A nanoparticle, a sustained-release hydrogel containing the same, and an ultrasound system and application thereof

Abstract

The application discloses a kind of nanoparticles, sustained-release hydrogel containing it and its ultrasonic system and application, the nanoparticle described in the application is formed by zinc nitrate hexahydrate and dimethyl imidazole form metal organic framework encapsulation hematoporphyrin monomethyl ether and plasmid expressing ZBP1.Form the application's ultrasonic regulation multifunctional hydrogel platform, the accurate control of ROS release is realized by ultrasound to achieve double treatment effect, while synergistic key gene regulation and improvement of microenvironment, cope with tumor treatment and bone regeneration problem in breast cancer bone metastasis.The application develops a kind of hydrogel loaded MOF multifunctional system with biological safety, has excellent antitumor and promotes the dual effect of bone repair, has in-situ injection and sustained-release type, and treatment ingredients are highly concentrated in target area, so as to significantly improve the curative effect and reduce side effect.

Description

Technical Field

[0001] This invention belongs to the field of ultrasonic dynamics, specifically relating to a nanoparticle, a sustained-release hydrogel containing the nanoparticle, and their ultrasonic systems and applications. Background Technology

[0002] In the treatment of breast cancer bone metastases, simultaneously eliminating tumor cells and promoting bone regeneration has been a pressing challenge. In recent years, reactive oxygen species (ROS), as important intracellular signaling molecules, have demonstrated unique bidirectional regulatory roles in tumor treatment and tissue repair, showing promise as an effective means to overcome this challenge. Abnormal accumulation of high concentrations of ROS in tumor cells can cause irreversible damage to proteins, nucleic acids, lipids, cell membranes, and organelles, ultimately leading to programmed cell death (PCD).

[0003] Low concentrations of reactive oxygen species (ROS) can accelerate bone repair by regulating multiple signaling pathways in osteoblasts and increasing chromatin accessibility to osteogenic genes, thereby promoting the proliferation, differentiation, and mineralization of bone marrow mesenchymal stem cells (BMSCs). This bidirectional regulatory effect makes ROS an ideal therapeutic molecule, but how to achieve precise regulation, especially under the dual needs of tumor treatment and bone repair, still requires further exploration. Ultrasound, as a non-invasive therapeutic method, has excellent controllability and high targeting. With the aid of sonosensitive agents, ROS generation can be released on demand in space and time, and the release amount can be precisely controlled by controlling the ultrasound frequency, flexibly addressing different treatment needs. Simultaneously, ultrasound has significant deep penetration capabilities and avoids phototoxicity, making it an ideal choice for treating deeper tumor tissues in vivo.

[0004] However, while simply regulating ROS concentration can exert anti-tumor and bone repair effects to some extent, its therapeutic efficacy is still limited. Firstly, most sonosensitive agents suffer from poor chemical stability, short circulation times, and limited tumor accumulation, affecting treatment efficacy. Secondly, the hypoxic tumor microenvironment and changes in key gene phenotypes lead to increased tolerance of tumor cells to oxidative stress, adapting to high ROS concentrations and thus limiting the killing effect of ROS. ZBP1 is a Z-DNA and Z-RNA binding protein. As a cytoplasmic nucleic acid sensor, it can trigger different forms of cell death and inflammatory responses. Studies have found that ZBP1 is usually expressed at low levels in genomically unstable tumors such as triple-negative breast cancer, leading to increased tolerance of tumor cells to oxidative stress and insufficient immune response, thus increasing the difficulty of treatment. Upregulating ZBP1 gene expression can effectively activate its downstream cell death pathways, increase the sensitivity of tumor cells to ROS, and thus enhance the therapeutic effect. It is worth noting that the role of ZBP1 in bone repair should not be overlooked. Studies have shown that ZBP1 can promote β-catenin nuclear translocation, regulate Wnt signaling, and thus promote osteogenic differentiation of BMSCs. Therefore, effectively upregulating gene expression might help achieve a dual effect of anti-tumor and bone regeneration. However, naked nucleic acids are very fragile and are rapidly degraded by serum nucleases in the blood. Finding a safe and effective delivery vector is crucial for gene regulation. Metal-organic frameworks (MOFs) are porous coordination polymers constructed by bridging metal ions or metal clusters with organic linkers. They possess open porous structures, high loading capacity, and good stability, allowing for biomimetic mineralization of biomolecules through one-pot encapsulation, making them a promising platform for biomedical applications. In recent years, MOFs have been studied for the delivery or controlled release of DNA, small interfering RNA (siRNA), and nucleic acid aptamers, demonstrating high efficiency in intracellular gene delivery and expression. Simultaneously, MOF encapsulation can protect the stability of sonosensitive agents during transport, and their metal ions or organic ligands can further promote the activation of sonosensitive agents. For example, metal ions in MOFs (such as Zn) 2+Under ultrasound stimulation, the generation of ROS can be promoted, thereby enhancing the effect of sonodynamic therapy. For hypoxic microenvironments, CaO2 has excellent oxygen release properties, continuously releasing oxygen in a weakly acidic environment, which not only helps improve the oxygenation environment of the tumor area but also promotes bone repair. Hydrogels, due to their excellent biocompatibility, controllability, sustained drug release, stimulus responsiveness, and tissue repair capabilities, have become an ideal choice in recent years for drug delivery, tissue regeneration, and combination therapy. Through rational design, the needs of simultaneously regulating ROS levels, ZBP1 gene expression, and improving the hypoxic microenvironment can be met. Furthermore, therapeutic components can be released simultaneously in the target area, thereby enhancing the therapeutic effect and overcoming the limitations of single-treatment methods. Compared to intravenous injection and oral administration, it can minimize the adverse effects of systemic drug exposure, showing great application prospects and clinical translation potential. The inventors have conducted in-depth research and experimentation on hydrogels as a bidirectional multifunctional material delivery medium.

