Multi-path injectable anti-tumor gel system as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of a multi-way injectable anti-tumor gel system. The preparation method comprises the following steps: (1) preparing PCN-224; (2) preparing DOX (at) PCN; (3) preparation of Tph-HA; and (4) dropwise adding DOX-coated PCN and GOx into the Tph-HA aqueous solution, uniformly mixing, dropwise adding a copper nitrate aqueous solution, and stirring at room temperature to obtain the multi-way injectable anti-tumor gel system within 10 seconds. The problems that traditional hydrogel is low in strength, poor in biocompatibility, complex in preparation process and the like are solved. The hydrogel prepared by the invention has good biocompatibility, self-healing property and injectable property, can deliver chemotherapeutic drugs to apoptosis cells and release a large amount of Cu < 2 + > to induce copper death, can be reduced into Cu < + > by glutathione to carry out Fenton-like reaction, and utilizes the photosensitizer to carry out photodynamic and photo-thermal synergistic anti-tumor. The hydrogel is quickly transferred to tumor tissues through in-situ injection, so that the method is an efficient multi-way anti-tumor combined therapy and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and functional materials, and in particular relates to a multi-channel injectable anti-tumor gel system and a preparation method and application thereof. Background Art

[0002] Cancer is one of the leading causes of death worldwide. Global cancer statistics in 2022 show that the number of new cancer cases is as high as 20 million, and about 9.7 million people died of cancer. According to estimates by the World Health Organization's International Agency for Research on Cancer, about 20% of people will develop cancer in their lifetime, and about 11.1% of men and 8.3% of women will die from cancer. In the past, clinical attempts to treat tumors have been made through surgery, radiotherapy, chemotherapy, and immunotherapy, but the use of traditional methods has problems such as high recurrence rates, large side effects, and irreversible damage to normal tissues. Therefore, it is urgent to integrate new tumor treatment strategies to treat tumors.

[0003] Doxorubicin (DOX) is a typical chemotherapy drug that blocks the synthesis of nucleic acids in tumor cells. It can cause mitochondrial swelling and change mitochondrial function, thereby enhancing the level of oxidative stress in cells, overproducing reactive oxygen species (ROS), and leading to cell apoptosis. However, the clinical application of DOX is limited by serious side effects such as cardiotoxicity and bone marrow suppression. By designing a stimulus-responsive targeted drug delivery system, it can be used as an alternative to DOX. It can be used to selectively target and release drugs in response to specific stimuli in the tumor microenvironment (TME), thereby improving the efficacy of DOX and reducing systemic toxicity.

[0004] Photodynamic therapy (PDT) is a non-invasive method that uses the powerful oxidative ability of ROS to cause apoptosis of tumor cells. It is minimally invasive, accurately eliminates local tumors, and avoids damage to healthy tissues. In recent years, nano-metal porphyrin organic frameworks (nMOFs) have been widely used for their 1 O2 is used for PDT due to its advantages such as easy diffusion, avoidance of self-quenching and high drug loading. Porous coordination network-224 (PCN-224) is a porphyrin-based Zr-MOF with the characteristics of biodegradability, large surface area and stability in a wide pH range. It can convert O2 into cytotoxic singlet oxygen ( 1 O2). However, most malignant tumors are in a state of hypoxia, and PDT is easily limited by the depth and range of light penetration, which affects the therapeutic effect of PDT. If it is used in combination with other anti-tumor therapies, it can effectively improve the anti-tumor efficacy.

[0005] Glucose oxidase (GOx) is an endogenous oxidoreductase widely present in organisms. It is a dimeric glycoprotein composed of two identical polypeptide chain subunits and two non-covalently bound flavin adenine dinucleotide (FAD) coenzymes. GOx can catalyze the conversion of D-glucose in tumor cells into glucuronic acid and hydrogen peroxide (H2O2), consuming glucose and oxygen in the tumor. In this catalytic reaction, GOx can use molecular oxygen as an electron acceptor to effectively catalyze the oxidation of β-D-glucose to D-glucose-δ-lactone, and then hydrolyze it to glucuronic acid. At the same time, FAD in GOx is reduced to FADH2, and then oxidized by oxygen to generate FAD and H2O2. However, it is difficult to completely eliminate tumors by GOx alone because it cannot block the continuous supply of oxygen and nutrients in the capillaries. Therefore, it can be combined with other treatment methods to fight tumors in order to achieve the best therapeutic effect.

