Q7R-cu coordination hydrogel, and preparation method and application thereof

Abstract

This invention belongs to the field of drug delivery and functional hydrogel materials technology, and discloses a Q7R-Cu coordination hydrogel, its preparation method, and its applications. The Q7R-Cu coordination hydrogel provided by this invention, by dissolving Q7R under alkaline conditions and inducing it to undergo a coordination reaction with copper ions under weakly acidic to neutral conditions to form a three-dimensional network structure, not only improves the physicochemical properties and local delivery effect of Q7R, but also enables the local release of Cu. 2+ It induces copper death in tumor cells, thereby activating dendritic cell and T cell-mediated anti-tumor immune responses, and can also induce remote effects and immune memory, providing a new local delivery and synergistic treatment strategy for liver cancer, which has high scientific value and potential application prospects.

Description

Technical Field

[0001] This invention relates to the field of drug delivery and functional hydrogel materials technology, and in particular to a Q7R-Cu coordination hydrogel, its preparation method and application. Background Technology

[0002] Primary liver cancer is the fourth most common malignant tumor and the second leading cause of death in my country, accounting for more than half of the world's cases. Hepatocellular carcinoma (HCC) accounts for 85-90% of these cases, seriously threatening the health and lives of the Chinese people. Most HCC patients in my country develop the disease on the basis of chronic liver disease and cirrhosis. The onset is often insidious, with few early symptoms, leading to most patients being diagnosed at an advanced stage. For patients with advanced liver cancer, systemic drug therapy is the primary choice, with molecularly targeted drugs such as sorafenib and lenvatinib being first-line treatments. However, clinical practice shows that the objective response rate of these targeted drugs is generally less than 30%, and they are often accompanied by serious side effects such as hypertension and hand-foot syndrome.

[0003] Quercetin-7-O-rhamnoside (Q7R) is an active ingredient in the traditional Chinese medicine *Pteris vittata* that exerts its anti-hepatocellular carcinoma effects by inducing apoptosis in liver cancer cells and inhibiting tumor angiogenesis. However, Q7R faces several limitations in practical applications: firstly, it has poor water solubility and low bioavailability; secondly, it is rapidly metabolized in vivo with a short half-life; and thirdly, it lacks tumor targeting, making it difficult to achieve effective therapeutic concentrations at the tumor site after systemic administration. These pharmacokinetic defects severely limit the clinical translation and application of Q7R.

[0004] Copper death is a copper-dependent, programmed cell death mechanism driven by mitochondrial respiratory chain abnormalities and protein toxicity stress, and it is closely related to metabolic abnormalities and uncontrolled proliferation in liver cancer. Copper ions, as a core regulator of copper death, can directly bind to esterified proteins in the tricarboxylic acid cycle, leading to their abnormal aggregation and dysfunction, thereby triggering mitochondrial metabolic collapse and cell death. Furthermore, copper ions can promote the excessive generation of reactive oxygen species and inhibit antioxidant systems (such as the glutathione pathway), thus enhancing the sensitivity of tumor cells to copper death. Copper death not only directly kills tumor cells, but the intense endoplasmic reticulum stress and mitochondrial dysfunction it induces can further initiate immunogenic cell death (ICD), activating anti-tumor immune responses. ICD is a key mechanism regulating the tumor immune microenvironment, characterized by the release of a series of danger-associated molecular patterns (DAMPs) from dying cells. These include calreticulin (CRT) exposed on the cell surface, high-mobility group box 1 (HMGB1) released extracellularly, and large amounts of secreted adenosine triphosphate (ATP). These signaling molecules collectively constitute the "eat me" signaling pathway, promoting the phagocytosis, processing, and presentation of tumor antigens by dendritic cells (DCs), thereby activating antigen-specific cytotoxic T cells (CD8+). + T cells) and helper T cells (CD4) + T cells (CTCs) mediate specific anti-tumor immunity and may generate distant effects and immune memory. Studies have shown that the lipoylated protein toxicity stress and mitochondrial membrane potential breakdown accumulated during copper death can significantly enhance endoplasmic reticulum stress levels, activate eukaryotic translation initiation factor 2α and downstream signaling pathways, which is an important prerequisite for CRT translocation to the cell membrane. Simultaneously, the massive burst of reactive oxygen species and mitochondrial DNA leakage caused by copper death can further promote the release of HMGB1 and ATP secretion, thereby efficiently recruiting and activating antigen-presenting cells. Therefore, copper death-based therapies not only directly eliminate tumor cells but may also reshape the immunosuppressive tumor microenvironment through ICDs, reverse T cell exhaustion, and thus stimulate a powerful anti-tumor immune response. Summary of the Invention

