Multi-shell drug-loaded microsphere, preparation method thereof and application of multi-shell drug-loaded microsphere in bone infection treatment

Abstract

The invention relates to the technical field of biological materials, in particular to a multi-shell drug-loaded microsphere, a preparation method thereof and application of the multi-shell drug-loaded microsphere in bone infection treatment. A multi-shell drug-loaded microsphere comprises: a core body formed by calcium phosphate loaded with an osteogenic drug; the degradable shell wraps the core body, and the shell contains antibiotics; the surface of the shell is coated with the metal-polyphenol complex network. According to the technical scheme provided by the invention, the osteogenesis medicine is loaded in the core body, and the antibiotics are integrated in the shell, so that partitioned loading of the medicine is realized. And the shell is made of a degradable material, so that the osteogenic drug and the antibiotics can be slowly released along with the degradation process of the osteogenic drug and the antibiotics. In the structural design, the shell forms a physical barrier for the core body, and the release rate of the osteogenesis medicine is effectively limited, so that the overall release time of the two medicines is synergistically prolonged.

Description

Technical Field

[0001] This application relates to the field of biomaterials technology, and more specifically, to a multi-shell drug-loaded microsphere, its preparation method, and its application in the treatment of bone infections. Background Technology

[0002] The content in this section provides only background information related to this application and may not constitute prior art.

[0003] The standard clinical treatment for infected bone defects includes thorough debridement, combined with local or systemic antibiotics to control the source of infection, followed by surgical repair and reconstruction of the secondary bone defect. Post-debridement, systemic intravenous antibiotics are usually administered to control potential systemic infection; simultaneously, local sustained-release drug carriers (such as antibiotic-loaded bone cement or controlled-release microspheres) are often used to increase the local drug concentration and enhance its antibacterial efficacy.

[0004] Currently widely used antibiotic-loaded bone cement is primarily based on polymethyl methacrylate (PMMA) matrix. However, PMMA bone cement loaded with antibiotics generally exhibits a burst release effect, meaning a large amount of antibiotic is rapidly released within a short initial period. This directly leads to a significantly shortened duration of drug release, making it difficult to achieve effective long-term local antibacterial activity.

[0005] Polylactic-co-glycolic acid copolymer (PLGA), a biomedical material with good biocompatibility and tunable degradation, has been widely used as a tissue engineering scaffold and drug carrier. However, in the treatment of infected bone defects, it is usually only used to load a single antibiotic component, lacking consideration for tissue regeneration function. Therefore, designing controlled-release systems with multiple drug loading is of great significance for the treatment of infected bone defects. Summary of the Invention

[0006] The summary section of this application is intended to provide a brief overview of the concepts, which will be described in detail in the detailed description section below. This summary section is not intended to identify key or essential features of the claimed technical solutions, nor is it intended to limit the scope of the claimed technical solutions.

[0007] Some embodiments of this application propose a multi-shell drug-loaded microsphere, its preparation method, and its application in the treatment of bone infections, in order to solve the technical problems mentioned in the background section above.

[0008] As a first aspect of this application, some embodiments of this application provide a multi-shell drug-loaded microsphere, comprising:

[0009] The nucleus is formed from calcium phosphate loaded with osteogenic drugs;

[0010] A biodegradable outer shell enclosing the core, the outer shell containing antibiotics;

[0011] And a metal-polyphenol complex network coating the surface of the outer shell.

[0012] The technical solution provided in this application achieves partitioned drug loading by loading osteogenic drugs into the core and integrating antibiotics into the shell. The shell is made of a biodegradable material, allowing the osteogenic drugs and antibiotics to be slowly released during their degradation process. In this structural design, the shell forms a physical barrier to the core, effectively limiting the release rate of the osteogenic drugs, thereby synergistically prolonging the overall release time of the two drugs. The metal-polyphenol complex network coating the surface of the shell can regulate the degradation kinetics of the shell, further optimizing the sustained-release behavior of the drugs.

[0013] Furthermore, the volume ratio of the core to the shell is less than 1:1.

[0014] In this application, the volume ratio of the core to the shell is less than 1:1, which allows the core to be uniformly dispersed and encapsulated within the shell during the preparation of multi-shell drug-loaded microspheres. This enables the antibiotics in the shell to be preferentially released during material degradation to achieve antibacterial effects, followed by the release of the core and osteogenic drugs to promote tissue regeneration and repair.

