Bone-targeted nano-vesicle for treating osteoporosis as well as preparation method and application of bone-targeted nano-vesicle

Abstract

The invention provides a bone-targeted nano-vesicle for treating osteoporosis as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The bone targeting nano vesicle disclosed by the invention is a bone marrow mesenchymal stem cell nano vesicle loaded with melatonin and / or a bone marrow mesenchymal stem cell nano vesicle modified by (AspSerSer) 6 and loaded with melatonin. The bone targeting nano vesicles prepared by the invention not only can recover the balance of osteoblasts / osteoclasts, but also can regulate circadian rhythm and recover a normal outflow function, so that the purpose of preventing or treating osteoporosis is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a bone-targeted nanocapsule for treating osteoporosis, and a preparation method and application thereof. Background Art

[0002] Bone homeostasis is directly maintained by the balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. This balance is disturbed by excessive resorption or insufficient formation, which leads to bone loss and deterioration of bone tissue microarchitecture, namely osteoporosis. In bone tissue, apoptosis is the most common form of cell death. Although apoptosis does not produce a large number of proinflammatory factors like other forms of cell death, apoptosis-related products still need to be cleared by efferocytosis. The efferocytosis of various apoptotic cells in the bone microenvironment, including osteoblasts, osteoclasts, and bone marrow mesenchymal stem cells (BMSCs), is crucial for regulating bone formation and bone resorption, and is an important mechanism for maintaining bone homeostasis. Recent studies have demonstrated that osteoblast efferocytosis mediated by macrophages (BMDMs) is an important factor affecting bone formation. 40%-70% of osteoblasts undergo apoptosis and are subsequently efferocytized. F4 / 80 + and CD68 + The reduction of macrophages and the inhibition of gene expression of key efferocytosis factors by pharmacological intervention lead to the failure of apoptotic osteoblast clearance, which in turn triggers a proinflammatory response and bone loss. In addition, due to the interruption of efferocytosis, the new mineralization and osteogenic differentiation of osteoblasts are also inhibited. In elderly individuals, apoptotic osteoblasts accumulate in the bone marrow microenvironment (BMME) due to the upregulation of the immune escape molecule CD47 or the impairment of macrophage efferocytosis due to aging. When macrophage efferocytosis is dysfunctional, BMSCs can act as non-professional phagocytes to clear apoptotic cells. However, the conduct of efferocytosis has been shown to inhibit their osteogenic differentiation, which further exacerbates bone loss. Therefore, restoring macrophage efferocytosis is essential to maintain the balance of bone metabolism in the aged BMME.

[0003] A variety of physiological processes are controlled by the circadian clock, and an increasing number of studies have shown that the circadian clock is closely related to bone metabolism. Circadian clock disorders increase with age and lead to a variety of diseases, including bone diseases. Circadian clock disorders are mainly manifested as disordered expression of core circadian clock genes, which further leads to weakening of circadian clock oscillators in tissues. The absence or weakening of core circadian clock gene expression is common in elderly individuals and has been reported to interfere with bone metabolism homeostasis. Like other senescent cells, senescent macrophages also exhibit a disordered circadian clock. Currently, most drugs used to treat osteoporosis attempt to restore the balance of osteoblasts and osteoclasts, but ignore the importance of restoring the circadian clock and macrophage efferocytosis. Therefore, there is an urgent need to understand the mechanism between circadian clock disorders and impaired efferocytosis, and studying drugs to treat osteoporosis from the perspective of regulating circadian rhythms and restoring bone immune balance is a technical problem that needs to be solved at this stage. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a bone-targeted nanovesicle for treating osteoporosis, and a preparation method and application thereof. The bone-targeted nanovesicle can not only restore the balance of osteoblasts / osteoclasts, but also regulate the circadian rhythm and restore normal outflow function, thereby achieving the purpose of preventing and / or treating osteoporosis.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a bone-targeted nanovesicle for treating osteoporosis, wherein the bone-targeted nanovesicle is any one of the following nanovesicles: (1) bone marrow mesenchymal stem cell nanovesicles loaded with melatonin; (2) (AspSerSer)6-modified bone marrow mesenchymal stem cell nanovesicles loaded with melatonin; (3) a combination of (1) and (2).

[0007] Preferably, the bone marrow mesenchymal stem cell nanovesicles are obtained by extruding bone marrow mesenchymal stem cells.

[0008] Preferably, the bone marrow mesenchymal stem cells are bone marrow mesenchymal stem cells that overexpress RANK and CXCR4.

