the rational design of the supramolecular macrocyclic structure so that it can successfully bind to the surface of proteases, thereby increasing the intracellular level of proteases.

[0037] The positive effects of the supramolecular macrocyclic engineered protein regulator capable of regulating the tumor hypoxia-autophagy microenvironment disclosed by the present invention, its preparation method and application are as follows: it can greatly improve the bioavailability of catalase and hydroxychloroquine, enhance the radiosensitization effect of radioimmunotherapy, and provide new ideas for the development of tumor radiotherapy strategies. Description of the Drawings

[0038] Figure 1 It is the property characterization of the supramolecular macrocyclic engineered protein regulator CAT-SAC4A@HCQ; where A is the transmission electron microscope photograph; B is the characterization of the binding ability of CAT-SAC4A and HCQ; C is the characterization of the oxygen production performance of CAT-SAC4A@HCQ; Figure 2Effect of CAT-SAC4A@HCQ on alleviating hypoxia in 4T1 cells; A is the confocal microscopy image of the expression of HIF1-α in 4T1 cells after different treatments; B is the quantitative analysis of HIF1-α. Figure 3 Regulation of the expression of autophagy-related proteins by CAT-SAC4A@HCQ in 4T1 cells; A is the WB band expression of autophagy-related proteins in 4T1 cells after different treatments; B is the quantitative analysis of the expression of autophagy-related proteins in 4T1 cells after different treatments. Figure 4 Radiosensitization ability of CAT-SAC4A@HCQ; A is the colony formation of breast cancer 4T1 cells treated with CAT, CAT-SAC4A and CAT-SAC4A@HCQ under different irradiation doses (0, 2, 4 and 6 Gy), and B is the clonogenic survival curve is plotted. Figure 5 Biological safety test of CAT-SAC4A@HCQ; Figure 6 Schematic diagram of the structure of CAT-SAC4A@HCQ and its preparation flow chart; Figure 7 Process of SAC4A generating p-aminobenzenesulfonic acid and aminocalixarene under the action of azoreductase. Detailed implementation manners

[0039] The present invention will be described below through specific implementation examples. Unless otherwise specified, the technical means used in the present invention are all methods well known to those skilled in the art. In addition, the implementation examples should be understood as illustrative and not limiting the scope of the present invention. The essence and scope of the present invention are only defined by the claims. For those skilled in the art, without departing from the essence and scope of the present invention, various changes or modifications to the material components and dosages in these implementation examples also belong to the protection scope of the present invention. The sources of the raw materials and reagents used in the present invention are as follows: Hydroxychloroquine (HCQ) was purchased from Merck KGaA, 1-bromo-2,3-epoxypropane was purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd., N,N′-dimethylformamide (DMF) and ether were both purchased from J&K Scientific Ltd., catalase (CAT), 25,26,27,28-tetrahydroxycalix[4]arene (C4A), and p-aminobenzenesulfonic acid were all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Examples

[0040] The solid-phase synthesis method for preparing the supramolecular macrocyclic engineered protein regulator is a conventional technical means in the art. The present invention provides an exemplary illustration in the specific implementation manner, but its specific synthesis method is not limited thereto, and it does not exclude that CAT-SAC4A@HCQ synthesized by other means can be used for the preparation of the radiotherapy sensitizer described in the present invention.

[0041] The preparation method of CAT-SAC4A@HCQ is as follows: (1) Synthesis of SAC4A: SAC4A was synthesized by diazo coupling reaction using 25, 26, 27, 28-tetrahydroxycalix[4]arene (C4A) and p-aminobenzenesulfonic acid as raw materials. The steps are as follows: 1) Dissolve p-aminobenzenesulfonic acid (1.73 g, 10 mmol) and concentrated hydrochloric acid (2 mL, 37%) in 15 mL of water in a round-bottom flask. After cooling the solution to 2 °C in an ice-water bath, slowly add an aqueous solution of sodium nitrite (10 mL, 10 mmol) while controlling the temperature below 5 °C.

