Intratumoral in situ cross-linked hydrogel nanocomposite and its preparation method and application

Abstract

The present invention discloses an intratumoral in situ cross-linked hydrogel nanocomposite and its preparation method and application, the method comprising the steps of adding MAA dropwise to a HA solution while simultaneously stirring at high speed, then dialyzing with a dialysis bag at 0°C for a period of time, and lyophilizing to obtain HAMA; adding a potassium permanganate solution to the HAMA solution and mixing, then dialyzing with a dialysis bag, and lyophilizing to obtain a HAMA-MnO2 composite material; dissolving the HAMA-MnO2 composite material in ultrapure water, adding lactate oxidase, and mixing to obtain an intratumoral in situ cross-linked hydrogel nanocomposite. The intratumoral in situ cross-linked hydrogel nanocomposite of the present invention can form a hydrogel locally in the tumor through a cross-linking reaction of hyaluronic acid and calcium ions, thereby achieving a local sustained release effect of the drug, and combining the synergistic effect of manganese dioxide and lactate oxidase to effectively regulate the tumor microenvironment, thereby improving the effect of radioimmunotherapy and promoting the apoptosis of tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of hydrogel nanocomposites, and specifically relates to an intratumoral in situ cross-linked hydrogel nanocomposite that targets the acidity and hypoxia dual regulation of the tumor microenvironment, as well as a preparation method and application thereof. Background Art

[0002] The tumor microenvironment (TME) is a complex ecosystem, the concept of which originates from Virchow's 19th-century proposal of the relationship between inflammation and cancer and Paget's "seed and soil" theory. Shaped by the activities of cancer cells, the TME comprises a variety of cell types, the extracellular matrix, vascular networks, and immune cells, all of which collectively influence tumor growth, invasion, and response to therapy. In particular, immune cells within the TME, such as tumor-associated macrophages (TAMs), play a key role in tumor immune evasion and resistance to immunotherapy.

[0003] Tumor-associated macrophages contribute to tumor immune escape and treatment resistance by secreting immunosuppressive factors and promoting angiogenesis, while M1 macrophages have anti-tumor activity. Radiotherapy and immunotherapy are the two mainstays of cancer treatment, but their effectiveness is often affected by the TME. The acidic and hypoxic properties of the TME can lead to radioresistance, as hypoxic cells are insensitive to radiation, while the acidic environment may inhibit the activity of immune cells and reduce the effectiveness of immunotherapy. Therefore, regulatory strategies targeting the tumor microenvironment, such as improving hypoxia and acidity and promoting the polarization of tumor-associated macrophages to the M1 type, have become important research directions for improving the efficacy of radiotherapy and immunotherapy.

[0004] In the field of tumor microenvironment regulation and enhanced immunotherapy, researchers are exploring various strategies to enhance the immune system's ability to kill tumors. These strategies aim to improve the tumor microenvironment, making it more conducive to the activity and proliferation of immune cells, thereby improving the effectiveness of immunotherapy. These strategies include macrophage polarization regulation, inhibition of tumor-associated macrophage recruitment, and tumor-associated macrophage exhaustion. As a new type of biomaterial, hydrogel has also shown its unique advantages and application prospects in regulating tumor-associated macrophages and improving the tumor microenvironment. Currently published studies on hydrogel composite materials that improve the tumor microenvironment can be roughly divided into the following types:

[0005] (1) Thermosensitive hydrogel material composites, such as the Gel / (REG+NG / LY) hydrogel system reported by Li et al. that can be used to amplify immune activation, and the LPR@CHG nanoparticle complex reported by Gao et al. that can reprogram the tumor immune niche;

[0006] (2) Ion-crosslinked hydrogel material composites, such as Shen et al. developed a sodium alginate hydrogel composed of elesclomol-Cu and galactose, which can form a gel with physiological concentrations of calcium ions, and Ding et al. reported a tumor microenvironment and near-infrared (NIR) light dual-responsive prodrug hydrogel APPF for synergistic cancer immunotherapy;

[0007] (3) Postoperative implantation of hydrogel material complexes, such as PSBMA hydrogel-encapsulated doxorubicin Gel@M / CuO, is used for adjuvant treatment after breast cancer surgery, and hyaluronic acid hydrogel-encapsulated CAR-T helps enhance the distribution of CAR-T cells in the surgical bed.