[0015] Furthermore, in the metal-polyphenol complex, the metal is one of the ions such as copper, zinc, magnesium, and silver, and the polyphenol is one of the ions such as tannic acid, dopamine, gallic acid, and epigallocatechin gallate.

[0016] In this application, the metal-polyphenol complex network can slowly release metal ions. These metal ions, along with the antibiotics released from the shell, synergistically act on the affected area to kill bacteria. Once the infection in the affected area is controlled, the osteogenic drug is slowly released from the core, at which point it exhibits a better osteogenic promoting effect. Thus, the multi-shell drug-loaded microsphere structure in this application allows for the release of antibacterial drugs first, followed by osteogenic drugs, resulting in a better synergistic effect.

[0017] Furthermore, the osteogenic drug is one of icariin, alendronate sodium, or strontium ranelate; the calcium phosphate is one of hydroxyapatite, tricalcium phosphate, or biphasic calcium phosphate.

[0018] In this application, the calcium phosphate is one of hydroxyapatite, tricalcium phosphate, or biphasic calcium phosphate; it has a loose and porous structure, which can stably load osteogenic drugs and then release the osteogenic drugs.

[0019] As a second aspect of this application, some embodiments of this application provide a preparation method for preparing the aforementioned multi-shell drug-loaded microspheres; the preparation method includes:

[0020] Drug-loaded calcium phosphate was prepared by mixing calcium phosphate with osteogenic drugs.

[0021] Polylactic acid glycolic acid copolymer, antibiotics and drug calcium phosphate were mixed in a solvent to generate a mixed solution. The solvent in the mixed solution was removed to prepare core-shell structured microspheres. The solvent was dichloromethane.

[0022] Core-shell structured microspheres were immersed in a polyphenol solution, filtered, transferred to a metal ion solution, and dried to obtain multi-shell drug-loaded microspheres.

[0023] In the technical solution provided in this application, an oil-in-water (polyvinyl alcohol-water solution) system (polylactic acid-glycolic acid copolymer containing antibiotics-dichloromethane solution) is selected to encapsulate (calcium phosphate loaded with osteogenic drugs). After mixing and stirring, the polylactic acid-glycolic acid copolymer can fully coat the calcium phosphate to form core-shell structured drug-loaded microspheres. The core-shell structured drug-loaded microspheres are first soaked in a polyphenol solution, which allows polyphenolic substances to adhere to the surface of the core-shell structured drug-loaded microspheres. When soaked in a metal ion solution, the polyphenols attached to the core-shell structured drug-loaded microspheres can combine with metal ions to generate a metal-polyphenol complex network that encapsulates the multi-shell drug-loaded surface.

[0024] Furthermore, the mass-to-volume ratio of polylactic acid-glycolic acid copolymer to dichloromethane is 0.02–0.1;

[0025] The mass ratio of antibiotic to polylactic acid-glycolic acid copolymer is 0.005 to 0.02.

[0026] The volume ratio of the calcium phosphate core loaded with osteogenic drugs to the antibiotic-containing polylactic-co-glycolic acid copolymer shell is less than 1:1.

[0027] The mass-to-volume ratio of polylactic-co-glycolic acid copolymer (PLGA) to dichloromethane is 0.02–0.1, and the mass ratio of antibiotic to PLAC is 0.005–0.02. The volume ratio of the calcium phosphate core loaded with osteogenic drugs to the PLAC shell containing antibiotics is less than 1:1. This allows both PLAC and antibiotics to dissolve in the solvent, ensuring that the calcium phosphate loaded with osteogenic drugs is fully dispersed and encapsulated within the PLAC containing antibiotics. As dichloromethane evaporates, the PLAC solidifies, forming core-shell microspheres with calcium phosphate loaded with osteogenic drugs as the core and PLAC containing antibiotics as the shell. This characteristic allows for the preferential release of antibiotics from the shell during material degradation, achieving antibacterial effects, followed by the release of the core and osteogenic drugs to promote tissue regeneration and repair.