[0005] The purpose of this invention is to provide a Q7R-Cu coordination hydrogel, its preparation method and application, to solve the problems of poor water solubility, low bioavailability, and limited improvement in efficacy caused by simply increasing the dosage of Q7R in the prior art.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing Q7R-Cu coordination hydrogel, comprising the following steps: Mix Q7R solution with Cu 2+ The solutions were mixed, the pH was adjusted to 5.0-7.4, and coordination crosslinking was performed to obtain Q7R-Cu coordination hydrogel; Among them, the Q7R solution is in contact with Cu-containing 2+ Before mixing the solutions, adjust the pH of the Q7R solution to 8.5-9.5.

[0007] Furthermore, in the preparation method, the Q7R solution is prepared by mixing quercetin-7-O-rhamnoside, a solubilizer, and a solvent.

[0008] Furthermore, in the preparation method, the co-solvent in the formulation of the Q7R solution includes one or more of dimethyl sulfoxide, dimethylformamide, and propylene glycol, and the co-solvent accounts for 0.5-2% of the sum of the volumes of the co-solvent and the solvent. The solvent includes water and / or PBS buffer.

[0009] Furthermore, in the preparation method, the mixing of quercetin-7-O-rhamnoside, the solubilizer, and the solvent is carried out by vortex mixing, and the mixing time is 5-60 seconds.

[0010] Furthermore, in the preparation method, the concentration of Q7R in the Q7R solution is 10~30 mmol / L; the Cu-containing solution... 2+ Cu in solution 2+ The concentration is 3~8 mmol / L; The Q7R solution and the Cu-containing solution 2+ The volume ratio of the solution is 1.5:1 to 3:1.

[0011] Furthermore, in the preparation method, the Cu-containing... 2+ In solution, Cu 2+ Including CuSO4 and / or CuCl2.

[0012] Furthermore, in the preparation method, the Q7R solution contains Cu 2+ The solution is mixed by vortex mixing, and the mixing time is 5~60s.

[0013] Furthermore, in the preparation method, the temperature of the coordination crosslinking is 20~37℃, and the time of the coordination crosslinking is 3~10min.

[0014] The present invention also provides a Q7R-Cu coordination hydrogel.

[0015] The present invention also provides the application of Q7R-Cu coordination hydrogel in the preparation of anti-liver cancer drugs, wherein the liver cancer is hepatocellular carcinoma.

[0016] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: The Q7R-Cu coordination hydrogel (Q7R-Cu@gel) provided by this invention constructs a three-dimensional network structure system through the coordination interaction between Q7R and copper ions, enabling efficient accumulation and sustained release of drugs at the tumor site. Simultaneously, this system exhibits good injectability and structural stability, making it suitable for use as a local delivery carrier. During tumor treatment, Q7R-Cu@gel can enhance anti-tumor effects by exogenously delivering copper ions and promoting their accumulation in tumor tissue; it also improves the retention and release behavior of Q7R at the tumor site, increasing treatment efficiency and reducing systemic toxicity. Furthermore, this hydrogel can improve the tumor microenvironment and enhance anti-tumor therapeutic effects, demonstrating promising application prospects.

