A composite nanoparticle targeting mesenchymal stem cells and application thereof in preparation of a drug for promoting vascularized bone regeneration

Abstract

This invention relates to a ZIF-8-based composite nanoparticle with a core-shell structure. The core is a zeolite imidazole ester backbone ZIF-8 carrier, and the pores of the ZIF-8 core are loaded with a photothermal agent. Liposomes are coated on the outer surface of the core as a shell, and CD90 antibodies are coupled to the surface of the liposomes. The composite nanoparticle targets mesenchymal stem cells, thereby more effectively delivering zinc ions into mesenchymal stem cells (MSCs) to regulate MMP-10 (matrix metalloproteinase-10) expression. The composite nanoparticle can be used to prepare drugs or medical dressings for treating bone defects and / or promoting vascularized bone regeneration.

Description

Technical Field

[0001] This invention relates to composite nanoparticles, and more specifically, to a composite nanoparticle that targets mesenchymal stem cells and can be used for vascularized bone regeneration. Background Technology

[0002] Bone regeneration, especially the repair of large-area bone defects, has become a major challenge in clinical medicine. These problems are often accompanied by local blood flow disorders and hypoxic microenvironments. These two factors hinder the homing and differentiation processes of host cells, thus limiting the bone tissue's ability to repair itself.

[0003] With the rapid development of stem cell technology, mesenchymal stem cell (MSC) therapy has shown significant clinical potential in promoting bone tissue regeneration. MSCs possess the ability to self-renew and differentiate into various cell types, accelerating the bone repair process. Cultured MSCs can be delivered to bone defect sites via local injection or other methods, utilizing their paracrine function or direct differentiation capacity to promote tissue regeneration. However, current MSC transplantation strategies still face significant challenges, especially after MSCs are transferred from the oxygen-rich in vitro culture environment to the extremely hypoxic in vivo microenvironment. This often leads to reduced proliferation, survival, and differentiation capacity, thus limiting the stability and effectiveness of treatment.

[0004] To address these issues, there is an urgent need to develop novel materials or methods to optimize the survival and function of MSCs. Therefore, composite nanoparticles based on the ZIF-8 structure, due to their excellent biocompatibility and ability to regulate the local microenvironment, can become an important candidate material in this research direction.

[0005] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] In one aspect, the present invention provides a ZIF-8-based composite nanoparticle having a core-shell structure, wherein the core is a zeolite imidazole ester backbone ZIF-8 carrier, the pores of the ZIF-8 core are loaded with a photothermal agent, and liposomes are wrapped on the outer surface of the core as a shell, and CD90 antibody is coupled to the surface of the liposomes.

[0007] In some embodiments, the composite nanoparticles, wherein the ZIF-8 carrier is formed from zinc ions and 2-methylimidazole anions, preferably self-assembled, and more preferably assembled from zinc nitrate hexahydrate and 2-methylimidazole.

[0008] In another aspect, the present invention provides a method for preparing composite nanoparticles, comprising:

[0009] 1) ZIF-8 carrier was prepared from zinc nitrate hexahydrate and 2-methylimidazole;

[0010] 2) A photothermal agent, preferably indocyanine green (ICG), is loaded into the pores of the ZIF-8 core to prepare ZIF-8-ICG;

[0011] 3) DSPE-PEG-CD90 is prepared by reacting CD90 antibody with DSPE-PEG-NHS. Then, the DSPE-PEG-CD90 is mixed with soybean phosphatidylcholine and cholesterol, preferably by thin-film hydration, to prepare liposomes.

[0012] 4) ZIF-8-ICG was mixed into the liposome solution to prepare composite nanoparticles.

[0013] In another aspect, the present invention provides a pharmaceutical composition or medical dressing comprising the composite nanoparticles described in any of the foregoing embodiments, and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition or medical dressing can be used to treat bone defects or promote vascularized bone regeneration.

[0014] In another aspect, the present invention provides the use of the composite nanoparticles described in any of the foregoing claims in the preparation of medicaments or medical dressings for treating bone defects and / or promoting vascularized bone regeneration.

[0015] In another aspect, the present invention provides a method for treating bone defects and / or promoting vascularized bone regeneration, comprising applying the composite nanoparticles described in any of the preceding claims to a subject in need, and irradiating the application site with near-infrared light some time after application (e.g., 1-7 days).

[0016] In this invention, the composite nanoparticles described in any of the foregoing claims can actively target MSCs in the bone defect area and regulate them in stages. In the first stage, zinc ions are released in a controlled manner to promote early angiogenesis. In the second stage, photothermal effects triggered by near-infrared light promote later osteoogenesis, thereby synergistically and efficiently completing the entire process of vascularized bone regeneration.

