A icariin-hydroxyapatite nanoparticle and a preparation method and application thereof

Abstract

The application discloses icariin-hydroxyapatite nanoparticles and a preparation method and application thereof, and belongs to the technical field of biomedical materials. The icariin-hydroxyapatite nanoparticles provided by the application comprise hydroxyapatite and icariin chemically bonded to the hydroxyapatite. The in-situ coprecipitation method is adopted to prepare the icariin-hydroxyapatite nanoparticles, and icariin is introduced in the process of hydroxyapatite nucleation and crystal growth, so that the icariin directly participates in the construction of an inorganic skeleton. In the process, the icariin is chemically bonded to the inside and the surface interface of the hydroxyapatite, so that the interface complexing and partial embedding between the drug and the inorganic matrix are realized.

Description

Technical Field

[0001] This application belongs to the field of biomedical materials technology, and in particular relates to an icariin-hydroxyapatite nanoparticle, its preparation method and application. Background Technology

[0002] With changing lifestyles and an aging population, the prevalence of osteoporosis continues to rise, and the age of onset is trending younger. Problems such as decreased bone mass and bone density further exacerbate the clinical challenges of bone defect repair. Bone density, cortical bone thickness, and bone mass are key anatomical factors determining the success of bone grafting. Natural bone tissue consists of nano-hydroxyapatite (Ca... 10 Hydroxyapatite (HA) is an ordered composite of inorganic minerals such as PO4)6(OH)2 and high molecules such as collagen, providing a theoretical basis for the biomimetic design of artificial repair materials. Hydroxyapatite is highly similar to the inorganic phase of human bone in terms of chemical composition, crystal structure, and physicochemical properties, possessing excellent osteoconductivity, biocompatibility, and bioactivity. However, the inherent defects of pure HA materials, such as high brittleness, low flexural strength, poor toughness, and slow degradation, severely limit its application in the field of bone transplantation.

[0003] To enhance the bone repair efficacy of hyaluronic acid (HA), existing technologies typically introduce icariin (ICA) for functional modification, leveraging its osteoproliferative and angiogenesis-promoting activities to compensate for HA's functional deficiencies. However, existing methods often rely on the physical encapsulation of ICA within a polymer matrix, which presents the following problems: ICA merely adheres to the material surface and fails to participate in the inorganic mineralization process of HA; furthermore, the preparation process often involves organic solvents and cross-linking agents, resulting in a complex system; simultaneously, the drug-HA matrix is ​​only physically bonded, with weak interfacial adhesion, leading to low drug loading and poor drug stability, severely impacting the efficacy of modified HA materials in bone defect repair.

[0004] It should be noted that the above content is not necessarily prior art, nor is it intended to limit the scope of protection of this application. Summary of the Invention

[0005] This application discloses an icariin-hydroxyapatite nanoparticle, its preparation method and application, aiming to solve the technical problems of weak binding force between icariin and hydroxyapatite, resulting in low drug loading and poor drug stability.

[0006] To achieve the above objectives, the technical solution of this application is: The first aspect of this application provides icariin-hydroxyapatite nanoparticles, comprising: hydroxyapatite and icariin chemically bonded to the hydroxyapatite.

[0007] Preferably, in conjunction with the first aspect, the mass ratio of the hydroxyapatite to the icariin is (89:11)-(93:7).

[0008] Preferably, in conjunction with the first aspect, the icariin-hydroxyapatite nanoparticles have a spherical morphology and a particle size of 40-60 nm.

[0009] The second aspect of this application provides a method for preparing the icariin-hydroxyapatite nanoparticles described in the first aspect, the method comprising: Icariin was dissolved in an ethanol solution containing polyvinylpyrrolidone and mixed with a calcium source to obtain a premixed solution. The aqueous solution of the phosphate source was reacted with the premixed solution, and the reaction product was separated and washed to obtain the icariin-hydroxyapatite nanoparticles.

[0010] In conjunction with the second aspect, preferably, the amount of calcium source and phosphate source added satisfies the molar ratio of calcium ions to phosphate ions being 1.67:1.

[0011] Preferably, in conjunction with the second aspect, the calcium source includes one or more of calcium chloride, calcium nitrate, calcium sulfate, or calcium acetate; The phosphoric acid source includes one or more of sodium monohydrogen phosphate dihydrate, ammonium dihydrogen phosphate, and dipotassium hydrogen phosphate.

