Nano-particles, slow-release hydrogel containing nano-particles and ultrasonic system and application of nano-particles and slow-release hydrogel

Abstract

The invention discloses nanoparticles, sustained-release hydrogel containing the nanoparticles, an ultrasonic system of the sustained-release hydrogel and application of the sustained-release hydrogel. The nanoparticles are formed by packaging hematoporphyrin monomethyl ether and plasmids for expressing ZBP1 through a metal organic framework formed by zinc nitrate hexahydrate and dimethylimidazole. According to the ultrasonic regulation and control multifunctional hydrogel platform, accurate control over ROS release is achieved through ultrasound so as to achieve the dual treatment effect, meanwhile, key gene regulation and control and microenvironment improvement are coordinated, and the problems of tumor treatment and bone regeneration in breast cancer bone metastasis are solved. The invention develops a hydrogel-loaded MOF multifunctional system with biosafety, the hydrogel-loaded MOF multifunctional system has excellent dual effects of resisting tumors and promoting bone repair, and has in-situ injectability and a slow release type, and therapeutic components are highly concentrated in a target area, so that the curative effect is remarkably improved, and side effects are reduced.

Description

Technical Field

[0001] The present invention belongs to the field of ultrasonic dynamics, and in particular relates to a nanoparticle, a sustained-release hydrogel containing the nanoparticle, and an ultrasonic system and application thereof. Background Art

[0002] In the treatment of breast cancer bone metastasis, how to simultaneously eliminate tumor cells and promote bone regeneration remains a pressing challenge. In recent years, reactive oxygen species (ROS), as important intracellular signaling molecules, have demonstrated unique bidirectional regulatory effects in tumor therapy and tissue repair, potentially offering a promising approach to addressing 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 ROS can promote the proliferation, differentiation, and mineralization of bone marrow mesenchymal stem cells (BMSCs) by regulating multiple signaling pathways in osteoblasts and increasing the accessibility of chromatin to osteogenic genes, thereby accelerating bone repair. This bidirectional regulatory effect makes ROS an ideal therapeutic molecule, but how to achieve its precise regulation, especially under the dual needs of tumor treatment and bone repair, still requires further exploration. Ultrasound, as a non-invasive treatment method, has excellent controllability and high targeting. In combination with sonosensitizers, the generation of ROS can be released on demand in space and time, and the amount of ROS released can be precisely controlled by controlling the ultrasound frequency to flexibly respond to different treatment needs. At the same time, ultrasound has significant deep penetration ability and avoids phototoxicity, making it an ideal choice for treating tumor tissues deeper in the body.

