A method for preparing a metal-polyphenol injectable hydrogel and the resulting product and applications

Abstract

The application discloses a preparation method of metal-polyphenol injectable hydrogel and the obtained product and application, and relates to the following steps: reacting hyaluronic acid with EDC and sulf-NHS, then adding DA and an organic base to react to obtain HA-DA; uniformly mixing HA-DA and taxifolin in a solution environment to obtain a precursor mixed solution; reacting the precursor mixed solution with a metal salt solution to form a network structure, thereby obtaining the metal-polyphenol injectable hydrogel. The application has the advantages of simple and easy process, low energy consumption, environmental friendliness, convenience for large-scale production, good biocompatibility, in-situ gelation and photothermal responsiveness of the obtained hydrogel, high efficient photothermal-immune combined treatment effect on liver cancer, reduction of systemic adverse reactions through local administration, excellent tumor killing, immune activation and anti-recurrence and metastasis functions in in-vitro and in-vivo experiments.

Description

Technical Field

[0001] This invention relates to a method for preparing a metal-polyphenol injectable hydrogel, the resulting product, and its applications, specifically to a method for preparing a metal-polyphenol injectable hydrogel with photothermal-immunotherapy combined effects, the resulting product, and its applications. Background Technology

[0002] Liver cancer (HCC) is the most common malignant tumor worldwide, ranking fourth in incidence and third in mortality. Previously, HCC treatment employed a comprehensive model based on surgical interventions, including radiofrequency ablation, interventional therapy, targeted therapy, chemotherapy, and radiotherapy. However, postoperative recurrence rates are high, and the effectiveness of existing treatments is limited. Immunotherapy is a treatment method that controls and kills tumor cells by stimulating immune function and enhancing anti-tumor immunity in the tumor microenvironment; the application of immune checkpoint inhibitors (ICIs) has become a current research hotspot. The probability of severe toxic reactions in immunotherapy is lower than in traditional HCC treatments, and patients tolerate immunotherapy better than with traditional treatments. In recent years, immunotherapy for HCC has shown positive effects in clinical settings. However, immunotherapy and targeted therapy for HCC suffer from low response rates and significant systemic adverse reactions, which may be related to immunosuppression in the tumor microenvironment and high drug off-target rates. Therefore, finding new and effective treatment methods and elucidating their mechanisms is of great significance for advancing the clinical treatment of HCC.

[0003] Photothermal therapy (PTT) converts external light energy into heat, increasing the temperature of the tumor area and killing tumor cells. During tumor ablation, tumor cells undergo immunogenic death, inducing a systemic tumor-specific cellular immune response and promoting T lymphocyte infiltration in tumor tissue, thereby effectively inhibiting local tumor cell invasion and distant metastasis. Furthermore, the antigens generated during tumor cell ablation, under the action of immune adjuvants, can act as "in situ tumor vaccines," inducing a long-term, systemic anti-tumor immune response in the host, significantly enhancing the killing effect on residual tumor cells in the body. Studies by numerous scholars both domestically and internationally have found that the combined use of PTT and immune checkpoint inhibitors can induce a strong systemic immune response, significantly enhancing the anti-tumor effect. To improve therapeutic efficacy, researchers often favor combination therapy; photothermal / immunotherapy, as a rapidly developing treatment method in recent years, has shown promising application prospects in tumor treatment.

[0004] In cancer treatment, severe systemic adverse reactions remain an unavoidable problem in the use of anti-tumor drugs. Combination therapy often induces more frequent and severe adverse reactions, even fatal ones. Postoperative local administration can significantly increase drug targeting efficacy while greatly reducing the drug concentration in the circulating blood, thereby reducing systemic toxicity. Currently, the main clinical method of local administration is implantation (such as fluorouracil implants), which has been proven to effectively reduce postoperative tumor recurrence rates. However, the polymeric framework of these implants cannot be degraded and absorbed by the human body, leading to serious postoperative complications such as local inflammation, bleeding, anastomotic leakage, and intestinal obstruction.

