Preparation method and application of immune nanocomposite hydrogel with anti-tumor effect

Abstract

The present invention discloses a preparation method and application of an immune nanocomposite hydrogel with anti-tumor effect, wherein dopamine-modified hyaluronic acid is added to water to form a dopamine-modified hyaluronic acid aqueous solution, δ-ALA@PLGA microspheres are then added, dopamine is added, sodium periodate is added, dopamine polymerization is induced under stirring, and then the mixture is allowed to stand to form a polydopamine hydrogel; sodium alginate and aCD47@CaCO3 nanoparticles are added to water to obtain a sodium alginate solution; the sodium alginate solution is added to the polydopamine hydrogel, calcium chloride solution is added, and the mixture is allowed to stand. The present invention can achieve the production of hydrogel by simple mixing, has a short reaction cycle, a high yield, no by-products, is easy to realize industrial production, and can effectively improve work efficiency. The composite hydrogel can be used as a new bioactive material for tumor treatment and can be used as a gel patch that adheres to the lesion and releases drugs for a long time.

Description

Technical Field

[0001] The present invention relates to the fields of polymer material technology and biomedical materials, and in particular to a preparation method and application of an immune nanocomposite hydrogel with anti-tumor effect. Background Art

[0002] Hydrogels are hydrophilic 3D network polymers with excellent biocompatibility, high water content, and tunable structural properties, making them widely used in the biomedical field. These gels not only embed bioactive molecules (such as drugs, proteins, or antibodies) into precursor solutions for targeted delivery, but also provide an environment similar to the extracellular matrix. Their porous structure allows for the exchange of nutrients and metabolites and cell migration, thereby promoting tissue regeneration and achieving better therapeutic effects. Hydrogels have attracted widespread attention in the fields of localized tissue defects and cancer treatment due to their excellent biocompatibility and drug-loading capacity. In the field of cancer treatment, hydrogels must meet the following three requirements: (i) the materials used to construct the hydrogels must have good biocompatibility; (ii) the prepared hydrogels must exhibit strong tissue adhesion and exhibit strong retention at the tumor site after entry into the body; and (iii) they must exhibit high drug loading rates and tumor microenvironment-responsive drug release. However, current hydrogels often have limited functionality and poor strength, resulting in several challenges in practical applications and significant room for development. Summary of the Invention

[0003] In response to the above problems in the prior art, the purpose of the present invention is to provide a method for preparing and applying an immune nanocomposite hydrogel with anti-tumor effects. The method is simple, the conditions are mild, and it is easy to industrialize. The prepared gel has no toxic side effects on healthy tissues, can only be induced to release by the specific microenvironment of tumor tissue, and can kill tumor cells under light. In addition, the induced immune response can effectively inhibit tumor recurrence.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] A method for preparing an immune nanocomposite hydrogel with anti-tumor effect comprises the following steps:

[0006] Dopamine-modified hyaluronic acid was added to water and stirred evenly to form a dopamine-modified hyaluronic acid aqueous solution, and then δ-ALA@PLGA microspheres were added and stirred evenly to obtain a mixed solution;

[0007] Add dopamine to the mixed solution, adjust the pH to 8-9, then add sodium periodate, induce dopamine polymerization under stirring, and then let it stand to form a polydopamine hydrogel;

[0008] Sodium alginate and aCD47@CaCO3 nanoparticles were added to water and stirred to obtain a sodium alginate solution;

[0009] Sodium alginate solution is added to polydopamine hydrogel, calcium chloride solution is added, and the mixture is allowed to stand to obtain an immune nanocomposite hydrogel with anti-tumor effect.

[0010] Furthermore, dopamine-modified hyaluronic acid is prepared by the following process: dissolving hyaluronic acid in ultrapure water, adjusting the pH to 5.5, adding dopamine hydrochloride under nitrogen protection, stirring in the dark, adding EDC solution and NHS solution, stirring in the dark at room temperature to obtain a precipitate; dissolving the precipitate in water, transferring it to a dialysis bag with a molecular cutoff of 3500 for dialysis, freeze-drying after dialysis, and drying in the dark at room temperature to obtain dopamine-modified hyaluronic acid.

[0011] Furthermore, the molecular weight of hyaluronic acid is 100,000 to 1,000,000 molecules; and the concentration of dopamine-modified hyaluronic acid is 20 mg / mL.

[0012] Furthermore, the mass ratio of δ-ALA@PLGA microspheres to dopamine-modified hyaluronic acid was 2:1.

[0013] Furthermore, δ-ALA@PLGA microspheres were prepared by the following process: poly(lactic acid-co-glycolic acid) was dissolved in dichloromethane, followed by adding δ-ALA solution, ultrasonicating at a power of 500 W, then adding PVA solution, ultrasonicating at a power of 900 W, and stirring to obtain δ-ALA@PLGA microspheres.

[0014] Furthermore, the standing time is 10-24 hours; and the mass ratio of dopamine-modified hyaluronic acid, dopamine, and sodium periodate is 2:10:(1-1.5).

[0015] Furthermore, aCD47@CaCO3 nanoparticles were prepared by the following process: Tris-hydrochloric acid buffer containing CaCl2 was mixed with 4-hydroxyethylpiperazineethanesulfonate aqueous buffer containing aCD47 antibody and Na2CO3, stirred, centrifuged, and freeze-dried to obtain aCD47@CaCO3 nanoparticles.

[0016] Furthermore, the mass concentration of sodium alginate in the sodium alginate solution is 2-4%; the mass concentration of the calcium chloride solution is 5%-10%.

[0017] Furthermore, the mass ratio of aCD47@CaCO3 nanoparticles to sodium alginate is 1:15; the mass ratio of sodium alginate to calcium chloride is 1-4:1.

[0018] The invention discloses an application of an immune nanocomposite hydrogel with anti-tumor effect in preparing a gel for tumor treatment.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The gel matrix used in the present invention is hyaluronic acid and sodium alginate, which are widely available, have good biocompatibility, are degradable in vivo, and the degradation products are non-toxic. They are suitable for use in the field of medical biology. The present invention uses hyaluronic acid and sodium alginate as substrates, respectively, and utilizes the polymerization of dopamine on dopamine-modified hyaluronic acid to prepare polydopamine hydrogel; sodium alginate is cross-linked with calcium ions to form a calcium alginate gel while being compounded with polydopamine hydrogel to obtain an immune nanocomposite hydrogel with anti-tumor effect. The composite hydrogel has uniform pores and lamellae, has good drug loading capacity, and can provide conditions for drug loading and specific release. The experimental process of the present invention is simple and easy to operate, has a short reaction cycle, high yield, substantially no loss of raw materials, and is tolerant to experimental conditions. The present invention does not produce organic waste liquid during operation, and meets the preparation conditions of green and environmentally friendly. The present invention does not require large and expensive instruments and equipment, and has low production cost.