[0004] However, while simply regulating ROS concentration can exert anti-tumor and bone repair effects to a certain extent, its therapeutic efficacy is still limited. First, most sonosensitizers suffer from poor chemical stability, short circulation time, and limited tumor accumulation, which compromise therapeutic efficacy. Second, the hypoxic tumor microenvironment and changes in key genetic phenotypes can enhance tumor cell tolerance to oxidative stress, allowing them to adapt to high ROS concentrations and thereby limit their cytotoxic effects. ZBP1 is a Z-DNA and Z-RNA binding protein. As a cytoplasmic nucleic acid sensor, it can trigger various forms of cell death and inflammatory responses. Studies have found that ZBP1 expression is often low in genomically unstable tumors, such as triple-negative breast cancer, leading to increased tumor cell tolerance to oxidative stress and a deficient immune response, making treatment more challenging. Upregulating ZBP1 gene expression can effectively activate downstream cell death pathways, increasing tumor cell sensitivity to ROS and thus enhancing therapeutic efficacy. Notably, the role of ZBP1 in bone repair cannot be ignored. Studies have shown that ZBP1 can promote β-catenin nuclear translocation and regulate Wnt signaling, thereby promoting the osteogenic differentiation of BMSCs. Therefore, if the expression of this gene can be effectively upregulated, it may help to achieve the dual effects of anti-tumor and bone regeneration. However, naked nucleic acids are very fragile and will be rapidly degraded by serum nucleases in the blood. Finding a safe and effective delivery carrier is the key to gene regulation. Metal-organic frameworks (MOFs) are porous coordination polymers constructed by bridging metal ions or metal clusters with organic linkers. They have an open porous structure, high load capacity and good stability. Biomacromolecules can be used to achieve biomimetic mineralization in MOFs through a one-pot encapsulation method, 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, and have shown efficient performance in intracellular gene delivery and expression. At the same time, MOF encapsulation can protect the stability of the sonosensitizer during transportation, and its metal ions or organic ligands will further promote the activation of the sonosensitizer. For example, metal ions in MOF (such as Zn 2+) Under the stimulation of ultrasound, it can promote the generation of ROS, thereby enhancing the effect of sonodynamic therapy. As for the hypoxic microenvironment, CaO2 has good oxygen release performance and can continuously release oxygen in a weakly acidic environment, which not only helps to improve the oxygenation environment of the tumor area, but also promotes bone repair. Hydrogel has become an ideal choice in the fields of drug delivery, tissue regeneration and combined therapy in recent years due to its excellent biocompatibility, controllability, drug sustained release, stimulus responsiveness and tissue repair ability. Through reasonable design, the needs of simultaneously regulating ROS levels, ZBP1 gene expression and improving the hypoxic microenvironment are met. In addition, the therapeutic ingredients can be released simultaneously in the target area, thereby enhancing the therapeutic effect and overcoming the limitations of a single treatment method. At the same time, compared with intravenous injection, oral administration and other forms, it can minimize the adverse effects of systemic exposure to drugs, and has great application prospects and clinical transformation potential. The inventors have conducted in-depth research and attempts on hydrogels as a bidirectional multifunctional material delivery medium.

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

[0006] 1. In the treatment process of breast cancer bone metastasis, current treatment options usually focus on a single treatment goal, such as focusing on 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, existing technologies ignore the impact of changes in the tumor microenvironment and genetic phenotype on the oxidative stress tolerance of tumor cells. Simply regulating ROS may not be able to effectively kill tumor cells.

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

[0009] In order to overcome the shortcomings of the above-mentioned 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 therapeutic system was developed and designed to meet the dual needs of simultaneous tumor cell clearance and bone defect repair through precise drug delivery and multiple therapeutic mechanisms.

[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. Use in situ injection to increase the local concentration of nanomedicines, thereby minimizing unnecessary damage to normal tissues caused by nanomedicines during anti-tumor treatment.

[0013] In order to solve the above technical problems, the present invention provides a nanoparticle, a sustained-release hydrogel containing the nanoparticle, and an ultrasound system and application thereof. The present invention develops an innovative ultrasound-regulated multifunctional hydrogel platform, which uses ultrasound to achieve precise control of ROS release to achieve dual therapeutic effects, while coordinating key gene regulation and improvement of the microenvironment, and simultaneously addressing the problems of tumor treatment and bone regeneration in breast cancer bone metastasis. The upregulation of ZBP1 gene expression developed by the present invention and the provision of a continuous oxygen supply to the hypoxic microenvironment can increase the responsiveness of tumor cells to ROS, induce cell necrosis, and greatly enhance the anti-tumor efficacy. The present invention develops a biosafe hydrogel-loaded MOF multifunctional system with excellent dual effects of anti-tumor and promoting bone repair, with in situ injectability and sustained release, and the therapeutic components are highly concentrated in the target area, thereby significantly improving the efficacy and reducing side effects.

[0014] In order to solve the above technical problems, the first aspect of the present invention provides a nanoparticle, which is formed by a metal organic framework (ZIF-8) formed by zinc nitrate hexahydrate and dimethylimidazole to encapsulate hematoporphyrin monomethyl ether and a plasmid containing a ZBP1 sequence.

[0015] The metal organic framework (ZIF-8) described in the present 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 of the present invention is a plasmid containing the ZBP1 gene sequence, and the skeleton of the plasmid can be a plasmid skeleton conventionally used in the art.

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

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

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

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

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

[0022] In a preferred embodiment, the ZBP1 gene is a human gene or a 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 the 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 dimethylimidazole 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 the present invention provides a method for preparing nanoparticles, comprising adding hematoporphyrin monomethyl ether to a 2-methylimidazole aqueous solution, and dropwise adding a Zn(NO3)2·6H2O aqueous solution thereto.