[0009] The present invention provides a method for preparing bone-targeted nanovesicles, comprising the following steps: (1) dispersing bone marrow mesenchymal stem cells in a PBS solution, then squeezing, centrifuging for the first time to collect a precipitate, resuspending the precipitate, centrifuging again, collecting the precipitate, and obtaining bone marrow mesenchymal stem cell nanovesicles; (2) mixing the bone marrow mesenchymal stem cell nanovesicles with melatonin by ultrasound, then incubating the mixture at 36-38° C. for 0.5-1.5 h, centrifuging and collecting the precipitate, and obtaining bone marrow mesenchymal stem cell nanovesicles loaded with melatonin; (3) The melatonin-loaded bone marrow mesenchymal stem cell nanovesicles are incubated with (AspSerSer)6 coupled with SPE-PEG-NHS at 3-5°C overnight, the precipitate is collected by centrifugation, washed and suspended in water to obtain (AspSerSer)6-modified melatonin-loaded bone marrow mesenchymal stem cell nanovesicles; (4) the melatonin-loaded bone marrow mesenchymal stem cell nanovesicles are mixed with (AspSerSer)6-modified melatonin-loaded bone marrow mesenchymal stem cell nanovesicles to obtain a nanovesicle composition.

[0010] Preferably, the pore sizes of the membranes used for the extrusion are 4-6 μm and 0.5-1.5 μm respectively.

[0011] Preferably, the first centrifugation parameter in step (1) is: 0.5×10 5 -1.5×10 5 g, 5-10 min; the parameters for the second centrifugation are: molecular weight cutoff 80-120 kDa, 900-1100 g, 8-12 min.

[0012] Preferably, the amount of bone marrow mesenchymal stem cell nanovesicles used in step (2) is 10 9 -10 11 The amount of melatonin used is 490-510 mg.

[0013] Preferably, the ultrasonic parameters in step (2) are: amplitude 18-22%, 28-32s on / off cycle 2.5-3.5min, repeated 5-7 times, with a cooling interval of 1.5-2.5min between each cycle; the centrifugation parameters in steps (2) and (3) are: first centrifugation at 1100-1300rpm for 8-12min, then at 1×10 4 -2×10 4 Centrifuge at 200 g for 25-35 min.

[0014] Preferably, the concentration of the melatonin-loaded bone marrow mesenchymal stem cell nanovesicles in step (3) is 10 11 -10 13 The DSPE-PEG-NHS-conjugated (AspSerSer)6 was used at a concentration of 8-12 μM.

[0015] The present invention also provides the use of the bone-targeted nanovesicle or the bone-targeted nanovesicle obtained by the preparation method in the preparation of a drug for preventing and / or treating osteoporosis.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention proposes for the first time to prepare melatonin-loaded bone marrow mesenchymal stem cell nanovesicles (Mel@RCNVs) using BMSCs that overexpress nuclear factor-κB receptor activator (RANK) and CXC motif chemokine receptor 4 (CXCR4), and then modify Mel@RCNVs with (AspSerSer)6 to obtain (AspSerSer)6-modified melatonin-loaded bone marrow mesenchymal stem cell nanovesicles (Mel@OST-RCNVs). Mel@RCNVs can specifically eliminate RANK ligand (RANKL) in the bone microenvironment, thereby inhibiting osteoclastogenesis. After Mel@RCNVs are modified with (AspSerSer)6, they will be redistributed to the bone formation surface and activate the RANKL reverse signal in osteoblasts, thereby promoting osteogenesis. At the same time, these nanovesicles have been shown to be phagocytosed by bone marrow-derived macrophages (BMDMs), restoring their key circadian clock gene expression and endocytosis function. The nanovesicle preparation flow chart and reaction mechanism are shown in Figure 1 Therefore, BMSCs were liberated from non-professional efferocytosis and their osteogenic / adipogenic transition was corrected. In summary, the present invention proposes a promising bone-targeted nanovesicle for the treatment of SOP by regulating the circadian clock and restoring normal efferocytosis function. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Flow chart of the preparation of bone-targeted nanovesicles and schematic diagram of the reaction mechanism for the treatment of SOP.