[0017] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0018] Figure 1 A transmission electron microscope image of the ZIF-8-ICG core is shown.

[0019] Figure 2Transmission electron microscopy (TEM) image of the composite nanoparticles CD90@ZIF-8-ICG is shown (the scale bar in the figure represents 100 nm).

[0020] Figure 3 The particle size of nanoparticles was determined using dynamic light scattering (DLS).

[0021] Figure 4 The diagram shows the changes in zeta potential of nanoparticles at different preparation stages.

[0022] Figure 5 The ultraviolet-visible-near-infrared absorption spectrum of the nanoparticles is shown.

[0023] Figure 6 The image shows the photothermal heating effect of nanoparticles under near-infrared light irradiation.

[0024] Figure 7 The flow cytometry results of the nanoparticles are shown.

[0025] Figure 8 The CCK-8 assay results for the composite nanoparticles are shown.

[0026] Figure 9 The flow cytometry results of apoptosis in nanoparticles are shown.

[0027] Figure 10 A fluorescence microscope image of the composite nanoparticles co-cultured with cells is shown (the scale bar in the figure represents 50 μm).

[0028] Figure 11 The average fluorescence intensity of intracellular zinc ions as measured by fluorescence method is shown.

[0029] Figure 12 The results of ELISA and Western Blot analysis of MMP-10 protein expression are shown.

[0030] Figure 13 A microscopic image of composite nanoparticles promoting angiogenesis is shown.

[0031] Figure 14 The results of real-time quantitative PCR for bone-related genes (Alp, Runx2, Osx, and Opn) are shown.

[0032] Figure 15 Histochemical staining images of osteogenic differentiation and mineralization are shown.

[0033] Figure 16 The results of apoptosis detection after 7 consecutive days of light exposure are shown.

[0034] Figure 17The results of flow cytometry analysis of local tissue in the bone defect area are shown.

[0035] Figure 18 The results of in vivo fluorescence imaging are shown.

[0036] Figure 19 The image shows a temperature change graph recorded by an infrared thermal imager.

[0037] Figure 20 The image shows a Micro-CT 3D reconstruction after skull defect repair.

[0038] Figure 21 The quantitative analysis of new bone volume and bone mineral density after skull defect repair is shown.

[0039] Figure 22 The H&E staining results are shown (the upward scale bar in the figure represents 1000 μm, the downward scale bar in the figure represents 100 μm, and B represents bone).

[0040] Figure 23 The results of CD31 immunofluorescence staining are shown.

[0041] Figure 24 The results of RUNX2 immunohistochemical staining are shown (the scale bar in the figure represents 100 μm).

[0042] Figure 25 The results of routine blood tests and blood biochemistry tests are shown.

[0043] Figure 26 Immunohistochemical staining images of TNF-α and IL-1β in local tissue of bone defect are shown. Detailed Implementation

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] The terms “some embodiments,” “certain embodiments,” or “other embodiments” used in this specification mean that a particular feature, structure, or characteristic described in relation to that embodiment is included in at least some embodiments of the invention, but not necessarily in all embodiments.

[0047] As used herein, ranges and quantities may be expressed as “about” a specific value or range. “About” also includes the exact quantity. Typically, the term “about” includes quantities expected to be within experimental error, such as within 15%, 10%, or 5%. The chapter headings used herein are for organizational purposes only and should not be construed as limiting the topics described.

[0048] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0049] This invention provides a ZIF-8-based composite nanoparticle with a core-shell structure. The core is a zeolite imidazole ester backbone ZIF-8 carrier, and the pores of the ZIF-8 core are loaded with a photothermal agent. The outer surface of the core is coated with liposomes as a shell, and the surface of the liposomes is coupled with CD90 antibody.

[0050] In some embodiments, the ZIF-8 carrier is composed of Zn 2+ It is a porous material with a topological structure composed of imidazole ligands, which has the advantages of regular pore size, high specific surface area and unsaturated metal sites.

[0051] In some embodiments, the ZIF-8 carrier is prepared from zinc ions and 2-methylimidazole anions, preferably by self-assembly, and more preferably by self-assembly of zinc nitrate hexahydrate and 2-methylimidazole. In this invention, the core function of the ZIF-8 carrier is to act as a biodegradable carrier, releasing bioactive zinc ions within cells.

[0052] In some embodiments, the photothermal agent is indocyanine green (ICG), Prussian blue, gold nanorods, copper sulfide nanoparticles, etc., preferably indocyanine green (ICG). In this invention, the photothermal agent indocyanine green (ICG) can be loaded into the pores of the ZIF-8 core. ICG is an FDA-approved near-infrared dye that can convert light energy into heat energy under 808nm laser irradiation.

