Supramolecular hydrogel carrier, supramolecular hydrogel wrapping illicit and glucose oxidase and preparation method and application of supramolecular hydrogel carrier and supramolecular hydrogel wrapping illicit and glucose oxidase

Abstract

The invention discloses a supramolecular hydrogel carrier, supramolecular hydrogel wrapping illicit and glucose oxidase as well as a preparation method and application of the supramolecular hydrogel. The supramolecular hydrogel carrier is prepared from the following raw materials: sodium carboxymethyl cellulose (CMC-Na), cuprous oxide modified by beta-cyclodextrin, (ferrocenyl methyl) trimethyl ammonium bromide and the balance of deionized water; wherein the sodium carboxymethyl cellulose is a substance for forming a network structure of the supramolecular hydrogel carrier. The supramolecular hydrogel can be used as a carrier of the illlisimos and the glucose oxidase for treating tumor wounds, has injectable, proper viscosity, porous structure and antibacterial properties, and is beneficial to killing tumor cells, promoting tumor wound closure and promoting tissue regeneration and remodeling.

Description

Technical Field

[0001] The present invention belongs to the technical field of polymer materials. Specifically, it relates to a supramolecular hydrogel carrier, a supramolecular hydrogel encapsulating elesclomol (ES) and glucose oxidase (GOx), and a preparation method and use thereof. Background Art

[0002] The healing process of tumor wounds is a complex and crucial process, involving tumor cell infiltration and the repair and regeneration of skin tissues.

[0003] The clinical manifestations of breast cancer are the appearance of hard masses in the breast. The masses cannot be pushed away, and the breast is painless, without heat, and the skin color remains unchanged. In the late stage, the breast is prone to ulceration, suppuration of blood, etc. The infiltration and ulceration of the chest skin in breast cancer are also one of the acute and severe complications of malignant tumors. In severe cases, the ulcerated wound does not heal for a long time, and the necrotic tissue is easily invaded by various miscellaneous bacteria and microorganisms, leading to septicemia, pyosepticemia, and the rapid growth of tumors resulting in death. Its clinical manifestations are local necrosis and infection of the chest skin tumor, with ulceration, bleeding, and suppuration, accompanied by a strong foul smell, which brings many inconveniences to the lives of patients and their families. The long-term use of methods such as chemotherapy, radiotherapy, and laser is also one of the reasons for tumor ulceration in breast cancer patients. The ulcerated tumor wound not only increases the pain of patients but also increases the difficulty of treatment. Therefore, exploring and developing more effective and simpler treatments to relieve the pain of breast cancer skin ulceration is a current challenge.

[0004] In recent years, various cancer treatment methods have emerged in an endless stream, such as photothermal therapy, photodynamic therapy, immunotherapy, cell therapy, starvation therapy, etc. In recent years, chemodynamic therapy (CDT) has attracted extensive attention in the academic circles at home and abroad. CDT uses the slightly acidic and excessive H 2 O 2 in the tumor microenvironment (TME) as a condition, and transition metals (such as Fe, Co, Ni, Cu, Mn) as catalysts. Through the Fenton / Fenton-like reaction, it catalyzes H 2 O 2 to generate strongly oxidizing reactive oxygen species such as hydroxyl radicals (·OH), thereby inducing cell death, and having unique advantages such as endogenous stimulus response, tumor specificity, and intelligent regulation of TME. As a new type of biomaterial, supramolecular hydrogels bring breakthroughs to disease treatment. They can perform targeted clearance of tumors through minimally invasive injection and cooperate with multi-modal combined actions, showing broad application prospects in tumor treatment.

[0005] Transition metal elements, including copper, can play an important role in the field of tumor treatment. They can serve as components of metalloenzymes involved in cell metabolism and as signaling molecules to regulate tumor proliferation and metastasis, and they are also components of metal-based anticancer drugs. Recent studies have shown that excessive copper can regulate programmed cell death (PCD) in cancer cells, namely cuprotosis.

[0006] However, there are few reports on the construction of Fenton reaction catalytic carriers based on supramolecular hydrogels for CDT. Based on the above research purposes and significance, the present invention designs and constructs a cuprotosis hydrogel with antitumor and wound healing promoting functions, hoping to achieve the purpose of treating cancer through the synergistic cooperation of multiple modes. Summary of the Invention

[0007] To solve the above problems, the present invention provides a supramolecular hydrogel carrier, a supramolecular hydrogel encapsulating elesclomol and glucose oxidase, and their preparation methods and uses. The supramolecular hydrogel carrier itself can continuously generate hydrogen peroxide (H 2 O 2 ), promoting tumor cell killing and rapid healing of tumor wound surfaces; when the supramolecular hydrogel carrier becomes a supramolecular hydrogel encapsulating elesclomol and glucose oxidase, it can not only continuously generate hydrogen peroxide (H 2 O 2 ) to promote cuprotosis of tumor cells, but also promote the more rapid healing of tumor wounds and improve the treatment effect. The synthesis methods of the supramolecular hydrogel carrier and the drug-loaded supramolecular hydrogel of the present invention are very simple and easy to implement. The drug-loaded supramolecular hydrogel of the present invention can be used to treat tumor wounds, providing new ideas for the development of biomaterials and tumor wound healing.