[0028] Furthermore, the concentration of metal ions in the metal solution is 1-10 mmol / L, the concentration of polyphenol solution is 1-5 mmol / L, and the pH value is 8-9.

[0029] In the technical solution provided in this application, the concentration of metal ions is 1-10 mmol / L, and the concentration of polyphenol solution is 1-5 mmol / L, so that the polyphenols attached to the surface of the core-shell structure microspheres can bind to the appropriate concentration of metal ions, avoiding the binding of excessive metal ions and the generation of cytotoxicity.

[0030] Furthermore, the molar ratio of lactic acid units to glycolic acid units in the polylactic acid-glycolic acid copolymer is 15:85-85:15, and the molecular weight of the polylactic acid-glycolic acid copolymer is 30-100 kDa.

[0031] In this application, the molar ratio of lactic acid units to glycolic acid units is 15:85-85:15, which can regulate the degradation rate of polylactic acid-glycolic acid copolymer.

[0032] Furthermore, the fabrication method of core-shell structured microspheres includes the following steps:

[0033] Dissolve polylactic acid-glycolic acid copolymer in dichloromethane, add antibiotics and drug calcium phosphate to dichloromethane and mix thoroughly to prepare a mixed solution;

[0034] The mixed solution was transferred to a polyvinyl alcohol solution, and the solution was stirred until the dichloromethane was completely evaporated to obtain a core-shell solution.

[0035] The core-shell solution was freeze-dried to obtain core-shell structured microspheres.

[0036] Furthermore, the multi-shell drug-loaded microspheres include the following steps:

[0037] The core-shell structured microspheres were immersed in a polyphenol solution, stirred, and then allowed to stand.

[0038] Core-shell structured microspheres were filtered out from a polyphenol solution and then transferred to a metal solution for isothermal shaking to obtain a metal mixed solution;

[0039] The metal mixture solution was freeze-dried to obtain multi-shell drug-loaded microspheres.

[0040] As a second aspect of this application, the aforementioned multi-shell drug-loaded microspheres and the multi-shell drug-loaded microspheres prepared by the aforementioned preparation method are used in the treatment of bone infections.

[0041] The technical solution of this application embodiment has at least the following advantages and beneficial effects:

[0042] (1) Multi-component controlled release effect: The proposed method uses calcium phosphate loaded with osteogenic drugs as the core, PLGA (polylactic-co-glycolic acid copolymer) containing antibiotics as the shell, and is surface modified with metal-polyphenol complexes to construct a multi-shell sustained-release microsphere system, which can realize the sequential release of multiple functional components such as trace metal elements, antibiotics, and osteogenic drugs. These functional components are beneficial for clearing infection and promoting tissue regeneration.

[0043] (2) Synergistic antibacterial effect of ions and drugs: The early release of metal ions is conducive to the rapid elimination of pathogens by synergistic antibiotics, reducing the risk of antibiotic resistance; as PLGA degrades, the continuous release of small amounts of antibiotics can ensure a stable antibacterial effect and prevent infection recurrence.

[0044] (3) Synergistic promotion of tissue regeneration: Calcium phosphate, as a biomaterial with osteoinductive properties, has a good effect on promoting bone repair. The osteogenic drugs loaded on it also have osteogenic activity. The two work synergistically to promote tissue regeneration.

[0045] (4) Based on the excellent drug loading performance of PLGA and calcium phosphate, the system can make appropriate adjustments to the type and dosage of the loaded drug to achieve different loading and application effects. Attached Figure Description

[0046] Figure 1 The image shows a scanning electron microscope (SEM) image of the multi-shell drug-loaded microspheres prepared in Embodiment 1.

[0047] Figure 2 The image shows a scanning electron microscope (SEM) image of the multi-shell drug-loaded microspheres prepared in Embodiment 2.

[0048] Figure 3 This is a scanning electron microscope image of the multi-shell drug-loaded microspheres prepared in Embodiment 3.

[0049] Figure 4 The multi-shell drug-loaded microspheres prepared in Embodiment 2 were subjected to Cu in a phosphate buffer solution (pH 6.0). 2+ The cumulative release chart over 2 days.

[0050] Figure 5 The multi-shell drug-loaded microspheres prepared in Embodiment 2 were subjected to Cu in a phosphate buffer solution (pH 6.0). 2+ The cumulative release chart over 28 days.

