Injectable nano composite hydrogel as well as preparation method and application thereof

Abstract

The invention discloses injectable nano-composite hydrogel as well as a preparation method and application of the injectable nano-composite hydrogel. The injectable nano-composite hydrogel at least comprises hydrogel, a free drug NHWD-870 containing cis-platinum loaded MnO2 and a liposome Cis / Mn-coated Lipo-IL11, wherein the liposome Cis / Mn-coated Lipo-IL11 is composed of il11r alpha targeting lipid particles; the liposome Cis / Mn (at) Lipo-IL11 is encapsulated in the hydrogel, so that the injectable nano composite hydrogel Cis / Mn / NH (at) PGP is formed. The injectable nano composite hydrogel can simultaneously deliver an effective MYC inhibitor (NHWD-870) and a targeted il11r alpha liposome (Cis / Mn at Lipo-IL11) containing cis-platinum loaded MnO2, and after in-situ administration, the NHWD-870 effectively degrades MYC, down-regulates CCL2 and IL13 cell factors, and triggers M1 type activation of macrophages. Meanwhile, Cis / Mn-coated Lipo-IL11 is delivered in a targeted mode to react with excessive intratumor GSH to generate Mn < 2 + >, generation of excessive reactive oxygen is induced through a fenton-like reaction, the Cis / Mn-coated Lipo-IL11 and cis-platinum induce death of immunogenic cells together, and maturation of the dendritic cells is promoted. Through cooperative regulation of MYC and ICD levels, an immune microenvironment is remodeled, immune infiltration is enhanced, and the compound can be used for treating MYC amplification type osteosarcoma.

Description

Technical Field

[0001] The present invention relates to the technical field of new biological materials, and in particular to an injectable nanocomposite hydrogel, a preparation method and application thereof. Background Art

[0002] Osteosarcoma (OS) is one of the most common primary malignant tumors in childhood and poses a significant threat to adolescent health. Over the past 40 years, the 5-year survival rate for OS has not substantially improved. This is primarily due to the high heterogeneity, invasiveness, and metastatic nature of OS, as well as the limited effectiveness of conventional radiotherapy and chemotherapy for OS. Multiple lines of evidence suggest that the oncogene MYC promotes the development, progression, and metastasis of various malignancies, including glioblastoma, triple-negative breast cancer, and OS. Furthermore, MYC can influence the tumor immune microenvironment through multiple pathways. Previous studies have reported that MYC amplification occurs in nearly a quarter of OS patients. MYC-amplified osteosarcoma has been identified as the most malignant subtype, with a 5-year survival rate of less than 40%. These tumors are also associated with low immune infiltration, frequent chemotherapy resistance, and lung metastasis. The use of small molecule drugs for the treatment of osteosarcoma is a potential targeted therapy, but these small molecule drugs have significant side effects.

[0003] Advances in nanotechnology have brought new vitality to the efficacy of cancer treatment. The small size of nanoparticles enables them to penetrate biological barriers and deliver drugs to tumor sites, thereby achieving higher therapeutic efficiency. Nanoparticles can also be functionally targeted, preferentially delivering drugs to tumor sites and minimizing side effects. Nanocomposite hydrogels have a three-dimensional structure, and the incorporation of multifunctional nanomaterials can achieve diverse antitumor properties. A recognized advantage of hydrogel-based medical interventions is their ability to control in situ degradation, which can be used for the precise and long-term release of individual components within nanocomposite hydrogels. Similar technologies have also been explored in the field of surgical systems. TME-responsive nanomedicines have been shown to safely and effectively enhance specific and localized immune responses in tumor tissues, thereby improving drug safety and efficacy while reducing immune-related side effects. High levels of reactive oxygen species (ROS) are a fundamental characteristic of tumor tissue and are also prominently expressed in surgical systems. Based on this characteristic, various antitumor strategies have been developed, including: further increasing ROS levels in tumor cells to reduce drug resistance; and constructing ROS-responsive nanomedicines to enhance drug accumulation within tumors. However, due to the insufficient supply of ROS in the local tumor, the effect of ROS-responsive nanomedicines in promoting tumor regression is not ideal. Summary of the Invention

[0004] The purpose of the present invention is to overcome the defect that the existing nanomedicine treatment effect for osteosarcoma is poor.

[0005] In order to achieve the above object, the present invention provides an injectable nanocomposite hydrogel, which comprises at least a hydrogel, a liposome Cis / Mn@Lipo-IL11 composed of a free drug NHWD-870 containing cisplatin loaded with MnO2 and il11rα targeting liposomes;

[0006] The liposome Cis / Mn@Lipo-IL11 is encapsulated in the hydrogel to form an injectable nanocomposite hydrogel Cis / Mn / NH@PGP.

[0007] Preferably, the liposome Cis / Mn@Lipo-IL11 can induce the production of excessive reactive oxygen species.

[0008] Preferably, the injectable nanocomposite hydrogel Cis / Mn / NH@PGP can promote the apoptosis of tumor cells.

[0009] Preferably, the injectable nanocomposite hydrogel Cis / Mn / NH@PGP gradually degrades under high ROS conditions in the tumor microenvironment and releases the MYC-targeting therapeutic drugs NHWD-870 and Cis / Mn@Lipo-IL11.

[0010] Preferably, the mass ratio of the free drug NHWD-870 containing cisplatin loaded with MnO2 to the il11rα targeting liposome is 1:1.

[0011] Preferably, the mass ratio of the hydrogel to liposome Cis / Mn@Lipo-IL11 is 10:1.

[0012] The present invention also provides a method for preparing an injectable nanocomposite hydrogel, which comprises at least the following steps:

[0013] Step 1, preparation of MnO2 nanoparticles:

[0014] After ethanol, deionized water, and ammonia solution are uniformly mixed, tetraethyl orthosilicate is added, and the mixture is centrifuged to obtain SiO2 nanoparticles; the SiO2 nanoparticles are dispersed in deionized water, an aqueous solution of KMnO4 is added dropwise thereto, and the mixture is ultrasonically treated and centrifuged to obtain SiO2@MnO2 nanoparticles; the SiO2@MnO2 nanoparticles are dispersed in a sodium carbonate solution, and the hollow structure MnO2 nanoparticles are obtained after centrifugation;

[0015] Step 2, preparation of DSPE-PEG-IL-11:

[0016] dissolving the IL-11 peptide in N,N-dimethylformamide to obtain an IL-11 peptide solution, dissolving DSPE-PEG-NHS in N,N-dimethylformamide to obtain a DSPE-PEG-NHS solution, adding the DSPE-PEG-NHS solution to the IL-11 peptide solution to obtain a mixed solution, and dialyzing the mixed solution against a dichloromethane solution to obtain DSPE-PEG-IL-11;

[0017] Step 3, preparation of liposome Cis / Mn@Lipo-IL11:

[0018] (2,3-Dioleylphosphatidylethanolamine) triethylammonium salt, cholesterol, and DSPE-PEG-IL-11 were dissolved in chloroform, and the chloroform was removed by rotary evaporation to obtain a lipid membrane. A PBS solution containing MnO2 nanoparticles was added to the lipid membrane to obtain Mn@Lipo-IL11, which was then mixed with a cisplatin solution to obtain liposomes Cis / Mn@Lipo-IL11 encapsulating MnO2 and cisplatin.