[0021] The polydopamine hydrogel in this invention not only provides drug loading but also provides photothermal therapy. The calcium alginate gel contains an antibody encapsulated in nanoparticles, providing a time gradient for release and tumor microenvironment-specific release. This increases the pH of the tumor microenvironment, thereby inducing macrophage polarization and an immune response. The composite hydrogel of this invention has no toxic side effects on healthy tissues and provides targeted treatment only at the tumor site, effectively avoiding side effects associated with treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a physical picture of the composite hydrogel prepared in Example 3 of the present invention;

[0023] Figure 2 These are scanning electron microscope images of the composite gel prepared in Example 3 of the present invention, wherein (a) is a morphology of the polydopamine hydrogel, (b) is a morphology of the PLGA microspheres loaded in the pores of the polydopamine hydrogel, (c) is a morphology of the calcium alginate hydrogel, and (d) is a morphology of the aCD47@CaCO3 nanoparticles loaded in the calcium alginate hydrogel sheet structure.

[0024] Figure 3 The photothermal performance of the polydopamine hydrogel prepared in Example 1 at different powers, where (a) is the temperature rise of the polydopamine hydrogel at different powers, and (b) is the photothermal image of the polydopamine hydrogel at different powers observed using a thermal imager at the 15th minute;

[0025] Figure 4This is the PLGA release of the polydopamine hydrogel prepared in Example 1 of the present invention under continuous light irradiation of different powers.

[0026] Figure 5 This is the long-term light-controlled release of the polydopamine hydrogel prepared in Example 1 of the present invention at a power of 1.25W.

[0027] Figure 6 This is the daily release amount of the polydopamine hydrogel prepared in Example 1 of the present invention after long-term light-controlled release at a power of 1.25 W.

[0028] Figure 7 pH changes of the buffer solution during the release of aCD47@CaCO3 prepared in Example 2 of the present invention from the gel.

[0029] Figure 8 The release rate of the calcium alginate gel loaded with aCD47@CaCO3 prepared in Example 2 of the present invention changes over time. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the embodiments and drawings, but is not limited thereto.

[0031] The invention relates to a multi-step method for preparing a hydrogel with a porous structure and the construction and application of biomedical materials based on the hydrogel.

[0032] A method for preparing an immune nanocomposite hydrogel with anti-tumor effect comprises the following steps:

[0033] By inducing dopamine-modified hyaluronic acid to polymerize, a black polydopamine-hyaluronic acid photothermal gel is formed and placed in a mold. Then, a sodium alginate aqueous solution is poured into the other half of the gel mold, and a calcium chloride solution is slowly added. The gel is allowed to stand for about 30 minutes to form a hyaluronic acid-calcium alginate composite hydrogel.

[0034] Specifically, the following steps are included:

[0035] 1) Preparation of dopamine-modified hyaluronic acid:

[0036] Weigh 1.0 g of HA (hyaluronic acid, molecular weight 100,000 to 1,000,000 molecules) and dissolve it in 50 mL of ultrapure water. Adjust the pH to 5.5 with 1 mol / L hydrochloric acid. Place under nitrogen for 2 hours. Add a predetermined amount of dopamine hydrochloride (0.5 times the molar amount of HA). Stir in the dark for 30 minutes. Then, sequentially add EDC solution (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EDC added in an amount twice the molar amount of dopamine) and NHS solution (N-hydroxysuccinimide, NHS added in an amount 1.5 times the molar amount of dopamine). Stir in the dark for 12 hours at room temperature to obtain a precipitate. Dissolve the precipitate in water and transfer it to a dialysis bag with a molecular weight cutoff of 3500 for dialysis. After 3 days of dialysis, freeze-dry it and dry it in the dark at room temperature to obtain dopamine-modified hyaluronic acid (HA-DOPA) and store it for future use.

[0037] Weigh 100 mg of PLGA (polylactic acid-co-glycolic acid) and dissolve it in 1 ml of dichloromethane, then add 100 μl of 0.05 mg / μl δ-ALA (5-aminolevulinic acid) solution. Place the mixed solution in an ultrasonic cell crusher and ultrasonicate it at a power of 500 W, with an interval of 9 seconds for ultrasonication and 4 seconds for rest, and repeat three times. Take 10 ml of a 1% PVA (polyvinyl alcohol) solution, quickly add it to the above solution, and place it in an ultrasonic cell crusher again for ultrasonication at a power of 900 W, with an interval of 9 seconds for ultrasonication and 4 seconds for rest, and repeat three times. Place the sonicated solution in a fume hood and stir for 3 hours. After the solvent evaporates, wash with water to obtain δ-ALA@PLGA microspheres, freeze-dry them, and store them in a 4°C freezer for later use.

[0038] 2) Add dopamine-modified hyaluronic acid to water and stir to form a dopamine-modified hyaluronic acid aqueous solution. Then, add δ-ALA@PLGA microspheres and stir to mix thoroughly. The concentration of dopamine-modified hyaluronic acid is 20 mg / mL, and the mass ratio of δ-ALA@PLGA microspheres to HA-DOPA is 2:1.

[0039] 3) Dopamine was added to the aqueous solution of dopamine-modified hyaluronic acid mixed with δ-ALA@PLGA microspheres. The pH was then adjusted to 8-9 with a NaOH solution. Sodium periodate was then added and stirred at 300-600 rpm for 10 minutes to induce dopamine polymerization. The mixture was then allowed to stand for 10-24 hours to form a polydopamine hydrogel. The mass ratio of dopamine-modified hyaluronic acid, dopamine, and sodium periodate was 2:10:(1-1.5).

[0040] 4) Mix 1 ml of Tris-HCl buffer (1 mmol / L, pH 7.6) containing 100 mmol / L CaCl₂ with 1 ml of HEPES (4-hydroxyethylpiperazineethanesulfonic acid) saline buffer (50 mmol / L, pH 7.1, 140 mmol / L NaCl) containing 100 μg aCD47 antibody and 10 mmol / L Na₂CO₃. Stir at 4°C for 12 hours. Remove excess ions and antibodies by centrifugation at 14,800 rpm for 5 minutes. Freeze-dry the resulting aCD47@CaCO₃ nanoparticles and store them in a -20°C freezer until use.

