CD301b + - EXO and preparation method thereof, and CD301b-loaded EXO + - Bone-targeting microspheres of EXO and preparation method and application thereof

Abstract

The application discloses a CD301b + EXO and a preparation method thereof, and a bone-targeting microsphere loaded with the CD301b + EXO and an application thereof. The bone-targeting microsphere loaded with the CD301b + EXO is modified by a functional component of alendronate, is endowed with excellent bone targeting capability, can significantly improve the enrichment and retention of the exosome in a bone defect area, and realizes sustained and controllable release in a bone defect environment. The application fully plays the triple repair function of the CD301b + EXO, and realizes the synergistic promotion of the whole bone healing process by combining the bone targeting and slow-release characteristics of the microsphere, significantly improves the repair efficiency and quality, and has high clinical transformation value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a CD301b + -EXO and its preparation method, CD301b loading + -EXO bone-targeting microspheres, their preparation methods, and applications. Background Technology

[0002] Bone defects are a common and significant clinical challenge, with causes including trauma, tumor resection, infection, and orthopedic surgery. Minor injuries can heal through the body's self-repair mechanism, but critical-sized or disease-related bone defects often exceed the natural regeneration limits of bone tissue, requiring exogenous intervention. Currently, autologous bone grafting remains the gold standard for bone repair, possessing excellent biocompatibility and osteogenic conductivity. However, its clinical application is significantly limited by donor shortages, significant surgical trauma, and the risk of infection. While allogeneic and artificial bone substitutes can partially alleviate donor shortages, drawbacks such as immune rejection, infection, and insufficient osteogenic capacity still make it difficult to meet clinical needs.

[0003] Existing bone repair materials generally lack sufficient bioactive components, with most possessing only a single function, such as anti-inflammation, angiogenesis, or osteogenic effects. However, bone healing is an ordered and complex process involving "inflammation regulation—angiogenesis—bone formation," each stage requiring specific bioactive effects. Traditional strategies often employ multiple active factors or cells in combination to cover different repair stages, but this not only increases the complexity and cost of preparation but may also introduce uncontrollable risks due to interactions between components, affecting the repair efficacy.

[0004] Recent studies have shown that successful bone repair depends on the coordinated regulation of the osteoconductive properties of materials and the local immune microenvironment. Macrophages, as core immune regulatory cells, play crucial roles in inflammation resolution, angiogenesis, and osteogenic differentiation. Although the classic M1 / M2 dichotomy model explains the functional diversity of macrophages to some extent, it still cannot fully encompass their complex behavior in bone regeneration. Recent research has discovered that CD301b... + Macrophages exhibit significant advantages over traditional M2 macrophages in immune regulation, angiogenesis, and osteogenic induction. However, direct use of CD301b... + Macrophage therapy still faces many bottlenecks, including limited cell sources, difficulties in in vitro expansion, low survival rates after transplantation, and poor targeted delivery efficiency, which seriously restrict its clinical translational value.

[0005] Exosomes (EXOs), as cell-secreted nanovesicles (30-150 nm), can carry various bioactive molecules such as proteins and nucleic acids, and can inherit and transfer the functions of parental cells, thus demonstrating advantages in cell-free therapy in regenerative medicine. Studies have shown that macrophages mainly regulate bone homeostasis by secreting exosomes. Summary of the Invention

[0006] In order to overcome the shortcomings and disadvantages of the prior art, the primary objective of this invention is to provide a CD301b + -EXO (from CD301b) + Preparation method of macrophage exosomes.

[0007] A second objective of this invention is to provide CD301b prepared by the above-described preparation method. + -EXO (from CD301b) + (Exosomes from macrophages). This invention is the first to identify and systematically elucidate exosomes derived from CD301b. + Macrophage exosomes (CD301b) + -EXO). This exosome naturally inherits the core functions of its parent cell, possessing multiple biological activities such as immunomodulation, angiogenesis, and osteogenic induction. It can cover key aspects of bone repair from a single source, avoiding the complexity of multi-component synergistic strategies, demonstrating outstanding application advantages and significant clinical translational potential. In addition to direct application, CD301b... + -EXO can also be combined with a variety of biomaterials to further enhance its bone repair effects. It can be loaded into natural or synthetic polymer hydrogels, microspheres, nanofiber scaffolds, or inorganic / organic composite scaffolds, leveraging the three-dimensional structure of the materials to achieve precise positioning, stable delivery, and controlled release, while maintaining the bioactivity of exosomes. Furthermore, it utilizes the inherent mechanical and microenvironmental regulation properties of the materials to further amplify its multiple functions. For example, hydrogels possess excellent injectability and self-healing properties, making them suitable for the malleable filling of irregular bone defects; microspheres can achieve controlled degradation and time-sequential release, highly matching the dynamic process of bone repair. To further enhance the bone repair effect, CD301b... + -EXO is better utilized in the field of regenerative medicine. This invention also prepares a CD301b-loaded... + -EXO bone-targeting microspheres (A-SCH / OSA@EXO microspheres).

[0008] A third objective of this invention is to provide a load cell CD301b. + -Preparation method of EXO bone-targeting microspheres (A-SCH / OSA@EXO microspheres). This preparation method involves CD301b +The method utilizes EXO loaded onto bone-targeting microspheres. This preparation method, while employing microfluidic technology, also leverages pH-responsive Schiff base bonds formed by oxidized sodium alginate (OSA) and alendronate-grafted succinylated chitosan (A-SCH) to achieve intelligent and controllable release of exosomes in an acidic microenvironment. This prolongs the duration of action and maintains bioactivity, thereby significantly enhancing the local retention and functional performance of exosomes.

[0009] A fourth objective of this invention is to provide a CD301b-loaded material prepared by the above-described preparation method. + -EXO bone-targeting microspheres (A-SCH / OSA@EXO microspheres). Loaded with CD301b + -EXO's bone-targeting microspheres (A-SCH / OSA@EXO microspheres) have uniform particle size and stable dispersion. By introducing bisphosphonate groups, the material is endowed with bone-targeting properties.

[0010] The fifth objective of this invention is to provide the aforementioned load CD301b + Application of EXO-targeted microspheres (A-SCH / OSA@EXO microspheres). The A-SCH / OSA@EXO microspheres prepared in this invention are applied to the field of bone defects, not only improving the enrichment efficiency of exosomes in the bone defect area but also achieving delayed release and significantly prolonging the biological action time. Through this "CD301b" + The combined strategy of "EXO + bone-targeting microspheres" integrates the multiple biological functions of exosomes with the targeted delivery capabilities of materials, overcoming the limitations of existing materials such as single activity, complex composition, and uncoordinated regulation. This provides a new, efficient, stable strategy with promising clinical translation prospects for bone defect repair. The A-SCH / OSA@EXO microspheres described in this invention achieve a synergistic effect between the material delivery advantages and the multifunctionality of exosomes, effectively covering the entire bone repair process. They exhibit superior repair effects compared to traditional single active factors or multi-component combinations, demonstrating higher clinical translational value.

[0011] The primary objective of this invention is achieved through the following technical solution:

[0012] A CD301b + -EXO (from CD301b) + The preparation method of macrophage exosomes includes the following steps:

[0013] (1) CD301b + Preparation of macrophages: RAW264.7 macrophages were stimulated with interleukin-4 and cultured in culture medium. Cells were collected and single-cell suspensions were prepared. The single-cell suspensions were incubated with an antibody mixture at room temperature in the dark. After centrifugation and discarding of the supernatant, the cells were washed, resuspended, filtered, and sorted to obtain CD301b.+ Macrophages;

[0014] (2) CD301b + -Preparation of EXO: The CD301b obtained in step (1) is processed... + Macrophages were cultured in medium after stimulation with interleukin-4. After culture, they were sequentially centrifuged at low temperature. The supernatant was then collected, and exosomes were enriched by a first ultracentrifugation. The resulting precipitate was pre-cooled and resuspended, followed by a second ultracentrifugation, and the resulting precipitate was resuspended to prepare CD301b. + -EXO, which originates from CD301b + Macrophage exosomes;

[0015] The mass concentration of interleukin-4 in steps (1) and (2) is 20 ng / mL; the culture medium is a high-glucose DMEM complete medium containing 10% (v / v) exosome-free FBS and 1% (v / v) penicillin-streptomycin;

[0016] The specific steps of differential centrifugation under low temperature environment described in step (2) are as follows: First, centrifuge at 300~400×g for 10~15 minutes at 4~10℃ to remove live cells, then centrifuge at 2000~3000×g for 10~15 minutes to remove dead cells, and then centrifuge at 10000~12000×g for 30~40 minutes to remove cell debris and collect the supernatant.