[0012] Preferably, in conjunction with the second aspect, the reaction of an aqueous solution of a phosphoric acid source with the premixed solution includes: The aqueous solution of the phosphate source was added dropwise to the premixed solution to carry out the reaction at a temperature of 20-30 °C for 6-8 h.

[0013] Preferably, in conjunction with the second aspect, the icariin dissolved in an ethanol solution containing polyvinylpyrrolidone comprises: Icariin was added to an ethanol system containing polyvinylpyrrolidone and stirred at 20-30 °C until the solid was completely dissolved, resulting in a clear icariin ethanol solution.

[0014] In conjunction with the second aspect, preferably, the separation and washing include: Centrifuge the solution after the mineralization reaction and collect the precipitate; The precipitate was washed with ethanol and then freeze-dried to obtain the icariin-hydroxyapatite nanoparticles.

[0015] The third aspect of this application provides the application of the icariin-hydroxyapatite nanoparticles described in the first aspect or the icariin-hydroxyapatite nanoparticles prepared by the preparation method described in the second aspect in the preparation of bone repair materials.

[0016] Compared with the prior art, the advantages or beneficial effects of the embodiments of this application include at least the following: The icariin-hydroxyapatite nanoparticles provided in this application were prepared using an in-situ co-precipitation method. This method involves introducing icariin during the nucleation and crystal growth of hydroxyapatite, allowing it to directly participate in the construction of the inorganic framework. In this process, icariin chemically bonds to the interior and surface interfaces of hydroxyapatite, achieving interfacial composite and partial encapsulation between the drug and the inorganic matrix. This structure significantly improves drug loading stability and capacity, giving the material broad application prospects in the field of bone repair. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Transmission electron microscope image of Embodiment 1 provided in this application; Figure 2 XRD pattern of Embodiment 1 provided in this application; Figure 3 Infrared spectral comparison diagrams of the embodiments and comparative examples provided in this application; Figure 4 Comparison of ultraviolet spectra of embodiments and comparative examples provided in this application; Figure 5 Example 1 provided in this application and Calcein-AM / PI fluorescence staining image of rat BMSC cells; Figure 6 Example 1 provided in this application and its effect on the proliferation of rat BMSC cells; Figure 7 Micro-CT images of bone repair experiments provided in Example 1 of this application. Detailed Implementation

[0019] The technical solutions in the embodiments of this application are described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of this application, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0020] In the following description of this embodiment, the term "and / or" is used to describe the association relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, B existing alone, and A and B existing simultaneously. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0021] In the following description of this embodiment, the term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0022] Those skilled in the art should understand that, in the following description of the embodiments of this application, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0023] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms "a" and "the" as used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0024] It should be noted that all raw materials and / or reagents in the embodiments of this application were purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0025] In a first aspect, this application provides an icariin-hydroxyapatite nanoparticle, comprising: hydroxyapatite and icariin chemically bonded to the hydroxyapatite.

[0026] It should be noted that in this application, icariin (ICA) is stably bound to hydroxyapatite (HA) through chemical bonds (such as Ca-O coordination bonds, hydrogen bonds, or covalent bonds), effectively avoiding early burst release of the drug in the body fluid environment, significantly prolonging the drug release cycle, and achieving long-acting sustained release. The chemical bonding method anchors ICA within the inorganic framework during HA mineralization, greatly enhancing its resistance to external pH fluctuations, enzymatic hydrolysis, and mechanical interference, ensuring the structural integrity of the active ingredient during in vivo transport and storage.

[0027] It should be noted that the chemical bonding process, which avoids high-temperature calcination or the introduction of organic cross-linking agents, ensures that the osteogenic and angiogenic activities of ICA are not compromised under the mild preparation conditions. No additional polymeric carriers or cross-linking agents are required, thus avoiding the risks of in vivo toxicity and batch stability issues associated with complex components.

[0028] In this embodiment, the preferred mass ratio of hydroxyapatite to icariin is (89:11)-(93:7). Controlling this mass ratio allows for a balance between drug loading and crystal integrity. When the ICA content is below 5%, it promotes insufficient bone activity; when it is above 15%, excessive ICA interferes with the normal growth of the HA crystal lattice, leading to decreased crystallinity and deterioration of mechanical properties. This mass ratio range ensures a high drug loading rate of 7-11% while maintaining the integrity of the HA crystal structure.

