A hafnium-based bisphosphonate coacervate hydrogel sustained release system, and a preparation method and application thereof

Abstract

The application provides a hafnium-based bisphosphonate condensate hydrogel sustained-release system and a preparation method and application thereof, and belongs to the field of bioengineering and material technology. A hafnium tetrachloride solution is added dropwise into a mixed solution containing a bisphosphonate drug and a chemotherapeutic drug, a reaction solution is obtained through liquid-liquid phase separation, and the obtained precipitate is the hafnium-based bisphosphonate condensate hydrogel sustained-release system after stirring and centrifugation. The hafnium-based bisphosphonate condensate hydrogel sustained-release system prepared by the preparation method is disclosed, and the application of the hafnium-based bisphosphonate condensate hydrogel sustained-release system in the preparation of a bone sarcoma radiotherapy sensitization agent and a drug delivery system is disclosed. The hafnium-based bisphosphonate condensate hydrogel sustained-release system prepared by the application has the ideal characteristics of easy preparation, super-high drug loading, pH-dependent controlled release, 'fine tuning' of the release rate, avoidance of drug waste, improvement of the treatment effect and control of adverse side effects.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and materials technology, and particularly relates to a hafnium-based bisphosphonate aggregate hydrogel sustained-release system, its preparation method and application. Background Technology

[0002] Osteosarcoma (OS) is the most common type of malignant bone tumor, occurring primarily in children and adolescents, and has extremely high rates of disability and mortality. Standard clinical treatment includes surgical resection combined with neoadjuvant chemotherapy and radiotherapy. However, positive surgical margins (R1 / R2 resection) and the development of chemotherapy resistance have become key adverse prognostic factors limiting improvements in overall survival. Clinical evidence shows that incomplete resection can increase the risk of local recurrence in trunk osteosarcoma by 3-5 times. These patients may benefit from radiotherapy. Radiotherapy triggers tumor cell death by inducing DNA damage and activating immune-mediated antitumor effects (such as dendritic cell-mediated T cell activation). Notably, high-dose radiotherapy produces immunosuppression, accelerating primary tumor progression and promoting distant metastasis—a significant drawback. In recent years, significant progress has been made in the development of radiosensitizers, achieving equivalent or even stronger tumor-killing effects at lower radiotherapy doses. These include various types such as small molecules, large molecules, and nanomaterials. However, these radiosensitizers still face many challenges and limitations in further clinical application. Existing radiosensitizers generally suffer from core defects such as low drug loading, poor tissue penetration, difficulty in precisely controlling the release rate, and lack of pH-responsive controlled release capability. This results in insufficient drug retention at the tumor site and short duration of action, leading to serious drug waste and significant adverse side effects due to systemic exposure. Furthermore, a single radiosensitizer can only focus on the single function of radiosensitizing and cannot synergistically address multiple issues such as tumor microenvironment immunosuppression, chemotherapy resistance, and bone destruction. It is difficult to achieve the multi-effect integrated treatment goal of "sensitization-chemotherapy-immunomodulation-bone protection" and cannot effectively reverse the immune escape phenomenon caused by high-dose radiotherapy.

[0003] M2 tumor-associated macrophages (M2-TAMs) in the tumor microenvironment (TME) are considered key effector cells mediating immunosuppression. Evidence suggests that M2-TAMs suppress T cell function by upregulating programmed death-ligand 1 (PD-L1) expression or secreting immunosuppressive cytokines such as IL-10 and TGF-β. Animal model studies have shown that targeted clearance of M2-TAMs can significantly restore the function of cytotoxic T lymphocytes (CTLs) and improve the tumor immune microenvironment. Bisphosphonates (BPs), with significant bone-targeting properties, are widely used clinically to alleviate bone erosion symptoms in osteosarcoma patients. Current research has found that BPs can reverse the tumor immunosuppressive state by regulating macrophage polarization, promoting the transformation of M2-type macrophages into pro-inflammatory M1-type macrophages, thereby achieving a shift from an "immunely cold tumor" to an "immunely hot tumor." This synergistic effect of immunomodulation and osteoprotection provides an important theoretical basis for the application of alendronate (ALN) in the treatment of osteosarcoma. However, bisphosphonates have low bioavailability and rapid metabolism when used alone, making it impossible to maintain an effective concentration for a long time. They also lack radiosensitizing activity, making it difficult to form a synergistic effect with radiotherapy and chemotherapy. At the same time, when traditional chemotherapy drugs are administered alone, they are easily cleared from the body, resulting in insufficient drug concentration at the tumor site and strong systemic toxicity, which seriously limits the therapeutic effect.