[0053] In some embodiments, the CD90 antibody is an anti-CD90 monoclonal antibody. In some embodiments, the CD90 antibody is attached to liposomes via a grafting agent (DSPE-PEG-NHS) with N-hydroxysuccinimide (NHS) as the reactive group.

[0054] In some embodiments, the liposomes are prepared by mixing CD90 antibody with DSPE-PEG-NHS to prepare DSPE-PEG-CD90, and then mixing the DSPE-PEG-CD90 with soybean phosphatidylcholine and cholesterol, preferably by thin-film hydration, to form liposomes.

[0055] In some embodiments, the composite nanoparticles prepared by the present invention target mesenchymal stem cells, preferably by delivering zinc ions into mesenchymal stem cells (MSCs) to regulate the expression of MMP-10 (matrix metalloproteinase-10), and preferably, the average particle size of the composite nanoparticles is 50-250 nanometers, for example, 100-200 nanometers.

[0056] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cell. They possess all the common characteristics of stem cells, namely, the ability to self-replicate and differentiate into multiple lineages. They are also widely available, for example, from bone marrow, umbilical cord blood, umbilical cord tissue, placental tissue, and adipose tissue.

[0057] Bone marrow mesenchymal stem cells (BMSCs) are a type of non-hematopoietic stem cell found in the bone marrow. They are not only present in the bone marrow but also in skeletal muscle, the periosteum, and bone trabeculae.

[0058] This invention provides a ZIF-8-based composite nanoparticle, comprising:

[0059] 1) ZIF-8 carrier was prepared from zinc nitrate hexahydrate and 2-methylimidazole;

[0060] 2) A photothermal agent, preferably indocyanine green (ICG), is loaded into the pores of the ZIF-8 core to prepare ZIF-8-ICG;

[0061] 3) DSPE-PEG-CD90 is prepared by reacting CD90 antibody with DSPE-PEG-NHS. Then, the DSPE-PEG-CD90 is mixed with soybean phosphatidylcholine and cholesterol, preferably by thin-film hydration, to form liposomes.

[0062] 4) The ZIF-8-ICG and the liposomes are mixed to form composite nanoparticles CD90@ZIF-8-ICG.

[0063] In some embodiments, in step 1) above, the zinc nitrate hexahydrate and 2-methylimidazole are dissolved in methanol.

[0064] In some embodiments, the ZIF-8-ICG, after preparation, is centrifuged and washed several times with methanol for subsequent use.

[0065] In some embodiments, in step 3), the pH of the reaction between the CD90 antibody and DSPE-PEG-NHS is 7.5-8.5, for example, pH 8.0, and then the solution is obtained by dialysis to obtain a DSPE-PEG-CD90 solution.

[0066] In some embodiments, in step 3), preferably, SPC (soybean phosphatidylcholine) and cholesterol are dissolved in chloroform, evaporated under reduced pressure to form a thin film, and then DSPE-PEG-CD90 is added to prepare liposomes. Then, ZIF-8-ICG is mixed into the liposome solution, ultrasonically treated, and extruded through a polycarbonate membrane to prepare composite nanoparticles CD90@ZIF-8-ICG. Preferably, the pore size of the polycarbonate membrane is 50-150 nm, preferably 80-120 nm, for example, 100 nm.

[0067] In some embodiments, the prepared composite nanoparticles CD90@ZIF-8-ICG can be added with a cryoprotectant and can be freeze-dried for later use.

[0068] The present invention provides a pharmaceutical composition comprising the aforementioned composite nanoparticles and a pharmaceutically acceptable carrier, preferably, the pharmaceutical composition for treating bone defects or promoting vascularized bone regeneration.

[0069] The present invention provides a medical dressing containing the aforementioned composite nanoparticles and a pharmaceutically acceptable carrier. Preferably, the medical dressing is used to treat bone defects or promote vascularized bone regeneration.

[0070] In some embodiments, "promoting vascularized bone regeneration" means that during the repair or regeneration of bone defects, the composite nanoparticles of this invention accelerate angiogenesis (vascularization), thereby more effectively promoting new bone formation (bone regeneration). Skeleton is a highly vascularized tissue; the formation and maintenance of new bone depend on a rich blood supply. Vascularization is a prerequisite for successful bone regeneration, and VEGF (vascular endothelial growth factor) is a commonly used pro-angiogenic factor.

[0071] This invention provides the application of the composite nanoparticles of this invention in the preparation of drugs or medical dressings for treating bone defects and / or promoting vascularized bone regeneration. Preferably, the composite nanoparticles are used in the preparation of drugs or medical dressings for early vascularization and / or late osteogenic processes in bone repair. Preferably, the composite nanoparticles enhance the tube-forming function of vascular endothelial cells. Preferably, the composite nanoparticles promote osteogenic differentiation and / or mineralization of MSCs, and have improved osteogenic capacity.