[0008] In summary, the hydrogel nanosystem has significant advantages in the treatment of tumor microenvironment, provides new strategies and methods for cancer treatment, and indicates that its application prospects in the biomedical field are broader.

[0009] Hyaluronic acid is a water-soluble natural polysaccharide that can react with multivalent cations (such as Ca 2+ ) cross-linking, at physiological concentrations of Ca 2+ Hydrogels are formed rapidly under certain conditions. Hydrogels composed of cross-linked polymer networks are often used for local controlled release of therapeutic drugs. Hydrogels can be used as efficient drug carriers to load chemotherapy drugs, biological agents or other therapeutic agents, and act directly on the tumor microenvironment through local injection or implantation. This method of administration can increase the concentration of drugs at the tumor site while reducing systemic side effects. Recently, various hydrogel-based nanocomposites have attracted attention and have been designed for tumor treatment. Hyaluronic acid has excellent biocompatibility and can be degraded in the body, reducing long-term effects on the human body. In addition, the softness and plasticity of nanohydrogels enable them to better integrate with tumor tissue and improve therapeutic effects.

[0010] These hydrogel drug delivery systems not only have the functions of each component, but also have synergistic effects. Among them, there are many basic research applications of materials such as those that relieve hypoxia and low pH in the tumor microenvironment. Hypoxia and low pH are key characteristics of the tumor microenvironment, which have a significant impact on tumor growth, invasion, metastasis and treatment response. Hypoxia and low pH environments inhibit the function of immune cells, promote the aggregation of immunosuppressive cells, and help tumors escape immunity. In addition, these conditions increase the resistance of tumor cells to radiotherapy and certain chemotherapy drugs, affecting the distribution and activity of drugs. Therefore, increasing oxygen concentration or adjusting pH are potential strategies for treating tumors by improving the tumor microenvironment. Summary of the Invention

[0011] In response to the problems existing in the prior art, the present invention provides an intratumoral in situ cross-linked hydrogel nanocomposite that targets the acidity and hypoxia of the tumor microenvironment and its preparation method and application. The hydrogel nanocomposite is formed by manganese dioxide, lactate oxidase and hyaluronic acid through a specific preparation method. It can play a synergistic role in the tumor microenvironment, regulate the tumor microenvironment, improve low pH and hypoxia conditions, and promote the polarization of tumor-associated macrophages to M1, thereby improving the effect of radioimmunotherapy.

[0012] In order to solve the above technical problems, the present invention is implemented through the following technical solutions:

[0013] A method for preparing an intratumoral in situ cross-linked hydrogel nanocomposite comprises the following steps:

[0014] Step 1: Completely dissolve hyaluronic acid (HA) in ultrapure water, add methacrylic anhydride (MAA) dropwise while stirring at high speed, and then stir at high speed for a period of time at 0°C;

[0015] Step 2: dialyzing the solution obtained in step 1 for a period of time using a dialysis bag with a specific molecular weight cut-off, and freeze-drying to obtain methacrylated hyaluronic acid (HAMA);

[0016] Step 3: dissolving the methacrylated hyaluronic acid (HAMA) obtained in step 2 in ultrapure water, then adding potassium permanganate solution (KMnO4) and mixing, and reducing potassium permanganate to manganese dioxide using the energy of the double bond;

[0017] Step 4: dialyzing the mixed solution obtained in step 3 for a period of time using a dialysis bag with a specific molecular weight cut-off, and freeze-drying to obtain a methacrylated hyaluronic acid-manganese dioxide composite material (HAMA-MnO2);

[0018] Step 5: dissolving the methacrylated hyaluronic acid-manganese dioxide composite material (HAMA-MnO2) obtained in step 4 in ultrapure water, adding lactate oxidase (LOX), and stirring thoroughly to obtain an intratumoral in situ cross-linked hydrogel nanocomposite, i.e., HAMA-MnO2 / LOX;

[0019] The HAMA-MnO2 / LOX can be combined with physiological concentrations of Ca 2+ Solution cross-linking generates hydrogels with MnO2 nanoparticles and LOX.