[0017] Preferably, the antibody mixture in step (1) is a mixture of APC-labeled anti-mouse F4 / 80 antibody diluted 400 times and PE-labeled anti-mouse CD301b antibody diluted 200 times.

[0018] Preferably, the specific conditions for the first and second ultracentrifugation in step (2) are 4℃~10℃, 100000~120000 ×g ultracentrifugation for 70~90 minutes.

[0019] The second objective of this invention is achieved through the following technical solution:

[0020] A CD301b + -EXO (from CD301b) + The macrophage exosomes were prepared by the above method.

[0021] The third objective of this invention is achieved through the following technical solution:

[0022] A load CD301b + The preparation method of EXO bone-targeting microspheres (A-SCH / OSA@EXO microspheres) includes the following steps:

[0023] (a) Preparation of oxidized sodium alginate (OSA): Sodium periodate aqueous solution was poured into anhydrous ethanol solution of sodium alginate (SA) and stirred magnetically, dialyzed, and freeze-dried to prepare oxidized sodium alginate (OSA). The mass concentration of the sodium periodate aqueous solution was 0.099 g / mL to 0.12 g / mL, the mass concentration of the sodium alginate (SA) anhydrous ethanol solution was 0.2 g / mL to 0.16 g / mL, and the volume ratio of the sodium periodate aqueous solution to the sodium alginate (SA) anhydrous ethanol solution was 1:1 to 1.5.

[0024] (b) Preparation of succinylated chitosan (SCH): Chitosan (CH), methanol solution and succinic anhydride were added sequentially to a lactic acid aqueous solution and stirred magnetically at room temperature until homogeneous. Then, NaOH was added dropwise until the pH of the solution was 7.00~7.11. The precipitate was collected by filtration, dissolved in deionized water, dialyzed, and lyophilized to obtain succinylated chitosan (SCH). The mass-volume ratio of chitosan (CH), lactic acid aqueous solution, methanol solution and succinic anhydride was 0.5g:44.25mL:160mL:150mg.

[0025] (c) Synthesis of alendronate-grafted succinylated chitosan (A-SCH): Aldehyde-activated alendronate (ALE-CHO) and succinylated chitosan (SCH) were reacted in pure water by stirring. The resulting mixture was dialyzed and freeze-dried to obtain A-SCH. The mass ratio of ALE-CHO to SCH was 50 mg: 250 mg.

[0026] (d) Load CD301b + Preparation of EXO bone-targeting microspheres: Aqueous and oil phases were injected into a microfluidic device at different flow rates to form monodisperse spherical droplets, which were collected in an oxidized sodium alginate (OSA) solution, crosslinked at low temperature, washed sequentially with anhydrous ethanol and deionized water, and centrifuged to prepare CD301b-loaded microspheres. + -EXO bone-targeting microspheres, wherein the flow rate of the aqueous phase is 2-4 μL / min, the flow rate of the oil phase is 40-80 μL / min, and the mass concentration of the oxidized sodium alginate (OSA) solution is 25-30 mg / mL; the aqueous phase contains CD301b. + -EXO alendronate-grafted succinylated chitosan aqueous solution; the oil phase is mineral oil.

[0027] Preferably, the dialysis time in steps (a) and (b) is three days, and the dialysis bag used for dialysis has a molecular weight cutoff of 3500 Da.

[0028] Preferably, the lactic acid aqueous solution in step (b) is a 5% (v / v) lactic acid aqueous solution, and the concentration of NaOH is 8M~10M.

[0029] Preferably, the aldehyde-activated alendronate (ALE-CHO) described in step (c) is prepared by reacting alendronate (ALE) with 50% (v / v) glutaraldehyde at a mass-volume ratio of 1.2 g: 30 mL to 35 mL at 45 to 50 °C for 12 to 16 hours.

[0030] Preferably, the aqueous phase in step (d) contains 0.5 mg / mL CD301b. + -EXO is an A-SCH aqueous solution, wherein the A-SCH concentration is 10~8 mg / mL; the oil phase is mineral oil containing 5% (v / v) Span 80; the low-temperature crosslinking temperature is 4~8℃, the low-temperature crosslinking time is 12~24 hours, the centrifugation speed is 3000~3500 rpm, and the centrifugation time is 5~10 minutes.

[0031] The fourth objective of this invention is achieved through the following technical solution:

[0032] A load CD301b + -EXO bone-targeting microspheres (A-SCH / OSA@EXO microspheres) were prepared by the above-described method.

[0033] The fifth objective of this invention is achieved through the following technical solution:

[0034] A load CD301b + - Application of EXO's bone-targeting microspheres (A-SCH / OSA@EXO microspheres) in the field of bone defect repair.

[0035] The load CD301b described in this invention + -EXO's bone-targeting microspheres utilize CD301b + The combination of EXO's multifunctional characteristics with the bone-targeted, controllable release microsphere delivery system not only significantly improves the efficiency and integrity of bone defect repair, but also solves key problems in existing technologies such as single function, low exosome delivery efficiency, and short local retention time, demonstrating clear clinical application prospects and industrialization potential.

[0036] This invention has significant advantages and positive effects compared to existing technologies:

[0037] (1) Improved synergy and reliability of treatment: This invention achieves synergistic and integrated regulation of the entire bone repair process, fundamentally avoiding the complexity of multi-factor compounding; the beneficial effects of this invention are mainly due to the use of CD301b + -EXO as a core functional component brings about this technical feature; CD301b +EXO naturally inherits the triple biological functions of its parent cells: "immunomodulation, angiogenesis, and osteogenic induction." It can simultaneously regulate the three key stages necessary for bone healing—inflammatory resolution, vascularization, and bone formation—in the form of a single component. Compared with the strategy of physically mixing multiple single-function active factors (such as BMP-2 for osteoogenesis, VEGF for angiogenesis, and IL-4 for anti-inflammation) in existing technologies, it completely avoids the complexity of multi-factor compatibility, the uncontrollability of interactions between different factors, and the difficulty in matching release kinetics, thus significantly improving the synergy and reliability of treatment.

[0038] (2) Intelligent bone-targeted delivery: The CD301b loaded in this invention + EXO's bone-targeting microspheres (A-SCH / OSA@EXO microspheres) utilize the A-SCH / OSA microsphere carrier, and the specific binding of sodium alendronate and hydroxyapatite, to achieve active targeted enrichment of exosomes at bone defect sites, avoiding clearance from non-target organs throughout the body, improving local bioavailability, and are in fact intelligent bone-targeting delivery.

[0039] (3) Controlled release and stability optimization: The CD301b loading described in this invention + The preparation method of EXO's bone-targeting microspheres (A-SCH / OSA@EXO microspheres) utilizes microfluidic technology to prepare microspheres, achieving uniform particle size and stable dispersion. Simultaneously, it leverages the pH-responsive Schiff base bond formed between OSA and A-SCH to achieve intelligent and controllable release of exosomes in an acidic microenvironment, prolonging the duration of action and maintaining bioactivity, thereby significantly improving the local retention and functional performance of exosomes. This environmentally responsive release characteristic, compared to simple sustained release, better matches the dynamic process of bone repair, further enhancing repair efficiency.

[0040] (4) Enhanced Functional Synergy: The load CD301b described in this invention + EXO's bone-targeting microspheres (A-SCH / OSA@EXO microspheres) not only provide physical support for the three-dimensional structure of the microspheres, but also promote bone defect repair, enabling CD301b... + -EXO's multiple functions have been further amplified. Attached Figure Description

[0041] Figure 1 CD301b - -EXO and CD301b + -Graph showing the isolation, identification, and transcriptomic analysis of EXO; where A represents CD301b. +Schematic diagram of fluorescence-activated cell sorting (FACS) for macrophage subsets; B shows flow cytometry analysis of RAW264.7 cells after 72 hours of induction with or without IL-4, showing CD301b. + The representation diagram; C is CD301b - -EXO, CD301b + Representative TEM images of -EXO and M2-EXO; D represents CD301b determined by NTA. - -EXO, CD301b + - Particle size distribution of EXO and M2-EXO; E represents Western blot analysis of RAW264.7 cells and CD301b. - -EXO and CD301b + - Plot showing the expression of exosome markers (CD63, CD81, CD9, and TSG101) in EXO; F shows Western blot analysis of RAW264.7 cells, M2-EXO, and CD301b. + - Expression of exosome markers (CD63, CD81, CD9, and TSG101) in EXO; G represents CD301b. - -EXO and CD301b + - Volcano plot of differentially expressed genes obtained from EXO transcriptome sequencing, where red data points represent upregulated genes, blue data points represent downregulated genes, and gray data points represent genes with no significant changes; H represents CD301b. - -EXO and CD301b + -GO enrichment analysis of significantly upregulated differentially expressed genes in EXO transcriptome sequencing;

