An injectable hydrogel, its preparation method and application

Abstract

This invention provides an injectable hydrogel, its preparation method, and its application, belonging to the field of biomedicine. The preparation method of the injectable hydrogel provided by this invention includes the following steps: mixing an oxidized dextran solution with a PIKFYVE kinase inhibitor solution to perform a first Schiff base reaction, obtaining a mixture; mixing the mixture with cell membrane fusion liposomes and a gelatin solution to perform a second Schiff base reaction, obtaining the injectable hydrogel; the preparation method of the cell membrane fusion liposomes includes: mixing dimyristoylphosphatidylcholine, cholesterol, triptolide, and a solvent, followed by solvent removal, hydration, and a first extrusion to obtain liposomes; mixing the liposomes with the cell membrane of mouse tongue squamous cell carcinoma cells and performing a second extrusion to obtain cell membrane fusion liposomes. The injectable hydrogel prepared by the method provided by this invention has no significant systemic toxicity and exhibits good water solubility, low toxicity, and long-lasting antitumor effects.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to an injectable hydrogel, its preparation method, and its application. Background Technology

[0002] Oral squamous cell carcinoma (OSCC) is the most common malignant solid tumor of the head and neck, accounting for more than 90% of head and neck malignancies. Despite continuous advancements in medical technology, the overall 5-year survival rate remains below 50%, with extremely poor prognosis for patients in advanced stages. Tongue squamous cell carcinoma, due to its unique anatomical location and rich blood supply and lymphatic network, exhibits extremely strong local invasiveness and lymph node metastasis, making clinical treatment even more challenging. Currently, the main clinical treatment for OSCC is surgical resection, supplemented by radiotherapy and chemotherapy. However, the tongue plays a crucial role in core physiological functions such as chewing, swallowing, and speech. Extensive surgical resection can easily lead to severe functional impairment and disfigurement, significantly reducing the patient's quality of life. Furthermore, traditional radiotherapy and chemotherapy lack tumor targeting, and while killing tumor cells, they can easily cause severe liver and kidney toxicity, bone marrow suppression, oral mucositis, and other adverse reactions. Long-term use can also induce multidrug resistance in tumors, ultimately leading to tumor recurrence and metastasis.

[0003] In recent years, immune checkpoint blockade (ICB) therapy has brought breakthroughs to cancer treatment. However, OSCC is a typical immune-cold tumor, which lacks effector T cell infiltration in its microenvironment and is enriched with a large number of M2 tumor-associated macrophages (TAMs), regulatory T cells (Tregs), and inhibitory cytokines, forming a deep immunosuppressive barrier. As a result, the overall response rate of ICB therapy in OSCC patients is less than 20%, which severely limits its clinical application.

[0004] Pyroptosis is a pro-inflammatory programmed cell death mediated by Gasdermin (GSDM) family proteins. Its core characteristic is the aggregation of the N-terminal fragment of the GSDM protein into pores on the cell membrane, leading to cell swelling and rupture, releasing large amounts of pro-inflammatory cytokines and damage-associated molecular patterns (DAMPs). It is a key strategy for transforming "immunely cold tumors" into "hot tumors" and reversing the immunosuppressive microenvironment. Tripterygium wilfordii extract (TPL) is a natural diterpenoid epoxide extracted from the traditional Chinese medicine Tripterygium wilfordii, possessing broad-spectrum and potent anti-tumor activity. TPL can induce pyroptosis in head and neck cancer cells by activating the Caspase-3 / GSDME pathway. However, TPL suffers from poor water solubility, a narrow therapeutic window, and high systemic toxicity, limiting its clinical application. More importantly, when tumor cells experience plasma membrane damage, they initiate a rapid membrane repair mechanism mediated by the endosomal sorting complex (ESCRT), actively clearing GSDM pores to resist pyroptosis, significantly weakening its efficacy and subsequent immune activation effects. Summary of the Invention

[0005] The purpose of this invention is to provide an injectable hydrogel, its preparation method, and its applications. The injectable hydrogel prepared by the method provided by this invention has good water solubility, low toxicity, and long-lasting anti-tumor (oral squamous cell carcinoma) effects.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing an injectable hydrogel, comprising the following steps: (1) Mix the oxidized dextran solution with the PIKFYVE kinase inhibitor solution and carry out the first Schiff base reaction to obtain a mixture; (2) The mixture obtained in step (1) is mixed with cell membrane fusion liposomes and gelatin solution to carry out a second Schiff base reaction to obtain an injectable hydrogel; the concentration of cell membrane fusion liposomes in the injectable hydrogel is 1~100 ng / mL; The method for preparing cell membrane fusion liposomes in step (2) includes: (a) After mixing myristoyl phosphatidylcholine, cholesterol, triptolide and solvent, the mixture was subjected to solvent removal, hydration and first extrusion in sequence to obtain liposomes; (b) The liposomes obtained in step (a) are mixed with the cell membrane of mouse oral squamous cell carcinoma cells and then subjected to a second extrusion to obtain cell membrane fusion liposomes; the drug loading of triptolide in the cell membrane fusion liposomes is 5-30%.

[0007] Preferably, in step (1), the molar ratio of oxidized dextran in the oxidized dextran solution to PIKFYVE kinase inhibitor in the PIKFYVE kinase inhibitor solution is (1000~100000):1.

[0008] Preferably, in step (1), the ratio of the mass of oxidized dextran to the volume of the solvent in the oxidized dextran solution is (0.5~5) g: 100 mL.

[0009] Preferably, the concentration of the PIKFYVE kinase inhibitor solution is 1~5mM.

[0010] Preferably, the molar ratio of the cell membrane fusion liposome in step (2) to the oxidized dextran in the oxidized dextran solution in step (1) is 1:(2~100).

[0011] Preferably, the molar ratio of gelatin in the gelatin solution in step (2) to oxidized dextran in the oxidized dextran solution in step (1) is 1:(1~3).

[0012] Preferably, in step (2), the ratio of the mass of gelatin to the volume of solvent in the gelatin solution is (5~25) g: 100 mL.

[0013] Preferably, in step (a), the molar ratio of myristoylphosphatidylcholine, cholesterol and triptolide is (2~3):1:(0.02~0.3).

[0014] The present invention also provides an injectable hydrogel prepared by the preparation method described in the above technical solution.

[0015] The present invention also provides the application of the injectable hydrogel described above in the preparation of a therapeutic drug for oral squamous cell carcinoma.

[0016] This invention provides a method for preparing an injectable hydrogel, comprising the following steps: (1) mixing an oxidized dextran solution with a PIKFYVE kinase inhibitor solution to perform a first Schiff base reaction to obtain a mixture; (2) mixing the mixture obtained in step (1) with cell membrane fusion liposomes and gelatin solution to perform a second Schiff base reaction to obtain an injectable hydrogel; the concentration of cell membrane fusion liposomes in the injectable hydrogel is 1~100 ng / mL; the method for preparing cell membrane fusion liposomes in step (2) comprises: (a) mixing myristoylphosphatidylcholine, cholesterol, triptolide and solvent, and then sequentially performing solvent removal, hydration and a first extrusion to obtain liposomes; (b) mixing the liposomes obtained in step (a) with the cell membrane of mouse tongue squamous cell carcinoma cells and then performing a second extrusion to obtain cell membrane fusion liposomes; the drug loading of triptolide in the cell membrane fusion liposomes is 5~30%.This invention utilizes a homologous tumor cell membrane (the cell membrane of mouse oral squamous cell carcinoma MOC1) to prepare cell membrane fusion liposomes (CLip), which completely preserve the homologous adhesion proteins of the MOC1 cell membrane, achieving targeted delivery to oral squamous cell carcinoma cells and significantly improving the intracellular uptake efficiency of triptolide (TPL). Simultaneously, the liposome preparation effectively encapsulates TPL, solving its poor water solubility problem, and exhibits almost no toxicity to normal endothelial cells in vitro, while achieving highly efficient killing of MOC1 tumor cells, thus broadening the therapeutic scope of TPL. The window reduces the risk of systemic toxicity. By utilizing the Schiff base reaction, the aldehyde groups of oxidized dextran can be cross-linked with the amino groups of gelatin to construct a hydrogel network. On the other hand, the PIKFYVE kinase inhibitor is directly coupled to the oxidized dextran backbone via primary amino groups, solving the problem of burst release of small molecule inhibitors. The resulting hydrogel exhibits excellent shear-thinning properties, allowing for smooth injection via a syringe needle. After injection, it rapidly gels in situ at physiological temperatures, adaptively filling irregular gaps in tongue tumors and possessing good tissue adhesion, effectively resisting saliva washout and tongue body erosion. Frequent movement leads to drug loss, significantly prolonging the drug's residence time at the tumor site, increasing local therapeutic concentration, and reducing systemic drug exposure. The injectable hydrogel maintains stable Schiff base bonds and structural integrity under physiological conditions (pH 7.4), resulting in low drug leakage. However, in the slightly acidic environment of the tumor (pH 6.0), Schiff base bonds undergo protonation hydrolysis, leading to gradual hydrogel degradation and the release of CLIP and PIKFYVE kinase inhibitors. By limiting the concentration of CLIP in the injectable hydrogel and the TPL concentration within CLIP, the release of CLIP can be controlled. TPL generates an active N-terminal fragment by specifically cleaving GSDME via Caspase-3, forming pyroptosis pores on the cell membrane. Meanwhile, PIKFYVE kinase inhibitors block ESCRT-III complex-mediated membrane repair by inhibiting PIKFYVE kinase, transforming reversible membrane damage in tumor cells into irreversible lytic pyroptosis. This increases the release of pro-inflammatory factors such as IL-1β and HMGB1, as well as DAMPs, achieving spatiotemporal synergy between pyroptosis induction and membrane repair blockade, amplifying the pyroptosis response while ensuring the safety of normal cells (low toxicity).The results of the examples show that the CLIp and PIKFYVE kinase inhibitors in the injectable hydrogel prepared by the method provided in this invention can effectively promote the maturation of DC cells in tumor-draining lymph nodes, drive M2 macrophages to repolarize to anti-tumor M1 type, significantly increase the proportion and infiltration of CD8+ effector T cells in peripheral blood and tumor tissue, and effectively reduce the proportion of Tregs in the tumor microenvironment, successfully reversing the immunosuppressive microenvironment of OSCC and achieving a long-term anti-tumor immune response. In a mouse orthotopic oral squamous cell carcinoma model, the injectable hydrogel achieved near-complete tumor regression, maintained the feeding function of mice, effectively improved weight loss, and stabilized the 30-day survival rate of mice to 80%. At the same time, no pathological damage was observed in the H&E staining of important organs of mice, and serum liver and kidney function indicators (ALT, AST, UREA, CREA) were maintained within the normal physiological range. There was no obvious systemic toxicity, and it has good water solubility, low toxicity, and long-term anti-tumor (oral squamous cell carcinoma) effect. Attached Figure Description