[0006] Hydrogel is a 3D hydrophilic polymer network. It is widely used in the field of biomedical materials because of its many advantages such as high water absorption, good swelling, drug loading capacity and structural stability. At present, the commonly used cross-linking methods for hydrogels are mainly chemical cross-linking and physical cross-linking. However, traditional hydrogels lack strength, and are prone to permanent breakage during deformation by chemical cross-linking with covalent bonds. In addition, their internal structure is simple and lacks special functions, which greatly limits their application. Injectable hydrogels can be constructed through physical interactions such as hydrogen bonds, ionic interactions and electrostatic interactions. Their cross-linked structure is dynamically reversible and has the characteristics of sol-gel transition.

[0007] HA is a natural anionic linear mucopolysaccharide and a component of the extracellular matrix (ECM). Its disaccharide units have carboxyl, hydroxyl and N-acetyl groups, which can be cross-linked with different functional groups. Copper is a key redox cofactor for many enzyme reactions in the human body and has good biosafety and unique multiple biological activities. HA-based injectable hydrogels can quickly locate and deliver drugs to tumor tissues and then release them directly into tumor cells. It has the characteristics of improving drug endocytosis and tissue penetration, increasing the therapeutic concentration of drugs in local tissues and prolonging the retention time of drugs in tumors. It has excellent biocompatibility, minimally invasive delivery capabilities and a highly adaptable structure with regulatory functions, and can be used as a local drug delivery system for tumor treatment. Summary of the invention

[0008] The present invention provides a multi-pathway injectable anti-tumor gel system and its preparation method and application to solve the problems of low hydrogel strength, poor biocompatibility and complex preparation process in the prior art. 2+Metal coordination cross-linking and physical adsorption are used to encapsulate anti-tumor drugs into the hydrogel. The operation is simple and in situ injection into the tumor site has a highly effective tumor growth inhibition effect and low toxic side effects.

[0009] The technical solution adopted by the present invention comprises the following steps:

[0010] (1) Synthesis of PCN-224: Tetrakis(4-carboxyphenyl)porphine (TCPP), zirconium oxychloride octahydrate (ZrOCl2·8H2O), benzoic acid (BA) and N,N'-dimethylformamide (DMF) were dispersed uniformly by ultrasonication, and then stirred in an oil bath at 90°C for 5 h. The resulting solution was collected by centrifugation and purified to obtain nano-scale PCN-224.

[0011] (2) Synthesis of DOX@PCN: Doxorubicin (DOX) and the obtained PCN-224 were mixed evenly in deionized water, stirred and reacted at room temperature in the dark for 24 h, and the obtained solution was collected by centrifugation and purified to obtain DOX@PCN;

[0012] (3) Synthesis of Tph-HA: 3,3'-disulfanediyldi(propionylhydrazide) (TPH) and hyaluronic acid (HA) were mixed evenly to obtain a clear solution, the pH value of the solution was adjusted to 5.5, and then N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCꞏHCl) were added to activate the carboxyl group. After stirring at room temperature for 36 hours, the reaction was dialyzed with a dialysis bag for 72 hours, and the resulting solution was freeze-dried to obtain a white flocculent Tph-HA;

[0013] (4) Preparation of GDP@HA-Cu hydrogel: DOX@PCN aqueous solution and glucose oxidase (GOx) aqueous solution were added dropwise to Tph-HA aqueous solution. After mixing evenly, copper nitrate aqueous solution was added and stirred at high speed at room temperature to form an injectable anti-tumor hydrogel within 10 s.

[0014] Furthermore, in the step (1), the mass ratio of TCPP, ZrOCl2·8H2O and BA is 1-3:2-5:27-28.

[0015] Furthermore, in step (1), the ultrasonic dispersion time is 2 min and the stirring speed is 300 rpm.