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

[0028] In a preferred embodiment, the ZBP1 gene is a human gene or a 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 the 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 dimethylimidazole 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] The third aspect of the present invention protects a sustained-release hydrogel raw material, which includes a hybrid gel formed by methacryloylated gelatin and methacryloylated sodium alginate, and the nanoparticles as described in the first aspect of the present invention or the nanoparticles as described in the first aspect of the present invention do not include a plasmid containing a ZBP1 sequence.

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

[0034] In a preferred embodiment, in the raw materials, the mass ratio of the methacrylated gelatin to the methacrylated sodium alginate 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 the 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] The third aspect of the present invention provides a sustained-release hydrogel, which can be 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 405 nm and 5 W, 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 methacrylated sodium alginate solution containing the nanoparticles described in the first aspect of the present invention as a B solution, and a methacrylated gelatin solution as an A solution, (2) mixing the A solution and the B solution 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 405 nm and 5 W, 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, in the polymer solution, the mass ratio of the methacrylated gelatin to the methacrylated sodium alginate 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, in the polymer solution, the final concentration of the nanoparticles 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, in the polymer solution, the final concentration of CaO2 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] Another aspect of the present invention further provides nanoparticles obtained by the preparation method described in the second aspect of the present invention.

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

[0047] On the other hand, the present invention also provides an ultrasound system, which includes a system formed by ultrasound and the nanoparticles as described in the first aspect of the present invention or the sustained-release hydrogel as described in the fourth aspect of the present invention, and the ultrasound system uses the ultrasound to irradiate the nanoparticles or the sustained-release hydrogel.

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

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

[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 irradiation time is 3-15 min, for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min or 15 min.

[0053] In a specific embodiment, when the ultrasound system is used for anti-tumor treatment, the ultrasound meets the following conditions: 1.5Wcm -2 , 50% duty cycle, and irradiation time is 5min.

[0054] In a specific embodiment, when the ultrasound system is used for osteogenesis, the ultrasound meets the following conditions: 0.3Wcm -2 , 50% duty cycle, and irradiation time is 15min.

[0055] On the other hand, the present invention also provides the nanoparticles as described in the first aspect of the present invention, the nanoparticles obtained by the preparation method as described in the second aspect of the present invention, the sustained-release hydrogel raw material as described in the third aspect of the present invention, the sustained-release hydrogel as described in the fourth aspect of the present invention, the sustained-release hydrogel obtained by the preparation method as described in the fifth aspect of the present invention, or the use of the ultrasonic system as described above in the preparation of anti-tumor / cancer drugs.

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

[0057] On the other hand, the present invention also provides a nanoparticle as described in the first aspect of the present invention, a nanoparticle obtained by the preparation method as described in the second aspect of the present invention, a sustained-release hydrogel raw material as described in the third aspect of the present invention, a sustained-release hydrogel as described in the fourth aspect of the present invention, a sustained-release hydrogel obtained by the preparation method as described in the fifth aspect of the present invention, or the use of the ultrasonic system as described above in the preparation of drugs for bone repair.

[0058] In a preferred embodiment, 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.

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

[0060] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

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

[0062] The positive progress effect of the present invention is:

[0063] 1. The present invention has developed an innovative ultrasound-regulated multifunctional hydrogel platform. This system uses ultrasound to precisely control ROS release to achieve a dual therapeutic effect, while synergizing key gene regulation and microenvironmental improvement to simultaneously address the issues of tumor treatment and bone regeneration in breast cancer bone metastasis.

[0064] 2. The method developed by the present invention upregulates ZBP1 gene expression and provides a continuous oxygen supply to the hypoxic microenvironment, which can increase the responsiveness of tumor cells to ROS, induce cell necrosis and apoptosis, and greatly enhance the anti-tumor efficacy.