[0072] The present invention also provides a method for promoting vascularized bone regeneration, the method comprising applying the composite nanoparticles to a subject in need and irradiating the application site with near-infrared light a period of time after application (e.g., 1-7 days).

[0073] In this invention, "NIR irradiation" refers to the use of near-infrared (NIR) light to irradiate live animals or humans to achieve a certain experimental or therapeutic purpose. Here, NIR usually refers to light with a wavelength range of about 700–1000 nanometers.

[0074] In some embodiments, the composite nanoparticles of the present invention target and deliver zinc ions into mesenchymal stem cells to regulate MMP-10 expression and promote angiogenesis. Preferably, the mechanism of action is a two-stage intelligent regulation. In the first stage, the composite nanoparticles of the present invention can promote angiogenesis. After being targeted by CD90 and internalized by MSCs in the bone defect area, the composite nanoparticles are degraded by ZIF-8 to release Zn. 2+ This leads to Zn2 in the cytoplasm of MSCs. + Enrichment inhibits ZIP6 protein and activates the JAK1 / STAT1 / MMP-10 pathway, promoting MMP-10 secretion and thus enhancing the tube-forming function of vascular endothelial cells. In some embodiments, in vitro experiments showed that conditioned medium containing BMSCs treated with the composite nanoparticles of the present invention promoted endothelial cell tube formation comparable to the positive control group supplemented with 20 ng / mL VEGF. In a second stage, the composite nanoparticles of the present invention can promote osteogenic formation; in some embodiments, after using the composite nanoparticles of the present invention, osteogenic formation was promoted using an 808 nm near-infrared laser (1.5 W / cm²). 2 Irradiation of the bone defect area for 3 minutes (approximately 40-43°C) produces a mild photothermal effect of ICG within the composite nanoparticles. This heat can promote osteogenic differentiation of MSCs. In vitro osteogenic induction followed by alkaline phosphatase and Alizarin Red staining demonstrates that the photothermal effect of the composite nanoparticles of this invention can promote osteogenic differentiation and mineralization of BMSCs. In vivo RUNX2 immunohistochemical staining of bone defect tissue demonstrates that the composite nanoparticles of this invention can also promote osteoblast differentiation in vivo.

[0075] In some embodiments, the technical solution corresponding to the composite nanoparticles "CD90@ZIF-8-ICG combined with NIR irradiation" prepared by the present invention achieves better levels of new bone volume and mineral density in all groups, and is significantly superior to all other comparative examples, including the CD90@ZIF-8-ICG group without NIR intervention.

[0076] In some embodiments, the present invention ingeniously combines ion stimulation with photothermal stimulation. The ICG in the core of the composite nanoparticle generates a mild photothermal effect (approximately 40–43°C), which can promote osteogenic differentiation of MSCs. The core of the composite nanoparticle is coated with a layer of modified liposomes, and the surface of the liposomes is coupled with a CD90 antibody. Through this antibody, the entire composite nanoparticle can be guided to effectively and precisely target MSCs in the bone defect area.

[0077] The composite nanoparticles prepared by this invention can promote the orderly development of two key stages of bone repair: early vascularization and late osteoogenesis, which is more in line with the physiological bone repair process.

[0078] The composite nanoparticles or pharmaceutical compositions provided by this invention can be administered to patients using methods well known to those skilled in the art, such as intra-arterial, intravenous, or percutaneous injection, preferably by injection into the area of ​​bone defect in the patient. The dosage and method of administration vary depending on the patient's weight and age, and can be selected as needed by those skilled in the art.

[0079] The following embodiments are provided to illustrate the principles and practice of the embodiments disclosed herein more clearly to those skilled in the art, and should not be construed as limiting the scope of any claimed embodiments.

[0080] Example 1: Preparation and characterization of composite nanoparticles (CD90@ZIF-8-ICG)

[0081] 1. Preparation of kernel ZIF-8-ICG:

[0082] 330 mg of 2-methylimidazole (Adamas Reagent Co., Ltd., Shanghai) and 150 mg of zinc nitrate hexahydrate (Adamas Reagent Co., Ltd., Shanghai) were dissolved in 10 ml of methanol respectively. The zinc nitrate solution was slowly added dropwise to the 2-methylimidazole solution, followed by the addition of 5 mg of indocyanine green (ICG, Shanghai Aladdin Biochemical Technology Co., Ltd.). The mixture was stirred at room temperature for 1 hour. The reaction product was collected by centrifugation at 12,000 rpm for 10 minutes, washed three times with methanol, and finally resuspended in ultrapure water to obtain the core ZIF-8-ICG.