[0005] In recent years, injectable hydrogels have been widely used in regenerative medicine, tissue engineering, and cancer treatment. Their main characteristic is that they form a gel in situ immediately after injection at the tumor site, allowing for easy loading of various drugs. Post-operatively, drug-loaded hydrogels are injected onto the operating table, where they adhere to the surgical wound to form films or coatings of different shapes and functions, directly contacting residual tumor cells to enhance targeting effects. They also possess hemostatic and wound-healing properties. When loaded with photosensitizers and combined with photothermal transdermal (PTT), they can not only remove some residual tumor tissue after surgery but also induce a strong anti-tumor immune response in the host. However, their disadvantages include complex preparation processes and a lack of photothermal response. Therefore, developing injectable hydrogels that are simple to prepare and possess photothermal effects is a major challenge. Metal-polyphenol network framework hydrogels, prepared by chelating polyphenols with metal ions, possess excellent photothermal performance and are simple to manufacture, making them ideal drug delivery materials. In addition to hemostasis and wound-healing promotion, combining them with PTT can also prevent surgical wound infection.

[0006] Currently, there are no reports of combining metal-polyphenol injectable hydrogels with photothermal therapy to achieve a dual synergistic treatment of "local killing and immune activation" after liver cancer surgery. Summary of the Invention

[0007] Addressing the bottlenecks in postoperative treatment of liver cancer, this invention provides a method for preparing a metal-polyphenol injectable hydrogel and the resulting product. This invention utilizes the complexation reaction of dopamine-modified hyaluronic acid and phenolic hydroxyl groups in paclitaxel with metal ions to prepare a metal-polyphenol network hydrogel. Through material self-assembly and combined with photothermal therapy, a dual synergistic therapeutic effect of "local killing and immune activation" can be achieved in liver cancer surgery. This hydrogel exhibits highly efficient photothermal-immunotherapy for liver cancer and can be injected into the tumor site postoperatively, forming a gel in situ immediately, reducing systemic adverse reactions and providing a novel treatment option for liver cancer surgery.

[0008] The specific technical solution of this invention is as follows: A method for preparing a metal-polyphenol injectable hydrogel, the method comprising the following steps: (1) Hyaluronic acid is reacted with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysulfosuccinimide (sulf-NHS) to activate the carboxyl group of hyaluronic acid. Then, dopamine hydrochloride (DA) and an organic base are added to react and obtain catechol-terminated hyaluronic acid (HA-DA). (2) The catechol-terminated hyaluronic acid (HA-DA) obtained in step (1) is uniformly mixed with paclitaxel (Pic) in a solution environment to obtain a precursor mixture; (3) Mix the precursor mixture with the metal salt solution, then add guar gum, and react to form a network structure to obtain a metal-polyphenol injectable hydrogel.

[0009] Furthermore, in step (1), the weight-average molecular weight of hyaluronic acid is 400,000 to 800,000 Daltons, for example, 400,000 Daltons, 450,000 Daltons, 500,000 Daltons, 550,000 Daltons, 600,000 Daltons, 650,000 Daltons, 700,000 Daltons, 750,000 Daltons, and 800,000 Daltons.

[0010] Furthermore, in step (1), the mass ratio of hyaluronic acid (HA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and N-hydroxysulfosuccinimide (sulf-NHS) is 1:2-3:1-1.5.

[0011] Further, in step (1), the organic base is triethylamine. Hyaluronic acid is first activated by reacting with EDC and sulf-NHS, and then reacted with dopamine hydrochloride and the organic base to achieve covalent bonding of HA and DA, obtaining the HA-DA conjugate. The mass ratio of hyaluronic acid to dopamine hydrochloride (DA) is 1:0.5-0.6. The ratio of hyaluronic acid to triethylamine organic base is 100mg:3-5µL.

[0012] Further, in step (1), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution is first added to the hyaluronic acid solution, and the reaction is carried out for 15-30 min. Then, N-hydroxysulfosuccinimide solution is added, and the reaction is carried out for 1-1.5 h. Then, dopamine hydrochloride solution and organic base are added, and the reaction is carried out for 20-21 h under inert gas protection. After the reaction is completed, the solution is dialyzed and lyophilized to obtain HA-DA powder. The solvents for the hyaluronic acid solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution, N-hydroxysulfosuccinimide solution, and dopamine hydrochloride solution are all phosphate buffer solutions with a pH of 5-6.

[0013] Furthermore, in step (2), the mass ratio of HA-DA to picolinate (Pic) is 3-4:1. Specifically: the lyophilized HA-DA is dissolved in water to form a solution, picolinate is dissolved in water to form a solution, the picolinate solution is added to the HA-DA solution, and the mixture is stirred evenly to obtain the precursor mixture. The concentration of the HA-DA solution is 20-40 mg / mL, and the concentration of the picolinate solution is 40-50 mg / mL.