[0016] Furthermore, the centrifugal collection and purification process in step (1) is as follows: centrifuging at a rotation speed of 15000 rpm for 15 minutes, collecting the precipitate and washing it with DMF and ethanol three times respectively.

[0017] Furthermore, in the step (2), the mass ratio of PCN-224 to DOX is 2-8:0.1-6.4.

[0018] Furthermore, the centrifugal collection and purification process in step (2) is as follows: centrifuging at a rotation speed of 15000 rpm for 15 minutes, collecting the precipitate and washing it three times with deionized water.

[0019] Furthermore, the solvent used to adjust the pH value in step (3) is 1M hydrochloric acid or 1M sodium hydroxide solution.

[0020] Furthermore, in step (3), the mass ratio of TPH, HA, NHS, and EDCꞏHCl is 1.1-1.2:1-1.1:0.069:0.096.

[0021] Furthermore, in the step (4), the mass ratio of DOX@PCN, GOx, Tph-HA and copper nitrate is 2-3: 0.27-0.6: 13-40: 7.7-31.4.

[0022] The advantages and positive effects of the present invention are as follows:

[0023] (1) The multi-pathway injectable anti-tumor gel system provided by the present invention adopts a supramolecular complexation method for preparing hydrogels, through Cu 2+ The metal coordination between the hydrazine groups on Tph-HA forms a physical cross-linking network. At the same time, the added DOX@PCN nanoparticles become cross-linking points inside the hydrogel through electrostatic attraction, thereby enhancing the strength of the hydrogel. 2+ The coordination reaction also endows the hydrogel with a variety of physicochemical functions such as good tensile properties, self-healing properties and injectability.

[0024] (2) The DOX@PCN loaded in the multi-pathway injectable anti-tumor gel system of the present invention can release DOX to cause cell apoptosis. The nano-metal organic framework PCN-224 can produce 1 O2 and accompanied by hyperthermia. At the same time, GOx catalyzes the conversion of D-glucose in tumor cells into glucuronic acid and H2O2, and the endogenous H2O2 is reduced by glutathione (GSH) to Cu + The Cu 2+ The accumulation of copper induces copper death. Compared with single therapy, the combination therapy of anti-tumor by integrating multiple pathways and administering through in situ injection can achieve good therapeutic effects.

[0025] (3) The reaction conditions of the present invention are mild and can be carried out at neutral pH and room temperature. The reaction time is ultra-fast and can be completed within 10 seconds. The operation is simple, the biocompatibility is good, the anti-tumor effect is excellent, and it has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the preparation process diagram of GDP@HA-Cu hydrogel.

[0027] Figure 2 FTIR graphs of PCN-224, DOX@PCN and DOX prepared in Example 1.

[0028] Figure 3 This is the SEM image of PCN-224 prepared in Example 1.

[0029] Figure 4 This is the SEM image of DOX@PCN prepared in Example 1.

[0030] Figure 5 3. The DLS graphs of PCN-224 and DOX@PCN prepared in Example 1.

[0031] Figure 6 This is the SEM image of the GDP@HA-Cu hydrogel prepared in Example 1.

[0032] Figure 7 This is the strain scanning diagram of the GDP@HA-Cu hydrogel prepared in Example 1.

[0033] Figure 8 This is the alternating strain scanning diagram of the GDP@HA-Cu hydrogel prepared in Example 1.

[0034] Fig. 9 This is a shear viscosity characterization diagram of the GDP@HA-Cu hydrogel prepared in Example 1.

[0035] Fig.10 This is a digital photograph of the injectability and self-healing properties of the GDP@HA-Cu hydrogel prepared in Example 1.

[0036] Fig.11 It is a cell activity diagram after the HA-Cu hydrogel prepared in Example 4 was co-incubated with human umbilical vein endothelial cells (HUVEC), triple-negative breast cancer cells (MDA-MB-231), human non-small cell lung cancer cells (A549) and mouse breast cancer cells (4T1).