[0004] Therefore, developing a drug delivery system that combines high drug loading capacity, good tissue permeability, pH-dependent precise controlled release, and "fine-tunable" release rate, and can achieve multiple effects such as radiosensitization, chemotherapy synergy, immunomodulation, and bone protection, is of great significance for solving the bottleneck problems in the current treatment of osteosarcoma, improving treatment efficacy, and reducing side effects. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a hafnium-based bisphosphonate aggregate hydrogel sustained-release system, its preparation method, and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing a hafnium-based bisphosphonate aggregate hydrogel sustained-release system, comprising the following steps:

[0008] Hafnium tetrachloride solution was added dropwise to a mixed solution containing bisphosphonate drugs and chemotherapy drugs. The reaction solution was obtained by liquid-liquid phase separation. After stirring and centrifugation under certain temperature conditions, the resulting precipitate was the hafnium-based bisphosphonate aggregate hydrogel sustained-release system.

[0009] Beneficial Effects: The hafnium-based bisphosphonate aggregate hydrogel sustained-release system prepared in this invention uses bisphosphonates (BPs) as organic drug ligands, reacting with metal hafnium ions at a certain temperature to obtain a pH-dependent, high-drug-loading, and highly tissue-permeable sustained-release system. BPs can inhibit osteoclast formation, differentiation, maturation, and activity, and promote apoptosis. Hafnium ions, as a high-Z (atomic number) element, can increase the sensitivity to radiotherapy, generating more reactive oxygen species under low-dose radiotherapy conditions. The metal ions and ligands synergistically treat osteosarcoma, thereby improving immune escape induced by high-dose radiotherapy. Loading osteosarcoma chemotherapeutic drugs further enhances anti-osteosarcoma activity.

[0010] Furthermore, the bisphosphonate drug is selected from one or more of etidronate, pamidronate, alendronate, zoledronic acid, and risedronate.

[0011] Furthermore, the chemotherapy drug is selected from one or more of methotrexate, doxorubicin, and cisplatin.

[0012] Furthermore, the molar ratio of hafnium tetrachloride to bisphosphonate drug and chemotherapy drug in the hafnium tetrachloride solution is (1000~2500):(1000~2500):1.

[0013] Furthermore, the concentration of the hafnium tetrachloride solution is 1~100 mmol / L.

[0014] Furthermore, the concentration of the bisphosphonate drug in the mixed solution is 1~100 mmol / L.

[0015] Furthermore, the concentration of the chemotherapeutic drug in the mixed solution is 0.001~0.1 mmol / L.

[0016] Furthermore, the reaction temperature of the stirring reaction is 4~60℃, and the reaction time is 1~6h.

[0017] The present invention also provides a hafnium bisphosphonate aggregate hydrogel sustained-release system prepared by the above preparation method.

[0018] The present invention also provides the application of the above-mentioned hafnium bisphosphonate aggregate hydrogel sustained-release system in the preparation of a radiosensitizer for the treatment of osteosarcoma.

[0019] The present invention also provides the application of the above-mentioned hafnium bisphosphonate aggregate hydrogel sustained-release system in the preparation of a drug delivery system for the treatment of osteosarcoma.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] This invention provides a hafnium-based bisphosphonate aggregate hydrogel sustained-release system, its preparation method, and its applications. This invention combines hafnium ions with bisphosphonate drugs and loads them with chemotherapeutic drugs for osteosarcoma to obtain a hydrogel sustained-release system. Compared with other radiosensitization and drug delivery systems, the hafnium-based bisphosphonate aggregate hydrogel sustained-release system prepared by this invention has ideal characteristics such as ease of preparation, ultra-high drug loading capacity, pH-dependent controlled release, "fine-tuned" release rate, avoidance of drug waste, improved therapeutic effect, and control of adverse side effects. Attached Figure Description

[0022] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0023] Figure 1 The sustained-release curves of the hafnium etidronate aggregate hydrogel sustained-release system prepared in Example 1 are shown, where a is the release rate of etidronate and b is the release rate of methotrexate.