[0017] Figure 1 This is a dynamic light scattering image of the cell membrane fused liposomes prepared in Example 1 of the present invention; Figure 2 This is a Zeta potential diagram of the cell membrane fused liposomes prepared in Example 1 of the present invention; Figure 3 This is an SDS-PAGE gel electrophoresis image of Lip, Cm, and CLIp in Example 1 of the present invention; Figure 4 This is a SEM image of the injectable hydrogel after lyophilization in Example 1 of the present invention; Figure 5 The bar chart shows the effect of different concentrations of TPL, Lip, and CLIp on the viability of MOC1 cells in Example 1 of this invention. Figure 6 The bar chart shows the effect of different concentrations of TPL, Lip, and CLIp on the viability of HUVEC cells in Example 1 of this invention. Figure 7 These are morphological images of MOC1 cells in the control group, Lip, CLIp, and CLIp+YM drug administration group in the test examples of this invention. Figure 8 This is a graph showing the expression of pyroptosis-related proteins in MOC1 cells of the control group, Lip, CLIp, and CLIp+YM drug-treated groups in the test examples of this invention. Figure 9 These are fluorescence images of MOC1 cells in the control group, Lip, CLIp, and CLIp+YM drug-treated groups in the test examples of this invention. Figure 10The fluorescence intensity distribution of ROS in MOC1 cells of the control group, Lip, CLIp, and CLIp+YM drug-treated group in the test examples of this invention is shown. Figure 11 This is a bar chart showing the average fluorescence intensity of ROS in MOC1 cells of the control group, Lip, CLIp, and CLIp+YM drug-treated groups in the test examples of this invention. Figure 12 These are confocal images of mitochondria in MOC1 cells from the control group, Lip, CLIp, and CLIp+YM drug administration group in the test examples of this invention. Figure 13 This is a bar chart showing the LDH release in the control group, Lip, CLIp, and CLIp+YM administration group in the test examples of this invention; Figure 14 This is a bar chart showing the IL-1β release in the control group, Lip, CLIp, and CLIp+YM administration group in the test examples of this invention; Figure 15 This is a bar chart showing the HMGB1 release in the control group, Lip, CLIp, and CLIp+YM administration group in the test examples of this invention; Figure 16 This is a GO enrichment analysis diagram of differentially expressed genes in the Control and CLIp dosing groups in the test examples of this invention; Figure 17 This is a GO enrichment analysis diagram of differentially expressed genes in the CLIp and CLIp+YM drug administration groups in the test examples of this invention; Figure 18 This is a KEGG enrichment analysis diagram of differentially expressed genes in the Control and CLIp dosing groups in the test examples of this invention; Figure 19 This is a KEGG enrichment analysis diagram of differentially expressed genes in the CLIp and CLIp+YM drug administration groups in the test examples of this invention; Figure 20 The CD86 levels in the control group, Lip, CLIp, and CLIp+YM administration group in the test examples of this invention are... + In F4 / 80 + Flow cytometry results in the image; Figure 21 The CD86 levels in the control group, Lip, CLIp, and CLIp+YM administration group in the test examples of this invention are... + Cells at F4 / 80 + A proportional histogram of cells; Figure 22 This is a bar chart showing the relative expression levels of the Arg-1 gene in the control group, Lip, CLIp, and CLIp+YM treatment group in the test examples of this invention. Figure 23This is a bar chart showing the relative expression levels of the Tnf-a gene in the control group, Lip, CLIp, and CLIp+YM treatment group in the test examples of this invention. Figure 24 The CD11c values ​​in the control group, Lip, CLIp, and CLIp+YM administration group in the test examples of this invention are... + CD80 in cells + CD86 + Image showing the result of double-positive ripening; Figure 25 The CD11c values ​​in the control group, Lip, CLIp, and CLIp+YM administration group in the test examples of this invention are... + CD80 in cells + CD86 + A histogram showing the proportion of double-positive mature DCs; Figure 26 These are bioluminescence images of mice with orthotopic tongue squamous cell carcinoma in the test examples of this invention after different treatments (Control group, GO group, GOC group, GOCY group); Figure 27 These are representative images of tongue tumors in mice after treatment in the Control group, GO group, GOC group, and GOCY group of the test examples of this invention; Figure 28 This is a graph showing the weight change of mice with orthotopic tongue squamous cell carcinoma in the Control group, GO group, GOC group, and GOCY group during the treatment cycle in the test examples of this invention. Figure 29 This is a survival curve of mice with orthotopic tongue squamous cell carcinoma in the Control group, GO group, GOC group, and GOCY group during the treatment cycle in the test examples of this invention; Figure 30 This is a Western Blot result of related proteins in mouse tumor tissues after treatment in the Control group, GO group, GOC group, and GOCY group of the test examples of this invention; Figure 31 This is a bar chart showing the changes in serum IL-1β concentration after treatment in the Control group, GO group, GOC group, and GOCY group in the test examples of this invention. Figure 32 This is a bar chart showing the changes in serum HMGB1 concentration after treatment in the Control group, GO group, GOC group, and GOCY group in the test examples of this invention. Figure 33 The flow cytometry results of dendritic cells in tumor draining lymph nodes (TDLNs) after treatment in the Control group, GO group, GOC group, and GOCY group in the test examples of this invention are shown. Figure 34This is a bar chart showing the proportion of mature dendritic cells in tumor draining lymph nodes (TDLNs) after treatment in the Control group, GO group, GOC group, and GOCY group in the test examples of this invention. Figure 35 The CD8+ levels after treatment in the Control group, GO group, GOC group, and GOCY group in the test examples of this invention. + Flow cytometry results of T cells; Figure 36 The CD8+ levels after treatment in the Control group, GO group, GOC group, and GOCY group in the test examples of this invention. + Bar chart showing the proportion of T cells; Figure 37 CD8+ levels in peripheral blood (PB) after treatment in the Control group, GO group, GOC group, and GOCY group of the test cases of this invention. + Flow cytometry results of T cells; Figure 38 CD8+ levels in peripheral blood (PB) after treatment in the Control group, GO group, GOC group, and GOCY group of the test cases of this invention. + Bar chart showing the proportion of T cells; Figure 39 The flow cytometry results of M2-like macrophages in tumor tissues after treatment in the Control group, GO group, GOC group, and GOCY group in the test examples of this invention are shown. Figure 40 This is a bar chart showing the proportion of M2-like macrophages in tumor tissue after treatment in the Control group, GO group, GOC group, and GOCY group in the test examples of this invention. Figure 41 The flow cytometry results of regulatory T cells in tumor tissues after treatment in the Control group, GO group, GOC group, and GOCY group of the test examples of this invention are shown. Figure 42 This is a bar chart showing the proportion of regulatory T cells in tumor tissue after treatment in the Control group, GO group, GOC group, and GOCY group in the test examples of this invention. Figure 43 These are CD8 immunofluorescence images of tumor tissues after treatment in the Control group, GO group, GOC group, and GOCY group in the test examples of this invention; Figure 44 This is a bar chart showing the CD8 immunofluorescence values ​​of tumor tissues after treatment in the Control group, GO group, GOC group, and GOCY group in the test examples of this invention. Figure 45 These are H&E staining images of major organs after treatment in the Control group, GO group, GOC group, and GOCY group in the test examples of this invention; Figure 46This is a bar chart showing the levels of alanine aminotransferase (ALT), a liver function marker, after treatment in the Control, GO, GOC, and GOCY groups in the test examples of this invention. Figure 47 This is a bar chart showing the levels of aspartate aminotransferase (AST) as a liver function marker after treatment in the Control group, GO group, GOC group, and GOCY group in the test examples of this invention. Figure 48 This is a bar chart showing the creatinine levels, a renal function marker, after treatment in the Control group, GO group, GOC group, and GOCY group in the test examples of this invention. Figure 49 This is a bar chart showing the urea levels, a renal function marker, after treatment in the Control, GO, GOC, and GOCY groups in the test examples of this invention. Detailed Implementation