[0017] The process of preparing Q7R-Cu coordination hydrogel in this invention is carried out under mild conditions, without the need for high temperature and high pressure treatment, and is simple and easy to implement. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0019] Figure 1 These are gelation state diagrams of the hydrogel systems in Example 1 and Comparative Examples 1-3; Figure 2 SEM images of the hydrogel systems of Example 1 and Comparative Examples 1-3; Figure 3 EDS energy dispersive spectroscopy (EDS) spectra of the hydrogel systems in Example 1 and Comparative Examples 1-3; Figure 4 The rheological properties of the hydrogel systems in Example 1 and Comparative Examples 1-3 are shown in the diagram. Figure 5 Raman spectra of the hydrogel systems in Example 1 and Comparative Examples 1-3; Figure 6 The drug release curves of the hydrogel systems in Example 1 and Comparative Examples 1-3 at pH 7.4 are shown. Figure 7 Figure 1 shows the effect of the hydrogel systems of Example 1 and Comparative Examples 1-3 on the proliferation of liver cancer cells and vascular endothelial cells. Figure 8 The graph shows the effect of different volumes of the hydrogel system from Example 1 on the tumor growth curves of tumor-bearing mice. Figure 9The graph shows the detection results of dendritic cell maturation and T cell activation induced by hydrogel systems of different volumes in Example 1. Figure 10 This is a diagram illustrating the effect of the hydrogel system in inducing distant tumor inhibition in Example 1. Figure 11 The graph shows the inhibitory effects of Example 1, Comparative Example 1, Comparative Example 4, and iliximab on the proliferation of liver cancer cells. Detailed Implementation

[0020] This invention provides a method for preparing Q7R-Cu coordination hydrogel, comprising the following steps: Mix Q7R solution with Cu 2+ The solutions were mixed, the pH was adjusted to 5.0-7.4, and coordination crosslinking was performed to obtain Q7R-Cu coordination hydrogel; Among them, the Q7R solution is in contact with Cu-containing 2+ Before mixing the solutions, adjust the pH of the Q7R solution to 8.5-9.5.

[0021] In this invention, the Q7R solution is prepared by mixing quercetin-7-O-rhamnoside, a solubilizer, and a solvent. The co-solvent preferably includes one or more of dimethyl sulfoxide, dimethylformamide, and propylene glycol, more preferably includes dimethyl sulfoxide, dimethylformamide, or propylene glycol, and more preferably is dimethyl sulfoxide; The co-solvent preferably accounts for 0.5-2% of the sum of the volumes of the co-solvent and the solvent, more preferably 0.8-1.2%, and even more preferably 1%; The solvent preferably includes water and / or PBS buffer, and more preferably water; The preferred method for mixing quercetin-7-O-rhamnoside, the solubilizer, and the solvent is vortex mixing; the preferred mixing time is 5-60 s, more preferably 20-40 s, and even more preferably 30 s.

[0022] In this invention, the reagent used to adjust the pH of the Q7R solution to 8.5-9.5 preferably includes NaOH and / or KOH, and more preferably NaOH.

[0023] In this invention, the pH of the Q7R solution adjusted to 8.5-9.5 is more preferably 8.8-9.2, and more preferably 9. The amount of reagent used is not limited; adjustment to the desired pH is sufficient.

[0024] In this invention, the Cu-containing 2+ In solution, Cu 2+ Preferably, it includes CuSO4 and / or CuCl2, more preferably CuCl2, such as CuSO4·5H2O or CuCl2·2H2O.

[0025] In this invention, for the Cu-containing 2+ The method for preparing the solution is not limited; any method well-known in the art may be used. Specifically, in the examples, the solution contains Cu... 2+ The solution is an aqueous solution.

[0026] In this invention, the concentration of Q7R in the Q7R solution is preferably 10-30 mmol / L, more preferably 15-25 mmol / L, and even more preferably 20 mmol / L.

[0027] In this invention, the Cu-containing 2+ Cu in solution 2+ The preferred concentration is 3-8 mmol / L, more preferably 4-6 mmol / L, and even more preferably 5 mmol / L.

[0028] In this invention, the Q7R solution and the Cu-containing solution 2+ The volume ratio of the solution is preferably 1.5:1 to 3:1, more preferably 2:1 to 2.5:1, and even more preferably 2:1.

[0029] In this invention, the reagent used to adjust the pH to 5.0-7.4 is preferably an acid. The acid preferably includes hydrochloric acid.

[0030] In this invention, the pH is preferably adjusted to 5.5-7.0, more preferably 6.0-7.0, and even more preferably 6.5. The amount of acid used is not limited; it is sufficient to adjust to the desired pH.

[0031] In this invention, the Q7R solution contains Cu 2+ The preferred method for mixing the solution is vortex mixing; the preferred mixing time is 5-60 s, more preferably 20-40 s, and even more preferably 30 s.

[0032] In this invention, the temperature for coordination crosslinking is preferably 20~37℃, more preferably 20~30℃, and even more preferably 25℃; the time for coordination crosslinking is preferably 3~10 min, more preferably 4~6 min, and even more preferably 5 min. The coordination crosslinking process can be carried out by standing.

