A bone-targeting platform based on black phosphorus and amorphous calcium carbonate, its preparation method and application

Abstract

This invention belongs to the field of biomedical technology and discloses a bone-targeting platform based on black phosphorus and amorphous calcium carbonate, its preparation method, and its applications. In this invention, amorphous calcium carbonate (ACC) is coated onto the surface of bone apatite (BP) to form the core (BA) of a nano-therapeutic platform. Subsequently, a polydopamine (PDA) layer is coated on the surface, and a bone-targeting ligand (polyacrylic acid-aldehyde PA) is further coupled to the PDA layer. After BA@PDA-PA precisely accumulates in bone tissue, it reacts to the acidic microenvironment and undergoes "autolysis-like" degradation, rapidly releasing BP and calcium carbonate. 2+ Released BP can scavenge ROS in the bone microenvironment and improve the bone immune microenvironment. Furthermore, BP and Ca... 2+ As a "raw material" for bone matrix mineralization, it synergistically promotes osteoogenesis and inhibits osteoclast activity, ultimately achieving a precise and multi-effect treatment for osteoporosis (OP).

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to a bone-targeting platform based on black phosphorus and amorphous calcium carbonate, its preparation method, and its application. Background Technology

[0002] Osteoporosis (OP) is a systemic skeletal disease characterized by decreased bone mineral density (BMD) and destruction of bone microstructure, leading to decreased bone strength and increased risk of fractures, often accompanied by severe bone defects and various complications. While drug treatment has achieved some efficacy, due to the lack of targeted therapy, drugs need to be used long-term, which can lead to complications such as malignant transformation of reproductive organs, cardiovascular disease, and pathological fractures.

[0003] The dynamic metabolic balance between bone formation and bone resorption is essential for maintaining bone health. Increased bone resorption and decreased bone formation both lead to an imbalance in bone metabolism, which can further cause osteoporosis (OP). Therefore, regulating this dynamic balance is key to treating OP. Furthermore, OP is often accompanied by a severe inflammatory response within the affected bone tissue. During this process, high levels of reactive oxygen species (ROS) not only promote the differentiation and maturation of osteoclast precursors but also inhibit the proliferation, differentiation, and activity of osteoblasts, thereby significantly enhancing bone resorption activity and reducing bone formation. In addition, mature osteoclasts can secrete large amounts of protons (H2O) at the bone resorption interface. + This creates a localized acidic microenvironment (pH ~ 4.5), accelerating bone calcium loss and interfering with normal signaling between osteoblasts and osteoclasts, further weakening bone strength. This pathological microenvironment occurs repeatedly and alternately, forming a vicious cycle that ultimately exacerbates the progression of osteoporosis. Therefore, it is necessary to develop long-acting and stable medication strategies with bone-targeting properties that both regulate the bone immune microenvironment and coordinate the promotion of bone repair.

[0004] In recent years, the development and application of nanomaterials have brought new directions to the treatment of osteoporosis. Among them, black phosphorus (BP), as a novel two-dimensional nanomaterial, possesses good biocompatibility and excellent ROS scavenging ability. Furthermore, its degradation product, phosphate ions, is a key raw material for bone mineralization and can play a crucial role in bone regeneration, creating favorable conditions for improving the bone immune microenvironment and promoting bone regeneration. However, osteoporotic bone tissue is already "severely calcium deficient," and using BP alone is insufficient to exert an effective anti-osteoporosis effect. Therefore, organically combining BP with calcium delivery materials is a scientifically sound choice for osteoporosis treatment. Among these, amorphous calcium carbonate (ACC) possesses unique acid-responsive degradation characteristics and high calcium loading capacity, enabling rapid decomposition and release of calcium under acidic conditions. 2+BP is a highly promising calcium delivery carrier. Therefore, effectively combining BP and ACC can synergistically promote bone growth by regulating the deteriorating bone immune microenvironment, thereby achieving effective treatment for osteoporosis. However, both BP and ACC are easily and rapidly degraded in the physiological environment and lack bone-targeting properties, meaning that they are mostly or completely degraded before reaching bone tissue, making it difficult to exert a long-term therapeutic effect.

[0005] Sea cucumbers, as typical benthic organisms, can migrate directionally to eutrophic seabed areas by sensing specific chemical signals. They also possess a "hardening-in-the-tide" self-protection mechanism, enhancing their structural integrity and self-defense capabilities. Under extreme stimuli, they can rapidly release nutrient molecules through autolysis, creating a transient eutrophic microenvironment to promote the recovery of the surrounding ecosystem. Inspired by the biological behavior of sea cucumbers, this invention develops a bone-targeting nanotherapy platform (BA@PDA-PA) that is both pH- and ROS-responsive. Summary of the Invention

[0006] In view of this, the present invention provides a bone-targeting platform based on black phosphorus and amorphous calcium carbonate, its preparation method and application, and develops a bone-targeting and pH / ROS dual-responsive nanotherapy platform (BA@PDA-PA).

[0007] One objective of this invention is to provide a method for preparing a bone-targeting platform based on black phosphorus and amorphous calcium carbonate, the specific steps of which are as follows:

[0008] Step 1. Preparation of BP Synthesized by liquid-phase exfoliation: Bone apatite crystals were ground and dispersed in N-methylpyrrolidone. The mixture was intermittently sonicated in a cell disruptor under ice bath (0°C) conditions. The supernatant was then collected by centrifugation and centrifuged again to obtain BP. Step 2. Preparation of BA Preparation by gas diffusion method: CaCl2 powder was dissolved in anhydrous ethanol, then BP was added and dispersed, then deionized water was added and ultrasonically dispersed to obtain a mixed solution; the mixed solution was transferred to a beaker sealed with a Paramec membrane, the beaker was placed in a sealed container containing NH4HCO3, and the reaction was carried out at room temperature; finally, the product was collected by centrifugation, washed, freeze-dried and stored to obtain BA; Step 3. Preparation of BA@PDA BA was added to a Tris-HCl buffer solution (pH 8.5, 10 mM) containing dopamine hydrochloride, stirred and centrifuged at room temperature, the precipitate was washed with anhydrous ethanol and deionized water, and then freeze-dried to obtain BA@PDA; Step 4. Preparation of bone-targeting ligand PA Polyacrylic acid and sodium alendronate trihydrate were dissolved in borate buffer (0.1M, pH 8.5). After stirring for 10 min, borate buffer (0.1M, pH 8.5) containing 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride was added. The pH was adjusted to 7.5 with NaOH solution, and the reaction was stirred. Finally, the mixture was purified by dialysis and freeze-dried to obtain a white foamy bone-targeting ligand PA. Step 5. Preparation of BA@PDA-PA BA@PDA was prepared into an aqueous solution of 1 mg / mL; PA was prepared into an aqueous solution of 0.1 g / mL; while stirring, the PA solution was slowly added dropwise to the BA@PDA solution. After reacting overnight, the mixture was centrifuged, washed, and the precipitate was freeze-dried and stored at -20℃ to obtain the bone-targeting platform BA@PDA-PA.

[0009] Preferably, in step 1, the solid-liquid ratio of bone apatite to N-methylpyrrolidone is 1 mg: 1 mL.

[0010] Preferably, in step 1, the grinding is done manually, and the dispersion is as follows: the concentration of the bone apatite crystals in N-methylpyrrolidone is 0.1 g / 100 mL; The cell disruptor is designed for intermittent operation, with the operating conditions being 900W for 4 seconds on and 4 seconds off. The centrifugation was performed at 8000 r / min for 20 min; the second centrifugation was performed at 14000 r / min for 20 min.

[0011] Preferably, in step 2, the solid-liquid ratio of CaCl2 to anhydrous ethanol is 2 mg:1 mL, the mass ratio of CaCl2 to BP is 50 mg:1 mg, the solid-liquid ratio of CaCl2 to deionized water is 1 mg:1 μL, and the mass ratio of CaCl2 to NH4HCO3 is 40 mg:1 g.

[0012] Preferably, in step 2, the beaker sealed with the Paramec membrane has several pores for gas exchange, and gas exchange is carried out at room temperature and pressure (without using a special atmosphere); the reaction is carried out at room temperature for 48 hours, and the centrifugation is carried out at 10000 r / min for 10 min.

[0013] Preferably, in step 3, the concentration of the Tris-HCl buffer solution containing dopamine hydrochloride is 0.1 mol / L; and the mass ratio of BA to dopamine hydrochloride is 1 mg:1 mg.

[0014] Preferably, in step 3, the stirring time is 3 hours.

[0015] Preferably, in step 4, the mass ratio of polyacrylic acid to sodium alendronate trihydrate is 2g:65g; The solid-liquid ratio of the polyacrylic acid to the borate buffer is 0.002 g: 100 mL; The concentration of the borate buffer containing 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride is 0.1 g / mL; The mass ratio of the polyacrylic acid to 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride is 2g:55g; The concentration of the NaOH solution is 2M.