[0020] Furthermore, in step 1, the high-speed stirring time is 24 hours.

[0021] Furthermore, in step 2, the molecular weight cut-off of the dialysis bag is 8000-14000 kDa, and the dialysis time is 48 hours.

[0022] Furthermore, in step 3, when the color of the potassium permanganate solution (KMnO4) changes from purple to brown when added, it indicates that the potassium permanganate in the mixed solution has been completely reduced to manganese dioxide.

[0023] Furthermore, in step 4, the molecular weight cut-off of the dialysis bag is 3000 kDa, and the dialysis time is 48 h.

[0024] An intratumor in situ cross-linked hydrogel nanocomposite is prepared by adopting the above-mentioned method for preparing the intratumor in situ cross-linked hydrogel nanocomposite.

[0025] A novel application of the above-mentioned intratumoral in situ cross-linked hydrogel nanocomplex for synergistically regulating the immune response and tumor-associated macrophage polarization state in the tumor microenvironment.

[0026] Furthermore, it is especially used to synergistically improve the low pH and hypoxia conditions in the tumor microenvironment, and promote the polarization of tumor-associated macrophages to M1, thereby increasing or decreasing the effect of radioimmunotherapy.

[0027] A new application of the above-mentioned intratumoral in situ cross-linked hydrogel nanocomposite is used as a delivery system or drug loading system for anti-cancer drugs including chemotherapy drugs and biological agents.

[0028] Furthermore, the anticancer drug delivery system or drug loading system can form a hydrogel with MnO2 nanoparticles and LOX in the tumor through the cross-linking reaction between its own hyaluronic acid and calcium ions, and play a synergistic role in the tumor microenvironment, improving low pH and hypoxia conditions, and promoting the polarization of tumor-associated macrophages to M1, thereby helping to improve the effect of radioimmunotherapy and promote the apoptosis of tumor cells.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The intratumoral in situ cross-linked hydrogel nanocomposite (particles) of the present invention is prepared from manganese dioxide, lactate oxidase and hyaluronic acid by a specific preparation method. The preparation process is relatively simple, easy to mass produce, and reduces production costs.

[0031] 2. The intratumoral in situ cross-linked hydrogel nanocomposite (particles) of the present invention can form a hydrogel locally in the tumor through the cross-linking reaction of hyaluronic acid and calcium ions, thereby achieving a local sustained release effect of the drug and improving the targeting and therapeutic effect of the drug.

[0032] 3. The intratumoral in situ cross-linked hydrogel nanocomplex (particles) of the present invention combines the synergistic effects of manganese dioxide, lactate oxidase and hyaluronic acid, which can effectively regulate the immune response and the polarization state of tumor-associated macrophages in the tumor microenvironment, especially effectively improve the low pH and hypoxia problems of the tumor microenvironment, promote the polarization of tumor-associated macrophages to M1, thereby improving the effect of radioimmunotherapy and promoting the apoptosis of tumor cells.

[0033] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the invention and to implement it according to the contents of the description, the following preferred embodiments of the present invention are described in detail with reference to the accompanying drawings. The specific implementation methods of the present invention are given in detail by the following embodiments and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0035] Figure 1 This is a diagram showing the basic properties characterization results of the HAMA-MnO2 nanocomposite prepared in Example 1 of the present invention.

[0036] Figure 2 HAMA-MnO2 / Ca obtained in Example 2 of the present invention 2+ Figure 2. Gel structure characterization results of hydrogel.