[0008] In the first aspect, the present invention provides a supramolecular hydrogel carrier, and the raw materials of the supramolecular hydrogel carrier are composed of sodium carboxymethyl cellulose (CMC-Na), β-cyclodextrin-modified cuprous oxide, (ferrocenylmethyl) trimethylammonium bromide, and the balance of deionized water; wherein, the sodium carboxymethyl cellulose is the substance forming the network structure of the supramolecular hydrogel carrier.

[0009] In the supramolecular hydrogel carrier of the present invention, (ferrocenylmethyl) trimethylammonium bromide (Fer) and cuprous oxide (CuO) are designed into the hydrogel material. The supramolecular hydrogel carrier can serve as a drug carrier, undergo a phase change in response to ROS, thereby releasing Fer and CuO, and releasing Fe 2+ and Cu + , and the released Fe 2+ and Cu +It can trigger the Fenton reaction, generate a small amount of hydroxyl radicals, and play a role in tumor killing and antibacterial in the tumor wound environment. Using a supramolecular hydrogel carrier as the carrier of ilixadencel and glucose oxidase, the ilixadencel and glucose oxidase can be continuously and effectively delivered to the tumor wound surface, promoting tumor cell killing and facilitating faster healing of the tumor wound surface.

[0010] In one embodiment of the supramolecular hydrogel carrier of the present invention, the supramolecular hydrogel carrier is obtained by modifying cuprous oxide with β-cyclodextrin to obtain β-cyclodextrin-modified cuprous oxide, and then adding the β-cyclodextrin-modified cuprous oxide to an aqueous solution of (ferrocenylmethyl) trimethylammonium bromide-sodium carboxymethylcellulose in deionized water and stirring at room temperature to form a supramolecular hydrogel; wherein, the molar ratio of β-cyclodextrin to (ferrocenylmethyl) trimethylammonium bromide is 1:1, and the weight ratio of β-cyclodextrin-modified cuprous oxide: (ferrocenylmethyl) trimethylammonium bromide: sodium carboxymethylcellulose is 100:11:500, and the weight ratio of β-cyclodextrin-modified cuprous oxide: sodium carboxymethylcellulose: deionized water is 100:500:20000.

[0011] In the supramolecular hydrogel carrier of the present invention, the key to the gelation of the supramolecular hydrogel carrier lies not only in the molar ratio of β-cyclodextrin modifying cuprous oxide: (ferrocenylmethyl) trimethylammonium bromide being 1:1, but also in the weight ratio of β-cyclodextrin-modified cuprous oxide: sodium carboxymethylcellulose: deionized water being 100:500:20000. If the weight ratio of the β-cyclodextrin-modified cuprous oxide component, the sodium carboxymethylcellulose component, and the deionized water component does not reach 100:100:10000, it will not gel and will be in a solution state; if it exceeds 100:300:10000, it will be too gelled to be injectable and cannot achieve the desired effect; more importantly, only when β-cyclodextrin modifying cuprous oxide and (ferrocenylmethyl) trimethylammonium bromide are assembled in a host-guest molar ratio of 1:1 can the supramolecular hydrogel carrier be formed.

[0012] Second aspect, the present invention provides a method for preparing the supramolecular hydrogel carrier described in the first aspect above, comprising the following steps: (1) modifying cuprous oxide with β-cyclodextrin to obtain β-cyclodextrin-modified cuprous oxide; (2) dissolving the (ferrocenylmethyl) trimethylammonium bromide in deionized water, and then adding the sodium carboxymethylcellulose thereto to form a (ferrocenylmethyl) trimethylammonium bromide-sodium carboxymethylcellulose solution through electrostatic interaction; (3) adding the β-cyclodextrin-modified cuprous oxide obtained in step (1) to the (ferrocenylmethyl) trimethylammonium bromide-sodium carboxymethylcellulose deionized aqueous solution obtained in step (2), and stirring at room temperature to form the supramolecular hydrogel carrier through the host-guest interaction between β-cyclodextrin and (ferrocenylmethyl) trimethylammonium bromide; wherein, the weight ratio of β-cyclodextrin-modified cuprous oxide: (ferrocenylmethyl) trimethylammonium bromide: sodium carboxymethylcellulose is 100:11:500, and the molar ratio of β-cyclodextrin to (ferrocenylmethyl) trimethylammonium bromide is 1:1; the weight ratio of β-cyclodextrin-modified cuprous oxide: sodium carboxymethylcellulose: deionized water is 100:500:20000.

