A manganese-based composite hydrogel, its preparation method and application

Abstract

This invention relates to a manganese-based composite hydrogel, its preparation method, and its application. The preparation method specifically involves: adding oleic acid dropwise to a potassium permanganate solution to react, collecting the product to obtain manganese-based nanoparticles; then dispersing them in water and slowly adding them dropwise to a polyallylamine solution, reacting under weakly alkaline conditions, collecting the precipitate to obtain NF-NH2; then dissolving NF-NH2 in a biotin solution to obtain a precipitate; adding the precipitate to a lipoic acid solution to obtain manganese oxide nanoparticles modified with biotin and lipoic acid; dissolving lipoic acid monomer in an alkaline solution to form a mixed solution of lipoic acid and sodium lipoate, followed by ring-opening polymerization and hydrogen bonding crosslinking to form a lipoic acid hydrogel, thus obtaining the manganese-based composite hydrogel. The manganese-based composite hydrogel provided by this invention can be applied to anti-tumor drugs. It is mainly used as a postoperative wound repair drug for melanoma and osteosarcoma, effectively preventing tumor recurrence and metastasis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical and chemical formulation technology, specifically to a manganese-based composite hydrogel, its preparation method, and its application. Background Technology

[0002] Tumor recurrence is a major factor affecting the survival of cancer patients, including primary recurrence and distant recurrence. According to large-sample case reports from both domestic and international sources, the primary recurrence rate of osteosarcoma is approximately 8-15%, while the distant recurrence rate is 25-35%. Primary recurrence is extremely rare in cutaneous melanoma, but the regional lymph node and distant recurrence rates are as high as 80%. The 5-year survival rate for osteosarcoma recurrence patients is only 10-20%, while it is less than 5% for melanoma.

[0003] Currently, surgery remains the primary treatment for osteosarcoma and melanoma. Numerous clinical and experimental studies have shown that surgery itself may promote tumor recurrence and metastasis due to the imbalance of the immune microenvironment caused by surgical trauma. On the one hand, surgery itself can release malignant cells into the circulating blood or lymphatic vessels, while upregulating angiogenesis and the expression of growth factors, promoting the formation and development of micrometastases. On the other hand, the inflammatory response induced by early postoperative wound healing and changes in the tumor immune microenvironment also increase the risk of recurrence and metastasis. For example, surgery causes an increase in the neutrophil trapping network, which traps circulating tumor cells and promotes their proliferation and metastasis; the increased inflammation levels caused by surgical trauma can inhibit the activity of natural killer (NK) cells, while promoting macrophage differentiation into pro-tumor phenotypes, forming an inhibitory immune microenvironment and promoting immune escape of tumor cells. In summary, the postoperative local microenvironment affects long-term prognosis, and regulating the postoperative tumor immune microenvironment is a feasible strategy to inhibit recurrence. However, current clinical methods for regulating the postoperative local immune microenvironment are very limited.

[0004] Currently, drugs for regulating the tumor immune microenvironment mainly consist of small molecule inhibitors and antibodies, such as monoclonal antibodies / inhibitors of CTLA-4 and PD-1. However, these face challenges such as high toxicity, low efficacy, and significant side effects. Patients with low-immunogenic tumors (such as osteosarcoma) have low response rates, while patients with high-immunogenic tumors (such as melanoma) may experience immune-related adverse reactions. Recent studies show that, compared to systemic administration, local immunomodulation is more helpful in overcoming postoperative immune tolerance and reducing systemic toxicity after tumor surgery.

[0005] Biomaterials have shown significant advantages in local drug delivery. For example, biodegradable and antioxidant biopolymer hydrogels can be used as carriers for local treatment at the tumor resection site. On the one hand, covalent / non-covalent modification can enable the gel to adhere to different tissues such as postoperative prostheses, bone, and skin under different conditions, meeting the diverse needs of bone and skin surgeries; simultaneously promoting bone regeneration and skin wound repair. On the other hand, the high level of reactive oxygen species (ROS) in the postoperative tumor microenvironment can regulate the inflammatory microenvironment of the tumor and control the release of immunomodulatory drugs. Thioctic acid-based hydrogels have good antioxidant and anti-inflammatory efficiencies and can regulate ROS and inflammation levels at the postoperative tumor wound site in situ, creating a favorable environment for the recovery of cell and tissue function and preventing the activation of malignant tumor cells.

[0006] In addition, various biomaterials, including nanoparticles, scaffolds, and implants, have been developed to induce the destruction of primary cancer cells, release tumor-associated antigens, or precisely target specific immune cell types through surface modification to improve anti-tumor immune responses. Among these, nano-metal oxides can effectively activate the immune microenvironment to achieve tumor therapy. Manganese-based nanomaterials, due to their excellent oxidizing capacity, can not only consume large amounts of GSH in the tumor microenvironment, leading to the inactivation of GPX, a key ferroptosis molecule, and thus inducing immunogenic cell death represented by ferroptosis; they can also improve the effector function of tumor-associated macrophages and dendritic cells (DCs), enhancing anti-tumor immune responses.