[0042] Figure 2 CD301b + - Comparative diagram of the promoting effects of M2-EXO on macrophage M2 polarization, angiogenesis, and osteogenic differentiation in vitro; where A represents the effects of M2-EXO and CD301b on macrophage M2 polarization, angiogenesis, and osteogenic differentiation in lipopolysaccharide (LPS)-induced RAW264.7 cells. + A) Representative immunofluorescence images of iNOS (green, M1 labeled) and CD206 (red, M2 labeled) after EXO treatment, with cell nuclei stained with DAPI (blue); B) Quantitative analysis of iNOS and CD206 fluorescence intensity in RAW264.7 cells; C) Control group, M2-EXO, and CD301b. + - A representative fluorescence image of matrix gel HUVECs tubes after EXO treatment; D is a quantitative analysis image of vascular network parameters; E is a control group, M2-EXO, and CD301b. +- ALP staining of BMSCs 6 days after EXO osteogenic induction; F represents control group, M2-EXO, and CD301b. + - A representative ARS staining image of mineralized nodules in BMSCs 12 days after EXO osteogenic induction; G is a quantitative analysis image of calcium deposition measured by spectrophotometry at 562 nm after ARS dissolution;

[0043] Figure 3 Figures show the preparation and characterization of SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres; where A is the Fourier transform infrared (FT-IR) spectrum of SA and OSA; B is the FT-IR spectrum of CH, SCH, and A-SCH; C is the FT-IR spectrum of SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres; and D is the nuclear magnetic resonance (NMR) spectrum of ALE, SCH, and A-SCH. 31 P-spectrum; E shows the scanning electron microscope (SEM) morphology of SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres; F shows the autofluorescence images of SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres under standard DAPI, FITC, and TRITC filters; G shows the injectability of SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres; H shows the particle size distribution statistics of SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres; I shows the fluorescence imaging of SCH / OSA microspheres and A-SCH / OSA microspheres attached to the surface of hydroxyapatite (HAp) sheets; J shows the quantitative analysis of fluorescence intensity corresponding to image I; K shows microspheres loaded with DIR-labeled exosomes (A-SCH / OSA@EXO). DIR SEM comparison of A-SCH / OSA and blank microspheres without exosome loading (A-SCH / OSA) on frozen sections; L represents A-SCH / OSA@BSA. RBITC BSA release kinetics curves of microspheres in simulated body fluids under weakly acidic (pH 5.0) or physiological conditions (pH 7.4);

[0044] Figure 4Cell compatibility diagrams of SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres are shown. A represents the live / dead cell staining (live cells show green fluorescence, dead cells show red fluorescence) after co-culturing SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres with BMSCs, HUVECs, and RAW264.7 cells for 3 days, respectively. B represents the cell compatibility of SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres with SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres as detected by CCK-8 assay. A) BMSCs cell proliferation activity after 1 and 3 days of co-culturing with SCH / OSA@EXO microspheres; B) HUVECs cell proliferation activity after 1 and 3 days of co-culturing with SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres, as detected by CCK-8 assay; C) RAW264.7 cells proliferation activity after 1 and 3 days of co-culturing with SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres, as detected by CCK-8 assay.

[0045] Figure 5 This image shows the validation potential of SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres in promoting macrophage polarization, angiogenesis, and osteogenic differentiation in vitro. Specifically, A shows the immunofluorescence staining of CD206 (red), iNOS (green), and cell nuclei (blue) in lipopolysaccharide (LPS)-induced RAW264.7 cells after treatment with SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres; B shows the quantitative analysis of iNOS and CD206 fluorescence intensity; C shows a representative image of matrix gel HUVECs tube formation after treatment with SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres; D shows the quantitative analysis of vascular network parameters; and E shows the results of treatment with control group, SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres for 14 days. Alizarin Red (ARS) staining of calcium deposition in BMSCs; F represents the quantitative analysis of calcium deposition (absorbance at 562 nm after ARS dissolution);

[0046] Figure 6The images show the bone repair effects of the control group, SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres in a rat skull defect model. Specifically, A shows representative Micro-CT 3D and 2D reconstructed images of the skull defect areas in each group (n = 5) 10 weeks after implantation of the control group, SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres, with red circles indicating new bone formation; B shows the quantitative statistical analysis of bone mineral density based on Micro-CT data for the control group, SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres; and C shows the quantitative statistical analysis of bone volume fraction for the control group, SCH / OSA microspheres, A-SCH / OSA microspheres, and A-SCH / OSA@EXO microspheres.

[0047] Figure 7 The images show hematoxylin-eosin staining of major organs (heart, liver, spleen, kidney, and lung) 10 weeks after implantation of control group, SCH / OSA, A-SCH / OSA, and A-SCH / OSA@EXO microspheres, respectively. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. All materials used in the embodiments of the present invention can be purchased commercially.

[0049] Example 1

[0050] 1.CD301b - -EXO and CD301b + -Identification and transcriptomic analysis of EXO

[0051] 1.1 RAW264.7 Cell Culture

[0052] The mouse macrophage cell line RAW264.7 was purchased from Wuhan Pronosei Biotechnology Co., Ltd., and routinely cultured in high-glucose DMEM complete medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin. It was incubated at 37℃ in a 5% (v / v) CO2 incubator. When the cell confluence reached 70%–80%, it was passaged using the following steps: ① Aspirate the original medium using a Pasteur tube or pipette; ② Gently wash the cells twice with sterile PBS; ③ Add fresh complete medium, gently pipette with a 1 mL pipette tip until the cells are completely detached, and collect the cell suspension; ④ Seed the cell suspension at a 1:5 ratio into new culture dishes for continued culture. For long-term culture, cells were passaged every 48 hours.

[0053] 1.2 CD301b - With CD301b + Macrophage sorting

[0054] RAW264.7 macrophages were induced for 72 hours with 20 ng / mL IL-4. Cells were cultured in high-glucose DMEM complete medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin. After induction, cells were collected and single-cell suspensions were prepared. A surface marker staining was performed using the following antibodies to form an antibody mixture: APC-labeled anti-mouse F4 / 80 antibody (1:400 dilution) and PE-labeled anti-mouse CD301b antibody (1:200 dilution). The single-cell suspension and antibody mixture were incubated at room temperature in the dark for 30 minutes, centrifuged at 3000 rpm for 5 minutes, and the supernatant was discarded. The cells were washed once more. Finally, the cells were resuspended in 500 μL PBS and filtered through a 40 μm cell sieve into flow cytometry tubes. Figure 1 A and Figure 1 As indicated by B in the data, CD301b was successfully sorted using a BD FACSAria II flow cytometer. - With CD301b + Macrophage subsets, quantitative analysis showed that CD301b was present in the IL-4-induced macrophage population. + The percentage of positive cells reached 40.7%.

[0055] 1.3 Preparation of macrophage-derived exosomes

[0056] CD301b - With CD301b + Macrophages and IL-4 (20 ng / mL)-stimulated RAW264.7 cells were cultured for 72 hours in high-glucose DMEM medium containing 10% (v / v) exosome-free FBS and 1% (v / v) penicillin-streptomycin. Conditioned media from each cell type were collected and centrifuged separately using the following methods: 300–400 ×g for 10–15 minutes at 4°C to remove live cells, 2000–3000 ×g for 10–15 minutes to remove dead cells, and 10000–12000 ×g for 30–40 minutes to remove cell debris. The supernatant was then ultracentrifuged at 100000–120000 ×g for 70–90 minutes at 4–10°C to enrich exosomes. The resulting precipitate was resuspended in pre-chilled PBS and washed again by centrifugation at 100000–120000 ×g for 70–90 minutes. The final precipitate was resuspended in PBS, and the three groups of exosomes obtained were: CD301b -Macrophage-derived exosomes (CD301b) - -EXO), CD301b + Macrophage-derived exosomes (CD301b) + The exosomes derived from M2 macrophages (M2-EXO) and M2 macrophages (M2-EXO) were aliquoted and stored at -80°C for later use.