[0029] In this embodiment, the icariin-hydroxyapatite nanoparticles have a spherical morphology, with a preferred particle size of 40-60 nm. This 40-60 nm nanometer size falls within the optimal particle size window for phagocytosis, facilitating efficient internalization by osteoblasts and bone marrow mesenchymal stem cells, thereby enhancing the intracellular delivery efficiency of ICA. Simultaneously, this size effectively evades clearance by the reticuloendothelial system, prolonging in vivo circulation time. Compared to rod-shaped or needle-shaped HA, the spherical particles have a smaller specific surface energy, are less prone to aggregation, and can form a uniformly dispersed repair layer at bone defect sites, avoiding excessively high or low local drug concentrations. They also impart a high specific surface area to the material, exposing more active sites to promote protein adsorption and cell adhesion, and providing a favorable microstructure for the osteoconductive properties of HA. The degradation rate of nanoparticles within this size range matches the ingrowth rate of new bone tissue, facilitating a dynamic balance between material degradation and bone regeneration.

[0030] It should be noted that hydroxyapatite shares similarities with the phosphate system of bone in terms of chemical composition and structure, exhibiting good tissue compatibility, bioactivity, osteoconductivity, and osteoinductive properties. Icariin, as an extract of traditional Chinese medicine, plays an important role in anti-inflammatory and antioxidant stress responses. It also significantly promotes bone repair and regeneration. This application combines hydroxyapatite and icariin to form a nanoscale structure, complementing each other's advantages. It utilizes the bone-repairing effects of hydroxyapatite and alleviates inflammatory responses at bone injury sites through sustained-release icariin. This preparation method significantly increases the surface area of ​​the material, facilitating the exertion of biological activity by both components, enabling targeted drug delivery to bone tissue, saving costs, and reducing drug waste. Furthermore, the rougher surface of the nanocrystals allows for easier cell adhesion at bone injury sites, further promoting osteogenic effects.

[0031] Secondly, this application provides a method for preparing the icariin-hydroxyapatite nanoparticles described in the first aspect, the preparation method comprising: Icariin was dissolved in an ethanol solution containing polyvinylpyrrolidone and mixed with a calcium source to obtain a premixed solution. The aqueous solution of the phosphate source was reacted with the premixed solution, and the reaction product was separated and washed to obtain the icariin-hydroxyapatite nanoparticles.

[0032] It should be noted that this application employs in-situ co-precipitation to prepare the material, that is, ICA is introduced during the HA nucleation and crystal growth stages, allowing icariin to directly participate in the construction of the inorganic framework, achieving chemical bonding and crystal embedding. Furthermore, the entire process is completed in an ethanol-water system at room temperature (20-30 ℃), without the need for high-temperature calcination, high pressure, or inert gas protection. The operation is simple, energy-efficient, and suitable for industrial production.

[0033] It should be noted that the preparation method used in this application uses only ethanol and water as solvents, which is green and environmentally friendly, avoids the risks caused by crosslinking agent residues, and has higher biosafety.

[0034] It should be noted that polyvinylpyrrolidone (PVP) serves as both a co-solvent and a crystal growth regulator. On the one hand, it guides HA to preferentially grow along specific crystal planes to form spherical nanomorphic morphologies. On the other hand, it effectively inhibits nanoparticle aggregation through steric hindrance, ensuring that the product particle size is uniformly distributed in the range of 40-60 nm.

[0035] In this embodiment, the amounts of calcium and phosphate sources added are preferably 1.67:1, where the molar ratio of calcium ions to phosphate ions is 1.67:1. 1.67 is the theoretical calcium-to-phosphorus ratio (Ca / P) of hydroxyapatite in human bone. This molar ratio ensures that the synthesized product is highly consistent with the inorganic phase of natural bone in terms of chemical composition, which is beneficial for forming a good chemical bonding interface with the host bone after implantation. Deviations from 1.67 can lead to the formation of calcium-deficient HA or calcium phosphate impurities (such as β-TCP, CaO, etc.), affecting material purity and degradation behavior. By limiting this molar ratio, the product is ensured to be a single, pure HA crystalline phase, thus guaranteeing performance stability.