[0016] Preferably, in step 4, the dialysis purification conditions are dialysis at 2000 kDa in aqueous solution.

[0017] Preferably, the above freeze-drying is carried out under vacuum conditions at -80°C.

[0018] Preferably, in step 5, the volume ratio of the PA solution to the BA@PDA solution is 0.1 mL: 20 mL.

[0019] The second objective of this invention is to provide a bone-targeting platform.

[0020] The third objective of this invention is to provide an application of a bone-targeting platform in drug preparation.

[0021] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows: This invention addresses key issues in osteoporosis treatment, such as poor drug targeting, insufficient regulation of the pathological microenvironment, and the inability of traditional monotherapy to simultaneously address the imbalance between bone resorption and formation. Inspired by the biological behavior of sea cucumbers, a novel nano-therapeutic platform, BA@PDA-PA, with bone-targeting capabilities has been successfully designed and constructed for in vivo treatment of osteoporosis. The encapsulation of PDA allows BA to effectively "harden" in complex body fluid environments, achieving stable delivery of therapeutic factors. Modification with bone-targeting ligands endows the material with bone-targeting properties, enabling efficient delivery of ROS-responsive BP and pH-responsive ACC to bone tissue, thereby sequentially releasing Ca2+. 2+ And BP. By actively improving the bone immune microenvironment, they synergistically promote bone growth and ultimately exert an anti-osteoporosis effect.

[0022] This invention not only provides a new strategy for the treatment of osteoporosis based on dual-response bone-targeting nanocarriers, but its "biomimetic repair" design concept also provides new ideas for the development of next-generation intelligent bone repair materials, which has important theoretical reference value and translational potential for the field of bone regeneration medicine. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 Preparation and characterization of BA@PDA-PA materials. (A) TEM image of BP. (B) HAADF-STEM image of BP. (C) AFM image of BP. (D) Raman spectra of BP and bulk black phosphorus (BP). (E) SEM images of BP, BA, BA@PDA, and BA@PDA-PA. (F) TEM and EDS images of BA@PDA-PA. (G) FTIR spectra of BP, BA, BA@PDA, and BA@PDA-PA. (H) Zeta potentials of BP, BA, BA@PDA, and BA@PDA-PA. (I) TG spectra of BP, BA, BA@PDA, and BA@PDA-PA. (J) XPS spectrum of BA@PDA-PA. (K) High-resolution XPS spectra of P 2p, Ca 2p, N 1s, and Na 1s in BA@PDA-PA.

[0025] Figure 2 Acid responsiveness and targeting performance of BA@PDA-PA. (A) Ca released by BA@PDA-PA in PBS solutions at different pH values. 2+ Total amount. (B) BA@PDA-PA at different pH values ​​(7.5 and 4.5) for 72 h Ca 2+ Release curves. (C) Acidity neutralization capacity of BA@PDA-PA. The pH value increased significantly when NaOH (0.1 M) or BA@PDA-PA was added to PBS solution (pH 4.5 or 6.5). (D) pH changes when HCl (1%) was added to BA@PDA-PA, H2O and CaCO3 solutions. (E) SEM images of BA@PDA-PA under acidic (pH 4.5) conditions from 0 to 60 min. (F) Fluorescence microscopy images of BA@PDA-PA distribution on bone surface at different pH values. (G) Representative SEM images of BA@PDA-PA interacting with HAP surface at different time points. (H) Fluorescence intensity of the supernatant after stirring BA@PDA-PA in HAP solution for different durations. (I) In vivo fluorescence images of mice at different time points after injection of BA@PDA-PA.

[0026] Figure 3ROS scavenging capacity of BA@PDA-PA. (AC) Effects of different concentrations of BA@PDA-PA on ABTS. +· UV-Vis spectral analysis of the scavenging ability of DPPH· and ·OH free radicals (Illustration: ABTS after treatment with different concentrations of BA@PDA-PA) +· (D) Color changes of DPPH· and ·OH free radical solutions. (E, F) Scavenging rate of ·OH by BA@PDA-PA at different concentrations. - ESR spectra. (G,H) SOD and CAT-like activities of BA@PDA-PA at different concentrations. (IK) Effects of different concentrations of BP, BA, BA@PDA, and BA@PDA-PA on ABTS. +· Scavenging efficiency of DPPH· and ·OH. (L,M) SOD and CAT-like activities of different concentrations of BP, BA, BA@PDA, and BA@PDA-PA. (N) Fluorescence images of RAW264.7 cells stained with DCFH-DA after treatment with different concentrations of BA@PDA-PA. (O) Flow cytometry analysis of the ROS scavenging ability of different concentrations of BA@PDA-PA in RAW264.7 cells.

[0027] Figure 4 Immunomodulatory effects of BP, BA, BA@PDA, and BA@PDA-PA. (A) Schematic diagram of the inflammatory regulation mechanism of nanoparticles. (BE) ELISA results of IL-6, TNF-α, IL-10, and TGF-β in LPS-induced RAW264.7 cells after different treatments. (F) Flow cytometry analysis of CD206 and CD86 expression in RAW264.7 cells of different treatment groups. (G) Immunofluorescence micrographs of CD206 and CD86 in RAW264.7 cells of different treatment groups. (H) Quantitative analysis of CD86 fluorescence intensity during flow cytometry. (I) Quantitative analysis of CD206 fluorescence intensity during flow cytometry. (J) Quantitative analysis of CD86 in immunofluorescence images. (K) Quantitative analysis of CD206 in immunofluorescence images.

[0028] Figure 5 To illustrate the anti-inflammatory mechanism of BA@PDA-PA. (A) PCA results from sequencing of two groups of RAW264.7 cells. (B) Volcano plot of upregulated and downregulated genes in RAW264.7 cells. (C) Related heatmap. (D) GO analysis. (E) KEGG pathway enrichment analysis. (F) GSEA results.

[0029] Figure 6The role of BA@PDA-PA in promoting osteogenic formation and inhibiting osteoclastogenesis. (A) ALP staining of MC3T3-E1 cells. (B) ARS staining of MC3T3-E1 cells. (C) Quantitative results of ALP staining. (D) Quantitative results of ARS staining. (E) mRNA expression of OCN in MC3T3-E1 cells. (F) mRNA expression of RUNX2 in MC3T3-E1 cells. (G) TRAP staining images after different treatments. (H) Quantitative analysis of the number of TRAP-positive cells. (I) Schematic diagram of the effects of nanoparticles on promoting osteogenic formation and inhibiting osteoclastogenesis.

[0030] Figure 7 The therapeutic effect of BA@PDA-PA on an osteoporotic mouse model. (A) Schematic diagram of the in vivo experimental protocol. (B) Changes in body weight of mice in different treatment groups. (C) Micro-CT images of the distal femoral cancellous bone microstructure after treatment in each group. (DH) Quantitative analysis of Tb.Th, Tb.Sp, BV / TV, BS / TV, and Tb.N parameters in the distal femur after treatment in each group.

[0031] Figure 8 Histological staining of mouse femur. (A) HE staining of distal mouse femur. (B) Masson staining of distal mouse femur. (C) Quantitative results of the ratio of newly formed bone area to total bone area in Masson staining. (D) RUNX2 immunohistochemical staining. (E) Quantitative analysis of RUNX2 positive areas in immunohistochemical staining. (F) TRAP staining of distal mouse femur. (G) Quantitative results of the ratio of TRAP positive areas to total bone area in TRAP staining. (H) Immunofluorescence staining image of distal mouse femur. (I) Quantitative analysis of CD86 in immunofluorescence staining. (J) Immunofluorescence staining image of distal mouse femur. (K) Quantitative analysis of CD206 in immunofluorescence staining.

[0032] Figure 9 (A) AFM image of BP and (B) corresponding thickness distribution.

[0033] Figure 10 The UV-Vis absorption spectra are for different concentrations of BP.

[0034] Figure 11 The intensity ratio of BP in NMP after storage for different times under dark conditions.

[0035] Figure 12 The results of TEM image and element mapping analysis for (A)BA and (B)BA@PDA.

[0036] Figure 13 Here is the FTIR spectrum of PA.

[0037] Figure 14 (A) MC3T3-E1 cells co-cultured with different concentrations of BA@PDA-PA for 1, 2 and 3 days, (B) cell viability of MC3T3-E1 cells co-cultured with different materials for 1, 2 and 3 days, and (C) staining results of cells cultured with different concentrations of BA@PDA-PA.

[0038] Figure 15 (A) RAW264.7 cells co-cultured with different concentrations of BA@PDA-PA for 1, 2, and 3 days. (B) Cell viability of RAW264.7 cells co-cultured with different materials for 1, 2, and 3 days. (C) Live / dead staining of RAW264.7 cells treated with different materials.