[0018] This invention provides a method for preparing an injectable hydrogel, comprising the following steps: (1) Mix the oxidized dextran solution with the PIKFYVE kinase inhibitor solution and carry out the first Schiff base reaction to obtain a mixture; (2) The mixture obtained in step (1) is mixed with cell membrane fusion liposomes and gelatin solution to carry out a second Schiff base reaction to obtain an injectable hydrogel.

[0019] In this invention, an oxidized dextran solution is mixed with a PIKFYVE kinase inhibitor solution to undergo a first Schiff base reaction to obtain a mixture.

[0020] In one embodiment of the present invention, the solvent in the oxidized dextran solution can be deionized water; the mass ratio of oxidized dextran to solvent volume in the oxidized dextran solution can be (0.5~5) g:100 mL, specifically 0.5 g:100 mL, 1 g:100 mL, 1.5 g:100 mL, 2 g:100 mL, 2.5 g:100 mL, 3 g:100 mL, 3.5 g:100 mL, 4 g:100 mL, 4.5 g:100 mL, or 5 g:100 mL. Using the oxidized dextran solution in solution form and limiting it to the above concentrations in the present invention is more conducive to mixing and subsequent Schiff base reactions.

[0021] As one embodiment of the present invention, the preparation method of the oxidized dextran may include: adding an aqueous solution of sodium periodate to an aqueous solution of dextran to carry out an oxidation reaction, then adding ethylene glycol to terminate the oxidation, and then performing dialysis and freeze-drying in sequence to obtain oxidized dextran.

[0022] In one embodiment of the present invention, the molar ratio of dextran in the dextran aqueous solution to sodium periodate in the sodium periodate aqueous solution can be 1:(1~2). In another embodiment of the present invention, the concentration of the dextran aqueous solution can be 0.05~0.2 g / mL; the concentration of the sodium periodate aqueous solution can be 0.05~0.2 g / mL. By limiting the molar ratio of dextran in the dextran aqueous solution to sodium periodate in the sodium periodate aqueous solution, and the concentrations of the dextran aqueous solution and the sodium periodate aqueous solution, within the above-mentioned ranges, the present invention can better achieve the oxidation of dextran.

[0023] In an embodiment of the present invention, the sodium periodate aqueous solution is added to the dextran aqueous solution at a rate of 4 mL / min.

[0024] In one embodiment of the present invention, the oxidation reaction can be carried out under stirring and light-protected conditions. The present invention does not specifically limit the stirring speed; stirring speeds commonly used by those skilled in the art can be used. In one embodiment of the present invention, the temperature of the oxidation reaction can be 20-30°C, or even 25°C; the time of the oxidation reaction can be 5.5-6.5 hours, or even 6 hours. Limiting the temperature and time of the oxidation reaction to the above ranges allows the reaction to proceed fully.

[0025] In an embodiment of the present invention, the ratio of the volume of ethylene glycol added to the total volume of the sodium periodate aqueous solution and the dextran aqueous solution is 1:60.

[0026] In one embodiment of the present invention, the addition of ethylene glycol to terminate oxidation can be carried out under stirring conditions; the stirring time can be 1 hour.

[0027] In one embodiment of the present invention, the dialysis can be performed in deionized water; the molecular cutoff of the dialysis can be 3500 Da; and the dialysis time can be 72 h.

[0028] This invention does not impose any particular limitation on the temperature and time of the freeze-drying process; any freeze-drying temperature and time commonly used by those skilled in the art can be employed. In one embodiment of this invention, the freeze-drying temperature is -50 to -30°C, and the time is 48 to 72 hours.

[0029] In one embodiment of the present invention, the solvent in the PIKFYVE kinase inhibitor solution can be dimethyl sulfoxide; the PIKFYVE kinase inhibitor in the PIKFYVE kinase inhibitor solution can be YM201636; the concentration of the PIKFYVE kinase inhibitor solution can be 1~5mM, specifically 1mM, 2mM, 3mM, 4mM or 5mM. Limiting the concentration of the PIKFYVE kinase inhibitor solution to the above range in the present invention is more conducive to the Schiff base reaction.

[0030] In one embodiment of the present invention, the molar ratio of oxidized dextran in the oxidized dextran solution to PIKFYVE kinase inhibitor in the PIKFYVE kinase inhibitor solution can be (1000~100000):1, specifically 1000:1, 5000:1, 10000:1, 20000:1, 40000:1, 50000:1, 75000:1, or 100000:1. By limiting the molar ratio of oxidized dextran in the oxidized dextran solution to PIKFYVE kinase inhibitor in the PIKFYVE kinase inhibitor solution to the above range, the present invention can better control the concentration of PIKFYVE kinase inhibitor in cancer cells.

[0031] The present invention does not have any special limitations on the operation of mixing the oxidized dextran solution with the PIKFYVE kinase inhibitor solution; the mixing methods and parameters commonly used by those skilled in the art can be adopted.

[0032] In one embodiment of the present invention, the temperature of the first Schiff base reaction can be 20-30°C, 22-28°C, or 25°C; the time of the first Schiff base reaction can be 10-20 min or 15 min. Limiting the temperature and time of the first Schiff base reaction to the above ranges allows the reaction to proceed sufficiently.

[0033] After obtaining the mixture, the present invention mixes the mixture with cell membrane fusion liposomes and gelatin solution to carry out a second Schiff base reaction to obtain an injectable hydrogel.

[0034] In this invention, the method for preparing the cell membrane fusion liposome includes: (a) After mixing myristoyl phosphatidylcholine, cholesterol, triptolide and solvent, the mixture was subjected to solvent removal, hydration and first extrusion in sequence to obtain liposomes; (b) The liposomes obtained in step (a) are mixed with the cell membrane of mouse oral squamous cell carcinoma cells and then subjected to a second extrusion to obtain cell membrane fused liposomes.

[0035] This invention involves mixing dimyristic phosphatidylcholine, cholesterol, triptolide, and a solvent, followed by solvent removal, hydration, and a first extrusion to obtain liposomes.

[0036] In one embodiment of the present invention, the molar ratio of myristoyl phosphatidylcholine, cholesterol, and triptolide can be (2~3):1:(0.02~0.3), (2.2~2.8):1:(0.05~0.25), (2.4~2.6):1:(0.1~0.2), or (2.4~2.5):1:(0.1~0.15). The present invention limits the molar ratio of myristoyl phosphatidylcholine, cholesterol, and triptolide to the above range, allowing them to self-assemble into liposomes and encapsulate triptolide within the liposomes, thereby improving the hydrophilicity of triptolide.

[0037] In this invention, the solvent can be an organic solvent. In an embodiment of this invention, the organic solvent is chloroform. This invention does not have a particular limitation on the amount of solvent added, as long as it is sufficient to dissolve dimyristoylphosphatidylcholine, cholesterol, and triptolide.

[0038] In one embodiment of the present invention, the solvent removal method may be vacuum rotary evaporation; the vacuum rotary evaporation is carried out under water bath conditions; the temperature of the vacuum rotary evaporation is 37°C.

[0039] In one embodiment of the present invention, the hydration can be performed by ultrasonic hydration in ultrapure water; the hydration temperature can be 30°C, and the hydration time can be 15~30 min.

[0040] In one embodiment of the present invention, the first extrusion can be repeated by passing the polycarbonate membrane with filter pore sizes of 1000nm, 400nm and 100nm in sequence; the number of extrusions for each filter pore size polycarbonate membrane can be 15 to 30 times.

