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Sea cucumber-inspired dual-responsive black phosphorus/amorphous calcium-based bone-targeting nanoplatform for synergistic therapy of osteoporosis.
Shen X, Lan Z, Zhu Y, Zhong T, Li B, Chen J, Zhu Y, Ye J, Wang X.
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The core pathological mechanism of osteoporosis (OP) resides in the imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Furthermore, the accumulation of reactive oxygen species (ROS) and pro-inflammatory factors in bone tissue induces disorders in the immune microenvironment, which substantially elevates the risk of fracture. Inspired by the biological behaviors of sea cucumbers, we designed and constructed a bone-targeting platform (BA@PDA-PA) based on black phosphorus (BP) and amorphous calcium carbonate (ACC). This platform enables precise targeted therapy for diseased bone tissue while improving drug bioavailability. Upon reaching the target site, BA@PDA-PA can respond to the acidic microenvironment of OP lesions, and then release BP and calcium ions (Ca2+) on demand. Specifically, BP efficiently scavenges local excessive ROS and improves the immune microenvironment. Meanwhile, the Ca2+ released from ACC degradation, along with the low phosphorus oxides derived from BP breakdown, work together to promote osteoblast proliferation and differentiation while inhibiting osteoclast activity. This establishes a synergistic therapeutic mechanism involving "targeted delivery, microenvironment regulation, and bone metabolism remodeling." In vivo experimental results demonstrate that this platform significantly improves the pathological bone microstructure of OP mice, thereby providing a promising therapeutic strategy for OP treatment.
82360220 Innovative Research Group Project of the National Natural Science Foundation of China, 82360424 Innovative Research Group Project of the National Natural Science Foundation of China, 32560229 Innovative Research Group Project of the National Natural Science Foundation of China, 20224BAB216050 the Youth Fund Project of Natural Science Foundation of Jiangxi Province, 20224BAB216031 the Youth Fund Project of Natural Science Foundation of Jiangxi Province, 20243BCE51164 the Ganpo Talent Support Program-Leading Academic and Technical Personnel in Major Disciplines of Jiangxi Province, 20242BAB23078 the Natural Science Foundation of Jiangxi Province for Distinguished Young Scholars, 20243BCE51143 he Ganpo Talent Support Program-Leading Academic and Technical Personnel in Major Disciplines of Jiangxi Province, 2025ZD011 the Key Project of Science and Technology Innovation of Jiangxi Health Commission, 20212BBG73004 the Key Research and Development Program of Jiangxi Province, PYJX20230001 the Interdiscipline Innovation Fund Project of Nanchang University, 2024SSY07061 the Jiangxi Province Key Laboratory of Bioengineering Drugs
Scheme 1. Schematic diagram of the synthesis process and application of BA@PDA-PA in the treatment of OP. (A) Design schematic diagram of BA@PDA-PA: (I) The biological behaviors of sea cucumbers. (II) Schematic diagram of the synthesis process of BA@PDA-PA. (B) Mechanism of BA@PDA-PA in the treatment of OP in mice: (I) In the bone microenvironment of OP, excessively differentiated mature osteoclasts adhere to the bone surface and secrete a large amount of H+; BA@PDA-PA neutralizes the excess H+, releases BP and Ca2+, and ameliorates the acidic microenvironment of OP. (II) The released BP effectively scavenges excess ROS in the inflammatory microenvironment, promotes macrophage polarization from the M1 to M2 phenotype, regulates the immune microenvironment, and subsequently degrades into non-toxic PxOy after treatment. (III) The released BP cooperate with Ca2+ to promote osteoblast-mediated bone formation and inhibit osteoclastogenesis, reversing OP
Fig. 1. Formation and characterization. (A) Diagram of the synthesis of BA@PDA-PA. (B) TEM image of BP. (C) HRTEM image of BP. (D) AFM image of BP. (E) Raman spectra of BP and bulk BP. (F) SEM images of BP, BA, BA@PDA and BA@PDA-PA. (G) TEM and mapping images of BA@PDA-PA. (H) FTIR spectra of BP, BA, BA@PDA and BA@PDA-PA. (I) Zeta potentials of BP, BA, BA@PDA and BA@PDA-PA. (J) TGA curves of BP, BA, BA@PDA and BA@PDA-PA. (K) XPS survey spectrum of BA@PDA-PA. (L) High-resolution XPS spectra of P 2p, Ca 2p, N 1 s and Na 1 s in BA@PDA-PA