[0005] The current mainstream technical solutions (or the closest technical solutions) have the following drawbacks / deficiencies:

[0006] 1. In the treatment of breast cancer bone metastases, current treatment plans usually focus on a single treatment goal, such as tumor treatment or bone repair, and lack a comprehensive treatment plan that takes both into account.

[0007] 2. In the process of regulating ROS to exert anti-tumor effects, current technologies neglect the influence of changes in the tumor microenvironment and gene phenotype on the oxidative stress tolerance of tumor cells. Simply regulating ROS may not be effective in killing tumor cells.

[0008] 3. The intravenous injection method and the presence of the bone-blood barrier make it difficult for traditional nanomedicines to achieve an effective therapeutic dose at the tumor site, and at the same time, nanomedicines may cause unnecessary damage to normal tissues. Summary of the Invention

[0009] To overcome the shortcomings of the existing technical solutions, we need to solve the following problems:

[0010] 1. To address the dual needs of tumor treatment and bone repair, a dual-function treatment system will be developed and designed. Through precise drug delivery and multiple treatment mechanisms, the system can simultaneously meet the dual requirements of tumor cell elimination and bone defect repair.

[0011] 2. Develop therapeutic strategies that can precisely regulate ROS generation, while taking into account the influence of the tumor microenvironment, and enhance the sensitivity of tumor cells to ROS through gene expression regulation (e.g., upregulating ZBP1) to improve therapeutic efficacy.

[0012] 3. By using in-situ injection, the local concentration of nanomedicines can be increased, thereby minimizing unnecessary damage to normal tissues caused by nanomedicines during anti-tumor treatment.

[0013] To address the aforementioned technical problems, this invention provides nanoparticles, a sustained-release hydrogel containing them, an ultrasound system, and their applications. This invention develops an innovative ultrasound-controlled multifunctional hydrogel platform. This system achieves precise control of ROS release through ultrasound to achieve dual therapeutic effects, while simultaneously regulating key genes and improving the microenvironment, addressing both tumor treatment and bone regeneration issues in breast cancer bone metastases. The invention upregulates ZBP1 gene expression and provides a continuous oxygen supply to a hypoxic microenvironment, which can enhance the responsiveness of tumor cells to ROS, induce necrosis and apoptosis, and significantly enhance anti-tumor efficacy. This invention develops a biosafety-compliant hydrogel-loaded MOF multifunctional system with excellent dual effects of anti-tumor and bone repair promotion. It features in-situ injection and sustained-release properties, with therapeutic components highly concentrated in the target area, thereby significantly improving efficacy and reducing side effects.

[0014] To address the aforementioned technical problems, the first aspect of this invention provides a nanoparticle formed by encapsulating hematoporphyrin monomethyl ether and a plasmid containing the ZBP1 sequence within a metal-organic framework (ZIF-8) formed by zinc nitrate hexahydrate and dimethylimidazole.

[0015] The metal-organic framework (ZIF-8) described in this invention is a three-dimensional network structure formed by zinc ions and 2-methylimidazole through coordination bonds, and has a highly ordered crystal structure and permanent pores.

[0016] The plasmid containing the ZBP1 sequence described in this invention is a plasmid containing the ZBP1 gene sequence, and the backbone of the plasmid can be a plasmid backbone conventionally used in the art.

[0017] In a preferred embodiment, the ZBP1 gene is mouse ZBP1, with GenBank number NM_021394.2 and NCBI Gene ID 58203.

[0018] In a preferred embodiment, the ZBP1 gene is rat ZBP1, with GenBank number NM_133564.1 and NCBI Gene ID 171091.

[0019] In a preferred embodiment, the ZBP1 gene is human ZBP1, whose NCBI Gene ID is 81030.

[0020] In a preferred embodiment, the plasmid containing the ZBP1 gene was purchased from Yunzhou Biotechnology.

[0021] The ZBP1 gene (Z-DNA binding protein 1, also known as DAI, DLM-1, or ZBP) described in this invention is a protein-encoding gene whose main function is to regulate immune responses, cell death, and inflammatory signaling pathways by recognizing specific nucleic acid structures (such as left-handed Z-configuration double-stranded DNA and RNA). The ZBP1 gene is located on human chromosome 20, specifically at 20q13.31, and encodes a protein consisting of 429 amino acids with a molecular weight of 46.3 kDa.

[0022] In a preferred embodiment, the ZBP1 gene is a human or mouse gene.

[0023] In a preferred embodiment, the mass ratio of zinc nitrate hexahydrate to dimethylimidazole is 1:(5-20), for example 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20.

[0024] In a preferred embodiment, the mass ratio of hematoporphyrin monomethyl ether to plasmid containing the ZBP1 sequence is 1:(0.5-5), for example 1:0.5, 1:0.6, 1:0.8, 1:1, 1:1.2, 4:5, 1.1.5, 1:2, 1.2.5, 1:3, 1.3.5, 1:4, 1.4.5, or 1:5.

[0025] In a preferred embodiment, the mass ratio of hematoporphyrin monomethyl ether to dimethyl imidazole is 1:(800-2000), for example 1:900, 1:950, 1:1000, 1:1100, 1:1200, 1:1300, 1:1400, 1:1500, 1:1600, 1:1200, 1:1700, 1:1800, 1:1900, or 1:2000.

[0026] The second aspect of this invention protects a method for preparing nanoparticles, comprising adding hematoporphyrin monomethyl ether to an aqueous solution of 2-methylimidazole and then adding an aqueous solution of Zn(NO3)2·6H2O dropwise thereto.

[0027] In a preferred embodiment, the Zn(NO3)2·6H2O contains an aqueous solution of a plasmid with the ZBP1 sequence.

[0028] In a preferred embodiment, the ZBP1 gene is a human or mouse gene.

[0029] In a preferred embodiment, the mass ratio of zinc nitrate hexahydrate to dimethylimidazole is 1:(5-20), for example 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20.