[0065] 3. The present invention has developed a biosafe hydrogel-loaded MOF multifunctional system with excellent dual effects of anti-tumor and promoting bone repair. It has in situ injectability and sustained release. The therapeutic ingredients are highly concentrated in the target area, thereby significantly improving the efficacy and reducing side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 Preparation process of ultrasound-controlled oxygen-producing MOF multifunctional hydrogel platform (GA@CaMP) and scanning electron microscopy (SEM) detection.

[0067] Figure 2 Fluorescence microscopy, CCK8 assay, and Western blot analysis were used to examine the effect of the ROS-promoting nanoplatform (MHP) on inducing ROS production and necroptosis in 4T1 tumor cells under ultrasound. (a) Fluorescence microscopy was used to examine ROS production in 4T1 tumor cells in each group. (bc) Fluorescence microscopy was used to examine live / dead staining and quantitative analysis of 4T1 tumor cells in each group. (d) CCK8 assay was used to examine cell viability in 4T1 tumor cells in each group. (e) Western blot analysis was used to examine the expression of necroptosis-specific proteins in 4T1 tumor cells in each group.

[0068] Figure 3 In vivo antitumor efficacy evaluation of the GA@CaMP hydrogel platform. (a) Schematic diagram of the in vivo primary tumor treatment process. (b) Average tumor growth curves for each group of mice. (c) Tumor weights for each group of mice. (d) Tumor inhibition rates for each group of mice. (e) Photos of tumors in each group of mice. (f) H&E images of tumors in each group of mice.

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

[0070] Figure 5 Evaluation of the efficacy of the GA@CaMP hydrogel platform in promoting bone regeneration and repair in vivo. (a) Schematic diagram of the in vivo bone defect repair process. (b) Microcomputed tomography (micro-CT) scan of skull repair in rats in each group. (c) Immunofluorescence staining of RUNX2 at the skull defect site in rats in each group. (d) Immunohistochemistry of HIF-1α at the skull defect site in rats in each group. DETAILED DESCRIPTION

[0071] Based on the dual nature of ultrasound regulating the generation level of reactive oxygen species (ROS) to kill tumors and promote bone repair, the stable protective properties of the MOF structure, the continuous oxygen production characteristics of CaO2, and the targeted sustained release of hydrogel as a carrier, the inventors conceived a multifunctional nano-delivery combination scheme combining CaO2 and hydrogel to promote ROS nano-platforms to synergistically improve the tumor hypoxic microenvironment, in order to provide a multifunctional targeted delivery system with high biosafety and the ability to precisely control the release of ROS. Therefore, the present invention includes the following core technical solutions:

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

[0073] Preferably, the MHP nanosystem is composed of hematoporphyrin monomethylether (H), ZBP1 plasmid (P) and metal organic framework MOF (ZIF-8, M). 2+ Before the coordination of ions and 2-methylimidazole (2-MIM), pDNA and hematoporphyrin monomethyl ether are mixed in their respective aqueous solutions. Zinc nitrate solution is then added dropwise to the imidazole complex solution to achieve in situ encapsulation of pDNA and hematoporphyrin monomethyl ether within ZIF-8. This results in a more robust encapsulation, rather than relying on unstable surface adsorption, significantly improving the stability of the encapsulation material, enhancing ROS resistance, and plasmid transfection efficiency.

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

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

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

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

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

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

[0080] (3) The MHP and CaO2 obtained in step (1) and step (2) were added 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] Alternatively, the preparation of MHP nanoparticles in step (1) above comprises the following steps: slowly adding hematoporphyrin monomethyl ether (HMME, 10 μL, 2 mg / mL) to 125 μL of an aqueous solution containing 23.75 mg of 2-methylimidazole under mechanical stirring at room temperature. Simultaneously, adding 25 μL of an aqueous solution containing 25 μg of pDNA (DNase-free) to 125 μL of an aqueous solution containing 2.4 mg of Zn(NO₃)₂·6H₂O, and stirring to mix evenly. After 10 minutes, a zinc nitrate complex solution is added dropwise to the 2-methylimidazole complex aqueous solution, until 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, dissolving them in double distilled water (ddH2O) containing lithium acylphosphonate (LAP, 0.3% w / v) at 10% w / v to produce precursor solutions A and B. Weighing an appropriate amount of MHP and fully dissolving it in the above-mentioned precursor solution B, while taking an appropriate amount of CaO2 and dissolving it in the precursor solution A. The two solutions are thoroughly stirred and mixed at a ratio of m(GelMA:AlgMA) = 7:3, so that the MHP concentration in the final composite solution reaches 200μg / mL and the CaO2 concentration reaches 600μg / mL, thereby obtaining a GA@CaMP polymer solution. The polymer solution is placed in a specific mold and photocrosslinked with ultraviolet light (405nm, 5W) for 5-10s to form a GA@CaMP platform.