[0037] Figure 3 This is a graph showing the test results of the HAMA-MnO2 / LOX composite material in Example 3 of the present invention on the degradation of lactic acid and the repolarization of tumor-associated macrophages.

[0038] Figure 4 This is a graph showing the test results of the hypoxia-regulating effect of the HAMA-MnO2 / LOX composite material on the tumor microenvironment in Example 4 of the present invention.

[0039] Figure 5 This is a test result diagram of the effect of the HAMA-MnO2 / LOX composite material on the function of immune cells in the tumor microenvironment in Example 5 of the present invention.

[0040] Figure 6 The HAMA-MnO2 / LOX composite material prepared by the present invention and the physiological concentration of Ca 2+ Schematic diagram of the reaction process for solution cross-linking to generate hydrogels with MnO2 nanoparticles and LOX. DETAILED DESCRIPTION

[0041] The following will be described in detail with reference to the accompanying drawings to better understand the purpose, features and advantages of the invention. It should be understood that the embodiments shown in the accompanying drawings are not intended to limit the scope of the invention, but are only intended to illustrate the essential spirit of the technical solution of the invention.

[0042] In the following description, for the purpose of illustrating the various disclosed embodiments, certain specific details are set forth in order to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the relevant art will recognize that the embodiments may be practiced without one or more of these specific details. In other cases, well-known devices, structures, and techniques associated with this application may not be shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0043] Unless the context requires otherwise, throughout the specification and claims, the word "comprise" and variations such as "include" and "have" should be construed in an open, inclusive sense, that is, should be interpreted to mean "including, but not limited to."

[0044] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any manner in one or more embodiments.

[0045] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be noted that the term "or" is generally employed in its sense including "and / or" unless the context clearly dictates otherwise.

[0046] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

[0047] In the field of tumor treatment, the acidic and hypoxic properties of the tumor microenvironment have always been a hot topic of research. These properties not only promote tumor invasion and metastasis, but also lead to immunosuppression and radioresistance of tumor-associated macrophages, which seriously hinder the therapeutic effect. Tumor-associated macrophages play a complex role in the tumor microenvironment, and their polarization state has a significant impact on tumor progression and treatment response. The present invention designs an intratumoral in situ cross-linked hydrogel complex. This strategy aims to reverse the hypoxic and acidic microenvironment of the tumor, thereby promoting the polarization of tumor-associated macrophages to the M1 type. The implementation of this strategy is expected to significantly improve the efficacy of combined radioimmunotherapy and bring new breakthroughs to tumor treatment.

[0048] Example 1

[0049] Synthesis of HAMA-MnO2 composite materials:

[0050] First, hyaluronic acid HA is completely dissolved in ultrapure water to obtain an HA solution (10 mg / mL, 200 mL). Then, while stirring at high speed, methacrylic anhydride MAA (1.035 g / m, 5 mL) is added dropwise to the HA solution (10 mg / mL, 200 mL) and stirred at high speed at 0°C for 24 hours. Subsequently, the HAMA is dialyzed for 48 hours using a dialysis bag with a molecular weight cutoff of 8000-14000 kDa, and lyophilized to obtain methacrylated hyaluronic acid powder HAMA, which is stored for later use. Next, the HAMA powder is dissolved in ultrapure water to obtain a HAMA solution (10 mg / mL, 50 mL), and potassium permanganate solution KMnO4 (1.05 mg / mL, 1 mL) is added and mixed and stirred. The energy of the double bond is used to reduce potassium permanganate to manganese dioxide. When the color of the KMnO4 solution changes from purple to brown, it indicates that the potassium permanganate has been completely reduced to manganese dioxide. Finally, the mixed solution was dialyzed for 48 hours using a dialysis bag with a molecular weight cutoff of 3000 kDa, and freeze-dried to obtain a methacrylated hyaluronic acid-manganese dioxide composite material, namely, HAMA-MnO2 nanocomposite.