[0030] In a preferred embodiment, the mass ratio of hematoporphyrin monomethyl ether to plasmid containing the ZBP1 sequence is 1:(0.5-5), for example 1:0.5, 1:0.6, 1:0.8, 1:1, 1:1.2, 4:5, 1.1.5, 1:2, 1.2.5, 1:3, 1.3.5, 1:4, 1.4.5, or 1:5.

[0031] In a preferred embodiment, the mass ratio of hematoporphyrin monomethyl ether to dimethyl imidazole is 1:(800-2000), for example 1:900, 1:950, 1:1000, 1:1100, 1:1200, 1:1300, 1:1400, 1:1500, 1:1600, 1:1200, 1:1700, 1:1800, 1:1900, or 1:2000.

[0032] A third aspect of the present invention protects a sustained-release hydrogel material comprising a hybrid gel formed of methacrylamide gelatin and methacrylamide sodium alginate, and nanoparticles as described in the first aspect of the present invention, or nanoparticles as described in the first aspect of the present invention excluding plasmids containing the ZBP1 sequence.

[0033] In a preferred embodiment, the raw material further includes oxygen-producing CaO2.

[0034] In a preferred embodiment, the mass ratio of the methacrylamide gelatin to the methacrylamide sodium alginate in the raw materials is (3-20):3, for example, 1:1, 4:3, 5:3, 2:1, 7:3, 8:3, 3:1, 10:3, 11:3, 4:1, 13:3, 14:3, 5:1, 16:3, 17:3, 6:1, 19:3, or 20:3.

[0035] In a preferred embodiment, the final concentration of nanoparticles in the raw material is 100–500 μg / mL, for example, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, or 500 μg / mL.

[0036] In a preferred embodiment, the final concentration of CaO2 in the raw material is 200–1000 μg / mL, for example, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL, or 1000 μg / mL.

[0037] A third aspect of the present invention provides a sustained-release hydrogel, which is formed by irradiating the sustained-release hydrogel raw material as described in the third aspect of the present invention with ultraviolet light.

[0038] In a preferred embodiment, the ultraviolet light is 405nm and 5W, and the irradiation time is 5-10 seconds.

[0039] The fifth aspect of the present invention provides a method for preparing a sustained-release hydrogel, comprising the following steps: (1) preparing a sodium alginate solution containing nanoparticles as described in the first aspect of the present invention as solution B, and a gelatin solution containing methacrylamide as solution A; (2) mixing solution A and solution B to obtain a polymer solution; and (3) irradiating the polymer solution with ultraviolet light to form a sustained-release hydrogel.

[0040] In a preferred embodiment, the ultraviolet light is 405nm and 5W, and the irradiation time is 5-10 seconds.

[0041] In a preferred embodiment, in step (1), solution A contains CaO2.

[0042] In a preferred embodiment, the mass ratio of the methacrylamide gelatin to the methacrylamide sodium alginate in the polymer solution is (3-20):3, for example, 1:1, 4:3, 5:3, 2:1, 7:3, 8:3, 3:1, 10:3, 11:3, 4:1, 13:3, 14:3, 5:1, 16:3, 17:3, 6:1, 19:3, or 20:3.

[0043] In a preferred embodiment, the final concentration of nanoparticles in the polymer solution is 100–500 μg / mL, for example, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, or 500 μg / mL.

[0044] In a preferred embodiment, the final concentration of CaO2 in the polymer solution is 200–1000 μg / mL, for example, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL, or 1000 μg / mL.

[0045] In another aspect, the present invention provides nanoparticles obtained by the preparation method described in the second aspect of the present invention.

[0046] In another aspect, the present invention provides a sustained-release hydrogel obtained by the preparation method described in the fifth aspect of the present invention.

[0047] Another aspect of the present invention provides an ultrasonic system comprising an ultrasonic wave and a system formed by nanoparticles as described in the first aspect of the present invention or a sustained-release hydrogel as described in the fourth aspect of the present invention, wherein the ultrasonic system uses the ultrasonic wave to irradiate the nanoparticles or the sustained-release hydrogel.

[0048] In a preferred embodiment, the ultrasound wave satisfies one or more of the following conditions:

[0049] 1.0-5.0MHz; for example, 1.0MHz, 2.0MHz, 3.0MHz, 4.0MHz, or 5.0MHz;

[0050] 0.1-5.0W cm -2 For example, 1W cm -2 1.5W cm -2 2W cm -2 2.5W cm -2 3W cm -2 3.5W cm -2 4.5W cm -2 4.5W cm -2 Or 5W cm -2 ;

[0051] And / or, 10%-70% duty cycle, such as 20% duty cycle, 30% duty cycle, 40% duty cycle, 50% duty cycle, 60% duty cycle, or 70% duty cycle;

[0052] The ultrasonic radiation time is 3-15 minutes, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes or 15 minutes.

[0053] In a specific implementation, when the ultrasound system is used for anti-tumor purposes, the ultrasound meets the following condition: 1.5W / cm². -2 50% duty cycle, radiation time is 5 minutes.

[0054] In one specific implementation, when the ultrasound system is used for osteogenic purposes, the ultrasound meets the following condition: 0.3 W / cm². -2 50% duty cycle, radiation time is 15 minutes.

[0055] In another aspect, the present invention also provides nanoparticles as described in the first aspect of the present invention, nanoparticles obtained by the preparation method described in the second aspect of the present invention, sustained-release hydrogel raw materials as described in the third aspect of the present invention, sustained-release hydrogels as described in the fourth aspect of the present invention, sustained-release hydrogels obtained by the preparation method described in the fifth aspect of the present invention, or the use of an ultrasound system as described above in the present invention in the preparation of antitumor / cancer drugs.

[0056] In a preferred embodiment, the tumor / cancer includes prostate cancer, colon cancer, rectal cancer, ovarian cancer, kidney cancer, breast cancer, glioblastoma, melanoma, malignant melanoma, or lung cancer.