[0039] Figure 16 (A) Staining results after treatment with different concentrations of BA@PDA-PA. (B) L929 cells co-cultured with different concentrations of BA@PDA-PA for 1, 2 and 3 days.

[0040] Figure 17 Hemolysis rate and optical images of different concentrations of BA@PDA-PA.

[0041] Figure 18 SEM images of BA@PDA-PA reacting under weakly acidic conditions (pH 4.5) for 0-60 minutes.

[0042] Figure 19 SEM images of BA@PDA-PA after treatment with different pH values.

[0043] Figure 20 (A) Representative SEM images of HAP targeted by BA@PDA-PA at different time intervals. (B) Representative SEM images of HAP targeted by BA@PDA at different time intervals.

[0044] Figure 21 ABTS for (A)BA@PDA-PA at different concentrations +· The scavenging efficiency. (B) DPPH scavenging efficiency of BA@PDA-PA at different concentrations.

[0045] Figure 22 Quantitative analysis results of ROS removal by flow cytometry.

[0046] Figure 23 The mRNA expression levels of M1 pro-inflammatory cytokine (TNF-α) and M2 anti-inflammatory cytokine (TGF-β) in RAW264.7 cells treated with different materials are shown in (A).

[0047] Figure 24H&E staining of the heart, liver, spleen, lungs, and kidneys of mice in each group.

[0048] Figure 25 Serum ALT, ALP, and AST levels in mice from different groups.

[0049] Figure 26 Microscopic CT images of the trabecular bone structure of each group of lumbar vertebrae.

[0050] Figure 27 Quantitative analysis of lumbar spine parameter Tb in patients with acute lumbar spine injury (AE). BV / TV and BS / TV in each group after treatment with Tb.Th, Tb.Sp, and Tb.N.

[0051] Figure 28 Histological staining of mouse vertebral bodies. (A) HE staining of the third vertebral body of a mouse. (B) Masson staining of the mouse vertebral body. (C) TRAP staining of the mouse vertebral body. (D) Quantitative results of the ratio of new bone area to total bone area in Masson staining. (E) Quantitative results of TRAP-positive cells in TRAP staining.

[0052] The data are expressed as mean ± standard deviation (n≥3), where nsp>0.05. p<0.05, p<0.01, p < 0.001. Green fluorescence represents live cells, and red fluorescence represents dead cells. Detailed Implementation

[0053] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] The core pathological mechanism of osteoporosis (OP) lies in the imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, accompanied by immune microenvironment dysregulation caused by the accumulation of reactive oxygen species (ROS) and inflammatory factors in bone tissue, ultimately significantly increasing the risk of fracture. This invention designs and constructs a bone-targeting nanocomposite material (BA@PDA-PA) based on black phosphorus (BP) and amorphous calcium carbonate (ACC), enabling precise targeted therapy of diseased bone tissue, thereby improving drug utilization. Upon entering the target area, BA@PDA-PA can release BP and calcium ions (Ca) on demand in response to the acidic microenvironment of OP. 2+), among which BP can efficiently remove excess ROS locally and improve the immune microenvironment; ACC degrades Ca 2+ The non-toxic phosphate generated from BP degradation synergistically promotes osteoblast proliferation and differentiation and inhibits osteoclast activity, forming a synergistic therapeutic mechanism of "targeted delivery - microenvironment regulation - bone metabolic remodeling". In vivo experimental results show that the material of this invention can significantly improve the bone microstructure of OP mice, providing a new approach for OP treatment.

[0055] This invention forms the foundation (BA) of a nanotherapy platform by encapsulating ACC on the surface of BP, and then coating it with a polydopamine (PDA) coating to effectively "harden" it in complex body fluid environments, ensuring stable delivery of therapeutic components. A bone-targeting ligand (PAA-Ald, PA) is connected to effectively improve the bone targeting of BA. After precise enrichment into bone tissue, BA@PDA-PA can actively respond to the existing acidic microenvironment and undergo "autolysis," rapidly releasing BP and Ca. 2+ BP can improve the bone immune microenvironment by clearing ROS from the bone microenvironment, thereby effectively improving the inflammatory bone microenvironment. Furthermore, the phosphorus and calcium released by this nanocarrier provide "raw materials" for bone matrix mineralization, synergistically promoting osteoporosis and inhibiting osteoclast activity, ultimately achieving precise and multi-effect treatment of osteoporosis. This "biomimetic repair" design concept holds promise for providing new insights into multi-mechanism combined treatment of osteoporosis.

[0056] The materials used in this invention are sourced from the following sources: BP was purchased from Jiangsu Pioneer Nanotechnology Co., Ltd. (Jiangsu, China). Calcium chloride, ammonium bicarbonate, sodium hydroxide, dexamethasone (≥98%), ascorbic acid (≥98%), sodium β-glycerophosphate, and hydroxyapatite (HAP) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Dopamine hydrochloride (DA·HCl), sodium alendronate trihydrate (Ald), polyacrylic acid (PAA, molecular weight = 2,000), N-methylpyrrolidone (NMP), fluorescein 5-isothiocyanate (FITC), 2,2'-azidobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), 2,2-diphenyl-1-trinitrohydrazine (DPPH), and 3,3',5,5'-tetramethylbenzidine (TMB) were purchased from Maclean Biotechnology Co., Ltd. (Shanghai, China). Anhydrous ethanol was purchased from Xilong Technology Co., Ltd. (China). Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich. Hoechst 33342, Cell Count Kit-8 (CCK-8), 2',7'-dichlorofluorescein diacetate (DCFH-DA), Calcein-AM, propidium iodide (PI), 4,6-diamidinyl-2-phenylindole (DAPI), and alkaline phosphatase (ALP) staining kits were purchased from Beyotime Biotechnology Co., Ltd. (Jiangsu, China). Alizarin Red (ARS) staining kits and tartrate acid phosphatase (TRAP) staining kits were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Dulbecco modified Eagle medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Thermo Fisher Scientific (USA). 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) was purchased from J&K Scientific.

[0057] Example 1: Preparation method of bone targeting platform based on black phosphorus and amorphous calcium carbonate Step 1. Preparation of BP BP was synthesized via liquid-phase exfoliation. 20 mg of blocky BP crystals were ground and dispersed in 20 mL of NMP. The mixture was placed in an ice bath and continuously sonicated in a cell disruptor (4 s on, 4 s off, 900 W) for 16 h. The mixture was then centrifuged (8000 rpm, 20 min) to remove unexfoliated particles, and the supernatant was collected and centrifuged again (14000 rpm, 20 min) to obtain BP.

[0058] Step 2. Preparation of BA BA was prepared by gas diffusion. 200 mg of CaCl2 powder was dissolved in 100 mL of anhydrous ethanol, and then the collected BP was dispersed in it (CaCl2 to BP mass ratio 50:1). 200 μL of deionized water was added, and the mixture was ultrasonically dispersed until homogeneous. The solution was poured into a beaker and sealed with a sealing film (leaving several pores for gas exchange). The beaker was placed in a sealed container containing 5 g of NH4HCO3, and the solution was allowed to react at room temperature for 48 h. The product was collected by centrifugation (10000 rpm, 10 min), washed three times with anhydrous ethanol, and then freeze-dried for storage.

[0059] Step 3. Preparation of BA@PDA Add 10 mg BA to a Tris-HCl buffer solution (pH 8.5, 10 mM) containing 10 mg DA·HCl, stir for 3 h at room temperature, collect the solution, centrifuge at 10000 rpm for 10 min, wash twice with anhydrous ethanol and deionized water, collect the precipitate, and then freeze-dry to obtain BA@PDA.

[0060] Step 4. Preparation of bone-targeting ligand PA 0.002 g PAA and 0.065 g Ald were dissolved in 100 mL borate buffer (0.1 M, pH 8.5). After stirring for 10 min, borate buffer (0.1 M, pH 8.5) containing 0.055 g DMTMM was added, with a DMTMM concentration of 0.1 g / mL in the borate buffer. The final pH was adjusted to 7.5 with 2 M NaOH. The reaction was carried out with stirring for 24 h. The product was purified by dialysis (2000 kDa, aqueous solution) and lyophilized to obtain a white, foamy bone-targeting ligand PAA-Ald, i.e., PA.

[0061] Step 5. Preparation of BA@PDA-PA The obtained BA@PDA was prepared into a 1 mg / mL aqueous solution, and then 1 mL of PA aqueous solution (0.1 g / mL) was slowly added dropwise while stirring. The volume ratio of PA solution to BA@PDA solution was 0.1 mL:20 mL. After reacting overnight, the mixture was centrifuged (11,000 rpm, 15 min). The product was washed twice with anhydrous ethanol and deionized water. The precipitate was freeze-dried and stored at -20°C.