[0019] Step 4, preparation of hydrogel:

[0020] 3-amino-4-methoxybenzoic acid and gelatin were dissolved in deionized water, and APS was added as an oxidative polymerization catalyst to obtain PAMB-gelatin; PAMB-gelatin and TK-NH2 were dissolved in DMSO to obtain PAMB-G-TK; PAMB-G-TK was mixed with four-arm polyethylene glycol succinyl glutarate for self-crosslinking to obtain a hydrogel;

[0021] Step 5, injectable nanocomposite hydrogel Cis / Mn / NH@PGP:

[0022] The liposome Cis / Mn@Lipo-IL11 was encapsulated in the hydrogel to obtain the injectable nanocomposite hydrogel Cis / Mn / NH@PGP.

[0023] Preferably, in step 3, the molar ratio of (2,3-dioleylphosphatidylethanolamine) triethylammonium salt: cholesterol: DSPE-PEG-IL-11 is 10:10:1.

[0024] The present invention also provides an application of an injectable nanocomposite hydrogel, wherein the injectable nanocomposite hydrogel is used for preparing a drug for treating osteosarcoma.

[0025] Preferably, the injectable nanocomposite hydrogel is used to prepare a drug for treating MYC-amplified osteosarcoma.

[0026] Compared with the prior art, the beneficial effects of the present invention include at least:

[0027] An injectable nanocomposite hydrogel of the present invention comprises at least a hydrogel, a free drug NHWD-870 containing cisplatin-loaded MnO2, and a liposome Cis / Mn@Lipo-IL11 composed of il11rα-targeted liposomes. The liposome Cis / Mn@Lipo-IL11 is encapsulated in the hydrogel to form an injectable nanocomposite hydrogel Cis / Mn / NH@PGP. The injectable nanocomposite hydrogel can simultaneously deliver an effective MYC inhibitor (NHWD-870) and a targeted il11rα liposome containing cisplatin-loaded MnO2 (Cis / Mn@Lipo-IL11). After in situ administration, NHWD-870 effectively degrades MYC, downregulates CCL2 and IL13 cytokines, and triggers macrophage M1 activation. At the same time, the targeted delivery of Cis / Mn@Lipo-IL11 reacts with excess intratumoral GSH to generate Mn 2+ , induces excessive reactive oxygen species (ROS) production through a fenton-like reaction, and together with cisplatin, induces immunogenic cell death (ICD), promoting dendritic cell maturation. Through the synergistic regulation of MYC and ICD levels, it reshapes the immune microenvironment and enhances immune infiltration, potentially treating MYC-amplified osteosarcoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the preparation and application of the injectable nanocomposite hydrogel Cis / Mn / NH@PGP of the present invention.

[0029] Figure 2 Preparation and characterization of the injectable nanocomposite hydrogel Cis / Mn / NH@PGP of the present invention:

[0030] (a) FTIR spectra of Lipo and Lipo-il11;

[0031] (b) XPS spectrum of Mn@Lipo-IL11;

[0032] (c) Particle size distribution of Lipo-IL-11, Mn@Lipo-IL11, and Cis / Mn@Lipo-IL11;

[0033] (d) TEM images of lipoo-il11, MnO2, and Cis / Mn@Lipo-IL11;

[0034] (e) (f) EDX and Cis elemental mapping analysis of Mn@Lipo-IL11;

[0035] (g) Macroscopic and SEM images of 2% PAMB / 1% PEG-SG and 3% PAMB / 1.5% PEG-SG hydrogels containing Cis / Mn@Lipo-IL11+NH.

[0036] (h)(i) Rheological properties of 3% PAMB / 1.5% PEG-SG hydrogel;

[0037] (j) Changes in shear viscosity of 3% PAMB / 1.5% PEG-SG hydrogel with increasing shear rate;

[0038] (k) Effect of hydrogel concentration on DPPH clearance after 60 min;

[0039] (l) Degradation rate of hydrogel under different H2O2 concentrations.

[0040] Figure 3 Evaluation of the in vitro targeting ability and anti-tumor performance of the injectable nanocomposite hydrogel Cis / Mn / NH@PGP of the present invention:

[0041] (a) Detection of MYC expression in different OS cells;

[0042] (b) IL11Ra expression in tumor cells and normal cells;

[0043] (c) (d) Flow cytometry detection of lipin and lipin after incubation of L929 and 143B cells for 8 h

[0044] -IL-11 cellular uptake, including fluorescence intensity and cellular uptake percentage;

[0045] (e) Representative fluorescence images of L929 and 143B cells co-incubated with Lipo and Lipo-IL11 for 8 h

[0046] light images;

[0047] (f) (g) Representative fluorescence images and quantification of DCFH-DA staining in 143B cells after 24 h of treatment

[0048] analyze;

[0049] (h)(i) Live / dead staining of 143B cells after treatment with Calcein-AM / PI kit for 24 h;

[0050] (j) (k) Apoptosis of cells after 24 h of treatment;

[0051] (l) (m) Wound healing assay to evaluate the in vitro migration of 143B cells after 24 h of treatment;

[0052] (n)(o) The in vitro invasiveness of 143B cells after treatment for 24 h was evaluated using the Transwell assay.

[0053] Figure 4 Evaluation of the In Vitro Macrophage Polarization and ICD Induction of the Present Invention:

[0054] (a) Schematic diagram of the co-culture system of 143B cells, RAW264.7 cells, and DCs;

[0055] (b)(c) ELISA analysis of CCL2 and IL13 expression levels in 143B cells;

[0056] (d)-(f) Representative flow cytometry images and M1 macrophages (CD86 + ) and M2 macrophages (CD206 + ) positive percentage;

[0057] (g) Immunofluorescence images of RAW264.7 cells showed that M2 macrophages (CD206 + , green), M1 macrophages (CD86 + , red) and nuclei (DAPI, blue);

[0058] (h) Western blot detection of Arg1, CD206, and iNOS in RAW264.7 cells. (i) Western blot detection of CRT and HMGB1 in K7M2 cells.