[0057] 1.4 EXO identification

[0058] 1.4.1 Observation of Ultrastructure

[0059] Take 10 μL of freshly extracted CD301b + -EXO, CD301b - -EXO and M2-EXO were dropped onto a clean sealing film surface. A 200-mesh copper mesh (carbon film side down) was gently placed on the droplet and allowed to absorb at room temperature for 2 minutes. Residual liquid was slowly absorbed from the edge of the mesh using filter paper. The sample was then transferred to a 2% (v / v) uranium acetate-oxygenate staining droplet for negative staining for 2 minutes. Excess staining was removed again, and the sample was dried at room temperature for 10 minutes. After the sample was completely dry, the morphological characteristics of exosomes were observed using a transmission electron microscope (TEM) at an accelerating voltage of 80 kV. At least five different fields of view were observed for each sample to ensure representativeness of the results. Figure 1 As shown in C, CD301b + -EXO, CD301b - Both -EXO and M2-EXO exhibit a typical cup-shaped morphology and a clear bilayer lipid membrane structure.

[0060] 1.4.2 Particle Size Analysis

[0061] Detection of CD301b using nanoparticle tracking analysis (NTA) + -EXO, CD301b - Particle size distribution of -EXO and M2-EXO. The three EXO suspensions were diluted to 1 mL with PBS, mixed, and equilibrated at room temperature for 2 minutes. Samples were injected into the detection cell at a constant flow rate (30 μL / min) using a microinjection pump. Three videos (60 seconds / video) were captured for each sample, and particle size distribution characteristics were analyzed using a NanoSight NS300 system. Figure 1 As shown in D, the NTA results indicate that CD301b + -EXO, CD301b - -EXO and M2-EXO have diameters ranging from 70 to 200 nm, with the main peak at approximately 100 nm.

[0062] 1.4.3 Surface Marker Detection

[0063] To confirm that the extracted substance was EXO, Western blotting (WB) was used to detect the expression of EXO-specific surface markers CD9, CD63, CD81, and TSG101. Figure 1 E and Figure 1 As shown in F, all three EXO strains stably express the EXO signature proteins CD9, CD63, CD81, and TSG101.

[0064] 1.5 CD301b - -EXO and CD301b + -EXO RNA sequencing analysis

[0065] To identify CD301b - -EXO and CD301b + Differential expression of RNA transcripts among the EXO groups was determined by whole-genome transcriptional sequencing performed by Beijing Aovisen Gene Technology Co., Ltd. RNA was extracted using the TRIzol method and treated with RNase-free DNase I. RNA degradation and contamination were monitored on 1% (w / v) agarose gels. RNA was quantified using an Agilent 2100 bioanalyzer, and its quality and integrity were assessed using a NanoDrop spectrophotometer. Sequencing libraries were then constructed using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® according to the manufacturer's recommendations, and library fragments were purified using the AMPure XP system. Finally, PCR products were purified using the AMPure XP system, library quality was assessed on an Agilent Bioanalyzer 2100 system, and sequencing was performed using the Illumina NovaSeq 6000 platform.

[0066] The sequencing data were aligned to the reference genome sequence using STAR software. The number of reads aligned to each gene was counted using HTSeq. Gene expression levels were estimated using the number of comparable fragments per million kilobase transcripts. Differentially expressed genes (DEGs) were analyzed between the two conditions / groups using the DESeq R package. A corrected p-value (q-value) < 0.05 and |log2 (fold change)| ≥ 1 were set as the threshold for significant differential expression. Based on the Wallenius noncentrocentric hypergeometric distribution, gene ontology (GO) enrichment analysis of differentially expressed genes was performed using the GOseq R package. Simultaneously, the significance of enrichment of differentially expressed genes in the KEGG pathway was tested using KOBAS software. Figure 1 The G results show that, compared with CD301b - Compared to EXO, CD301b + -EXO revealed a significant differential gene expression profile, identifying 8,909 significantly upregulated genes and 2,469 significantly downregulated genes. For example... Figure 1 As shown in H, functional enrichment analysis of differentially expressed genes using Gene Ontology (GO) revealed that CD301b... + -EXO's functional genes are mainly enriched in three key pathways: "macrophage activation", "angiogenesis regulation" and "positive regulation of osteogenic differentiation", suggesting that it may play multiple roles in coordinating immune regulation, promoting angiogenesis and inducing bone regeneration.

[0067] Example 2

[0068] 2.1 Immunofluorescence staining detection of CD301b + -Expression of RAW264.7 macrophage polarization-related proteins by EXO

[0069] The immune microenvironment plays a crucial role in bone regeneration. To investigate CD301b... + -EXO's immunomodulatory function, causing RAW264.7 cells to grow at a rate of 5 × 10⁻⁶. 5 RAW264.7 cells were seeded at a density of 1 cell / well onto sterile glass slides in 24-well plates. After cell adhesion, the cells were stimulated with LPS (100 ng / mL) for 12 hours to establish an inflammation model. Subsequently, M2-EXO (40 μg / mL) or CD301b was added. +-EXO (40 μg / mL), with cells cultured in conventional tissue culture plates as a blank control. After 2 days of incubation, cells were fixed with 4% (v / v) paraformaldehyde for 30 min, gently washed twice with PBS, permeabilized with 0.1% Triton X-100 for 20 min, washed with PBS, and blocked with 1% bovine serum albumin for 30 min. Cells were incubated overnight at 4°C with anti-iNOS (1:200) and anti-CD206 (1:200) primary antibodies, respectively. After elution with PBS, cells were incubated for 40 min with a mixture of fluorescent secondary antibodies Alexa Fluor 488 (1:200) and Alexa Fluor 568 phalloidin (1:40). Finally, the cells were mounted with a DAPI-containing antifluorescence quencher, and images were acquired and observed using a confocal fluorescence microscope. The expression levels of iNOS and CD206 were quantitatively analyzed using ImageJ software. Figure 2 A in and Figure 2 The B section shows that, compared to the M2-EXO group, CD301b + -EXO exhibits a stronger dual regulatory capacity: it not only significantly inhibits the expression of the M1 phenotypic marker inducible nitric oxide synthase (iNOS), but also enhances the fluorescence intensity of the M2 phenotypic marker CD206.

[0070] 2.2 Detection of CD301b in matrix gelation tube formation experiment + -EXO promotes angiogenesis in HUVECs

[0071] To investigate CD301b + The regulatory effect of EXO on angiogenesis was evaluated in this study using a matrix gel in vitro tube formation assay. First, the matrix gel was placed in a 4°C freezer overnight to allow for complete liquefaction. Simultaneously, pipettes, pipette tips, and 48-well plates were pre-chilled to ensure the matrix gel remained liquid during the procedure. The matrix gel was diluted 2:1 with high-glucose DMEM basal medium, and 130 μL of the diluted matrix gel was added to each well of the pre-chilled 48-well plate. The plate was kept on ice to ensure uniform spreading, and then transferred to a 37°C incubator for 30 minutes to 1 hour to allow the matrix gel to completely solidify. HUVECs were routinely digested, centrifuged, and resuspended in high-glucose DMEM basal medium at 1.6 × 10⁻⁶ cells / well. 5 Inoculate the cells onto the solidified matrix gel at a density of cells / well, and simultaneously add 40 μg / mL of M2-EXO or CD301b. +-EXO was used to treat cells, with the untreated group serving as a control. After incubating the plates at 37°C in a 5% (v / v) CO2 incubator for 6 hours, the culture medium was carefully aspirated, and Calcein AM working solution was added for staining in the dark for 15 minutes. After washing with high-glucose DMEM medium, cell morphology was observed under a 10x fluorescence microscope, and three fields of view were randomly selected for photographic recording. Finally, ImageJ software with an angiogenesis analysis plugin was used to quantitatively analyze parameters such as the number of luminal junctions, the number of luminal grids, and the number of branch points to evaluate the effect of different treatment groups on the angiogenesis ability of hUVECs. Figure 2 As shown in C, CD301b + -EXO significantly enhances the complexity of the tubular networks formed by HUVECs. For example... Figure 2 Quantitative analysis of D in the study confirmed that key parameters of vascular structure, including the total number of connections, average vascular area, and average vascular length, were significantly improved.

[0072] 2.3. CD301b + Effects of EXO on osteogenic differentiation of BMSCs

[0073] 2.3.1 ALP staining

[0074] BMSCs were divided into 5 × 10 4 Cells were seeded at a density of 10 cells / well in 12-well plates. When cell confluence reached 80%, the medium was replaced with osteogenic induction medium (OIM). Cells were randomly divided into three groups: control group, 40 μg / mL M2-EXO group, and 40 μg / mL CD301b group. + - EXO group: Change culture medium and EXO every 3 days. After 5-6 days of induction, wash cells twice with PBS, fix with 4% (v / v) paraformaldehyde for 15 minutes, prepare working solution according to the BCIP / NBT alkaline phosphatase chromogenic kit instructions, stain at room temperature in the dark for 30 minutes, then stop the reaction with distilled water, and observe and photograph under an inverted microscope. Figure 2 As shown in E, CD301b + -EXO induced ALP staining intensity higher than M2-EXO, indicating that it has a stronger ability to initiate early osteogenic processes.