[0041] Sodium alginate and aCD47@CaCO3 nanoparticles are added to water and stirred to mix evenly to prepare a sodium alginate solution. The mass ratio of aCD47@CaCO3 nanoparticles to sodium alginate is 1:15, and the mass concentration of sodium alginate in the sodium alginate solution is 2-4%.

[0042] A sodium alginate solution is added to the other side of the mold where the polydopamine hydrogel is placed, and a calcium chloride solution with a mass concentration of 5%-10% is slowly added to the sodium alginate solution. After the addition is completed, the solution is allowed to stand for about 20-60 minutes. After the calcium alginate hydrogel is gelled, an immune nanocomposite hydrogel for tumor treatment is obtained; the mass ratio of sodium alginate to calcium chloride is 1-4:1.

[0043] Among them, δ-ALA@PLGA microspheres loaded in polydopamine hydrogel were added by physical mixing during the dissolution of dopamine-modified hyaluronic acid, and aCD47@CaCO3 loaded in calcium alginate hydrogel was added by physical mixing during the dissolution of sodium alginate.

[0044] Wherein, the mass ratio of dopamine-modified hyaluronic acid to sodium alginate is 1:1-1.5.

[0045] The composite porous gel prepared by the invention has uniformly distributed pores and has good drug-loading capacity.

[0046] Application of the drug-loaded hydrogel prepared by the above method in preparing a gel for tumor treatment.

[0047] The following describes it in detail through specific embodiments.

[0048] Example 1

[0049] (1) Weigh 1.0 g of HA (hyaluronic acid) and dissolve it in 50 mL of ultrapure water. Adjust the pH to 5.5 with 1 mol / L hydrochloric acid solution. Protect the mixture with nitrogen for 2 h, add a certain amount of dopamine hydrochloride (the amount added is 0.5 times the molar amount of hyaluronic acid), stir in the dark for 30 min, then add EDC solution (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, the amount of EDC added is 2 times the molar amount of dopamine) and NHS solution (N-hydroxysuccinimide, the amount of NHS added is 1.5 times the molar amount of dopamine) in sequence, stir in the dark at room temperature for 12 h to obtain a precipitate. Dissolve the precipitate in water, transfer it to a dialysis bag with a molecular weight cutoff of 3500, and dialyze it. After dialysis for 3 days, freeze-dry it, dry it in the dark at room temperature, and obtain dopamine-modified hyaluronic acid HA-DOPA, which is stored for future use.

[0050] Weigh 100 mg of PLGA (polylactic acid-co-glycolic acid) and dissolve it in 1 ml of dichloromethane, then add 100 μl of 0.05 mg / μl δ-ALA (5-aminolevulinic acid) solution. Place the mixed solution in an ultrasonic cell crusher and ultrasonicate it at a power of 500 W, with an interval of 9 seconds for ultrasonication and 4 seconds for rest, and repeat three times. Take 10 ml of a 1% PVA (polyvinyl alcohol) solution, quickly add it to the above solution, and place it in an ultrasonic cell crusher again for ultrasonication at a power of 900 W, with an interval of 9 seconds for ultrasonication and 4 seconds for rest, and repeat three times. Place the sonicated solution in a fume hood and stir for 3 hours. After the solvent evaporates, wash with water to obtain δ-ALA@PLGA microspheres, freeze-dry them, and store them in a 4°C freezer for later use.

[0051] (2) 20 mg of dopamine-modified hyaluronic acid was added to 1 ml of ultrapure water and stirred to form a dopamine-modified hyaluronic acid aqueous solution. Then, 40 mg of δ-ALA@PLGA microspheres were added and stirred to mix evenly.

[0052] (3) 100 mg of dopamine was added to the dopamine-modified hyaluronic acid aqueous solution mixed with δ-ALA@PLGA microspheres, and then the pH was adjusted to 8-9 with NaOH solution. 10 mg of sodium periodate was added, and the mixture was stirred at a speed of 300-600 r / min for 10 min to induce dopamine polymerization. The mixture was then allowed to stand for 12 h to form a polydopamine hydrogel.

[0053] The polydopamine hydrogel obtained under these conditions has a good gelling effect, a distinct pore structure, and a uniform pore size distribution.

[0054] See also Figure 2 (a) and (b). Figure 2 (a) is a scanning electron microscope image of the polydopamine hydrogel prepared in Example 1. It can be seen that the prepared polydopamine hydrogel has uniform pores and good structure; Figure 2 (b) shows the δ-ALA@PLGA microspheres loaded in the pores of the polydopamine hydrogel prepared in Example 1 observed under a scanning electron microscope, verifying that the microspheres can be successfully loaded in the pores of the polydopamine hydrogel.

[0055] Photothermal performance test of hydrogel 1:

[0056] 1 mg of the polydopamine hydrogel described in Example 1 was added with 1 ml of PBS solution with a pH of 6.8, and placed under an 808 nm laser with a power of 1.25 W. The temperature change of the gel was measured at 0, 1, ..., 14, and 15 min using an infrared thermal imager. Figure 3 The test results show that at this power, the temperature of the hydrogel rises by about 20°C in the 15th minute, and the photothermal performance is good.

[0057] Photothermal performance test of hydrogel 2:

[0058] 1 mg of the polydopamine hydrogel described in Example 1 was added with 1 ml of PBS solution with a pH of 6.8, and placed under an 808 nm laser with a laser power of 0.5 W. The temperature change of the gel was measured at 0, 1, ..., 14, and 15 min using an infrared thermal imager. Figure 3 The test results of (a) and (b) show that since the power of 0.5W is not the appropriate power, at this power, the temperature of the hydrogel rises by about 5°C in the 15th minute, and the photothermal performance is poor.

[0059] Photothermal performance test of polydopamine hydrogel 3:

[0060] 1 mg of the polydopamine hydrogel described in Example 1 was added with 1 ml of PBS solution with a pH of 6.8, and placed under an 808 nm laser with a power of 2 W. The temperature change of the gel was measured at 0, 1, ..., 14, and 15 min using an infrared thermal imager. Figure 3 The test results show that at this power, the temperature of the hydrogel rises by about 40°C in the 15th minute, and its photothermal performance is excellent. However, this temperature rise may cause unnecessary damage to the surrounding tissues, so this temperature is not selected as the implementation temperature during the gel treatment process, that is, the power of 2W is not the appropriate power.

[0061] The results of the photothermal performance test 1, the photothermal performance test 2 and the photothermal performance test 3 of the polydopamine hydrogel determined that the appropriate power was 1.25 W, and the local temperature rise achieved at this power was conducive to killing tumor cells.