[0024] Figure 2 The sustained-release curves of the hafnium pamidronate aggregate hydrogel sustained-release system prepared in Example 2 are shown, where a is the release rate of pamidronate and b is the release rate of doxorubicin.

[0025] Figure 3 The sustained-release curves of the hafnium-based alendronate aggregate hydrogel sustained-release system prepared in Example 3 are shown, where a is the release rate of alendronate and b is the release rate of cisplatin.

[0026] Figure 4 The sustained-release curves of the hafnium zoledronic acid condensate hydrogel sustained-release system prepared in Example 4 are shown, where a is the release rate of zoledronic acid and b is the release rate of methotrexate.

[0027] Figure 5 The sustained-release curves of the hafnium-based risedronate aggregate hydrogel sustained-release system prepared in Example 5 are shown, where a is the release rate of risedronate and b is the release rate of doxorubicin.

[0028] Figure 6 The drug loading rate of bisphosphonate drugs in the hafnium-based bisphosphonate aggregate hydrogel sustained-release systems prepared in Examples 1-5;

[0029] Figure 7 The drug loading rate of the chemotherapeutic drugs in the hafnium bisphosphonate aggregate hydrogel sustained-release systems prepared in Examples 1-5;

[0030] Figure 8 The results of DAPI fluorescence staining of γ-H2AX after treatment of mouse osteosarcoma osteoblasts (K7M2-wt) in each group;

[0031] Figure 9 The results of Goldner trichrome staining are shown for each group after treatment of mouse orthotopic tibial osteosarcoma models under radiotherapy conditions. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] This invention provides a method for preparing a hafnium-based bisphosphonate aggregate hydrogel sustained-release system, comprising the following steps:

[0038] Hafnium tetrachloride solution was added dropwise to a mixed solution containing bisphosphonate drugs and chemotherapy drugs. The reaction solution was obtained by liquid-liquid phase separation. After stirring and centrifugation under certain temperature conditions, the resulting precipitate was the hafnium-based bisphosphonate aggregate hydrogel sustained-release system.

[0039] This invention provides a simple and mild one-step method for preparing hydrogel sustained-release systems, achieving in-situ drug loading during gel formation, avoiding subsequent drug loading steps, improving drug loading efficiency and uniformity, and resulting in a gel with pH-responsive drug release, high tissue permeability, and sustained-release properties. This invention utilizes the dissociation of hafnium tetrachloride in water to release hafnium ions (Hf). 4+ It coordinates with the phosphonic acid groups of bisphosphonate molecules to form a three-dimensional network cross-linked structure. Liquid-liquid phase separation promotes local concentration increase, accelerates coordination reaction, forms micron / nano-scale condensates, provides mild thermodynamic conditions for the reaction, promotes coordination network stabilization, and embeds chemotherapeutic drug molecules.

[0040] In a preferred embodiment of the present invention, the bisphosphonate drug is selected from one or more of etidronate, pamidronate, alendronate, zoledronic acid, and risedronate. The PCP structure in the bisphosphonate molecule used in the present invention has an affinity for bone-strengthening minerals (hydroxyapatite) to achieve bone targeting, and its phosphonate group forms a stable coordination bond with hafnium ions to construct a gel framework.

[0041] In a preferred embodiment of the present invention, the chemotherapeutic drug is selected from one or more of methotrexate, doxorubicin, and cisplatin. The chemotherapeutic drug used in the present invention is embedded in the gel network through physical encapsulation or weak interactions (such as hydrogen bonding and hydrophobic interactions), and is gradually released under gel degradation or pH response, maintaining a high local concentration and reducing systemic toxicity.