[0042] 2) Slowly add the obtained solution to a solution of 25, 26, 27, 28-tetrahydroxycalix[4]arene (C4A, 1.0 g, 2.36 mmol) and sodium acetate (2.46 g, 30 mmol) in MeOH - DMF (26 mL, 5∶8, V∶V) to obtain a red suspension.

[0043] 3) After stirring at room temperature for 2 h, add hydrochloric acid solution (37%) until the solution is adjusted to pH = 1. After heating to 60 °C and reacting for 30 min, filter the mixture and wash it with water and MeOH to obtain a light red solid SAC4A in quantitative yield.

[0044] (2) To synthesize CAT-SAC4A, react SAC4A with 1-bromo-2,3-epoxypropane to obtain SAC4A-epoxy. The steps are as follows: 1) Dissolve 35 mg of SAC4A in 4 mL of DMF, and successively add 400 mg of Na 2 CO 3 (100 mg / mL) and 1-bromo-2,3-epoxypropane (1 g, 7.3 mmol), and stir in the dark at room temperature for 24 h.

[0045] 2) After the reaction, remove the insoluble Na 2 CO 3 .

[0046] 3) Take the supernatant and add it dropwise to an excess of diethyl ether to precipitate and remove the unbound 1-bromo-2,3-epoxypropane. Then collect the precipitate and dry it in vacuo to obtain SAC4A-epoxy.

[0047] (3) Prepare CAT-SAC4A by binding SAC4A-epoxy to CAT through the reaction between the amino group of CAT and SAC4A-epoxy. The steps are as follows: 1) Accurately weigh 100 mg of CAT powder and add it to a 100 mL round-bottom flask. Then add it to 25 mL of Na 2 CO 3 buffer solution (100 mM) and stir at low speed until CAT is completely dissolved.

[0048] 2) Subsequently, add 479 μL of 50 mg / mL SAC4A-epoxy dropwise and stir in the dark at room temperature for 24 h.

[0049] 3) Separate the product by dialysis against water (MWCO 10000), then ultrafiltration (MWCO 30000) and desalting with a desalting column (MWCO 7000).

[0050] Mix the HCQ methanol solution with the same concentration as SAC4A in CAT-SAC4A together and gently invert to mix evenly to obtain CAT-SAC4A@HCQ.

[0051] See the appendix Figure 1 , which gives the characterization results of the physicochemical properties of the prepared supramolecular macrocyclic engineered protein regulator CAT-SAC4A@HCQ. The steps are as follows: 1) Drop CAT-SAC4A onto a 300-mesh carbon support film copper grid. After standing for 4 min, gently suck out the excess sample with filter paper. Then drop 10 μL of uranyl acetate double stain onto the carbon support film copper grid with the sample. After standing for 4 min, suck out the excess stain with filter paper and dry it in vacuo for later use. Subsequently, study the microstructure of CAT-SAC4A by transmission electron microscopy.

[0052] 2) Determine the binding affinity between CAT-SAC4A and HCQ by fluorescence titration. As Figure 1 shown in B, the binding constant (Ka) between SAC4A and HCQ is (1.83 ± 0.36) × 10 6 M -1 , and this excellent binding affinity shows the good drug-loading potential of CAT-SAC4A.

[0053] 3) Measure the oxygen production ability of CAT, CAT-SAC4A, and CAT-SAC4A@HCQ using a portable dissolved oxygen meter (Leici, model JPBJ-608). The oxygen production curve ( Figure 1 C) Both CAT-SAC4A and CAT-SAC4A@HCQ exhibited similar oxygen production efficiency to natural CAT, being able to continuously catalyze hydrogen peroxide to produce oxygen, demonstrating their potential to relieve tumor hypoxia in vivo.