[0062] Drug release performance test of polydopamine hydrogel 1:

[0063] 1 mg of the polydopamine hydrogel in Example 1 was added to 1 ml of a PBS solution having a pH of 6.8, and the mixture was placed under an 808 nm laser with a power of 1.25 W. The supernatant was collected at 0, 15, 30, 45, 60, 120, 150, and 180 min, and the drug release was measured by a fluorescence spectrophotometer. Figure 4 The experimental results show that at a power of 1.25W, the drug release rate is ultimately about 14%. The drug release effect at this power is average, but considering the side effects of higher temperature rise on tissues at a power of 2W, this power was ultimately used as the implementation power for the photothermal gel.

[0064] Drug release performance test of polydopamine hydrogel 2:

[0065] 1 mg of the polydopamine hydrogel in Example 1 was added to 1 ml of a PBS solution having a pH of 6.8, and the mixture was placed under an 808 nm laser with a power of 0.5 W. The supernatant was collected at 0, 15, 30, 45, 60, 120, 150, and 180 min, and the drug release was measured by a fluorescence spectrophotometer. Figure 4 The experimental results show that at a power of 0.5 W, the drug release rate is only about 4% due to the lower power compared to the embodiment. The drug release effect is poor at this power.

[0066] Drug release performance test of polydopamine hydrogel 3:

[0067] 1 mg of the polydopamine hydrogel in Example 1 was added to 1 ml of a PBS solution having a pH of 6.8, and the mixture was placed under an 808 nm laser with a power of 2 W. The supernatant was collected at 0, 15, 30, 45, 60, 120, 150, and 180 min, and the drug release was measured by a fluorescence spectrophotometer. Figure 4 The experimental results showed that at a power of 2 W, the drug release rate was approximately 52%, indicating good drug release. However, considering the side effects of the higher temperature rise at 2 W on tissues, this power was not ultimately used for the photothermal gel.

[0068] Controlled drug release performance test of polydopamine hydrogel 4:

[0069] 1 mg of the polydopamine hydrogel in Example 1 was added to 1 ml of a PBS solution with a pH of 6.8, and placed under an 808 nm laser with a laser power of 1.25 W. The solution was irradiated for 15 minutes and allowed to stand for 24 hours for a week of long-term controlled release. The supernatant was collected before and after each irradiation, and the drug release was measured by a fluorescence spectrophotometer. Figure 5 The experimental results show that under the long-term controlled release of 1.25W power, the drug release rate is finally about 72%, which can achieve the purpose of long-term treatment in the body. Figure 6 The release situation after daily illumination is shown. It can be seen that after daily illumination, the hydrogel has a drug release rate of about 1.5%-5.3%, which illustrates the feasibility of light-controlled release.

[0070] The drug release performance test 1, drug release performance test 2, drug release performance test 3 and drug release performance test 4 of the polydopamine hydrogel indicate that the drug-loaded polydopamine hydrogel can achieve controllable release, and can achieve continuous and rapid release under continuous light, and can also achieve long-term slow release under long-term intermittent light, which is beneficial to achieve the purpose of controllable tumor treatment in clinical treatment.

[0071] Example 2

[0072] 1 ml of Tris-HCl buffer (1 mmol / L, pH 7.6) containing 100 mmol / L CaCl₂ was mixed with 1 ml of HEPES saline buffer (50 mmol / L, pH 7.1, 140 mmol / L NaCl) containing 100 μg of CD47 antibody and 10 mmol / L Na₂CO₃ and stirred at 4°C for 12 hours. Excess ions and antibodies were removed by centrifugation at 14,800 rpm for 5 minutes. The resulting aCD47@CaCO₃ nanoparticles were freeze-dried and stored in a -20°C freezer until use.

[0073] Sodium alginate and aCD47@CaCO3 nanoparticles were added to 1 ml of ultrapure water and stirred to mix evenly to prepare a sodium alginate solution with a sodium alginate concentration of 3%. The mass ratio of aCD47@CaCO3 nanoparticles to sodium alginate was 1:15.

[0074] In order to test the performance of calcium alginate gel, in the present invention, about 200 μl of a 10% calcium chloride solution was slowly added dropwise to the sodium alginate solution. After the addition was completed, the solution was allowed to stand for 30 minutes to obtain calcium alginate gel.

[0075] See also Figure 2 In (c) and (d), Figure 2 (c) shows the lamellar structure of the calcium alginate gel synthesized in Example 2 observed under a scanning electron microscope. Figure 2 (d) shows the morphology of aCD47@CaCO3 nanoparticles loaded in the calcium alginate gel sheet structure synthesized in Example 2. The small particles in the gel are calcium carbonate nanoparticles, indicating that the calcium carbonate nanoparticles are successfully loaded.

[0076] pH adjustment performance test of calcium alginate hydrogel:

[0077] 1 mg of the calcium alginate hydrogel synthesized in Example 2 was added to 5 ml of a PBS solution having a pH of 6.8, and the pH value of the supernatant was measured by a pH meter at 0, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 10, 24, and 120 h. Figure 7 The experimental results showed that the pH of the supernatant rose to above 7.4 in just 1 hour and could still maintain this level for 120 hours, indicating that the hydrogel has a good effect on pH regulation, which is beneficial for regulating the pH of the tumor site and inducing macrophage polarization in clinical treatment.

[0078] Antibody release performance test of calcium alginate hydrogel:

[0079] 1 mg of the calcium alginate hydrogel synthesized in Example 2 was added with 1 ml of a PBS solution having a pH of 6.8. The supernatant was collected on days 0, 1, ..., 15, and 16, and the antibody release rate was measured using a fluorescence spectrophotometer. Figure 8 The experimental results show that the antibody can eventually achieve a release rate of about 73%. At the same time, the coating of calcium carbonate nanoparticles effectively prevents the waste caused by the rapid release of drugs. The antibodies achieve the purpose of treating tumors by inducing a series of immune responses in the body.

[0080] Example 3

[0081] The polydopamine hydrogel loaded with δ-ALA@PLGA microspheres prepared in Example 1 was placed in a mold. The sodium alginate solution mixed with aCD47@CaCO3 nanoparticles prepared in Example 2 was then added to the other side of the mold containing the polydopamine hydrogel. A 10% calcium chloride solution was slowly added dropwise. After the addition was complete, the solution was allowed to stand for approximately 30 minutes until the calcium alginate gel formed. This yielded an immunonanocomposite hydrogel for tumor treatment. The immunonanocomposite hydrogel, formed by combining the two gels from Examples 1 and 2, avoids repeated dosing during tumor treatment, facilitating their combined function in clinical treatment and achieving a better synergistic therapeutic effect.