[0057] In another aspect, the present invention also provides nanoparticles as described in the first aspect of the present invention, nanoparticles obtained by the preparation method described in the second aspect of the present invention, sustained-release hydrogel raw materials as described in the third aspect of the present invention, sustained-release hydrogels as described in the fourth aspect of the present invention, sustained-release hydrogels obtained by the preparation method described in the fifth aspect of the present invention, or the application of the ultrasound system described above in the present invention in the preparation of osteogenic repair drugs.

[0058] In a preferred embodiment, the osteogenic repair is caused by tumor bone metastasis, or the osteogenic repair is induced by osteogenic differentiation of bone marrow mesenchymal stem cells.

[0059] Unless otherwise specified, the mass ratios mentioned above in this invention are weight ratios or concentration ratios.

[0060] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0061] Unless otherwise specified, all reagents and raw materials used in this invention are commercially available.

[0062] The positive and progressive effects of this invention are as follows:

[0063] 1. This invention develops an innovative ultrasound-controlled multifunctional hydrogel platform. This system achieves precise control of ROS release through ultrasound to achieve dual therapeutic effects, while simultaneously regulating key genes and improving the microenvironment, addressing both tumor treatment and bone regeneration issues in breast cancer bone metastases.

[0064] 2. The method developed in this invention, which upregulates ZBP1 gene expression and provides a continuous oxygen supply to a hypoxic microenvironment, can improve the responsiveness of tumor cells to ROS, induce necrosis and apoptosis, and greatly enhance the anti-tumor therapeutic effect.

[0065] 3. This invention develops a biosafety-compliant hydrogel-loaded MOF multifunctional system with excellent dual effects of anti-tumor and bone repair promotion. It features in-situ injection and sustained-release properties, and the therapeutic components are highly concentrated in the target area, thereby significantly improving efficacy and reducing side effects. Attached Figure Description

[0066] Figure 1 This document describes the preparation process of an ultrasound-controlled oxygen-generating MOF multifunctional hydrogel platform (GA@CaMP) and its detection using scanning electron microscopy (SEM).

[0067] Figure 2 The effects of the ROS-promoting nanoplatform (MHP) on inducing ROS production and necrosis-induced apoptosis in 4T1 tumor cells under ultrasound were detected by fluorescence microscopy, CCK8 assay, and Western blot. (a) Fluorescence microscopy was used to detect ROS production in 4T1 tumor cells of each group. (bc) Fluorescence microscopy was used to detect the liveness and death staining of 4T1 tumor cells of each group and related quantitative analysis was performed. (d) CCK8 assay was used to detect the cell viability of 4T1 tumor cells of each group. (e) Western blot analysis was used to analyze the expression of necrosis-induced apoptosis-specific proteins in 4T1 tumor cells of each group.

[0068] Figure 3 Evaluation of the in vivo antitumor efficacy of the GA@CaMP hydrogel platform. (a) Schematic diagram of the in vivo primary tumor treatment process. (b) Mean tumor growth curves of mice in each group. (c) Tumor weight of mice in each group. (d) Tumor inhibition rate of mice in each group. (e) Photographs of tumors of mice in each group. (f) H&E images of tumors of mice in each group.

[0069] Figure 4 To evaluate the in vitro osteogenic differentiation-promoting efficacy of the GA@CaMP hydrogel platform. (a) Alkaline phosphatase (ALP) staining of BMSCs after 7 days of treatment. (b) Alizarin Red S (ARS) staining of BMSCs after 14 days of treatment. (c) ALP activity of BMSCs after 7 days of treatment. (d) Quantitative analysis of osteogenic mineralization in BMSCs after 7 days of treatment.

[0070] Figure 5 To evaluate the efficacy of the GA@CaMP hydrogel platform in promoting bone regeneration and repair in vivo. (a) Schematic diagram of the bone defect repair process in vivo. (b) Micro-computed tomography (micro-CT) scans of cranial bone repair in rats of each group. (c) RUNX2 immunofluorescence staining at cranial bone defects in rats of each group. (d) HIF-1α immunohistochemistry at cranial bone defects in rats of each group. Detailed Implementation

[0071] Based on the dual nature of ultrasound-regulated reactive oxygen species (ROS) generation—capable of killing tumors while promoting osteogenic repair—the stable protective properties of MOF structures, the sustained oxygen production characteristics of CaO2, and the targeted sustained-release properties of hydrogel as a carrier, the inventors have conceived a multifunctional nanodelivery combination scheme that combines CaO2 and hydrogel to synergistically improve the hypoxic tumor microenvironment through a ROS nanoplatform. This aims to provide a multifunctional targeted delivery system with high biosafety and precise controlled release of ROS. Therefore, this invention includes the following core technical solutions:

[0072] One aspect of this invention is to propose a multifunctional targeting platform for ultrasonically controlled release of ROS, comprising hematoporphyrin monomethyl ether (HMME), a plasmid expressing ZBP1, and a metal-organic framework (MOF) as a carrier, forming an MHP nanosystem, and a sustained-release hydrogel for in-situ targeting.

[0073] Preferably, the above-mentioned MHP nanosystem is composed of hematoporphyrin monomethyl ether (H), ZBP1 plasmid (P), and metal-organic framework MOF (ZIF-8, M), and is synthesized using a classic one-pot method in the presence of metallic Zn. 2+ Before coordination with ions and 2-methylimidazole (2-MIM), pDNA and hematoporphyrin monomethyl ether are mixed separately in their respective aqueous solutions. Then, zinc nitrate solution is added dropwise to the imidazole complex solution, thus encapsulating the pDNA and hematoporphyrin monomethyl ether in situ within ZIF-8. This results in a more robust encapsulation, rather than relying on unstable surface adsorption, significantly improving the stability of the encapsulated material and enhancing ROS performance and plasmid transfection efficiency.

[0074] CaO2 was prepared using a classic method developed by predecessors.

[0075] Furthermore, the aforementioned multifunctional targeted platform for promoting ultrasound-controlled release of ROS also includes an injectable sustained-release hydrogel.