[0054] Appendix Figure 2 The hypoxia-relieving situation of the supramolecular macrocyclic engineered protein regulator CAT-SAC4A@HCQ in 4T1 cells is presented as follows: 1) Seed 4T1 cells at a density of 3×10 5 in a confocal microscope culture dish and incubate for 24 h.

[0055] 2) After the cells are fully adhered, discard the original culture medium and re-add the culture medium containing CAT (4 μM), CAT-SAC4A (20 μM), and CAT-SAC4@HCQ (20 μM / 20 μM) and incubate for 24 h.

[0056] 3) Add PBS to wash the cell surface and fix with 4% paraformaldehyde solution at room temperature for 30 min.

[0057] 4) Re-add PBS to wash the cell surface and permeabilize with pre-cooled ice methanol solution at -20°C for 15 min.

[0058] 5) After washing the cells with PBS again, block the cells with 1% BSA solution at room temperature for 1 h.

[0059] 6) Discard the BSA solution, wash the cells with PBS, add the primary antibody solution of HIF1-α diluted with 1% BSA, and incubate overnight at 4°C.

[0060] 7) Discard the primary antibody solution, wash 3 times with PBS, add the fluorescent secondary antibody solution diluted with 1% BSA, and incubate at room temperature in the dark for 1 h.

[0061] 8) Discard the secondary antibody solution, wash 3 times with PBS, continue to add 500 μL of DAPI stain, and stain at room temperature in the dark for 10 min.

[0062] 9) Wash the cells with PBS, then add PBS buffer to soak at 4°C, and place on the sample stage of a confocal fluorescence microscope to take fluorescence photos.

[0063] Figure 2This is the situation of CAT-SAC4A@HCQ alleviating hypoxia in 4T1 cells. As can be seen from the figure, under hypoxic conditions, untreated 4T1 cells showed strong green fluorescence signals of HIF-1α, while cells treated with CAT-SAC4 or CAT-SAC4@HCQ showed significantly reduced fluorescence intensity, which confirmed that the catalytic oxygen generation of CAT effectively alleviated tumor hypoxia.

[0064] Appendix Figure 3 The regulation of the expression of autophagy-related proteins by the supramolecular macrocyclic engineered protein regulator CAT-SAC4A@HCQ in 4T1 cells was detected by Western blot as follows: 1) 4T1 cells in the logarithmic growth phase were seeded in a 6-cm-diameter culture dish and cultured in an incubator for 24 h.

[0065] 2) After treatment with drug-containing media of CAT, CAT-SAC4A, and CAT-SAC4@HCQ at a final concentration of 20 μM for 24 h, they were replaced with fresh media, irradiated with γ-rays (6 Gy), and cultured for another 24 h.

[0066] 3) The cells were digested with trypsin and collected, and lysed in RIPA lysis buffer containing PMSF (1 mM) at 4 °C for 30 minutes. Centrifuged at 12,000 rpm for 10 minutes at 4 °C to obtain the supernatant containing total cellular proteins. The extracted proteins were further quantified using a BCA protein assay kit.

[0067] 4) Subsequently, different samples of equal amounts of protein (50 μg) were separated by SDS-PAGE gel and electrophoretically transferred to a PVDF membrane.

[0068] 5) The non-specific binding sites were blocked using a TBST solution containing 5% non-fat milk. Then, the PVDF membrane was incubated overnight with primary antibodies (such as anti-ATG5 antibody (Abmart), anti-Beclin-1 antibody (Abmart), anti-LC3 antibody (CST), anti-p62 antibody (Abmart)).

[0069] 6) After the incubation with the primary antibody, the membrane was incubated with a horseradish peroxidase-conjugated anti-rabbit antibody for 1 h, and the luminescence was detected using an enhanced chemiluminescence (ECL) detection reagent.

[0070] GAPDH was detected as a housekeeping protein control.