[0051] Figure 6 This is a photograph of the multi-shell drug-loaded microspheres prepared in Embodiment 2 mixed with physiological saline and extruded through a 23G syringe.

[0052] Figure 7 The image shows colonies of the multi-shell drug-loaded microspheres prepared in Implementation Method 2 after culturing Staphylococcus aureus for 1 day on a plate.

[0053] Figure 8 The image shows colonies of the multi-shell drug-loaded microspheres prepared in Implementation Method 2 after culturing Enterobacteriaceae for 1 day and then plating them.

[0054] Figure 9 The multi-shell drug-loaded microspheres prepared in Implementation Method 2 were co-cultured with Staphylococcus aureus and Escherichia coli for 15 days, and the antibacterial rate of the multi-shell drug-loaded microspheres against the two bacteria is shown in the following graph.

[0055] Figure 10 The image shows alkaline phosphatase staining after the multi-shell drug-loaded microspheres prepared in Implementation Method 2 were co-cultured with bone marrow mesenchymal stem cells for 14 days.

[0056] Figure 11 The multi-shell drug-loaded microspheres prepared in Implementation Method 2 were used in a rat infectious bone defect model. Micro-CT images were obtained 4 weeks after implantation. Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below in conjunction with specific embodiments. The same reference numerals in the accompanying drawings represent the same components. It should be noted that the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the described embodiments of this application without creative effort are within the scope of protection of this application.

[0058] Compared to the embodiments shown in the accompanying drawings, feasible embodiments within the scope of this application may have fewer components, other components not shown in the drawings, different components, differently arranged components, or components with different connections, etc. Furthermore, two or more components in the drawings may be implemented in a single component, or a single component shown in the drawings may be implemented as multiple separate components.

[0059] Unless otherwise defined, the technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this application pertains. The terms “first,” “second,” and similar terms used in this specification and claims do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Similarly, the terms “an” or “a” and similar terms do not necessarily indicate a quantity limitation. Terms such as “upper” and “lower” are used only to indicate relative positional relationships, and these relative positional relationships may change accordingly when the absolute position of the described object changes.

[0060] refer to Figure 1Example 1: A multi-shell drug-loaded microsphere comprising: a core, an outer shell, and a metal-polyphenol complex network. The core is calcium phosphate loaded with an osteogenic drug; the outer shell encapsulates the core, is a biodegradable shell, and contains an antibiotic; the metal-polyphenol complex network coats the surface of the outer shell.

[0061] The osteogenic drug is one of the following: icariin, alendronate sodium, or strontium ranelate;

[0062] Calcium phosphate is one of hydroxyapatite, tricalcium phosphate, or diphasic calcium phosphate;

[0063] The antibiotic is one of the following: rifampin, vancomycin, clindamycin, gentamicin, amikacin, norfloxacin, levofloxacin, etc.

[0064] The metal element in the metal-polyphenol complex network is one of copper, zinc, magnesium, silver, etc., and the polyphenol is one of tannic acid, dopamine, gallic acid, epigallocatechin gallate ester.

[0065] The volume ratio of the core to the shell is less than 1:1.

[0066] Multi-shell drug-loaded microspheres use calcium phosphate loaded with osteogenic drugs as the core, polylactic acid-glycolic acid copolymer containing antibiotics as the shell, and are surface-modified with a metal-polyphenol complex network. Thus, when injected into the human body, they can slowly release antibiotics, metal ions and osteogenic drugs, thereby continuously applying drugs to the affected area.

[0067] Example 2: Based on Example 1, Example 2 provides a preparation method for preparing multi-shell drug-loaded microspheres. The preparation method includes the following steps:

[0068] Step 1: Prepare drug calcium phosphate loaded with osteogenic drugs by mixing calcium phosphate with osteogenic drugs.

[0069] Step 1 includes the following steps:

[0070] Step 11: Prepare a calcium-phosphorus solution using calcium nitrate and diammonium hydrogen phosphate, maintaining the Ca2+ level. 2+ The concentration was 0.06 mol / L, and the Ca:P molar ratio was 1.6. Nitric acid was added to the calcium-phosphorus solution to adjust the pH. The pH adjustment was complete when the solution became clear. Then, cyclohexanehexacarboxylic acid and urea were added until the concentration of cyclohexanehexacarboxylic acid reached 5 mmol / L and the concentration of urea reached 0.6 mol / L. The mixture was stirred thoroughly to obtain an intermediate calcium-phosphorus solution.