[0076] In a preferred embodiment, the above-mentioned in-situ injected sustained-release hydrogel is a hybrid gel composed of methacrylamide gelatin (GelMA) and methacrylamide sodium alginate (AlgMA). The ROS-promoting nanoplatform MHP and oxygen-generating CaO2 are mixed in their prepolymer solution and irradiated with ultraviolet light for 5-10 seconds to form the GA@CaMP multifunctional hydrogel platform.

[0077] A first aspect of the present invention is to provide a method for preparing the above-mentioned ultrasound-controlled release ROS multifunctional targeting platform, which includes the following steps:

[0078] (1) MHP nanoparticles were prepared by a one-pot synthesis method;

[0079] (2) CaO2 nanoparticles were prepared using the classic preparation methods of predecessors;

[0080] (3) Add the MHP and CaO2 obtained in steps (1) and (2) to the GelMA / AlgMA hybrid hydrogel at concentrations of 200 μg / mL and 600 μg / mL, respectively, to prepare the GA@CaMP multifunctional hydrogel platform.

[0081] Optionally, the preparation steps of MHP nanoparticles in step (1) above include: at room temperature, hematoporphyrin monomethyl ether (HMME, 10 μL, 2 mg / mL) is slowly added to 125 μL of an aqueous solution containing 23.75 mg of 2-methylimidazole under mechanical stirring. Simultaneously, 25 μL of an aqueous solution containing 25 μg of pDNA (DNase-free) is added to 125 μL of an aqueous solution containing 2.4 mg of Zn(NO3)2·6H2O, and the mixture is stirred until homogeneous. After 10 minutes, zinc nitrate composite solution is added dropwise to the 2-methylimidazole composite aqueous solution. The solution becomes turbid. After stirring for 30 minutes, the product is collected by centrifugation at 10,000 rpm and washed three times with anhydrous ethanol to obtain MHP nanoparticles.

[0082] The preparation steps of the GA@CaMP multifunctional hydrogel platform in step (3) above include: weighing GelMA and AlgMA respectively, and dissolving them at 10% w / v in double-distilled water (ddH2O) containing lithium acylphosphonate (LAP, 0.3% w / v) to produce precursor solution A and precursor solution B. Weigh an appropriate amount of MHP and dissolve it fully in the above precursor solution B, and at the same time take an appropriate amount of CaO2 and dissolve it in precursor solution A. Mix the two solutions thoroughly according to the ratio of m(GelMA:AlgMA) = 7:3, so that the MHP concentration in the final composite solution reaches 200 μg / mL and the CaO2 concentration reaches 600 μg / mL, thus obtaining the GA@CaMP polymer solution. Place the polymer solution in a specific mold and perform photocrosslinking with ultraviolet light (405nm, 5W) for 5-10s to obtain the GA@CaMP platform.

[0083] Furthermore, the above method also includes the following steps:

[0084] (4) The GA@CaMP multifunctional hydrogel platform obtained in step (3) was examined by scanning electron microscopy to confirm that MHP and CaO2 were successfully encapsulated inside the hydrogel system.

[0085] Another aspect of this invention provides the use of the aforementioned ultrasound-controlled ROS multifunctional targeting platform in the preparation of antitumor / cancer drugs. The tumors / cancers include, but are not limited to, malignant cancers selected from the group consisting of: prostate cancer, colon cancer, rectal cancer, ovarian cancer, kidney cancer, breast cancer, glioblastoma, melanoma, malignant melanoma, and lung cancer. It can also be used for subsequent osteogenic repair therapy for tumor bone metastases.

[0086] This invention proposes a multifunctional targeted platform for ultrasound-controlled release of ROS, comprising a hydrogel carrier, a MOF-based ROS-promoting nanoplatform, and oxygen-producing CaO2 nanoparticles. The ROS-promoting nanoplatform can flexibly generate different levels of ROS under irradiation conditions at different ultrasound frequencies for tumor killing and bone defect repair, respectively. CaO2, acting in a hypoxic microenvironment, synergistically enhances the dual therapeutic effects. This invention develops a highly biosafe multifunctional injectable hydrogel (GelMA / AlgMA) loaded with the ROS-promoting nanoplatform MHP and oxygen-producing CaO2 nanoparticles. Under ultrasound irradiation, it induces necrosis and apoptosis in tumor cells, activates an anti-tumor immune response, and inhibits tumor recurrence. Subsequently, it activates osteoblast progenitor cell proliferation and differentiation, accelerating the bone defect repair process. The in-situ injection method minimizes unnecessary damage to normal tissues caused by the nanomedicine.

[0087] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments, unless otherwise specified, were performed according to conventional methods and conditions, or as selected in the product instructions:

[0088] Example 1: Preparation of an ultrasound-controlled oxygen-generating MOF multifunctional hydrogel platform (GA@CaMP).

[0089] Preparation of MHP: Hematoporphyrin monomethyl ether (HMME) and ZBP1 plasmid (pDNA, Yunzhou Biotechnology) were synthesized using a one-pot synthesis method with a metal-organic framework (ZIF-8) as the structural support. The raw materials for synthesis included zinc nitrate hexahydrate and dimethylimidazole. At room temperature, under mechanical stirring, 10 μL of hematoporphyrin monomethyl ether (HMME, 2 mg / mL) was slowly added to 125 μL of an aqueous solution containing 23.75 mg of 2-methylimidazole. Simultaneously, 25 μL of an aqueous solution containing 25 μg of pDNA (DNase-free) was added to 125 μL of an aqueous solution containing 2.4 mg of Zn(NO3)2·6H2O, and the mixture was stirred until homogeneous. Ten minutes later, zinc nitrate solution was added dropwise to the 2-methylimidazole complex aqueous solution. The solution became turbid. After stirring for 30 minutes, the product was collected by centrifugation at 10,000 rpm. A light pink precipitate, MHP, was observed. After washing three times, the product was freeze-dried for later use.