[0041] After obtaining the liposomes, the present invention mixes the liposomes with the cell membrane of mouse oral squamous cell carcinoma cells and performs a second extrusion to obtain cell membrane fused liposomes.

[0042] In an embodiment of the present invention, the method for preparing the cell membrane of mouse oral squamous cell carcinoma cells is as follows: MOC1 cells in the logarithmic growth phase are collected, cell membrane fragments are extracted using hypotonic lysis and differential centrifugation, and the cells are resuspended and stored at -40°C.

[0043] In an embodiment of the present invention, the mass ratio of the membrane protein to the liposome is 1:500, based on the membrane protein in the cell membrane of mouse oral squamous cell carcinoma cells.

[0044] The present invention does not have any particular limitations on the mixing method and parameters of the liposomes and the cell membrane of mouse oral squamous cell carcinoma cells; any mixing method and parameters commonly used by those skilled in the art can be used.

[0045] In this invention, the operation and parameter selection range of the second extrusion is the same as that of the first extrusion, and will not be described again here.

[0046] In this invention, the drug loading of triptolide in the cell membrane fusion liposome is 5-30%, and can specifically be 7.2%, 14.4%, 21.6% or 28.8%.

[0047] In this invention, the concentration of triptolide in the cell membrane fusion liposome can be 6.9~41.7μM, specifically 10μM, 20μM, 30μM or 40μM. Limiting the drug loading and corresponding concentration of triptolide in the cell membrane fusion liposome to the above range allows for better coordination with PIKFYVE kinase inhibitors for targeting cancer cells, while ensuring the low toxicity of the hydrogel.

[0048] In one embodiment of the present invention, the molar ratio of the cell membrane fusion liposome to the oxidized dextran in the oxidized dextran solution can be 1:(2~100), specifically 1:2, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100. By limiting the molar ratio of the cell membrane fusion liposome to the oxidized dextran in the oxidized dextran solution to the above range, the present invention can better obtain hydrogels that cooperate with PIKFYVE kinase inhibitors for action on cancer cells.

[0049] In one embodiment of the present invention, the molar ratio of gelatin in the gelatin solution to oxidized dextran in the oxidized dextran solution can be 1:(1~3), specifically 1:1, 1:2, or 1:3. Limiting the molar ratio of gelatin in the gelatin solution to oxidized dextran in the oxidized dextran solution to the above range allows for better hydrogel formation.

[0050] In one embodiment of the present invention, the mass ratio of gelatin to solvent in the gelatin solution can be (5~25) g:100 mL, specifically 5 g:100 mL, 10 g:100 mL, 15 g:100 mL, 20 g:100 mL, or 25 g:100 mL; the solvent in the gelatin solution can be ultrapure water. Limiting the concentration of the gelatin solution to the above range in the present invention facilitates the smooth progress of the Schiff base reaction.

[0051] The present invention does not impose any special limitations on the mixing method and parameters of the mixture with cell membrane fused liposomes and gelatin solution; the mixing method and parameters commonly used by those skilled in the art can be used.

[0052] In one embodiment of the present invention, the temperature of the second Schiff base reaction can be 30-40°C, or 35-37°C; the reaction time can be 10-15 min, or 12-13 min. Limiting the temperature and time of the second Schiff base reaction to the above ranges allows for a more complete reaction.

[0053] In this invention, the concentration of cell membrane fusion liposomes in the injectable hydrogel is 1~100 ng / mL, preferably 10~80 ng / mL, more preferably 10~50 ng / mL, and even more preferably 10~20 ng / mL. Limiting the concentration of cell membrane fusion liposomes in the hydrogel to the above range in this invention is compatible with the concentration of triptolide in the cell membrane fusion liposomes, ensuring the low toxicity of the injectable hydrogel.

[0054] This invention utilizes a homologous tumor cell membrane (the cell membrane of mouse oral squamous cell carcinoma MOC1) to prepare cell membrane fusion liposomes (CLip), which completely preserve the homologous adhesion proteins of the MOC1 cell membrane, achieving targeted delivery to oral squamous cell carcinoma cells and significantly improving the intracellular uptake efficiency of triptolide (TPL). Simultaneously, the liposome preparation effectively encapsulates TPL, solving its poor water solubility problem, and exhibits almost no toxicity to normal endothelial cells in vitro, while achieving highly efficient killing of MOC1 tumor cells, thus broadening the therapeutic scope of TPL. The window reduces the risk of systemic toxicity. By utilizing the Schiff base reaction, the aldehyde groups of oxidized dextran can be cross-linked with the amino groups of gelatin to construct a hydrogel network. On the other hand, the PIKFYVE kinase inhibitor is directly coupled to the oxidized dextran backbone via primary amino groups, solving the problem of burst release of small molecule inhibitors. The resulting hydrogel exhibits excellent shear-thinning properties, allowing for smooth injection via a syringe needle. After injection, it rapidly gels in situ at physiological temperatures, adaptively filling irregular gaps in tongue tumors and possessing good tissue adhesion, effectively resisting saliva washout and tongue body erosion. Frequent movement leads to drug loss, significantly prolonging the drug's residence time at the tumor site, increasing local therapeutic concentration, and reducing systemic drug exposure. The injectable hydrogel maintains stable Schiff base bonds and structural integrity under physiological conditions (pH 7.4), resulting in low drug leakage. However, in the slightly acidic environment of the tumor (pH 6.0), Schiff base bonds undergo protonation hydrolysis, leading to gradual hydrogel degradation and the release of CLIP and PIKFYVE kinase inhibitors. By limiting the concentration of CLIP in the injectable hydrogel and the TPL concentration within CLIP, the release of CLIP can be controlled. TPL generates an active N-terminal fragment by specifically cleaving GSDME via Caspase-3, forming pyroptosis pores on the cell membrane. Meanwhile, PIKFYVE kinase inhibitors block ESCRT-III complex-mediated membrane repair by inhibiting PIKFYVE kinase, transforming reversible membrane damage in tumor cells into irreversible lytic pyroptosis. This increases the release of pro-inflammatory factors such as IL-1β and HMGB1, as well as DAMPs, achieving spatiotemporal synergy between pyroptosis induction and membrane repair blockade, amplifying the pyroptosis response while ensuring the safety of normal cells (low toxicity).

[0055] The present invention also provides an injectable hydrogel prepared by the preparation method described in the above technical solution.

[0056] In this invention, the injectable hydrogel undergoes protonation hydrolysis of Schiff base bonds in a tumor microacidic environment (pH 6.0), and the hydrogel is gradually degraded, releasing cell membrane fusion liposomes (CLip) and PIKFYVE kinase inhibitors.

[0057] In one embodiment of the present invention, the concentration of triptolide in the injectable hydrogel can be 1~50 nM, specifically 1 nM, 2 nM, 4 nM, 5 nM, 8 nM, 10 nM, 15 nM, 20 nM, 30 nM, 40 nM, or 50 nM; the concentration of the releaseable PIKFYVE kinase inhibitor in the injectable hydrogel can be 0.1~50 μM, specifically 0.1 μM, 1 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, or 50 μM. By limiting the concentrations of triptolide and the releaseable PIKFYVE kinase inhibitor in the injectable hydrogel to the above ranges, the present invention can reduce systemic toxicity while ensuring antitumor efficacy.

[0058] The present invention also provides the application of the injectable hydrogel described above in the preparation of a therapeutic drug for oral squamous cell carcinoma.

[0059] In one embodiment of the present invention, when the injectable hydrogel is used in the preparation of a therapeutic drug for oral squamous cell carcinoma, the injectable hydrogel can be used directly in the form of an injection; the single injection volume of the injectable hydrogel injection can be 10~40μL.