[0083] Furthermore, the above method further comprises the following steps:

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

[0085] Another aspect of the present invention provides the use of the multifunctional, targeted platform for ultrasound-controlled ROS release in the preparation of anti-tumor / cancer drugs. Such 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. The platform can also be used for subsequent osteoblastic repair therapy of tumor bone metastases.

[0086] The present invention proposes a multifunctional targeting platform for promoting ultrasonic controlled release of ROS, including a hydrogel carrier, a MOF-encapsulated ROS-promoting nanoplatform and oxygen-producing CaO2 nanoparticles. The ROS-promoting nanoplatform can flexibly produce different levels of ROS under the irradiation conditions of different ultrasonic frequencies for killing tumors and repairing bone defects, respectively, while CaO2 can create an oxygen-deficient microenvironment and synergistically enhance the bidirectional therapeutic effect. The present invention has developed a highly biosafe multifunctional injectable hydrogel (GelMA / AlgMA) loaded with a ROS-promoting nanoplatform MHP and oxygen-producing CaO2 nanoparticles, which, under ultrasonic irradiation, induces necroptosis in tumor cells, activates anti-tumor immune response, and inhibits tumor recurrence. It then activates the proliferation and differentiation of osteoblast precursor cells and accelerates the bone defect repair process. Through the in situ injection method, the unnecessary damage caused by nanomedicines to normal tissues is minimized.

[0087] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications:

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

[0089] MHP preparation: Hematoporphyrin monomethyl ether (HMME) and ZBP1 plasmid (pDNA, Yunzhou Bio) were synthesized using a metal-organic framework (ZIF-8) as a structural support using a one-pot synthesis method. The starting materials for the synthesis include zinc nitrate hexahydrate and dimethylimidazole. At room temperature, hematoporphyrin monomethyl ether (10 μL, 2 mg / mL) was slowly added to 125 μL of an aqueous solution containing 23.75 mg of 2-methylimidazole under mechanical stirring. Simultaneously, 25 μL of a DNase-free aqueous solution containing 25 μg of pDNA was added to 125 μL of an aqueous solution containing 2.4 mg of Zn(NO₃)₂·6H₂O and stirred to mix thoroughly. After 10 minutes, zinc nitrate complex 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 was observed, which was MHP. After washing three times, it was freeze-dried for later use.

[0090] Preparation of CaO2: Under ultrasound, 0.1g of CaCl2 and 0.35g of PVP were thoroughly dissolved in 15mL of anhydrous ethanol to yield a clear, transparent liquid. Subsequently, 1mL of NH4OH was added with continuous stirring. After 30 minutes, 200μL of H2O2 solution was slowly added dropwise to the mixture, yielding a light blue, milky-white solution. The product was collected by centrifugation at 16,000rpm for 30 minutes, washed three times with ethanol, and freeze-dried for later use.

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

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

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

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

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

[0096] 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to detect the production of ROS in cells. 5 Cells were seeded into 6-well plates and incubated at 37°C for 24 hours to allow the cells to adhere to the plates. The 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). After grouping, each group was incubated at 37°C for 6 hours. Then, the culture medium was replaced with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The US irradiation group was irradiated with US (1.0 MHz, 1.5 W / cm 2 , 50% duty cycle, 5 min), and then incubated at 37°C for 30 min. The ROS generation in the cells of each treatment group was observed under a fluorescence microscope (displaying green fluorescence).