[0036] In this embodiment, the calcium source is preferably one or more of calcium chloride, calcium nitrate, calcium sulfate, or calcium acetate; more preferably, calcium chloride. The phosphate source is preferably one or more of sodium monohydrogen phosphate dihydrate, ammonium dihydrogen phosphate, or dipotassium hydrogen phosphate; more preferably, sodium monohydrogen phosphate dihydrate, to improve the purity of hydroxyapatite.

[0037] In this embodiment, the aqueous solution of the phosphate source is added dropwise to the premixed solution for reaction. The preferred temperature is 20-30 °C, and the preferred reaction time is 6-8 h. The dropwise addition of the phosphate source allows for slow contact between calcium and phosphorus ions, ensuring a stable crystal growth process. The low-temperature reaction throughout avoids the oxidation or degradation of active groups such as phenolic hydroxyl and carbonyl groups in the ICA, ensuring the biological properties of the product.

[0038] In this embodiment of the application, the dissolution of icariin in an ethanol solution containing polyvinylpyrrolidone includes: adding icariin to an ethanol system containing polyvinylpyrrolidone, stirring at 20-30 °C until the solid is completely dissolved, and obtaining a clear icariin ethanol solution.

[0039] In this embodiment, the separation and washing process includes: centrifuging the solution after the mineralization reaction to collect the precipitate; washing the precipitate with ethanol; and freeze-drying to obtain the icariin-hydroxyapatite nanoparticles. Centrifuging the solution after the mineralization reaction effectively separates unreacted free ICA and unnucleated calcium phosphate precursors, ensuring the distinction between chemically bonded ICA and free ICA in the final product and improving product purity. Freeze-drying maintains nano-dispersion, facilitating the preparation of injectable bone repair gels or composite scaffolds.

[0040] It should be noted that the preparation method employed in this application enables ICA to directly participate in the structure formation during the nucleation and growth of nHAP, achieving interfacial composite or partial embedding between the drug and the inorganic framework, thereby significantly improving drug loading stability and capacity. Simultaneously, the uniform distribution of the drug in the inorganic phase facilitates a smoother and more controllable release behavior. Although the introduction of organic molecules may have some impact on crystal growth, the stability of the reaction process can be effectively ensured by adjusting the experimental parameters of the reaction system, without affecting the application performance of the material. Furthermore, this method combines material synthesis with drug loading, significantly simplifying the preparation process and improving the reproducibility and scalability of the system. Therefore, this application uses an in-situ co-precipitation method to construct ICA-nHAP nanoparticles and conducts a preliminary evaluation of their feasibility for application in bone repair through in vitro cell experiments.

[0041] Thirdly, this application provides the application of the icariin-hydroxyapatite nanoparticles described in the first aspect or the icariin-hydroxyapatite nanoparticles prepared by the method described in the second aspect in the preparation of bone repair materials. The icariin-hydroxyapatite nanoparticles of this application possess good tissue compatibility, bioactivity, bone repair promotion, and osteoinductive properties. The bone repair materials prepared from these nanoparticles are suitable for multiple clinical scenarios and have broad market application prospects.

[0042] The technical solution of this application will be further described below with reference to specific embodiments, but the scope of protection of this application is not limited to the following embodiments.

[0043] Example 1 The preparation method of A1-icariin-hydroxyapatite nanoparticles (A1-ICA-nHAP) provided in this embodiment specifically includes: S101: Premix 0.2 M CaCl2 solution, 1% PVP anhydrous ethanol solution, and 2% icariin anhydrous ethanol solution and stir thoroughly to obtain a premixed solution. S102: Place the premixed solution in a magnetic stirrer and stir at 600 rpm. During this process, slowly add an aqueous solution of sodium hydrogen phosphate dihydrate dropwise. The solution will change from a pale yellow to a bright yellow during the addition. During the reaction, Ca... 2+ / PO4 3- The molar ratio was controlled at 1.67:1. The reaction was allowed to proceed for 6 hours. The mixture was then centrifuged and the precipitate was washed 2-3 times with 50% ethanol solution. The precipitate was then freeze-dried to obtain Al-ICA-nHAP.