[0059] (j) CRT immunofluorescence image of K7M2 cells (green);

[0060] (k) Quantification of fluorescence signals of CRT staining in K7M2 cells;

[0061] (l) Extracellular ATP levels in K7M2 cells;

[0062] (o) Western blot detection of STING pathway activation markers in DCs;

[0063] Figure 5 Evaluation of the anti-tumor performance of the injectable nanocomposite hydrogel of the present invention on the 143b-OS model in vivo:

[0064] (a)(b) Representative fluorescence images of mice after injection of Lipo and Lipo-il11 (labeled with Cy5.5) and quantitative analysis of fluorescence signal intensity;

[0065] (c) Schematic diagram of the treatment of the mouse orthotopic 143b osteosarcoma model;

[0066] (d)(e) Representative fluorescence images and fluorescence intensity analysis of ROS levels in tumors;

[0067] (f) Evaluation of the changes in tumor volume after different treatments in each group;

[0068] (g) Reconstructed microCT image of the joint of tumor-bearing mice;

[0069] (h)-(j) Representative images of tumor tissue H&E staining, TUNEL immunofluorescence staining, and Ki-67 immunohistochemical staining;

[0070] (k) Representative images of MYC immunofluorescence staining in tumors.

[0071] Figure 6 Evaluation of the in vivo anti-tumor properties of the injectable nanocomposite hydrogel of the present invention in the OS model containing subcutaneous K7M2:

[0072] (a) Treatment of OS model in mice with subcutaneous K7M2;

[0073] (b) Measurement of tumor volume changes after different treatments in each group;

[0074] (c) Immunofluorescence image of CRT in tumor;

[0075] (d) Immunofluorescence image of HMGB1 in tumors;

[0076] (e) (f) Representative flow cytometry images and quantitative analysis of M1 marker expression (F4 / 80 + / CD86 + )

[0077] and M2 marker expression (F4 / 80 + / CD206 + ) and CD86 + / CD206 + ratio;

[0078] (g) (h) Representative flow cytometry images and quantitative analysis of mature DCs;

[0079] (i)(j) CD4 in tumor + 、CD8 + Immunofluorescence staining of cells.

[0080] Figure 7 Characterization of Cis / Mn@Lipo-IL11 prepared in the present invention:

[0081] (a) 1H NMR spectra of Lipo and Lipo-IL-11;

[0082] (b) FTIR spectra of PAMB-G and PAMB-G-TK;

[0083] (c) 1H NMR spectra of PAMB-G and PAMB-G-TK.

[0084] Figure 8 The hydrodynamic diameter and zeta potential of the MnO2 nanoparticles of the present invention are:

[0085] (a) Hydrodynamic diameter diagram;

[0086] (b) Zeta potential diagram.

[0087] Figure 9 This is a comparison chart of zeta potential measurements of different materials of the present invention in aqueous solution.

[0088] Figure 10 This is the cisplatin release profile of Cis / Mn@Lipo-IL11 prepared in the present invention in the presence or absence of GSH.

[0089] Figure 11 These are gelatin state images of PAMB-G-TK and 4-arm-PEG-SG hydrogels with different ratios according to the present invention.

[0090] Figure 12 The photos show the injectability and plasticity of PAMB-G-TK and 4-arm-PEG-SG hydrogels with different ratios of the present invention.

[0091] Figure 13 The release behavior of Lipo-IL11 in the Cis / Mn@PGP hydrogel prepared in the present invention under different concentrations of H2O2.

[0092] Figure 14 The expression level of MYC protein after 143B cells were treated with different materials:

[0093] (a) Western Blot image; (b) Quantitative analysis of relative expression: Ⅰ: Control, Ⅱ: Lipo-IL11, Ⅲ: Mn@Lipo-IL11, Ⅳ: Cis / Mn@Lipo-IL11, Ⅴ: Cis / Mn@Lipo-IL11+NH, Ⅵ: Cis / Mn / NH@PGP. *P<0.05, **P<0.01, ***P<0.001.

[0094] Figure 15 Fluorescence images of major organs and tumors in mice treated with Lipo and Lipo-IL11 (labeled with Cy5.5) for 24 hours.

[0095] Figure 16 In vivo therapeutic effect of the injected hydrogel on the 143b-OS model:

[0096] (a) Representative photos of nude mouse legs on day 18;

[0097] (b) Representative photos of nude mouse legs on day 24: Ⅰ: Control, Ⅱ: Lipo-IL11, Ⅲ:

[0098] Mn@Lipo-IL11,Ⅳ:Cis / Mn@Lipo-IL11,Ⅴ:Cis / Mn@Lipo-IL11+NH,

[0099] Ⅵ: Cis / Mn / NH@PGP.*P<0.05, **P<0.01, ***P<0.001.

[0100] Figure 17 Representative images of tumor tissues in the 143b OS model after 24 days of treatment with different materials: Ⅰ: Control, Ⅱ: PGP, Ⅲ: Mn@PGP, Ⅳ: Cis / Mn@PGP, Ⅴ: Mn / NH@PGP, Ⅵ: Cis / Mn / NH@PGP.

[0101] Figure 18 The weight changes of 143b OS model nude mice after treatment with different materials: Ⅰ: Control, Ⅱ: PGP, Ⅲ: Mn@PGP, Ⅳ: Cis / Mn@PGP, Ⅴ: Mn / NH@PGP, Ⅵ: Cis / Mn / NH@PGP. Figure 19 H&E staining of the main organs (heart, liver, spleen, lung, and kidney) of nude mice after 24 days of treatment with different materials. Ⅰ: Control, Ⅱ: PGP, Ⅲ: Mn@PGP, Ⅳ: Cis / Mn@PGP, Ⅴ: Mn / NH@PGP, Ⅵ: Cis / Mn / NH@PGP.

[0102] Figure 20 Fourteen days after treatment with different materials, routine blood tests of nude mice were performed, including red blood cell count (RBC), mean corpuscular hemoglobin (MCH), platelet distribution width (PDW), hemoglobin concentration (HGB), and white blood cell count (WBC). Ⅰ: Control, Ⅱ: PGP, Ⅲ: Mn@PGP, Ⅳ: Cis / Mn@PGP, Ⅴ: Mn / NH@PGP, Ⅵ: Cis / Mn / NH@PGP.

[0103] Figure 21 In vivo degradation behavior of Cis / Mn / NH@PGP hydrogel after subcutaneous injection into mice at 0, 1, 3, and 7 days:

[0104] (a) Representative images of hydrogel degradation after subcutaneous injection; (b) Quantitative analysis of the percentage of hydrogel remaining.

[0105] Figure 22Representative images of K7M2-OS model tumor tissues after 24 days of treatment with different materials: I: Control, II: PGP, III: Mn@PGP, IV: Cis / Mn@PGP, V: Mn / NH@PGP, VI: Cis / Mn / NH@PGP.

[0106] Figure 23 The changes in subcutaneous tumor weight in the k7m2-OS model after 24 days of treatment with different materials. Ⅰ: Control, Ⅱ: PGP, Ⅲ: Mn@PGP, Ⅳ: Cis / Mn@PGP, Ⅴ: Mn / NH@PGP, Ⅵ: Cis / Mn / NH@PGP.