[0014] Furthermore, the polyphenol compound selected in this invention is a combination of catechol-terminated hyaluronic acid and picrol. Picrol is a natural polyphenol compound mainly found in blueberries, grapes, nuts (such as almonds), and plants of the *Pterocarpus* genus, especially in high concentrations in blueberries. As a methylated derivative of resveratrol, picrol exhibits enhanced bioavailability and stability, with its biological activities encompassing multiple functions including antioxidant, anti-inflammatory, hypoglycemic, neuroprotective, and antitumor effects. In its antitumor role, picrol exerts significant effects through multiple pathways. This invention is the first to discover that picrol inhibits the immunosuppressive factor PGE2, highlighting its potential as a natural anticancer compound.

[0015] Furthermore, in step (3), the metal salt is a copper salt, and the metal salt is a sulfate or chloride of the metal.

[0016] Furthermore, in step (3), the volume ratio of the precursor mixture to the metal salt solution is 10-13:1.

[0017] Furthermore, in step (3), the molar ratio of metal ions to paclitaxel is 4-5:1.

[0018] Furthermore, in step (3), after mixing the precursor mixture with the metal salt solution, guar gum is added and stirred until completely dissolved. The mixture is then allowed to stand for approximately 2-3 hours to form a network structure through the chelation reaction of phenolic hydroxyl groups with copper ions, thus obtaining the final hydrogel. The mass ratio of HA-DA to guar gum is 1:1-1.5, for example, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, or 1:1.5.

[0019] This invention utilizes the complexation reaction between phenolic hydroxyl groups and metal ions to prepare a metal-polyphenol network injectable hydrogel. This injectable hydrogel can form in situ gel at the tumor surgical site, allowing for local injection without off-target issues and with low adverse reactions; it also falls within the scope of this invention.

[0020] This invention relates to an injectable hydrogel combined with photothermal-immunotherapy (PTT), which demonstrates highly effective photothermal-immunotherapy for liver cancer. The injectable hydrogel combined with PTT achieves: 1. direct photothermal killing of residual tumor cells; 2. inhibition of PGE2 production by paclitaxel, activating anti-tumor immunity. This hydrogel can minimize tumor recurrence and metastasis while reducing systemic adverse reactions, providing a novel treatment option for liver cancer surgery. The application of this metal-polyphenol network injectable hydrogel in the preparation of postoperative treatment drugs for liver cancer is also within the scope of this invention.

[0021] This invention utilizes the complexation reaction of phenolic hydroxyl groups with metal ions to prepare a metal-polyphenol network hydrogel. This hydrogel not only possesses excellent biocompatibility, in-situ gelling properties, and photothermal responsiveness, but also reduces systemic adverse reactions through local administration. Both in vitro and in vivo experiments have demonstrated excellent tumor-killing, immune-activating, and anti-recurrence / metastasis functions, exhibiting highly effective photothermal-immunotherapy for liver cancer. Compared with existing technologies, this invention has the following beneficial effects: 1. Preparation advantages: The hydrogel of this invention is based on the rapid coordination reaction of phenolic hydroxyl groups and metal ions, which does not require complex equipment, has mild reaction conditions (room temperature, aqueous solution system), and the preparation process is simple and easy to carry out. It has low energy consumption, the raw materials used have good biocompatibility, are environmentally friendly, and are easy to scale up for production.

[0022] 2. Precise local drug delivery: The injectable hydrogel of this invention can be adapted to the shape of the surgical wound. After gelling in situ, it can directly contact the residual tumor cells, resulting in a high local drug concentration and a low drug concentration in the circulating blood. Local drug delivery significantly reduces systemic adverse reactions.

[0023] 3. Dual Synergistic Therapy: In terms of photothermal killing, under irradiation with an 808 nm near-infrared laser (1.5 W / cm²), the metal-polyphenol network hydrogel can convert light energy into heat energy, raising the local temperature of the tumor to 60-70 ℃, achieving a photothermal killing effect. Ablation directly kills residual tumor cells; in terms of immune activation, in the tumor microenvironment, paclitaxel can inhibit COX-2, further inhibiting PGE2. COX-2 catalyzes the production of prostaglandin E2 (PGE2) from arachidonic acid. PGE2 inhibits the function of cytotoxic T lymphocytes (CTLs) by binding to EP2 / EP4 receptors and promotes the expansion of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), thereby inhibiting the anti-tumor immune response and reducing the negative regulatory effect of immunity. At the same time, photothermal therapy induces ICD, releasing tumor antigens. The ICD effect promotes the maturation of dendritic cells and induces CD4⁺ / CD8⁺ T cell infiltration, overcoming the tumor-suppressive immune effect, reversing the tumor microenvironment, activating systemic anti-tumor immunity, and achieving the effect of preventing tumor recurrence and metastasis after liver cancer surgery. Attached Figure Description

[0024] Figure 1 The image shows the UV-Vis absorption spectrum of the HA-DA composite material prepared in Example 1 (detection wavelength range 200~400nm).