[0097] Live-death staining and CCK8 assays were used to evaluate the ability of the ROS-promoting nanoplatform (MHP) to kill tumor cells in vitro. Western blot was used to detect the expression of specific proteins that cause cell necroptosis. First, 4T1 cells (5×10 5 Cells were seeded into 6-well plates and incubated at 37°C for 24 hours to allow the cells to adhere to the plates. The 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). After grouping, each group was incubated at 37°C for 6 hours, washed with PBS three times, and then the US irradiation group was irradiated with US (1.0 MHz, 1.5 W / cm 2, 50% duty cycle, 5 minutes). Then, a mixed solution of calcein-acetoxymethyl ester (Calcein-AM) and propidium iodide (PI) was added to all wells and stained at 37°C for 15 minutes. The cells were observed under an inverted fluorescence 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. For CCK8 detection, 4T1 cells (1×10 4 Cells were seeded in 96-well plates (100 μL / well) and treated as above. The prepared CCK-8 solution (100 μL / well) was added and incubated at 37°C for 1 hour. The absorbance at 450 nm was measured on a microplate reader. Western blot analysis: Cells were treated as above. After each treatment, the cells were incubated at 37°C for 12 hours. The cells were then lysed and protein extracted for Western blot analysis. Equal amounts of total protein were separated by 10% SDS PAGE and transferred to PVDF membranes. After blocking, the membranes were 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 antibodies at room temperature for 1 hour. Finally, after washing three times with TBST, the target proteins were visualized using an enhanced chemiluminescence (ECL) kit.

[0098] Finally, the efficacy of ROS-promoting nanoplatform (MHP) in inducing tumor cells to produce ROS and necroptosis under ultrasound is shown as follows: Figure 2 As shown, from Figure 2 It can be seen that 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 anti-tumor therapeutic effect of the ultrasound-controlled oxygen-producing MOF multifunctional hydrogel platform (GA@CaMP).

[0100] To further evaluate the in vivo anti-tumor effect of ultrasound-controlled oxygen-producing MOF multifunctional hydrogel platform (GA@CaMP), an orthotopic tumor mouse model was constructed and 4T1 tumor cells (1×10 6 ) were injected into the third pair of left mammary fat pads of female BALB / c mice (4-5 weeks old). 3 The 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@CaM+US and (G6) GA@CaMP+US. The US irradiation group was irradiated with US (1.0 MHz, 1.5 W / cm 2, 50% duty cycle, 5 minutes). The above drugs were injected only on day 1. The ultrasound treatment group was irradiated with ultrasound on days 2, 4, 6, and 8. During days 0-14, the body weight and tumor volume of mice were measured every 2 days. The formula (tumor length) × (tumor width) was used. 2 / 2 The tumor volume was calculated and a tumor growth curve was drawn. The mouse tumors were collected, weighed, and stained with hematoxylin-eosin (H&E). Figure 3 It can be seen that under ultrasound irradiation, the GA@CaMP gel platform has a significant anti-tumor effect.

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

[0102] To examine the effects of different treatments on osteogenic differentiation of BMSCs, we performed a Transwell assay to simulate a co-culture environment. Specifically, BMSCs (2 × 10 4 / well) were seeded in the lower chamber of Transwell, the hydrogel group was placed in the upper chamber (100vl), and the CON group added an equal amount of PBS to the upper chamber to replace it. Incubate at 37°C for 24 hours to allow the cells to attach to the well plate. The culture medium of all groups was replaced with osteogenic induction medium (DMEM complete medium supplemented with 10mM β-glycerophosphate disodium salt, 50μg / mL L-ascorbic acid and 10nM dexamethasone), which was changed every three days. The cells were then divided into the following groups: (G1) control, (G2) GA@CaMP, (G3) GA@CaM+US and (G4) GA@CaMP+US. The US irradiation group was irradiated with US (1.0MHz, 0.3W / cm 2, 50% duty cycle, 15 minutes). On the 7th day of co-culture, the cells were stained with a BCIP / NBTALP colorimetric 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 fixed at 4°C for 20 minutes. The cells were then washed three times with PBS and stained with 1× BCIP / NBT staining solution. The cells were incubated at room temperature in the dark for 30 minutes and washed three times with PBS. The staining was observed under an optical microscope and photographed. To determine alkaline phosphatase (ALP) activity, 100 μL of Western and IP cell lysis buffer (without inhibitors) was added to the lower chamber, followed by centrifugation to obtain the supernatant. The live ALP activity was determined according to the instructions of the BCA protein detection kit. On the 14th day of co-culture, Alizarin Red S (ARS) staining was performed to detect the formation of mineralized matrix. The operation steps are the same as those for ALP detection. After the cells are fixed and washed, the prepared 2% ARS staining solution is added to each well and incubated at room temperature for 30 minutes. The staining solution is aspirated and the cells are washed three times with sterile PBS. The color reaction is terminated and the mineralized matrix is ​​observed under an optical microscope. Subsequently, 1 ml of 10% cetylpyridinium chloride solution is added to each well. After incubation for 15 minutes, the supernatant is aspirated into a 96-well plate and the absorbance at a wavelength of 562 nm is measured using a microplate reader (Thermo 3001, Thermo Scientific, USA) to quantify the amount of mineralized matrix. Figure 4 It can be seen that under ultrasound irradiation, the GA@CaMP gel platform is conducive to the progression of BMSCs towards osteogenic differentiation and significantly improves the expression of osteogenic markers.