[0075] 2.3.2 Alizarin Red Staining

[0076] BMSCs were divided into 5 × 10 4 Cells were seeded at a density of 10 cells / well in 12-well plates, and replaced with OIM when cell confluence reached 80%. Cells were randomly assigned to three groups: control, 40 μg / mL M2-EXO, and 40 μg / mL LCD301b. +- EXO group: Culture medium and EXO were changed every 3 days. After 14 days of induction, the nodules were washed with PBS, fixed with 4% (v / v) paraformaldehyde for 15 minutes, incubated with Alizarin Red S staining solution at room temperature for 15 minutes, washed with PBS, and observed under an inverted fluorescence microscope. The stained nodules were then dissolved with 10% (w / v) cetylpyridinium chloride (CPC), and the absorbance was measured at 562 nm using a multi-mode microplate reader for quantitative analysis. Figure 2 As shown in F, ARS staining results indicate that the formation of mineralized nodules gradually increased in each group, in the following order: control group <M2-EXO<CD301b + -EXO. (e.g.) Figure 2 The quantitative analysis of G further validated this trend.

[0077] Example 3

[0078] 3. Preparation and characterization of microspheres

[0079] 3.1 Preparation of microspheres

[0080] 3.1.1 OSA Preparation

[0081] Partially oxidized OSA was prepared by oxidation with sodium periodate: 4 g of SA was weighed into a beaker containing 20–25 mL of anhydrous ethanol and stirred thoroughly for 20–30 minutes. 1.98–2.40 g of sodium periodate was added to a beaker containing 20 mL of deionized water and stirred thoroughly for 20–30 minutes. Then, the dissolved sodium periodate was poured into 20 mL of SA in anhydrous ethanol and magnetically stirred for 6 hours. Finally, the solution was dialyzed against deionized water for three days (dialysis bag molecular weight cutoff: 3500 Da), with the water changed at least twice daily. The lyophilized liquid was then used as OSA. SA and OSA were characterized by FT-IR. Figure 3 As shown in A, the OSA sample was at 1725 cm⁻¹. -1 A weaker aldehyde characteristic peak appears at this location.

[0082] 3.1.2 Preparation of SCH

[0083] Prepare a 5% (v / v) lactic acid aqueous solution. Weigh 0.5 g of chitosan (CH) and add it to 40 mL of a well-stirred 5% (v / v) lactic acid aqueous solution. Stir magnetically for 4 hours. Then add 160 mL of methanol solution and continue stirring for 20-30 minutes to ensure the chitosan is fully dispersed in the methanol solution. Add 150 mg of succinic anhydride and stir rapidly with a magnetic stirrer at room temperature for 24 hours. Then, while stirring, add 8 M-10 M NaOH dropwise to adjust the pH of the solution to 7.00-7.11. During this process, a precipitate will gradually form. Filter the liquid through filter paper. After filtration, transfer the precipitate from the filter paper to a 200 mL beaker, add 100 mL of deionized water, and stir magnetically at 800 rpm overnight until the precipitate dissolves. Finally, dialyze the solution with deionized water for three days (dialysis bag molecular weight cutoff: 3500 Da), changing the water at least twice a day. Freeze-dry the dialyzed liquid to obtain SCH. Characterize CH and SCH by FTIR. like Figure 3 As shown in B, the amide I band (C=O stretching vibration, 1647 cm⁻¹) is clearly visible in the FT-IR spectrum. -1 ) and amide II band (NH bending vibration, 1554 cm -1 The characteristic peaks confirmed the successful acylation reaction. It is worth noting that, as... Figure 3 As shown in B, the A-SCH sample at 3530 cm⁻¹ -1 The presence of a significant NH stretching vibration peak further corroborates the formation of amide bonds.

[0084] 3.1.3 Synthesis of A-SCH

[0085] Weigh 1.2 g of alendronate (ALE) and dissolve it in 30 mL–35 mL of 50% (v / v) glutaraldehyde solution. Stir the mixture at 45–50 °C for 12–16 hours. After the reaction, allow the mixture to stand for 20 minutes–1 hour. Wash the product with a large amount of cold acetone to obtain a brown precipitate. Dry the precipitate thoroughly in a ventilated environment to obtain aldehyde-activated ALE (ALE-CHO). Separately, under stirring, dissolve 250 mg of SCH in 150 mL of ultrapure water until completely dissolved. Then add 50 mg of ALE-CHO and stir continuously at 37 °C for 12 hours. Dialyze the resulting mixture to deionized water (molecular weight cutoff: 3500 Da) for 72 hours, then freeze-dry to obtain yellow porous A-SCH, which is stored at 4 °C. Nuclear magnetic resonance (NMR) was used to analyze the precipitate. 31 P-spectroscopy characterizes whether bisphosphonates have been grafted onto A-SCH. For example... Figure 3As shown in D, both ALE and A-SCH exhibit characteristic chemical shifts at 17.6 ppm, directly proving that a covalent bond has formed between ALE and SCH.

[0086] 3.1.4 Preparation of A-SCH / OSA@EXO microspheres

[0087] Monodisperse hydrogel microspheres were prepared using a microfluidic device. Aqueous phases (8–10 mg / mL SCH, 8–10 mg / mL LA-SCH, or containing 0.5 mg / mL CD301b) were used. + The oil phase (8–10 mg / mL A-SCH from EXO) was injected into the microfluidic chip at controlled flow rates of 2–4 μL / min and 40–80 μL / min, respectively. This caused the aqueous phase to form monodisperse spherical droplets under the shear force of the oil phase. The resulting emulsion droplets were collected in a 25–30 mg / mL OSA solution and crosslinked in a shaker at 4 °C for 12 hours. The microspheres were washed sequentially with anhydrous ethanol (to remove residual oil) and rinsed three times with deionized water. After centrifugation (3000–3500 rpm, 5–10 min), the purified microspheres (SCH / OSA, A-SCH / OSA, and A-SCH / OSA@EXO) were stored at -20 °C to 80 °C for subsequent research.

[0088] 3.2 Testing of the physicochemical properties of the microspheres

[0089] 3.2.1 FT-IR Detection of Microspheres

[0090] Samples were prepared using the potassium bromide tableting method: lyophilized microspheres were mixed with potassium bromide at a ratio of 1:100 (w / w), thoroughly ground into a uniform and fine powder, then tableted. FT-IR was used for analysis of the chemical composition and functional group characteristics of the microspheres. Figure 3 As shown in C, all microsphere samples were at 1643 cm⁻¹. -1 The peaks at all locations showed distinct imine bond (-C=N-) characteristic absorption peaks, confirming that intermolecular crosslinking was achieved through the Schiff base reaction.

[0091] 3.2.2 Morphological observation of microspheres

[0092] The prepared SCH / OSA, A-SCH / OSA, and A-SCH / OSA@EXO microsphere samples were resuspended in deionized water. A small amount of the dispersed microsphere solution was dropped onto a circular glass slide, air-dried at room temperature, and then sputtered with gold. The surface morphology and structural characteristics of the microspheres were then observed using a scanning electron microscope (SEM). Figure 3The E results showed that the prepared microspheres had smooth surfaces, regular morphology, and uniform particle size distribution.

[0093] 3.2.3 Detection of microsphere particle size distribution and fluorescence properties

[0094] Three groups of microsphere samples—SCH / OSA, A-SCH / OSA, and A-SCH / OSA@EXO—were resuspended in deionized water. Small amounts of the dispersion were then added dropwise to 24-well plates, and their fluorescence characteristics were observed using an inverted fluorescence microscope. Microsphere imaging was recorded under different fluorescence channels by sequentially switching between three standard filters: DAPI, FITC, and TRITC. Particle size statistical analysis was performed on ≥200 microspheres in each group using NanoMeasure software, and microsphere size distribution maps were plotted using Origin software. Fluorescence microscopy confirmed that these microspheres were regularly spherical and possessed autofluorescence properties; for example... Figure 3 As shown in Figure F, under DAPI, FITC, and TRITC filters, it emits blue, green, and red fluorescence, respectively. It is worth noting that, as... Figure 3 As shown in Figure G, all microspheres can be successfully injected through a 32G fine-bore injection needle (110μm inner diameter) without clogging, and the structure remains intact after injection.

[0095] like Figure 3 As shown in Figure H, particle size statistical analysis using Nano Measure software revealed that the average diameters of the three microspheres—SCH / OSA, A-SCH / OSA, and A-SCH / OSA@EXO—were 59 ± 9.5 μm, 53 ± 10.4 μm, and 53 ± 7.4 μm, respectively.