[0051] See also Figure 1 As shown, Figure 1 The figure shows the basic properties characterization results of the HAMA-MnO2 nanocomposite prepared in this example. In order to synthesize the HAMA-MnO2 nanocomposite, this example first synthesized methacrylated hyaluronic acid (HAMA). Figure 1 The hydrogen spectrum of A shows that the active hydrogens of carboxylic acid, hydroxyl and amino groups on HAMA disappear, and the 32nd olefinic hydrogen on HAMA exists (around 6.28ppm), and the 4th alkyl hydrogen affected by two oxygens is located around 5.8ppm. The singlet peaks of alkyl groups on HAMA are all in high field, and the remaining alkyl hydrogens are split between 3.45ppm and 4ppm due to the mutual influence of each other. Figure 1 As shown in the infrared spectrum of B, it can be seen from the infrared spectrum of HAMA that 3302cm -1 The stretching vibration absorption peak of NH and OH in HA is 1655 cm -1 The peak at 1381 cm is the stretching vibration absorption peak of the C=C double bond in methacrylic acid. -1 The absorption peak at the center is the bending vibration absorption peak of -CH3 in methacrylic acid. After wrapping MnO2, the infrared absorption peak of the product remains basically unchanged, the peak position is the same, but the peak intensity is significantly enhanced. This is because the hydrogen bonding force between HAMA and MnO2 leads to the enhancement of the absorption peak intensity.

[0052] Example 2

[0053] HAMA-MnO2 composite gelation and hydrogel-related characterization:

[0054] The intratumoral colloid formation performance of the HAMA-MnO2 composite material was measured. The HAMA-MnO2 composite material obtained in Example 1 at different concentrations (0, 1, 5, 10, 20 mg / mL, 2 mL) was mixed with the physiological concentration of Ca 2+ The solution (1.8mmoL / , 1mL) was mixed and allowed to stand for 5 minutes. When the concentration reached 10mg / mL, HAMA-MnO2 and Ca 2+ Cross-linking occurs to form a hydrogel, and the reaction principle is as follows Figure 6 Then HAMA-MnO2 / Ca 2+ The gel was swelled in a large amount of water for 24 hours and then freeze-dried. The freeze-dried gel was fractured by liquid nitrogen and photographed using a scanning electron microscope to observe the swelling pores and drug loading of the hydrogel. HAMA-MnO2 (10 mg / mL, 50 mL) and FITC-NH2 (1.018 mg / mL, 1 mL) were stirred in the dark for 2 hours, then dialyzed in a dialysis bag with a molecular weight cutoff of 3000 kDa for 24 hours in the dark. After freeze-drying, it was fully dissolved in ultrapure water. HAMA-MnO2 grafted with FITC (10.4 mg / mL, 50 μL) was injected into the subcutaneous tumors of mice. After 24 hours, the tumors were sliced ​​and the gel formation in the tumors was observed using a fluorescence microscope.

[0055] See also Figure 2 As shown, Figure 2 The HAMA-MnO2 / Ca obtained in this example 2+ The hydrogel structure characterization results are shown in the figure. Figure 2 The appearance photo of B shows that HAMA-MnO2 has the ability to react with physiological concentrations of Ca 2+ The solution is cross-linked to form a hydrogel. Figure 2 The scanning electron microscopy image of A shows that in HAMA-MnO2 / Ca 2+ The scanning electron microscope images of HAMA-MnO2 / Ca 2+ MnO2 nanoparticles with a size of about 40 nm can be found in the SEM images. Figure 2 As shown in Figure C, using PBS as a control, both HAMA and HAMA-MnO2 formed a porous gel structure in the tumor and remained in the tumor for more than 24 hours.