[0033] The present invention also provides a Q7R-Cu coordination hydrogel.

[0034] In this invention, the Q7R-Cu coordination hydrogel is injectable, flows under external force, and returns to a gel state under static conditions; the Q7R-Cu coordination hydrogel exhibits pH-responsive release behavior within the pH range of 1.5 to 7.4; and the Q7R-Cu coordination hydrogel remains non-flowing in an aqueous environment by being inverted at 45° for 1 minute.

[0035] In the Q7R-Cu coordination hydrogel provided by this invention, Q7R serves as both an active pharmaceutical ingredient and a coordination building block, forming a hydrogel (@gel) system with a three-dimensional porous network structure under the action of metal ions. This system exhibits excellent gelling ability, mechanical stability, injectability, self-healing properties, thixotropy, and pH-responsive release behavior. Compared to Q7R itself, the hydrogel enhances the local retention capacity of Q7R and reduces system exposure. Compared to other metal ion coordination systems, Q7R-Cu@gel demonstrates superior mechanical stability, sustained-release performance, and antitumor activity. Specifically, Q7R-Cu@gel can induce copper death in tumor cells through the local release of copper ions. Simultaneously, Q7R can target and inhibit DHRS13, further enhance CPT1A enzyme activity, reprogram fatty acid oxidation metabolism, promote lipid droplet changes and mitochondrial ROS accumulation, thereby enhancing the sensitivity of tumor cells to copper death and further inducing immunogenic cell death, activating dendritic cell and T cell-mediated antitumor immune responses. The results show that this synergistic mechanism of "metabolic regulation-copper death-immune activation" can significantly inhibit the progression of liver cancer and has good safety and translational potential.

[0036] This invention also provides the application of Q7R-Cu coordination hydrogel in the preparation of anti-liver cancer drugs.

[0037] In this invention, the liver cancer is preferably hepatocellular carcinoma.

[0038] In this invention, the anti-liver cancer drug is administered locally, including but not limited to injection. The Q7R-Cu coordination hydrogel delivery system, due to its ability to form a drug reservoir in the tumor region via local injection, achieves controlled release and long-lasting retention, significantly increasing local drug concentration and reducing systemic toxicity.

[0039] In this invention, there are no limitations on the source of raw materials or other parameters and conditions; commercially available products and methods known in the art can be used.

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Unless otherwise specified, all reagents used in the examples and comparative examples were commercially available. Quercetin-7-O-rhamnoside (Vicchi Biotechnology, 22007-72-3); copper chloride, ferric chloride, and zinc chloride were conventional metal coordination raw materials; HepG2 cells and Huh7 cells were used for liver cancer cells; HUVECs cells were used for normal cells; and C57BL / 6 mice were used to establish a liver cancer model.

[0042] Example 1

[0043] This embodiment provides a method for preparing Q7R-Cu@gel, including the following steps: To prepare a 20 mmol / L Q7R solution: Accurately weigh 0.8968 g of Q7R powder and dissolve it completely in 100 mL of deionized water containing 1 vt% cosolvent (dimethyl sulfoxide). Adjust the pH of the system to 9 dropwise with NaOH solution and vortex for 30 s until the Q7R is completely dissolved to obtain a clear and homogeneous Q7R solution. Prepare a 5 mmol / L Cu-containing solution 2+ Solution: Weigh 0.0852 g CuCl2·2H2O and dissolve it in 100 mL of deionized water to obtain a solution containing Cu. 2+ Solution; During gel preparation, follow the Q7R solution: containing Cu 2+ The solutions were mixed and added at a volume ratio of 2:1, and each group of samples was thoroughly vortexed for 30 seconds. After mixing, the mixture was slowly titrated with HCl solution to precisely adjust the pH to 7. After pH adjustment, the mixture was placed at 37°C and allowed to stand for 5 minutes. The gel was formed by pH-induced metal coordination self-assembly reaction, and Q7R-Cu@gel was prepared. The prepared gel was sealed and stored at 4°C in the dark for later use.

[0044] Comparative Example 1

[0045] Comparative Example 1 provides a Q7R system, the preparation method of which is basically the same as that of Example 1, except that Cu is not added. 2+ The Q7R system was obtained by dissolving Q7R in deionized water containing 1 wt% cosolvent (dimethyl sulfoxide).