[0060] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0061] Example 1 A method for preparing an injectable hydrogel comprises the following steps: (1) An oxidized dextran solution (the mass ratio of oxidized dextran to the volume of deionized water is 1 g: 100 mL) is mixed with a PIKFYVE kinase inhibitor solution (YM201636, 5 mM) and subjected to a first Schiff base reaction (25 °C, 15 min) to obtain a mixture; the molar ratio of oxidized dextran in the oxidized dextran solution to that of the PIKFYVE kinase inhibitor solution is 10000:1; the oxidized dextran is prepared by: mixing 20 g of oxidized dextran with 100 mL of deionized water. 0.1 g / mL sodium periodate aqueous solution was added (at a rate of 4 mL / min) to 10 mL dextran aqueous solution (at a concentration of 0.1 g / mL). The oxidation reaction was carried out under stirring and light-protected conditions (25 °C, 6 h). Then, 500 μL of ethylene glycol was added and stirred for 1 h to quench the reaction. After that, the mixture was dialyzed in deionized water (molecular cutoff of 3500 Da, dialyzing time of 72 h) and freeze-dried (freeze-drying temperature of -40 °C, time of 60 h) to obtain oxidized dextran. (2) The mixture obtained in step (1) is mixed with cell membrane fusion liposomes and gelatin solution (the mass ratio of gelatin to the volume ratio of ultrapure water is 12g:100mL) and subjected to a second Schiff base reaction (37℃, 10min) to obtain an injectable hydrogel (denoted as GOCY); the molar ratio of the cell membrane fusion liposomes to the oxidized dextran in the oxidized dextran solution in step (1) is 1:50; the molar ratio of the gelatin in the gelatin solution to the oxidized dextran in the oxidized dextran solution is 1:2; the concentration of cell membrane fusion liposomes in the injectable hydrogel is 10ng / mL; The method for preparing cell membrane fusion liposomes in step (2) includes: (a) Dimyristic phosphatidylcholine, cholesterol, and triptolide were mixed in a molar ratio of 2.3:1:0.1 with a solvent (chloroform, with a volume ratio of chloroform to the total molar amount of dimyristic phosphatidylcholine, cholesterol, and triptolide of 5 mL:5 mol). The solvent was removed by rotary evaporation under reduced pressure in a water bath at 37°C. The mixture was then ultrasonically hydrated in ultrapure water at 30°C for 20 min. The mixture was then repeatedly extruded through polycarbonate membranes with pore sizes of 1000 nm, 400 nm, and 100 nm, with each pore size being extruded 20 times to obtain liposomes (denoted as Lip). (b) Based on the membrane proteins in the cell membrane of mouse oral squamous cell carcinoma cells (denoted as Cm), the membrane proteins were mixed with the liposomes obtained in step (1) at a mass ratio of 1:500, and then repeatedly extruded through polycarbonate membranes with filter pore sizes of 1000 nm, 400 nm and 100 nm. The extrusion times for each filter pore size polycarbonate membrane were 20 times to obtain cell membrane fusion liposomes (denoted as CLIp). The drug loading of triptolide in the cell membrane fusion liposomes was 14.4% (corresponding to a concentration of 20 μM). The concentration of triptolide in the injectable hydrogel prepared in Example 1 was 4 nM, and the concentration of the releaseable PIKFYVE kinase inhibitor was 1 μM.

[0062] Comparative Example 1 A method for preparing an injectable hydrogel includes: mixing an oxidized dextran solution (the mass ratio of oxidized dextran to the volume of deionized water is 1 g: 100 mL) with a gelatin solution (the mass ratio of gelatin to the volume of ultrapure water is 12 g: 100 mL) and performing a Schiff base reaction (37 °C, 10 min) to obtain an injectable hydrogel (denoted as GO); the molar ratio of gelatin in the gelatin solution to oxidized dextran in the oxidized dextran solution is 1:2. The preparation of the oxidized dextran is the same as in Example 1.

[0063] Comparative Example 2 A method for preparing an injectable hydrogel comprises the following steps: (1) An oxidized dextran solution (the mass ratio of oxidized dextran to the volume of deionized water is 1 g: 100 mL) is mixed with cell membrane fusion liposomes and gelatin solution (the mass ratio of gelatin to the volume of ultrapure water is 12 g: 100 mL) and subjected to a Schiff base reaction (37 °C, 10 min) to obtain an injectable hydrogel (denoted as GOC); the molar ratio of the cell membrane fusion liposomes to the oxidized dextran in the oxidized dextran solution in step (1) is 1:50; the molar ratio of the gelatin in the gelatin solution to the oxidized dextran in the oxidized dextran solution is 1:2; the concentration of the cell membrane fusion liposomes in the injectable hydrogel is 10 ng / mL; The preparation method of the oxidized dextran and cell membrane fused liposomes is the same as in Example 1. The concentration of triptolide in the injectable hydrogel prepared in Comparative Example 1 was 4 nM.

[0064] Test case (a) Physicochemical and biological properties (modified protein) testing of CLIp and GOCY The particle size and effective surface charge of the cell membrane fusion liposomes prepared in Example 1 were tested. The dynamic light scattering (DLS) image of the cell membrane fusion liposomes prepared in Example 1 is shown below. Figure 1 As shown, the Zeta potential diagram is as follows: Figure 2 As shown.

[0065] from Figure 1 and Figure 2 It can be seen that the hydrated particle size of CLIp is approximately 140.7 nm, and the Zeta potential is approximately -17.53 mV.

[0066] The protein profile of CLIp was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Pure MOC1 cell membranes (Cm) were harvested by centrifugation at 15000g for 20 min. The precipitate was then resuspended in 20 µL of RIPA lysis buffer and lysed on ice for 30 min. After centrifugation at 15000g for 20 min, the precipitate was discarded. 5× (5-fold dilution, final concentration 1×) loading buffer was added to the protein-containing supernatant, and the mixture was boiled at 95°C for 10 min. After natural cooling, Cm and CLIp from Example 1 were loaded onto a 12% sodium dodecyl sulfate-polyacrylamide gel at an equal protein concentration of 1 μg / well. Lip served as a protein-free negative control. Electrophoresis was performed at 120V for 60 min, stopping when the blue blot reached the bottom of the gel. The gel was then stained with a rapid silver staining kit, and the protein bands on the gel were observed by photography. The SDS-PAGE gel electrophoresis images of Lip, Cm, and CLIp are shown below. Figure 3 As shown.

[0067] from Figure 3 It can be seen that CLIp completely preserved all the characteristic protein bands of the MOC1 cell membrane, confirming that the membrane proteins were not degraded or lost during the preparation process.

[0068] The injectable hydrogel prepared in Example 1 was freeze-dried and observed using a scanning electron microscope. The SEM image of the freeze-dried injectable hydrogel is shown below. Figure 4 As shown.

[0069] from Figure 4 It can be seen that the freeze-dried injectable hydrogel exhibits a typical three-dimensional porous network structure.

[0070] (II) Cytotoxicity detection MOC1 cells were cultured in IMDM / F12 medium with 10% FBS and 1% antibiotics at 37°C in a 5% CO2 incubator; human umbilical vein endothelial cells (HUVECs) were cultured in DMEM complete medium.

[0071] The killing ability of each drug-treated group against MOC1 cells was determined using a CCK-8 assay kit: MOC1 cells were seeded into 96-well plates and cultured for 24 h. The old culture medium was then discarded, and different concentration gradients of TPL, Lip, and CLIp (equivalent to TPL concentrations of 0.1 nM, 0.2 nM, 0.5 nM, 1.0 nM, 1.5 nM, 2.0 nM, 4.0 nM, and 8.0 nM) were added. A cell-free blank control group and a positive control group with drug-free culture medium were also included. After 24 h of culture, the drug-containing culture medium was aspirated, and 100 μL of 10% CCK-8 solution was added to each well. Air bubbles were avoided during the process. The 96-well plates were incubated at 37°C in the dark for 1 h, with continuous observation. Once the culture medium turned orange-yellow, the optical density (OD) of each well was measured at 450 nm using a microplate reader. To verify the biosafety of TPL loaded onto liposomes, HUVECs were used as a normal cell model, and the procedure was the same as above. The bar chart shows the effect of different drug administration groups (different concentrations of TPL, Lip, and CLIp) on the viability of MOC1 cells. Figure 5 As shown in the bar chart, the effects of different drug administration groups (different concentrations of TPL, Lip, and CLIp) on the viability of HUVEC cells are as follows: Figure 6 As shown, where This means p < 0.05. This means p < 0.01. This means p < 0.001. This means p < 0.0001.

[0072] from Figure 5 and Figure 6 It can be seen that free TPL exhibits strong non-specific toxicity to both MOC1 and HUVEC, with HUVEC survival rate <5% at a concentration of 4 nM; while CLIp at a concentration of 4 nM showed a killing rate of >70% for MOC1 cells and a survival rate of >85% for HUVEC cells, indicating that loading TPL onto liposomes can improve the therapeutic window of TPL and increase the therapeutic concentration of TPL to 4 nM, confirming that CLIp has targeted killing ability and excellent biocompatibility.

[0073] (III) Observation of pyroptosis morphology and detection of related proteins MOC1 cells were seeded into 6-well plates. After attachment, Lip, CLIp, and CLIp+YM201636 (labeled CLIp+YM, containing 4 nM TPL and 1 μM YM201636) were added for 24 h, respectively. Culture medium alone served as the control group. The cells were observed under an inverted microscope (scale bar: 10 μm) in bright field. The morphological images of the obtained MOC1 cells are shown below. Figure 7 As shown.