[0007] Therefore, in response to the high level of inflammation in surgical wounds after tumor surgery (such as melanoma) and the suppressed function of DC cells and effector T cells in the postoperative immune microenvironment, we synthesized a manganese-based composite hydrogel by loading manganese oxide nanoparticles onto a natural biomolecule lipoic acid-based hydrogel. On the one hand, the composite hydrogel can firmly adhere to the surgical resection site, keeping the wound moist, while clearing high levels of ROS in the postoperative wound microenvironment, regulating inflammation, accelerating wound healing, and reducing the activation of tumor cells by inflammatory factors. On the other hand, as the gel degrades, the released nanoparticles target tumor cells in the blood or lymphatic circulation, inducing ferroptosis and enhancing the release of tumor antigens, while promoting the antigen-presenting function of DC cells, thereby systematically activating the anti-tumor immune response and inhibiting tumor recurrence and metastasis. Summary of the Invention

[0008] This invention addresses the limitations of current methods for regulating the local immunosuppressive microenvironment after surgery by providing a multifunctional manganese-based composite hydrogel. This manganese-based composite hydrogel promotes wound repair after skin tumor surgery while exerting an anti-tumor immune response, effectively inhibiting tumor recurrence and metastasis.

[0009] Specifically, this invention utilizes the natural small-molecule antioxidant lipoic acid (LA) in an alkaline solution to undergo ring-opening polymerization and hydrogen bonding cross-linking to form a hydrogel with highly efficient self-healing, antioxidant, and anti-inflammatory properties as a carrier. Manganese dioxide nanoparticles are prepared using an in-situ collapse self-assembly method via a Baeyer test for unsaturation reaction of potassium permanganate (KMnO4) and oleic acid (OA) at the O / W emulsion interface. Furthermore, biotin (B) and LA molecules are modified onto the nanoparticle surface by grafting amino groups, resulting in manganese dioxide nanoparticles (NBL) with tumor-targeting capabilities that can further participate in the cross-linking polymerization reaction of the lipoic acid-based hydrogel. By supporting NBL nanoparticles, the gel strength is enhanced, and gel degradation is delayed, resulting in a high-performance nano-manganese dioxide hydrogel composite material, NBL@Gel. PLAS This product is applied to the inflammatory and immune microenvironment of postoperative wounds after skin tumor surgery, aiming to eliminate high levels of ROS in the wound, accelerate wound repair, and reduce the activation of tumor cells by oncogenic factors. Furthermore, the nanoparticles released as the gel slowly degrades can target residual tumor cells, activate anti-tumor immune responses, and inhibit tumor recurrence and metastasis.

[0010] The present invention provides a method for preparing a manganese-based composite hydrogel, comprising the following steps:

[0011] 1) Oleic acid was added dropwise to potassium permanganate solution to carry out the reaction, the product was collected and purified to obtain manganese-based nanoparticles;

[0012] 2) After dispersing the manganese-based nanoparticles in water, a polyallylamine solution was added dropwise to react and the precipitate was collected to obtain NF-NH2; then, NF-NH2 was dissolved in a biotin solution to obtain a precipitate, which is the biotin-modified manganese oxide nanoparticles.

[0013] 3) Add biotin-modified manganese oxide nanoparticles to a lipoic acid solution to obtain a precipitate, thus obtaining biotin- and lipoic acid-modified manganese oxide nanoparticles.

[0014] 4) Thioctic acid monomers are ring-opening polymerized and hydrogen-bonded to form thioctic acid hydrogels, which are then loaded with manganese oxide nanoparticles modified with biotin and thioctic acid to obtain manganese-based composite hydrogels.

[0015] Furthermore, in step 1), the mass ratio of potassium permanganate to oleic acid is 1:5 to 15, preferably 1:8 to 10; and most preferably 1:9. The solvent for the potassium permanganate solution is a conventional solvent, such as water.

[0016] The oleic acid was added at a rate of approximately 2 d / s.

[0017] The reaction takes 5 to 7 hours, preferably 6 hours. The reaction is carried out at room temperature with stirring.

[0018] The purification was carried out using conventional methods in the art; the optimized method was as follows: after the reaction was completed, the product (precipitate) was recovered by centrifugation; the precipitate was washed with alternating ethanol and water, centrifuged repeatedly, the precipitate was collected, dissolved in water, dispersed with ultrasonic assistance, centrifuged at low speed first to remove larger particles, and the resulting solution was centrifuged at high speed to collect the final product, which is manganese-based nanoparticles (NF).