[0082] See also Figure 1 The composite hydrogel prepared in the present invention includes polydopamine hydrogel and calcium alginate gel. Figure 1 The black gel on the right side is the polydopamine hydrogel prepared in Example 1. Figure 1 The transparent gel on the left is the calcium alginate gel synthesized in Example 2.

[0083] Example 4

[0084] (1) Weigh 1.0 g of HA (hyaluronic acid) and dissolve it in 50 mL of ultrapure water. Adjust the pH to 5.5 with 1 mol / L hydrochloric acid solution. Protect the mixture with nitrogen for 2 h, add a certain amount of dopamine hydrochloride (the amount added is 0.5 times the molar amount of hyaluronic acid), stir in the dark for 30 min, then add EDC solution (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, the amount of EDC added is 2 times the molar amount of dopamine) and NHS solution (N-hydroxysuccinimide, the amount of NHS added is 1.5 times the molar amount of dopamine) in sequence, stir in the dark at room temperature for 12 h to obtain a precipitate. Dissolve the precipitate in water, transfer it to a dialysis bag with a molecular weight cutoff of 3500, and dialyze it. After dialysis for 3 days, freeze-dry it, dry it in the dark at room temperature, and obtain dopamine-modified hyaluronic acid HA-DOPA, which is stored for future use.

[0085] Weigh 100 mg of PLGA (polylactic acid-co-glycolic acid) and dissolve it in 1 ml of dichloromethane, then add 100 μl of 0.05 mg / μl δ-ALA (5-aminolevulinic acid) solution. Place the mixed solution in an ultrasonic cell crusher and ultrasonicate it at a power of 500 W, with an interval of 9 seconds for ultrasonication and 4 seconds for rest, and repeat three times. Take 10 ml of a 1% PVA (polyvinyl alcohol) solution, quickly add it to the above solution, and place it in an ultrasonic cell crusher again for ultrasonication at a power of 900 W, with an interval of 9 seconds for ultrasonication and 4 seconds for rest, and repeat three times. Place the sonicated solution in a fume hood and stir for 3 hours. After the solvent evaporates, wash with water to obtain δ-ALA@PLGA microspheres, freeze-dry them, and store them in a 4°C freezer for later use.

[0086] (2) 20 mg of dopamine-modified hyaluronic acid was added to 1 ml of ultrapure water and stirred to form a dopamine-modified hyaluronic acid aqueous solution. Then, 40 mg of δ-ALA@PLGA microspheres were added and stirred to mix evenly.

[0087] (3) 100 mg of dopamine was added to the dopamine-modified hyaluronic acid aqueous solution mixed with the above-mentioned δ-ALA@PLGA microspheres, and then the pH was adjusted to 8 using NaOH solution. 15 mg of sodium periodate was then added, and the mixture was stirred at a speed of 300 r / min for 10 min to induce dopamine polymerization. The mixture was then allowed to stand for 10 h to form a polydopamine hydrogel.

[0088] (4) 1 ml of Tris-HCl buffer (1 mmol / L, pH 7.6) containing 100 mmol / L CaCl₂ was mixed with 1 ml of HEPES saline buffer (50 mmol / L, pH 7.1, 140 mmol / L NaCl) containing 100 μg aCD47 antibody and 10 mmol / L Na₂CO₃. The mixture was stirred at 4°C for 12 hours. Excess ions and antibodies were removed by centrifugation at 14,800 rpm for 5 minutes. The aCD47@CaCO₃ nanoparticles were obtained after freeze-drying and stored in a -20°C freezer until use.

[0089] (5) Sodium alginate and aCD47@CaCO3 nanoparticles were added to 1 ml of ultrapure water and stirred to obtain a sodium alginate solution with a sodium alginate concentration of 2%. The mass ratio of aCD47@CaCO3 nanoparticles to sodium alginate was 1:15.

[0090] (6) The polydopamine hydrogel loaded with δ-ALA@PLGA microspheres was placed in a mold, and then the sodium alginate solution mixed with aCD47@CaCO3 nanoparticles was added to the other side of the mold where the polydopamine hydrogel was placed. Calcium chloride solution with a mass concentration of 5% was slowly added dropwise. After the addition was completed, it was allowed to stand for about 20 minutes. After the calcium alginate gel was formed, the immune nanocomposite hydrogel for tumor treatment was obtained.

[0091] Example 5

[0092] (1) Weigh 1.0 g of HA (hyaluronic acid) and dissolve it in 50 mL of ultrapure water. Adjust the pH to 5.5 with 1 mol / L hydrochloric acid solution. Protect the mixture with nitrogen for 2 h, add a certain amount of dopamine hydrochloride (the amount added is 0.5 times the molar amount of hyaluronic acid), stir in the dark for 30 min, then add EDC solution (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, the amount of EDC added is 2 times the molar amount of dopamine) and NHS solution (N-hydroxysuccinimide, the amount of NHS added is 1.5 times the molar amount of dopamine) in sequence, stir in the dark at room temperature for 12 h to obtain a precipitate. Dissolve the precipitate in water, transfer it to a dialysis bag with a molecular weight cutoff of 3500, and dialyze it. After dialysis for 3 days, freeze-dry it, dry it in the dark at room temperature, and obtain dopamine-modified hyaluronic acid HA-DOPA, which is stored for future use.

[0093] Weigh 100 mg of PLGA (polylactic acid-co-glycolic acid) and dissolve it in 1 ml of dichloromethane, then add 100 μl of 0.05 mg / μl δ-ALA (5-aminolevulinic acid) solution. Place the mixed solution in an ultrasonic cell crusher and ultrasonicate it at a power of 500 W, with an interval of 9 seconds for ultrasonication and 4 seconds for rest, and repeat three times. Take 10 ml of a 1% PVA (polyvinyl alcohol) solution, quickly add it to the above solution, and place it in an ultrasonic cell crusher again for ultrasonication at a power of 900 W, with an interval of 9 seconds for ultrasonication and 4 seconds for rest, and repeat three times. Place the sonicated solution in a fume hood and stir for 3 hours. After the solvent evaporates, wash with water to obtain δ-ALA@PLGA microspheres, freeze-dry them, and store them in a 4°C freezer for later use.

[0094] (2) 20 mg of dopamine-modified hyaluronic acid was added to 1 ml of ultrapure water and stirred to form a dopamine-modified hyaluronic acid aqueous solution. Then, 40 mg of δ-ALA@PLGA microspheres were added and stirred to mix evenly.