[0062] 1.1. pH responsiveness of BA@PDA-PA 5 mg of BA@PDA-PA was mixed with 10 mL of phosphate-buffered saline (PBS) solutions at different pH values ​​(pH 4.5, pH 5.5, pH 6.5, pH 7.5) and stirred for 1 h. The solutions from each group were collected and dropped onto a silicon wafer for sample preparation. The morphological changes of BA@PDA-PA were further observed using SEM. In addition, calcium ions (Ca) were detected using ICP-AES. 2+ The content of ) was also determined. Similarly, the morphological changes of BA@PDA-PA and the Ca content were observed by SEM at different time points after collecting the solution. 2+ Release amount. See also Figure 2 A, B, E.

[0063] To demonstrate the acid-neutralizing ability of this material, PBS solution (pH 4.5 or 6.5) was titrated with BA@PDA-PA (1 mg / mL) or NaOH (0.1 M) solution (10 μL each time) under stirring. The pH was continuously monitored using a pH meter (PHB-1, SANXIN, China) after each titration. Additionally, BA@PDA-PA (1 mg / mL), deionized water, and CaCO3 (1 mg / mL) solutions were titrated with 1% (v / v) HCl (10 μL each time), and the resulting pH changes were recorded. See also Figure 2 C, D.

[0064] 1.2. BA@PDA-PA in vitro bone fragment co-culture experiment To simulate the pH response of the material in vivo, BA@PDA-PA and fresh bone sections were co-cultured in vitro for 7 days in cell culture containing fetal bovine serum (FBS) (10% FBS in DMEM complete medium). After sterilization (75% ethanol, 2 h) and freeze-drying, the bone sections were immersed in FITC-labeled material, designated BA@PDA-PA-FITC, and incubated at 37°C in the dark for 12 h. The samples were then washed three times with physiological saline solution. The treated bone sections were then placed in 96-well plates containing DMEM complete medium, with the solution changed every 2 days. During this period, the FITC fluorescence signal of BA@PDA-PA was observed using an inverted fluorescence microscope, and images were captured on days 1, 3, and 7. On day 7, H2-rich... + Prepare a buffer solution (pH 4.5) and immediately record sequential images to observe fluorescence changes. See also Figure 2 F.

[0065] 1.3. In vitro and in vivo bone targeting capability assessment of BA@PDA-PA To evaluate the targeting ability of the nanoparticles, 1 mg / mL of BA@PDA or BA@PDA-PA was mixed with 10 mg of HAP in 10 mL of deionized water and stirred for different times. Subsequently, the supernatant and precipitate were collected at different time points by centrifugation. The supernatant was used for fluorescence spectroscopy measurements, while the precipitate was washed three times with deionized water, dried, and characterized by SEM. For in vivo bone targeting ability testing, FITC-labeled BA@PDA-PA (1 mg / mL) saline solution was intraperitoneally injected into 10-week-old female C57BL / 6 mice. After different time intervals, the mice were euthanized and their skeletons were fully exposed for imaging (IVIS, PerkinElmer). All experimental procedures were performed in accordance with institutional animal care guidelines and were approved by the Animal Ethics Committee of Nanchang University (NCULAE-20221228021, Nanchang, China).

[0066] See Figure 2 G, H, I and Figure 18 , 19 20.

[0067] 1.4. ABTS free radical (ABTS) +· ) Cleanup test ABTS +· The scavenging activity was assessed based on the method reported by Liu et al. A 7 mM ABTS solution was reacted with 2.45 mM potassium persulfate overnight in the dark. Then, it was diluted with deionized water to achieve a suitable absorbance at 734 nm. 1 mL of BA@PDA-PA solutions at different concentrations (0, 5, 10, 20, 50, and 100 μg / mL) were mixed with 1 mL of diluted ABTS. +· The solution was allowed to react in the dark for 10 min. The color change of the solution was observed, and the absorbance at 734 nm was measured using a UV-Vis spectrophotometer. (ABTS) +· The clearance rate is calculated as follows:

[0068] Where A0 represents pure ABTS +· The absorbance of the solution, A1 represents ABTS treated with BA@PDA-PA. +· Absorbance of the solution. See also Figure 3 A, I and Figure 21 A.

[0069] 1.5. DPPH radical (DPPH·) scavenging test First, prepare a 0.2 mM DPPH·anhydrous ethanol solution. Mix 100 μL of BA@PDA-PA solutions of different concentrations (0, 5, 10, 20, 50, and 100 μg / mL) with 100 μL of the DPPH· solution and react in the dark for 30 min. Then, observe the color change of the solution and collect its absorbance at 517 nm using a UV spectrophotometer. The DPPH· scavenging rate is calculated using the following formula:

[0070] Where A0 represents the absorbance of the DPPH solution without sample, and A1 represents the absorbance of the DPPH solutions treated with different samples. See also Figure 3 B, J and Figure 21 B.

[0071] 1.6. ·OH scavenging activity of BA@PDA-PA The TMB colorimetric method was used to test the ·OH scavenging activity. ·OH scavenges through the reaction of H2O2 with Fe. 2+ The TMB is generated by the classic Fenton reaction, which converts TMB to its oxidized form (oxTMB), which has a characteristic absorption peak at 652 nm. BA@PDA-PA solutions containing 250 mM TMB, 2 mM H₂O₂, 1 mM MFeSO₄, and different concentrations (0, 5, 10, 20, 50, and 100 μg / mL) were prepared in a sodium acetate-acetic acid (NaAc-HAc) buffer solution (0.5 M, pH 4.5) and incubated in the dark for 5 min. The absorption peak at 652 nm was detected by UV-Vis spectroscopy.

[0072] See Figure 3 C, D, E, F, K, G, H, L, M.

[0073] 1.7. Detection of Intracellular Reactive Oxygen Species (ROS) Scavenging Mouse mononuclear macrophages (RAW264.7) were divided into groups of 2 × 10⁶ cells per well. 4 Cells were seeded per well in 24-well plates and incubated for 24 hours. They were then co-cultured for 24 hours in DMEM medium containing LPS (200 ng / mL) and different concentrations of samples. Finally, the cells were incubated for 30 minutes in medium containing DCFH-DA and Hoechst 33342, washed twice with PBS, and the production of intracellular reactive oxygen species (ROS) was monitored under an inverted fluorescence microscope. See also Figure 3 N, O and Figure 22 .

[0074] 1.8. Cell viability The cytotoxicity of BA@PDA-PA nanoparticles was assessed using a CCK-8 assay kit. RAW264.7 cells, mouse fibroblasts (L929), and mouse embryonic pre-osteoblasts (MC3T3-E1) were seeded in 96-well plates at a cell density of 5 × 10⁶ cells per well. 3 Cells were exposed to different concentrations of BA@PDA-PA nanoparticles for 1, 2, and 3 days. After treatment, DMEM medium containing 10% (v / v) CCK-8 solution was added to each well, and the cells were incubated at 37°C for 1 h. Finally, the absorbance at 450 nm was measured. See [link to other instructions] Figure 14 A, B; Figure 15 A, B; Figure 16 A.

[0075] 1.9. Live / Dead Cell Staining The three types of cells were spaced at 3 × 10⁻⁶ cells per well. 4 Cells were seeded in 24-well plates and incubated for 24 h. The existing medium was then replaced with DMEM complete medium containing different concentrations of the sample. Co-incubation was continued for 24 h. After incubation, live / dead cell staining was performed using a live / dead cell staining kit. Calcein-AM was used for live cell staining, and PI was used for dead cell staining; incubation was 30 min. Cells were then washed twice with PBS and observed using an inverted fluorescence microscope. See [link to relevant documentation]. Figure 14 C Figure 15 C Figure 16 B.

[0076] 1.10. Hemolysis test Blood was extracted from mice and centrifuged at 1500 rpm for 15 min. Red blood cells were diluted with physiological saline. 100 μL of the diluted suspension was then mixed with 1.1 mL of saline solution and deionized water, respectively, as negative and positive controls. Additionally, equal volumes of red blood cell suspension were mixed with 1.1 mL of BA@PDA-PA solutions of different concentrations as experimental groups and incubated in a 37 ℃ water bath for 1 h. After incubation, the supernatant was collected, and the absorbance at 540 nm was measured using a microplate reader. The hemolysis rate was calculated using the following formula:

[0077] Where A1 represents the absorbance of the experimental group, A2 represents the absorbance of the negative control group, and A3 represents the absorbance of the positive control group. See also Figure 16 .

