Nerve growth factor-loaded biomimetic prussian blue nanocomplexes for reversing osteoporosis via promoting osteoblast precursor cell proliferation and differentiation

Abstract

The fundamental issue of osteoporosis (OP) is osteoblast decrease due to oxidative stress and the subsequent disruption of the osteogenic and osteoclastic dynamic balance. How to promote the proliferation and osteogenic differentiation of osteoblast precursor cells (MC3T3-E1) in an oxidative microenvironment is a great challenge for improving OP. In this study, Prussian blue nanoparticles (PB NPs) were initially functionalized with a polydopamine (PDA) coating. Nerve growth factor (NGF) was subsequently immobilized on the PDA layer, followed by the hybrid membrane coating composed of red blood cell membrane (RBCm) and MC3T3-E1 cell membrane (MC3T3m), thereby constructing a biomimetic Prussian blue nanocomplex loaded with NGF (MPDN NPs). In vitro studies have shown that the nanodrug restored the impaired proliferation viability of MC3T3-E1 cells and inhibit their apoptosis by scavenging reactive oxygen species (ROS), and further cooperate with NGF to promote osteogenic differentiation. In vivo studies have demonstrated that the nanodrug significantly inhibited bone loss and promote bone regeneration in osteoporotic mice. Moreover, this nanodrug with excellent safety both in vitro and in vivo showed the long half-life in the bloodstream and high accumulation in the bone. In summary, this strategy addresses the fundamental issue of decreased osteoblast in OP and offers a novel approach for preventing and treating OP.

Keywords: Nerve growth factor; Osteoblast precursor cells; Osteogenic differentiation; Osteoporosis; Prussian blue nanoparticles; Reactive oxygen species.

Graphical abstract

Scheme

Synthesis process of MPDN NPs and the mechanism of anti-osteoporosis.

Fig. 1

The "high apoptosis, low osteogenesis" microenvironment induced by ROS at osteoporotic sites and drug concentration screening. (A) Representative images of H&E staining of tibia tissue. (B) Representative images of DHE staining (red) of tibia tissue. (C) Representative immunohistochemistry staining of Bcl2 and Bax in tibia sections. (D) Representative immunofluorescence double staining of OCN (red) and TUNEL (green) positive cells in tibia sections. (E–F) Cell viability of the MC3T3-E1 cells incubated with NGF or PB NPs for 24 h. (G) Flow cytometry identification of ROS in MC3T3-E1 cells incubated with PB NPs (20, 25, 30, 35 μg/mL). (H) ALP and ARS staining of MC3T3-E1 cells treated with NGF (0, 50, 100 ng/mL). (I) ALP activity of MC3T3-E1 cells treated with NGF (0, 50, 100 ng/mL). n = 3.

Fig. 2

Characterization of MPDN NPs. (A) Schematic representation of the synthesis of MPDN NPs. (B) TEM images of P, PD and MPDN NPs. (C) NGF entrapment efficiency of MPDN at the different ratio and stirring time. (D–E) Diameter and potential of P, PD, PDN and MPDN NPs. (F) Stability of MPDN NPs in water, PBS, total medium with 10 % FBS (0, 24, 48, 72 h). (G-I)The element content of C, N, S, Fe in P, PD, PDN NPs. (J) CLSM images of hybrid membrane. (red: RBCm, green: MC3T3m). (K) SDS-PAGE of the membrane proteins (1: MC3T3m, 2: RBCm, 3: Hybrid membrane, 4: MPDN NPs).

Fig. 3

In vitro cellular uptake behavior and in vivo biodistribution. (A) Fluorescence images of cellular uptake of MPDN NPs by MC3T3-E1 cells. (B) Fluorescence images of cellular uptake of MPDN NPs by RAW264.7 cells. (C) Representative fluorescence images of blood samples collected from mice after administration of Ce6 or M@PD^Ce6 at preset time point (0, 0.5, 2, 4, 8, 12, 24 h). (D) The pharmacokinetics curves of plasma samples. (E–F) Fluorescence distribution and quantitative fluorescence analysis of lower limbs bone after 24 h post-injection. (G–H) Fluorescence distribution and quantitative fluorescence analysis of main organs after 24 h post-injection. n = 3.