[0019] The present invention further proposes that, in step 2), the mass ratio of manganese-based nanoparticles (NF) to polyallylamine (PAH) is 1:2 to 8, preferably 1:4 to 6, and most preferably 1:5.

[0020] The concentration of the manganese-based nanoparticles dispersed in water is 1.8–2.2 mg / mL.

[0021] The pH value of the weakly alkaline conditions is 7-8; conventional acid-base adjusters such as 1M NaOH can be used.

[0022] The reaction time for step 2) is 25 to 35 minutes; preferably, it is carried out at room temperature.

[0023] After the reaction, centrifugation was performed to obtain a precipitate, which was then washed with water at least twice to obtain NF-NH2.

[0024] Biotin is dissolved in an organic solvent; preferably, dimethyl sulfoxide (DMSO).

[0025] Biotin, generally referring to vitamin H, also known as vitamin B7 or coenzyme R, is a water-soluble vitamin. Due to its high affinity and specificity, it has been widely used in biochemistry and medicine. Biotin can specifically bind to biotin receptors on cell surfaces with very high binding strength. In tumor-targeted drug delivery, biotin can be covalently or non-covalently modified with the surface of drugs or drug carriers. After entering the body, it binds to biotin receptors on the surface of tumor cells, enabling precise delivery of drugs to the tumor cell surface, thus achieving tumor-targeted drug delivery.

[0026] The biotin is first dissolved in DMSO, and then N,N'-diisopropylcarbodiimide (DIC) is added for activation to obtain a biotin solution.

[0027] The mass ratio of NF-NH2 to biotin is 1:20 to 80, preferably 1:35 to 45; and most preferably 1:40.

[0028] In this process, NF-NH2 is dissolved in the above biotin DMSO solution, stirred overnight at room temperature, centrifuged at high speed, and the resulting precipitate is washed with water at least twice to obtain biotin-modified manganese oxide nanoparticles (NB).

[0029] The present invention further proposes that, in step 3), the mass ratio of NB to lipoic acid (LA) is 1:10 to 50; preferably 1:20 to 30, and most preferably 1:25.

[0030] The thioctic acid solution uses a conventional solvent in the art, preferably tetrahydrofuran (THF). Specifically, the thioctic acid is hydrolyzed in tetrahydrofuran (THF) and activated by the addition of DIC.

[0031] Preferably, the concentration of the thioctic acid solution is 0.012 to 0.018 mg / mL.

[0032] In steps 2) and 3), the mass percentage concentration of DIC is 5%.

[0033] In steps 2) and 3), the activation time for adding DIC is approximately 30 minutes.

[0034] In step 3), NB is added to the THF solution of the above-mentioned lipoic acid and stirred overnight at room temperature. The product is harvested by high-speed centrifugation, and the precipitate is washed with water at least twice to obtain manganese oxide nanoparticles (NBL) modified with biotin and lipoic acid.

[0035] The centrifugation speed provided by this invention is approximately 1000 rpm at low speed and approximately 12000 rpm at high speed, with a centrifugation time of 5-10 minutes.

[0036] The present invention further proposes that, in step 4), the thioctic acid monomer is dissolved in the alkaline solution under the following conditions: at a temperature of 42-48°C, the reaction is carried out for 14-16 minutes to form a mixed solution of thioctic acid and sodium thiocate.

[0037] The alkaline solution is a sodium bicarbonate solution, preferably with a concentration of 0.475 M. The molar ratio of lipoic acid to sodium bicarbonate is 1:0.475.

[0038] The ring-opening polymerization and hydrogen bonding crosslinking are specifically carried out as follows: NBL is added to a mixed solution of thioctic acid and sodium thiocate, the mixture is heated in an oil bath to 60-70°C, and then the polymerization reaction is carried out for 2-6 hours.

[0039] Another object of the present invention is to provide a manganese-based composite hydrogel prepared by any of the above-described preparation methods.

[0040] The NBL concentration in the manganese-based composite hydrogel prepared by the above method is 0.5-7.5 mg / mL, preferably 5 mg / mL.

[0041] This invention provides a preferred embodiment in which the manganese-based composite hydrogel is prepared by the following method:

[0042] 1) Oleic acid was added dropwise to potassium permanganate solution and reacted at room temperature for 5-7 hours. The product was collected and purified to obtain manganese-based nanoparticles.

[0043] The mass ratio of potassium permanganate to oleic acid is 1:8-10.

[0044] 2) The manganese-based nanoparticles were dispersed in water and then slowly added dropwise to a polyallylamine solution. The reaction was carried out under weakly alkaline conditions, and the precipitate was collected to obtain NF-NH2. Biotin was first dissolved in DMSO and then activated with N,N'-diisopropylcarbodiimide to obtain a biotin solution. Then, NF-NH2 was dissolved in the biotin solution to obtain a precipitate, which was the biotin-modified manganese oxide nanoparticles.