[0013] Third aspect, the present invention provides a supramolecular hydrogel encapsulating ilesimod and glucose oxidase, wherein the supramolecular hydrogel is composed of the supramolecular hydrogel carrier described in the first aspect above encapsulating ilesimod and glucose oxidase.

[0014] In one embodiment of the supramolecular hydrogel encapsulating ilesimod and glucose oxidase of the present invention, the supramolecular hydrogel carrier encapsulating ilesimod and glucose oxidase is obtained by modifying cuprous oxide with β-cyclodextrin to obtain β-cyclodextrin-modified cuprous oxide, and after adding the β-cyclodextrin-modified cuprous oxide to the (ferrocenylmethyl) trimethylammonium bromide-sodium carboxymethylcellulose deionized aqueous solution to form a supramolecular hydrogel carrier, then mixing with ilesimod and glucose oxidase to form a supramolecular hydrogel encapsulating ilesimod and glucose oxidase; wherein, the molar ratio of β-cyclodextrin to (ferrocenylmethyl) trimethylammonium bromide is 1:1, the weight ratio of β-cyclodextrin-modified cuprous oxide: (ferrocenylmethyl) trimethylammonium bromide: sodium carboxymethylcellulose is 100:11:500, the weight ratio of β-cyclodextrin-modified cuprous oxide: sodium carboxymethylcellulose: deionized water is 100:500:20000, and the concentrations of ilesimod and glucose oxidase are 0.5 mg / ml.

[0015] Fourth aspect, the present invention provides a method for preparing the supramolecular hydrogel encapsulating elesclomol and glucose oxidase described in the third aspect above, comprising the following steps: (1) modifying cuprous oxide with β-cyclodextrin to obtain β-cyclodextrin-modified cuprous oxide; (2) dissolving (ferrocenylmethyl)trimethylammonium bromide in deionized water, adding sodium carboxymethyl cellulose, and forming a (ferrocenylmethyl)trimethylammonium bromide-sodium carboxymethyl cellulose solution through electrostatic interaction; (3) adding the β-cyclodextrin-modified cuprous oxide obtained in step (1) to the (ferrocenylmethyl)trimethylammonium bromide-sodium carboxymethyl cellulose deionized aqueous solution obtained in step (2), stirring at room temperature, and forming the supramolecular hydrogel carrier through the host-guest interaction between β-cyclodextrin and (ferrocenylmethyl)trimethylammonium bromide; (4) uniformly stirring the supramolecular hydrogel carrier with elesclomol and glucose oxidase with a glass rod at a speed of 100 revolutions per minute, and binding elesclomol and glucose oxidase together at a ratio of 0.5 mg / mL to form the supramolecular hydrogel encapsulating elesclomol and glucose oxidase; wherein, the weight ratio of β-cyclodextrin-modified cuprous oxide: (ferrocenylmethyl)trimethylammonium bromide: sodium carboxymethyl cellulose is 100:11:500.

[0016] Fifth aspect, the present invention provides the use of the supramolecular hydrogel encapsulating elesclomol and glucose oxidase described in the third aspect above in the preparation of a medicament for treating tumor wounds.

[0017] In one embodiment of the use of the supramolecular hydrogel encapsulating elesclomol and glucose oxidase in the preparation of a medicament for treating tumor wounds, the tumor wound is a skin ulcer caused by a tumor.

[0018] In an alternative embodiment, the tumor is breast cancer.

[0019] In the present invention, "β-CD" represents β-cyclodextrin; "Fer" represents (ferrocenylmethyl)trimethylammonium bromide; "CuO-CD" represents β-cyclodextrin-modified cuprous oxide.

[0020] The present invention has the following beneficial effects:

[0021] Considering the characteristics of tumor wounds, the main strategies for their treatment focus on killing tumor cells and reducing infections. In clinical treatment, due to the limitations of traditional treatments, there is an urgent need to find a new alternative therapy. The combination of cuproptosis and hydrogels has created a composite hydrogel system with excellent mechanical properties and tumor-killing ability, and also provided a new method for wound healing and skin dressings.