[0107] Figure 24 For tumor H&E staining:

[0108] (a) H&E staining of subcutaneous tumors in mice treated with different materials;

[0109] (b) TUNEL immunofluorescence staining: Ⅰ: Control, Ⅱ: PGP, Ⅲ: Mn@PGP, Ⅳ:

[0110] Cis / Mn@PGP,Ⅴ:Mn / NH@PGP,Ⅵ:Cis / Mn / NH@PGP. DETAILED DESCRIPTION

[0111] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0112] Currently, ROS-responsive nanomedicines are not effective in treating osteosarcoma, and exogenous or endogenous stimulation must be introduced to enhance ROS supply. Manganese dioxide (MnO2) has TME-responsive degradation properties and effectively releases Mn under the action of excess glutathione (GSH) in the microenvironment. 2+ , and generate hydroxyl radicals (-OH). Therefore, the introduction of Mn into the nanotherapeutic system 2+ It can increase the ROS level in the tumor microenvironment, thereby regulating the drug release in ROS-responsive nanosystems and reducing the drug resistance of cancer cells.

[0113] Based on the above, the present invention provides an injectable nanocomposite hydrogel Cis / Mn / NH@PGP, which simultaneously delivers an effective MYC inhibitor (NHWD-870) and targeted il11rα liposomes containing cisplatin loaded with MnO2 (Cis / Mn@Lipo-IL11), establishing a targeted chemotherapy-chemodynamic therapy-immunotherapy triple therapy to treat MYC-amplified osteosarcoma. After in situ administration, NHWD-870 effectively degrades MYC, downregulates CCL2 and IL13 cytokines, and triggers macrophage M1 activation. At the same time, the targeted delivery of Cis / Mn@Lipo-IL11 reacts with excess intratumoral GSH to generate Mn 2+ , inducing excessive reactive oxygen species (ROS) production through a fenton-like reaction, and together with cisplatin, inducing immunogenic cell death (ICD), promoting dendritic cell maturation. Through the synergistic regulation of MYC and ICD levels, the immune microenvironment is reshaped and immune infiltration is enhanced. In an osteosarcoma-bearing model, the nanocomposite hydrogel can significantly enhance tumor T cell infiltration, induce effective anti-tumor immunity, and reduce lung metastasis. Therefore, the research results of the present invention reveal a powerful strategy for targeted combination therapy of MYC-amplified osteosarcoma.

[0114] Experimental Materials

[0115] Tetraethyl orthosilicate (TEOS), potassium permanganate (KMnO₄), ammonium hydroxide (approximately 25–28% ammonia solution), calcium chloride dihydrate (CaCl₂·2H₂O), ammonium bicarbonate (NH₂CO₄), m-xylene, ethanol, methanol, dimethyl sulfoxide (DMSO), hydrochloric acid (HCl), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), 2,2-diphenyl-1-picrylhydrayl (DPPH), 3-amino-4-methoxybenzoic acid (AMB), and ammonium persulfate (APS) were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd., China. Four-arm polyethylene glycol succinyl glutarate (4-arm-PEG-SG) was obtained from Xi'an Ruixi Biotechnology Co., Ltd. Cisplatin and NHWD-870 (CAS: 2115742-03-3) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). All cell culture media were purchased from Life Technologies (China). All antibodies were provided by Biolegend (USA) and Abcam (USA). All detection kits were purchased from Beyotime Biotechnology (Shanghai, China) and R&D Systems (USA). Solvents were provided by Sinopharm Group (Shanghai, China).

[0116] like Figure 1 As shown, the present invention provides a method for preparing an injectable nanocomposite hydrogel, comprising:

[0117] Preparation of MnO2 nanoparticles: 14 mL of ethanol, 2 mL of deionized water, and 50 μL of ammonia solution were added to a round-bottom flask and stirred at 50°C for 5 minutes using a magnetic stirrer. Then, 500 μL of TEOS was added dropwise to the mixture and stirred for 2 hours. The mixture was then centrifuged at 14,800 rpm for 5 minutes to obtain SiO2 nanoparticles. The SiO2 nanoparticles were dispersed in 10 mL of deionized water, and then 20 mL of an aqueous solution containing 80 mg of KMnO4 was added dropwise under ultrasonication to prepare MnO2 nanoparticles. After complete addition of KMnO4, the mixture was sonicated continuously for 1 hour and stirred at room temperature overnight. The reaction mixture was then centrifuged at 14,800 rpm for 5 minutes, and the supernatant was removed to obtain SiO2@MnO2 nanoparticles. The resulting nanoparticles were dispersed in a sodium carbonate solution (4.29 g, 20 μL) and stirred at 60°C overnight. After centrifugation at a speed of 14800 rpm for 5 minutes, the precipitate was washed with deionized water to obtain MnO2 nanoparticles with a hollow structure.

[0118] Synthesis of DSPE-PEG-IL-11: I. The copolymer DSPE-PEG-IL-11 was prepared by reacting DSPE-PEG-NHS with IL-11 peptide (China Polypeptide Co., Ltd., QYAOBIO, Shanghai, China). 200 mg of DSPE-PEG-NHS was dissolved in 1 mL of N,N-dimethylformamide (DMF), and 10.58 mg of IL-11 peptide was dissolved in 0.1 mL of DMF. Under nitrogen, the DSPE-PEG-NHS solution was slowly added to the IL-11 peptide solution at 30°C for 30 minutes. After continuous stirring for 24 hours, the mixed solution was dialyzed against DMF and dichloromethane (DCM). Finally, the product DSPE-PEG-IL-11 was recovered by cold ether precipitation and vacuum drying. The synthesis of DSPE-PEG-IL-11 was characterized by Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR).

[0119] Preparation of Cis / Mn@Lipo-IL11 liposomes: MnO2- and cisplatin-loaded liposomes were prepared using a thin film hydration method. (2,3-Dioleylphosphatidylethanolamine) triethylammonium salt (DOTAP), cholesterol, and DSPE-PEG-IL-11 were dissolved in chloroform at a molar ratio of 10:10:1. The chloroform was removed by rotary evaporation to obtain a lipid film. Lipo-IL11 and Cy5.5-labeled Lipo were prepared. Subsequently, 2 ml of MnO2-containing PBS solution was added to the dried lipid film to obtain MnO2-loaded liposomes (Mn@Lipo-IL11). The liposomes were then extruded using a liposome extruder to obtain uniformly sized unilamellar liposomes. The liposomes were freeze-dried and mixed with a 30 μM cisplatin solution. Finally, hydration was performed to obtain liposomes encapsulating MnO2 and cisplatin, designated Cis / Mn@Lipo-IL11.