Fig. 2. The acid-responsiveness and targeting performance of BA@PDA-PA. (A) The total amount of Ca2+ released by BA@PDA-PA in PBS with various pH values. (B) Ca2+ release from BA@PDA-PA at different pH (7.5 and 4.5) for 72 h. (C) Acidity neutralization by BA@PDA-PA. (D) pH value changes during the HCl (1%, v/v) titration to BA@PDA-PA, deionized water, and CaCO3. (E) The SEM images of BA@PDA-PA at the mild acidic (pH 4.5) condition for 0–60 min. (F) Fluorescence microscopic analysis regarding BA@PDA-PA distribution on bone surface at different pH values. (G) Representative SEM images of the interaction between the surface of BA@PDA-PA and HAP at various time points. (H) Fluorescence intensity of the supernatant of BA@PDA-PA after stirring in HAP solution for different time points. (I) In vivo fluorescence images at diverse times post-injection of BA@PDA-PA in mice. FL: Fluorescence imaging mode; BF: Bright field imaging mode. Data are means ± S.D. (n ≥ 3)
Fig. 3. ROS scavenging capacity of BA@PDA-PA. (A-C) UV–vis detection of the scavenging ability of BA@PDA-PA at different concentrations on (A) ABTS+·, (B) DPPH·, and (C) ·OH radicals (inset: the color change of ABTS+·, DPPH·, and ·OH radical solutions after treatment with different concentrations of BA@PDA-PA). (D) ·OH clearance rate of BA@PDA-PA at different concentrations. (E, F) ESR spectra of ·OH and ·O2− formed by BA@PDA-PA. (G, H) SOD-like, CAT-like activity of BA@PDA-PA at different concentrations. (I-K) ABTS+·, DPPH·, and ·OH clearance efficiency of BP, BA, BA@PDA, BA@PDA-PA. (L, M) SOD-like, CAT-like activity of BP, BA, BA@PDA, BA@PDA-PA. (N) Fluorescence images of RAW264.7 cells stained with DCFH-DA at different concentrations of BA@PDA-PA. (O) Flow cytometry analysis of ROS clearance in RAW264.7 cells at different concentrations of BA@PDA-PA. Data are means ± S.D. (n ≥ 3), *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 4. Immunomodulatory effects of BP, BA, BA@PDA and BA@PDA-PA. (A) The scheme of inflammation regulation of nanoparticles. (B-E) ELISA assays of IL-6, TNF-α, IL-10 and TGF-β in LPS-induced RAW264.7 cells after different treatments. (F) Flow cytometry analysis of the expression of CD206 and CD86 in RAW264.7 cells with different treatments. (G) Immunofluorescence images of CD206 and CD86 in RAW264.7 cells with different treatments. (H) Quantitative analysis of the fluorescence intensity of CD86 in flow cytometry. (I) Quantitative analysis of the fluorescence intensity of CD206 in flow cytometry. (J) Quantitative analysis of CD86 in immunofluorescence images. (K) Quantitative analysis of CD206 in immunofluorescence images. Data are means ± S.D. (n ≥ 3), *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 5. The anti-inflammation mechanism of BA@PDA-PA. (A) PCA results of RAW264.7 cells sequencing in two groups. (B) Volcano plot displaying up-regulated and down-regulated genes in RAW264.7 cells. (C) Related heatmap. (D) GO analysis. (E) KEGG pathway enrichment analysis. (F) GSEA results
Fig. 6. The role of BA@PDA-PA in promoting osteogenesis and inhibiting osteoclastogenesis. (A) ALP staining of MC3T3-E1 cells. (B) ARS staining of MC3T3-E1 cells. (C) Quantitative results to ALP. (D) Quantitative results to ARS. (E) mRNA expression of OCN and (F) 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 effect of nanoparticles in promoting osteogenesis and inhibiting osteoclasts. Data are means ± S.D. (n ≥ 3), *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 7. The therapeutic effect of BA@PDA-PA on mice model of OP. (A) Schematic diagram of the in vivo experimental plan. (B) Body weight changes of mice with various treatments. (C) Micro-CT images of the distal femoral trabecular microstructure of each group after treatment. (D-H) Quantitative analysis of Tb.Th, Tb.N, BV/TV, BS/TV and Tb.Sp parameters of the distal femur of each group after treatment. Data are means ± S.D. (n = 5), ns > 0.05, *p < 0.05, ***p < 0.001, ****p < 0.0001
Fig. 8. Histological staining of mice femora. (A) HE staining of the distal femur of mice. (B) Masson staining of the distal femur of mice. (C) Quantitative results of the ratio of the new bone area to the total bone area in Masson staining. (D) Immunohistochemistry staining of RUNX2. (E) Quantitative analysis of RUNX2 positive area in immunohistochemistry staining. (F) TRAP staining of the distal femur of mice. (G) Quantitative results of the TRAP positive area to the total bone area in TRAP staining. (H) Immunofluorescence staining images of CD86 in the distal femur of mice. (I) Quantitative analysis of CD86 in immunofluorescence staining. (J) Immunofluorescence staining images of CD206 in the distal femur of mice. (K) Quantitative analysis of CD206 in immunofluorescence staining. Data are means ± S.D. (n = 5), ***p < 0.001