[0090] Preparation of CaO2: Under ultrasonication, 0.1 g CaCl2 and 0.35 g PVP were fully dissolved in 15 mL of anhydrous ethanol to obtain a clear and transparent liquid. Then, 1 mL of NH4OH was added under continuous stirring. After 30 min, 200 μL of H2O2 solution was slowly added dropwise to the mixed solution, resulting in a pale blue-white milky solution. The product was collected by centrifugation at 16,000 rpm for 30 min, washed three times with ethanol, and freeze-dried for later use.

[0091] Preparation of GA@CaMP: GelMA and AlgMA were weighed separately and dissolved at 10% w / v in double-distilled water (ddH2O) containing lithium acylphosphonate (LAP, 0.3% w / v) to produce precursor solution A and precursor solution B. An appropriate amount of MHP was weighed and fully dissolved in precursor solution B, while an appropriate amount of CaO2 was dissolved in precursor solution A. The two solutions were thoroughly mixed at a ratio of m(GelMA:AlgMA) = 7:3, until the final composite solution had an MHP concentration of 200 μg / mL and a CaO2 concentration of 600 μg / mL, thus obtaining the GA@CaMP polymer solution. The polymer solution was placed in a specific mold and photocrosslinked with ultraviolet light (405 nm, 5 W) for 5-10 s to form the GA@CaMP platform. The preparation process and scanning electron microscope (SEM) results of GA@CaMP are shown below. Figure 1 As shown.

[0092] Preparation method of CaMP: The difference from the preparation of GA@CaMP is that there is no hydrogel as a carrier. CaMP polymer is formed by dissolving CaO2 and MHP in PBS at final concentrations of 600 μg / mL and 200 μg / mL, respectively.

[0093] Preparation method of GA@MP: The difference from the preparation of GA@CaMP is that CaO2 is no longer doped in the precursor solution A.

[0094] Preparation method of GA@CaM: The difference from the preparation of GA@CaMP is that plasmid ZBP1 is not added when preparing nanoparticles to obtain M. Then, referring to the preparation process of GA@CaMP, M and CaO2 are doped into the gel.

[0095] Example 2: Evaluation of the efficacy of the ROS-promoting nanoplatform (MHP) in in vitro inducing ROS production and necrosis-induced apoptosis in tumor cells.

[0096] The ROS production of cells was detected using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. 4T1 cells (5 × 10⁻⁶) were used. 5 Cells were seeded per well in 6-well plates and incubated at 37°C for 24 hours to allow cell attachment. Cells were then divided into the following groups: control group (G1), US only (G2), MH (G3), MHP (G4), MP+US (G5), and MHP+US ​​(G6). Each group was incubated at 37°C for a total of 6 hours. The culture medium was then replaced with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The US irradiation group underwent US irradiation (1.0 MHz, 1.5 W / cm²). 2 50% duty cycle, 5 minutes), then continue incubation at 37°C for 30 minutes, and observe intracellular ROS generation in each treatment group under a fluorescence microscope (showing green fluorescence).

[0097] The ability of the ROS-promoting nanoplatform (MHP) to kill tumor cells in vitro was evaluated using live / dead staining and CCK8 assays. Western blot was used to detect the expression of specific proteins involved in necrosis and apoptosis. First, 4T1 cells (5 × 10⁶ cells) were... 5 Cells were seeded in 6-well plates (number per well) and incubated at 37°C for 24 hours to allow cell attachment. Cells were then randomly divided into the following groups: control group (G1), US only (G2), MH (G3), MHP (G4), MH + US (G5), and MHP + US (G6). Each group was incubated at 37°C for a total of 6 hours. After washing three times with PBS, the US irradiation group underwent US irradiation (1.0 MHz, 1.5 W / cm²). 2(50% duty cycle, 5 minutes). Then, a mixed solution of Calcein-AM and propidium iodide (PI) was added to all wells, and staining was performed at 37°C for 15 minutes. Cells were observed under a fluorescence inverted microscope. The excitation filter was set to 488 nm to observe live cells (green) detected by Calcein-AM, and to 561 nm to observe dead cells (red) detected by PI. CCK8 assay was performed on 4T1 cells (1×10⁻⁶). 4 Cells were seeded in 96-well plates (number of cells / well) and treated as above. Prepared CCK-8 solution (100 μL / well) was added, and all groups were incubated at 37°C for 1 hour. The absorbance was measured at 450 nm using a microplate reader. For Western blot analysis, cells were treated as above, and each group was incubated at 37°C for another 12 hours. Cells were then lysed to extract protein for Western blot analysis. Equal amounts of total protein were separated by 10% SDS-PAGE, transferred to PVDF membranes, blocked, and incubated with ZBP1, p-MLKL, and MLKL rabbit monoclonal antibodies at room temperature for 1 hour. After washing three times with TBST, the membranes were incubated with goat anti-rabbit IgG antibody at room temperature for 1 hour. Finally, after washing three times with TBST, the target protein was developed using an enhanced chemiluminescence (ECL) kit.

[0098] Ultimately, the efficacy of the ROS-promoting nanoplatform (MHP) in inducing ROS production and necrosis / apoptosis in tumor cells under ultrasound is as follows: Figure 2 As shown, from Figure 2 As can be seen, under the action of ultrasound, the ROS-promoting nanoplatform MHP can stimulate the production of a large amount of ROS and effectively activate the ZBP1 / MLKL-dependent necroptosis pathway, significantly killing tumor cells.

[0099] Example 3: Evaluation of the in vivo antitumor therapeutic effect of an ultrasound-controlled oxygen-generating MOF multifunctional hydrogel platform (GA@CaMP).