[0095] (3) 100 mg of dopamine was added to the dopamine-modified hyaluronic acid aqueous solution mixed with δ-ALA@PLGA microspheres, and then the pH was adjusted to 9 with NaOH solution. 12 mg of sodium periodate was added, and the mixture was stirred at 600 r / min for 10 min to induce dopamine polymerization. The mixture was then allowed to stand for 24 h to form a polydopamine hydrogel.

[0096] (4) 1 ml of Tris-HCl buffer (1 mmol / L, pH 7.6) containing 100 mmol / L CaCl₂ was mixed with 1 ml of HEPES saline buffer (50 mmol / L, pH 7.1, 140 mmol / L NaCl) containing 100 μg aCD47 antibody and 10 mmol / L Na₂CO₃. The mixture was stirred at 4°C for 12 hours. Excess ions and antibodies were removed by centrifugation at 14,800 rpm for 5 minutes. The aCD47@CaCO₃ nanoparticles were obtained after freeze-drying and stored in a -20°C freezer until use.

[0097] (5) Sodium alginate and aCD47@CaCO3 nanoparticles were added to 1 ml of ultrapure water and stirred to obtain a sodium alginate solution with a sodium alginate concentration of 3%. The mass ratio of aCD47@CaCO3 nanoparticles to sodium alginate was 1:15.

[0098] (6) The polydopamine hydrogel loaded with δ-ALA@PLGA microspheres was placed in a mold, and then the sodium alginate solution mixed with aCD47@CaCO3 nanoparticles was added to the other side of the mold where the polydopamine hydrogel was placed. Calcium chloride solution with a mass concentration of 10% was slowly added dropwise. After the addition was completed, it was allowed to stand for about 60 minutes. After the calcium alginate gel was formed, the immune nanocomposite hydrogel for tumor treatment was obtained.

[0099] Example 6

[0100] (1) Weigh 1.0 g of HA (hyaluronic acid) and dissolve it in 50 mL of ultrapure water. Adjust the pH to 5.5 with 1 mol / L hydrochloric acid solution. Protect the mixture with nitrogen for 2 h, add a certain amount of dopamine hydrochloride (the amount added is 0.5 times the molar amount of hyaluronic acid), stir in the dark for 30 min, then add EDC solution (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, the amount of EDC added is 2 times the molar amount of dopamine) and NHS solution (N-hydroxysuccinimide, the amount of NHS added is 1.5 times the molar amount of dopamine) in sequence, stir in the dark at room temperature for 12 h to obtain a precipitate. Dissolve the precipitate in water, transfer it to a dialysis bag with a molecular weight cutoff of 3500, and dialyze it. After dialysis for 3 days, freeze-dry it, dry it in the dark at room temperature, and obtain dopamine-modified hyaluronic acid HA-DOPA, which is stored for future use.

[0101] Weigh 100 mg of PLGA (polylactic acid-co-glycolic acid) and dissolve it in 1 ml of dichloromethane, then add 100 μl of 0.05 mg / μl δ-ALA (5-aminolevulinic acid) solution. Place the mixed solution in an ultrasonic cell crusher and ultrasonicate it at a power of 500 W, with an interval of 9 seconds for ultrasonication and 4 seconds for rest, and repeat three times. Take 10 ml of a 1% PVA (polyvinyl alcohol) solution, quickly add it to the above solution, and place it in an ultrasonic cell crusher again for ultrasonication at a power of 900 W, with an interval of 9 seconds for ultrasonication and 4 seconds for rest, and repeat three times. Place the sonicated solution in a fume hood and stir for 3 hours. After the solvent evaporates, wash with water to obtain δ-ALA@PLGA microspheres, freeze-dry them, and store them in a 4°C freezer for later use.

[0102] (2) 20 mg of dopamine-modified hyaluronic acid was added to 1 ml of ultrapure water and stirred to form a dopamine-modified hyaluronic acid aqueous solution. Then, 40 mg of δ-ALA@PLGA microspheres were added and stirred to mix evenly.

[0103] (3) 100 mg of dopamine was added to the dopamine-modified hyaluronic acid aqueous solution mixed with the above-mentioned δ-ALA@PLGA microspheres, and then the pH was adjusted to 9 with NaOH solution. Then 13 mg of sodium periodate was added, and the mixture was stirred at a speed of 500 r / min for 10 min to induce dopamine polymerization. The mixture was then allowed to stand for 18 h to form a polydopamine hydrogel.

[0104] (4) 1 ml of Tris-HCl buffer (1 mmol / L, pH 7.6) containing 100 mmol / L CaCl₂ was mixed with 1 ml of HEPES saline buffer (50 mmol / L, pH 7.1, 140 mmol / L NaCl) containing 100 μg aCD47 antibody and 10 mmol / L Na₂CO₃. The mixture was stirred at 4°C for 12 hours. Excess ions and antibodies were removed by centrifugation at 14,800 rpm for 5 minutes. The aCD47@CaCO₃ nanoparticles were obtained after freeze-drying and stored in a -20°C freezer until use.

[0105] (5) Sodium alginate and aCD47@CaCO3 nanoparticles were added to 1 ml of ultrapure water and stirred to mix evenly to prepare a sodium alginate solution with a sodium alginate concentration of 4%. The mass ratio of aCD47@CaCO3 nanoparticles to sodium alginate was 1:15.

[0106] (6) The polydopamine hydrogel loaded with δ-ALA@PLGA microspheres was placed in a mold, and then the sodium alginate solution mixed with aCD47@CaCO3 nanoparticles was added to the other side of the mold where the polydopamine hydrogel was placed. Calcium chloride solution with a mass concentration of 7% was slowly added dropwise. After the addition was completed, it was allowed to stand for about 40 minutes. After the calcium alginate gel was formed, the immune nanocomposite hydrogel for tumor treatment was obtained.

[0107] Comparative Example 1

[0108] Weigh 1.0 g of HA (hyaluronic acid) and dissolve it in 50 mL of ultrapure water. Adjust the pH to 5.5 with 1 mol / L hydrochloric acid. Place under nitrogen for 2 hours, then add 0.2 g of dopamine hydrochloride. Stir in the dark for 30 minutes, then sequentially add EDC solution (10 mg / ml) and NHS solution (4 mg / ml). Stir in the dark at room temperature overnight (12 hours). Transfer the solution to a dialysis bag with a molecular weight cutoff of 3500 and dialyze. After 3 days of dialysis, freeze-dry to obtain HA-DOPA. Store in a dry place at room temperature in the dark until use.