[0056] Example 3

[0057] HAMA-MnO2 / LOX composite material's effect on lactic acid degradation and repolarization of tumor-associated macrophages:

[0058] Establishment of co-culture model of tumor-associated macrophages and tumor cells: The two cells were co-cultured using Transwell chambers. Five groups were divided into five groups, and the lower layer was plated with a uniform concentration of BMDM (2*10 6 pcs / well), different concentrations of CT26 (0 pcs / well, 3.8*10 5 Pieces / hole, 1.12*10 6 / hole, 1.86*10 6 After iron wall incubation for 24 h, the supernatant was collected and lactate concentration was measured using a UV-visible spectrophotometer. BMDM from the lower layer was collected and macrophage polarization was measured using flow cytometry. The group with the best polarization was selected for repolarization experiments.

[0059] To test the repolarization ability of HAMA-MnO2 / LOX on macrophages: the upper layer of CT26 cells was removed, and HAMA, HAMA-MnO2, HAMA / LOX, and HAMA-MnO2 / LOX freeze-dried powder were added to the upper layer. LPS was used as a positive control and co-cultured with BMDM for 24 hours. The supernatant was then collected and the lactate concentration was measured using a UV-visible spectrophotometer. The repolarization effect of the composite material was detected by flow cytometry for CD86. + CD206 showed that macrophages were polarized to M1 type. + This demonstrates that macrophages are polarized toward the M2 type.

[0060] See also Figure 3 As shown, Figure 3 The figure shows the test results of the HAMA-MnO2 / LOX composite material on the degradation of lactic acid and the repolarization of tumor-associated macrophages. Figure 3 As shown in A, HAMA-MnO2 showed good biocompatibility in 3T3, BMDM and CT26 cells, and 200 μg / mL was used in subsequent experiments. We then tested the lactate concentration in the cell culture supernatant. Figure 3 As shown in B, as the concentration of tumor cells increases, the concentration of lactate also increases. Figure 3 As shown in Figure C, after using the composite material, it was found that the composite material containing LOX had a significantly better degradation effect on lactic acid than the group without LOX. Figure 3 As shown in D, as the concentration of CT26 increases, the proportion of BMDM polarized toward M2 increases. Figure 3 As shown in E, the M2 ratio of the HAMA-MnO2 / LOX composite material group was the lowest, indicating that this group had the best repolarization effect on tumor-associated macrophages.

[0061] Example 4

[0062] Hypoxia-regulating effect of HAMA-MnO2 / LOX composite material on tumor microenvironment:

[0063] The [Ru(dpp)3]Cl2 oxygen probe was used to examine the tumor microenvironmental regulation of the HAMA-MnO2 / LOX composite. At the end of the treatment cycle, mice were injected with a [Ru(dpp)3]Cl2 oxygen probe (15 mg / L, 20 μL) into subcutaneous tumors (CT26). After 24 hours, the tumors were observed using a confocal microscope. A stronger green fluorescence signal indicates a higher oxygen content within the tumor. The animals were divided into seven groups: a blank control group, a radiotherapy group, a radiotherapy plus HAMA group, a radiotherapy plus HAMA-MnO2 group, a radiotherapy plus HAMA / LOX group, a radiotherapy plus MnO2 / LOX group, and a radiotherapy plus HAMA-MnO2 / LOX group.

[0064] See also Figure 4 As shown, Figure 4 The results of the HAMA-MnO2 / LOX composite material's hypoxic regulation on the tumor microenvironment are shown in Figure 2. The results show that the intensity of green fluorescence in the MnO2-treated group was much stronger than that in the non-MnO2-treated group and the control group, indicating that the MnO2-treated group has a better regulatory effect on the hypoxic characteristics of the tumor microenvironment.

[0065] Example 5

[0066] Sensitization effect of HAMA-MnO2 / LOX composite material on radiotherapy:

[0067] To investigate the radiosensitization effect of the HAMA-MnO2 / LOX composite material, the animals were divided into seven groups: a blank control group, a radiotherapy group, a radiotherapy plus HAMA group, a radiotherapy plus HAMA-MnO2 group, a radiotherapy plus HAMA / LOX group, a radiotherapy plus MnO2 / LOX group, and a radiotherapy plus HAMA-MnO2 / LOX group. Tumor cell survival was assessed using TUNEL staining; more pronounced apoptosis indicates stronger green fluorescence. Tumor size was also recorded.