[0083] 2. Preparation of CD90@ZIF-8-ICG composite nanoparticles

[0084] 3 mg of CD90 antibody (Proteintech) was reacted with 1.11 mg of DSPE-PEG-NHS (Xi'an Ruixi Biotechnology) in 3 ml of aqueous solution at pH 8.0. After the reaction was completed, the solution containing DSPE-PEG-CD90 conjugate was obtained by dialysis purification.

[0085] Subsequently, the liposome shell was prepared using a thin-film hydration method: 8.0 mg of SPC (soybean phosphatidylcholine) and 2.0 mg of cholesterol (Xi'an Ruixi Biotechnology) were dissolved in 3 ml of chloroform and evaporated under reduced pressure to form a uniform lipid film. Then, 2 ml of the above-mentioned DSPE-PEG-CD90 conjugate solution was added to hydrate the film, thus preparing a liposome solution with CD90 antibody modified on the surface.

[0086] Finally, the ZIF-8-ICG core prepared in step 1 was mixed into the above liposome solution, subjected to ultrasonic treatment, and extruded through a 100nm pore size polycarbonate membrane (Whatman, Cardiff, UK) to uniformly coat the liposome shell onto the core surface. After adding a cryoprotectant, it was freeze-dried to obtain the final product CD90@ZIF-8-ICG composite nanoparticles.

[0087] 3. Characterization of composite nanoparticles

[0088] Morphological and structural observation Nanoparticles were observed using a transmission electron microscope (TEM). Figure 1 The transmission electron microscope image of the prepared ZIF-8-ICG core shows that the core exhibits a uniform spherical morphology. Figure 2 The transmission electron microscope image of the prepared composite nanoparticle CD90@ZIF-8-ICG clearly shows that it has a core-shell structure, which indicates that the liposome shell is successfully coated on the surface of the ZIF-8-ICG core.

[0089] Particle size and zeta potential analysis Particle size and zeta potential were determined using dynamic light scattering (DLS). Figure 3 As shown, the average hydration kinetic particle size of the core (ZIF-8-ICG) was 123.29±6.10 nm. After coating with a liposome shell, the average particle size of the final product (CD90@ZIF-8-ICG) increased to 139.98±8.17 nm, which further confirms the successful coating of the ZIF-8-ICG core with a liposome shell. Figure 4 The graph shows the zeta potential changes of nanoparticles at different preparation stages. The potential of the final product is similar to that of liposomes, but significantly different from that of the core. This also indicates that the liposome shell was successfully coated on the surface of the ZIF-8-ICG core.

[0090] Successful loading and photothermal performance of ICG Detection was performed using ultraviolet-visible-near-infrared absorption spectroscopy and photothermal experiments. Figure 5 The image shows the UV-Vis-NIR absorption spectrum of the nanoparticles. The results show that, unlike pure ZIF-8, CD90@ZIF-8-ICG exhibits a characteristic absorption peak of ICG at around 800 nm, proving that ICG has been successfully loaded into the core. Figure 6 This demonstrates the photothermal heating effect of nanoparticles under near-infrared light irradiation, using an 808nm laser (1.0W / cm²). 2 Under irradiation, the temperature of the CD90@ZIF-8-ICG solution can rise significantly within 3 minutes, demonstrating its excellent photothermal conversion capability.

[0091] Successful modification of CD90 antibody : Detection of CD90 antibodies on the surface of nanoparticles using flow cytometry. Figure 7 The flow cytometry results show that CD90@ZIF-8-ICG exhibits a very high CD90 positivity rate compared to unmodified particles, demonstrating that the CD90 antibody was successfully conjugated to the surface of the nanoparticles.

[0092] Example 2: In vitro biological function verification of composite nanoparticles CD90@ZIF-8-ICG

[0093] A series of in vitro cell experiments verified the dual-stage regulatory function of the composite nanoparticles (CD90@ZIF-8-ICG) of this invention in promoting angiogenesis in the first stage and osteogenic differentiation in the second stage, and evaluated their biocompatibility.

[0094] 1. Cell Culture and Experimental Grouping

[0095] Bone marrow mesenchymal stem cells (BMSCs): Femurs from 6-8 week old male C57BL / 6 mice were collected after bone marrow flushing and filtration through a 70 μm cell sieve. Cells were cultured in α-MEM medium containing 20% ​​fetal bovine serum, 5% penicillin / streptomycin, and 5% glutamine at 37°C and 5% CO2. Second-generation (P2) cells were used in all experiments.

[0096] Human umbilical vein endothelial cells (HUVECs): purchased from Jiangsu Cyagen Biosciences and cultured according to standard methods.