[0019] Figure 2Preparation and characterization of Mel@RCNVs and Mel@OST-RCNVs. (A) Schematic diagram of the preparation of Mel@RCNVs and Mel@OST-RCNVs; (B) Immunofluorescence staining images of original bone marrow mesenchymal stem cells (control group) and bone marrow mesenchymal stem cells overexpressing RANK and CXCR4 stained with DAPI (blue), anti-RANK (green) and anti-CXCR4 (red) antibodies, scale bar = 10 μm; (C) Schematic diagram of the structure of RCNVs and immunofluorescence staining images, scale bar = 10 μm; (D) Quantitative analysis of the number of CXCR4 and RANK on NVs from original bone marrow mesenchymal stem cells (control group) and bone marrow mesenchymal stem cells overexpressing RANK and CXCR4, data are expressed as mean ± standard deviation, n = 5; (E) Quantitative analysis of RCNVs protein concentration and particle yield, RCNVs are from 10 6 The cells were prepared from 100 cells, and the data are expressed as mean ± SD, n = 5; (F) Transmission electron microscopy images of RCNVs, Mel@RCNVs and Mel@OST-RCNVs, scale bar = 100 nm; (G) Fluorescence image of Mel@OST-RCNVs (Cy3-labeled (AspSerSer)6 modified) captured by RANKL-coupled immunomagnetic beads, scale bar = 50 μm; (H) Cumulative release curve of Mel from Mel@RCNVs and Mel@OST-RCNVs; (I) Western blotting results of RANK and CXCR4 in RCNVs, Mel@RCNVs and Mel@OST-RCNVs and NVs of original bone marrow mesenchymal stem cells (control group); (J) Changes in diameter and zeta potential of Mel@RCNVs and Mel@OST-RCNVs in PBS, n = 3.

[0020] Figure 3 Diameter distribution results of RCNVs, Mel@RCNVs and Mel@OST-RCNVs detected by dynamic light scattering (DLS).

[0021] Figure 4 The results of cell viability determination of bone marrow-derived macrophages (BMDMs) after incubation with Mel@RCNVs and Mel@OST-RCNVs for 24 hours. The data are expressed as mean ± standard deviation, n = 3.

[0022] Figure 5 The hemolysis rate of samples incubated with Mel@RCNVs and Mel@OST-RCNVs. The data are expressed as mean ± standard deviation, n = 3.

[0023] Figure 6Results of bone-targeting ability determination of bone-targeted nanovesicles. (A) Schematic design of bone-targeting ability study; (B) Biodistribution of different nanovesicles (labeled with DiR) in mice after tail vein injection, with nanovesicles from primary bone marrow mesenchymal stem cells as control. (C) Quantitative analysis of fluorescence intensity reflecting the distribution of DiR-labeled nanovesicles in vivo, n=3. (D) Colocalization of nanovesicles (labeled with DiO, green fluorescence) and bone formation surface (labeled with XO, red fluorescence), scale bar = 50 μm; (E) Fluorescence staining images of Col1a1 (green fluorescence) and different nanovesicles (labeled with DiR, red fluorescence), scale bar = 50 μm; (F) Fluorescence staining images of CD68 (green fluorescence) and different nanovesicles (labeled with DiR, red fluorescence), scale bar = 50 μm, data are expressed as mean ± standard deviation, n = 3, NS means no significant difference, * means p < 0.05, ** means p < 0.01; *** means p < 0.001.

[0024] Figure 7 Results of the in vitro anti-osteoclastogenesis assay of Mel@RCNVs. (A) Schematic design of the RANKL clearance assay; (B) Relative concentrations of RANKL after incubation with RCNVs, Mel@RCNVs, and Mel@OST-RCNVs (with or without pre-incubation with RANK and CXCR4 antibodies), with nanovesicles from primary bone marrow mesenchymal stem cells as control, n=5; (C) Quantitative analysis of relative c-Fos mRNA expression, n=5; (D) Quantitative analysis of relative Nfatc1 mRNA expression, n=5; (E) Relative Mmp9 Quantitative analysis of mRNA expression, n = 5; (F) Quantitative analysis of relative Ctskm RNA expression, n = 5; (G I) Concentrations of p-ERK, p-JNK and p-p38 determined by ELISA (n = 5); (J) Quantitative analysis of TRAP-positive areas (n = 3); (K) Representative microscopic images of TRAP staining, scale bar = 100 μm; (L) Quantitative analysis of fusion index (n = 3); NS means no significant difference, * means p < 0.05, ** means p < 0.01; *** means p < 0.001, # means p < 0.0001.

[0025] Figure 8 The results show that the neutralization effect of Mel@RCNVs on RANKL is dose-dependent. The data are expressed as mean ± standard deviation, n = 3.