[0074] from Figure 7 It can be seen that the untreated Control group MOC1 cells adhered to the wall in a spindle or polygonal shape with uniform cytoplasm; while Lip or CLIp treatment caused some cells to become rounded and detach, and bubbles characteristic of pyroptosis could be observed on the cell membrane surface (red arrows); and the most obvious cell morphological changes were observed in the CLIp+YM combined treatment group, with cells significantly increased in volume, becoming spherical, and forming the largest number of pyroptosis bubbles.

[0075] Centrifuge and vortex the desired protein and protein markers. Load 7 μL of sample into each well, add electrophoresis buffer, and adjust the voltage to 120 V. Once the bromophenol blue reaches the bottom of the gel, electrophoresis is complete. Pre-activate the polyvinylidene fluoride (PVDF) membrane by soaking it in methanol for 1-2 minutes. Remove the gel, assemble the sandwich structure, ensuring no air bubbles between the gel and membrane, and place it in the transfer tank. Add pre-cooled transfer buffer and transfer at a constant current of 230 mA for 60 minutes under ice bath conditions. After transfer, remove the PVDF membrane and desorb it using 5% TBST desaturation solution. The membrane was blocked with milk powder at room temperature for 1.5 h, then washed three times with TBST. Primary antibody dilution buffer for the relevant protein was added, and the membrane was incubated overnight at 4°C. The next day, the primary antibody was recovered, and the membrane was washed three times with TBST for 15 min each time. HRP-labeled goat anti-rabbit secondary antibody (1:10000) was added, and the membrane was incubated at room temperature for 1 h. The membrane was washed three times with TBST again. ECL ultrasensitive luminescent substrate solution (a 1:1 mixture of solution A and solution B) was uniformly added to the PVDF membrane surface. The membrane was developed and photographed using a chemiluminescence system. The expression of pyroptosis-related proteins in MOC1 cells is shown in the figure below. Figure 8 As shown.

[0076] from Figure 8 It can be seen that the CLIp group significantly upregulated the expression of Cle-Caspase-3 and GSDME-N; while in the CLIp+YM201636 combination group, the expression of Cle-Caspase-3 and GSDME-N was further increased, confirming that YM201636 successfully blocked ESCRT-mediated membrane repair and amplified the pyroptosis signal.

[0077] (iv) Detection of ROS and mitochondrial membrane potential Cells were seeded in confocal microscopy dishes. After drug treatment, the culture medium was aspirated, and serum-free medium diluted with the reactive oxygen species probe DCFH-DA (final concentration 10 μM) was added. The cells were incubated at 37°C for 30 min, washed with PBS, and observed using a laser confocal microscope (CLSM) with excitation wavelength of 488 nm and emission wavelength of 525 nm. Fluorescence images of MOC1 cells after different treatments are shown below. Figure 9 As shown.

[0078] For quantitative analysis by flow cytometry, MOC1 cells were seeded in 6-well plates. After treatment with the drugs in each group, the cells were digested and collected. The cells were then incubated with the DCFH-DA probe for 30 min, washed, resuspended, and analyzed by flow cytometry. The average fluorescence intensity of the FITC channel was recorded and statistically analyzed. The fluorescence intensity distribution of ROS in MOC1 cells after different treatments is shown in the figure below. Figure 10 As shown in the figure, the average fluorescence intensity bar chart is as follows: Figure 11 As shown in Table 1, the average fluorescence intensity of ROS in MOC1 cells after different treatments is presented in Table 1.

[0079] Table 1. Data on the mean ROS fluorescence intensity in MOC1 cells after different treatments.

[0080] from Figures 9-11 As can be seen, the CLIp+YM group cells were filled with bright green fluorescence, while the control group showed extremely weak fluorescence, and the Lip and CLIp groups showed only a small amount of fluorescence. Flow cytometry results and statistical analysis showed that the mean fluorescence intensity (MFI) of ROS in the control group was approximately 1400, which increased to around 4800 after CLIp treatment, while the value in the CLIp+YM group reached as high as 8000. The generation of ROS confirmed that CLIp successfully delivered TPL and played a role. The addition of YM201636 further increased the ROS level.

[0081] For mitochondrial membrane potential detection, MOC1 cells were seeded in confocal dishes. After drug treatment, the supernatant was discarded, JC-1 staining working solution was added, and the cells were mixed and incubated at 37°C for 20 min. The staining solution was then removed, and the cells were washed twice with the JC-1 staining buffer provided in the kit. Imaging was performed using a CLSM scanner, acquiring red and green channels separately. Confocal images of mitochondria in MOC1 cells after different treatments are shown below. Figure 12 As shown.

[0082] Since excessive ROS production typically disrupts mitochondrial integrity, we assessed mitochondrial membrane potential using JC-1 staining. Figure 12As can be seen, the Control group exhibited bright red fluorescence, indicating intact mitochondrial function. After treatment, compared to Lip, the CLIp group showed overlapping red and green fluorescence, indicating damaged mitochondria. In the CLIp+YM group, the red fluorescence almost disappeared, showing obvious green fluorescence, proving that it induced the strongest mitochondrial damage. This complete red-to-green phenomenon indicates that the mitochondrial membrane potential was reversed, which is a result of ROS accumulation.

[0083] (v) Detection of the release of pro-inflammatory cytokines The cell membrane rupture rate was detected using a lactate dehydrogenase (LDH) kit, and the concentrations of interleukin-1β (IL-1β) and high-mobility group box 1 (HMGB1) in the cell supernatant were detected using an ELISA kit. The LDH release bar charts obtained after different treatments (control group, Lip group, CLIp group, and CLIp+YM201636 group) are shown below. Figure 13 As shown in Table 2; the IL-1β release histogram is shown in... Figure 14 As shown in Table 3; the HMGB1 release histogram is shown in... Figure 15 As shown in Table 4, the data is as follows.

[0084] Table 2 LDH release data after different treatments

[0085] Table 3. IL-1β release data after different treatments

[0086] Table 4 HMGB1 release data after different treatments

[0087] From Tables 2-4 and Figures 13-15 It can be seen that the LDH release rate in the CLIp+YM201636 combination group was as high as 80%, and the concentrations of IL-1β and HMGB1 were more than twice that of the CLIp group, confirming that membrane repair blockade significantly amplified the release of pyroptosis-mediated pro-inflammatory factors and DAMPs.

[0088] (vi) Transcriptomics analysis Cells were divided into three groups: Control group, CLIp group, and a combined treatment group (CLip+YM). Old culture medium was discarded, and cells were washed with pre-chilled sterile PBS buffer. Then, 1 mL of pre-chilled Trizol reagent was added to each well, and the cells were repeatedly pipetted on ice until no visible cell clumps were observed. The cells were incubated at room temperature for 5 min. The lysis buffer was transferred to RNase-free 1.5 mL EP tubes, and 200 μL of chloroform was added to each tube. The tubes were vigorously shaken for 15–30 s and incubated at room temperature for 5 min. Subsequently, the cells were centrifuged at 12000 g for 15 min at 4 °C. The supernatant was carefully aspirated and transferred to a new EP tube. An equal volume of pre-chilled isopropanol was added, and the cells were mixed by inverting. The cells were centrifuged at 12000 g for 10 min, and the supernatant was discarded. RNA was washed with 75% ethanol prepared with DEPC water. RNA-seq sequencing and enrichment analysis were performed. The GO enrichment analysis of differentially expressed genes in Control and CLIp cells is shown in the figure below. Figure 16 As shown in the figure, the GO enrichment analysis diagram of differentially expressed genes in CLIp and CLIp+YM is as follows. Figure 17 As shown in the figure, the KEGG enrichment analysis of differentially expressed genes in Control and CLIp is as follows. Figure 18 As shown in the figure, the KEGG enrichment analysis diagram of differentially expressed genes in CLIp and CLIp+YM is as follows. Figure 19 As shown.

[0089] from Figures 16-19 It can be seen that CLIp treatment significantly enriched inflammatory response, innate immune activation, and pyroptosis-related pathways; the CLIp+YM201636 combination group significantly downregulated membrane repair and autophagy regulation-related pathways, and further enriched immune activation and antigen presentation-related pathways, confirming the pyroptosis amplification and immune activation effects of the combination strategy at the gene level.

[0090] (vii) Macrophage repolarization experiment RAW264.7 cells were loaded at a rate of 2 × 10⁻⁶. 5 RAW264.7 cells were seeded at a density of 2 mL / well in 6-well plates. After adhesion, the old culture medium was discarded and replaced with complete culture medium containing recombinant mouse IL-4 (final concentration 10 ng / mL). The plates were then incubated at 37°C for 24 h to induce RAW264.7 cells to polarize into M2 macrophages with an immunosuppressive phenotype. The polarized M2 macrophages were then re-inoculated at a density of 2 × 10⁻⁶ cells / well. 5 Seeds were planted at a density of cells / mL into new 6-well plates and cultured overnight to allow adhesion. The old culture medium was discarded, and the cells were gently washed twice with PBS. Subsequently, the MOC1 pyroptosis medium from each group was added to the corresponding M2 macrophage wells and co-incubated for 24 h to evaluate the ability of different groups of MOC1-treated pyroptosis-releasing factors to induce macrophage repolarization from M2 to M1.