[0045] The mass ratio of manganese-based nanoparticles to polyallylamine is 1:4-6; the mass ratio of NF-NH2 to biotin is 1:35-45.

[0046] 3) Lipoic acid is dissolved in tetrahydrofuran and activated with DIC to obtain a lipoic acid solution; biotin-modified manganese oxide nanoparticles are added to the tetrahydrofuran solution of lipoic acid, stirred overnight at room temperature to obtain a precipitate, thus obtaining manganese oxide nanoparticles modified with biotin and lipoic acid.

[0047] Among them, the mass ratio of biotin-modified manganese oxide nanoparticles to lipoic acid is 1:20-30;

[0048] 4) Dissolve the lipoic acid monomer in an alkaline solution and react at 42-48°C for 14-16 minutes to form a mixed solution of lipoic acid and sodium lipoate; then add NBL to the mixed solution of lipoic acid and sodium lipoate, heat in an oil bath to 60-70°C, and carry out a polymerization reaction for 2-6 hours to obtain a manganese-based composite hydrogel.

[0049] The manganese-based composite hydrogel provided by this invention can be applied to anti-tumor drugs. It is mainly used as a postoperative wound repair agent for melanoma and osteosarcoma. This manganese-based composite hydrogel can effectively prevent tumor recurrence and metastasis.

[0050] The manganese-based composite hydrogel provided by this invention is applied to the inflammatory microenvironment of postoperative wounds after skin tumor surgery, aiming to eliminate high levels of ROS in the wound, accelerate wound repair, and reduce the activation of tumor cells by oncogenic factors. Furthermore, the nanoparticles released as the gel slowly degrades can target residual tumor cells, activate anti-tumor immune responses, and inhibit tumor recurrence and metastasis. Attached Figure Description

[0051] Figure 1 Image A is a TEM image of manganese oxide nanoparticles; image B is a SEM image of manganese-based composite hydrogel.

[0052] Figure 2 Photos of manganese-based composite hydrogels with different modifications after tilting for 0.5, 1, and 12 hours;

[0053] Figure 3 Rheological property curves of manganese-based composite hydrogels with different modifications;

[0054] Figure 4 Comparative diagram of in vitro cell compatibility experiments of manganese-based composite hydrogels with different modifications;

[0055] Figure 5 Comparison of the intracellular ROS scavenging capabilities of manganese-based composite hydrogels with different modifications;

[0056] Figure 6 Comparative diagram of in vivo degradation experiments of manganese-based composite hydrogels with different modifications;

[0057] Figure 7 Comparison of tumor accumulation in vivo with different modified manganese-based composite hydrogels;

[0058] Figure 8 Comparative diagram showing the in vitro induction of immunogenic cell death by manganese-based composite hydrogels with different modifications. Detailed Implementation

[0059] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0060] Example 1

[0061] This embodiment provides a method for preparing a manganese-based composite hydrogel, as detailed below:

[0062] 1) Preparation of manganese-based nanoparticles

[0063] Weigh 0.8 g of potassium permanganate and dissolve it in a beaker containing 300 mL of deionized water. Stir for 0.5 h to ensure the solid is fully dissolved. Add 8 mL of oleic acid dropwise to the potassium permanganate solution at a rate of 2 drops / s, adjusting the stirring speed to 800 g. Stir the reaction at room temperature for 6 h. Recover the manganese oxide nanoparticle solution and centrifuge at 12000 rpm for 5 min. Discard the supernatant. Wash the resulting precipitate repeatedly with alternating ethanol-water centrifugation 3-4 times to remove residual oleic acid. Collect the precipitate, dissolve it in water, and disperse it with ultrasonic assistance. Centrifuge at 1000 rpm for 5 min to remove larger particles. Centrifuge the supernatant at 12000 rpm for 7 min to collect the precipitate, thus obtaining manganese-based nanoparticles (NF). Figure 1 A is a TEM image of manganese oxide nanoparticles.

[0064] 2) Preparation of biotin-modified manganese oxide nanoparticles

[0065] Weigh 10 mg of NF and disperse it in 5 mL of deionized water. Dissolve 50 mg of polyallylamine (PAH) in a round-bottom flask containing 10 mL of deionized water to prepare a 5 mg / mL PAH solution. Slowly add the above 5 mL NF solution dropwise to the PAH solution while stirring at 300 g. Adjust the pH to 7-8 with 1 M NaOH. After reacting for 30 minutes, centrifuge the reaction solution at 12000 rpm for 10 minutes. Wash the precipitate twice with water to obtain NF-NH2. Dissolve 4 mM biotin (B) in 18 mL of DMSO and activate with 800 μL of N,N'-diisopropylcarbodiimide (DIC) for 30 minutes. Add 12 mg of NF-NH2 to the above biotin DMSO solution and stir overnight at 500 g. Centrifuge the reaction solution at 13000 rpm for 15 minutes. Wash the precipitate twice with water to obtain biotin-modified manganese oxide nanoparticles (NB).