[0022] 1. The supramolecular hydrogel carrier of the present invention can be in a tumor microenvironment (pH is acidic, rich in H 2 O2 ) Continuously release ilisomide and glucose oxidase under [conditions], kill tumor cells through cuproptosis and starvation therapy, and then promote the healing of tumor wounds.

[0023] 2. The β-cyclodextrin-modified cuprous oxide enables the loading of Cu in the hydrogel material + , and (ferrocenylmethyl) trimethylammonium bromide enables the loading of Fe in the hydrogel material 2+ , in the tumor microenvironment (acidic and with an excessive concentration of H 2 O 2 ), this Cu + and Fe 2+ can be released from the hydrogel, so that it can react with H 2 O 2 to generate hydroxyl radicals through the Fenton reaction.

[0024] 3. The supramolecular hydrogel of the present invention has properties such as injectability, adhesiveness, porous structure, and biocompatibility, and can absorb excessive exudate from the tumor wound and promote the healing of the tumor wound by surface adhesion.

[0025] 4. As one of the raw materials, the molecule of (ferrocenylmethyl) trimethylammonium bromide can perform molecular assembly with β-cyclodextrin-modified cuprous oxide, and the host-guest molar ratio can be as high as 1:1; and (ferrocenylmethyl) trimethylammonium bromide can bind to negatively charged carboxymethyl cellulose sodium (CMC-Na) through electrostatic interaction, and then can form a hydrogel with β-cyclodextrin-modified cuprous oxide through coordination, making it possible to obtain the supramolecular hydrogel.

[0026] 5. The method for preparing the supramolecular hydrogel of the present invention forms the supramolecular hydrogel through molecular assembly and intermolecular electrostatic force, with few steps, easily obtainable reaction conditions, and a very simple method. Description of the Drawings

[0027] Figure 1 is the scanning electron micrograph of the CCFC supramolecular hydrogel.

[0028] Figure 2 is the Fourier infrared spectrum of compound 6-β-CD-OTS, ALA-β-CD, ALA-β-CD-Cu 2 O.

[0029] Figure 3 a-3c are the gelation pictures of supramolecular hydrogels of different ratios of Cu 2 O-CD and CMC-Na; Figure 3 d and 3e are the pictures of the adhesiveness test of the supramolecular hydrogel; Figure 3 f is the picture of the injectability test of the supramolecular hydrogel.

[0030] Figure 4 It is the SEM energy spectrum surface scan image of the CCFC supramolecular hydrogel.

[0031] Figure 5 It is the spectrum for detecting the generation of hydroxyl radicals by the TMB (3,3',5,5'-tetramethylbenzidine) method. Figure 5 a is the UV-visible absorption spectrum of the amount of hydroxyl radicals generated by the reaction of supramolecular hydrogels with different concentrations and 3,3',5,5'-tetramethylbenzidine (TMB) at pH = 4.8; Figure 5 b is the changing trend of the amount of hydroxyl radicals generated by the supramolecular hydrogel (20 μg / mL) over time.

[0032] Figure 6 For the solution of H 2 O 2 (0.01 - 0.05 mol / L), it is the UV-visible absorption spectrum of the supramolecular hydrogel (20 μg / mL) and TMB at pH = 4.8.

[0033] Figure 7 For Cu 2 O, Cu 2 O-CD, Cu 2 O-CD-Fer, it is the Zeta potential characterization diagram.

[0034] Figure 8 It is the rheological test of the supramolecular hydrogel. Figure 8 a is the curve of the storage modulus G' and the loss modulus G'' changing with the shear stress; Figure 8 b is the curve of the viscosity changing with the shear rate; Figure 8 c is the curve of the storage modulus G' and the loss modulus G'' changing with the oscillatory shear stress.

[0035] Figure 9 It is the Western blot result of the supramolecular hydrogel encapsulating elesclomol and glucose oxidase in triple-negative breast cancer cell lines BT549 and 4T1.

[0036] Figure 10 It is the photo of mouse tumor wound healing.

[0037] Figure 11 It is the in vivo imaging result and analysis of mouse tumors. Figure 11 a is the bioluminescence image of in vivo imaging of mice, Figure 11 b is the quantification of the bioluminescence image. Specific embodiments

[0038] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, and these modifications and substitutions all fall within the protection scope of the present invention.

[0039] The main instrument devices and main raw materials and reagents used in the following embodiments are shown in Table 1 and Table 2 respectively as follows.