[0097] Experimental grouping: Control and experimental groups were set up according to different experimental objectives, and the specific grouping is described in detail in each experiment. The working concentration of CD90@ZIF-8-ICG was determined to be 30 μg / mL after screening.

[0098] 2. Biosafety assessment

[0099] To verify the biocompatibility of the material, the CCK-8 (Cell Counting Kit-8) method and apoptosis assay were used for detection.

[0100] Cell viability assay BMSCs were co-cultured with different concentrations of CD90@ZIF-8-ICG (10, 30, 50, 70, 90 μg / mL) for 24 hours, and cell viability was detected using a CCK-8 assay kit.

[0101] Figure 8 The results of CCK-8 assay (where ns represents not significant, *p<0.05, **p<0.01, and the same applies below) show that when the concentration is less than or equal to 30 μg / mL, the prepared composite nanoparticles have no significant effect on the cell viability of BMSCs, demonstrating that they have good biocompatibility within this concentration range.

[0102] Apoptosis detection After co-culturing BMSCs with different concentrations of CD90@ZIF-8-ICG (0, 10, 30, 50 μg / mL) for 24 hours, cell apoptosis was detected using the FITC Annexin V apoptosis detection kit and flow cytometry.

[0103] Figure 9 The flow cytometry results for apoptosis also showed that at a concentration of 30 μg / mL, the nanoparticles did not induce significant apoptosis.

[0104] 3. Phase 1 Functional Validation: Targeted Delivery and Angiogenesis

[0105] Targeting and cellular endocytosis To verify the targeting ability of the CD90 antibody, Cy5-labeled CD90@ZIF-8-ICG was co-cultured with PKH67-labeled BMSCs and RAW264.7 cells for 24 hours and observed using a fluorescence microscope.

[0106] Figure 10 The image shows a fluorescence microscope image of co-cultured cells. As can be seen from the image, most of the red nanoparticles (Cy5 labeled) are internalized by green BMSCs (PKH67 labeled), demonstrating its excellent targeting ability to BMSCs.

[0107] Intracellular zinc ion release and MMP-10 upregulation After co-culturing BMSCs with CD90@ZIF-8-ICG, the intracellular zinc ion concentration and MMP-10 protein secretion level were detected using a zinc ion probe and ELISA.

[0108] Figure 11The study showed the average fluorescence intensity of intracellular zinc ions after co-culturing BMSCs with PBS (control group) or CD90@ZIF-8-ICG (experimental group), as measured by fluorescence method. The data showed that the zinc ion level in the CD90@ZIF-8-ICG treatment group was significantly higher than that in the control group.

[0109] Figure 12 The results of Western Blot and ELISA quantification of MMP-10 protein expression are shown, respectively. The data show that the level of MMP-10 secreted by BMSCs was significantly upregulated after treatment with the composite nanoparticles of this invention.

[0110] Angiogenesis function :

[0111] Conditioned culture medium containing CD90@ZIF-8-ICG was collected and co-cultured with HUVECs for in vitro tube formation experiments. Untreated BMSCs and VEGF-supplemented groups were set up as negative and positive controls, respectively.

[0112] Figure 13 The image shows a microscopic image of the in vitro tubular formation experiment. The results indicate that the conditioned medium containing BMSCs treated with the composite nanoparticles of this invention effectively promoted the formation of tubular networks by endothelial cells, comparable to the positive control group supplemented with 20 ng / mL VEGF, and significantly superior to the untreated BMSC group. This demonstrates that the first-stage function of the composite nanoparticles of this invention—targeted delivery and angiogenesis promotion—indirectly promotes angiogenesis by regulating BMSCs.

[0113] 4. Second-stage functional verification: photothermal effect and promotion of osteodifferentiation

[0114] Osteogenesis-promoting assay: BMSCs were cultured in osteogenic induction medium, and 30 μg / mL of CD90@ZIF-8-ICG was added. For the next 7 days, the cells were treated daily with an 808 nm near-infrared laser (1.0 W / cm²). 2 Irradiate for 3 minutes.

[0115] Figure 14 The results of real-time quantitative PCR for osteogenic-related genes (Alp, Runx2, Osx, Opn) show that the expression levels of all key osteogenic genes were significantly upregulated after treatment with the composite nanoparticles of this invention and NIR light.

[0116] Figure 15Histochemical staining images of osteogenic differentiation and mineralization: alkaline phosphatase (ALP) staining after 7 days of culture (top) and alizarin red staining after 21 days of culture (bottom). The results show that the experimental group treated with "composite nanoparticles + NIR" had significantly higher osteogenic differentiation capacity and calcium salt deposition level than the control group, proving the strong promoting effect of photothermal effect on osteogenic formation.