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

[0104] To evaluate the in vivo osteoreplasty efficacy of the GA@CaMP hydrogel platform, a critical skull defect model was constructed in rats. Six-week-old male Sprague Dawley rats were deeply anesthetized with 4% chloral hydrate. Subsequently, the skin of the head was incised, and tissue was bluntly dissected until the skull was fully exposed. The periosteum was neatly incised and carefully protected. A 5-mm-diameter circular critical skull defect was created on the right side of the midline. The rats were randomly divided into four groups: (G1) control, (G2) GA@CaMP, (G3) GA@CaM+US, and (G4) GA@CaMP+US. The sterilized gel platform was then implanted into the defect area. Rats in the CON group, which did not receive any filler material, served as controls. Finally, the rat scalp was carefully sutured layer by layer, and cefuroxime was administered intraperitoneally daily for 3 consecutive days to prevent potential infection. The US-irradiated group underwent US irradiation (1.0 MHz, 0.3 W / cm2) on days 1, 5, 10, and 14 postoperatively. 2, 50% duty cycle, 15 minutes). After the 8th week, the rats were euthanized and the skulls were obtained. The samples were fixed in 4% paraformaldehyde, and the mineralization of the defect area was analyzed by micro-computed tomography (micro-CT). The skull tissue was decalcified and embedded in paraffin to prepare tissue sections. Runx2 immunofluorescence staining was performed to determine the degree of bone repair, and HIF-1α immunohistochemistry was performed to evaluate the relief of tissue hypoxia. Figure 5 As shown in the results, under ultrasound irradiation, the GA@CaMP gel platform can effectively promote matrix maturation and mineralization during bone repair and remodeling, enhance the expression of osteogenesis-related proteins, alleviate the hypoxic microenvironment, and jointly accelerate the regeneration of bone defects.

Claims

1. A nanoparticle, which is formed by forming a metal organic framework with zinc nitrate hexahydrate and dimethylimidazole to encapsulate hematoporphyrin monomethyl ether and a plasmid containing a ZBP1 sequence.

2. The nanoparticle according to claim 1, wherein The gene corresponding to the ZBPI sequence is a human gene or a mouse gene; preferably, the mass ratio of zinc nitrate hexahydrate to dimethylimidazole is 1:(5-20), for example, 1:10; the mass ratio of hematoporphyrin monomethyl ether to the plasmid containing the ZBP1 sequence is 1:(0.5-5), for example, 4:5; and / or, the mass ratio of hematoporphyrin monomethyl ether to dimethylimidazole is 1:(800-2000), for example, 1:1200.

3. A method for preparing nanoparticles, characterized in that: Hematoporphyrin monomethyl ether is added to a 2-methylimidazole aqueous solution, and a Zn(NO3)2·6H2O aqueous solution is added dropwise thereto; preferably, the Zn(NO3)2·6H2O contains an aqueous solution of a plasmid containing the ZBP1 sequence.