[0071] Step 12: The calcium phosphate intermediate solution was transferred to a polytetrafluoroethylene reactor and reacted at 95°C for 5 hours. After the reaction was completed, the precipitate was collected. The precipitate was calcined at 800°C for 2 hours to obtain calcium phosphate microspheres.

[0072] Step 13: Mix 50 mg of calcium phosphate microspheres with 2 mg of osteogenic drug solution and shake at 37°C for 2 hours. Remove the unloaded osteogenic drug and freeze-dry at -50°C for 12 hours to obtain osteogenic drug-loaded calcium phosphate.

[0073] Preferably, the pH of the calcium phosphate solution is adjusted to 2-3 with nitric acid;

[0074] Preferably, the concentration range of cyclohexanehexacarboxylic acid is 0.5-10 mmol / L.

[0075] Preferably, the concentration of urea is 0.2-1 mol / L.

[0076] Preferably, the hydrothermal reaction takes place in a polytetrafluoroethylene reactor at a temperature of 90-180°C for 2-12 hours.

[0077] Preferably, the calcination temperature of the precipitate is 600-1150℃, the heating rate is 2-10℃ / min, and the holding time is 1-4 hours.

[0078] Preferably, the mass ratio of the osteogenic drug to calcium phosphate is 0.01 to 0.1.

[0079] Preferably, when mixing 50 mg of calcium phosphate microspheres with 2 mg of osteogenic drug solution, the constant temperature oscillation is 35-40°C, the oscillation frequency is 50-120 rpm, and the time is 1-4 hours.

[0080] Preferably, the freeze-drying temperature is -45 to 60°C and the time is 8 to 24 hours.

[0081] Preferably, the osteogenic drug is one of icariin, alendronate sodium, or strontium ranelate.

[0082] Preferably, the calcium phosphate is one of hydroxyapatite, tricalcium phosphate, or biphasic calcium phosphate.

[0083] Step 2: Mix polylactic acid glycolic acid copolymer, antibiotic and drug calcium phosphate in a solvent to generate a mixed solution, remove the solvent from the mixed solution to prepare core-shell structured microspheres, the solvent being dichloromethane.

[0084] Step 2 includes the following steps:

[0085] Step 21: Dissolve the polylactic acid-glycolic acid copolymer in dichloromethane, add antibiotics and calcium phosphate to the dichloromethane and mix well to prepare a mixed solution.

[0086] The molar ratio of lactic acid units to glycolic acid units in the polylactic acid-glycolic acid copolymer is 15:85-85:15, and the molecular weight of the polylactic acid-glycolic acid copolymer is 30-100 kDa.

[0087] The mass-to-volume ratio of polylactic acid glycolic acid copolymer to dichloromethane is 0.02–0.1; the mass-to-volume ratio of antibiotic to polylactic acid glycolic acid copolymer is 0.005–0.02.

[0088] The antibiotic is one of the following: rifampin, vancomycin, clindamycin, gentamicin, amikacin, norfloxacin, or levofloxacin.

[0089] Step 22: Transfer the mixed solution to a 0.1-1% polyvinyl alcohol solution and stir the solution until the dichloromethane has completely evaporated to obtain a core-shell solution.

[0090] Step 23: Freeze-dry the core-shell solution to obtain core-shell structured microspheres.

[0091] The specific preparation method of core-shell structured microspheres is as follows:

[0092] 100 mg of polylactic-co-glycolic acid copolymer (PLA) with a molecular weight of 30 kDa and a lactic acid to glycolic acid ratio of 50:50 was dissolved in 3 mL of dichloromethane. 1.5 mg of the antibiotic rifampicin (RF) and 10 mg of the drug calcium phosphate were added to the dichloromethane and mixed thoroughly to obtain a homogeneous solution. The solution was then transferred to 50 mL of 0.1–1% polyvinyl alcohol solution and stirred at 400 rpm for 4 hours until the dichloromethane evaporated. The mixture was collected and freeze-dried at -50°C for 12 hours to obtain core-shell structured microspheres.