[0100] To further evaluate the in vivo antitumor effect of the ultrasound-controlled oxygen-generating MOF multifunctional hydrogel platform (GA@CaMP), an orthotopic tumor mouse model was constructed, and 4T1 tumor cells (1×10⁻⁶) were introduced into the platform. 6 Injected into the fat pad of the third pair of left mammary glands in female BALB / c mice (4-5 weeks old). When the tumor volume reaches approximately 125-150 mm... 3 At that time, tumor-bearing mice were randomly divided into 6 groups and given different treatments: (G1) control, (G2) CaMP+US, (G3) GA@CaMP, (G4) GA@MP+US, (G5) GA@CaMP+US, and (G6) GA@CaMP+US. The US irradiation group received US irradiation (1.0 MHz, 1.5 W / cm²). 2(50% duty cycle, 5 minutes). The above drugs were injected only on day 1. The ultrasound treatment group received ultrasound irradiation on days 2, 4, 6, and 8. During days 0-14, mouse body weight and tumor volume were measured every two days, calculated using the formula (tumor length) × (tumor width). 2 / 2 The tumor volume was calculated and a tumor growth curve was plotted. Mouse tumors were collected, weighed, and stained with hematoxylin and eosin (H&E). From Figure 3 As can be seen, the GA@CaMP gel platform has a significant anti-tumor effect under ultrasound irradiation.

[0101] Example 4: Evaluation of the efficacy of an ultrasound-controlled oxygen-producing MOF multifunctional hydrogel platform (GA@CaMP) in in vitro induction of osteogenic differentiation of bone marrow mesenchymal stem cells.

[0102] To investigate the effects of different treatments on osteogenic differentiation of BMSCs, we performed a Transwell assay to simulate a co-culture environment. Specifically, BMSCs (2 × 10⁶ cells / year) were subjected to different treatments. 4 Cells were seeded in the lower chamber of a Transwell plate (100 vl / well) for the gel group and in the upper chamber for the CON group (100 vl / well). An equal volume of PBS was added to replace the upper chamber. Cells were incubated at 37°C for 24 hours to allow them to adhere to the plate. The culture medium for all groups was then replaced with osteogenic induction medium (DMEM complete medium supplemented with 10 mM β-glycerophosphate disodium salt, 50 μg / mL L-ascorbic acid, and 10 nM dexamethasone) every three days. Cells were then grouped as follows: (G1) control, (G2) GA@CaMP, (G3) GA@CaM+US, and (G4) GA@CaMP+US. The US-irradiated group was irradiated with US (1.0 MHz, 0.3 W / cm²). 2(50% duty cycle, 15 minutes). On day 7 of co-culture, cells were stained using the BCIP / NBTALP staining kit. The upper chamber was removed and the liquid in the lower chamber was aspirated. 4% paraformaldehyde fixative was added to the lower chamber, and the cells were fixed at 4°C for 20 min. The cells were then washed three times with PBS, stained with 1×BCIP / NBT staining solution, and incubated at room temperature in the dark for 30 min. The cells were washed three times with PBS, and the staining was observed and photographed under an optical microscope. To determine alkaline phosphatase (ALP) activity, 100 μL of Western blotting and IP cell lysis buffer (without inhibitors) were added to the lower chamber. The supernatant was then collected by centrifugation, and ALP activity was measured according to the instructions of the BCA protein detection kit. On day 14 of co-culture, Alizarin Red S (ARS) staining was performed to detect the formation of the mineralized matrix. The procedure was the same as for ALP detection. After cell fixation and washing, 2% ARS staining solution was added to each well, and the cells were incubated at room temperature for 30 min. The staining solution was then removed, and the cells were washed three times with sterile PBS to terminate the colorimetric reaction. The mineralized matrix was observed under an optical microscope. Subsequently, 1 ml of 10% hexadecylpyridine chloride solution was added to each well, and after incubation for 15 min, the supernatant was aspirated into a 96-well plate. The absorbance at 562 nm was measured using a microplate reader (Thermo 3001, Thermo Scientific, USA) to quantify the amount of mineralized matrix. Figure 4 As can be seen, under ultrasound irradiation, the GA@CaMP gel platform is conducive to the progression of BMSCs toward osteogenic differentiation and significantly improves the expression of osteogenic markers.

[0103] Example 5: Evaluation of the in vivo osteogenic repair effect of the ultrasound-controlled oxygen-generating MOF multifunctional hydrogel platform (GA@CaMP).

[0104] To evaluate the in vivo osteoproliferative effect of the GA@CaMP hydrogel platform on bone repair, a rat model of critical skull defects was constructed. Six-week-old male Sprague Dawley rats were deeply anesthetized with 4% chloral hydrate, and the scalp was then incised, with tissue bluntly dissected until the skull was fully exposed. The periosteum was neatly incised and carefully protected. A circular critical defect with a diameter of 5 mm was created on the right side of the midline of the rat skull. The rats were randomly divided into four groups: (G1) control, (G2) GA@CaMP, (G3) GA@CaMP+US, and (G4) GA@CaMP+US. The sterilized gel platform was then implanted into the defect area. The CON group used rats without any filling material as controls. Finally, the rat scalp was carefully sutured layer by layer, and cefuroxime antibiotics were administered intraperitoneally daily for 3 consecutive days to prevent potential infection. The US irradiation group received US irradiation (1.0 MHz, 0.3 W / cm²) on days 1, 5, 10, and 14 postoperatively. 2(50% duty cycle, 15 minutes). After week 8, rats were euthanized and their skulls were harvested. Samples were fixed in 4% paraformaldehyde, and the defect areas were analyzed for mineralization using micro-computed tomography (micro-CT). The skull tissue was decalcified, embedded in paraffin, and sectioned for Runx2 immunofluorescence staining to assess the degree of bone repair, and HIF-1α immunohistochemistry to evaluate the relief of tissue hypoxia. Figure 5 As shown, under ultrasound irradiation, the GA@CaMP gel platform can effectively promote matrix maturation and mineralization during bone repair and remodeling, enhance the expression of osteogenic-related proteins, alleviate the hypoxic microenvironment, and jointly accelerate the regeneration of bone defects.