[0037] Fig.12 This is a picture of in vitro orthotopic tumor tissue on the 14th day after 4T1 tumor-bearing mice were treated with different drugs. DETAILED DESCRIPTION

[0038] In order to further illustrate the present invention and its advantages, the technical scheme of the present invention will be further illustrated below in conjunction with embodiments, and it should be understood that these embodiments are only a part of embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work premise belong to the scope of protection of the present invention. The methods used in the embodiments are conventional methods in the art unless otherwise specified. Raw materials and reagents used in the present invention are all commercially available products.

[0039] Example 1

[0040] The preparation method of the multi-channel injectable anti-tumor gel system comprises the following steps:

[0041] (1) Synthesis of PCN-224: 25 mg of tetrakis(4-carboxyphenyl)porphine (TCPP), 75 mg of zirconium oxychloride octahydrate (ZrOCl2•8H2O), 700 mg of benzoic acid (BA) and 25 mL of N,N'-dimethylformamide (DMF) were added to a round-bottom flask. After being dispersed uniformly by ultrasonication, the mixture was stirred in an oil bath at 90 °C for 5 h. The resulting solution was centrifuged at 15,000 rpm for 15 min. The precipitate was collected and washed three times with DMF and ethanol to obtain nano-sized PCN-224.

[0042] (2) Synthesis of DOX@PCN: 3.2 mg of doxorubicin (DOX) and 8 mg of PCN-224 were mixed evenly in 4 mL of deionized water, placed in a round-bottom flask, and stirred for reaction at room temperature in the dark for 24 h. The resulting solution was centrifuged at 15,000 rpm for 15 min, and the precipitate was collected and washed three times with deionized water to obtain DOX@PCN.

[0043] (3) Synthesis of Tph-HA: 1.19 g of 3,3'-disulfanediyldi(propionylhydrazide) (TPH) and 1 g of hyaluronic acid (HA) were mixed to obtain a clear solution. The pH value of the solution was adjusted to 5.5 with 1 M hydrochloric acid or 1 M sodium hydroxide solution, and then 69 mg of N-hydroxysuccinimide (NHS) and 96 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCꞏHCl) were added to activate the carboxyl group. After stirring at room temperature for 36 h, the reaction was dialyzed for 72 h using a dialysis bag with a molecular weight cutoff of 2500 Da. The resulting solution was freeze-dried to obtain a white flocculent Tph-HA.

[0044] (4) Preparation of GDP@HA-Cu hydrogel: 0.2 mL of 10 mg / mL DOX@PCN aqueous solution and 0.05 mL of 12 mg / mL glucose oxidase (GOx) aqueous solution were added dropwise to 0.75 mL of 40 mg / mL Tph-HA aqueous solution. After mixing evenly, 0.043 mL of 3 mol / L copper nitrate aqueous solution was added. The mixture was stirred at high speed at room temperature to form an injectable anti-tumor hydrogel within 10 s. The resulting composite hydrogel was designated as GDP@HA-Cu-1.

[0045] Example 2

[0046] The preparation method of the multi-channel injectable anti-tumor gel system comprises the following steps:

[0047] (1) Synthesis of PCN-224: 50 mg of tetrakis(4-carboxyphenyl)porphine (TCPP), 50 mg of zirconium oxychloride octahydrate (ZrOCl2•8H2O), 700 mg of benzoic acid (BA) and 25 mL of N,N'-dimethylformamide (DMF) were added to a round-bottom flask. After being dispersed uniformly by ultrasonication, the mixture was stirred in an oil bath at 90 °C for 5 h. The resulting solution was centrifuged at 15,000 rpm for 15 min. The precipitate was collected and washed three times with DMF and ethanol to obtain nano-sized PCN-224.

[0048] (2) Synthesis of DOX@PCN: 0.1 mg doxorubicin (DOX) and 2 mg PCN-224 were mixed evenly in 4 mL deionized water, placed in a round-bottom flask, and stirred for reaction at room temperature in the dark for 24 h. The resulting solution was centrifuged at 15,000 rpm for 15 min, and the precipitate was collected and washed three times with deionized water to obtain DOX@PCN.