[0044] Example 2 The preparation method of A2-icariin-hydroxyapatite nanoparticles (A2-ICA-nHAP) provided in this embodiment specifically includes: S201: Premix 0.2 M CaCl2 solution, 1% PVP anhydrous ethanol solution, and 5% icariin anhydrous ethanol solution and stir thoroughly to obtain a premixed solution. S202: Place the premixed solution in a magnetic stirrer and stir at 600 rpm. During this process, slowly add an aqueous solution of sodium hydrogen phosphate dihydrate dropwise. The solution will change from a pale yellow to a bright yellow during the addition. During the reaction, Ca... 2+ / PO4 3- The molar ratio was 1.67:1. After reacting for 7 hours, the mixture was centrifuged and the precipitate was washed 2-3 times with 50% ethanol solution. The precipitate was then freeze-dried to obtain A2-ICA-nHAP.

[0045] Example 3 The preparation method of A3-icariin-hydroxyapatite nanoparticles (A3-ICA-nHAP) provided in this embodiment specifically includes: S301: Premix 0.5 M CaCl2 solution, 1% PVP anhydrous ethanol solution, and 10% icariin anhydrous ethanol solution and stir thoroughly to obtain a premixed solution. S302: Place the premixed solution in a magnetic stirrer and stir at 600 rpm. During this process, slowly add an aqueous solution of sodium phosphate dropwise. The solution will change from a pale yellow to a bright yellow during the addition. During the reaction, Ca... 2+ / PO4 3-The molar ratio was 1.67:1. After reacting for 8 hours, the mixture was centrifuged and the precipitate was washed 2-3 times with 50% ethanol solution. The precipitate was then freeze-dried to obtain A3-ICA-nHAP.

[0046] Example 4 The preparation method of A4-icariin-hydroxyapatite nanoparticles (A3-ICA-nHAP) provided in this embodiment specifically includes: S401: Premix 0.5 M CaSO4 solution, 1% PVP anhydrous ethanol solution, and 10% icariin anhydrous ethanol solution and stir thoroughly to obtain a premixed solution. S402: Place the premixed solution in a magnetic stirrer and stir at 600 rpm. During this process, slowly add an aqueous solution of sodium phosphate dropwise. The solution will change from a pale yellow to a bright yellow during the addition. During the reaction, Ca... 2+ / PO4 3- The molar ratio was 1.67:1. After reacting for 7 hours, the mixture was centrifuged and the precipitate was washed 2-3 times with 50% ethanol solution. The precipitate was then freeze-dried to obtain A4-ICA-nHAP.

[0047] Example 5 The preparation method of A5-icariin-hydroxyapatite nanoparticles (A5-ICA-nHAP) provided in this embodiment specifically includes: S501: Premix 0.1 M Ca(NO3)2 solution, 1% PVP anhydrous ethanol solution and 1% icariin anhydrous ethanol solution and stir thoroughly to obtain a premixed solution. S502: Place the premixed solution in a magnetic stirrer and stir at 600 rpm. During this process, slowly add an aqueous solution of disodium hydrogen phosphate dropwise. The solution will change from a pale yellow to a bright yellow during the addition. During the reaction, Ca... 2 + / PO4 3- The molar ratio was 1.67:1. After reacting for 7 hours, the mixture was centrifuged and the precipitate was washed 2-3 times with 50% ethanol solution. The precipitate was then freeze-dried to obtain A5-ICA-nHAP.

[0048] Meanwhile, to verify the impact of the preparation methods in the above embodiments on the overall performance, this application provides the following comparative examples for detailed explanation.

[0049] Comparative Example 1 This comparative example uses a blank control, with unmodified hydroxyapatite pure phase as the control sample, denoted as nHAP.

[0050] Comparative Example 2 This comparative example uses a blank control, with the unmodified pure phase of icariin as the control sample, denoted as ICA.

[0051] To verify the influence of the various substances added in the preparation process used in this application on the morphology, the icariin-hydroxyapatite nanoparticles prepared in the examples were subjected to transmission electron microscopy. Figure 1 As shown.

[0052] according to Figure 1 As shown, the overall morphology consists of short, elliptical particles, forming uniformly sized, dispersed nanoparticles of 40-60 nm in size, with some aggregation, possibly due to the low solubility of icariin. This indicates that icariin-hydroxyapatite nanoparticles can be prepared via in-situ co-precipitation.