[0025] Figure 2 The rheological properties (storage modulus, loss modulus) of the hydrogel prepared in Example 2.

[0026] Figure 3 The rheological properties (relationship between shear rate and viscosity) of the hydrogel prepared in Example 2.

[0027] Figure 4 The image shows a scanning electron microscope (SEM) characterization of the hydrogel prepared in Example 2.

[0028] Figure 5 The images show thermal images of the HPC injectable hydrogel prepared in Example 2 under laser irradiation at different power densities.

[0029] Figure 6 This is a short-term photothermal heating curve of the HPC injectable hydrogel prepared in Example 2 under laser irradiation.

[0030] Figure 7 The graph shows the long-term photothermal stability of the HPC injectable hydrogel prepared in Example 2 under constant power laser irradiation.

[0031] Figure 8 The figure shows the results of cytotoxicity experiments on Heapl-6 cells at different concentrations of hydrogel extract.

[0032] Figure 9 Flow cytometry analysis of CRT in Hepa1-6 cells from different treatment groups.

[0033] Figure 10 This is an ELISA analysis of ATP levels in Hepa1-6 cells from different treatment groups.

[0034] Figure 11 This is a graph showing the ELISA analysis of HMGB1 in Hepa1-6 cells under different treatment groups.

[0035] Figure 12 The inhibition curve of paclitaxel on COX-2 (COX-2 enzyme activity assay kit).

[0036] Figure 13 This is an ELISA analysis of the negative immunomodulatory effect of paclitaxel on PGE2.

[0037] Figure 14 Flow cytometry analysis of BMDCs isolated from different treatment groups and stimulated to mature DC cells. Detailed Implementation

[0038] The following description illustrates exemplary embodiments of the present invention, including various details to aid understanding, and should be considered merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the invention. Similarly, for clarity and brevity, descriptions of well-known functions, operations, and structures are omitted in the following description.

[0039] Unless otherwise defined, the technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, the present invention describes materials and methods hereinafter. In case of conflict, the definitions included herein shall prevail.

[0040] Example 1: Preparation of HA-DA Weigh 500 mg of hyaluronic acid (HA, molecular weight 400,000-800,000) and add it to 25 mL of phosphate buffered saline (PBS, pH 5-6, concentration 0.01 M). Stir every 30 min at room temperature until the HA is completely dissolved to obtain the HA solution. Weigh 1195 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), dissolve it in 4 mL of PBS, and transfer it to the HA solution. React at room temperature for 20 min. Weigh 541.75 mg of N-hydroxysulfosuccinimide (sulf-NHS), dissolve it in 3 mL of PBS, and add it to the above reaction system. Continue the reaction at 25 °C for 1 h. Weigh 278.75 mg of dopamine hydrochloride (DA), dissolve it in 4 mL of PBS, add it to the reaction flask, and then add 20 μL of triethylamine. Purge with nitrogen for protection and react at room temperature for 20 h. After the reaction, the HA-DA mixed solution was collected and dialyzed four times in pure water (8 h each time). After dialysis, the solution was freeze-dried to obtain HA-DA powder. The obtained HA-DA powder was dissolved in ultrapure water to prepare a 0.5 mg / mL solution, and the solution was scanned using a UV spectrophotometer in the wavelength range of 200-400 nm. The results are as follows. Figure 1 As shown, the HA-DA solution exhibits a distinct characteristic absorption peak at 280 nm, which is attributed to the π→π* transition of the catechol group in DA. Pure HA shows no absorption at this point, confirming that DA has been successfully grafted onto the HA molecular chain.

[0041] Example 2: Preparation of Metal-Polyphenol Network Injectable Hydrogel Weigh the HA-DA lyophilized powder obtained in Example 1, dissolve it in ultrapure water, and prepare an HA-DA solution with a concentration of 33.3 mg / mL (calculated as HA-DA). Weigh picolinol (Pic), dissolve it in water, and prepare a Pic stock solution with a concentration of 42.2 mg / mL (weigh 9.54 mg picolinol and add 226 μL of water).

[0042] Take 500 μL of HA-DA solution (containing approximately 16.65 mg of HA-DA), add 113 μL of Pic stock solution (containing 4.77 mg of paclitaxel), stir for 30 seconds to mix evenly, and obtain the precursor mixture.