[0040] Table 1 Main instrument devices for experiments

[0041]

[0042] Table 2 Main raw materials and reagents

[0043]

[0044] Example

[0045] 1. Synthesis of β-cyclodextrin-modified cuprous oxide (Cu 2 O-CD)

[0046] (1) Synthesis of mono-(6-O-p-toluenesulfonyl)-β-cyclodextrin (6-β-CD-OTS)

[0047] Add 1.5 L of distilled water and 270 g of β-CD (β-cyclodextrin) into a 5 L beaker to obtain a white turbid liquid. Stir continuously. Dissolve 25.7 g of NaOH in 77 mL of distilled water, and slowly add it to the beaker containing the white turbid liquid after cooling. The solution becomes clear and shows a light yellow color. Dissolve 39 g of TSCI (p-toluenesulfonyl chloride) in 128 mL of acetonitrile, and slowly add it to the clear and light yellow reaction system, 1 - 2 drops per second. After dropping, stir at room temperature for 2 h, and a small amount of white precipitate is produced. Filter by suction and discard the precipitate to obtain a light yellowish-green solution. Adjust the light yellowish-green solution to pH 7 - 8 with 2 M HCl (25.7 mL of concentrated HCl added to 250 mL of water), a white precipitate is precipitated. After fully stirring, put it in the refrigerator and cool overnight, and filter by suction under reduced pressure to obtain a white precipitate. The white precipitate is recrystallized twice again, and then filtered by suction and dried to obtain a white solid, which is 6-β-CD-OTS (mono-(6-O-p-toluenesulfonyl)-β-cyclodextrin).

[0048] (2) Synthesis of mono-(6-allylamine-6-deoxy)-β-cyclodextrin (ALA-β-CD)

[0049] 1.97 g (1.53 mmol) of 6-β-CD-OTS and 30 mL of 0.306 mmol allylamine were added to a dry round-bottom flask, and the reaction was refluxed under condensation at 70 °C for 6 h. After the solution was cooled to room temperature, it was diluted with 30 mL of methanol. After adding 150 mL of acetonitrile, a pale yellow solid was precipitated, and after filtration and drying, ALA-β-CD was obtained.

[0050] (3) 0.1212 g of ALA-β-CD was added to 20 mL of deionized water and stirred continuously. 0.75 g of Cu(NO 3 ) 2 ·3H 2 O was dissolved in 1.5 mL of water, 0.088 g of NaOH was dissolved in 2.2 mL of water, and the above solutions were added slowly in turn. 0.1057 g of ascorbic acid was dissolved in 5 mL of water and added dropwise to the above system. After dropping, it was stopped immediately to obtain an orange-yellow suspension. After centrifugation at 5000 r / min for 10 min, an orange-yellow solid was obtained, which was centrifuged and washed with deionized water and dried to obtain β-cyclodextrin-modified cuprous oxide (Cu 2 O-CD). Store at 4 °C.

[0051] 2. Synthesis and drug loading of supramolecular hydrogel carrier (Cu 2 O-CD / Fer / CMC-Na, CCFC)

[0052] 5 mg of Cu 2 O-CD was added to 5 mL of water, and ultrasonicated for 3 min to obtain a mother liquor with a concentration of 1 mg / mL. 1 mL of the mother liquor was taken, 1 mg of (ferrocenylmethyl)trimethylammonium iodide was added, diluted to 10 mL with water, mixed evenly, 0.3 g of CMC-Na was added, and stirred continuously until the white powder completely disappeared. After standing, a gel was formed to obtain CCFC hydrogel.

[0053] Elisomol and glucose oxidase were stirred evenly with the supramolecular gel at a concentration of 0.5 mg / mL with a glass rod at a speed of 100 revolutions per minute to form a supramolecular hydrogel loaded with elisomol and glucose oxidase.

[0054] 3. Establishment of a mouse tumor wound model

[0055] First, all mice (BALB / c mice, female, 4 - 6 weeks old) were raised for 3 days to adapt to the environment. 1×10 64T1 cells were subcutaneously injected into the right axillary back of mice. When the tumor diameter reached 10 mm, the mice were randomly divided into 6 groups: CMC group (the group treated with sodium carboxymethylcellulose for the tumor wound), CCFC group (the group treated with CCFC hydrogel carrier for the tumor wound), CCFC + elesclomol group (the group treated with hydrogel carrier and elesclomol for the tumor wound), CCFC + glucose oxidase group (the group treated with hydrogel carrier and glucose oxidase for the tumor wound), CCFC + elesclomol + glucose oxidase group (the group treated with hydrogel carrier, elesclomol and glucose oxidase for the tumor wound), positive drug group (the group treated with elesclomol and copper chloride for the tumor wound). The mice were anesthetized with sodium pentobarbital (10 mg / mL), and the full-thickness skin on the surface of the tumor (diameter: 8 mm) was excised using sterile instruments to expose the tumor surface, simulating a tumor ulcerated wound. Then, the mice in different groups were treated accordingly. The dressing was changed twice a day, morning and evening. The gel was thinly coated on the wound surface. The wound healing was recorded on the 1st, 7th, and 14th days after modeling, and in vivo imaging and analysis were performed on the 7th, 16th, 19th, and 27th days after tumor cell injection. The results of Western blot in this study were statistically analyzed by one-way analysis of variance among three groups; the P value in in vivo animal imaging analysis was calculated by two-way analysis of variance. A P < 0.05 was considered statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