[0049] (3) Synthesis of Tph-HA: 1.15 g of 3,3'-disulfanediyldi(propionylhydrazide) (TPH) and 1.05 g of hyaluronic acid (HA) were mixed to obtain a clear solution. The pH value of the solution was adjusted to 5.5 with 1 M hydrochloric acid or 1 M sodium hydroxide solution, and then 69 mg of N-hydroxysuccinimide (NHS) and 96 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCꞏHCl) were added to activate the carboxyl group. After stirring at room temperature for 36 h, the reaction was dialyzed for 72 h using a dialysis bag with a molecular weight cutoff of 2500 Da. The resulting solution was freeze-dried to obtain a white flocculent Tph-HA.

[0050] (4) Preparation of GDP@HA-Cu hydrogel: 0.3 mL of 10 mg / mL DOX@PCN aqueous solution and 0.01 mL of 27 mg / mL glucose oxidase (GOx) aqueous solution were added dropwise to 0.69 mL of 29 mg / mL Tph-HA aqueous solution. After mixing evenly, 0.022 mL of 3 mol / L copper nitrate aqueous solution was added. The mixture was stirred at high speed at room temperature to form an injectable anti-tumor hydrogel within 10 s. The resulting composite hydrogel was designated as GDP@HA-Cu-2.

[0051] Example 3

[0052] The preparation method of the multi-channel injectable anti-tumor gel system comprises the following steps:

[0053] (1) Synthesis of PCN-224: 75 mg of tetrakis(4-carboxyphenyl)porphine (TCPP), 125 mg of zirconium oxychloride octahydrate (ZrOCl2•8H2O), 675 mg of benzoic acid (BA) and 25 mL of N,N'-dimethylformamide (DMF) were added to a round-bottom flask. After being dispersed uniformly by ultrasonication, the mixture was stirred in an oil bath at 90 °C for 5 h. The resulting solution was centrifuged at 15,000 rpm for 15 min. The precipitate was collected and washed three times with DMF and ethanol to obtain nano-sized PCN-224.

[0054] (2) Synthesis of DOX@PCN: 1.6 mg doxorubicin (DOX) and 2 mg PCN-224 were mixed evenly in 4 mL deionized water, placed in a round-bottom flask, and stirred for reaction at room temperature in the dark for 24 h. The resulting solution was centrifuged at 15,000 rpm for 15 min, and the precipitate was collected and washed three times with deionized water to obtain DOX@PCN.

[0055] (3) Synthesis of Tph-HA: 1.1 g of 3,3'-disulfanediyldi(propionylhydrazide) (TPH) and 1.1 g of hyaluronic acid (HA) were mixed to obtain a clear solution. The pH value of the solution was adjusted to 5.5 with 1 M hydrochloric acid or 1 M sodium hydroxide solution, and then 69 mg of N-hydroxysuccinimide (NHS) and 96 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCꞏHCl) were added to activate the carboxyl group. After stirring at room temperature for 36 h, the reaction was dialyzed for 72 h using a dialysis bag with a molecular weight cutoff of 2500 Da. The resulting solution was freeze-dried to obtain a white flocculent Tph-HA.

[0056] (4) Preparation of GDP@HA-Cu hydrogel: 0.2 mL of 10 mg / mL DOX@PCN aqueous solution and 0.05 mL of 12 mg / mL glucose oxidase (GOx) aqueous solution were added dropwise to 0.75 mL of 40 mg / mL Tph-HA aqueous solution. After mixing evenly, 0.065 mL of 2 mol / L copper nitrate aqueous solution was added. The mixture was stirred at high speed at room temperature to form an injectable anti-tumor hydrogel within 10 s. The resulting composite hydrogel was designated as GDP@HA-Cu-3.

[0057] Example 4

[0058] 0.25 mL of deionized water was added to 0.75 mL of 40 mg / mL Tph-HA aqueous solution. After mixing evenly, 0.043 mL of 3 mol / L copper nitrate aqueous solution was added dropwise. The mixture was stirred at high speed at room temperature to form HA-Cu hydrogel within 10 s.

[0059] The preparation process of hydrogel is shown in Figure 1 .

[0060] The embodiments were tested, and the test conditions all followed the relevant national standards.