[0043] Preparation of the metal salt solution: Weigh 392.9 mg of copper sulfate pentahydrate (CuSO4·5H2O), add 1 mL of ultrapure water to dissolve, and obtain a solution with a Cu²⁺ concentration of 100 mg / mL (calculated as Cu²⁺). Take 53 μL of this Cu²⁺ solution (containing 5.3 mg of Cu²⁺) and add it to the above precursor mixture, and stir for 30 seconds.

[0044] Then, 0 mg, 12.525 mg, 16.7 mg, and 20.875 mg of guar gum were added respectively (corresponding to HA-DA to guar gum mass ratios of 1:0, 1:0.75, 1:1, and 1:1.25, respectively), stirred until completely dissolved, and allowed to stand for 2 hours to react, resulting in four metal-polyphenol network injectable hydrogels, denoted as HA-G0, HA-G... 0.75 HA-G1, HA-G 1.25 .

[0045] Linear viscoelastic region tests were performed on the four metal-polyphenol network injectable hydrogels mentioned above, with a frequency scan range of 0.1-100 rad / s. The obtained storage modulus (G') and loss modulus (G'') are as follows: Figure 2 As shown, the results indicate that HA-G 1.25 The highest G' value and the widest plateau region indicate that its three-dimensional network structure is the most stable, confirming the formation of a stable hydrogel.

[0046] Change HA-G0, HA-G 0.75 HA-G1, HA-G 1.25 Steady-state shear tests were conducted on the hydrogel within a shear rate range of 0.1–100 s⁻¹, and the results are as follows: Figure 3 As shown in the figure, the viscosity of the hydrogel decreases with increasing shear rate, exhibiting typical shear-thinning characteristics, which proves that it has good injectability.

[0047] The hydrogel HA-G1.25 was frozen overnight at -80°C, and then freeze-dried for 48 hours. The freeze-dried sample was subjected to liquid nitrogen fracture followed by gold sputtering, and its cross-sectional morphology was observed using scanning electron microscopy (SEM) at an accelerating voltage of 10 kV. The results are as follows: Figure 4 As shown in the figure, the HA-G1.25 hydrogel exhibits a typical three-dimensional porous network structure with pore sizes ranging from 50 to 150 µm. This structure is beneficial for drug loading and extracellular matrix simulation.

[0048] Example 3: Photothermal Performance Testing of Injectable Hydrogels 3.1. Photothermal conversion capacity test: Take 200 µL of freshly prepared HA-G 1.25 Hydrogels were placed in 48-well plates. Three experimental groups were set up, irradiated for 10 min for each group with 808 nm near-infrared lasers at power densities of 0.5 W / cm², 1.0 W / cm², and 1.5 W / cm², respectively, with three replicates per group. Temperature changes in the central region of the hydrogel were recorded in real time using an infrared thermal imager, with recordings every 30 seconds, and temperature-time curves were plotted. Results are shown below. Figure 5 (Thermal image) and Figure 6 As shown in the heating curves, after 10 minutes of irradiation, the highest temperatures of the 0.5, 1.0, and 1.5 W / cm² groups reached approximately 33.2℃, 47.8℃, and 64.0℃, respectively. This hydrogel exhibits excellent photothermal conversion capabilities and shows a power density dependence.

[0049] 3.2. Photothermal stability test: Take 200 µL of freshly prepared HA-G 1.25 Hydrogels were placed in 48-well plates. The plates were irradiated with an 808 nm near-infrared laser at 1.5 W / cm² for 10 min (laser on), then the laser was turned off, and the plates were allowed to cool naturally to room temperature (approximately 25°C). This constituted one cycle. This process was repeated for 5 cycles. The temperature was continuously recorded using an infrared thermal imager, and the maximum temperature rise in each cycle (ΔT = T_max - T_initial) was calculated. Figure 7 As shown, in the first cycle, ΔT was approximately 40°C (rising from 25°C to 65°C). After five heating-cooling cycles, the hydrogel's ΔT still reached approximately 37°C, decreasing by only 7.5%, indicating its good photothermal stability.

[0050] Example 4: Cytotoxicity Test The sample used in this embodiment is the extract of the HA-G1.25 hydrogel prepared in Example 2.