[0042] In a preferred embodiment of the present invention, the molar ratio of hafnium tetrachloride to bisphosphonate drug and chemotherapeutic drug in the hafnium tetrachloride solution is (1000~2500):(1000~2500):1. The molar ratio of hafnium to bisphosphonate affects the number of coordination sites and the integrity of the gel network. The lower proportion of chemotherapeutic drug avoids interference with the formation of the main network while ensuring the therapeutic dose.

[0043] In a preferred embodiment of the present invention, the concentration of the hafnium tetrachloride solution is 1~100 mmol / L. By controlling the hafnium ion concentration, the gel formation rate and structural compactness are affected, avoiding excessively high concentrations that lead to rapid precipitation and uneven structure.

[0044] In a preferred embodiment of the present invention, the concentration of the bisphosphonate drug in the mixed solution is 1~100 mmol / L.

[0045] In a preferred embodiment of the present invention, the concentration of the chemotherapy drug in the mixed solution is 0.001~0.1 mmol / L.

[0046] In a preferred embodiment of the present invention, the reaction temperature of the stirring reaction is 4~60℃ and the reaction time is 1~6h.

[0047] For example, in an embodiment of the present invention, a method for preparing a hafnium-based bisphosphonate aggregate hydrogel sustained-release system includes the following steps:

[0048] (1) Prepare a hafnium tetrachloride solution of 1~100 mmol / L, and at the same time prepare a mixed solution of bisphosphonate drug (etidronate, pamidronate, alendronate, zoledronic acid or risedronate, concentration of 1~100 mmol / L) and chemotherapy drug (methotrexate, doxorubicin or cisplatin, concentration of 0.001~0.1 mmol / L). Then add 20 mL of hafnium tetrachloride solution dropwise to 20 mL of the mixed solution of bisphosphonate drug and chemotherapy drug, and obtain the reaction solution by liquid-liquid phase separation;

[0049] (2) Continue stirring the reaction solution and react at 4~60℃ for 1~6h. Then centrifuge and discard the supernatant to obtain the hafnium bisphosphonate aggregate hydrogel sustained-release system.

[0050] The hafnium tetrachloride solution is prepared by dissolving hafnium tetrachloride in water and stirring until homogeneous; the bisphosphonate drug solution is prepared by dissolving bisphosphonate in water and stirring until homogeneous; the chemotherapy drug solution is prepared by dissolving chemotherapy drug in dimethyl sulfoxide and stirring until homogeneous; and the mixed solution of bisphosphonate and chemotherapy drug is prepared by adding the chemotherapy drug solution to the bisphosphonate solution and stirring until homogeneous.

[0051] This invention also provides a hafnium-based bisphosphonate aggregate hydrogel sustained-release system prepared by the above-described method. The invention yields a multifunctional hydrogel sustained-release system with pH responsiveness, high drug loading capacity, bone targeting, and radiosensitization capabilities. It enables synergistic drug release and treatment of bisphosphonates, chemotherapeutic drugs, and hafnium ions. The gel network is constructed based on Hf-bisphosphonate coordination bonds and exhibits pH-sensitive dissociation properties (accelerated release in an acidic tumor microenvironment). Hafnium, as a high-zir element, enhances radiosensitivity, and bisphosphonates regulate the immune microenvironment.

[0052] This invention also provides an application of the above-mentioned hafnium bisphosphonate aggregate hydrogel sustained-release system in the preparation of a radiosensitizer for the treatment of osteosarcoma.

[0053] This invention also provides an application of the above-mentioned hafnium bisphosphonate aggregate hydrogel sustained-release system in the preparation of a drug delivery system for the treatment of osteosarcoma.

[0054] All raw materials used in the embodiments of this invention were commercially available. Hafnium tetrachloride, etidronate, pamidronate, alendronate, zoledronic acid, risedronate, methotrexate, doxorubicin and cisplatin were purchased from Aladdin, and K7M2-wt cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0055] The technical solution of the present invention will be further illustrated by the following embodiments.