[0061] Fourier transform infrared spectrometer (FTIR) was used to collect FTIR spectra of DOX, PCN-224 and DOX@PCN for component analysis. The results are as follows: Figure 2 As shown, the infrared absorption spectra of PCN-224 and DOX@PCN prepared in Example 1 are basically consistent with those reported in the literature, indicating that the preparation of PCN-224 and DOX@PCN is successful.

[0062] The morphology of PCN-224 and DOX@PCN was analyzed using scanning electron microscopy (SEM). Figure 3 As shown, the PCN-224 and DOX@PCN prepared in Example 1 are both spherical particles with uniform particle size.

[0063] The particle sizes of PCN-224 and DOX@PCN were measured by dynamic light scattering (DLS). Figure 4 As shown, the average particle size of PCN-224 prepared in Example 1 is 158 nm, and the average particle size of DOX@PCN is 167.6 nm.

[0064] The GDP@HA-Cu hydrogel was analyzed by scanning electron microscopy (SEM). Figure 5 As shown, the hydrogel prepared in Example 1 has a three-dimensional porous microstructure, and the average pore size is about 62 μm.

[0065] The rheological properties of the GDP@HA-Cu hydrogel prepared in Example 1 were measured using a rheometer. Figure 6 , Figure 7 and Figure 8 shown. Figure 6 This is the strain scan of GDP@HA-Cu hydrogel. When the strain reaches 305%, the hydrogel network collapses. Figure 7 This is the alternating strain scan of GDP@HA-Cu hydrogel. When the applied shear strain is switched from 1% to 450% and repeated three times, G' and G" can still recover to their initial values, proving that the hydrogel has good self-healing properties. Figure 8 This is the shear viscosity characterization diagram of GDP@HA-Cu hydrogel. With the increase of shear rate, the viscosity of the hydrogel continues to decrease, indicating that the hydrogel has excellent injectability.

[0066] The injectability and self-healing properties of the GDP@HA-Cu hydrogel prepared in Example 1 were further studied by injection experiments and self-healing experiments. Fig. 9 As shown, the hydrogel can be squeezed out from the needle of the syringe smoothly, continuously and without breaking. When two cut hydrogels are put together, they can adhere into one hydrogel in a short time without applying external force and can be picked up with tweezers, which further demonstrates that the hydrogel has good injectability and self-healing properties.

[0067] The effect of the HA-Cu hydrogel prepared in Example 4 on the cell activity of human umbilical vein endothelial cells (HUVEC), triple-negative breast cancer cells (MDA-MB-231), human non-small cell lung cancer cells (A549) and mouse breast cancer cells (4T1) was evaluated by MTT experiment. After the cells were cultured overnight at 37°C and 5% CO2, equal amounts of HA-Cu hydrogel extracts of different concentrations were added, and the cells were incubated again for 48 hours, and MTT was added to detect the cell survival rate. The results are shown in Figure 2. Fig.10 As shown, the cell viability of all experimental groups reached more than 90%, indicating that the hydrogel has good biocompatibility.

[0068] The anti-tumor effect of the GDP@HA-Cu hydrogel prepared in the present invention was evaluated using a mouse tumor model. The specific steps were as follows: 4T1 mouse breast cancer cells in the logarithmic growth phase were inoculated subcutaneously into the right buttocks of Balb / c female mice to establish a 4T1 tumor model. When the tumor volume grew to about 70 mm, the anti-tumor effect of the GDP@HA-Cu hydrogel was evaluated using a mouse tumor model. The anti-tumor effect of the GDP@HA-Cu hydrogel prepared in the present invention was evaluated using a mouse tumor model. The specific steps were as follows: 4T1 mouse breast cancer cells in the logarithmic growth phase were inoculated subcutaneously into the right buttocks of Balb / c female mice to establish a 4T1 tumor model. 3The mice were randomly divided into 5 groups, with 6 mice in each group, and intratumoral injection was performed: PBS group, DOX group, GOx group, GDP@Gel group and GDP@Gel+NIR group. The GDP@Gel+NIR group was irradiated with 660nm near-infrared light on the 1st, 3rd, 5th and 7th days of administration. The weight and tumor size of the mice were measured every two days. The mice were killed on the 14th day and the tumor tissues were removed. The results are shown in Fig.11 As shown, the tumors in the GDP@Gel+NIR group were significantly smaller than those in the other four groups, and the weight of the mice remained stable during the treatment, indicating that GDP@Gel hydrogel has low toxic side effects, high biosafety, and good anti-tumor effects.