[0068] To explore the immunomodulatory effect of HAMA-MnO2 / LOX composite materials on tumor microenvironment: After the treatment cycle, tumor tissues were taken and flow cytometry was used to detect the polarization expression of tumor-associated macrophages, CD86 + CD206 showed that macrophages were polarized to M1 type. + The results showed that macrophages were polarized toward the M2 type. The levels of other key cytokines such as IFN-γ, TGF-β, and IL-10 in the venous blood of mice were measured by ELISA to further analyze the effect of HAMA-MnO2 / LOX composite materials on the function of immune cells in the tumor microenvironment.

[0069] See also Figure 5 As shown, Figure 5 The figure shows the test results of the effect of HAMA-MnO2 / LOX composite material on the function of immune cells in the tumor microenvironment. Figure 5 A shows that in the subcutaneous tumors of mice, the apoptosis of the tumors in the LOX-treated group was more obvious than that in the other treatment groups and the control group, which indicates that the regulation of lactate has a direct or indirect inhibitory effect on the tumor. Before the addition of MnO2 and LOX, the blank control group, radiotherapy group, and radiotherapy plus HAMA group did not show obvious TUNEL fluorescence. Figure 5 As shown in B, the double-positive fluorescence intensity of CD206 and F480 showed a decreasing trend. The expression of M2 macrophages in the tumor tissue of the blank control group was the highest, and the expression of M2 macrophages in the radiotherapy plus HAMA-MnO2 / LOX group was the lowest. There was no particularly obvious change in M2 macrophages in the radiotherapy group. Figure 5 As shown in C, the changes in tumor volume showed a decreasing trend, and the tumor proliferation in the radiotherapy plus HAMA-MnO2 / LOX group was the slowest. Figure 5 D. Figure 5 As shown in E, the concentration of IFN-γ showed a decreasing trend, while the concentration of IL-10 showed an increasing trend. The results showed that the HAMA-MnO2 / LOX composite material had a good effect on radiosensitization and repolarization of tumor-associated macrophages.

[0070] In summary, the HAMA-MnO2 / LOX composite significantly enhanced the efficacy of radiotherapy and promoted tumor cell apoptosis by modulating the immune response and macrophage polarization in the tumor microenvironment. The application of this composite not only improves therapeutic efficacy but also may provide a new strategy for tumor immunotherapy. Future research will further explore its potential for clinical application, hoping to provide more effective treatment options for cancer patients.

[0071] It can be seen that the HAMA-MnO2 / LOX composite material prepared by the present invention is actually an intratumoral in situ cross-linked hydrogel nanocomposite that targets the acidity and hypoxia of the tumor microenvironment and can assist in the treatment of cancer. It is used to synergistically regulate the immune response and the polarization state of tumor-associated macrophages in the tumor microenvironment, especially to synergistically improve the low pH and hypoxia conditions in the tumor microenvironment, and promote the polarization of tumor-associated macrophages to M1, thereby increasing or decreasing the effect of radioimmunotherapy.

[0072] Furthermore, the HAMA-MnO2 / LOX composite material prepared by the present invention is expected to be used as a drug delivery system or drug carrier for anticancer drugs (chemotherapeutics, biologics, or other drugs). This drug delivery system or drug carrier system can form a hydrogel containing MnO2 nanoparticles and LOX through a cross-linking reaction between hyaluronic acid and calcium ions within the tumor. This hydrogel then acts synergistically in the tumor microenvironment, improving low pH and hypoxia conditions and promoting the polarization of tumor-associated macrophages toward M1, thereby enhancing the effectiveness of radioimmunotherapy and promoting tumor cell apoptosis.