[0120] Hydrogel Preparation: PAMB-gelatin (PAMB-G) was prepared by oxidative polymerization using an AMB to gelatin ratio of 1:10 (w / w). The steps were as follows: 1 g of gelatin and 100 mg of AMB were dissolved in 10 mL of deionized water and thoroughly mixed at 40°C. Then, 136.5 mg of APS was added as an oxidative polymerization catalyst. After stirring for 24 hours, the reaction solution was dialyzed against deionized water for 3 days and freeze-dried to obtain PAMB-G. PAMB-G-TK was synthesized by dissolving 300 mg of PAMB-G and 100 mg of TK-NH2 in DMSO. 0.25 g of EDC was then added and stirred at 37°C for 24 hours. Finally, PAMB-G-TK was obtained after extensive dialysis against PBS (pH 7.4) with a molecular weight cutoff (MWCO) of 3.5 kDa and freeze-dried. Finally, 3% PAMB-G-TK was mixed with 1.5% four-arm polyethylene glycol succinyl glutarate for self-crosslinking to form a hydrogel.

[0121] Finally, the liposome Cis / Mn@Lipo-IL11 is encapsulated in the hydrogel to obtain the injectable nanocomposite hydrogel of the present invention.

[0122] Preparation and characterization of Cis / Mn@Lipo-IL11

[0123] In order to enhance the accumulation of chemotherapy drugs at the tumor site and improve the drug delivery efficiency, MnO2 and cisplatin were integrated into IL11rα-targeted liposomes to obtain Cis / Mn@Lipo-IL11 nanoparticles. Figure 2 a and Figure 7 a) and proton nuclear magnetic resonance (1H NMR) spectra ( Figure 7b, c) detected the successful synthesis of lipoo-il11 and PAMB-G-TK. In addition, high-resolution Mn2p X-ray photoelectron spectroscopy (XPS) spectra confirmed the successful synthesis of mesoporous MnO2 ( Figure 2 b). Dynamic light scattering (DLS) and zeta potential measurements demonstrated the relevant physical properties of manganese dioxide. ( Figure 8 The average diameter of MnO2 is about 188.13±9.77nm, and the zeta potential shows a negative charge phenomenon. The loading of MnO2 in liposomes was studied using zeta potential. The results ( Figure 9 ) showed that after the successful loading of MnO2 (-17.3mV), the zeta potential of Mn@Lipo-IL11 decreased from 33.5mV to 22.7mV, while the zeta potential of Cis / Mn@Lipo-IL11 was comparable to that of Mn@Lipo-IL11. Subsequently, DLS and transmission electron microscopy (TEM) were used to observe the Cis / Mn@Lipo-IL11 nanoparticles, which were regular spherical and relatively uniform in size (average diameter of approximately 166.82±6.81nm). Compared with lipoo-il-11, the hydrodynamic diameter increased ( Figure 2 UV spectroscopy was used to further verify the encapsulation of cisplatin in Cis / Mn@Lipo-IL11 and its release in the tumor microenvironment in the presence of GSH ( Figure 10 ). The results showed that Cis / Mn@Lipo-IL11 encapsulated 83.6% of cisplatin. As expected, Cis / Mn@Lipo-IL11 exhibited GSH-dependent cisplatin release under simulated tumor cell physiological conditions. After exposure to GSH, the release rate of cisplatin reached 60.7±7.7%, while the release rate was only 28.1±1.7% without exposure to GSH. Elemental mapping and energy dispersive x-ray spectroscopy (EDX) analysis of Cis / Mn@Lipo-IL11 showed the presence of Mn and Pt elements, indicating that MnO2 and cisplatin were uniformly encapsulated ( Figure 2 e,f).

[0124] Preparation and characterization of nanocomposite hydrogels

[0125] In order to prepare the nanocomposite hydrogel, an addition reaction was carried out between the amino group of PAMB-G-TK and the succinimide functional group of 4-arm PEG-SG under mild conditions to form a hydrogel PGP with ROS responsive properties. The Cis / Mn@Lipo-IL11 liposomes were then encapsulated in the hydrogel PGP to form Cis / Mn / NH@PGP. Subsequently, scanning electron microscopy (SEM) observations of 3% PAMB-G-TK / 1.5% PEG-SG and 2% PAMB-G-TK / 1% PEG-SG hydrogels confirmed the formation of a three-dimensional network of the hydrogel and the successful encapsulation and uniform distribution of Cis / Mn@Lipo-IL11 nanoparticles within the porous structure of the hydrogel ( Figure 2 However, the hydrogel prepared by 2% PAMB-G-TK and 1% 4-arm-peg-sg was in a relaxed state and had weak plasticity after injection ( Figure 11 and 12 ). In addition, the rheological properties of the hydrogel were studied to verify its injectability. Figure 2 As shown in hj, the storage modulus (G') is greater than the loss modulus (G'), indicating that a stable hydrogel has been formed. In addition, the viscosity decreases significantly with the increase of shear rate, indicating that the hydrogel has good injectability. The 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazine (DPPH) detection kit was used to detect the ROS response characteristics. Within 60 minutes, the scavenging efficiency of the three hydrogels for DPPH increased with the increase of concentration, indicating that the scavenging of DPPH is sustained and effective ( Figure 2 Then, the weight loss of the hydrogel over time was monitored to study the degradation of the 3% PAMB-G-TK / 1.5% PEG-SG hydrogel induced by H2O2 ( Figure 2 l). After incubation with PBS, a slight decrease in the weight of the hydrogel was observed (≈23%), which may be due to the hydrolysis of ester bonds and gelatin matrix. However, with the increase of H2O2 concentration, the weight loss of the hydrogel gradually intensified. Incubation with 0.5mM H2O2 at 37°C resulted in a significant decrease in the mass of the hydrogel, with a loss of 91.97±4.02% at 18d, indicating ROS-responsive degradation of the hydrogel. To study the release of liposomes from the hydrogel, Cy5.5-labeled Cis / Mn@Lipo-IL11 nanoparticles were loaded into 3% PAMB-G-TK / 1.5% PEG-SG hydrogels and soaked in solutions with different H2O2 concentrations. The absorbance was measured at 680nm to obtain the in vitro release rate of Cis / Mn@Lipo-IL11 ( Figure 13). The results showed that the release rate of Cis / Mn@Lipo-IL11 in the hydrogel increased significantly with the increase of H2O2 concentration. After soaking in 0.5mM H2O2 for 18 days, the cumulative release rate of Cis / Mn@Lipo-IL11 was 90.31±3.02%, which was much higher than that of PBS solution without H2O2 (31.89±2.45%), indicating that the degree of hydrogel degradation was higher in the culture medium with added H2O2. This result is consistent with the results of H2O2-induced hydrogel degradation and strongly supports that liposome release depends on the ROS concentration in the hydrogel. Therefore, considering the injectability and ROS responsiveness, 3% PAMB-G-TK / 1.5% PEG-SG hydrogel will be the first choice for subsequent studies.