[0051] 4.1 Preparation of extract: Under aseptic conditions, take 1 mL of freshly prepared HA-G extract. 1.25Hydrogel was prepared by adding 9 mL of RPMI-1640 cell culture medium and incubating at 37°C for 24 hours. The supernatant was collected and sterilized by filtration through a 0.22 µm filter membrane to obtain a 10% (w / v) hydrogel stock solution. The stock solution was then diluted with RPMI-1640 medium to prepare 1%, 2%, and 5% dilutions, respectively.

[0052] 4.2 Cell Viability Assay: Hepa1-6 cells in logarithmic growth phase were digested with trypsin and seeded at a density of 5 × 10³ cells / well in 96-well plates with 100 µL of culture medium per well. Cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. After cell attachment, the supernatant was discarded. Experimental setups included a blank group (culture medium only), a control group (100 µL of fresh culture medium), and experimental groups (100 µL of 1%, 2%, 5%, and 10% hydrogel extract, respectively), with 5 parallel wells in each group. After another 24 hours of incubation, 10 µL of CCK-8 solution was added to each well, and incubation was continued for 2 hours. The absorbance (OD450) at 450 nm was measured using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = [(OD experimental group - OD blank group) / (OD control group - OD blank group)] × 100%.

[0053] The results are as follows Figure 8 As shown, when the concentration of the hydrogel extract was ≤5% (w / v), the survival rate of Hepa1-6 cells was higher than 90%. When the concentration reached 10% (w / v), the cell survival rate was still higher than 85%. These results indicate that the HA-G1.25 hydrogel has good biocompatibility.

[0054] Example 5: Photothermal induction of immunogenic cell death (ICD) in tumor cells by hydrogel The sample used in this embodiment is the HA-G prepared in Example 2. 1.25 Hydrogel.

[0055] 5.1 Experimental grouping and treatment: Hepa1-6 cells were divided into groups of 2×10⁻⁶ cells and treated with a PCR method. 5 The cells were seeded at a density of cells / well in 6-well plates and incubated overnight. The experiment was divided into four groups: Control group: only 2 mL of fresh culture medium was added; Laser group: 2 mL of fresh culture medium was added, and then irradiated with an 808 nm laser (1.5 W / cm²) for 10 min; HPC group (hydrogel group): 200 µL of freshly prepared HA-G1.25 hydrogel (transwell chamber) was added to each well, and 1.8 mL of culture medium was added to the bottom layer. The mixture was incubated for a total of 4 hours; HPC+Laser group (hydrogel+laser group): after incubating for 4 hours as the HPC group, the mixture was irradiated with an 808 nm laser (1.5 W / cm²) for 10 min.

[0056] 5.2 CRT Expression Detection: After the above treatments, cells were collected by trypsin digestion with EDTA-free solution. Cells were washed twice with PBS, and 5 µL of PE-labeled anti-CRT antibody (diluted 1:50) was added. Cells were incubated at 4°C in the dark for 30 minutes. After washing, the proportion of CRT-positive cells was detected by flow cytometry. Results are shown below. Figure 9 As shown, the CRT positivity rates in the Control group, Laser group, and HPC group were 5.2%, 8.5%, and 12.1%, respectively, while the CRT positivity rate in the HPC+Laser group significantly increased to 45.6%.

[0057] 5.3 ATP and HMGB1 Release Detection: After the above treatments, cell supernatants were collected. The amount of ATP released in the supernatant was detected using a chemiluminescence assay kit. The HMGB1 content in the supernatant was detected using an HMGB1 ELISA kit, following the manufacturer's instructions. Results are as follows: Figure 10 and Figure 11 As shown, the ATP release (approximately 120 nM) and HMGB1 content (approximately 2.3 µM) in the HPC+Laser group were significantly higher than those in the other three groups (ATP < 60 nM, HMGB1 < 1.2 µM in the other three groups). These results indicate that hydrogel combined with photothermal therapy (HPC+Laser) can effectively induce immunogenic cell death in tumor cells.

[0058] Example 6: Effects of picolinate (Pic) on the in vitro inhibition of COX-2 activity and PGE2 secretion function of Hepa1-6 6.1. Effect of Pic on COX-2 activity: Cyclooxygenase (COX), also known as prostaglandin reductase, is a bifunctional enzyme with both cyclooxygenase and catalase activities. It is the rate-limiting enzyme in the oxygenation of arachidonic acid (AA) to prostaglandins (PGs). There are two isoenzymes of COX: COX-1 and COX-2. COX-1 is the structural form, mainly found in tissues such as blood vessels, stomach, and kidneys, and participates in the regulation of vasomotor activity, platelet aggregation, gastric mucosal blood flow, gastric mucus secretion, and kidney function. COX-2 is the inducible form; under normal physiological conditions, COX-2 is almost not expressed or expressed very little, but its expression increases under stimulation by growth factors, cytokines, cytokine mediators, and carcinogens.