[0053] To verify the influence of the various substances added in the preparation process used in this application on the structure, the icariin-hydroxyapatite nanoparticles prepared in the examples and comparative examples were subjected to XRD, infrared, and ultraviolet tests. Figure 2-4 As shown.

[0054] according to Figure 2 It can be seen that after the introduction of ICA, the nHAP structure remained basically unchanged, but the overall diffraction intensity decreased, the crystallinity also decreased significantly, and the peak shape and width did not change significantly. This indicates that ICA molecules are bonded to the interior and surface of hydroxyapatite crystals through chemical forces (coordination bonds, hydrogen bonds), rather than entering the crystal lattice. Due to the reduced crystallinity, the bioactivity of ICA-nHAP is relatively improved, while its mechanical hardness and strength are relatively reduced, which is more conducive to the release of ICA and the degradation of nHAP.

[0055] according to Figure 3 It can be seen that, compared with pure ICA, the carbonyl (C=O) or aromatic ring skeleton vibration region (1600-1700 cm⁻¹) in ICA-nHAP is more pronounced. -1 The carbonyl group in ICA has changed, indicating that the aromatic ring skeleton and Ca in ICA have changed. 2+ Coordination occurred. Simultaneously, phosphate (PO4) ions... 3- Characteristic vibration zone (500-600 cm) -1 1000-1100 cm -1 The subtle changes indicate a shift in the chemical environment of the phosphate ions in ICA-nHAP, demonstrating that the two are bonded together via chemical bonds.

[0056] according to Figure 4 It can be seen that the absorbance of ICA-nHAP is generally lower than that of ICA, indicating that the electronic transition probability of the chromophore decreases when ICA combines with CaHA. At the absorption edge near 430 nm, the descent point of the ICA-nHAP curve shows a slight but discernible shift (blue shift) towards shorter wavelengths compared to the ICA curve, indicating an increase in the energy required for molecular excitation and a change in the chemical bonds between molecules.

[0057] In addition, the performance of the icariin-hydroxyapatite nanoparticles in various embodiments of this application was measured, and the results were obtained. Figure 5-6 The specific testing process, methods, and conditions are as follows: 1. Seed an appropriate amount of BMSC cells into a 24-well plate. When the cells proliferate to 80%, add blank culture medium, ICA-nHAP, nHAP, and ICA respectively, and treat for 24 hours.

[0058] 2. After 24 hours, the culture medium was precipitated and washed twice with PBS. 200 μL of working solution was added to each well and incubated at 37°C in the dark for 20 minutes.

[0059] 3. Place the processed cells in a high-content instrument for fluorescence imaging. Green cells are live cells, and red cells are dead cells.

[0060] according to Figure 5 The results showed that, using the kit to perform live / dead staining (green fluorescence for live cells, red fluorescence for dead cells) on rat bone marrow-derived mesenchymal stem cells under different treatments and performing high-content imaging and statistical analysis, the cell viability was consistently above 90%. This indicates that the material has no significant toxicity to cells.

[0061] according to Figure 6 It can be seen that by using CCK8 to detect the cell proliferation of rat BMSCs using different concentrations of materials, it can be found that the concentration of materials at 25-100 ug / mL has a promoting effect on the proliferation of rat BMSCs.

[0062] To further investigate the repair effect of icariin-hydroxyapatite nanoparticles in the various embodiments of this application on bone defects, the following tests were conducted: 1. Six-week-old male Wistar rats were used for modeling. The rats were first anesthetized with 50 mg / kg of acetaminophen. After anesthesia, the skin of the left hind limb was prepared. Then, the skin was bluntly incised and the muscle dissected using a scalpel. A 3 mm incision was made in the distal femur using a bone drill. A 3mm cavity defect was treated without filling in the control group, but filling was performed in the ICA-nHAP group. After filling, the muscle and skin were sutured together. Erythromycin ointment was applied to prevent infection.

[0063] 2. First, turn on the desktop high-resolution micro-CT scanner and preheat for 15 minutes. Simultaneously, induce anesthesia in the rats using an air anesthesia machine. After the machine has preheated, place the rat to be scanned inside the instrument, with its mouth and nose placed in the maintenance anesthesia sleeve. Secure the rat and perform the scan, ensuring it does not awaken during the process. After the scan is complete, remove the rat and place it in a cage to await awakening. Anesthesia is not required for imaging of the isolated femur.