[0078] 1.11. RAW264.7 Cell Polarization and Inflammatory Gene Expression RAW264.7 cells were planted at a density of 1 × 10⁶ cells per well. 6RAW264.7 cells were seeded at a density of [number] cells per well in 6-well plates and cultured for 24 h. Simultaneously, the cells were treated with various samples and LPS (200 ng / mL) for 24 h. Flow cytometry was used to assess the polarization status of RAW264.7 cells. Cells were labeled with fluorescently labeled CD86 antibody (Servicebio, China) for M1 and CD206 antibody (Servicebio, China) for M2, and stained at room temperature for 1 h. Cell nuclei were counterstained with 4',6-diamidinyl-2-phenylindole (DAPI). Finally, images were collected using an inverted fluorescence microscope. Total RNA was extracted from the cells using Trizol reagent (Beyotime, China), and cDNA was synthesized from the isolated RNA by reverse transcription. qRT-PCR was used to detect the expression levels of relevant inflammatory genes. ELISA was used to detect the protein expression levels of relevant inflammatory factors. See [link to relevant information]. Figure 3 and Figure 4 all, Figure 23 .

[0079] 1.12. Osteogenic Activity Assessment MC3T3-E1 cells were spaced at 5 × 10⁻⁶ cells per well. 3 Cells were seeded at a density of 100 μg / mL in 6-well plates and cultured for 14 or 21 days in complete medium supplemented with 50 μg / mL ascorbic acid, 10 mM β-glycerophosphate sodium, and 100 nM dexamethasone. After 14 days of co-culture, MC3T3-E1 cells were fixed with 4% (w / v) paraformaldehyde for 10 min, washed three times with PBS, and ALP activity was assessed using an ALP (alcoholic acid) assay kit. After 21 days of co-culture, cells were stained with ARS staining solution. Images of the stained samples were captured under a microscope, and semi-quantitative analysis was performed using ImageJ software to analyze ALP and ARS-positive regions. MC3T3-E1 cells were cultured for 14 days using the same method, and the expression of osteogenic-related genes was detected by qRT-PCR.

[0080] See Figure 5 A, B, C, D, E, F.

[0081] 1.13. Osteoclast formation assay RAW264.7 cells were planted at 3 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of [number] cells / well in 48-well plates, induced with 50 ng / mL RANKL, and co-incubated with different nanomaterials for 5 days. TRAP staining was performed using a TRAP staining kit, and TRAP-positive osteoclasts (with wine-red cytoplasm) were observed under an optical microscope.

[0082] See Figure 6 G, 6H, Figure 6 I.

[0083] 1.14. Animal Experiments All experiments were conducted in accordance with relevant guidelines and approved by the Animal Ethics Committee of Nanchang University (NCULAE-20221228021). Ovariectomy (OVX) was performed on 10-week-old female C57 mice using a standard aseptic surgical procedure. Simultaneously, five mice from the same batch underwent only adipose tissue removal, leaving the ovaries intact as the sham surgery group. Four weeks post-surgery, mice were randomly assigned to six groups: sham surgery group, ovariectomy group, ovariectomy + BP group, ovariectomy + BA group, ovariectomy + BA@PDA group, and ovariectomy + BA@PDA-PA group, with five mice in each group. Equal volumes of nanomaterials or saline were injected intraperitoneally every two days for four weeks. Animal weight was recorded weekly for eight weeks. Mice were euthanized at week 8, and bone correlation analysis was performed using micro-computed tomography (micro-CT).

[0084] 1.15. Micro-CT Scan After euthanasia, bone tissue (lumbar vertebrae and femur) of mice was collected and fixed with 10% (v / v) neutral buffered formalin. Micro-CT scans were performed to examine the microstructure of the vertebral bodies and left femoral trabeculae, and the acquired images were reconstructed using software. By analyzing the target regions, bone structural parameters such as the bone surface to tissue volume ratio (BS / TV), bone volume to tissue volume ratio (BV / TV), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), and trabecular number (Tb.N) were determined.

[0085] See 1.14 and 1.15 Figure 7 , Figure 26 and 27 .

[0086] 1.16. Histological staining Lumbar vertebrae and femurs were fixed in 10% (v / v) neutral buffered formalin for at least 48 h, followed by decalcification in 10% EDTA for one week. Specimens were embedded in paraffin and sectioned at 5 μm. Hematoxylin-eosin (HE) staining was used to observe morphological changes in tissues and trabecular regions. Masson staining was performed to assess new bone formation, while TRAP staining was used to quantify the number of TRAP-positive osteoclasts in specific regions. RUNX2 immunohistochemical staining was used to observe the expression of osteogenic markers within the tissues. Immunofluorescence staining was used to determine the number of CD86 and CD206-positive cells in selected regions. Semi-quantitative analysis was performed using ImageJ software.

[0087] See Figure 8 , Figure 24 , Figure 25 , Figure 28 .

[0088] 1.17. Statistical Analysis Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA, and t-tests were conducted using GraphPad Prism 9.0. A p-value less than 0.05 was considered statistically significant. p<0.05 p<0.01 p<0.001 p < 0.0001; when the p value is greater than 0.05, the difference is considered not statistically significant, expressed as p < 0.0001. ns p>0.05.

[0089] 2. Results and Discussion 2.1. Preparation and Characterization of BA@PDA-PA First, BP was obtained using a modified liquid-phase exfoliation method. The morphology of the BP was characterized by TEM and SEM. Figure 1 A and Figure 1 As shown in E1, BP particles are spherical with a diameter of approximately 7-10 nm. High-resolution TEM (HRTEM) images show a lattice spacing of 0.25 nm. Figure 1 B), corresponding to its (111) crystal plane, indicates that its internal crystal structure has not been significantly damaged. AFM further clarifies the morphology and height distribution of BP, with a thickness of approximately 1-3 nm. Figure 1 C and Figure 9 BP was also characterized using Raman spectroscopy. For example... Figure 1 As shown in Figure D, the three prominent peaks can be attributed to 364.24 cm⁻¹. -1 An out-of-plane phonon mode (A1) g ) and respectively at 440.95cm -1 and 468.58 cm -1 Two in-plane modes B 2g and A2 g Compared to body-sized BP, BP's A1 g B 2g and A2 g The models redshifted by approximately 2.8, 2.72, and 2.87 cm, respectively. -1 This demonstrates that the BP thickness is significantly reduced. UV-Vis absorption spectroscopy was used to test different concentrations of BP. Figure 10The spectra showed that, similar to other two-dimensional nanomaterials, BP exhibited broad absorption bands in the ultraviolet and near-infrared regions. Subsequently, to assess the stability of BP, it was dispersed in NMP organic solvent and stored in the dark at 4 °C for 7 days. Figure 11 As shown, it exhibits stable absorbance, indicating that NMP can effectively protect BP from the effects of oxygen and water, thereby increasing its stability.

[0090] Next, BP was combined with amorphous calcium carbonate (ACC) using an improved gas-phase diffusion method to obtain BP@ACC(BA), which was found to be uniformly spherical. Figure 1 E2). Characterization of BA using TEM and energy-dispersive X-ray spectroscopy (EDS) further confirmed the uniform presence of calcium (Ca) and phosphorus (P) elements in BA. Figure 12 A) proved that BP and ACC successfully combined. Subsequently, BA was coated with PDA to form BA@PDA. SEM images showed that the particle size of BA@PDA was increased compared to BA, and the adhesion was significantly increased, with more nanoparticles agglomerating. Figure 1 E3). TEM revealed that BA@PDA possesses a core-shell structure, and EDS further indicated that BA@PDA contains calcium (Ca), phosphorus (P), and nitrogen (N) elements. Figure 12 B). To endow BA@PDA with bone-targeting capabilities, PAA was covalently modified onto Ald to form the bone-targeting ligand PAA-Ald (PA), in which Ald specifically binds to HAP in the bone matrix. FTIR of PAA-Ald was observed at 1705 cm⁻¹. -1 There is a characteristic peak at 1185 cm⁻¹, which is attributed to the C=O stretching vibration of the carbonyl group in PAA; -1 The characteristic peak observed at this point corresponds to the P=O stretching vibration in Ald ( Figure 13 This demonstrates the successful synthesis of PAA-Ald. PA with carboxylic acid groups was anchored to the surface of BA@PDA via an amidation reaction, yielding the final material BA@PDA-PA. Figure 1 E4 and 1F). Then, BP, BA, BA@PDA, and BA@PDA-PA were characterized by FTIR. Figure 1 G). The results showed that the characteristic peak of P=O was observed in the pure BP sample, which may be due to a certain degree of oxidation on the BP surface. When ACC was combined with BP, the characteristic peak of C=O appeared. After further coating with PDA, the characteristic peak was observed at approximately 3400 cm⁻¹. -1 A broad absorption band is observed at 1200 cm⁻¹, belonging to the stretching vibration mode of the NH bond. When PA is combined, a band appears at 1200 cm⁻¹. -1 The presence of characteristic peaks for P=O bonds on both sides confirms the successful binding of PA. Next, Zeta potentials were performed on BP, BA, BA@PDA, and BA@PDA-PA, respectively. Figure 1 The results showed that BP has a negative charge because its surface forms a large number of hydroxyl (-OH) functional groups (i.e., P-OH groups) due to the chemical activity of phosphorus atoms, which carry a negative charge after partial dissociation. After binding with ACC, the Zeta potential turns positive, and after further coating with PDA, the potential reverses to negative, because the exposed -OH and catechol structures on its surface become negatively charged after deprotonation. After binding with PA, the negative charge of BA@PDA-PA is enhanced because PAA contains many negatively charged carboxyl groups that neutralize the positively charged amino groups in the PDA coating. Thermogravimetric analysis (TGA) of BP, BA, BA@PDA, and BA@PDA-PA revealed that pure BP begins to decompose at around 400 °C. Weight reductions of approximately 16.82 wt% and 3.8 wt% were measured for BA@PDA and BA@PDA-PA, respectively, indicating that the percentage of PDA in BA@PDA is approximately 16.82 wt% and the percentage of PA in BA@PDA-PA is approximately 3.8 wt%. Figure 1 I). XPS analysis of BA@PDA-PA then confirmed the presence of P, Ca, N, and Na elements. Figure 1 J). The peak at 132.48 eV in the high-resolution XPS spectrum belongs to the P 2p state; the two peaks at 348.94 eV and 352.47 eV correspond to the binding energies of Ca 2p3 / 2 and Ca 2p1 / 2, respectively, indicating that Ca exists in the +2 valence state; the peaks at 399.72 eV and 1071.40 eV belong to the N 1s and Na 1s states, respectively. Figure 1 K).