[0091] The repolarization status of RAW264.7 cells was quantitatively analyzed by detecting the M1-type macrophage marker CD86 and the M2-type macrophage marker CD206 on the macrophage surface using flow cytometry. The expression changes of the M2-type macrophage marker gene (Arg-1) and the M1-type macrophage marker gene (Tnf-a) were detected using RT-qPCR. The expression of CD86 under different treatments was also analyzed. + In F4 / 80 + The flow cytometry results are shown in the figure below. Figure 20 As shown; CD86 after different processing + Cells at F4 / 80 + A proportional bar chart of cells as shown Figure 21 As shown in Table 5, the data are presented in the bar charts for the relative expression levels of the Arg-1 gene after different treatments. Figure 22 As shown in Table 6, the data are presented in the bar charts for the relative expression levels of the Tnf-a gene after different treatments. Figure 23 As shown in Table 7, the data is as follows.

[0092] Table 5 CD86 after different treatments + Cells at F4 / 80 + Proportional data in cells

[0093] Table 6. Relative expression levels of the Arg-1 gene after different treatments.

[0094] Table 7. Relative expression levels of the Tnf-a gene after different treatments.

[0095] From Tables 5-7 and Figures 20-23 It can be seen that the CLIp+YM201636 combination group increased the CD86 positivity rate from 7.35% to 34.1%, downregulated Arg-1 expression by 80%, and upregulated Tnf-α expression by nearly 20-fold, confirming that the amplified pyroptosis response can efficiently drive M2 macrophages to repolarize to M1.

[0096] (viii) Dendritic cell maturation experiment Bone marrow cells were isolated from the femur and tibia of C57BL / 6 mice and induced to differentiate into immature BMDCs by GM-CSF (20 ng / mL) and IL-4 (10 ng / mL). After incubation for 24 h with conditioned medium containing pretreated MOC1 cells, cell morphology was observed using laser confocal microscopy, and CD11c was detected by flow cytometry. + CD80 in cells + CD86 + The proportion of double-positive mature DCs. CD11c after different treatments.+ CD80 in cells + CD86 + Double-positive maturation result image as shown Figure 24 As shown; CD11c after different treatments + CD80 in cells + CD86 + The proportion of double-positive mature DCs is shown in the bar chart. Figure 25 As shown in Table 8, the data is as follows.

[0097] Table 8. CD11c after different treatments + CD80 in cells + CD86 + Proportion of double-positive mature DCs

[0098] from Figures 24-25 As shown in Table 8, the combined conditioned medium can induce BMDC to form characteristic dendritic protrusions, CD80 + CD86 + The proportion of double-positive mature DCs increased from 11.0% to 49.1%, confirming that the amplified pyroptosis response can effectively promote DC cell maturation and lay the foundation for subsequent T cell activation.

[0099] (ix) Evaluation of anti-tumor effects MOC1-Luc cells stably expressing luciferase were constructed. MOC1-Luc cells in the logarithmic growth phase were collected, trypsinized, washed once with pre-chilled PBS, and resuspended in sterile HBSS buffer to adjust the final cell concentration to 4 × 10⁶ cells / year. 7 The concentration of insulin was 1 × 10⁻⁶ / mL, and it was kept on ice for later use. Mice were anesthetized by inhalation gas using an isoflurane anesthesia machine. Once the mice's breathing slowed, they were promptly removed. The tongue of the mouse was carefully pulled out using ophthalmic forceps, and insulin was slowly administered using an insulin syringe containing 1 × 10⁻⁶ / mL. 6 25 μL of MOC1-Luc cell suspension was precisely injected into one-third of the tongue of a mouse. After injection, the needle was left in place for 5-10 seconds before being slowly withdrawn to prevent cell fluid leakage, thus obtaining a model mouse (orthotopic tongue squamous cell carcinoma mouse). The model mice were randomly divided into 4 groups: Control group (physiological saline), GO group (comparative example 1), GOC group (comparative example 2, TPL concentration in hydrogel was 4 nM), and GOCY group (Example 1, TPL concentration in hydrogel was 4 nM, YM201636 concentration was 1 μM). Intratumoral injection of 20 μL was performed on days 0, 5, 9, 13, and 17.

[0100] Mice were intraperitoneally injected with D-Luciferin substrate on days 0, 5, 9, 13, 17, and 21. Seven minutes later, the mice were anesthetized with isoflurane. Bioluminescence images and corresponding average fluorescence intensity (p / s / cm) of the tumor sites were acquired and quantified using an in vivo bioluminescence system. 2 / sr), and plotted curves. Mouse body weight and tongue tumor morphology were recorded every 2 days; mouse survival was also recorded, and survival curves were plotted. Bioluminescence images of mice with orthotopic tongue squamous cell carcinoma after different treatments are shown below. Figure 26 As shown, representative images of tongue tumors in mice from different treatment groups are as follows: Figure 27 As shown in the figure, the weight change curves of mice with orthotopic tongue squamous cell carcinoma during different treatment cycles are as follows. Figure 28 As shown in the figure, the survival curves of mice with orthotopic tongue squamous cell carcinoma during different treatment cycles are as follows. Figure 29 As shown.

[0101] from Figures 26-29 It can be seen that the tumor fluorescence signal in the Control group and GO group continued to increase, and a huge tumor was visible on the tongue. The weight of the mice continued to decrease. The tumor growth in the GOC group was partially inhibited, and the 30-day survival rate was 40%. The tumor fluorescence signal in the GOCY group almost disappeared completely, the tongue morphology basically returned to normal, the weight of the mice was maintained at 19~20g, and the 30-day survival rate was as high as 80%, which confirms that GOCY hydrogel has excellent in vivo tumor inhibition effect.

[0102] (x) Molecular detection of tumor tissue After the treatment cycle was completed, the mice were euthanized, and about 20 mg of tumor tissue was taken from each group. The tissue was immediately placed in liquid nitrogen for 1 min to freeze and then ground into a fine powder. To prevent the powder from becoming wet and sticky, it was immediately transferred to a pre-cooled centrifuge tube, and 100 μL of RIPA lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor was added. The mixture was vortexed and lysed on ice for 30 min to extract the protein for Western blotting analysis.

[0103] At the end of the experiment, whole blood was collected from mice in different groups by cardiac puncture. After coagulation at room temperature, the blood was centrifuged at 12,000 rpm for 20 min at 4°C. The upper light yellow serum was separated. The ELISA kits for mouse IL-1β and HMGB1 were tested according to standard procedures. The OD values ​​were measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The concentrations of pro-inflammatory cytokines IL-1β and HMGB1 in the serum of each group of mice were calculated based on the standard curve.

[0104] Western blot results of related proteins in mouse tumor tissues after different treatments are shown in the figure below. Figure 30 As shown in the bar chart; the changes in serum IL-1β concentration after different treatments are shown in the following figure. Figure 31As shown in Table 9, the data are presented in the bar chart showing the changes in serum HMGB1 concentration after different treatments. Figure 32 As shown in Table 10.

[0105] Table 9. Data on changes in serum IL-1β concentration after different treatments.

[0106] Table 10. Data on changes in serum HMGB1 concentration after different treatments.

[0107] from Figures 30-32 As shown in Tables 9-10, Western blotting confirmed that Cle-Caspase-3 and GSDME-N were significantly overexpressed in tumor tissues after GOCY treatment. CHMP-4B, a key protein of the ESCRT-III component responsible for membrane repair, showed a compensatory increase after GOC treatment. However, in the GOCY group containing YM201636, its expression was inhibited, verifying its ability to inhibit membrane repair. Serum concentrations of IL-1β and HMGB1 were significantly increased.

[0108] (xi) Detection of immune microenvironment remodeling After treatment, multiple tissues (tumor tissue, tumor draining lymph nodes TDLNs, and peripheral blood) were extracted from mice in each group, and immune cells were measured using multicolor flow cytometry.

[0109] Tumor tissue: 500 μL of 10 mg / mL collagenase IV and 100 μL of 5 mg / mL deoxyribonuclease I were added to 4.4 mL of HBSS buffer to prepare a 5 mL digestion solution containing 1 mg / mL collagenase IV and 100 μg / mL DNase I. The tumor mass was added to the digestion solution and digested on a shaker at 37°C for 30 min. After digestion, the digestion solution was filtered through a 70 μm cell sieve, and the visible tumor tissue was ground with the end of a sterile syringe to obtain a single-cell suspension. Simultaneously, CD8 immunofluorescence staining was performed on the tumor tissue.