[0093] Step 3: Immerse the core-shell structured microspheres in a polyphenol solution, filter, transfer to a metal ion solution, and dry to obtain multi-shell drug-loaded microspheres.

[0094] Step 3 includes the following steps:

[0095] Step 31: Immerse the core-shell structured microspheres in a polyphenol solution, stir, and then let stand.

[0096] Step 32: Filter the core-shell structured microspheres from the polyphenol solution, and then transfer them to a metal solution for isothermal shaking to obtain a metal mixed solution;

[0097] Step 33: Freeze-dry the metal mixture solution to obtain multi-shell drug-loaded microspheres.

[0098] Furthermore, in the metal-polyphenol complex, the metal is one of the ions such as copper, zinc, magnesium, and silver, and the polyphenol is one of the ions such as tannic acid, dopamine, gallic acid, and epigallocatechin gallate.

[0099] Furthermore, the concentration of metal ions in the metal solution is 1-10 mmol / L, the concentration of polyphenol solution is 1-5 mmol / L, and the pH value is 8-9.

[0100] The specific preparation method of multi-shell drug-loaded microspheres is as follows:

[0101] 100 mg of core-shell microspheres were immersed in a 2 mg / mL tannic acid solution (pH = 8) and stirred overnight. After washing, the core-shell microspheres were transferred to a 5 mmol / L copper chloride solution and shaken at 37 °C for 2 hours. After freeze-drying at -50 °C for 12 hours, multi-shell drug-loaded microspheres were obtained.

[0102] Example 3: Experiments were conducted on the slow release of antibiotics and osteogenic drugs from multi-shell drug-loaded microspheres prepared using the preparation method provided in Example 2, as well as correlation tests on in vitro antibacterial activity.

[0103] Based on Example 2, the following five specific implementation methods are provided, namely, Implementation Method 1 to Implementation Method 5. The methods for preparing multi-shell drug-loaded microspheres in Implementation Method 1 to Implementation Method 5 are roughly the same as the preparation methods provided in Example 2, except that the corresponding parameters are different.

[0104] Specifically:

[0105] In Implementation Method 1, the steps for preparing multi-shell drug-loaded microspheres are as follows:

[0106] Step 1: Prepare a calcium phosphate solution using calcium nitrate and diammonium hydrogen phosphate, maintaining the Ca2+ content. 2+ The concentration was 0.06 mol / L, and the Ca:P molar ratio was 1.6. Nitric acid was added to the calcium-phosphorus solution to adjust the pH. The pH adjustment was complete when the solution became clear. Then, cyclohexanehexacarboxylic acid and urea were added until the concentration of cyclohexanehexacarboxylic acid reached 5 mmol / L and the concentration of urea reached 0.6 mol / L. The mixture was stirred thoroughly to obtain an intermediate calcium-phosphorus solution.

[0107] The calcium phosphate intermediate solution was transferred to a polytetrafluoroethylene reactor and reacted at 95°C for 5 hours. After the reaction was completed, the precipitate was collected. The precipitate was calcined at 800°C for 2 hours to obtain calcium phosphate microspheres.

[0108] 50 mg of calcium phosphate microspheres were mixed with 2 mg of the osteogenic drug icariin (ICA) solution and shaken at 37 °C for 2 hours. The unloaded osteogenic drug was removed, and the mixture was freeze-dried at -50 °C for 12 hours to obtain the osteogenic drug-loaded calcium phosphate.

[0109] Step 2: Dissolve 100 mg of polylactic-co-glycolic acid copolymer (PLACO) in 3 mL of dichloromethane. The PLACO has a molecular weight of 30 kDa and a lactic acid to glycolic acid ratio of 50:50. Add 1.5 mg of the antibiotic rifampicin (RF) and 10 mg of the drug calcium phosphate to the dichloromethane and mix thoroughly to obtain a mixed solution. Transfer the mixed solution to 50 mL of 0.1% polyvinyl alcohol solution and stir at 400 rpm for 4 hours until the dichloromethane evaporates. Collect and freeze-dry at -50°C for 12 hours to obtain core-shell structured microspheres.