[0046] Comparative Example 2

[0047] Comparative Example 2 provides a Q7R-Fe@gel, which differs from Example 1 in that 0.0852g CuCl2·2H2O is replaced with 0.1351g FeCl3·6H2O, resulting in a Fe-containing gel. 3+ The solution contains Cu 2+ The solution was replaced with one containing Fe.3+ The solution is prepared using the same steps as before.

[0048] Comparative Example 3

[0049] Comparative Example 3 provides a Q7R-Zn@gel, which differs from Example 1 in that 0.0852g CuCl2·2H2O is replaced with 0.1438g ZnSO4·7H2O, resulting in a Zn-containing gel. 2+ The solution contains Cu 2+ The solution was replaced with one containing Zn. 2+ The solution is prepared using the same steps as before.

[0050] Comparative Example 4

[0051] Comparative Example 4 provides a Cu 2+ The system is prepared in a manner similar to that of Example 1, except that Q7R is not added, and only Cu is used. 2+ Dissolved in deionized water, Cu is obtained. 2+ system.

[0052] To demonstrate the beneficial effects of the Q7R-Cu@gel prepared in Example 1, the hydrogel systems prepared in Examples 1 and Comparative Examples 1-3 were measured. In the accompanying figures, they are respectively labeled Q7R-Cu, Q7R, Q7R-Fe, and Q7R-Zn, as shown below: 1. The physicochemical properties of Q7R-based metal coordination hydrogels were determined. 1) Observe Q7R, Q7R-Cu@gel, Q7R-Fe@gel and Q7R-Zn@gel by inverting test tubes. Figure 1 As shown, each metal ion coordination system can form a uniform and stable hydrogel.

[0053] 2) The microstructure of Q7R-Cu@gel, Q7R-Fe@gel, and Q7R-Zn@gel was observed using scanning electron microscopy, such as... Figure 2 As shown, each group maintains a three-dimensional porous network structure, indicating that metal coordination does not disrupt the network framework formed by Q7R.

[0054] 3) Elemental energy dispersive spectroscopy (EDS) was used to detect each system, such as... Figure 3 As shown, the metallic element was successfully introduced into the corresponding system, indicating that Q7R and Cu... 2+ Fe 3+ and Zn 2+ Both can undergo coordination.

[0055] 4) Rheology is used to characterize each system, such as... Figure 4As shown, all hydrogels exhibit typical gel behavior, with certain mechanical stability and injectability; among them, the Q7R-Cu@gel group has the highest storage modulus and the best structural integrity.

[0056] 5) Raman spectroscopy was used to analyze the coordination interactions between Q7R and different metal ions, such as... Figure 5 As shown, the shift of the characteristic peak of Q7R-Cu@gel is the most significant, suggesting that Cu 2+ It has a stronger coordination effect with Q7R and a more stable binding.

[0057] 2. Determination of the release and degradation behavior of Q7R-based metal coordination @gels

[0058] In vitro release experiments were conducted on Q7R, Q7R-Cu@gel, Q7R-Fe@gel, and Q7R-Zn@gel at pH 7.4. Figure 6 As shown, all groups of hydrogels exhibited significant pH responsiveness; among them, the Q7R-Cu group showed a more sustained release behavior at pH 7.4, indicating that it is beneficial to reduce non-specific release and prolong the local drug action time.

[0059] 3. Effects of Q7R-based metal coordination @gel on the viability of liver cancer cells.

[0060] HepG2 cells, Huh7 cells, and HUVECs cells were seeded in 96-well plates. After cell attachment, different concentrations of Q7R, Q7R-Cu@gel, Q7R-Fe@gel, and Q7R-Zn@gel were added for treatment, and cell viability was measured using the CCK-8 assay.

[0061] The results are as follows Figure 7 As shown, all treatments exhibited concentration- and time-dependent inhibitory effects; among them, Q7R-Cu@gel showed the strongest killing effect on HepG2 and Huh7 cells, significantly better than Q7R, Q7R-Fe@gel and Q7R-Zn@gel; at the same time, Q7R-Cu@gel had relatively mild toxicity to HUVECs and showed a wide safety window under low and medium concentration conditions.