[0059] Follow the instructions for the COX-2 inhibitor screening kit. In the reaction wells of a 96-well plate, add buffer, heme, COX-2 enzyme (0.5 U / well), arachidonic acid substrate (10 µM), and different concentrations of paclitaxel solution (final concentrations of 5 nM, 10 nM, 50 nM, and 100 nM, in water), with three replicates for each concentration. A positive control group without Pic and an inhibition control group containing the COX-2 specific inhibitor celecoxib (1 µM) are also included. After reacting at room temperature for 5 minutes, add the chromogenic reagent and measure the absorbance at 590 nm. Calculate the inhibition rate of COX-2 enzyme activity for each Pic concentration and calculate the half-maximal inhibitory concentration (IC50) using nonlinear regression fitting.

[0060] The results are as follows Figure 12 As shown, Pic's inhibitory effect on COX-2 is concentration-dependent. Its IC50 was calculated to be 40 nM, comparable to the positive control celecoxib (IC50 ≈ 30 nM), confirming Pic as an effective COX-2 inhibitor.

[0061] 6.2 Effect of Pic on PGE2 secretion by tumor cells: Hepa1-6 cells were fed at a rate of 2 × 10⁻⁶ 5 Seeds were planted at a density of cells / well in 24-well plates and incubated overnight. The experiment was divided into two groups: Control group: 1 mL of fresh culture medium was added; Pic group: 1 mL of culture medium containing 20 µM Pic was added. Each group had 3 replicates.

[0062] After incubating at 37°C for 24 hours, the cell supernatant from each group was collected and centrifuged to remove particulate matter. The concentration of PGE2 in the supernatant was measured according to the PGE2 ELISA kit instructions. Results are as follows: Figure 13As shown, the PGE2 secretion level in the control group was approximately 33 pg / mL. The secretion level in the Pic group was significantly reduced to 26 pg / mL. This result demonstrates that Pic is an effective PGE2 inhibitor.

[0063] Example 7: In vitro isolation and stimulation maturation of BMDCs 7.1 Isolation and culture of BMDCs: Female C57BL / 6 mice aged 6-8 weeks were euthanized by cervical dislocation and then disinfected by immersion in 75% alcohol for 5 minutes. Under aseptic conditions, the tibia and femur were isolated, and the epiphyses at both ends were cut open. The bone marrow cavity was flushed with RPMI-1640 medium using a syringe, and the flushing fluid was collected. The flushing fluid was passed through a 70 µm cell sieve, and the filtrate was collected. The cells were centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in erythrocyte lysis buffer and lysed at room temperature for 3 minutes. The lysis was terminated by adding culture medium, and the cells were centrifuged again. The resulting bone marrow cells were resuspended in RPMI-1640 complete medium containing 10% FBS, 20 ng / mL IL-4, and 20 ng / mL GM-CSF, and incubated at 1×10⁻⁶ cells / mL. 6 Cells were seeded per well in a 6-well plate. Half the medium was changed every 2 days. On day 7, the suspended and loosely adherent cells were collected, which were the immature BMDCs.

[0064] 7.2 Stimulated maturation and flow cytometry detection of BMDCs: The experimental groups were as follows, with 3 replicates per group: PBS group (negative control): 1 mL PBS was added; Control group: 1 mL fresh culture medium was added; Laser group (laser group): 1 mL fresh culture medium was added, and the cells were irradiated with an 808 nm laser (1.5 W / cm²) for 10 min; HPC group (hydrogel group): 100 µL of freshly prepared HA-G1.25 hydrogel (to cover the cell layer) and 900 µL of culture medium were added; HPC+Laser group (hydrogel+laser group): treated with the HPC group for 4 hours, and then irradiated with an 808 nm laser (1.5 W / cm²) for 10 min; After treating Hepa1-6 cells according to the method in Example 5, the cell supernatant of each group (PBS, Laser, HPC, HPC+Laser) was collected as "ICD supernatant" for later use.

[0065] Take the immature BMDCs obtained on day 7 above, and use 1×10 6Seeds were planted at a density of cells / well in new 24-well plates. Supernatant from each treatment group (1 mL / well) was added. Incubation was performed at 37°C for 18 hours. BMDCs from each group were collected, washed with PBS, and then incubated at 4°C for 30 minutes in the dark with FITC-labeled anti-CD11c antibody, PE-labeled anti-CD80 antibody, and APC-labeled anti-CD86 antibody (each diluted 1:100). After washing, the proportion of CD80 and CD86 double-positive cells among CD11c-positive cells was determined by flow cytometry.