[0091] 2.2. In vitro cell compatibility evaluation Good biocompatibility of nanomaterials is a prerequisite for their in vivo application. Therefore, osteogenic MC3T3-E1 cells and RAW264.7 cells were first selected to evaluate the in vitro biocompatibility of BA@PDA-PA. Through CCK-8 assay and live / dead cell staining, it was determined that the viability of both cell types was greater than 80% at BA@PDA-PA concentrations below 100 μg / mL. No obvious red fluorescence staining was observed in the live / dead cell staining, and cell morphology and number were not significantly affected, indicating no significant cell death. This demonstrates that BA@PDA-PA has good biocompatibility. Figure 14 A and 14C, Figure 15 A and 15C). CCK-8 assays of different components of the material also showed no significant toxicity to cells. Figure 14 B and 15B). Additionally, L929 cells were selected as a representative of normal cells. The results showed that L929 cells exhibited good cell viability (B and 15B). Figure 16(A and 16B). Simultaneously, the blood compatibility of BA@PDA-PA was investigated using an in vitro hemolysis experiment. For example... Figure 17 As shown, erythrocytes treated with BA@PDA-PA showed no obvious hemolytic trend, and the hemolysis rate of different concentrations of BA@PDA-PA was less than 5%. In summary, this nanomaterial has good biocompatibility.

[0092] 2.3. pH responsiveness of BA@PDA-PA Osteoporosis is often associated with an imbalance in bone homeostasis. As the primary source of bone resorption, mature osteoclasts adhere to the bone surface and secrete large amounts of H+ ions to acidify the local extracellular microenvironment (pH ~ 4.5), leading to bone mineralization. Therefore, releasing key therapeutic ions in response to the excessively acidic microenvironment during the osteoporosis process can enhance the therapeutic effect of materials and regulate the microenvironment. To examine the pH-responsive performance of BA@PDA-PA, it was dispersed in PBS at different pH values, and the Ca2+ concentration in the ACC was measured. 2+ The release of Ca was detected by ICP emission spectroscopy. It was found that at pH 4.5, the released Ca... 2 + The largest quantity ( Figure 2 A). Additionally, the changes in Ca2+ over time during the degradation of BA@PDA-PA at different acidities were monitored. 2+ Release amount. For example... Figure 2 As shown in Figure B, in an acidic microenvironment (pH 4.5), Ca... 2+ Release was quite rapid within 12 h, followed by sustained release, but the release rate slowed significantly at pH 7.5. Its acid-neutralizing ability was then further verified by adding BA@PDA-PA to acidic PBS solution. Figure 2 As shown in Figure C, the pH value of the solution increased significantly after the addition of BA@PDA-PA, indicating its good acidity reversal performance. Furthermore, titration with a 1% HCl solution revealed that, compared to pure H₂O and CaCO₃, the BA@PDA-PA solution significantly prevented acidification. Figure 2 D). Meanwhile, representative SEM images of the reaction at different times under acidic conditions (pH 4.5) show that the spherical morphology gradually collapses with increasing time. Figure 2 E and Figure 18 Under the same reaction time, BA@PDA-PA remained intact in a neutral environment (pH 7.5), but its morphology gradually deteriorated as the pH decreased. Figure 19This demonstrated that BA@PDA-PA exhibits good pH response. Next, to simulate the stability and rapid pH response of BA@PDA-PA in vivo, FITC-labeled BA@PDA-PA (BA@PDA-PA-FITC) was co-cultured with fresh bone slices in serum-containing cell culture medium (DMEM complete medium containing 10% FBS) for 7 days in vitro. Figure 2 F). Fluorescence imaging results showed that after 7 days of culture, the nanoparticles were mainly distributed on the surface of the bone slices. However, when the pH value dropped to 4.5, the FITC fluorescence gradually disappeared, indicating that BA@PDA-PA exhibited good pH response and could trigger Ca2+ in an acidic microenvironment. 2+ With the release of BP, excess H is neutralized. + This improves the acidic microenvironment of OP, providing a foundation for subsequent treatment.

[0093] 2.4. Evaluation of bone targeting of BA@PDA-PA Osteoporosis typically manifests as a decrease in bone mass throughout the skeletal system. Targeting bone tissue is crucial for ensuring the precise and efficient delivery of materials into bone tissue in vivo. Therefore, the in vivo and in vitro bone tissue targeting capabilities of BA@PDA-PA were evaluated. The results indicate that the phosphonate groups in Ald can selectively chelate calcium in bone tissue. 2+ This demonstrates bone affinity. HAP is the most abundant mineral component of bone tissue, containing a large amount of calcium. Given the widespread distribution of HAP in bone tissue, HAP was chosen to simulate the in vivo bone microenvironment for in vitro targeting experiments. First, a certain amount of BA@PDA-PA was stirred with HAP for different times, followed by centrifugation to collect the precipitate and supernatant. SEM images were used to observe the adsorption of BA@PDA-PA on the HAP surface at different time points. With increasing time, the number of BA@PDA-PA particles bound to the HAP surface gradually increased (…). Figure 2 G and Figure 20 A). However, unmodified PA BA@PDA particles showed almost no adsorption on the HAP surface (A). Figure 20 B). BP exhibits certain photoluminescence properties, with maximum excitation and emission wavelengths of 370 nm and 447 nm, respectively. Therefore, by measuring the fluorescence intensity in the supernatant, if the substance successfully targets the HAP surface, the fluorescence intensity in the supernatant will decrease accordingly. Figure 2 As shown in Figure H, after stirring for 24 h, the fluorescence intensity of the supernatant decreased significantly and in a time-dependent manner. Subsequently, the in vivo bone-targeting performance of BA@PDA-PA was verified. The distribution of BA@PDA-PA in vivo at different time points was observed using an in vivo imaging system (IVIS). Fluorescence images showed that the fluorescence intensity in mouse bones initially increased and then decreased over time, demonstrating that BA@PDA-PA can effectively target bone tissue. Figure 2 I). The above results indicate that BA@PDA-PA has good bone-targeting properties, which is of great significance for subsequent in vivo treatment of osteoporosis and can effectively reduce the side effects of systemic administration.