[0126] In vitro antitumor activity of Cis / Mn / NH@PGP

[0127] First, western blotting was used to detect the expression levels of MYC and il11Rα in different cell lines. The results showed that MYC expression in tumor cells was highest in 143B OS cells, moderate in HOS cells, and extremely low in SJSA and U2OS cells ( Figure 3 In addition, il11Rα was detected to be highly expressed in human 143B OS cells, followed by L929, HUVECs, and NIH3T3 cells ( Figure 3 Based on the above findings, the present invention used 143B cells and L929 cells to verify the targeting specificity of lipo-il11 by flow cytometry. The results showed that in L929 cells, there was no significant difference in the cellular uptake of Lipo and Lipo-il11, while in 143B cells, the internalization rate of Lipo-il11 was 69.57±2.67%, which was 2.5 times that of Lipo ( Figure 3 In addition, the targeting ability of Lipo-il11 was also confirmed by confocal microscopy of 143B cells and L929 cells. After 8 hours of incubation, obvious red fluorescence signals were observed in 143B and L929 cells ( Figure 3 e), indicating that these nanoparticles were successfully taken up by cells. However, the present invention observed that internalization of Lipo and Lipo-il11 was more pronounced in 143B cells than in L929 cells. Notably, Lipo-il11 was most abundant in the cytoplasm of 143B cells, indicating that 143B cells have better uptake of Lipo-il11. Therefore, the results demonstrate that Lipo-il11 has good targeting specificity for MYC-amplified OS cells.

[0128] To evaluate the ability of Cis / Mn@Lipo-IL11 to generate ROS in vitro, the ROS fluorescent indicator 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was used to observe the intracellular ROS level, and the fluorescence intensity was analyzed ( Figure 3 f, g). Compared with the control group, the green fluorescence signal of the lipoo-il11 group increased slightly. The ROS level in the Mn@Lipo-IL11 group was significantly increased due to the fenton-like reaction of the incorporated MnO2, producing -OH. In contrast, in the Cis / Mn@Lipo-IL11 and Cis / Mn@Lipo-IL11+NH groups, cisplatin and Mn 2+ The combination induced higher oxidative stress, resulting in a significant increase in green fluorescence in the treated cells. However, there were more dead cells in the Cis / Mn@Lipo-IL11+NH group and the Cis / Mn / NH@PGP group ( Figure 3 Flow cytometry was used to detect the apoptosis rate of 143B cells under different culture conditions. After treatment with Cis / Mn / NH@PGP, the apoptosis rate of 143B cells reached 67.3%, which was significantly higher than that of cells cultured with Cis / Mn@Lipo-IL11 (42.1%) and Cis / Mn@Lipo-IL11+NH (46.2%). Figure 3 j, k). Thus, it can be seen that Cis / Mn / NH@PGP has significant anti-tumor effect on 143B cells in vitro. The results showed that the treatment group containing NHWD-870 led to a significant downregulation of MYC protein expression in 143B cells ( Figure 14 ), thereby significantly inhibiting the migration and invasion of 143B cells ( Figure 3 This was confirmed by wound healing and Transwell migration assays. These results suggest that Cis / Mn@Lipo-IL11 can synergize with NHWD-870 to enhance oxidative damage to tumor cells through targeted chemotherapy-chemotherapy synergistic therapy, significantly inhibiting the invasion and metastasis of OS cells.

[0129] In vitro immune activation of Cis / Mn / NH@PGP

[0130] Mouse mononuclear macrophages (RAW264.7) and dendritic cells (DCs) were co-cultured with 143B and K7M2 cells treated with different groups in a Transwell system for 24 hours to evaluate macrophage polarization and DC maturation. Figure 4 First, the secretion of CCL2 and IL13 in the culture supernatant of 143B cells was detected by ELISA. Compared with the other groups, the levels of tumor-promoting cytokines CCL2 and IL13 in the NHWD-870-loaded group were significantly reduced after treatment, especially in the Cis / Mn / NH@PGP group ( Figure 4 Next, flow cytometry was used to detect the expression of CD206 (m2 macrophage marker) and CD86 (m1 macrophage marker) in RAW264.7 cells. The results showed that the percentage of M1 macrophages increased from 5.22% (control group) to 23.50% (Cis / Mn / NH@PGP), and the percentage of M2 macrophages decreased to one-fourth of that in the control group ( Figure 4 To obtain further evidence, the present invention performed immunofluorescence staining on CD86 and CD206, as shown in FIG. Figure 4 As shown. CD86 detected strong red fluorescence signals in the Cis / Mn@Lipo-IL11+NH group and Cis / Mn / NH@PGP group, while it was not detected or weakly expressed in the PBS, lipoo-il11, Mn@Lipo-IL11 and Cis / Mn@Lipo-IL11 groups. In RAW264.7, the expression of CD206 (green fluorescence signal) showed the opposite trend. In addition, western blotting results showed that CD206 and Arg1 were downregulated, and iNOS was upregulated ( Figure 4 h). These results indicate that Cis / Mn / NH@PGP can promote the reprogramming of macrophage phenotype. Next, the enhancement of ICD induced by Cis / Mn / NH@PGP and its promotion of dendritic cell (DC) maturation were studied. Compared with the control group and lipoo-il11 group, the expression level of CRT in K7M2 cells in the Mn@Lipo-IL11, Cis / Mn@Lipo-IL11, Cis / Mn@Lipo-IL11+NH and Cis / Mn / NH@PGP groups was significantly upregulated ( Figure 4 On the contrary, the expression of HMGB1 in K7M2 cells in Mn@Lipo-IL11, Cis / Mn@Lipo-IL11, Cis / Mn@Lipo-IL11+NH and Cis / Mn / NH@PGP groups decreased, among which the decrease in Cis / Mn@Lipo-IL11+NH and Cis / Mn / NH@PGP groups was the most significant. Immunofluorescence staining was used to observe the expression of CRT in K7M2 cells ( Figure 4j, k). The CRT staining signals of the control group and lipoo-il11 group cells were weak, while the CRT staining signals of the Mn@Lipo-IL11, Cis / Mn@Lipo-IL11, Cis / Mn@Lipo-IL11+NH and Cis / Mn / NH@PGP group cells were obvious. The present invention further verified the extracellular ATP levels of K7M2 cells after different treatments using western blotting. For the Mn@Lipo-IL11 and Cis / Mn@Lipo-IL11 groups, the extracellular ATP levels were 1.6 times and 1.9 times that of the control group, respectively. In the Cis / Mn@Lipo-IL11+NH group and the Cis / Mn / NH@PGP group, the extracellular ATP levels were 2.5 times and 2.7 times that of the control group, respectively. When treated with lipo-il11 alone, the extracellular ATP level was not affected ( Figure 4 These results indicate that Cis / Mn / NH@PGP groups can induce stronger ICD effects through the synergistic action of CRT, HMGB1 and ATP. Therefore, flow cytometry was used to detect the mature DCs (CD80 + and CD86 + The proportion of mature cells increased significantly from 6.49±0.46% (control group) to 30.7±3.35% (Cis / Mn / NH@PGP group) ( Figure 4 m,n). Figure 4 As shown in Figure 3, Cis / Mn / NH@PGP treatment significantly upregulated the phosphorylation of STING, TBK1, and IRF3 in DCs, indicating that the STING pathway was activated. These results suggest that Cis / Mn / NH@PGP can promote the maturation of DCs by inducing ICD, thereby enhancing anti-tumor immune responses.