[0109] Weigh 20 mg HA-DOPA and 40 mg DOPA and dissolve them in 1 ml ultrapure water. After complete dissolution, use 1 mol / L NaOH solution to adjust the pH to 8-9, then add 10 mg sodium periodate, stir for 10 minutes, remove the magnet and let it stand for 12 hours to form an immune nanocomposite hydrogel with anti-tumor effect.

[0110] Because the amount of DOPA under this condition is less than the amount added in Example 1 and is insufficient to induce dopamine polymerization, the hydrogel obtained under this condition has a poor gelling effect, low gel strength and unstable structure.

[0111] Comparative Example 2

[0112] Weigh 1.0 g of HA and dissolve it in 50 mL of ultrapure water. Adjust the pH to 5.5 with 1 mol / L hydrochloric acid. Place under nitrogen for 2 hours, then add 0.2 g of dopamine hydrochloride. Stir in the dark for 30 minutes, then sequentially add EDC solution (10 mg / ml) and NHS solution (4 mg / ml). Stir overnight at room temperature in the dark. Transfer the solution to a dialysis bag with a molecular weight cutoff of 3500 and dialyze it for 3 days. After 3 days of dialysis, freeze-dry to obtain HA-DOPA. Store dry at room temperature in the dark until use.

[0113] Weigh 20 mg HA-DOPA and 60 mg DOPA and dissolve them together in 1 ml ultrapure water. After complete dissolution, use 1 mol / L NaOH solution to adjust the pH to 8-9, then add 10 mg sodium periodate, stir for 10 minutes, remove the magnet and let it stand for 12 hours to form a hydrogel.

[0114] Because the amount of DOPA under this condition is less than the amount added in Example 1 and is insufficient to induce dopamine polymerization, the hydrogel obtained under this condition has a poor gelling effect, unstable gel strength, and uneven pore size distribution.

[0115] Comparative Example 3

[0116] Weigh 1.0 g of HA and dissolve it in 50 mL of ultrapure water. Adjust the pH to 5.5 with 1 mol / L hydrochloric acid. Place under nitrogen for 2 hours, then add 0.2 g of dopamine hydrochloride. Stir in the dark for 30 minutes, then sequentially add EDC solution (10 mg / ml) and NHS solution (4 mg / ml). Stir overnight at room temperature in the dark. Transfer the solution to a dialysis bag with a molecular weight cutoff of 3500 and dialyze it for 3 days. After 3 days of dialysis, freeze-dry to obtain HA-DOPA. Store dry at room temperature in the dark until use.

[0117] Weigh 20 mg HA-DOPA and 80 mg DOPA and dissolve them in 1 ml ultrapure water. After complete dissolution, use 1 mol / L NaOH solution to adjust the pH to 8-9, then add 10 mg sodium periodate, stir for 10 minutes, remove the magnet and let it stand for 12 hours to form a hydrogel.

[0118] Because the amount of DOPA under this condition is less than the amount added in Example 1, it is insufficient to completely induce dopamine polymerization. Therefore, although the hydrogel obtained under this condition can be gelled, the pore size distribution is uneven.

[0119] Comparative Example 4

[0120] Weigh 1.0 g of HA and dissolve it in 50 mL of ultrapure water. Adjust the pH to 5.5 with 1 mol / L hydrochloric acid. Place under nitrogen for 2 hours, then add 0.2 g of dopamine hydrochloride. Stir in the dark for 30 minutes, then sequentially add EDC solution (10 mg / ml) and NHS solution (4 mg / ml). Stir overnight at room temperature in the dark. Transfer the solution to a dialysis bag with a molecular weight cutoff of 3500 and dialyze it for 3 days. After 3 days of dialysis, freeze-dry to obtain HA-DOPA. Store dry at room temperature in the dark until use.

[0121] Weigh 20 mg HA-DOPA and 120 mg DOPA and dissolve them together in 1 ml ultrapure water. After complete dissolution, use 1 mol / L NaOH solution to adjust the pH to 8-9, then add 10 mg sodium periodate, stir for 10 minutes, remove the magnet and let it stand for 12 hours to form an immune nanocomposite hydrogel with anti-tumor effect.

[0122] Because the amount of DOPA under this condition is higher than the amount added in Example 1, although dopamine polymerization can be induced, the hydrogel obtained under this condition has a better gelation effect, a clear pore structure, and good strength, but the pore size distribution is uneven.

[0123] Comparative Example 5

[0124] Weigh 1.0 g of HA and dissolve it in 50 mL of ultrapure water. Adjust the pH to 5.5 with 1 mol / L hydrochloric acid. Place under nitrogen for 2 hours, then add 0.2 g of dopamine hydrochloride. Stir in the dark for 30 minutes, then sequentially add EDC solution (10 mg / ml) and NHS solution (4 mg / ml). Stir overnight at room temperature in the dark. Transfer the solution to a dialysis bag with a molecular weight cutoff of 3500 and dialyze it for 3 days. After 3 days of dialysis, freeze-dry to obtain HA-DOPA. Store dry at room temperature in the dark until use.

[0125] Weigh 20 mg HA-DOPA and 100 mg DOPA and dissolve them together in 1 ml ultrapure water. After complete dissolution, use 1 mol / L NaOH solution to adjust the pH to 8-9, then add 5 mg sodium periodate, stir for 10 minutes, remove the magnet and let it stand for 12 hours to form an immune nanocomposite hydrogel with anti-tumor effect.

[0126] Because the amount of sodium periodate added under this condition is less than that added in Example 1 and is insufficient to induce dopamine polymerization, the hydrogel obtained under this condition has a poor gelling effect, low gel strength and unstable structure.

[0127] Comparative Example 6

[0128] Weigh 1.0 g of HA and dissolve it in 50 mL of ultrapure water. Adjust the pH to 5.5 with 1 mol / L hydrochloric acid. Place under nitrogen for 2 hours, then add 0.2 g of dopamine hydrochloride. Stir in the dark for 30 minutes, then sequentially add EDC solution (10 mg / ml) and NHS solution (4 mg / ml). Stir overnight at room temperature in the dark. Transfer the solution to a dialysis bag with a molecular weight cutoff of 3500 and dialyze it for 3 days. After 3 days of dialysis, freeze-dry to obtain HA-DOPA. Store dry at room temperature in the dark until use.

[0129] Weigh 20 mg HA-DOPA and 100 mg DOPA and dissolve them together in 1 ml ultrapure water. After complete dissolution, use 1 mol / L NaOH solution to adjust the pH to 8-9, then add 8 mg sodium periodate, stir for 10 minutes, remove the magnet and let it stand for 12 hours to form an immune nanocomposite hydrogel with anti-tumor effect.