[0056] Example 1

[0057] A method for preparing a hafnium-based bisphosphonate aggregate hydrogel sustained-release system includes the following steps:

[0058] (1) Prepare a 1 mmol / L hafnium tetrachloride solution, and at the same time prepare a mixed solution of bisphosphonate drug (etidronate, concentration of 1 mmol / L) and chemotherapy drug (methotrexate, concentration of 0.001 mmol / L). Then add 20 mL of hafnium tetrachloride solution dropwise to 20 mL of the mixed solution of bisphosphonate drug and chemotherapy drug. After liquid-liquid phase separation, the reaction solution is obtained. In this step, the molar ratio of hafnium tetrachloride, bisphosphonate drug and chemotherapy drug is 1:1:0.001.

[0059] (2) Continue stirring the reaction solution and react at 4°C for 1 hour. Then centrifuge and discard the supernatant to obtain the hafnium etiphosphonate aggregate hydrogel sustained-release system.

[0060] Example 2

[0061] A method for preparing a hafnium-based bisphosphonate aggregate hydrogel sustained-release system includes the following steps:

[0062] (1) Prepare a 25 mmol / L hafnium tetrachloride solution, and at the same time prepare a mixed solution of bisphosphonate drug (pamidronate, concentration of 25 mmol / L) and chemotherapy drug (doxorubicin, concentration of 0.01 mmol / L). Then add 20 mL of hafnium tetrachloride solution dropwise to 20 mL of the mixed solution of bisphosphonate drug and chemotherapy drug. After liquid-liquid phase separation, the reaction solution is obtained. In this step, the molar ratio of hafnium tetrachloride, bisphosphonate drug and chemotherapy drug is 25:25:0.01.

[0063] (2) Continue stirring the reaction solution and react at 25°C for 2 hours. Then centrifuge and discard the supernatant to obtain the hafnium pamidronate aggregate hydrogel sustained-release system.

[0064] Example 3

[0065] A method for preparing a hafnium-based bisphosphonate aggregate hydrogel sustained-release system includes the following steps:

[0066] (1) Prepare a 50 mmol / L hafnium tetrachloride solution, and at the same time prepare a mixed solution of bisphosphonate drug (alendronate, concentration of 50 mmol / L) and chemotherapy drug (cisplatin, concentration of 0.02 mmol / L). Then add 20 mL of hafnium tetrachloride solution dropwise to 20 mL of the mixed solution of bisphosphonate drug and chemotherapy drug. After liquid-liquid phase separation, the reaction solution is obtained. In this step, the molar ratio of hafnium tetrachloride, bisphosphonate drug and chemotherapy drug is 50:50:0.02.

[0067] (2) Continue stirring the reaction solution and react at 37°C for 3 hours. Then centrifuge and discard the supernatant to obtain the hafnium alenphosphonate aggregate hydrogel sustained-release system.

[0068] Example 4

[0069] A method for preparing a hafnium-based bisphosphonate aggregate hydrogel sustained-release system includes the following steps:

[0070] (1) Prepare a 75 mmol / L hafnium tetrachloride solution, and at the same time prepare a mixed solution of bisphosphonate drug (zoledronic acid, concentration of 75 mmol / L) and chemotherapy drug (methotrexate, concentration of 0.05 mmol / L). Then add 20 mL of hafnium tetrachloride solution dropwise to 20 mL of the mixed solution of bisphosphonate drug and chemotherapy drug. After liquid-liquid phase separation, the reaction solution is obtained. In this step, the molar ratio of hafnium tetrachloride, bisphosphonate drug and chemotherapy drug is 75:75:0.05.

[0071] (2) Continue stirring the reaction solution and react at 45°C for 6 hours. Then centrifuge and discard the supernatant to obtain the hafnium azole phosphonate aggregate hydrogel sustained-release system.

[0072] Example 5

[0073] A method for preparing a hafnium-based bisphosphonate aggregate hydrogel sustained-release system includes the following steps:

[0074] (1) Prepare a 100 mmol / L hafnium tetrachloride solution, and at the same time prepare a mixed solution of bisphosphonate drug (risedronate, concentration of 100 mmol / L) and chemotherapy drug (doxorubicin, concentration of 0.1 mmol / L). Then add 20 mL of hafnium tetrachloride solution dropwise to 20 mL of the mixed solution of bisphosphonate drug and chemotherapy drug. After liquid-liquid phase separation, the reaction solution is obtained. In this step, the molar ratio of hafnium tetrachloride, bisphosphonate drug and chemotherapy drug is 100:100:0.1.