[0096] 3.2.4 In vitro affinity experiment with hydroxyapatite to verify the targeting ability of A-SCH / OSA microspheres

[0097] The in vitro binding capacity of microspheres was evaluated using a hydroxyapatite (HAp) affinity assay. The procedure was briefly described as follows: One HAp disc (11 ± 1 mm in diameter, 3 ± 0.5 mm in thickness) was placed in each well of a 24-well plate. Equal volumes of prepared SCH microspheres (untargeted) and A-SCH microspheres (targeted) were dispersed separately in 0.5 mL of 0.9% (w / v) NaCl solution and added to the wells containing the HAp discs (three parallel wells per sample). The 24-well plate was incubated on a shaker. At 4, 16, and 24 hours, the HAp discs were removed from the wells and gently rinsed with 0.9% (w / v) NaCl solution to remove unbound microspheres. Subsequently, the microspheres adsorbed on the HAp discs were observed and images were acquired using an inverted fluorescence microscope. Finally, the images were analyzed quantitatively using ImageJ software. The bone-binding properties of A-SCH / OSA microspheres were systematically evaluated using a fluorescence tracer method. The experimental group (targeted A-SCH / OSA microspheres) and the control group (non-targeted SCH / OSA microspheres) were co-incubated with HAp tablets for 4 hours, 14 hours, and 24 hours, respectively. Observation under an inverted fluorescence microscope showed that... Figure 3 As shown in Figure I, A-SCH / OSA microspheres exhibit time-dependent fluorescence enrichment on the HAp surface, with significantly higher fluorescence intensity than the control group. Figure 3 Further quantitative verification using fluorescence quantification analysis showed that the HAP binding capacity of A-SCH / OSA microspheres at 4 hours, 14 hours, and 24 hours was 4.5 times, 10 times, and 16 times that of the SCH / OSA microsphere group, respectively. P <0.001).

[0098] 3.2.5 Encapsulation Verification Experiment of EXO in Microspheres

[0099] To verify CD301b + EXO was successfully encapsulated within A-SCH / OSA@EXO composite microspheres. First, exosomes were labeled with DIR near-infrared fluorescent dye. The specific steps were as follows: DIR dye working solution was added to the EXO suspension to adjust the final concentration to 10 μM. After vortexing and mixing, the mixture was incubated at 37°C in the dark for 30 minutes. After incubation, the mixture was diluted with PBS buffer and purified by ultracentrifugation (100,000–120,000 × g, 70–90 minutes) to remove unbound free dye. The precipitate was collected and resuspended in 200 μL of PBS to obtain DIR-labeled CD301b. + -EXO (EXO) DIR (For backup). Microfluidic technology is used to deliver EXO... DIRA-SCH / OSA@EXO was prepared by encapsulating it into A-SCH / OSA microspheres. DIR Microspheres were then sliced ​​into 5.0 μm thick samples using cryosectioning. CD301b was observed using SEM. + The distribution of -EXO within the microspheres. The results are shown in Figure 3, K, for A-SCH / OSA@EXO. DIR The microspheres have relatively rough cross-sections, forming granular aggregates, while the cross-sections of the A-SCH / OSA microsphere group are smoother. These experiments demonstrate from multiple perspectives that EXO was successfully encapsulated within the microspheres.

[0100] 3.2.6 EXO sustained-release experiment within microspheres

[0101] To track CD301b in A-SCH / OSA@EXO microspheres + - The release characteristics of EXO were studied using rhodamine B isothiocyanate-labeled bovine serum albumin (BSA). RBITC As a tracer molecule to replace CD301b + -EXO, preparation of A-SCH / OSA@BSA RBITC Microspheres. Aliquots of microspheres were placed into dialysis bags (molecular weight cutoff: 3500 Da) containing 2 mL of simulated body fluid (SBF) at pH 7.4 or pH 5. The dialysis system was immersed in 45 mL of release buffer at the same pH and continuously agitated (1000 rpm / min, 37°C). At predetermined time intervals (days 1, 3, 5, 7, 9, 11, 13, and 15), 1 mL of external release buffer was collected, and the same volume of fresh buffer was added. The collected buffer was quantified using a multi-plate reader (excitation wavelength: 560 nm, emission wavelength: 600 nm). Based on a pre-established BSA... RBITC Standard curve to determine the BSA released by microspheres RBITC Concentration, and denoted as C. n Then calculate BSA using the following formula (1). RBITC The cumulative release amount.

[0102] Cumulative release amount = (1)

[0103] : is the sum of the concentrations of all previous samples.

[0104] Example 4

[0105] 4. Biocompatibility testing of microspheres

[0106] 4.1 Live and Dead Staining

[0107] BMSCs (2×10 3 (number / well), HUVECs (3×10) 3 Cells per well) and RAW264.7 cells (5 × 10⁻⁶ cells / well) 3 Cells were seeded in the lower chamber of 24-well Transwell plates (8 μm pores) and cultured. After cell adhesion, three types of sterile microspheres (SCH / OSA, A-SCH / OSA, and A-SCH / OSA@EXO, 0.5 mg / well) were placed in the upper chamber of the Transwell plate. On day 3, live / dead cell staining was performed using the Calcein-AM / PI kit. 2 μL of PI and 0.5 μL of Calcein AM were added to 1 mL of culture medium to prepare the staining working solution. The 24-well plate medium was discarded, and the cells in the lower chamber were gently washed twice with PBS. 300 μL of the staining working solution was added, and the plates were incubated at 37°C in the dark for 30 minutes. Images were then observed and acquired using an inverted fluorescence microscope. Calcein AM labeled live cells (green fluorescence), and PI labeled dead cells (red fluorescence). Figure 4 The results of live / dead staining showed that after BMSCs, HUVECs and RAW264.7 cells were co-cultured with microspheres for 3 days, the proportion of live cells (green fluorescence) exceeded 95%, with only sporadic red fluorescence signals (dead cells).

[0108] 4.2 CCK-8 test

[0109] BMSCs (2×10 3 (number / well), HUVECs (3×10) 3 Cells per well) and RAW264.7 cells (5 × 10⁻⁶ cells / well) 3 Cells were seeded in the lower chamber of 24-well Transwell chambers (8 μm pores) and cultured. After cell adhesion, three types of sterile microspheres (SCH / OSA, A-SCH / OSA, and A-SCH / OSA@EXO, 0.5 mg / well) were placed in the upper chamber of the Transwell chamber. After 1 and 3 days of culture, CCK-8 working solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance of each well was then measured at 450 nm. The results showed that the proliferation of all cell types was time-dependent. Notably, as shown in Figure 4, B... Figure 4 C in Figure 4As shown in Figure D, on day 3, both A-SCH / OSA and A-SCH / OSA@EXO microspheres significantly promoted the proliferation of RAW264.7 cells, while no significant difference was observed in the proliferation levels of BMSCs and HUVECs among the groups.

[0110] Example 5

[0111] 5. Validation of the potential of A-SCH / OSA@EXO microspheres to promote macrophage polarization, angiogenesis, and osteogenic differentiation in vitro.

[0112] 5.1 Effects of microspheres on RAW264.7 macrophage polarization

[0113] RAW264.7 cells were cultured at a rate of 5 × 10⁻⁶. 5 Cells were seeded overnight on sterile slides in 24-well plates at a density of cells / well and induced to polarize to the M1 phenotype with 100 ng / mL lipopolysaccharide (LPS) for 12 hours. Various microspheres (SCH / OSA, A-SCH / OSA, and A-SCH / OSA@EXO, 0.5 mg / well) were then placed in the Transwell chambers of each well. Untreated cells served as controls. After 48 hours of culture, cells were fixed with 4% (v / v) paraformaldehyde, permeated with 0.1% Triton X-100 for 30 min, and blocked with 10% goat serum to prevent nonspecific staining. Cells were then incubated overnight at 4°C with primary antibodies: anti-iNOS (1:200) or anti-CD206 (1:200). After elution, the cells were incubated with either Alexa Fluor 488-labeled goat anti-rabbit IgG (1:400) or Alexa Fluor 568-labeled goat anti-rabbit IgG (1:400) and DAPI for nuclear labeling. Images were acquired using CLSM and analyzed using ImageJ software. Figure 5 Immunofluorescence analysis showed that LPS stimulation significantly induced intracellular high expression of the M1 biomarker CD86, while all microsphere groups effectively inhibited CD86 expression, with the A-SCH / OSA@EXO group showing the most significant effect. Notably, compared to the low expression of the M2 biomarker CD206 in the LPS group, all three microspheres induced specific high expression of CD206 in the perinuclear region, with the A-SCH / OSA@EXO group exhibiting the most significant fluorescence enhancement effect. Figure 5 The results of quantitative fluorescence analysis of B in the image were completely consistent with the image observation.