4. The preparation method according to claim 3, wherein The gene corresponding to the ZBPI sequence is a human gene or a mouse gene; preferably, the mass ratio of zinc nitrate hexahydrate to dimethylimidazole is 1:(5-20), for example, 1:10; the mass ratio of hematoporphyrin monomethyl ether to the plasmid containing the ZBP1 sequence is 1:(0.5-5), for example, 4:5; and / or, the mass ratio of hematoporphyrin monomethyl ether to dimethylimidazole is 1:(800-2000), for example, 1:1200.

5. A sustained-release hydrogel raw material, characterized in that: Comprising a hybrid gel formed by methacryloyl gelatin and methacryloyl sodium alginate, and the nanoparticles according to claim 1 or 2, or the nanoparticles according to claim 1 or 2 do not comprise a plasmid containing a ZBP1 sequence.

6. The sustained-release hydrogel material according to claim 5, characterized in that The raw materials also include oxygen-producing CaO2; preferably, each component of the raw materials meets one or more of the following conditions: The mass ratio of the methacrylated gelatin to the methacrylated sodium alginate is (3-20):3, for example 7:3; The final concentration of the nanoparticles in the raw material is 100 to 500 μg / mL, for example, 200 μg / mL; And / or, the final concentration of CaO2 in the raw material is 200-1000 μg / mL, for example 600 μg / mL.

7. A sustained-release hydrogel, characterized in that: The sustained-release hydrogel material according to any one of claims 4 to 6 is irradiated with ultraviolet light to form the sustained-release hydrogel. Preferably, the ultraviolet light is 405 nm and 5 W, and the irradiation time is 5-10 seconds.

8. A method for preparing a sustained-release hydrogel, characterized in that: The following steps are involved: (1) preparing a methacrylated sodium alginate solution containing the nanoparticles according to claim 1 or 2 as solution B, and a methacrylated gelatin solution 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, preferably, the ultraviolet light has an energy of 405 nm and a power of 5 W, and the irradiation time is 5-10 seconds.

9. The preparation method according to claim 8, wherein In step (1), solution A contains CaO2; preferably, 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 (3-20):3, for example 7:3; The final concentration of the nanoparticles is 100 to 500 μg / mL, for example, 200 μg / mL; And / or, the final concentration of CaO2 is 200-1000 μg / mL, for example 600 μg / mL.

10. An ultrasound system comprising a system formed by ultrasound waves and the nanoparticles according to claim 1 or 2 or the sustained-release hydrogel according to claim 7, wherein the nanoparticles or the sustained-release hydrogel are irradiated with the ultrasound waves.

11. The ultrasound system according to claim 10, wherein The ultrasonic wave satisfies one or more of the following conditions: 1.0-5.0MHz; 0.1-5.0W cm -2 For example, 1.5W cm -2 ; and / or, 10%-70% duty cycle, such as 50% duty cycle; The ultrasonic irradiation time is 3-15 min, for example, 5 min.

12. Use of the nanoparticles according to claim 1 or 2, the nanoparticles obtained by the preparation method according to claim 3 or 4, the sustained-release hydrogel raw material according to claim 5 or 6, the sustained-release hydrogel according to claim 7, the sustained-release hydrogel obtained by the preparation method according to claim 8 or 9, or the ultrasonic system according to claim 10 or 11 in the preparation of anti-tumor / cancer drugs; preferably, the tumor / cancer includes prostate cancer, colon cancer, rectal cancer, ovarian cancer, kidney cancer, breast cancer, glioblastoma, melanoma, malignant melanoma or lung cancer.

13. Use of the nanoparticles according to claim 1 or 2, the nanoparticles obtained by the preparation method according to claim 3 or 4, the sustained-release hydrogel raw material according to claim 5 or 6, the sustained-release hydrogel according to claim 7, the sustained-release hydrogel obtained by the preparation method according to claim 8 or 9, or the ultrasonic system according to claim 10 or 11 in the preparation of a drug for bone repair; preferably, the bone repair is caused by tumor bone metastasis, or the bone repair is the induction of osteogenic differentiation of bone marrow mesenchymal stem cells.

Images