[0117] 5. Security verification:

[0118] Figure 16 The results of apoptosis detection after 7 consecutive days of light exposure show that the mild photothermal therapy used in this invention does not cause additional apoptotic damage to cells, proving the safety of the second-stage operation.

[0119] Example 3: Verification of the in vivo bone defect repair effect of composite nanoparticles CD90@ZIF-8-ICG

[0120] The biocompatibility, targeting retention ability, and ultimate bone repair therapeutic effect of the composite nanoparticles (CD90@ZIF-8-ICG) of this invention were verified in vivo through experiments on a mouse skull defect model.

[0121] 1. Animal model establishment and experimental grouping

[0122] Laboratory animals: Male C57BL / 6 mice, 6–8 weeks old, purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animal experiments were approved by the Animal Ethics Committee of Beijing Stomatological Hospital (lot number: KQYY-202008-005).

[0123] Establishment of bone defect model: After anesthetizing mice, a full-thickness circular bone defect with a diameter of 4 mm was made in the midline of the skull.

[0124] Experimental grouping: Mice that successfully modeled the disease were randomly divided into 5 groups (n=6): Blank group: injected with PBS into the bone defect area; BMSC treatment group: injected with 1×10 6 BMSCs; CD90@ZIF-8-ICG group: 10 μg of the composite nanoparticles prepared in this invention were injected into the bone defect area; NIR group: PBS was injected into the bone defect area and then irradiated with near-infrared light; CD90@ZIF-8-ICG+NIR group: 10 μg of the composite nanoparticles prepared in this invention were injected into the bone defect area and then irradiated with near-infrared light.

[0125] Dosing was administered on day 5 after modeling. All animals were sacrificed one month after the intervention for further analysis.

[0126] 2. NIR light intervention

[0127] For the NIR group and the CD90@ZIF-8-ICG+NIR group, 808nm near-infrared laser (1.5W / cm²) was used daily for 7 consecutive days after drug administration. 2 Irradiate the bone defect area for 3 minutes each time.

[0128] 3. Targeting and photothermal effect verification

[0129] Figure 17 The results of flow cytometry analysis of local tissue in the bone defect area include CD105. + CD146 + It is a marker for MSCs, which can be identified by CD105. + CD146 + The results showed that approximately 57.64% of the nanoparticles were localized in mesenchymal stem cells (MSCs) 24 hours after injection, demonstrating their precise targeting ability in vivo.

[0130] Figure 18 The results of in vivo fluorescence imaging show that the nanoparticles can remain effectively in the bone defect area for at least one week after injection, providing sufficient time for subsequent treatment.

[0131] Figure 19 The temperature change graph recorded by the infrared thermal imager shows that during 7 consecutive days of light exposure, the composite nanoparticles of this invention can stably produce a mild local photothermal effect (about 41°C), which meets the temperature conditions for promoting bone formation in the second stage.

[0132] 4. Evaluation of bone defect repair effect

[0133] Micro-CT 3D and Quantitative Analysis: Figure 20 These are Micro-CT 3D reconstruction images of each group of skull defects after repair. Figure 21 It is a quantitative analysis of key indicators such as the volume of new bone and bone mineral density after the repair of skull defects in each group.

[0134] These results clearly and quantitatively demonstrate that the amount and quality of new bone formation in the CD90@ZIF-8-ICG+NIR group were the best among all groups, significantly superior to all other control groups, proving the excellent bone repair effect of the present invention.

[0135] 1. Histological analysis – early vascularization and later osteogenesis:

[0136] The repaired bone defect tissue was sectioned and stained to evaluate the repair quality at the cellular and tissue levels. Figure 22The results of H&E (hematoxylin and eosin) staining were shown. The CD90@ZIF-8-ICG+NIR group showed a large amount of mature new bone tissue and neovascularization at the defect margin, with tight tissue connection and the highest repair quality.

[0137] Figure 23 The results of CD31 immunofluorescence staining are used to label vascular endothelial cells. The CD90@ZIF-8-ICG+NIR group showed the richest and most mature neovascular network, demonstrating the powerful promoting effect of the present invention on early vascularization of bone repair.

[0138] Figure 24 The results of RUNX2 immunohistochemical staining are used to label osteogenic progenitor cells / osteoblasts. The CD90@ZIF-8-ICG+NIR group showed the highest number of RUNX2-positive cells, demonstrating the strong promoting effect of the present invention on osteogenic formation in the later stages of bone repair.

[0139] Example 4: In vivo biosafety evaluation of CD90@ZIF-8-ICG

[0140] The biosafety of the composite nanoparticles (CD90@ZIF-8-ICG) of this invention for in vivo application was evaluated by hematological tests and local tissue inflammation analysis.

[0141] 1. Experimental Methods

[0142] Experimental animals and administration: The same animal model and administration method as in Example 3 were used.