[0066] 3) Preparation of manganese oxide nanoparticles modified with biotin and lipoic acid

[0067] The preparation of NF-NH2 is the same as in step 2). 1.5 mM lipoic acid (LA) was dissolved in 20 mL of THF, and 400 μL of LDIC was added for activation for 30 min. 20 mg of NB was dissolved in the above lipoic acid THF solution, and the mixture was stirred overnight at 500 g. The reaction solution was centrifuged at 6000 rpm for 5 min, and the resulting precipitate was washed twice with water to obtain lipoic acid-modified manganese oxide nanoparticles (NBL).

[0068] 4) Preparation of biotin- and lipoic acid-modified manganese oxide nanoparticle composite hydrogels

[0069] At 45°C, 1 mmol of lipoic acid was mixed thoroughly with 1 mL of 0.475 M sodium bicarbonate solution, and then 50-750 μL of 10 mg / mL manganese oxide nanoparticles (NBL) were added. The mixture was stirred thoroughly to ensure uniform dispersion, and then reacted in an oil bath at 60°C for 3 h to obtain a biotin- and lipoic acid-modified manganese oxide nanocomposite hydrogel material (manganese-based composite hydrogel, NBL@Gel). PLAS ). Figure 1 B is a SEM image of the manganese-based composite hydrogel.

[0070] Comparative Example 1: Preparation of manganese oxide nanoparticles modified with lipoic acid

[0071] The preparation of NF-NH2 is the same as in step 2). 1.5 mM lipoic acid (LA) was dissolved in 20 mL of THF, and 400 μL of LDIC was added for activation for 30 min. 12 mg of NF-NH2 was dissolved in the above lipoic acid THF solution, and the mixture was stirred overnight at 500 g. The reaction solution was centrifuged at 6000 rpm for 5 min, and the resulting precipitate was washed twice with water to obtain lipoic acid-modified manganese oxide nanoparticles (NL).

[0072] Comparative Example 2: Preparation of Biotinylated Manganese Oxide Nanoparticle Composite Hydrogel

[0073] At 45°C, 1 mmol of lipoic acid was mixed thoroughly with 1 mL of 0.475 M sodium bicarbonate solution, and then 50-750 μL of manganese oxide nanoparticles (NB) with a concentration of 10 mg / mL were added. The mixture was stirred thoroughly to ensure uniform dispersion, and then reacted in an oil bath at 60°C for 3 h to obtain a biotin- and lipoic acid-modified manganese oxide nanocomposite hydrogel material, NB@Gel. PLAS .

[0074] Comparative Example 3: Preparation of Lipoic Acid Manganese Oxide Nanoparticle Composite Hydrogel

[0075] At 45°C, 1 mmol of lipoic acid was mixed thoroughly with 1 mL of 0.475 M sodium bicarbonate solution, and then 50-750 μL of 10 mg / mL manganese oxide nanoparticles (NL) were added. The mixture was stirred thoroughly to ensure uniform dispersion, and then reacted in an oil bath at 60°C for 3 h to obtain a biotin- and lipoic acid-modified manganese oxide nanocomposite hydrogel material, NL@Gel. PLAS .

[0076] Experiment Example 1: Tilting Experiment of Manganese-based Composite Hydrogels with Different Modifications

[0077] Manganese-based composite hydrogels with different manganese contents and modifications were tilted and placed for 0.5, 1 and 12 h respectively, and the macroscopic flowability of the gels was observed.

[0078] The results are as follows Figure 2 As shown, blank hydrogel PLAS With prolonged placement, there was almost no change. After 0.5 hours of tilting, NF@Gel PLAS ,NB@Gel PLAS The fluidity increases with increasing manganese loading. And NL@Gel PLAS NBL@Gel PLAS The fluidity did not change significantly. After 1 hour of tilting, with increasing manganese loading, NL@Gel... PLAS NBL@Gel PLAS The fluidity increased. After being tilted for 12 hours, NL@Gel with a manganese loading of 2 mg showed improved fluidity.PLAS , NBL@Gel PLAS has increased fluidity. NL@Gel with a manganese loading of 7.5 mg PLAS , NBL@Gel PLAS has significantly increased fluidity. NBL@Gel with a manganese loading of 5 mg PLAS has little change in fluidity.