[0062] 4. The anti-tumor and immune-activating effects of Q7R-Cu@gel in vivo.

[0063] Tumor-bearing mice were randomly divided into groups and administered saline or different volumes of Q7R-Cu@gel locally. Tumor growth was observed, and tumor volume was measured at the experimental endpoint. The results are as follows: Figure 8 As shown, the results of immune cell infiltration are as follows: Figure 9 As shown.

[0064] The results showed that Q7R-Cu@gel had a more significant tumor-suppressing effect than other groups, significantly reducing tumor volume and endpoint tumor weight; simultaneously, Q7R-Cu@gel enhanced dendritic cell maturation, antigen presentation, and CD8+. + T cell activation and a decrease in the proportion of Treg cells indicate that Q7R-Cu@gel can significantly enhance the anti-tumor immune response.

[0065] 5. The remote effect and immune memory function of Q7R-Cu@gel

[0066] In the bilateral tumor-bearing model, the results of local treatment with Q7R-Cu@gel on only the primary tumor were as follows: Figure 10 As shown in the figure. The results indicate that Q7R-Cu@gel can not only significantly inhibit the growth of the primary tumor, but also inhibit the growth of the contralateral tumor that was not directly treated, suggesting that it has a remote anti-tumor effect.

[0067] 6. Q7R-Cu@gel exhibits superior inhibitory effects on liver cancer cell proliferation compared to Q7R and Cu. 2+

[0068] In hepatocellular carcinoma-derived organoid models, Q7R and Cu were administered respectively. 2+ Q7R-Cu@gel and irismo, results as follows Figure 11 As shown in the figure. The results indicate that Q7R-Cu@gel has a better inhibitory effect on liver cancer proliferation than Q7R and Cu. 2+ And Illismo, hinting at Q7R and Cu 2+ It has a good synergistic effect in fighting liver cancer.

[0069] The aforementioned tests demonstrate that the Q7R-Cu@gel of Example 1 forms a uniform and stable gel state macroscopically and possesses a three-dimensional porous network structure microscopically. Elemental energy dispersive spectroscopy and potential analysis indicate that Cu was successfully introduced into the system and coordinated with Q7R. Rheological characterization shows that it exhibits typical gel behavior, with a storage modulus higher than its loss modulus. Furthermore, among various Q7R-based metal coordination hydrogels, the Q7R-Cu@gel group exhibits a higher storage modulus, better structural integrity, and better injectability.

[0070] The Q7R-Cu@gel in Example 1 exhibits good pH-responsive release behavior, with relatively slow release under acidic conditions and accelerated release and degradation under neutral and weakly alkaline conditions. Among them, the Q7R-Cu@gel group shows a more sustained release characteristic at pH 7.4, which can provide a basis for local tumor delivery.

[0071] The Q7R-Cu@gel in Example 1 showed a significant inhibitory effect on HepG2 and Huh7 liver cancer cells, and its inhibitory effect was superior to that of Q7R and Cu. 2+The Q7R-Fe@gel and Q7R-Zn@gel systems are also mentioned; at the same time, their toxicity to normal HUVECs cells is relatively mild, showing a good safety and therapeutic window.

[0072] In Example 1, Q7R-Cu@gel significantly inhibited tumor volume growth and reduced endpoint tumor weight in a mouse subcutaneous tumorigenesis model, and enhanced dendritic cell maturation, antigen presentation, and CD8+ expression. + T cell activation and a reduction in the proportion of immunosuppressive Treg cells indicate that it can effectively reshape the tumor immune microenvironment.

[0073] The Q7R-Cu@gel in Example 1 produced a remote antitumor effect in a bilateral tumor-bearing model and induced an increase in central memory T cells and effector memory T cells in a rechallenge model, suggesting that it can establish durable immune memory.

[0074] The Q7R system obtained in Comparative Example 1 has certain anti-tumor activity and can inhibit DHRS13 by targeting it, but its gelling ability, local retention ability, sustained release ability, and ability to induce copper death and immunogenic cell death are lower than those of Q7R-Cu@gel in Example 1.

[0075] Comparative Example 2's Q7R-Fe@gel can form a stable gel and has certain anti-tumor activity, but it is weaker than Q7R-Cu@gel in terms of mechanical properties, sustained release characteristics, and inhibitory effect on liver cancer cells.