[0110] Step 3: Immerse 100 mg of core-shell microspheres in a 2 mg / mL tannic acid solution (pH = 8) and stir overnight. After washing, transfer the core-shell microspheres to a 5 mmol / L copper chloride solution and shake at 37°C for 2 hours. After freeze-drying at -50°C for 12 hours, multi-shell drug-loaded microspheres are obtained.

[0111] In Embodiment 2: The specific steps of Embodiment 2 are the same as those of Embodiment 1, except that in step 2 of Embodiment 2, the mass of calcium phosphate added to dichloromethane is 50 mg.

[0112] In Embodiment 3: The specific steps of Embodiment 3 are the same as those of Embodiment 1, except that in step 2 of Embodiment 3, the mass of calcium phosphate added to dichloromethane is 100mg.

[0113] The test results for Implementation Methods 1 to 3 are as follows:

[0114] 1. Morphological structure:

[0115] The morphology of the multi-shell drug-loaded microspheres prepared according to Embodiments 1, 2, and 3 was observed using a scanning electron microscope. The results are as follows: Figure 1 , Figure 2 and Figure 3 As shown. Comparison Figure 1 , Figure 2 and Figure 3 It was observed that as the ratio of the drug calcium phosphate to PLGA increased, the multi-shell microspheres initially maintained good integrity, but significant amounts of rupture occurred at a 1:1 ratio. Furthermore, due to… Figure 3 It can be seen that the drug calcium phosphate is dispersed inside PLGA, which is conducive to the gradual release of osteogenic drugs along with the degradation of PLGA.

[0116] 2. Release behavior:

[0117] The multi-shell drug-loaded microspheres prepared in Embodiment 2 were immersed in a phosphate buffer solution with a pH of 6.0 for 28 days, and the copper ion content (Cu) was measured. 2+ Release curves of RF and ICA, such as Figure 4-5 As shown. By Figure 4It can be seen that within 48 hours, Cu 2+ It releases rapidly initially, followed by a slow release phase. Figure 5 It can be seen that the antibiotic RF exhibits a stable release trend after 28 days, while the osteogenic drug ICA begins release after 3 days, also showing a stable release behavior. These release behaviors are beneficial for early Cu... 2+ It works synergistically with RF for rapid sterilization, followed by slow release of RF to inhibit infection recurrence. Furthermore, the release of ICA after 3 days promotes tissue repair.

[0118] 3. Injectability:

[0119] The multi-shell drug-loaded microspheres prepared in Embodiment 2 were mixed with physiological saline, and an extrusion experiment was conducted using a 23G needle to evaluate their extrusion injectability. Figure 6 As shown. By Figure 6 As can be seen, multi-shell drug-loaded microspheres can be extruded via a 23G syringe. This excellent injectability provides an important basis for minimally invasive and irregular bone defect filling treatment.

[0120] 3. In vitro antibacterial effect:

[0121] Take the multi-shell drug-loaded microspheres prepared in Embodiment 2, and respectively mix them with 1×10 5 Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were co-cultured at CFU / mL, and plated after 1 day. Figure 7 and Figure 8 As shown. By Figure 7 and Figure 8 It can be seen that the multi-shell drug-loaded microspheres can inhibit the colony formation of S. aureus and E. coli within 1 day, indicating that they have good antibacterial activity.

[0122] Take the multi-shell drug-loaded microspheres prepared in Embodiment 2, and respectively mix them with 1×10 8 S. aureus (CFU / mL) was co-cultured with E. coli, and the long-term antibacterial activity was calculated after 15 days. Figure 9 As shown. By Figure 9 It can be seen that the multi-shell drug-loaded microspheres have an antibacterial rate of over 90% against both S. aureus and E. coli within 15 days, indicating that they have long-lasting antibacterial properties.

[0123] 4. In vitro osteogenic effects:

[0124] The multi-shell drug-loaded microspheres prepared in Implementation Method 2 were co-cultured with bone marrow mesenchymal stem cells at a density of 5104 cells / well. After 14 days, alkaline phosphatase staining was performed to evaluate osteogenic activity. The results are as follows: Figure 10 As shown. By Figure 10It can be seen that after co-culturing multi-shell drug-loaded microspheres with bone marrow mesenchymal stem cells for 14 days, they expressed a large amount of alkaline phosphatase (blue-purple), indicating that they have osteogenic activity.