[0064] 3. After the sample scanning was completed, 3D reconstruction was performed using software. The image was then saved after adjusting to the highest resolution for analysis. Results are as follows: Figure 7 As shown.

[0065] according to Figure 7 It was found that CT scans were performed on rats three days and eight weeks after mouse modeling. In vivo CT scans of mice were performed under isoflurane anesthesia and on a high-resolution micro-CT scanner. Eight weeks later, the femur of the modeled rats was harvested and separated, and scanned again on a high-resolution micro-CT scanner. It can be seen that the cortical bone in the material-filled group was thicker, and trabecular structures were formed within the bone shaft, fully demonstrating that the icariin-hydroxyapatite nanoparticles prepared in this application can promote the repair of defects in this area.

[0066] Therefore, the icariin-hydroxyapatite nanoparticles provided in this application are prepared by an in-situ co-precipitation method, that is, icariin is introduced into the nucleation and crystal growth process of hydroxyapatite, allowing it to directly participate in the construction of the inorganic framework. During this process, icariin is chemically bonded to the interior and surface interfaces of hydroxyapatite, achieving interfacial composite and partial encapsulation between the drug and the inorganic matrix. This structure significantly improves drug loading stability and capacity, giving the material broad application prospects in the field of bone repair.

[0067] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0068] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of this application.

Claims

1. An icariin-hydroxyapatite nanoparticle, characterized in that, include: Hydroxyapatite and icariin chemically bonded to the hydroxyapatite.

2. The icariin-hydroxyapatite nanoparticles according to claim 1, characterized in that, The mass ratio of the hydroxyapatite to the icariin is (89:11)-(93:7).

3. The icariin-hydroxyapatite nanoparticles according to claim 1, characterized in that, The morphology of the icariin-hydroxyapatite nanoparticles is spherical, with a particle size of 40-60 nm.

4. A method for preparing icariin-hydroxyapatite nanoparticles according to any one of claims 1-3, characterized in that, The preparation method includes: Icariin was dissolved in an ethanol solution containing polyvinylpyrrolidone and mixed with a calcium source to obtain a premixed solution. The aqueous solution of the phosphate source was reacted with the premixed solution, and the reaction product was separated and washed to obtain the icariin-hydroxyapatite nanoparticles.

5. The method for preparing icariin-hydroxyapatite nanoparticles according to claim 4, characterized in that, The amounts of calcium source and phosphate source added are such that the molar ratio of calcium ions to phosphate ions is 1.67:

1.

6. The method for preparing icariin-hydroxyapatite nanoparticles according to claim 4, characterized in that, The calcium source includes one or more of calcium chloride, calcium nitrate, calcium sulfate, or calcium acetate. The phosphoric acid source includes one or more of sodium monohydrogen phosphate dihydrate, ammonium dihydrogen phosphate, and dipotassium hydrogen phosphate.

7. The method for preparing icariin-hydroxyapatite nanoparticles according to claim 4, characterized in that, The reaction of an aqueous solution of a phosphate source with the premixed solution includes: The aqueous solution of the phosphate source was added dropwise to the premixed solution to carry out the reaction at a temperature of 20-30 °C for 6-8 h.

8. The method for preparing icariin-hydroxyapatite nanoparticles according to claim 4, characterized in that, The icariin dissolved in an ethanol solution containing polyvinylpyrrolidone comprises: Icariin was added to an ethanol system containing polyvinylpyrrolidone and stirred at 20-30 °C until the solid was completely dissolved, resulting in a clear icariin ethanol solution.

9. The method for preparing icariin-hydroxyapatite nanoparticles according to claim 4, characterized in that, The separation and washing process includes: Centrifuge the solution after the mineralization reaction and collect the precipitate; The precipitate was washed with ethanol and then freeze-dried to obtain the icariin-hydroxyapatite nanoparticles.

10. The use of the icariin-hydroxyapatite nanoparticles according to any one of claims 1-3 or the icariin-hydroxyapatite nanoparticles prepared by the preparation method according to any one of claims 4-9 in the preparation of bone repair materials.

Images