[0079] Rheological properties of manganese-based composite hydrogels with different modifications in Experimental Example 2

[0080] The rheological properties of manganese oxide nanoparticle composite hydrogels with different modifications were characterized using a rheometer (DHR-2). 500 μL of the composite hydrogel was placed between 20 mm parallel plates with a gap of 1000 μm and sealed with silicone oil around to prevent water evaporation. During the frequency sweep test, the strain was fixed at 1%, the temperature was at room temperature, and the frequency sweep range was 0.1 - 100 rad / s. At least five samples of each hydrogel sample were tested.

[0081] The test results are as Figure 3 shown. NF@Gel with a small amount of NF PLAS (0.5 / 2 mg NF), the storage modulus is greater than the loss modulus (G' > G"), and it is a viscoelastic solid; NF@Gel with a large amount of NF PLAS (5 / 7.5 mg NF), the storage modulus is less than the loss modulus (G' < G"), and it is a viscoelastic fluid; NB@Gel with different contents of NB PLAS , the storage modulus is greater than or close to the loss modulus (G' > G"), and it is a viscoelastic solid; NB@Gel with different contents of manganese PLAS and NBL@Gel PLAS , the storage modulus is greater than the loss modulus (G' > G"), and it is a viscoelastic solid. It shows that LA modification is beneficial to enhancing the crosslinking of the gel and nanoparticles. Compared with NB@Gel PLAS , NBL@Gel with manganese loadings of 5 and 7.5 mg PLAS has a relatively higher G' > G" intensity (≥102), indicating that co-modification with biotin and LA is more beneficial to the crosslinking of NF and the gel.

[0082] Cell compatibility of manganese-based composite hydrogels with different modifications in Experimental Example 3

[0083] The cell compatibility of manganese-based composite hydrogels with different modifications was verified by the MTT method within 5 days. Well-grown mouse epithelial-like fibroblasts (L929 cells) were seeded in 96-well plates at a density of 5×104 cells / well. Then, they were cultured under the conditions of 37 °C and 5% CO2 for 12 h, and the old culture medium in each well was removed. The manganese-based composite hydrogels Gel PLAS , NF@GelPLAS ,NB@Gel PLAS NL@Gel PLAS NBL@Gel PLAS Cells were soaked in 10 mL of cell culture medium (containing 5 mg / mL manganese) for 24 h to obtain an extract. Cells were then incubated with the extract for another 24 h. MTT solution (5 mg / mL) was added to each well of a 96-well plate (20 μL / well), and the plates were incubated for another 4 h. The culture medium was then discarded, and DMSO (150 μL / well) was added. The 96-well plate was placed on a microplate reader, shaken for 3 min, and the absorbance (OD value) of each well was measured at 490 nm. The average OD value of the three replicates was calculated as the OD value of each sample at different concentrations, and the relative cell viability was calculated. Results are as follows: Figure 4 As shown, there were no significant differences in cell viability among the hydrogel treatment groups on Day 1, Day 3, and Day 5, indicating good cell compatibility.

[0084] Experiment Example 4: Intracellular ROS scavenging ability of manganese-based composite hydrogels with different modifications

[0085] The intracellular ROS scavenging capacity of manganese-based composite hydrogels with different modifications was determined using a reactive oxygen species (ROS) detection kit. Healthy mouse epithelial-like fibroblasts (L929 cells) were used at a concentration of 1.0 × 10⁻⁶. 5 Seeds were planted in 24-well plates at a density of [number] cells / well. The plates were then incubated at 37°C and 5% CO2 for 12 hours, after which the old culture medium was removed from each well. Different modified manganese-based composite hydrogels were then seeded. PLAS NF@Gel PLAS ,NB@Gel PLAS NL@Gel PLAS NBL@Gel PLAS Cells were soaked in 10 mL of cell culture medium (containing 5 mg / mL manganese) for 24 h to obtain an extract. The cells were then incubated for 6 h with a gel extract containing Rosup reagent (1 μg / mL). After washing twice with sterile PBS, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was diluted 1:1000 with DMEM medium to a final concentration of 10 μm / L. The cell culture medium was removed, and 2 mL of DCFH-DA solution was added. The mixture was incubated for 20 min. Cells were washed three times with PBS to thoroughly remove any untreated DCFH-DA. Cells in 24-well plates were then observed and photographed using an inverted fluorescence microscope. Cells treated with Rosup alone were used as a positive control, and untreated cells were used as a negative control.

[0086] The results are as follows Figure 5As shown, the ROS fluorescence intensity was significantly increased in the ROS-up treatment group, indicating an increase in intracellular ROS levels. Co-culturing with different modified manganese-based composite hydrogel extracts significantly reduced the fluorescence intensity, indicating that each hydrogel group had the ability to scavenge ROS in L929 cells, potentially holding great potential for postoperative wound healing, especially NL@Gel. PLAS NBL@Gel PLAS The group with the lowest fluorescence intensity indicates that the LA-modified nanoparticle composite hydrogel has increased LA content, thereby enhancing its ability to scavenge intracellular reactive oxygen species. Example 5: In vivo degradation of manganese-based composite hydrogels with different modifications.