[0073] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing an intratumoral in situ cross-linked hydrogel nanocomposite, characterized in that: The following steps are involved: Step 1: Completely dissolve hyaluronic acid in ultrapure water, add methacrylic anhydride dropwise while stirring at high speed, and then stir at high speed for a period of time at 0°C; Step 2: dialyzing the solution obtained in step 1 for a period of time using a dialysis bag with a specific molecular weight cut-off, and freeze-drying to obtain methacrylated hyaluronic acid; Step 3: dissolving the methacrylated hyaluronic acid obtained in step 2 in ultrapure water, then adding potassium permanganate solution and mixing, and reducing the potassium permanganate to manganese dioxide using the energy of the double bond; Step 4: dialyzing the mixed solution obtained in step 3 for a period of time using a dialysis bag with a specific molecular weight cut-off, and freeze-drying to obtain a methacrylated hyaluronic acid-manganese dioxide composite material; Step 5: dissolving the methacrylated hyaluronic acid-manganese dioxide composite material obtained in step 4 in ultrapure water, adding lactate oxidase, and stirring thoroughly to obtain an intratumoral in situ cross-linked hydrogel nanocomposite, namely, HAMA-MnO2 / LOX; The HAMA-MnO2 / LOX can be combined with physiological concentrations of Ca 2+ Solution cross-linking generated a hydrogel with manganese dioxide nanoparticles and lactate oxidase.

2. The method for preparing the intratumoral in situ cross-linked hydrogel nanocomposite according to claim 1, characterized in that: In step 1, the high-speed stirring time is 24 hours.

3. The method for preparing the intratumoral in situ cross-linked hydrogel nanocomposite according to claim 1, characterized in that: In step 2, the molecular weight cut-off of the dialysis bag is 8000-14000 kDa, and the dialysis time is 48 h.

4. The method for preparing the intratumoral in situ cross-linked hydrogel nanocomposite according to claim 1, characterized in that: In step 3, when the color of the potassium permanganate solution changes from purple to brown, it means that the potassium permanganate in the mixed solution has been completely reduced to manganese dioxide.

5. The method for preparing the intratumoral in situ cross-linked hydrogel nanocomposite according to claim 1, characterized in that: In step 4, the molecular weight cut-off of the dialysis bag is 3000 kDa, and the dialysis time is 48 h.

6. An intratumoral in situ cross-linked hydrogel nanocomposite, characterized in that: The intratumoral in situ cross-linked hydrogel nanocomposite is prepared by the preparation method according to any one of claims 1 to 5.

7. Use of an intratumoral in situ cross-linked hydrogel nanocomposite prepared by the method for preparing an intratumoral in situ cross-linked hydrogel nanocomposite according to any one of claims 1 to 5 or the intratumoral in situ cross-linked hydrogel nanocomposite according to claim 6 in preparing a drug for treating tumors, characterized in that: Used to synergistically regulate the immune response in the tumor microenvironment and the polarization state of tumor-associated macrophages.

8. The use according to claim 7, characterized in that Used to synergistically improve the low pH and hypoxic conditions in the tumor microenvironment and promote the polarization of tumor-associated macrophages to M1.

9. Use of an intratumoral in situ cross-linked hydrogel nanocomposite prepared by the method for preparing an intratumoral in situ cross-linked hydrogel nanocomposite according to any one of claims 1 to 5 or the intratumoral in situ cross-linked hydrogel nanocomposite according to claim 6 in preparing a drug delivery system or a drug loading system, characterized in that: Used as a drug delivery system or drug carrier system for anti-cancer drugs including chemotherapy drugs and biological agents.

10. The use according to claim 9, characterized in that The drug delivery system or drug loading system of the anticancer drug can form a hydrogel with manganese dioxide nanoparticles and lactate oxidase in the tumor through the cross-linking reaction of its own hyaluronic acid and calcium ions, and play a synergistic role in the tumor microenvironment, improving low pH and hypoxia conditions, and promoting the polarization of tumor-associated macrophages to M1, thereby helping to improve the effect of radioimmunotherapy and promote the apoptosis of tumor cells.

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