[0131] In vivo antitumor activity of Cis / Mn / NH@PGP

[0132] The therapeutic effect of Cis / Mn / NH@PGP was evaluated in a nude mouse orthotopic tumor model. 143B cells were injected into the tibia of Balb / c nude mice to establish an orthotopic 143B tumor model. An in vivo imaging system (IVIS) was used to study the accumulation and biodistribution of liposomes and liposome-il-11 at the tumor site after intravenous administration. After liposome injection, the fluorescence signal at the tumor site of all mice gradually increased and reached a maximum at 24 hours, while the tumor accumulation in the Lipo group was much lower than that in the Lipo-il-11 group ( Figure 5 (a, b). Subsequently, the biodistribution of the liposomes during treatment was systematically monitored. 24-hour in vitro imaging showed that lipid IL-11 accumulated significantly in tumors, while lipid IL-11 accumulated less in major organs ( Figure 15These results reveal the important role of IL-11 peptide modification in promoting lipoprotein-IL-11 accumulation in 143B OS tumors. Figure 5 The treatment scheme shown in c was used to evaluate the in vivo therapeutic effect of Cis / Mn / NH@PGP by tumor size and bone destruction using the 143B-bearing OS model. To verify that the hydrogel produced ROS, the ROS fluorescence signal was detected in the tumor specimens ( Figure 5 d). In the tumor injected with PGP hydrogel, only weak ROS fluorescence signal was detected, which was similar to the control group. In contrast, obvious ROS fluorescence signal was observed in the tumor injected with Mn@PGP, indicating that Mn 2+ The generation of ROS is mediated by the fenton-like reaction. Due to the fenton-like reaction and oxidative stress induced by cisplatin, the fluorescence intensity of ROS generation in the Cis / Mn@PGP group was significantly increased, and the ROS generation in the Cis / Mn / NH@PGP group encapsulating NHWD-870 was even higher ( Figure 5 18 and 24 days after surgery, the tumor volumes of each group gradually increased as shown in the photos of the mouse hind legs ( Figure 16 On day 24, the hind leg tumors of mice in the control group and PGP hydrogel group were larger in outline, while the leg tumors of mice in the Cis / Mn / NH@PGP hydrogel group grew slowly. This indicates that the orthotopic 143BOS model was successfully established. The present invention further evaluated the tumor growth to study its anti-tumor effect within 24 days. As can be seen from the growth curve of the orthotopic bone tumor, the tumor growth of the control group and the PGP group was almost the same ( Figure 5 f). On the 24th day, the tumor volume of these groups increased dramatically and was larger than that of the other groups. This indicates that the anti-tumor effect of the PGP hydrogel itself is negligible. In contrast, the tumor volume of the Mn@PGP group and the Cis / Mn@PGP group also increased significantly, but the tumor volume was moderate compared with the control group and the PGP group. The experiment found that the Cis / Mn / NH@PGP group had a significant inhibitory effect on tumor growth starting from the 7th day, and the tumor volume was the smallest on the 24th day. In addition, the photos of the tumor-bearing tibia of mice after treatment also supported the above results ( Figure 17 ), showing that the tumor specimen size in the Cis / Mn / NH@PGP group was the smallest. Micro-computed tomography (micro-CT) was used to collect images of the tumor tibia and evaluate its inhibitory effect on bone destruction. Reconstructed images showed that the tibia of the Cis / Mn / NH@PGP group had the greatest integrity compared with the other groups ( Figure 5 ), which may be related to the superior anti-tumor efficacy of this group. Through H&E staining, TUNEL staining and Ki-67 staining of tumor tissues, it was found that the Cis / Mn / NH@PGP group had the strongest damage to tumor tissues, significantly inhibited tumor cell proliferation, and significantly enhanced cell apoptosis ( Figure 5 To further understand the targeted degradation ability of these nanocomposite hydrogels on MYC, tumor tissues were collected and immunofluorescence staining of MYC was performed 24 days after treatment. It was found that Cis / Mn / NH@PGP had a significant inhibitory effect on MYC protein ( Figure 5 These results indicate that Cis / Mn / NH@PGP hydrogel can increase ROS production, promote cell apoptosis, and inhibit MYC expression, thereby inhibiting tumor growth and bone destruction.

[0133] To further evaluate the in vivo biosafety of Cis / Mn / NH@PGP hydrogels, the body weight of mice treated with different methods was monitored. The results showed that even at the end of the treatment, the body weight had a stable change, indicating that these hydrogels have good biosafety ( Figure 18 In addition, the effects of the injectable hydrogel on the major organs of mice were evaluated by H&E staining ( Figure 19 Compared with the control group, the hydrogel group showed no significant changes. At the same time, blood biochemical analysis was performed to quantify the levels of various biochemical indicators in the blood of each group ( Figure 20 ). In terms of the main blood biochemical indices (RBC, MCH, PDW, HGB, WBC), there were no significant differences between the control and treatment groups. These findings support the obvious biosafety of the anti-tumor process based on the nanocomposite hydrogel. In addition, the degradation of Cis / Mn / NH@PGP in vivo was evaluated, and the residual amount of Cis / Mn / NH@PGP was less than 40% on day 7 ( Figure 21 ).

[0134] In vivo immunomodulatory properties of Cis / Mn / NH@PGP

[0135] To evaluate the immunomodulatory effect of Cis / Mn / NH@PGP hydrogel on subcutaneous OS tumors in mice, K7M2 cells were injected subcutaneously into Balb / c mice. Figure 6 As shown in a. Tumor growth was evaluated by tumor volume, tumor weight, histological staining and other indicators to study its anti-tumor effect. As expected, the tumor volume and weight of the Cis / Mn / NH@PGP group were the smallest after treatment ( Figure 6 b. Figure 22 and 23 ). H&E staining of tumor sections showed that the nuclear density of tumor cells in the Mn / NH@PGP group and the Cis / Mn / NH@PGP group was significantly reduced. In TUNEL staining, the tumor tissue treated with Cis / Mn / NH@PGP showed strong green fluorescence ( Figure 24 ), it can be seen from the fluorescence staining image of CRT that the fluorescence signal of Cis / Mn / NH@PGP group is the strongest, confirming the highest level of CRT ( Figure 6 CRT staining fluorescence intensity in Mn / NH@PGP and Cis / Mn@PGP groups was significantly higher than that in Mn@PGP and PGP groups. Compared with the Control and PGP groups, HMGB1 staining in Mn@PGP, Cis / Mn@PGP, Mn / NH@PGP and Cis / Mn / NH@PGP groups was significantly reduced ( Figure 6 These results confirmed that Cis / Mn / NH@PGP could induce and enhance the ICD-activated immune response.