Fig. 4

MPDN NPs scavenge ROS in vitro thereby promoting proliferation of MC3T3-E1 cells and inhibiting their apoptosis. (A) Representative fluorescence images of ROS in MC3T3-E1 cells. (B–C) Flow cytometry assay of ROS in MC3T3-E1 cells. (D) Anti-oxidant abilities of NGF, MPD, MPDN by DPPH assay. (E) Cell viability of the MC3T3-E1 cells incubated with MPDN and H₂O₂ for 24 h. (F–G) Representative fluorescence images and quantitative analysis of EdU positive MC3T3-E1 cells. (H–I) Representative fluorescence images and quantitative analysis of Ki67 in MC3T3-E1 cells. (J–K) Flow cytometry analysis of apoptosis in H₂O₂-treated MC3T3-E1 cells and quantitative analysis of the total apoptotic rate (early and late apoptotic cells in MC3T3-E1 cells. (L–M) Western blot assay of Bcl2 and Bax apoptosis-related proteins and quantitative analysis of the ratio of Bcl2/Bax. n = 3.

Fig. 5

MPDN NPs promote osteogenic differentiation of MC3T3-E1 cells in vitro oxidative microenvironment. (A–B) Photographs of ALP and ARS staining of MC3T3-E1 cells after 7 and 21 days of various treatments. (C–D) Quantitative analysis of ALP and ARS staining. (E–G) Representative fluorescence images of early (Runx2) and late (Col Ⅰ and OCN) osteogenesis-related proteins. (H–J) Quantitative fluorescence analysis of Runx2, Col Ⅰ and OCN. (K) Schematic diagram of pro-osteogenic differentiation process.

Fig. 6

MPDN NPs prevents bone loss in vivo. (A) Flowchart of animal experiments. (B) Representative micro-CT images of distal femur. n = 5. (C–G) BV/TV, Tb. Sp, Tb.N, SMI, Tb.Th of mouse tibia obtained from micro-CT data. n = 5. (H) Photograph of the three-point bending test. (I–J) Maximum load and break load of tibia. (n = 4). (K–M) H&E, Masson and Trap staining of tibia tissue. n = 3.

Fig. 7

MPDN NPs exert anti-osteoporotic effects by regulating the expression of multiple proteins in bones. (A) PCA plot of proteomic data of Control, Model, MPDN NPs groups. (B) Histogram of differentially expressed proteins. (C) Venn map of differentially expressed proteins. (D) KEGG pathway analysis of differentially expressed proteins (Control-vs-Model). (E) KEGG pathway analysis of differentially expressed proteins (Model-vs-MPDN). (F–I) GSEA plots of significant biological process associated with osteogenesis and ROS metabolism (Model-vs-MPDN). (J–K) GSEA plots of Chemokine and Hedgehog signaling pathway (Model-vs-MPDN). (L) Heat map of antioxidation-related proteins. (M) Heat map of cell growth-related proteins. (N) Heat map of osteogenesis-related proteins.

Fig. 8

MPDN NPs regulate the expression of antioxidation, cell growth, and osteogenesis-related proteins in bones of OP mice. (A) Immunohistochemistry staining of Foxo1 in the tibia tissue. (B) Immunofluorescence staining for Ki67 in the tibia tissue. (C–D) Immunohistochemistry staining of Bcl2 and Bax in the tibia tissue. (E) Immunofluorescence staining for Runx2 in the tibia tissue. (F–G) Immunohistochemistry staining of Col Ⅰ and OCN in the tibia tissue. n = 3.

Fig. 9

MPDN NPs have excellent safety in vitro and vivo. (A) Cytotoxicity of the MC3T3-E1 cells and HUVEC incubated with NGF, MPD and MPDN NPs for 24 h. (B) Morphological images of RBCs incubated with water, PBS, MPDN NPs (25, 50, 100 μg/mL) for 4 h. (C) The images and hemolysis rate of RBCs incubated with MPDN NPs (25, 50, 100 μg/mL) for 4 h. (D) Platelet aggregation assay of NGF, MPD and MPDN NPs. (E) Representative images of the zebrafish incubation process during 96 h. n = 6. (F–G) The heart rate and body length of zebrafish. n = 6. (H) H&E staining of main organs from different groups. (I) Complete blood count of WBC, RBC, HGB, and PLT levels. (J) Hepatotoxicity and nephrotoxicity by measuring plasma levels of CREA, UREA, ALT and AST. n = 3.