[0076] Comparative Example 3, Q7R-Zn@gel, can also form a gel and has certain biological activity, but it is weaker than Q7R-Cu@gel in inhibiting the growth of liver cancer cells, enhancing mitochondrial oxidative stress, and having a comprehensive anti-tumor effect.

[0077] Cu obtained in Comparative Example 4 2+ It has certain anti-tumor activity and can inhibit liver cancer proliferation by activating copper death-related pathways, but its ability to induce copper death and immunogenic cell death is lower than that of Q7R-Cu@gel in Example 1.

[0078] In summary, the Q7R-Cu@gel preparation method provided by this invention is simple, has mild reaction conditions, and is easy to operate. The prepared hydrogel can be directly stored and used, exhibiting good stability, injectability, and biocompatibility. The @gel is formed by dissolving Q7R under alkaline conditions and inducing its coordination reaction with copper ions under weakly acidic to neutral conditions to form a three-dimensional network structure. Compared with Q7R, Q7R-Fe@gel, and Q7R-Zn@gel, Q7R-Cu@gel shows superior structural stability, sustained-release performance, and antitumor activity. Q7R-Cu@gel not only improves the physicochemical properties and local delivery effect of Q7R but also enables the local release of Cu2+ This invention induces copper death in tumor cells, thereby activating dendritic cell and T cell-mediated anti-tumor immune responses, and can also induce remote effects and immune memory. This indicates that Q7R-Cu@gel possesses the characteristic of enabling exogenous delivery of copper ions through local administration and enrichment in hepatocellular carcinoma tissue, thereby enhancing local drug retention and achieving sustained release, exerting a synergistic anti-hepatocellular carcinoma effect, while reducing systemic exposure and toxic reactions. It has beneficial effects on improving the therapeutic efficacy of hepatocellular carcinoma and optimizing the hepatocellular carcinoma microenvironment, and is suitable for the preparation of drugs for treating hepatocellular carcinoma. Therefore, this invention provides a new local delivery and synergistic treatment strategy for hepatocellular carcinoma, with high scientific value and potential application prospects.

[0079] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a Q7R-Cu coordination hydrogel, characterized in that, Includes the following steps: Mix Q7R solution with Cu 2+ The solutions were mixed, the pH was adjusted to 5.0-7.4, and coordination crosslinking was performed to obtain Q7R-Cu coordination hydrogel; Among them, the Q7R solution is in contact with Cu-containing 2+ Before mixing the solutions, adjust the pH of the Q7R solution to 8.5-9.

5.

2. The preparation method according to claim 1, characterized in that, The Q7R solution is prepared by mixing quercetin-7-O-rhamnoside, a solubilizer, and a solvent.

3. The preparation method according to claim 2, characterized in that, In the preparation method of the Q7R solution, the co-solvent includes one or more of dimethyl sulfoxide, dimethylformamide, and propylene glycol, and the co-solvent accounts for 0.5-2% of the sum of the volumes of the co-solvent and the solvent. The solvent includes water and / or PBS buffer.

4. The preparation method according to claim 2 or 3, characterized in that, The method for mixing quercetin-7-O-rhamnoside, the solubilizer, and the solvent is vortex mixing, and the mixing time is 5-60 seconds.

5. The preparation method according to claim 1, characterized in that, The concentration of Q7R in the Q7R solution is 10~30 mmol / L; the Cu-containing solution... 2+ Cu in solution 2+ The concentration is 3~8 mmol / L; The Q7R solution and the Cu-containing solution 2+ The volume ratio of the solution is 1.5:1 to 3:

1.

6. The preparation method according to claim 1 or 5, characterized in that, The Cu-containing 2+ In solution, Cu 2+ Including CuSO4 and / or CuCl2.

7. The preparation method according to claim 6, characterized in that, The Q7R solution and Cu-containing 2+ The solution is mixed by vortex mixing, and the mixing time is 5~60s.

8. The preparation method according to claim 1, characterized in that, The coordination crosslinking temperature is 20~37℃, and the coordination crosslinking time is 3~10min.

9. A Q7R-Cu coordination hydrogel prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the Q7R-Cu coordination hydrogel according to claim 9 in the preparation of anti-liver cancer drugs, characterized in that, The liver cancer mentioned is hepatocellular carcinoma.

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