[0071] Appendix Figure 3This is the situation of CAT-SAC4A@HCQ regulating autophagy in 4T1 cells. As shown in the figure, in the CAT-SAC4 and CAT-SAC4@HCQ groups, Beclin 1, a key driver of early autophagy initiation and phagocyte assembly, was downregulated, indicating that the nanocatalytic drug alleviated hypoxia and inhibited protective autophagy. The downregulation of another important autophagy-related protein, ATG5, further supported this conclusion. In addition, the ratio of LC3-II / LC3-I, a marker of autophagic activity, increased significantly in the CAT-SAC4@HCQ group, accompanied by the accumulation of autophagic substrate p62, indicating impaired autophagic degradation.

[0072] Appendix Figure 4 The evaluation of the radiosensitization ability of the supramolecular macrocyclic engineered protein regulator CAT-SAC4A@HCQ on 4T1 cells was compared by a colony formation assay, and the steps are as follows: 1) 4T1 cells in the logarithmic growth phase were seeded into 12-well plates at a density of 500 cells per well and cultured in an incubator for 24 h.

[0073] 2) CAT, CAT-SAC4A, and CAT-SAC4@HCQ with the same drug concentration were added to the 12-well plates and cultured for another 24 h.

[0074] 3) After replacing with fresh medium, ionizing radiation with irradiation doses of 0, 2, 4, and 6 Gy was performed respectively, and then cultured in an incubator for another 7 days to wait for the formation of cell clone clusters, and the cell clone clusters in each group were counted.

[0075] 4) It can be seen that Figure 4 4T1 cells treated with CAT or CAT-SAC4A showed a similar colony formation survival trend to the control group after ionizing radiation, indicating that the radiosensitization effect could be ignored. On the contrary, compared with the CAT + RT and CAT-SAC4A + RT groups, the CAT-SAC4@HCQ + RT group showed a significant reduction in colony formation, demonstrating that CAT-SAC4@HCQ had better tumor cell killing ability in vitro. With the increase of radiation dose (0, 2, 4, and 6 Gy), the survival rate of 4T1 cells in the CAT-SAC4@HCQ group decreased significantly.

[0076] Appendix Figure 5 The biosafety of the supramolecular drug CAT-SAC4@HCQ was evaluated by blood routine and blood biochemistry, and the steps are as follows: 1) Healthy BALB / c mice at 5 - 6 weeks of age were randomly divided into 5 groups and were injected via the tail vein with PBS (200 μL), CAT (200 μL, 80 μM), CAT - SAC4A (200 μL, 400 μM), and CAT - SAC4A@HCQ (200 μL, 400 μM), respectively.

[0077] 2) On the 2nd and 7th days after injection, 100 μL of blood was collected from the retro - orbital venous plexus of the mice into an anticoagulation tube, gently shaken, and the changes in blood routine indexes were detected using an automatic hematology analyzer.

[0078] 3) On the 7th day after injection, the whole blood of the mice was placed in a 1.5 mL centrifuge tube. After the plasma coagulated, it was centrifuged in a centrifuge (3000 rpm, 5 min), and the upper - layer serum was aspirated for the next - step detection of liver and kidney function indexes.

[0079] 4) Through Figure 5 It can be seen that all blood parameters remained within the normal physiological range, and there were no significant differences among the groups. These findings emphasized the excellent biosafety characteristics and clinical translation potential of CAT - SAC4@HCQ.