[0056] Comparative Example 1

[0057] Prepare the supramolecular hydrogel encapsulating elesclomol and glucose oxidase in a similar manner to the example, except that the mass ratio of Cu 2 O-CD:CMC-Na is 1:1.

[0058] The product obtained is as Figure 3 shown in a, in a solution state and not forming a hydrogel.

[0059] Comparative Example 2

[0060] Prepare the supramolecular hydrogel encapsulating elesclomol and glucose oxidase in a similar manner to the example, except that the mass ratio of Cu 2 O-CD:CMC-Na is 1:3.

[0061] The product obtained is as Figure 3 shown in c, the product is too solidified and has poor injectability.

[0062] Experimental results:

[0063] Figure 1It is the scanning electron microscope image of the CCFC supramolecular hydrogel. By observing the microscopic morphology of the hydrogel through scanning electron microscopy, a three-dimensional cross-linked network structure can be observed, and the pore size ranges from 50 to 300 μm, which is most similar to the extracellular matrix (ECM) structure, indicating that the CCFC supramolecular hydrogel can be used as a potential carrier for drugs and can absorb excessive exudate from the wound surface.

[0064] Figure 2 For the Fourier transform infrared spectra of compound 6-β-CD-OTS, ALA-β-CD, and ALA-β-CD-Cu 2 O. The supramolecular hydrogel was characterized by Fourier transform infrared spectroscopy. The characteristic peak at 1640 cm -1 in the figure belongs to ALA-β-CD, and the characteristic peaks at 1030 cm -1 and 630 cm -1 belong to Cu 2 O-CD. The above infrared data results confirm the successful synthesis of Cu 2 O-CD.

[0065] Figure 3 a - 3c are the gelation pictures of supramolecular hydrogels of different ratios of Cu 2 O-CD and CMC-Na. When the mass ratio of Cu 2 O-CD:CMC-Na is less than 1:1 (as shown in Figure 3 a), it is in a solution state; when the mass ratio of Cu 2 O-CD:CMC-Na is about 1:2, it is in a gel state; when the mass ratio of Cu 2 O-CD:CMC-Na is greater than 1:3, it is too solidified, affecting the injectability of the supramolecular hydrogel. Figure 3 d, Figure 3 e are the adhesion tests of the supramolecular hydrogel of the present invention. The supramolecular hydrogel can adhere an object to the surface, indicating that the supramolecular hydrogel has good adhesion; Figure 3 f is the injectability test of the supramolecular hydrogel of the present invention, confirming its good injectability.

[0066] Figure 4 It is the scanning electron microscope energy spectrum surface scan image of the CCFC supramolecular hydrogel. Through scanning electron microscope energy spectrum, it can be observed that the CCFC supramolecular hydrogel contains C, N, O, and Cu elements.

[0067] Figure 5a is a spectrum for detecting the generation of hydroxyl radicals by the TMB (3,3',5,5'-tetramethylbenzidine) method. TMB can be oxidized by hydroxyl radicals to produce a blue compound with an absorption peak at 652 nm. By measuring the change in absorbance, the generation rate of hydroxyl radicals can be calculated. The basic steps for detecting the generation of hydroxyl radicals using the TMB method are as follows: 1. Prepare the TMB solution: Dissolve an appropriate amount of TMB in an organic solvent to prepare a 0.8 mM solution. 2. Prepare the reaction system: Mix the TMB solution with different amounts of the supramolecular hydrogel sample of the present invention and add an appropriate oxidant hydrogen peroxide to initiate the reaction. 3. Reaction: Place the reaction system at room temperature for 0 - 20 minutes to oxidize TMB to produce a blue compound. 4. Measure the absorbance: Use a spectrophotometer to measure the change in absorbance of the reaction solution at 652 nm. 5. Calculate the generation rate of hydroxyl radicals: Calculate the oxidation rate of TMB based on the change in absorbance, and thus deduce the generation rate of hydroxyl radicals. Figure 5 a is the ultraviolet-visible absorption spectrum of the amount of hydroxyl radicals generated by the reaction of supramolecular hydrogels with different concentrations and 3,3',5,5'-tetramethylbenzidine (TMB) under the condition of pH = 4.8. Figure 5 As can be seen from a, the increase in ultraviolet absorption intensity indicates that the CCFC supramolecular hydrogel can efficiently generate hydroxyl radicals, and with the increase in the concentration of the supramolecular hydrogel, the amount of generated hydroxyl radicals increases. Figure 5 b is the change trend of the amount of hydroxyl radicals generated by the supramolecular hydrogel (20 μg / mL) with time, which generally shows a proportional increase.