[0075] (2) Continue stirring the reaction solution and react at 60°C for 3 hours. Then centrifuge and discard the supernatant to obtain the hafnium phosphonate aggregate hydrogel sustained-release system.

[0076] Performance testing

[0077] The in vitro release kinetics of hafnium-based bisphosphonate aggregate hydrogels prepared in Examples 1-5 were studied using a Transwell diffusion system in a simulated physiological environment (37°C, PBS buffer at pH 4.5 or 7.4). 200 μL of sample was added to the upper chamber, and the release medium from the lower chamber was collected at different time points (6-96 hours). The hafnium ion concentration was determined by ICP-MS, and the release amounts of the corresponding bisphosphonates and chemotherapeutic drugs were analyzed by HPLC.

[0078] The sustained-release curves of the hafnium-based bisphosphonate aggregate hydrogel sustained-release systems prepared in Examples 1-5 are shown below. Figures 1-5 Where a represents the release rate of the bisphosphonate drug, and b represents the release rate of the hafnium-based bisphosphonate aggregate hydrogel. It can be seen that the hafnium-based bisphosphonate aggregate hydrogel prepared in this embodiment of the invention has the effect of slowly releasing ions and drugs, and exhibits pH-dependent characteristics.

[0079] The drug loading rates of the hafnium-based bisphosphonate aggregate hydrogel sustained-release systems prepared in Examples 1-5 are shown in the figures below. Figure 6 and Figure 7 ,in Figure 6 The drug loading rate of bisphosphonate drugs in the hafnium-based bisphosphonate aggregate hydrogel sustained-release systems in each embodiment is shown. Figure 7 The figures represent the drug loading rates of chemotherapeutic drugs in the hafnium-based bisphosphonate aggregate hydrogel sustained-release systems in each embodiment. It can be seen that the hafnium-based bisphosphonate aggregate hydrogels prepared in the embodiments of the present invention have extremely high drug loading capacities.

[0080] Comparative Example 1

[0081] Same as Example 4, except that the addition of methotrexate is omitted, and the specific steps are as follows:

[0082] (1) Prepare a 75 mmol / L hafnium tetrachloride solution and a bisphosphonate drug (zoledronic acid, concentration 75 mmol / L) solution. Then add 20 mL of hafnium tetrachloride solution to 20 mL of bisphosphonate drug solution and obtain the reaction solution by liquid-liquid phase separation.

[0083] (2) Continue stirring the reaction solution and react at 45°C for 6 hours. Then centrifuge and discard the supernatant to obtain the hafnium-zoledronic acid hydrogel sustained-release system.

[0084] Application Example 1

[0085] To verify the promoting effect of the hafnium azole phosphonate condensate hydrogel prepared in this invention on osteosarcoma cell apoptosis, the expression level of the DNA damage marker γ-H2AX was detected to clarify its induction effect on cell apoptosis.

[0086] The experiment used mouse osteosarcoma osteoblasts (K7M2-wt). The mouse osteosarcoma osteoblasts (K7M2-wt) were administered at a concentration of 10... 4 Cells were seeded in confocal culture dishes and, after adhesion, 10 μL of hafnium-zoledronic acid condensate hydrogel prepared in Example 4 (denoted as methotrexate@hafnium-zoledronic acid + radiotherapy group) was added. After co-culturing for 24 hours, the cells were irradiated with 8 Gy X-rays for 10 min to fix them. Immunofluorescence staining was performed using an antibody against the DNA damage marker γ-H2AX to detect cell apoptosis.

[0087] A blank control group was set up: only an equal volume of PBS buffer was added, and no drug treatment or radiotherapy was performed;

[0088] Radiotherapy group: No drugs were added, and they received only X-ray irradiation (irradiation dose 8 Gy, irradiation time 10 min).

