Bone-targeting engineered milk-derived extracellular vesicles for MRI-assisted therapy of osteoporosis

Abstract

The imbalance between osteoblasts and osteoclasts is the cause of osteoporosis. Milk-derived extracellular vesicles (mEVs), excellent drug delivery nanocarriers, can promote bone formation and inhibit bone resorption. In this study, we conjugated bone-targeting peptide (AspSerSer, DSS)₆ to mEVs by click chemistry and then loaded with SRT2104, a SIRT1 (silent mating-type information regulation 2 homolog 1) agonist that was proofed to help reduce bone loss. The engineered (DSS)₆-mEV-SRT2104 had the intrinsic anti-osteoporosis function of mEVs and SRT2104 to reverse the imbalance in bone homeostasis by simultaneously regulating osteogenesis and osteoclastogenesis. Furthermore, we labelled mEVs with MnB nanoparticles that can be used for the in vivo magnetic resonance imaging (MRI) visualization. The obtained nanocomposites significantly prevented bone loss in osteoporosis mice and increased bone mineral density, exhibiting superior bone accumulation under MRI. We believe the proposed (DSS)₆-mEV-SRT2104/MnB provides a novel paradigm for osteoporosis treatment and monitoring.

Keywords: MRI; SRT2104; bone-targeting peptide; milk-derived extracellular vesicles; osteoporosis.

Graphical abstract

Scheme 1.

Schematic illustration of the development of bone-targeting engineered milk-derived extracellular vesicles for MRI-assisted therapy of osteoporosis.

Figure 1.

Preparation and characterization of (DSS)₆-mEV-SRT2104/MnB. (A) Schematic illustration of the preparation procedure of (DSS)₆-mEV-SRT2104/MnB. (B) TEM images of mEVs. Scale bar = 100 nm. (C) Protein marker characterization of mEVs via Western blot. (D) Schematic illustration of the preparation of (DSS)₆-mEVs via click chemistry. (E) The characterization of (DSS)₆-mEVs by flow cytometry. (F) Schematic illustration of the synthesis of MnB NPs. (G) Hydrodynamic size of MnB NPs via DLS. (H) T₁-weighted MR images and 1/T₁ of MnB NPs via 3.0 T clinical MRI scanner. (I) T₁-weighted MR images of different samples and the corresponding SNR. (J) TEM images of (DSS)₆-mEV-SRT2104/MnB. Scale bar = 100 nm (ns, no significant; ***P < 0.001).

Figure 2.

The effects of mEV-SRT2104 on osteoblasts and osteoclasts in vitro. (A) Schematic illustration of the osteogenic differentiation of MC3T3-E1 cells. (B) ALP staining of MC3T3-E1 cells after treatment for 7 days and (C) the quantification analysis of staining integrated density according to (B). Scale bar = 200 μm. (D) Relative mRNA expression of RUNX2, ALP and OPN after treatment for 7 days. (E) Western blot assay of protein RUNX2 and OPN after treatment for 7 days. (F) Schematic illustration of the osteoclast differentiation of RAW264.7 cells. (G) TRAP staining of RAW264.7 cells after treatment for 5 days and (H) the quantification analysis of TRAP-positive cell area. Scale bar = 200 μm. (I) Relative mRNA expression of CTSK, RANK and NFATC1. (J) Western blot assay of CTSK protein (ns, no significance; *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3.

Bone-targeting capability of engineered mEVs in vitro and in vivo. (A) Schematic illustration of the culture of MC3T3-E1 cells. (B) Uptake of Dil-labelled (DSS)₆-mEVs with MCST3-E1 cells by flow cytometry. (C) Schematic illustration of the binding of (DSS)₆-mEV-SRT2104/MnB and mEV-SRT2104/MnB with hydroxyapatite. (D) T₁-weighted MR images of the supernatant after the binding of mEVs or (DSS)₆-mEVs with hydroxyapatite. (E) In vivo biodistribution of DiR-labelled (DSS)₆-mEVs or mEVs by IVIS. (F) In vivo biodistribution of mEV-SRT2104/MnB or (DSS)₆-mEV-SRT2104/MnB by MRI (ns, no significant; *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4.

Establishment of OVX mouse model and in vivo biocompatibility evaluation. (A) The schematic illustration of the in vivo studies. (B) Representative uterus images of mice 4 weeks after surgery and quantitative results of uterus’ weight. (C) Representative micro-CT images showing 3D microarchitecture of trabeculae in the distal femurs. (D–H) Characterizations of the BMD, BV/TV, Tb.N, Tb.Th and Tb.Sp of distal femurs 4 weeks after surgery. (I) H&E staining of the main organs in different groups after 6 weeks of treatment to OVX mice. Scale bar = 250 μm (**P＜0.01, ***P＜0.001).

Figure 5.

(DSS)₆-mEV-SRT2104 reduces bone loss in OVX mice by dual regulation of bone remodeling. (A) Representative micro-CT images showing 3D microarchitectures of trabeculae in the distal femurs. (B) Micro-CT quantitative analysis of BMD, BV/TV, Tb.N and Tb.Sp of distal femurs. (C) TRAP staining of bone sections. Scale bar = 250 μm. (D) H&E staining of bone sections of experimental mice. Scale bar = 250 μm. (E) Histomorphometric analysis of osteoclast surface per bone surface (Oc.S/BS). (F) Histomorphometric analysis of the trabecular bone area. (G–K) The relative serum protein expression evaluated by the Elisa test, including β-CTX (G), TRACP-5b (H), BALP (I), OCN (J) and P1NP (K) (ns, no significance; *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 6.

T₁-weighted MRI of OVX mice. (A) The femur T₁-weighted MR images were acquired from OVX mice administered with mEV-MnB, mEV-SRT2104/MnB or (DSS)₆-mEV-SRT2104/MnB at various time points during week 4 and 10. (B) SNR analysis of (a). (C) The ΔSNR from 4 to 24 h in the distal femur of OVX mice administered with (DSS)₆-mEV-SRT2104/MnB was assessed based on T₁-weighted MRI images (ns, no significance; *P ＜ 0.05).