[0068] Figure 6 For different addition amounts of H 2 O 2 (0.01 - 0.05 mol / L) solution, the ultraviolet-visible absorption spectrum of the supramolecular hydrogel (20 μg / mL) and TMB under the condition of pH = 4.8 shows that with the increase in the addition amount of H 2 O 2 solution, the amount of hydroxyl radicals generated by the supramolecular hydrogel increases accordingly.

[0069] Figure 7 For Cu 2 O, Cu 2 O-CD, Cu 2 O-CD-Fer Zeta potential characterization diagram, this diagram shows that Cu 2 O-CD-Fer can be electrostatically combined with CMC-Na.

[0070] Figure 8 It is the rheological test of the supramolecular hydrogel. Figure 8a is the curve of the storage modulus G' and the loss modulus G'' versus the shear stress, demonstrating that CCFC exists in a gel state. Figure 8 b is the curve of the viscosity versus the shear rate. The viscosity gradually decreases with the increase of the shear rate, proving that the CCFC supramolecular hydrogel has shear-thinning behavior. Figure 8 c is the curve of the storage modulus G' and the loss modulus G'' versus the oscillatory shear stress. When changing from a larger strain to a smaller strain, the modulus can recover, demonstrating that the CCFC supramolecular hydrogel has self-healing properties.

[0071] Figure 9 are the Western blot results of the supramolecular hydrogel encapsulating elesclomol and glucose oxidase in the triple-negative breast cancer cell lines BT549 and 4T1. The oligomerization of the DLAT protein and the loss of the iron-sulfur proteins ACO2, LIAS, and FDX1 indicate that cuproptosis has occurred in the cells.

[0072] Figure 10 are the photos of mouse tumor wound healing. Among them, both the CCFC + elesclomol group and the CCFC + elesclomol + glucose oxidase group have the effect of promoting wound healing, but the wound healing effect is more obvious when elesclomol and glucose oxidase are applied simultaneously.

[0073] Figure 11 are the in vivo imaging results of mouse tumors. Figure 11 a are the in vivo imaging photos of mouse tumors, Figure 11 b is its statistical quantification graph. Among them, both the CCFC + elesclomol group and the CCFC + elesclomol + glucose oxidase group have the effect of inhibiting tumor growth, but the effect of inhibiting tumor growth is more obvious when elesclomol and glucose oxidase are used simultaneously. This may be related to the starvation effect of glucose oxidase and the production of H 2 O 2 further promoting the Fenton reaction and thus generating more Cu 2+ triggering cuproptosis in tumor cells.

Claims

1. A supramolecular hydrogel carrier, characterized in that: The raw materials of the supramolecular hydrogel carrier are composed of sodium carboxymethyl cellulose CMC-Na, cuprous oxide modified with β-cyclodextrin, (ferrocenylmethyl)trimethylammonium bromide and the balance of deionized water; wherein the sodium carboxymethyl cellulose is a substance that forms the network structure of the supramolecular hydrogel carrier.

2. The supramolecular hydrogel carrier according to claim 1, characterized in that: The supramolecular hydrogel carrier is obtained by modifying cuprous oxide with β-cyclodextrin, and then adding the β-cyclodextrin-modified cuprous oxide into a deionized aqueous solution of (ferrocenylmethyl)trimethylammonium bromide-sodium carboxymethylcellulose, and stirring at room temperature to form a supramolecular hydrogel; Among them, the molar ratio of β-cyclodextrin and (ferrocenylmethyl)trimethylammonium bromide is 1:1, the weight ratio of β-cyclodextrin modified cuprous oxide: (ferrocenylmethyl)trimethylammonium bromide: sodium carboxymethyl cellulose is 100:11:500, and the weight ratio of β-cyclodextrin modified cuprous oxide: sodium carboxymethyl cellulose: deionized water is 100:500:20000.