[0026] Fig. 9Results of the in vitro osteogenesis-promoting effect of Mel@OST-RCNVs via RANKL reverse signaling. (A) Representative confocal images showing immunofluorescence staining of Runx2 and Osterix at days 3 and 7 during osteogenic differentiation (green: fluorescein isothiocyanate-labeled phalloidin; red: Runx2 or Osterix; blue: DAPI), scale bar = 20 μm. (BC) Quantitative analysis of Runx2 and Osterix fluorescence intensity. (D) Microscope photos of osteoblasts stained with Alizarin Red S at day 14, scale bar = 100 μm. (E) Quantification of optical density values ​​of Alizarin Red S staining, n = 5. (FI) Relative mRNA expression of Runx2, Osterix, Col1a1, and Alp genes after treatment with Mel@OST-RCNVs with or without sRANKL preincubation, n = 3. (J) Western blotting was used to detect the activation of PRAS40, S6K, and Akt in the PI3K-Akt-mTORC1 pathway in osteoblasts. Cells were stimulated with Mel@RCNVs and Mel@OST-RCNVs with or without preincubation of sRANKL (NS, no significant difference; *, p < 0.05, **, p < 0.01; ***, p < 0.001; #, p < 0.0001).

[0027] Fig.10 Results of melatonin-induced phagocytosis functional assay to restore circadian rhythm dependence. (A) Expression of Bmal1, Clock, Cry1, and Per1 genes in bone marrow-derived macrophages from young mice and BMDMs from old mice within 48 h, Young for BMDMs from young mice, Old for BMDMs from old mice, Young+siBmal1 for BMDMs from young mice treated with small interfering RNA of Bmal1 gene, Old+Mel for BMDMs from old mice treated with Mel, n=3; (B) Schematic design of phagocytosis assay; (C) Representative confocal images showing phagocytosis; green is FITC-phalloidin, red is apoptotic osteoblasts, blue is DAPI), scale bar=20 μm; (D) Quantitative analysis of phagocytosis index (NS, no significant difference; ***, p<0.001).

[0028] Fig.11Results of restoration of circadian rhythm and phagocytosis function of Mel@RCNVs / Mel@OST-RCNVs. (A) Expression of Bmal1, Clock, Cry1 and Per1 genes in bone marrow-derived macrophages from young mice and BMDMs from old mice within 48 h, Young is BMDMs from young mice, Old is BMDMs from old mice, RCNVs is BMDMs from old mice treated with RCNVs, Mel@RCNVs is BMDMs from old mice treated with Mel@RCNVs, Mel@OST-RCNVs is BMDMs from old mice treated with Mel@OST-RCNVs, n=3; (B) Representative confocal images showing phagocytosis (green: FITC-phalloidin; red: apoptotic osteoblasts; blue: DAPI), scale bar = 20 μm; (C) Quantitative analysis of phagocytosis index (NS, no significant difference; ***, p<0.001).

[0029] Fig.12 The results of the anti-osteoporosis effect of bone-targeted nanovesicles in senile osteoporosis (SOP) mice. Among them, (A) Schematic design of the animal treatment experiment in this study; (B) Bar graph showing the maximum load of the mouse tibia after treatment, n = 5; (C) Bar graph showing the stiffness of the mouse tibia after treatment; (D) Representative micro-computed tomography (μCT) images showing the bone microarchitecture of the distal femur of the mouse after three-dimensional digital reconstruction; (EH) Bar graphs showing bone density (BMD), trabecular volume fraction (Tb.BV / TV), trabecular number (Tb.N) and trabecular spacing (Tb.Sp) after treatment, respectively, = 5; (IM) Bar graphs showing the concentrations of RANKL, TRAP5b, β-CTX, P1NP and BAP in serum after treatment, respectively, n = 5; NS is no significant difference, * is p < 0.05, ** is p < 0.01; *** is p < 0.001, # is p < 0.0001.

[0030] Fig.13 The bar graphs show the concentrations of osteoprotegerin (OPG) (A) and tumor necrosis factor-α (TNF-α) in mouse plasma. The data are expressed as mean ± standard deviation, n = 5; NS means no significant difference; * means p < 0.05; ** means p < 0.01.