Claims

1. A sustained-release hydrogel, characterized in that, The invention comprises a hybrid gel formed from methacrylamide gelatin and methacrylamide sodium alginate, and nanoparticles formed by encapsulating hematoporphyrin monomethyl ether and plasmids containing the ZBP1 sequence in a metal-organic framework formed by zinc nitrate hexahydrate and dimethylimidazolium. The sustained-release hydrogel also includes CaO2.

2. The sustained-release hydrogel as described in claim 1, characterized in that, The components of the sustained-release hydrogel meet one or more of the following conditions: the mass ratio of the methacrylamide gelatin and the methacrylamide sodium alginate is (3~20):3; The final concentration of nanoparticles in the sustained-release hydrogel is 100~500μg / mL; And / or, the final concentration of CaO2 in the sustained-release hydrogel is 200~1000 μg / mL.

3. The sustained-release hydrogel as described in claim 2, characterized in that, The components of the sustained-release hydrogel meet one or more of the following conditions: the mass ratio of the methacrylamide gelatin and the methacrylamide sodium alginate is 7:3; The final concentration of nanoparticles in the sustained-release hydrogel is 200 μg / mL; And / or, the final concentration of CaO2 in the sustained-release hydrogel is 600 μg / mL.

4. The sustained-release hydrogel as described in claim 1, characterized in that, The gene corresponding to the ZBPI sequence is a human or mouse gene; the mass ratio of zinc nitrate hexahydrate to dimethylimidazole is 1:(5~20); the mass ratio of hematoporphyrin monomethyl ether to plasmid containing the ZBP1 sequence is 1:(0.5~5); and / or, the mass ratio of hematoporphyrin monomethyl ether to dimethylimidazole is 1:(800~2000).

5. The sustained-release hydrogel as described in claim 4, characterized in that, The mass ratio of zinc nitrate hexahydrate to dimethylimidazole is 1:10; the mass ratio of hematoporphyrin monomethyl ether to plasmid containing the ZBP1 sequence is 4:5; and / or, the mass ratio of hematoporphyrin monomethyl ether to dimethylimidazole is 1:1200.

6. The sustained-release hydrogel as described in claim 1, characterized in that, The sustained-release hydrogel can be formed by irradiation with ultraviolet light, wherein the ultraviolet light is 405 nm and 5 W, and the irradiation time is 5-10 seconds.

7. A method for preparing a sustained-release hydrogel, characterized in that, The steps include: (1) preparing a sodium alginate solution containing nanoparticles as described in any one of claims 1 to 6 as solution B, and a gelatin solution containing methacrylamide as solution A, wherein solution A also contains CaO2; (2) mixing solution A and solution B to obtain a polymer solution; and (3) irradiating the polymer solution with ultraviolet light to form a slow-release hydrogel, wherein the ultraviolet light is 405 nm and 5 W, and the irradiation time is 5-10 seconds.

8. The preparation method according to claim 7, characterized in that, In step (1), each component in the polymer solution satisfies one or more of the following conditions: the mass ratio of the methacrylamide gelatin to the methacrylamide sodium alginate is (3~20):3; The final concentration of the nanoparticles is 100~500μg / mL; And / or, the final concentration of CaO2 is 200~1000 μg / mL.

9. The preparation method according to claim 8, characterized in that, In step (1), each component in the polymer solution satisfies one or more of the following conditions: the mass ratio of the methacrylated gelatin to the methacrylated sodium alginate is 7:3; The final concentration of nanoparticles in the sustained-release hydrogel is 200 μg / mL; And / or, the final concentration of CaO2 in the sustained-release hydrogel is 600 μg / mL.

10. The preparation method according to claim 7, characterized in that, The method for preparing the nanoparticles also includes: adding hematoporphyrin monomethyl ether to an aqueous solution of 2-methylimidazolium, and then adding an aqueous solution of Zn(NO3)2·6H2O dropwise to it; wherein the Zn(NO3)2·6H2O is an aqueous solution of a plasmid containing the ZBP1 sequence.

11. The preparation method according to claim 10, characterized in that, The gene corresponding to the ZBPI sequence is a human or mouse gene; the mass ratio of zinc nitrate hexahydrate to dimethylimidazole is 1:(5~20); the mass ratio of hematoporphyrin monomethyl ether to plasmid containing the ZBP1 sequence is 1:(0.5~5); and / or, the mass ratio of hematoporphyrin monomethyl ether to dimethylimidazole is 1:(800~2000).

12. An ultrasound system comprising ultrasound waves and a system for forming a sustained-release hydrogel as described in any one of claims 1 to 6, wherein the sustained-release hydrogel is irradiated using the ultrasound waves.

13. The ultrasonic system as described in claim 12, characterized in that, The ultrasonic wave meets one or more of the following conditions: 1.0-5.0 MHz; 0.1-5.0 in cm -2 ; And / or, 10%-70% duty cycle; The ultrasonic radiation time is 3-15 minutes.

14. The ultrasonic system as claimed in claim 12, characterized in that, The ultrasound wave meets one or more of the following conditions: 1.5 W / cm² -2 ; and / or, 50% duty cycle; the ultrasonic radiation time is 5 min.

15. The use of the sustained-release hydrogel according to any one of claims 1 to 6, the sustained-release hydrogel obtained by the preparation method according to any one of claims 7 to 11, or the use of the ultrasound system according to any one of claims 12 to 14 in the preparation of antitumor / cancer drugs; wherein the tumor / cancer includes prostate cancer, colon cancer, rectal cancer, ovarian cancer, kidney cancer, breast cancer, glioblastoma, melanoma, or lung cancer.

16. The sustained-release hydrogel according to any one of claims 1 to 6, the sustained-release hydrogel obtained by the preparation method according to any one of claims 7 to 11, or the ultrasound system according to any one of claims 12 to 14, in the preparation of a drug for osteogenic repair; wherein the osteogenic repair is caused by tumor bone metastasis, or the osteogenic repair is the induction of osteogenic differentiation of bone marrow mesenchymal stem cells.

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