[0094] 2.5. ROS Scavenging Ability Evaluation Excessive ROS accumulation can promote osteoclastogenesis, thereby impairing bone healing and is a significant factor contributing to osteoporosis. Therefore, effectively removing ROS from the bone microenvironment is crucial. In vitro ABTS was first performed. +· Scavenging experiments with DPPH· showed that ABTS can be oxidized to stable ABTS. +· The solution exhibits a bright blue-green color and strong absorption at 734 nm. UV-Vis curves show that the absorbance gradually decreases and the solution color gradually fades with increasing BA@PDA-PA concentration. Quantitative results indicate that when the BA@PDA-PA concentration is 100 μg / mL, the scavenging rate can reach 75%. Figure 3 A and 21A). Similarly, DPPH· is purple in aqueous solution, but turns pale yellow when it scavenge free radicals and forms covalent bonds, exhibiting a characteristic absorption peak at 517 nm. The absorbance gradually decreases with increasing solution concentration, and a concentration of 100 μg / mL effectively scavenge over 70% of free radicals (A and 21A). Figure 3 B and Figure 21 B). ·OH is the most toxic and harmful ROS to biological systems, so the TMB method was first used to test the ·OH scavenging ability of BA@PDA-PA, where ·OH is scavenged by H2O2 and Fe. 2+ It is produced by the classic Fenton reaction between TMB and oxTMB, which can convert TMB to oxTMB and has a characteristic absorption at 652 nm. Figure 3 C and 3D results show that BA@PDA-PA can effectively remove ·OH. Electron spin resonance (ESR) spectroscopy further confirmed its ·OH removal ability, showing a multi-peak pattern of 1:2:2:1. The signal of the ·OH peak was significantly reduced after the addition of BA@PDA-PA. Figure 3 E). Similarly, ·O2 - The ESR signal was also reduced after adding BA@PDA-PA. Figure 3 F). In addition, the superoxide dismutase (SOD) and catalase (CAT)-like activities of the material were detected. For example... Figure 3The results of G and 3H show that BA@PDA-PA achieved a 60% SOD-like inhibition rate and approximately 45 U / mL CAT-like activity at a concentration of 100 μg / mL. These results indicate that BA@PDA-PA possesses good ROS scavenging ability. Subsequently, ROS scavenging was validated on different groups of materials, and the results showed that they all exhibited good ROS scavenging ability, with BP playing a major role in this process. Figure 3 IM).

[0095] The ROS scavenging ability of the materials was further investigated at the cellular level using the DCFH-DA fluorescent probe, which reacts with intracellular ROS to generate 2',7'-dichlorofluorescein (DCF), emitting green fluorescence. ROS production in RAW264.7 cells was induced using LPS as a pro-inflammatory stimulant. Figure 3 As shown in Figure N, the control group exhibited strong green fluorescence, while the green fluorescence significantly decreased with increasing BA@PDA-PA concentration, indicating a sharp decline in intracellular ROS levels. This trend was further confirmed by quantitative analysis of intracellular ROS levels using flow cytometry. Figure 3 O and Figure 22 ).

[0096] 2.6. Anti-inflammatory effects of BA@PDA-PA Osteoporosis is often accompanied by elevated levels of inflammation in bone tissue; therefore, reducing the inflammatory response is crucial for restoring bone metabolic balance and treating osteoporosis. Macrophages, a subset of immune cells, play a specific role in inflammation. Macrophages can polarize into various types depending on changes in their microenvironment, generally falling into two extreme phenotypes: the M1 phenotype, known as "classical activation," promotes inflammatory responses, while the M2 phenotype, known as "selective activation," promotes immune regulation and tissue remodeling. In inflammatory states, M1 macrophages are upregulated, producing large amounts of inflammatory mediators such as pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). M2 macrophages contribute to tissue regeneration and reduce inflammation by releasing anti-inflammatory cytokines such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β). Figure 4 A). When the balance between the M1 and M2 macrophage phenotypes is disrupted in normal bone regeneration, it may lead to immune dysregulation and accelerate the development of osteoporosis.

[0097] Therefore, this invention selected RAW264.7 cells to investigate the regulatory effect of BA@PDA-PA on inflammation. First, the secretion of representative inflammatory cytokines was measured using enzyme-linked immunosorbent assay (ELISA). The results showed that the levels of pro-inflammatory cytokines IL-6 and TNF-α were significantly downregulated in the BA@PDA-PA group, while the levels of anti-inflammatory cytokines IL-10 and TGF-β were significantly enhanced. Figure 4 BE). Flow cytometry results showed ( Figure 4 Compared with the control group, BA@PDA-PA treatment significantly reduced the expression of the M1 phenotypic marker (CD86) and increased the expression of the M2 macrophage marker (CD206). Subsequently, the polarization status of macrophages was further assessed by immunofluorescence staining. The results showed that the number of CD86-positive cells increased significantly under LPS stimulation, indicating a successful establishment of an inflammatory environment. In the BA@PDA-PA group, the fluorescence signal of CD86 was significantly weakened, while the fluorescence signal of CD206 was significantly enhanced. Figure 4 G and 4J-K). Furthermore, the mRNA expression of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine TGF-β in each group was detected by qRT-PCR. The results showed that BA@PDA-PA significantly inhibited the expression of pro-inflammatory cytokines and promoted the expression of anti-inflammatory cytokines (G and 4J-K). Figure 23 The above results all indicate that BA@PDA-PA can alleviate the inflammatory response and regulate the immune microenvironment to some extent by releasing BP.

[0098] 2.7. Anti-inflammatory mechanism of BA@PDA-PA To further elucidate the molecular pathway by which BA@PDA-PA exerts its anti-inflammatory effect, transcriptome sequencing (RNA-seq) was performed on RAW264.7 cells treated with different methods. Principal component analysis (PCA) results showed high correlation among samples within the same group, and significant differences between the LPS group (Control) and the LPS+BA@PDA-PA group (Treatment). Both groups of samples met the quality control requirements. Figure 5 A). Volcano plots and heatmaps showed significant differences in gene expression levels between the two groups; in the BA@PDA-PA group, 820 genes were downregulated and 516 genes were upregulated. Figure 5 BC). Gene ontology (GO) analysis of these differentially expressed genes showed that these differentially expressed genes are mainly related to immune responses. Figure 5 D). Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that differentially expressed genes were significantly associated with signaling pathways such as TNF, IL-17, and NF-κB, all of which are related to the occurrence and development of inflammation. Figure 5E). To confirm the expression status of these pathways, gene set enrichment analysis (GSEA) showed that the IL-17, TNF, and NF-κB signaling pathways were significantly inhibited after BA@PDA-PA treatment. Figure 5 F). Therefore, it can be concluded that BA@PDA-PA can effectively inhibit the expression of inflammation-related pathways in RAW264.7 cells, indicating its potential role in regulating immune responses and controlling inflammation.

[0099] 2.8. Ability to promote osteogenic formation and inhibit osteoclast formation in vitro An imbalance between osteoblasts and osteoclasts is a major cause of characteristic bone loss in osteoporosis. Therefore, this invention further investigates the regulatory role of materials in the osteogenic process. ALP is one of the marker enzymes of early osteogenic differentiation; ALP can react with the assay substrate to produce a color change, which is positively correlated with ALP activity. First, we co-cultured various materials with MC3T3-E1 cells in osteogenic medium for 14 days. ALP staining images revealed that the BA@PDA-PA group exhibited the highest ALP activity (…). Figure 6 A and 6C). Furthermore, cellular mineralization is an important marker of new bone maturation, and ARS staining is a decisive indicator for assessing the formation of osteoblast mineralization nodules. ARS staining images and quantitative analysis results 21 days after osteogenic induction showed that obvious mineralization nodules were observed in the BA@PDA-PA group, with the highest level of mineralization (A and 6C). Figure 6 B and 6D). Furthermore, qRT-PCR results showed a significant increase in the expression levels of osteogenic-related genes osteocalcin (OCN) and Runt-related transcription factor 2 (RUNX2), with more pronounced effects than the BP group alone. Figure 6 E and 6F). The above results indicate that the synergistic effect of BP and ACC is beneficial to osteogenic differentiation. Subsequently, the ability of BA@PDA-PA to inhibit osteoclast formation was further investigated. Osteoclasts are cells responsible for bone resorption, formed by the fusion of mononuclear precursors of monocyte-macrophage lineages. Nuclear factor κB ligand-activated receptor (RANKL) plays a central role in osteoclast differentiation and maturation, and can induce osteoclast differentiation. RAW264.7 cells were induced with RANKL for 5 days, and TRAP staining was used to assess osteoclast formation. The experimental results showed that a large number of osteoclasts stained purple-red appeared in the RANKL-containing medium, while the number of osteoclasts was significantly reduced in the BA@PDA-PA group ( Figure 6 Quantitative analysis also showed that BA@PDA-PA effectively inhibited osteoclast formation (G). Figure 6 H). Overall, BA@PDA-PA can effectively restore the imbalance between osteoblast and osteoclast formation in vitro, providing a basis for subsequent in vivo treatment of osteoporosis. Figure 6 I).