[0114] 5.2 Effects of microspheres on angiogenesis in HUVECs cells

[0115] The effect of microspheres on the in vitro lumen-forming ability of HUVECs was evaluated using a Transwell co-culture system. The simplified procedure was as follows: 250 μL of liquid matrix gel was added to the lower chamber of a pre-cooled 24-well Transwell plate and allowed to solidify completely at 37 °C for 30 minutes. HUVECs were then routinely digested, centrifuged, and cultured at 3 × 10⁻⁶ microspheres. 5 Cells were resuspended in serum-free medium at a density of 0.5 mg / well and evenly seeded onto the solidified matrix gel surface. Subsequently, microspheres (SCH / OSA, A-SCH / OSA, or A-SCH / OSA@EXO, at a concentration of 0.5 mg / well) suspended in serum-free DMEM were added to the upper chamber of a Transwell culture and co-cultured with HUVECs in the lower chamber for 4–6 hours. After culture, cells were stained with calcein-AM, and luminal structures were observed using an inverted fluorescence microscope. Quantitative analysis was performed using AngioTool software.

[0116] like Figure 5 The C-results showed that, compared with SCH / OSA microspheres, the A-SCH / OSA group significantly enhanced the tubular formation ability of HUVECs. It is noteworthy that, as... Figure 5 As shown in D, the A-SCH / OSA@EXO microsphere group exhibited the best angiogenesis performance, characterized by a densely branched tubular network, with significantly increased total number of connection points, percentage of vessel area, and average vessel length compared to other experimental groups.

[0117] 5.3 Effect of microspheres on osteogenic differentiation capacity of BMSCs

[0118] BMSCs were 5 × 10 4 Cells were seeded at a density of 10 cells / well in 12-well plates. When the cell confluence reached 80%, the medium was replaced with osteogenic induction medium. The wells were randomly divided into four groups: (1) OIM as the control group; (2) SCH / OSA microsphere group; (3) A-SCH / OSA microsphere group; (4) A-SCH / OSA@EXO. After induction for 12-14 days, mineralization was assessed using Alizarin Red S (ARS) staining. Cells were fixed and then incubated with ARS solution for 15 minutes. The stained samples were observed under an inverted fluorescence microscope. To quantify calcium deposition, the ARS-stained nodules were dissolved in 10% (w / v) hexadecylpyridine chloride, and the absorbance was measured at 562 nm using a multi-plate reader. Figure 5The results of alizarin red staining in [the relevant context] showed that, compared with the control group, each microsphere group significantly promoted the formation of mineralized nodules in BMSCs. Moreover, the staining area and intensity showed a gradient enhancement trend: SCH / OSA < A-SCH / OSA < A-SCH / OSA@EXO. It is worth noting that the A-SCH / OSA@EXO group had the darkest staining, and dense dark red calcium deposition plaques could be seen around the microspheres. As Figure 5 Quantitative analysis in [the relevant context] further confirmed that the amount of calcium salt deposition in the A-SCH / OSA@EXO group was significantly higher than that in the other groups.

[0119] Example 6

[0120] 6. Effect of A-SCH / OSA@EXO microspheres on the repair of rat skull defects

[0121] 6.1 Animal purchase and feeding

[0122] Twenty-four SPF-grade female SD rats, 6 - 8 weeks old, weighing 260 - 300 g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (certificate number: 210726220102060432). The feeding conditions for SD rats were a 12-hour light / dark cycle, with unrestricted access to water and food, and the temperature maintained at 23 ± 2°C. All animals were normally housed in the experimental animal facility for more than one week before the experiment to adapt to the environment. All animal studies were approved by the Animal Ethics Committee of the Affiliated Hospital of Guangdong Medical University (approval number: AHGDMU-LAC-B-202406-0039) and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals.

[0123] 6.2 Rat skull defect modeling and microsphere implantation

[0124] ① Material preparation

[0125] The microspheres used in the experiment were prepared by the microfluidic method. After soaking the microspheres in 75% (v / v) ethanol solution for 5 min, they were repeatedly washed with excess PBS to remove residual ethanol and were reserved for use;

[0126] ② Surgical implementation

[0127] SD rats were fasted for 12 hours prior to surgery and then anesthetized with intraperitoneal injection of afodin (300 mg / kg body weight). The rats were placed prone on the operating table, and skin preparation, routine disinfection, and draping were performed. An incision was made along the midline of the skull, the skin was cut open, and the skin and subcutaneous tissue were dissected layer by layer to reach the periosteum, clearly exposing the flattest part of the midline of the parietal bone. A circular defect with a diameter of 5.0 mm and a thickness of 1.0 mm was drilled using a trephine. Care was taken during drilling to avoid damage to the dura mater and brain tissue. During the operation, saline was instilled into the surgical area using a syringe to prevent tissue damage due to overheating of the trephine. Twenty-four rats were randomly divided into four groups: A: 6 rats served as a blank control, without implantation of any material; B: 6 rats implanted with SCH / OSA microspheres; C: 6 rats implanted with A-SCH / OSA microspheres; D: 6 rats implanted with A-SCH / OSA@EXO microspheres. After establishing the rat skull defect model, sterilized microspheres were immediately implanted into the bone defects of the rats according to the above-mentioned grouping to fill the defect area. After confirming proper placement of the microspheres and the absence of active bleeding, the incision was sutured layer by layer. After closing the surgical incision, it was disinfected with povidone-iodine, and the rats were intramuscularly injected with 200,000 units of penicillin sodium. The rats were placed in a warm, dry place and allowed free access to water for 2 hours after fully waking up. They were allowed free access to food on the first day after surgery. Ten weeks post-surgery, rat skull, heart, liver, spleen, lung, and kidney tissues were collected, fixed in 4% (v / v) paraformaldehyde solution for 48 hours, and then transferred to 75% (v / v) ethanol for subsequent experiments.

[0128] 6.3 Micro-CT Analysis

[0129] The fixed rat skull specimen was removed and scanned using Micro-CT with the following parameters: slice thickness: 48 μm, slice interval: 48 μm, pixel size: 48 μm, and mode: high voltage. 3D reconstruction of the defect area was performed using CT Analyser Version 1.13 software, and parameters such as total tissue volume (TV) / bone volume (BV) and bone mineral density (BMD) of the defect area were analyzed. Figure 6 As shown in Figure A, the blank control group showed only a small amount of new bone tissue at the periphery of the defect area, while the central area still exhibited significant bone defects, indicating that bone defects are difficult to heal spontaneously without external intervention. All experimental groups showed varying degrees of bone repair effects. While the SCH / OSA group showed a significant increase in bone mineral density (BMD) compared to the control group, its bone volume fraction (BV / TV) only showed a non-significant increase. Notably, the A-SCH / OSA group and the A-SCH / OSA@EXO group showed significantly improved repair efficacy: [Example data would be inserted here]. Figure 6 B in Figure 6Quantitative analysis of C showed that the BMD and BV / TV values ​​of both groups were significantly higher than those of the SCH / OSA group, and the A-SCH / OSA@EXO microsphere group showed the best repair effect, with BMD reaching 0.36±0.07 g / cm³ (an increase of 23.5% compared with the A-SCH / OSA group) and BV / TV reaching 31.5±3.5% (an increase of 13.5% compared with the A-SCH / OSA group), demonstrating a significant osteogenic promoting effect.

[0130] 6.4 Specimen decalcification and paraffin embedding

[0131] Rat skull, heart, liver, spleen, lung, and kidney tissues were placed in an automated tissue dehydrator and sequentially immersed in ethanol solutions with volume fractions of 75.0%–30 min, 75.0%–1 h, 85.0%–3 h 30 min, 85.0%–1 h, 95.0%–1 h, 95.0%–4 h, 100.0%–30 min, and 100.0%–30 min for gradient dehydration. The samples were then immersed in xylene II (100% xylene) for 17 min, xylene II for 17 min, wax for 20 min, and wax for 1 h 30 min. The samples were then embedded in an embedding machine.