[0136] Next, various immune cells in the obtained tumor tissues were studied. After Cis / Mn / NH@PGP treatment, the ratio of M1-like macrophages to M2-like macrophages and the proportion of mature DCs increased significantly ( Figure 6 This indicates that Cis / Mn / NH@PGP successfully activated the immune response. The increase in the Cis / Mn / NH@PGP group was higher than that in the Cis / Mn@PGP group, which may be attributed to the additional contribution of MYC degradation to immunotherapy. In addition, after treatment with Cis / Mn / NH@PGP, the immunofluorescence in tumor tissues was enhanced, and tumor-infiltrating CD4 + T cells and CD8 + T cells increased significantly ( Figure 6 Therefore, Cis / Mn / NH@PGP has a significant inhibitory effect on MYC and a significant promoting effect on ICD levels, which leads to a significant infiltration of T cells in tumor tissues for immunotherapy.

[0137] In summary, the injectable nanocomposite hydrogel Cis / Mn / NH@PGP of the present invention is composed of free NHWD-870 and il11rα targeted liposomes containing cisplatin-loaded MnO2 (Cis / Mn@Lipo-IL11). After intratumoral injection, the injectable nanocomposite hydrogel gradually degrades under high ROS conditions in the TME and releases targeted MYC therapeutic drugs (NHWD-870) and Cis / Mn@Lipo-IL11. The small molecule drug NHWD-870 is released to target MYC therapy, inhibit the production of downstream cytokines CCL2 and IL13, and promote the polarization of tumorigenic M2 macrophages to anti-tumor M1 macrophages. The mesoporous manganese dioxide-coated liposomes loaded with cisplatin target tumor cells through surface-modified il11rα. Subsequently, GSH in the TME promotes the activation of Mn 2+ The formation of , leading to Fenton-like reaction, produces ROS for chemodynamic therapy. In particular, the released cisplatin and Mn 2+Inducing ICD and promoting DC maturation. These mature DCs and M1 macrophages further activated T cells, improved overall immune infiltration, and inhibited tumor growth and metastasis. The results showed that the TME-responsive nanocomposite hydrogel had enhanced anti-tumor efficacy in MYC-amplified OS, providing further support for the rational combination therapy of MYC-amplified tumors.

[0138] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. An injectable nanocomposite hydrogel, characterized in that: The injectable nanocomposite hydrogel at least comprises a hydrogel, a free drug NHWD-870 containing cisplatin loaded with MnO2, and a liposome Cis / Mn@Lipo-IL11 composed of il11rα targeting liposomes; The liposome Cis / Mn@Lipo-IL11 is encapsulated in the hydrogel to form an injectable nanocomposite hydrogel Cis / Mn / NH@PGP.

2. The injectable nanocomposite hydrogel according to claim 1, characterized in that The liposome Cis / Mn@Lipo-IL11 can induce the production of excessive reactive oxygen species.

3. The injectable nanocomposite hydrogel according to claim 1, characterized in that The injectable nanocomposite hydrogel Cis / Mn / NH@PGP can promote the apoptosis of tumor cells.

4. The injectable nanocomposite hydrogel according to claim 1, characterized in that The injectable nanocomposite hydrogel Cis / Mn / NH@PGP gradually degrades under high ROS conditions in the tumor microenvironment and releases the MYC-targeting therapeutic drugs NHWD-870 and Cis / Mn@Lipo-IL11.

5. The injectable nanocomposite hydrogel according to claim 1, characterized in that In terms of mass ratio, the free drug NHWD-870 containing cisplatin loaded with MnO2:il11rα targeting liposomes=1:

1.

6. The injectable nanocomposite hydrogel according to claim 1, characterized in that In terms of mass ratio, the hydrogel:liposome Cis / Mn@Lipo-IL11=10:

1.

7. A method for preparing an injectable nanocomposite hydrogel, characterized in that: At least the following steps are included: Step 1, preparation of MnO2 nanoparticles: After ethanol, deionized water and ammonia solution are uniformly mixed, tetraethyl orthosilicate is added, and SiO2 nanoparticles are obtained by centrifugation; SiO2 nanoparticles are dispersed in deionized water, and an aqueous solution of KMnO4 is added dropwise thereto, and SiO2@MnO2 nanoparticles are obtained by centrifugation after ultrasonic treatment; SiO2@MnO2 nanoparticles are dispersed in a sodium carbonate solution, and hollow MnO2 nanoparticles are obtained by centrifugation; Step 2, preparation of DSPE-PEG-IL-11: dissolving the il-11 peptide in N,N-dimethylformamide to obtain an il-11 peptide solution, dissolving DSPE-PEG-NHS in N,N-dimethylformamide to obtain a DSPE-PEG-NHS solution, adding the DSPE-PEG-NHS solution to the il-11 peptide solution to obtain a mixed solution, dialyzing the mixed solution with a dichloromethane solution to obtain DSPE-PEG-IL-11; Step 3, preparation of liposome Cis / Mn@Lipo-IL11: Dissolve (2,3-dioleylphosphatidylethanolamine) triethylammonium salt, cholesterol and DSPE-PEG-IL-11 in chloroform, remove chloroform by rotary evaporation to obtain lipid membrane; add PBS solution containing MnO2 nanoparticles to the lipid membrane to obtain Mn@Lipo-IL11, mix Mn@Lipo-IL11 with cisplatin solution to obtain liposome Cis / Mn@Lipo-IL11 encapsulating MnO2 and cisplatin; Step 4, preparation of hydrogel: Dissolving 3-amino-4-methoxybenzoic acid and gelatin in deionized water, adding APS as an oxidative polymerization catalyst to obtain PAMB-gelatin; dissolving PAMB-gelatin and TK-NH2 in DMSO to obtain PAMB-G-TK; mixing the PAMB-G-TK with four-arm polyethylene glycol succinyl glutarate to obtain a hydrogel by self-crosslinking; Step 5, injectable nanocomposite hydrogel Cis / Mn / NH@PGP: The liposome Cis / Mn@Lipo-IL11 is encapsulated in the hydrogel to obtain the injectable nanocomposite hydrogel Cis / Mn / NH@PGP.

8. The preparation method according to claim 7, characterized in that: In terms of molar ratio, in step 3, the (2,3-dioleylphosphatidylethanolamine) triethylammonium salt: cholesterol: DSPE-PEG-IL-11 = 10:10:

1.

9. An application of an injectable nanocomposite hydrogel, characterized in that: The injectable nanocomposite hydrogel is used for preparing medicine for treating osteosarcoma.

10. The use according to claim 9, characterized in that The injectable nanocomposite hydrogel is used for preparing a drug for treating MYC amplified osteosarcoma.

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