Claims

1. A supramolecular macrocyclic engineered protein regulator CAT-SAC4A@HCQ, the structure of which is as follows: 1) Enzyme catalytic core: composed of catalase (CAT), which retains its natural catalytic activity and can decompose hydrogen peroxide into oxygen; 2) Supramolecular macrocyclic carrier: sulfonated azocalix[4]arene (SAC4A), which is covalently linked to the amino group on the surface of the catalase through its epoxy group to form a CAT-SAC4A complex; 3) Drug loading unit: Hydroxychloroquine (HCQ) is encapsulated in the cavity of the sulfonated azocalix[4]arene through host-guest interaction to form a CAT-SAC4A@HCQ complex; in, The azo group of the sulfonated azocalix[4]arene can be specifically reduced by azoreductase in the tumor microenvironment, resulting in the targeted release of hydroxychloroquine, while the catalytic activity of the catalase is not affected by the covalent modification; Structural features: 1) Covalent bonding: The epoxy group of SAC4A and the amino group of CAT form a stable covalent bond through a nucleophilic ring-opening reaction, ensuring the structural stability of the complex in a physiological environment; 2) Host-guest loading: The hydrophobic cavity of SAC4A efficiently encapsulates HCQ through π-π stacking and hydrophobic interaction, with a loading rate of ≥85%, and drug release depends on the enzyme responsiveness of the tumor microenvironment; 3) Functional synergy: CAT maintains catalytic oxygen production function and alleviates tumor hypoxia; SAC4A realizes the spatiotemporal controlled release of HCQ and blocks protective autophagy; the two synergistically induce oxidative stress and immunogenic cell death, reversing the immunosuppressive microenvironment.

2. The method for preparing the supramolecular macrocyclic engineered protein regulator CAT-SAC4A@HCQ according to claim 1, characterized in that Proceed as follows: (1) Synthesis of SAC4A: Synthesis of SAC4A SAC4A was synthesized by diazo coupling reaction using 25,26,27,28-tetrahydroxycalix[4]arene (C4A) and p-aminobenzenesulfonic acid as raw materials; the steps are as follows: 1) Dissolve 1.73 g of p-aminobenzenesulfonic acid and 2 mL of concentrated hydrochloric acid in 15 mL of water in a flask; cool the solution to 2°C in an ice-water bath, then slowly add 10 mL of sodium nitrite aqueous solution while keeping the temperature below 5°C; 2) The obtained solution was slowly added to a solution of 1.0 g of 25,26,27,28-tetrahydroxycalix[4]arene and 2.46 g of sodium acetate in 26 mL of MeOH-DMF: 5:8, V:V, to obtain a red suspension; 3) After stirring at room temperature for 2 h, 37% hydrochloric acid solution was added until the solution was adjusted to pH = 1; after heating to 60 °C for 30 min, the mixture was filtered and washed with water and MeOH to obtain light red solid SAC4A in quantitative yield; (2) SAC4A is reacted with 1-bromo-2,3-propylene oxide to obtain SAC4A-epoxy resin; the preparation process is as follows: 1) Dissolve 35 mg of SAC4A in 4 mL of DMF, add 400 mg of Na2CO3 and 1 g of 1-bromo-2,3-propylene oxide, and stir at room temperature in the dark for 24 h; 2) After the reaction, remove the insoluble Na2CO3 by centrifugation at 2000 rpm for 10 min; 3) Take the supernatant and add it dropwise to excess ether to precipitate and remove unbound 1-bromo-2,3-epoxypropane. Then collect the precipitate and dry it in a vacuum to obtain SAC4A-epoxy. (3) CAT-SAC4A is prepared by combining SAC4A-epoxy with CAT through the reaction between the amino group of CAT and SAC4A-epoxy. The preparation process is as follows: 1) Accurately weigh 100 mg of CAT powder into a flask, add it to 25 mL of Na2CO3 buffer at a concentration of 100 mM, and stir at low speed until CAT is completely dissolved; 2) Then, 479 μL of 50 mg / mL SAC4A-epoxy was added dropwise and stirred at room temperature in the dark for 24 h; 3) The product was isolated by dialysis against water using MWCO 10,000, followed by ultrafiltration using MWCO 30,000 and desalting using a MWCO 7000 desalting column; (4) Mix the methanol solution of HCQ with the same concentration as that of SAC4A in CAT-SAC4A and gently invert to mix to obtain CAT-SAC4A@HCQ.

3. Use of the supramolecular macrocyclic engineered protein regulator CAT-SAC4A@HCQ for regulating the tumor microenvironment according to claim 1 in the preparation of drugs for enhancing the effect of tumor radioimmunotherapy.

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