3. The method for preparing the supramolecular hydrogel carrier according to claim 1 or claim 2, characterized in that: The following steps are involved: (1) using β-cyclodextrin to modify cuprous oxide to obtain β-cyclodextrin-modified cuprous oxide; (2) dissolving the (ferrocenylmethyl)trimethylammonium bromide in deionized water, and then adding the sodium carboxymethyl cellulose thereto to form a (ferrocenylmethyl)trimethylammonium bromide-sodium carboxymethyl cellulose solution through electrostatic interaction; (3) adding the β-cyclodextrin-modified cuprous oxide obtained in step (1) to the (ferrocenylmethyl)trimethylammonium bromide-sodium carboxymethylcellulose deionized water solution obtained in step (2), stirring at room temperature, and forming the supramolecular hydrogel carrier through the host-guest interaction between β-cyclodextrin and (ferrocenylmethyl)trimethylammonium bromide; Among them, the weight ratio of β-cyclodextrin modified cuprous oxide: (ferrocenylmethyl)trimethylammonium bromide: sodium carboxymethyl cellulose is 100:11:500, the molar ratio of β-cyclodextrin and (ferrocenylmethyl)trimethylammonium bromide is 1:1; the weight ratio of β-cyclodextrin modified cuprous oxide: sodium carboxymethyl cellulose: deionized water is 100:500:20000.

4. A supramolecular hydrogel encapsulating ilisimol and glucose oxidase, characterized in that: The supramolecular hydrogel is composed of the supramolecular hydrogel carrier according to claim 1 or claim 2 encapsulating ilisimol and glucose oxidase.

5. The supramolecular hydrogel encapsulating ilisimol and glucose oxidase according to claim 4, characterized in that: The supramolecular hydrogel is obtained by modifying cuprous oxide with β-cyclodextrin, and the β-cyclodextrin-modified sodium hyaluronate is added into a deionized aqueous solution of (ferrocenylmethyl)trimethylammonium bromide-sodium carboxymethylcellulose to form a supramolecular hydrogel that can be used as a carrier, and then mixed with ilisimol and glucose oxidase to form a supramolecular hydrogel that encapsulates ilisimol and glucose oxidase; Among them, the molar ratio of β-cyclodextrin and (ferrocenylmethyl)trimethylammonium bromide is 1:1, the weight ratio of β-cyclodextrin-modified cuprous oxide: (ferrocenylmethyl)trimethylammonium bromide: sodium carboxymethyl cellulose is 100:11:500, the weight ratio of β-cyclodextrin-modified cuprous oxide: sodium carboxymethyl cellulose: deionized water is 100:500:20000, and the concentrations of ilisimol and glucose oxidase are 0.5 mg / ml.

6. The method for preparing the supramolecular hydrogel encapsulating ilisimol and glucose oxidase according to claim 4 or claim 5, characterized in that: The following steps are involved: (1) using β-cyclodextrin to modify cuprous oxide to obtain β-cyclodextrin-modified cuprous oxide; (2) dissolving (ferrocenylmethyl)trimethylammonium bromide in deionized water, adding sodium carboxymethyl cellulose, and forming a (ferrocenylmethyl)trimethylammonium bromide-sodium carboxymethyl cellulose solution through electrostatic interaction; (3) adding the β-cyclodextrin-modified cuprous oxide obtained in step (1) to the (ferrocenylmethyl)trimethylammonium bromide-sodium carboxymethylcellulose deionized water solution obtained in step (2), stirring at room temperature, and forming the supramolecular hydrogel carrier through the host-guest interaction between β-cyclodextrin and (ferrocenylmethyl)trimethylammonium bromide; (4) stirring the supramolecular hydrogel carrier, ilisimol and glucose oxidase uniformly with a glass rod at a speed of 100 rpm, and combining them together at a ratio of ilisimol and glucose oxidase concentration of 0.5 mg / ml to form the supramolecular hydrogel encapsulating ilisimol and glucose oxidase; Wherein, the weight ratio of β-cyclodextrin modified cuprous oxide: (ferrocenylmethyl)trimethylammonium bromide: sodium carboxymethyl cellulose is 100:11:

500.

7. The preparation method according to claim 6, characterized in that: In steps (1) to (3), β-cyclodextrin-modified cuprous oxide and (ferrocenylmethyl)trimethylammonium bromide are assembled at a host-guest molar ratio of 1:

1.

8. The preparation method according to claim 6, characterized in that: In steps (1) to (3), the weight ratio of β-cyclodextrin-modified cuprous oxide: sodium carboxymethyl cellulose: deionized water is 100:500:20000.

9. Use of the supramolecular hydrogel according to claim 4 or claim 5 in preparing a drug for treating tumor wounds.

10. The use according to claim 9, characterized in that The tumor wound surface is skin ulceration caused by the tumor.

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