[0110] Lymph nodes: Tumor draining lymph nodes (TDLNs) in the neck were taken and placed directly into a 70μm cell sieve. FC staining buffer was added and the cells were ground through the handle of a sterile syringe. FC staining buffer was added multiple times during the process to collect single cells.

[0111] Peripheral blood: Collect peripheral blood from mice in EDTA anticoagulant tubes and shake thoroughly to prevent clotting. Add 100 μL of whole blood to 900 μL of erythrocyte lysis buffer, lyse on ice for 20 min, add PBS buffer to stop lysis, centrifuge and discard the supernatant; the cell pellet at the bottom should contain no erythrocytes.

[0112] Experimental results were obtained using a flow cytometer after antibody staining by flow cytometry, with voltage and compensation adjusted. FlowJo software was used for gating and data analysis.

[0113] Flow cytometry results of dendritic cells in tumor draining lymph nodes (TDLNs) after different treatments are shown in the figure below. Figure 33 As shown in the bar chart of maturity ratio, Figure 34 As shown in Table 11, the data represent the CD8 levels after different treatments. + The flow cytometry results of T cells are shown in the figure below. Figure 35 As shown, the scale bar chart is as follows: Figure 36 As shown in Table 12, the data show the CD8 levels in peripheral blood (PB) after different treatments. + The flow cytometry results of T cells are shown in the figure below. Figure 37 As shown, the scale bar chart is as follows: Figure 38 As shown in Table 13; flow cytometry results of M2-like macrophages in tumor tissues after different treatments are shown in the figure. Figure 39 As shown, the scale bar chart is as follows: Figure 40 As shown in Table 14, the data are presented; flow cytometry results of regulatory T cells in tumor tissue after different treatments are shown in the figure. Figure 41 As shown, the scale bar chart is as follows: Figure 42 As shown in Table 15, the data are as follows; CD8 immunofluorescence images of tumor tissues after different treatments are shown in Table 15. Figure 43 As shown in the figure, the fluorescence value bar chart is as follows: Figure 44 As shown in Table 16.

[0114] Table 11. Data on the maturation rate of dendritic cells in tumor draining lymph nodes (TDLNs) after different treatments.

[0115] Table 12 CD8 after different treatments + T cell proportion data

[0116] Table 13 CD8 levels in peripheral blood (PB) after different treatments + T cell proportion data

[0117] Table 14. Proportion of M2-like macrophages in tumor tissues after different treatments.

[0118] Table 15. Proportion of regulatory T cells in tumor tissue after different treatments.

[0119] Table 16 CD8 immunofluorescence values ​​of tumor tissues after different treatments

[0120] from Figures 33-44 As shown in Tables 11-16, the DC maturity rate in TDLNs of the GOCY group increased to 46.4%, and CD8... + The proportion of T cells increased to 38.0%; CD8+ in peripheral blood... + The proportion of T cells was significantly increased; the proportion of M2 macrophages in tumor tissue decreased to 17.7%, and the proportion of Tregs decreased to 3.93%; CD8 immunofluorescence showed that CD8 in the tumor tissue of the GOCY group was significantly increased. + The T-cell infiltration rate was more than ten times higher than that of the control group, confirming the amplification effect of pyroptosis and a significant enhancement of T-cell infiltration in vivo. These results demonstrate that GOCY hydrogel achieves a deep remodeling of the tumor immunosuppressive microenvironment.

[0121] (xii) Safety assessment After treatment, important organs of mice, including the heart, liver, spleen, lungs, and kidneys, were stained with hematoxylin and eosin (H&E). Mouse serum was collected, and liver function indicators ALT (alanine aminotransferase) and AST (aspartate aminotransferase), and kidney function indicators UREA (urea) and CREA (creatinine) were detected by a fully automated biochemical analyzer.

[0122] H&E staining images of major organs after different treatments are shown below. Figure 45 As shown in the bar chart; the levels of the liver function marker alanine aminotransferase (ALT) after different treatments are shown in the figure. Figure 46 As shown in Table 17, the data are presented in the bar charts of liver function marker AST levels after different treatments. Figure 47 As shown in Table 18, the data are presented in the bar charts for creatinine levels, a renal function marker, after different treatments. Figure 48 As shown in Table 19, the data are presented in the bar charts for urea levels, a renal function marker, after different treatments. Figure 49 As shown in Table 20.

[0123] Table 17 Data on liver function markers, specifically alanine aminotransferase (ALT) levels, after different treatments.

[0124] Table 18 Data on liver function markers, specifically aspartate aminotransferase (AST) levels, after different treatments.

[0125] Table 19 Data on renal function markers, specifically creatinine levels, after different treatments.

[0126] Table 20 Data on renal function markers, specifically urea levels, after different treatments.

[0127] from Figures 45-49 As shown in Tables 17-20, no obvious pathological damage was observed in the important organs of the GOCY group mice, the tissue structure was intact, and there were no abnormalities such as inflammatory cell infiltration or necrosis. Serum ALT, AST, UREA, and CREA were all maintained within the normal physiological range and there was no significant difference from the control group, which confirms that GOCY hydrogel has no obvious liver and kidney toxicity and systemic toxicity and has excellent in vivo biological safety.

[0128] The injectable hydrogel prepared by the method provided in this invention contains CLIp and PIKFYVE kinase inhibitors, which, through in vitro and in vivo experiments, have been shown to effectively promote the maturation of DC cells in tumor-draining lymph nodes, drive M2 macrophages to repolarize into anti-tumor M1 macrophages, significantly increase the proportion and infiltration of CD8+ effector T cells in peripheral blood and tumor tissue, and effectively reduce the proportion of Tregs in the tumor microenvironment, successfully reversing the immunosuppressive microenvironment of OSCC and achieving a long-lasting anti-tumor immune response. In a mouse orthotopic oral squamous cell carcinoma model, the injectable hydrogel achieved near-complete tumor regression, maintained the feeding function of mice, effectively improved weight loss, and stabilized the 30-day survival rate of mice to 80%. At the same time, no pathological damage was observed in the H&E staining of important organs of mice, and serum liver and kidney function indicators (ALT, AST, UREA, CREA) were maintained within the normal physiological range. There was no obvious systemic toxicity, and it has good water solubility, low toxicity, and long-lasting anti-tumor effects.

[0129] 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 an injectable hydrogel, comprising the following steps: (1) Mix the oxidized dextran solution with the PIKFYVE kinase inhibitor solution and carry out the first Schiff base reaction to obtain a mixture; (2) The mixture obtained in step (1) is mixed with cell membrane fusion liposomes and gelatin solution to carry out a second Schiff base reaction to obtain an injectable hydrogel; the concentration of cell membrane fusion liposomes in the injectable hydrogel is 1~100 ng / mL; The method for preparing cell membrane fusion liposomes in step (2) includes: (a) After mixing myristoyl phosphatidylcholine, cholesterol, triptolide and solvent, the mixture was subjected to solvent removal, hydration and first extrusion in sequence to obtain liposomes; (b) The liposomes obtained in step (a) are mixed with the cell membrane of mouse oral squamous cell carcinoma cells and then subjected to a second extrusion to obtain cell membrane fusion liposomes; the drug loading of triptolide in the cell membrane fusion liposomes is 5-30%.

2. The production method according to claim 1, characterized by, In step (1), the molar ratio of oxidized dextran in the oxidized dextran solution to PIKFYVE kinase inhibitor in the PIKFYVE kinase inhibitor solution is (1000~100000):

1.

3. The preparation method according to claim 2, characterized in that, In step (1), the ratio of the mass of oxidized dextran to the volume of the solvent in the oxidized dextran solution is (0.5~5) g: 100 mL.

4. The production method according to claim 3, characterized by, The concentration of the PIKFYVE kinase inhibitor solution is 1~5mM.

5. The preparation method according to claim 2, characterized in that, The molar ratio of the cell membrane fusion liposome in step (2) to the oxidized dextran in the oxidized dextran solution in step (1) is 1:(2~100).

6. The preparation method according to claim 5, characterized in that, The molar ratio of gelatin in the gelatin solution in step (2) to oxidized dextran in the oxidized dextran solution in step (1) is 1:(1~3).

7. The preparation method according to claim 6, characterized in that, In step (2), the ratio of the mass of gelatin to the volume of solvent in the gelatin solution is (5~25) g: 100 mL.

8. The preparation method according to claim 1, characterized in that, In step (a), the molar ratio of dimyristoylphosphatidylcholine, cholesterol, and triptolide is (2~3):1:(0.02~0.3).

9. The injectable hydrogel prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the injectable hydrogel of claim 9 in the preparation of a therapeutic agent for oral squamous cell carcinoma.

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