[0066] The results are as follows Figure 14 As shown, CD80 in the PBS group, Laser group, and HPC group + CD86 + The cell percentages were 19.76%, 49.38%, and 50.48%, respectively. Compared with the Control group, the HPC+Laser group (with added ICD supernatant) had significantly higher CD80 counts. + CD86 + The cell proportion increased significantly to 68.97%, indicating that hydrogel combined with photothermal therapy can effectively promote the maturation of BMDCs by inducing ICD and releasing antigens and signaling molecules.

[0067] In conclusion, the dual synergistic strategy of photothermal therapy combined with immunotherapy employed by HPC+Laser injectable hydrogel has demonstrated great potential in postoperative treatment models of liver cancer, providing new ideas and methods for the clinical treatment of postoperative recurrence and metastasis of liver cancer.

[0068] The hydrogel of this invention not only has good biocompatibility, in-situ gelation and photothermal responsiveness, but can also be combined with PTT to achieve a dual synergistic therapeutic effect of "local killing and immune activation" after liver cancer surgery. Both in vitro and in vivo experiments have shown excellent tumor killing, immune activation and anti-recurrence and metastasis functions, providing a new solution for the treatment of liver cancer after surgery.

Claims

1. A method for preparing a metal-polyphenol injectable hydrogel, characterized in that: Includes the following steps: (1) Hyaluronic acid was reacted with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide to activate the carboxyl group of hyaluronic acid. Then, dopamine hydrochloride and an organic base were added to react and obtain catechol-terminated hyaluronic acid. (2) The catechol-terminated hyaluronic acid obtained in step (1) is uniformly mixed with paclitaxel in a solution environment to obtain a precursor mixture; (3) Mix the precursor mixture with the metal salt solution, then add guar gum, and react to form a network structure to obtain a metal-polyphenol injectable hydrogel.

2. The preparation method according to claim 1, characterized in that: In step (1), the weight-average molecular weight of hyaluronic acid is 400,000 to 800,000 Daltons; Preferably, the mass ratio of hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and N-hydroxysulfosuccinimide is 1:2-3:1-1.5; Preferably, the organic base is triethylamine; Preferably, the mass ratio of hyaluronic acid to dopamine hydrochloride is 1:0.5-0.6; Preferably, the ratio of hyaluronic acid to organic base is 100mg: 3-5µL.

3. The preparation method according to claim 1 or 2, characterized in that: In step (1), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution is first added to the hyaluronic acid solution and reacted for 15-30 min. Then, N-hydroxysulfosuccinimide solution is added and reacted for 1-1.5 h. Then, dopamine hydrochloride solution and organic base are added and reacted for 20-21 h under inert gas protection. Preferably, the solvents for the hyaluronic acid solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution, N-hydroxysulfosuccinimide solution and dopamine hydrochloride solution are all phosphate buffer solutions with pH=5~6.

4. The preparation method according to claim 1, characterized in that: In step (2), the mass ratio of catechol-terminated hyaluronic acid to paclitaxel is 3-4:

1.

5. The preparation method according to claim 1 or 4, characterized in that: In step (2), the catechol-terminated hyaluronic acid solution is mixed with the paclitaxel solution to obtain a precursor mixture; Preferably, the concentration of the catechol-terminated hyaluronic acid solution is 20-40 mg / mL, and the concentration of the paclitaxel solution is 40-50 mg / mL.

6. The preparation method according to claim 1, characterized in that: In step (3), the metal salt is a copper salt, which is a sulfate or chloride of a metal; Preferably, the molar ratio of metal ions to paclitaxel is 4-5:1; Preferably, the volume ratio of the precursor mixture to the metal salt solution is 10-13:

1.

7. The preparation method according to claim 1 or 6, characterized in that: In step (3), the mass ratio of catechol-terminated hyaluronic acid to guar gum is 1:1-1.

5.

8. The preparation method according to claim 1 or 6, characterized in that: In step (3), after adding guar gum, stir until completely dissolved, and then let stand for 2-3 hours.

9. The metal-polyphenol injectable hydrogel prepared according to any one of claims 1-8.

10. The use of the metal-polyphenol injectable hydrogel of claim 8 in the preparation of postoperative treatment drugs for liver cancer.

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