[0143] Hematological testing: 24 hours after injecting the nanoparticles into the mice, orbital blood was collected and sent to the Department of Laboratory Medicine of Beijing Stomatological Hospital for routine blood tests and blood biochemistry tests.

[0144] Local inflammation analysis: Mice were sacrificed one month after intervention, and local tissue from the bone defect area was taken, paraffin-embedded and sectioned, and immunohistochemical staining analysis was performed on the inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β).

[0145] 2. Experimental Results

[0146] Hematological test results:

[0147] Figure 25 The results are blood routine and blood biochemistry test results, respectively. Data shows that 24 hours after injection of the composite nanoparticles of this invention, all blood indicators in mice were within the normal physiological range, with no significant difference compared to the blank control group.

[0148] Results of local inflammation analysis:

[0149] Figure 26The images show immunohistochemical staining of TNF-α and IL-1β in local tissue of bone defects. The results showed that, compared with the control group, the expression levels of the inflammatory factors TNF-α and IL-1β in the experimental group injected with the composite nanoparticles of this invention were not increased.

[0150] 3. Conclusion

[0151] Hematological and local histological analyses indicate that the composite nanoparticles of this invention have good in vivo biosafety and do not cause significant systemic toxicity or local inflammatory reactions.

[0152] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. The application of composite nanoparticles in the preparation of drugs or medical dressings for treating bone defects and / or promoting vascularized bone regeneration, wherein, The composite nanoparticles have a core-shell structure. The core is a zeolite imidazole ester backbone ZIF-8 carrier. The pores of the ZIF-8 core are loaded with a photothermal agent. The outer surface of the core is coated with liposomes as a shell. The surface of the liposomes is coupled with CD90 antibody. The ZIF-8 carrier is formed by self-assembly of zinc nitrate hexahydrate and 2-methylimidazole. The photothermal agent is indocyanine green (ICG). The composite nanoparticles target mesenchymal stem cells.

2. The application according to claim 1, wherein, The CD90 antibody is attached to liposomes via a grafting agent DSPE-PEG-NHS with N-hydroxysuccinimide (NHS) as the reactive group.

3. The application according to claim 1, wherein, The liposomes are prepared by mixing CD90 antibody with DSPE-PEG-NHS to prepare DSPE-PEG-CD90, and then mixing the DSPE-PEG-CD90 with soybean phosphatidylcholine and cholesterol to form liposomes.

4. The application according to claim 1, wherein, The composite nanoparticles deliver zinc ions into mesenchymal stem cells to regulate the expression of matrix metalloproteinase-10.

5. The application according to claim 1, wherein, The average particle size of the composite nanoparticles is 50-250 nanometers.

6. The application according to claim 1, wherein, The average particle size of the composite nanoparticles is 100-200 nanometers.

7. The application according to claim 1, wherein, The composite nanoparticles are prepared by the following steps: 1) ZIF-8 carrier was prepared from zinc nitrate hexahydrate and 2-methylimidazole; 2) Photothermal agent is loaded into the pores of the ZIF-8 core to prepare ZIF-8-ICG; 3) DSPE-PEG-CD90 is prepared by reacting CD90 antibody with DSPE-PEG-NHS. Then, the DSPE-PEG-CD90 is mixed with soybean phosphatidylcholine and cholesterol and liposomes are prepared by membrane hydration. 4) The ZIF-8-ICG and the liposomes are mixed to form composite nanoparticles CD90@ZIF-8-ICG.

8. The application of composite nanoparticles in the preparation of drugs or medical dressings for early vascularization and / or late osteogenic processes in bone repair, wherein, The composite nanoparticles have a core-shell structure. The core is a zeolite imidazole ester backbone ZIF-8 carrier. The pores of the ZIF-8 core are loaded with a photothermal agent. The outer surface of the core is coated with liposomes as a shell. The surface of the liposomes is coupled with CD90 antibody. The ZIF-8 carrier is formed by self-assembly of zinc nitrate hexahydrate and 2-methylimidazole. The photothermal agent is indocyanine green (ICG). The composite nanoparticles target mesenchymal stem cells.

9. The application according to claim 8, wherein, The composite nanoparticles enhance the tube-forming function of vascular endothelial cells.

10. The application according to claim 8, wherein, The composite nanoparticles promote osteogenic differentiation and / or mineralization of MSCs, and have improved osteogenic capacity.

11. The application according to claim 8, wherein, It includes applying the composite nanoparticles to the object in need and irradiating the application site with near-infrared light some time after application.

12. The application according to claim 11, wherein, It includes applying the composite nanoparticles to the recipient and irradiating the application site with near-infrared light 1-7 days after application.

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