Claims

1. A method for preparing a multi-channel injectable anti-tumor gel system, characterized in that: The following steps are included (1) Synthesis of PCN-224: Tetrakis(4-carboxyphenyl)porphine (TCPP), zirconium oxychloride octahydrate (ZrOCl2•8H2O), benzoic acid (BA) and N,N'-dimethylformamide (DMF) were dispersed uniformly by ultrasonication, and then stirred in an oil bath at 90°C for 5 h. The resulting solution was collected by centrifugation and purified to obtain nano-scale PCN-224. (2) Synthesis of DOX@PCN: Doxorubicin (DOX) and the obtained PCN-224 were mixed evenly in deionized water, stirred and reacted at room temperature in the dark for 24 h, and the obtained solution was collected by centrifugation and purified to obtain DOX@PCN; (3) Synthesis of Tph-HA: 3,3'-disulfanediyldi(propionylhydrazide) (TPH) and hyaluronic acid (HA) were mixed evenly to obtain a clear solution, the pH value of the solution was adjusted to 5.5, and then N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCꞏHCl) were added to activate the carboxyl group. After stirring at room temperature for 36 hours, the reaction was dialyzed with a dialysis bag for 72 hours, and the resulting solution was freeze-dried to obtain a white flocculent Tph-HA; (4) Preparation of GDP@HA-Cu hydrogel: DOX@PCN aqueous solution and glucose oxidase (GOx) aqueous solution were added dropwise to Tph-HA aqueous solution. After mixing evenly, copper nitrate aqueous solution was added and stirred at high speed at room temperature to form an injectable anti-tumor hydrogel within 10 s.

2. The method for preparing the multi-channel injectable anti-tumor gel system according to claim 1, characterized in that: In the step (1), the mass ratio of TCPP, ZrOCl2•8H2O and BA is 1-3:2-5:27-28.

3. The method for preparing the multi-channel injectable anti-tumor gel system according to claim 1, characterized in that: In the step (1), the ultrasonic dispersion time is 2 min and the stirring speed is 300 rpm.

4. The method for preparing the multi-channel injectable anti-tumor gel system according to claim 1, characterized in that: The centrifugal collection and purification process in step (1) is as follows: centrifuge at a rotation speed of 15000 rpm for 15 minutes, collect the precipitate and wash it with DMF and ethanol three times respectively.

5. The method for preparing the multi-channel injectable anti-tumor gel system according to claim 1, characterized in that: The mass ratio of PCN-224 to DOX in the step (2) is 2-8:0.1-6.

4.

6. The method for preparing the multi-channel injectable anti-tumor gel system according to claim 1, characterized in that: The centrifugal collection and purification process in step (2) is as follows: centrifuge at a rotation speed of 15000 rpm for 15 minutes, collect the precipitate and wash it three times with deionized water.

7. The method for preparing the multi-channel injectable anti-tumor gel system according to claim 1, characterized in that: The solvent used to adjust the pH value in step (3) is 1M hydrochloric acid or 1M sodium hydroxide solution.

8. The method for preparing the multi-channel injectable anti-tumor gel system according to claim 1, characterized in that: In the step (3), the mass ratio of TPH, HA, NHS, and EDC / HCl is 1.1-1.2:1-1.1:0.069:0.

096.

9. The method for preparing the multi-channel injectable anti-tumor gel system according to claim 1, characterized in that: In the step (4), the mass ratio of DOX@PCN, GOx, Tph-HA and copper nitrate is 2-3:0.27-0.6:13-40:7.7-31.

4.

10. The hydrogel prepared by the method for preparing a multi-channel injectable anti-tumor gel system according to any one of claims 1 to 11, characterized in that: The hydrogel is administered by injection for multi-pathway anti-tumor treatment.

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