[0087] Different modified manganese-based composite hydrogels were subcutaneously implanted into the backs of Kunming mice to study their in vivo degradation. In short, after anesthetizing the mice, they were fixed on an operating table, and the dorsal region (from the tail to the back) was shaved and disinfected. Circularly cut composite hydrogels (8 mm in diameter and 2 mm in thickness) were implanted subcutaneously. The mice were sacrificed at predetermined times (0, 5, and 8 days) to observe the hydrogel degradation behavior. All procedures were performed under sterile conditions, and the implanted hydrogels were pre-sterilized by ultraviolet irradiation.

[0088] Experimental results are as follows Figure 6 As shown, on Day 5, Gel PLAS NF@Gel PLAS Fragmented into fragments, NL@Gel PLAS ,NB@Gel PLAS NBL@Gel PLAS Partial degradation occurred, but the gel remained intact. By Day 8, all groups of gels had completely degraded, indicating that our hydrogels can be completely absorbed by the body. The hydrogels co-modified with biotin and LA are beneficial to enhancing the cross-linking properties of the gels. The gels degraded in a slow release manner.

[0089] Experimental Example 6: Cellular Uptake and Intracellular Drug Distribution of Manganese-Based Composite Hydrogels with Different Modifications

[0090] Uptake of manganese-based composite hydrogels with different modifications was performed on B16 cell lines. For visualization of the nanoparticles, FITC-labeled nanoparticles with different modifications (NF and NBL) were used. Well-grown B16F10 cells were used at 1.0 × 10⁶ cells per cell line. 5Cells were seeded at a density of cells / well in 24-well plates and cultured at 37°C and 5% CO2 for 12 h. The old culture medium in each well was then removed. FITC-labeled manganese-based composite hydrogels NF@GelPLAS and NBL@GelPLAS (containing 5 mg / ml manganese) were then soaked in 10 mL of cell culture medium for 24 h to obtain leachates. The cells were then incubated with the leachates for another 8 h. Subsequently, the solution in each well was removed, and the cells were washed twice with cold PBS, fixed with 4% paraformaldehyde solution, and analyzed under a fluorescence microscope.

[0091] Experimental results are as follows Figure 7 As shown, with NF@Gel PLAS In comparison, NBL@Gel PLAS The intracellular fluorescence intensity was higher in the NBL@Gel group. PLAS The released nanoparticles can effectively target and internalize into cells and accumulate in the cytoplasm.

[0092] Experimental Example 7: Immunogenic cell death induced by manganese-based composite hydrogels with different modifications

[0093] The release of high-mobility group box 1 (HMGB1) and the expression of calreticulin (CRT) were detected by immunofluorescence to investigate the effect of manganese oxide nanogel-induced immunogenic cell death (ICD) on activating the immune system to fight tumors. In a 24-well plate, each well was filled with 1 × 10⁻⁶ cells. 5 B16F10 cells were seeded on coverslips and incubated overnight. Different modified manganese-based composite hydrogels were then applied. PLAS NF@Gel PLAS ,NB@Gel PLAS NBL@GelPLAS (containing 5 mg / ml manganese) was soaked in 10 mL of cell culture medium for 24 h to obtain the extract. The extract was then incubated for another 12 h. Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 for 10 min, blocked with 5% skim milk powder for 2 h, and then treated with anti-HMGB1 antibody and incubated overnight at 4°C. The next day, the cells were removed, incubated with fluorescent secondary antibody for 2 h, and stained with DAPI (1 mg / mL, 1 μL / well) for 5 min. Cells were washed three times with PBS before each subsequent step. Imaging was then performed using a confocal microscope. The experimental procedure for CRT expression was similar, except that calreticulin antibody was used for overnight incubation during primary antibody treatment.

[0094] Experimental results are as follows Figure 8As shown, a common induction of ICD in tumor cells is the release of HMGB1 from the cell nucleus and the high expression of calreticulin on the surface of dying cancer cells. The blank control PBS group showed very strong red fluorescence that overlapped with the cell nucleus, while NF@Gel... PLAS ,NB@Gel PLAS and NBL@Gel PLAS The group showed weak fluorescence intensity, indicating that it was treated with NF@Gel. PLAS ,NB@Gel PLAS and NBL@Gel PLAS All treatments enhanced the release of HMGB1 from the nucleus of B16 cells, especially NBL@Gel. PLAS The fluorescence intensity was the weakest, suggesting NBL@Gel PLAS The group may have a stronger ability to induce ICD in tumor cells. Furthermore, via NF@Gel... PLAS ,NB@Gel PLAS and NBL@Gel PLAS Significant expression of CRT was also observed in the treated cells, in stark contrast to the PBS group. These results indicate that NBL@Gel PLAS It has a significant ability to induce ICD in tumor cells.