[0130] Because the amount of sodium periodate added under this condition is less than that added in Example 1, it is insufficient to completely induce dopamine polymerization. Therefore, although the hydrogel obtained under this condition can form a gel, its strength is poor and the pore structure is not obvious.

[0131] Comparative Example 7

[0132] Weigh 1.0 g of HA and dissolve it in 50 mL of ultrapure water. Adjust the pH to 5.5 with 1 mol / L hydrochloric acid. Place under nitrogen for 2 hours, then add 0.2 g of dopamine hydrochloride. Stir in the dark for 30 minutes, then sequentially add EDC solution (10 mg / ml) and NHS solution (4 mg / ml). Stir overnight at room temperature in the dark. Transfer the solution to a dialysis bag with a molecular weight cutoff of 3500 and dialyze it for 3 days. After 3 days of dialysis, freeze-dry to obtain HA-DOPA. Store dry at room temperature in the dark until use.

[0133] Weigh 20 mg HA-DOPA and 100 mg DOPA and dissolve them together in 1 ml ultrapure water. After complete dissolution, use 1 mol / L NaOH solution to adjust the pH to 8-9, then add 18 mg sodium periodate, stir for 10 minutes, remove the magnet and let it stand for 12 hours to form an immune nanocomposite hydrogel with anti-tumor effect.

[0134] In Comparative Example 7, because the amount of sodium periodate added is higher than that in Example 1, dopamine polymerization can be completely induced. However, although the hydrogel obtained under this condition has a good gelation effect and good strength, the pore size distribution is uneven and it is not suitable for loading drugs.

[0135] Since whether or not the drug is loaded has no effect on the gelling effect of the gel, the present invention uses the preparation of the gel without drug loading in Examples 1-7 to illustrate that only when the amounts of DOPA and sodium periodate are used in the amounts of the present invention can the composite hydrogel with better performance of the present invention be obtained.

[0136] The present invention dissolves dopamine-modified hyaluronic acid and dopamine together and stirs them evenly. Sodium periodate is added under an alkaline environment. After high-speed stirring, the mixture is allowed to stand overnight to form a polydopamine hydrogel. The completely dissolved sodium alginate solution is added to the polydopamine hydrogel to form a gel. A calcium chloride solution is slowly added dropwise and allowed to stand to obtain a composite hydrogel. The loaded drug is added during the gelling process. The production of the hydrogel can be achieved by simple mixing. The present invention has a short reaction cycle, is very easy to meet reaction conditions, has a high yield, is free of by-products, is easy to realize industrial production, and can effectively improve work efficiency. The composite hydrogel can be used as a novel bioactive material for tumor treatment and can be used as a gel patch that adheres to lesions and releases drugs for a long time.

Claims

1. A method for preparing an immune nanocomposite hydrogel with anti-tumor effect, characterized in that: The following steps are involved: Dopamine-modified hyaluronic acid was added to water and stirred evenly to form a dopamine-modified hyaluronic acid aqueous solution, and then δ-ALA@PLGA microspheres were added and stirred evenly to obtain a mixed solution; Add dopamine to the mixed solution, adjust the pH to 8-9, then add sodium periodate, induce dopamine polymerization under stirring, and then let it stand to form a polydopamine hydrogel; Sodium alginate and aCD47@CaCO3 nanoparticles were added to water and stirred to obtain a sodium alginate solution; Sodium alginate solution is added to polydopamine hydrogel, calcium chloride solution is added, and the mixture is allowed to stand to obtain an immune nanocomposite hydrogel with anti-tumor effect; Dopamine-modified hyaluronic acid is prepared by the following process: dissolving hyaluronic acid in ultrapure water, adjusting the pH to 5.5, adding dopamine hydrochloride under nitrogen protection, stirring in the dark, then adding EDC solution and NHS solution, stirring in the dark at room temperature to obtain a precipitate; dissolving the precipitate in water, transferring it to a dialysis bag with a molecular weight cutoff of 3500 for dialysis, and freeze-drying after dialysis, and drying at room temperature in the dark to obtain dopamine-modified hyaluronic acid; The mass ratio of dopamine-modified hyaluronic acid, dopamine and sodium periodate is 2:10:(1-1.5), wherein δ-ALA is 5-aminolevulinic acid.

2. The method for preparing an immune nanocomposite hydrogel with anti-tumor effect according to claim 1, characterized in that: The molecular weight of hyaluronic acid is 100,000 to 1,000,000 molecules; the concentration of dopamine-modified hyaluronic acid is 20 mg / mL.

3. The method for preparing an immune nanocomposite hydrogel with anti-tumor effect according to claim 1, characterized in that: The mass ratio of δ-ALA@PLGA microspheres to dopamine-modified hyaluronic acid was 2:

1.

4. The method for preparing an immune nanocomposite hydrogel with anti-tumor effect according to claim 1, characterized in that: δ-ALA@PLGA microspheres were prepared by dissolving poly(lactic acid-co-glycolic acid) in dichloromethane, adding δ-ALA solution, sonicating at 500 W, then adding PVA solution, sonicating at 900 W, and stirring to obtain δ-ALA@PLGA microspheres.

5. The method for preparing an immune nanocomposite hydrogel with anti-tumor effect according to claim 1, characterized in that: The standing time for forming polydopamine hydrogel is 10-24h.

6. The method for preparing an immune nanocomposite hydrogel with anti-tumor effect according to claim 1, characterized in that: aCD47@CaCO3 nanoparticles were prepared by the following process: Tris-hydrochloric acid buffer containing CaCl2 was mixed with 4-hydroxyethylpiperazineethanesulfonate aqueous buffer containing aCD47 antibody and Na2CO3, stirred, centrifuged, and freeze-dried to obtain aCD47@CaCO3 nanoparticles.

7. The method for preparing an immune nanocomposite hydrogel with anti-tumor effect according to claim 1, characterized in that: The mass concentration of sodium alginate in the sodium alginate solution is 2-4%; the mass concentration of the calcium chloride solution is 5%-10%.

8. The method for preparing an immune nanocomposite hydrogel with anti-tumor effect according to claim 1, characterized in that: The mass ratio of aCD47@CaCO3 nanoparticles to sodium alginate is 1:15; the mass ratio of sodium alginate to calcium chloride is 1-4:

1.

9. Use of the immunonanocomposite hydrogel with anti-tumor effect prepared according to the method of any one of claims 1 to 8 in the preparation of a gel for tumor treatment.

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