[0132] 6.5 HE staining

[0133] Sectioning: Paraffin-embedded visceral tissue blocks were transversely sectioned into 5.0 μm thicknesses, unfolded in a 42℃ water bath, and mounted onto glass slides. The slides were then baked in a 60℃ incubator for 2 hours. Dewaxing and hydration: Sections were dewaxed by immersing each section in xylene I and xylene II for 15 minutes each, followed by rehydration in 100.0%, 100.0%, 95.0%, 85.0%, and 75.0% ethanol solutions and deionized water for 5 minutes each. Cell nuclei were stained with hematoxylin for 10 minutes, washed with deionized water for 5 minutes, differentiated with 1% hydrochloric acid alcohol for 3 seconds, and rinsed with running water for 10 minutes. Cytoplasm was stained with eosin for 30 minutes. The tissues were immersed for 5 seconds in 75.0%, 85.0%, 95.0%, and 95.0% ethanol solutions (v / v), 2 minutes each in 100.0% ethanol I and 100.0% ethanol II, and 2 minutes each in xylene I and xylene II. The tissues were then mounted with neutral resin and photographed under an optical microscope. Histological examinations were performed on major organs (including the heart, lungs, liver, spleen, and kidneys). Figure 7 As shown, H&E staining revealed no significant histological changes or inflammatory lesions in all groups, confirming the excellent biocompatibility of the various microspheres.

[0134] Based on the above examples, the following conclusions can be drawn:

[0135] (1) The experimental system can stably separate and enrich functional CD301b. + -EXO, whose morphology, particle size and marker proteins conform to the characteristics of exosomes, and has a clear expression profile related to immune / angiogenic / osteogenic functions (transcriptomics evidence).

[0136] (2) CD301b + -EXO promotes macrophage polarization toward reparative M2, enhances tube formation of human umbilical vein endothelial cells (HUVECs), and significantly promotes early (ALP) and late (mineralized nodule) osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs).

[0137] (3) The constructed payload CD301b + EXO's bone-targeting microspheres (A-SCH / OSA@EXO microspheres) have uniform particle size, good dispersibility and bone affinity (can specifically bind hydroxyapatite), and exhibit pH responsiveness—accelerating the release of exosomes in acidic (inflammatory) environments, thereby achieving targeted and environment-triggered release at damaged sites.

[0138] (4) Consistent in vitro and in vivo evidence: CD301b + -EXO loaded into bone-targeting microspheres (A-SCH / OSA@EXO) showed significantly better synergistic therapeutic effects than the control / single-component approach in terms of immune regulation (increasing CD206 and decreasing CD86), angiogenesis, and osteogenic effects. Ultimately, it achieved optimal bone defect repair in a rat skull defect model (highest BMD and BV / TV on Micro-CT, and histological findings showed a more dense and orderly bone matrix).

[0139] (5) Initial safety is good: No evidence of systemic toxicity was found during the dosing regimen and observation period in this study.

[0140] In summary, functional immune repair exosomes (CD301b) + The combination of -EXO with a controlled-release, bone-targeted microsphere delivery system is an effective multimodal bone repair strategy. By modulating the local immune microenvironment and simultaneously promoting angiogenesis and osteogenic differentiation, it significantly improves the repair effect of bone defects, with clear mechanistic support and potential clinical translational value.

[0141] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A CD301b + The preparation method of -EXO is characterized by, Includes the following steps, (1) CD301b + Preparation of macrophages: RAW264.7 macrophages were stimulated with interleukin-4 and cultured in culture medium. Cells were collected and single-cell suspensions were prepared. The single-cell suspensions were incubated with an antibody mixture at room temperature in the dark. After centrifugation and discarding of the supernatant, the cells were washed, resuspended, filtered, and sorted to obtain CD301b. + Macrophages; (2) CD301b + -Preparation of EXO: The CD301b obtained in step (1) is processed... + Macrophages were cultured in culture medium. After culture, they were sequentially centrifuged at low temperature. The supernatant was then collected, and exosomes were enriched by a first ultracentrifugation. The resulting precipitate was pre-cooled and resuspended, followed by a second ultracentrifugation. The resulting precipitate was then resuspended to prepare CD301b. + -EXO, which originates from CD301b + Exosomes of macrophages; The specific conditions for the first and second ultracentrifugations were 4℃~10℃, 100000~120000×g ultracentrifugation for 70~90 minutes. The mass concentration of interleukin-4 mentioned in step (1) is 20 ng / mL; The culture medium described in steps (1) and (2) is a high-glucose DMEM complete medium containing 10% by volume exosome-free FBS and 1% by volume penicillin-streptomycin; The specific steps of differential centrifugation under low temperature environment described in step (2) are as follows: First, centrifuge at 300~400×g for 10~15 minutes at 4~10℃ to remove live cells, then centrifuge at 2000~3000×g for 10~15 minutes to remove dead cells, and then centrifuge at 10000~12000×g for 30~40 minutes to remove cell debris and collect the supernatant.

2. The CD301b according to claim 1 + The preparation method of -EXO is characterized by, The antibody mixture in step (1) is a mixture of APC-labeled anti-mouse F4 / 80 antibody diluted 400 times and PE-labeled anti-mouse CD301b antibody diluted 200 times.

3. A CD301b + -EXO, characterized in that, It is prepared according to any one of the preparation methods in claims 1 to 2.

4. A load cell as described in claim 3, specifically the CD301b. + -The method for preparing EXO bone-targeting microspheres is characterized by, Includes the following steps, (a) Preparation of oxidized sodium alginate: Sodium periodate aqueous solution was poured into anhydrous ethanol solution of sodium alginate and magnetically stirred, dialyzed, and freeze-dried to prepare oxidized sodium alginate. The mass concentration of the sodium periodate aqueous solution was 0.099 g / mL to 0.12 g / mL, the mass concentration of the sodium alginate anhydrous ethanol solution was 0.16 g / mL to 0.2 g / mL, and the volume ratio of the sodium periodate aqueous solution to the sodium alginate anhydrous ethanol solution was 1:1 to 1.

5. (b) Preparation of succinylated chitosan: Chitosan, methanol solution, and succinic anhydride were added sequentially to a lactic acid aqueous solution and stirred magnetically at room temperature until homogeneous. Then, NaOH was added dropwise until the pH of the solution was 7.00~7.

11. The precipitate was collected by filtration, dissolved in deionized water, dialyzed, and lyophilized to obtain succinylated chitosan. The mass-volume ratio of chitosan, lactic acid aqueous solution, methanol solution, and succinic anhydride was 0.5g:40mL:160mL:150mg; the lactic acid aqueous solution was a 5% (v / v) lactic acid aqueous solution. (c) Synthesis of alendronate-grafted succinylated chitosan: Aldehyde-activated alendronate and succinylated chitosan were reacted in pure water by stirring. The resulting mixture was dialyzed and freeze-dried to obtain alendronate-grafted succinylated chitosan. The mass ratio of aldehyde-activated alendronate to succinylated chitosan was 50 mg: 250 mg. The aldehyde-activated alendronate was prepared by reacting alendronate with 50% glutaraldehyde at a mass-volume ratio of 1.2 g: 30 mL to 35 mL at 45 to 50 °C for 12 to 16 hours. (d) Load CD301b + Preparation of EXO bone-targeting microspheres: Aqueous and oil phases were injected into a microfluidic device at different flow rates to form monodisperse spherical droplets, which were collected in an oxidized sodium alginate solution, crosslinked at low temperature, washed sequentially with anhydrous ethanol and deionized water, and centrifuged to obtain CD301b-loaded microspheres. + -EXO bone-targeting microspheres, wherein the flow rate of the aqueous phase is 2-4 μL / min, the flow rate of the oil phase is 40-80 μL / min, and the mass concentration of the oxidized sodium alginate solution is 25-30 mg / mL; the aqueous phase contains CD301b. + -EXO alendronate grafted succinylated chitosan aqueous solution.

5. The load CD301b according to claim 4 + -The method for preparing EXO bone-targeting microspheres is characterized by, The concentration of NaOH mentioned in step (b) is 8M~10M.

6. The load CD301b according to claim 4 + -The method for preparing EXO bone-targeting microspheres is characterized by, The aqueous phase described in step (d) contains 0.5 mg / mL CD301b. + -EXO alendronate-grafted succinylated chitosan aqueous solution, wherein the concentration of alendronate-grafted succinylated chitosan is 8~10 mg / mL; the oil phase is mineral oil containing 5% by volume of Span 80; the low-temperature crosslinking temperature is 4~8℃, the low-temperature crosslinking time is 12~24 hours, the centrifugation speed is 3000~3500 rpm, and the centrifugation time is 5~10 minutes.

7. A load CD301b + -EXO's bone-targeting microspheres, characterized by... It is prepared according to any one of the preparation methods in claims 4 to 6.

8. A load CD301b according to claim 7 + - Application of EXO bone-targeting microspheres in the preparation of bone defect repair drugs.

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