[0095] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a manganese-based composite hydrogel, characterized in that, Includes the following steps: 1) Oleic acid was added dropwise to potassium permanganate solution to carry out the reaction, the product was collected and purified to obtain manganese-based nanoparticles; 2) After dispersing the manganese-based nanoparticles in water, they are slowly added dropwise to a polyallylamine solution and reacted under weakly alkaline conditions. The precipitate is collected to obtain NF-NH2. Then, NF-NH2 is dissolved in a biotin solution to obtain a precipitate, which is biotin-modified manganese oxide nanoparticles. 3) Add biotin-modified manganese oxide nanoparticles to a lipoic acid solution to obtain a precipitate, thus obtaining biotin- and lipoic acid-modified manganese oxide nanoparticles. 4) After dissolving lipoic acid monomer in alkaline solution to form a mixed solution of lipoic acid and sodium lipoate, the solution is then subjected to ring-opening polymerization and hydrogen bonding crosslinking to form a lipoic acid hydrogel. Manganese oxide nanoparticles modified with biotin and lipoic acid are then loaded to obtain a manganese-based composite hydrogel.

2. The preparation method according to claim 1, characterized in that, In step 1), the mass ratio of potassium permanganate to oleic acid is 1:5~15; The reaction takes 5 to 7 hours and is carried out at room temperature.

3. The preparation method according to claim 1 or 2, characterized in that, In step 2), the mass ratio of manganese-based nanoparticles to polyallylamine is 1:2~8.

4. The preparation method according to claim 3, characterized in that, The mass ratio of NF-NH2 to biotin is 1:20~80; The biotin is first dissolved in DMSO, and then N,N'-diisopropylcarbodiimide is added for activation to obtain a biotin solution.

5. The preparation method according to claim 1, characterized in that, In step 3), the mass ratio of biotin-modified manganese oxide nanoparticles to lipoic acid is 1:10~50.

6. The preparation method according to claim 5, characterized in that, Biotin-modified manganese oxide nanoparticles were added to a tetrahydrofuran solution of lipoic acid and stirred overnight at room temperature. The lipoic acid is dissolved in tetrahydrofuran and activated with N,N'-diisopropylcarbodiimide (DIC).

7. The preparation method according to claim 1, characterized in that, In step 4), the thioctic acid monomer is dissolved in an alkaline solution and reacted at a temperature of 42-48°C for 14-16 minutes to form a mixed solution of thioctic acid and sodium thiocate. Then, manganese oxide nanoparticles (NBL) modified with biotin and thioctic acid are added to the mixed solution of thioctic acid and sodium thiocate, and the mixture is heated in an oil bath to 60-70°C for 2-6 hours to carry out the polymerization reaction.

8. The manganese-based composite hydrogel prepared by the preparation method according to any one of claims 1 to 7.

9. A manganese-based composite hydrogel, characterized in that, The concentration of manganese oxide nanoparticles modified with biotin and lipoic acid is 0.5~7.5 mg / mL; It is prepared by the following method: 1) Oleic acid was added dropwise to potassium permanganate solution and reacted at room temperature for 5-7 hours. The product was collected and purified to obtain manganese-based nanoparticles. The mass ratio of potassium permanganate to oleic acid is 1:8~10; 2) The manganese-based nanoparticles were dispersed in water and then slowly added dropwise to a polyallylamine solution. The reaction was carried out under weakly alkaline conditions, and the precipitate was collected to obtain NF-NH2. Biotin was first dissolved in DMSO and then activated with N,N'-diisopropylcarbodiimide to obtain a biotin solution. Then, NF-NH2 was dissolved in the biotin solution to obtain a precipitate, which was the biotin-modified manganese oxide nanoparticles. The mass ratio of manganese-based nanoparticles to polyallylamine is 1:4~6; the mass ratio of NF-NH2 to biotin is 1:35~45. 3) Lipoic acid is dissolved in tetrahydrofuran and activated with DIC to obtain a lipoic acid solution; biotin-modified manganese oxide nanoparticles are added to the lipoic acid tetrahydrofuran solution and stirred overnight at room temperature to obtain a precipitate, thus obtaining biotin- and lipoic acid-modified manganese oxide nanoparticles. Among them, the mass ratio of biotin-modified manganese oxide nanoparticles to lipoic acid is 1:20~30; 4) Dissolve the lipoic acid monomer in an alkaline solution and react at 42-48°C for 14-16 minutes to form a mixed solution of lipoic acid and sodium lipoate; then add NBL to the mixed solution of lipoic acid and sodium lipoate, heat in an oil bath to 60-70°C, and carry out a polymerization reaction for 2-6 hours to obtain a manganese-based composite hydrogel.

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