[0089] Methotrexate group: The same amount of free methotrexate (MTX) as the subsequent gel group was added, and no radiotherapy was performed;

[0090] Hafnium-zoledronic acid + radiotherapy group: 10 μL of the hafnium-zoledronic acid hydrogel sustained-release system prepared in Comparative Example 1 was added, and after incubation for 12 h, the group was irradiated with 8 Gy X-rays for 10 min.

[0091] The results of γ-H2AX staining with 4',6-diamidinyl-2-phenylindole (DAPI) after treatment of mouse osteosarcoma osteoblasts (K7M2-wt) in each group are as follows: Figure 8 As shown. According to Figure 8 The results show that treatment with the hafnium azole phosphonate aggregate hydrogel sustained-release system of Example 4 can significantly enhance the apoptosis effect of K7M2-wt cells.

[0092] Application Example 2

[0093] To confirm the anti-osteosarcoma effect of the hafnium azole phosphonate aggregate hydrogel prepared in this invention, a mouse tibial osteosarcoma model was established. BALB / c mice were selected, and 1x10⁻⁶ oz.5 ... 6 An orthotopic osteosarcoma model was established using K7M2-wt cells. The model was considered successfully established after tumor growth and the formation of clearly defined bone destruction lesions. Hafnium-zoledronic acid (HZZ) aggregate hydrogel prepared in Example 4 was used for local administration at a dose of 50 μL / week. After 4 weeks of administration, mice were euthanized, and the affected tibia was harvested for Goldner trichrome staining histological analysis to observe changes in tumor volume, degree of bone destruction, and intratumoral bone density. Separate control groups, radiotherapy groups, methotrexate groups, and hafnium-zoledronic acid + radiotherapy groups were also included, with treatment methods identical to those in Example 1.

[0094] The results of Goldner trichrome staining of mouse orthotopic tibial osteosarcoma models after treatment under radiotherapy conditions are as follows: Figure 9 As shown in the figure, B, M, and T represent bone tissue, bone marrow, and tumor, respectively. It can be seen that in this embodiment, after combined treatment, the hafnium zoledronic acid aggregate hydrogel loaded with methotrexate exhibited a more significant effect in inhibiting osteosarcoma growth and reducing bone destruction, demonstrating its potential as a targeted therapy for osteosarcoma.

[0095] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing a hafnium-based bisphosphonate aggregate hydrogel sustained-release system, characterized in that, Includes the following steps: Hafnium tetrachloride solution was added dropwise to a mixed solution containing bisphosphonate drugs and chemotherapy drugs. The reaction solution was obtained by liquid-liquid phase separation. After stirring and centrifugation, the resulting precipitate was the hafnium-based bisphosphonate aggregate hydrogel sustained-release system. The bisphosphonate drug is selected from one or more of etidronate, pamidronate, alendronate, zoledronic acid, and risedronate; The chemotherapy drug is selected from one or more of methotrexate, doxorubicin, and cisplatin; The molar ratio of hafnium tetrachloride to bisphosphonate drug and chemotherapy drug in the hafnium tetrachloride solution is (1000~2500):(1000~2500):1; The temperature of the stirring reaction is 4~60℃, and the reaction time is 1~6h.

2. The preparation method according to claim 1, characterized in that, The concentration of the hafnium tetrachloride solution is 1~100 mmol / L.

3. The preparation method according to claim 1, characterized in that, The concentration of the bisphosphonate drug in the mixed solution is 1~100 mmol / L; the concentration of the chemotherapeutic drug in the mixed solution is 0.001~0.1 mmol / L.

4. A hafnium bisphosphonate aggregate hydrogel sustained-release system prepared by the preparation method according to any one of claims 1-3.

5. The use of the hafnium bisphosphonate aggregate hydrogel sustained-release system as described in claim 4 in the preparation of a radiosensitizer for the treatment of osteosarcoma.

6. The use of the hafnium bisphosphonate aggregate hydrogel sustained-release system as described in claim 4 in the preparation of a drug delivery system for the treatment of osteosarcoma.

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