[0125] 5. Therapeutic effect of infectious bone defects in rats:

[0126] The multi-shell drug-loaded microspheres prepared in Example 2 were used to treat a rat femoral infection model with a diameter of 1 mm. Micro-CT imaging analysis was performed 4 weeks later, and the results are as follows: Figure 11 As shown. By Figure 11 It can be seen that multi-shell drug-loaded microspheres can promote bone defect healing within 4 weeks in a rat femoral infectious bone defect model, demonstrating their in vivo repair effect on infectious bone defects.

[0127] The above are merely preferred embodiments of this application and are not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A multi-shell drug-loaded microsphere, characterized in that, include: The nucleus is formed from calcium phosphate loaded with osteogenic drugs; A biodegradable outer shell enclosing the core, the outer shell containing antibiotics; And a metal-polyphenol complex network coating the surface of the outer shell.

2. The multi-shell drug-loaded microspheres according to claim 1, characterized in that, The volume ratio of the core to the shell is less than 1:

1.

3. The multi-shell drug-loaded microspheres according to claim 1, characterized in that, In metal-polyphenol complexes, the metal is one of the ions such as copper, zinc, magnesium, and silver, and the polyphenol is one of the ions such as tannic acid, dopamine, gallic acid, and epigallocatechin gallate.

4. The multi-shell drug-loaded microspheres according to claim 1, characterized in that, The osteogenic drug is one of icariin, alendronate sodium, or strontium ranelate; the calcium phosphate is one of hydroxyapatite, tricalcium phosphate, or biphasic calcium phosphate.

5. A preparation method for preparing multi-shell drug-loaded microspheres according to any one of claims 1 to 4, characterized in that, include: Drug-loaded calcium phosphate was prepared by mixing calcium phosphate with osteogenic drugs. Polylactic acid glycolic acid copolymer, antibiotics and drug calcium phosphate were mixed in a solvent to generate a mixed solution. The solvent in the mixed solution was removed to prepare core-shell structured microspheres. The solvent was dichloromethane. Core-shell structured microspheres were immersed in a polyphenol solution, filtered, transferred to a metal ion solution, and dried to obtain multi-shell drug-loaded microspheres.

6. The preparation method according to claim 5, characterized in that, The mass-to-volume ratio of polylactic acid-glycolic acid copolymer to dichloromethane is 0.02–0.1; The mass-to-volume ratio of antibiotics to polylactic acid-glycolic acid copolymer is 0.005 to 0.

02.

7. The preparation method according to claim 5, characterized in that, The concentration of metal ions in the metal solution is 1-10 mmol / L, the concentration of polyphenol solution is 1-5 mmol / L, and the pH value is 8-9.

8. The preparation method according to claim 5, characterized in that, The molar ratio of lactic acid units to glycolic acid units in polylactic acid-glycolic acid copolymer is 15:85-85:15, and the molecular weight of polylactic acid-glycolic acid copolymer is 30-100 kDa.

9. The preparation method according to claim 5, characterized in that, The preparation method of core-shell structured microspheres includes the following steps: Dissolve polylactic acid-glycolic acid copolymer in dichloromethane, add antibiotics and drug calcium phosphate to dichloromethane and mix thoroughly to prepare a mixed solution; The mixed solution was transferred to a polyvinyl alcohol solution, and the solution was stirred until the dichloromethane was completely evaporated to obtain a core-shell solution. The core-shell solution was freeze-dried to obtain core-shell structured microspheres; Multi-shell drug-loaded microspheres include the following steps: The core-shell structured microspheres were immersed in a polyphenol solution, stirred, and then allowed to stand. Core-shell structured microspheres were filtered out from a polyphenol solution and then transferred to a metal solution for isothermal shaking to obtain a metal mixed solution; The metal mixture solution was freeze-dried to obtain multi-shell drug-loaded microspheres.

10. The application of the multi-shell drug-loaded microspheres according to any one of claims 1 to 4 and the multi-shell drug-loaded microspheres prepared by the preparation method according to any one of claims 6 to 9 in the treatment of bone infections.

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