[0100] 2.9. Internal treatment of osteoporosis To further verify the role of the constructed nanomaterials in reversing osteoporosis in vivo, an ovariectomy (OVX)-induced osteoporosis mouse model was established. Mice with peripheral fat removed were designated as the Sham group, while mice with bilateral ovariectomy were divided into OVX, BP, BA, BA@PDA, and BA@PDA-PA groups. Modeling was performed for 4 weeks, followed by 4 weeks of treatment. Figure 7 A). During this period, changes in body weight were recorded in different groups of mice. (e.g.) Figure 7 As shown in Figure B, compared to the Sham group mice, the OVX group mice showed a significant increase in body weight, which may be due to changes in estrogen levels after ovarian removal, preliminarily demonstrating the successful establishment of the OVX-induced osteoporosis model. To verify the in vivo biosafety of BA@PDA-PA, HE staining was performed on major organs (heart, liver, spleen, lung, and kidney), as shown in Figure B. Figure 24 The results showed that, compared with the Sham group, no significant histopathological changes or cell damage were detected in the BA@PDA-PA group. The degree of liver function impairment was assessed by detecting liver function-related biomarkers alanine aminotransferase (ALT), ALP, and aspartate aminotransferase (AST). There were no significant differences in ALT, ALP, and AST between the BA@PDA-PA group and the control group, indicating no damage to liver function. Figure 25 Furthermore, all indicators in the blood routine examination of mice treated with BA@PDA-PA were within the normal range. These results preliminarily demonstrate that this nanocomposite material has good biocompatibility and holds promise as a nanomedicine for the treatment of osteoporosis.

[0101] Micro-CT was used to analyze bone quality in the distal femur of mice. Figure 7 Results showed that, compared to the Sham group, the OVX group exhibited significant trabecular bone loss and slowed bone growth, while the BA@PDA-PA group showed a significant increase in bone mass. Quantitative analysis of bone parameters showed that, compared to the Sham group, the OVX group had decreased Tb.Th, Tb.N, BV / TV, and BS / TV, while Tb.Sp increased, further demonstrating the successful establishment of the osteoporosis model. BA@PDA-PA treatment in mice increased Tb.Th, Tb.N, BV / TV, and BS / TV, and was superior to other experimental groups, while Tb.Sp significantly decreased (…). Figure 7DH). This demonstrates that BA@PDA-PA can target bone tissue and effectively promote bone formation in vivo, showing a significant therapeutic effect on osteoporosis. The poor therapeutic effects of the BP, BA, and BA@PDA groups may be due to a lack of targeting, making it difficult to accurately act on bone tissue and exert a therapeutic effect. Furthermore, micro-CT and related bone parameter analyses were performed on the lumbar spine of mice, showing a trend similar to the femoral treatment effect. Figure 26 and 27 ).

[0102] To further evaluate the efficacy of BA@PDA-PA nanoparticles in treating osteoporosis, histological staining of the femur and lumbar vertebrae of mice was performed to observe new bone formation. HE and Masson staining results of the femur after different treatments showed that, compared with the Sham group, the OVX group exhibited limited bone formation and significant bone loss, while the BA@PDA-PA treatment group showed more new bone formation. Figure 8 AC). Immunohistochemical staining results showed that the expression of the osteogenic marker RUNX2 was significantly increased in mice treated with BA@PDA-PA. Figure 8 D and 8E). Furthermore, TRAP staining revealed a significant decrease in osteoclast numbers in bone tissue treated with BA@PDA-PA compared to the OVX group, indicating that it can significantly inhibit osteoclast formation. Figure 8 F and 8G). Furthermore, histological staining of the lumbar vertebrae in mice also showed the same trend (F and 8G). Figure 28 The above results are consistent with the conclusions of in vitro experiments, indicating that BA@PDA-PA nanoparticles can effectively release calcium and phosphorus to promote osteogenic formation while inhibiting osteoclast formation, demonstrating great potential in the treatment of osteoporosis.

[0103] In addition, immunofluorescence staining was performed on the femur of mice to observe changes in inflammation levels during osteoporosis development and treatment. Staining images and quantitative analysis showed that, compared with the Sham group, the number of CD86-positive cells was significantly increased in the OVX group, while the number of CD86-positive cells was significantly decreased in the BA@PDA-PA treatment group. Figure 8 H and 8I), while compared with the OVX group, the number of CD206 positive cells increased in the BA@PDA-PA group ( Figure 8 J and 8K). These results indicate that osteoporosis is accompanied by chronic inflammation, promoting macrophage polarization towards the M1 type. BA@PDA-PA nanoparticles can continuously release BP, inhibiting M1 polarization and promoting M2 polarization, significantly reducing the level of inflammation in bone tissue and improving the bone immune microenvironment. In conclusion, BA@PDA-PA nanoparticles, by combining BP and ACC to jointly promote osteogenic formation and exert anti-inflammatory effects, hold promise for reversing osteoporosis.

[0104] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a bone-targeting platform based on black phosphorus and amorphous calcium carbonate, characterized in that, The specific steps are as follows: Step 1. Preparation of BP Bone apatite was ground and dispersed in N-methylpyrrolidone. The mixture was sonicated in a cell disruptor under ice bath conditions, and then the supernatant was collected by centrifugation and centrifuged again to obtain BP. Step 2. Preparation of BA CaCl2 powder was dissolved in anhydrous ethanol, then BP was added and dispersed. Deionized water was then added and ultrasonically dispersed to obtain a mixed solution. The mixed solution was transferred to a beaker and sealed with sealing film. The beaker was placed in a sealed container containing NH4HCO3 and reacted at room temperature. Finally, the product was collected by centrifugation, washed, and freeze-dried to obtain BA. Step 3. Preparation of BA@PDA BA was added to a Tris-HCl buffer solution containing dopamine hydrochloride, stirred and centrifuged at room temperature, the precipitate was washed with anhydrous ethanol and deionized water, and then freeze-dried to obtain BA@PDA; Step 4. Preparation of bone-targeting ligand PA Polyacrylic acid and sodium alendronate trihydrate were dissolved in borate buffer, and after stirring, borate buffer containing 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride was added. The pH was adjusted to 7.5 with NaOH solution, the reaction was stirred, and finally purified by dialysis and freeze-dried to obtain bone-targeting ligand PA. Step 5. Preparation of BA@PDA-PA BA@PDA was prepared into an aqueous solution of 1 mg / mL; PA was prepared into an aqueous solution of 0.1 g / mL; while stirring, the PA solution was slowly added dropwise to the BA@PDA solution. After reacting overnight, the mixture was centrifuged, washed, and the precipitate was freeze-dried and stored at -20℃ to obtain the bone-targeting platform BA@PDA-PA.

2. The method for preparing a bone-targeting platform based on black phosphorus and amorphous calcium carbonate according to claim 1, characterized in that, In step 1, the solid-liquid ratio of bone apatite to N-methylpyrrolidone is 1 mg: 1 mL.

3. The method for preparing a bone-targeting platform based on black phosphorus and amorphous calcium carbonate according to claim 1, characterized in that, In step 1, the cell disruptor is operated intermittently, with the operating conditions being 900W for 4 seconds on and 4 seconds off; the centrifugation is performed at 8000 r / min for 20 minutes. The second centrifugation was performed at 14000 r / min for 20 min.

4. The method for preparing a bone-targeting platform based on black phosphorus and amorphous calcium carbonate according to claim 1, characterized in that, In step 2, the solid-liquid ratio of CaCl2 to anhydrous ethanol is 2 mg:1 mL, the mass ratio of CaCl2 to BP is 50 mg:1 mg, the solid-liquid ratio of CaCl2 to deionized water is 1 mg:1 μL, and the mass ratio of CaCl2 to NH4HCO3 is 40 mg:1 g.

5. The method for preparing a bone-targeting platform based on black phosphorus and amorphous calcium carbonate according to claim 1, characterized in that, In step 2, the sealing film has several pores for gas exchange; the reaction time at room temperature is 48 hours.

6. The method for preparing a bone-targeting platform based on black phosphorus and amorphous calcium carbonate according to claim 1, characterized in that, In step 3, the concentration of dopamine hydrochloride in Tris-HCl buffer solution is 0.1 mol / L; the mass ratio of BA to dopamine hydrochloride is 1 mg:1 mg. The stirring time is 3 hours.

7. The method for preparing a bone-targeting platform based on black phosphorus and amorphous calcium carbonate according to claim 1, characterized in that, In step 4, the mass ratio of polyacrylic acid to sodium alendronate trihydrate is 2g:65g; the solid-liquid ratio of polyacrylic acid to borate buffer is 0.002g:100mL. The concentration of the borate buffer containing 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride is 0.1 g / mL; The mass ratio of the polyacrylic acid to 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride is 2g:55g; The dialysis purification was performed using 2000 kDa dialysis under aqueous solution conditions.

8. The method for preparing a bone-targeting platform based on black phosphorus and amorphous calcium carbonate according to claim 1, characterized in that, In step 5, the volume ratio of the PA solution to the BA@PDA solution is 0.1 mL: 20 mL.

9. The bone-targeting platform obtained by any one of the preparation methods according to claims 1-9.

10. The application of the bone-targeting platform according to claim 9 in drug preparation.

Images