[0031] Fig.14Results of bone-targeted nanovesicles restoring circadian rhythm-mediated phagocytosis and bone metabolic homeostasis in vivo. (A) Representative microscope images of bone tissue sections after TRAP staining, scale bar = 30 μm; (B) Representative immunofluorescence staining images and fluorescence intensity quantitative analysis of Runx2 in bone tissue sections (red: Runx2; blue: DAPI), scale bar = 100 μm; (C) Representative immunofluorescence staining images and fluorescence intensity quantitative analysis of OCN in bone tissue sections (red: OCN; blue: DAPI), scale bar = 100 μm; (D) Representative immunofluorescence staining images and fluorescence intensity quantitative analysis of Bmal1 and CD68 in bone tissue sections (red: CD68; green: Bmal1; blue: DAPI), scale bar = 100 μm; (E) Representative immunofluorescence staining images and fluorescence intensity quantitative analysis of Clock and CD68 in bone tissue sections (A) Representative immunofluorescence staining images and quantitative analysis of fluorescence intensity (red: CD68; green: Clock; blue: DAPI), scale bar = 100 μm; (F) Representative immunofluorescence staining images and quantitative analysis of fluorescence intensity of Mertk and CD68 in bone tissue sections (red: CD68; green: Mertk; blue: DAPI), scale bar = 100 μm; (G) Representative immunofluorescence staining images and quantitative analysis of fluorescence intensity of Mertk and CD90 in bone tissue sections (red: CD90; green: Mertk; blue: DAPI), scale bar = 100 μm; NS means no significant difference, * means p < 0.05, ** means p < 0.01; *** means p < 0.001, # means p < 0.0001; the color legend boxes in the figure represent the same meaning as Fig.13 Consistent.

[0032] Fig.15 The results of blood biochemical analysis of plasma samples of mice in each group. All values ​​were normalized to the values ​​of healthy mice at 12 weeks of age without drug treatment, n = 3; Scr is serum creatinine; BUN is blood urea nitrogen; TBil is total bilirubin; ALT is alanine aminotransferase; AST is aspartate aminotransferase.

[0033] Fig.16 The results of blood routine examination of whole blood samples of mice in all groups. All values ​​were normalized to the values ​​of 12-week-old healthy mice without drug treatment. DETAILED DESCRIPTION

[0034] The present invention provides a bone-targeted nanovesicle for treating osteoporosis, wherein the bone-targeted nanovesicle is any one of the following nanovesicles: (1) a bone marrow mesenchymal stem cell nanovesicle loaded with melatonin; (2) a bone marrow mesenchymal stem cell nanovesicle loaded with melatonin modified by (AspSerSer)6; (3) a combination of (1) and (2). The bone marrow mesenchymal stem cell nanovesicle loaded with melatonin of the present invention is referred to as Mel@RCNVs, the bone marrow mesenchymal stem cell nanovesicle loaded with melatonin modified by (AspSerSer)6 is referred to as Mel@OST-RCNVs, the combination of Mel@RCNVs and Mel@OST-RCNVs is referred to as Nano-Cocktail, and the bone marrow mesenchymal stem cell nanovesicle is referred to as RCNVs.

[0035] In the present invention, the bone marrow mesenchymal stem cell nanovesicles are obtained by squeezing bone marrow mesenchymal stem cells. The bone marrow mesenchymal stem cells described in the present invention are bone marrow mesenchymal stem cells that overexpress RANK and CXCR4. The bone marrow mesenchymal stem cells that overexpress RANK and CXCR4 described in the present invention are obtained by editing conventional bone marrow mesenchymal stem cells through gene editing.

[0036] The present invention also provides a method for preparing the bone-targeted nanovesicles, comprising the following steps: (1) dispersing bone marrow mesenchymal stem cells in a PBS solution, then squeezing, centrifuging for the first time to collect the precipitate, resuspending the precipitate, centrifuging again, collecting the precipitate, and obtaining bone marrow mesenchymal stem cell nanovesicles (RCNVs); (2) mixing the bone marrow mesenchymal stem cell nanovesicles with melatonin by ultrasound, then incubating the mixture at 36-38° C. for 0.5-1.5 h, centrifuging and collecting the precipitate, and obtaining bone marrow mesenchymal stem cell nanovesicles (Mel@RCNVs) loaded with melatonin; and (3) The bone marrow mesenchymal stem cell nanovesicles are incubated with (AspSerSer)6 coupled with SPE-PEG-NHS at 3-5°C overnight, the precipitate is collected by centrifugation, washed and suspended in water to obtain (AspSerSer)6-modified bone marrow mesenchymal stem cell nanovesicles loaded with melatonin (Mel@OST-RCNVs); (4) the bone marrow mesenchymal stem cell nanovesicles loaded with melatonin are mixed with the bone marrow mesenchymal stem cell nanovesicles loaded with melatonin modified with (AspSerSer)6 to obtain a nanovesicle composition (Nano-Cocktail). The Mel@RCNVs and Mel@OST-RCNVs described in the present invention are prepared in combination: the content of the active drug carrier Mel@RCNVs accounts for 5%-30% of the total weight, and the Mel@OST-RCNVs is supplemented to 100%.

[0037] In the present invention, the bone marrow mesenchymal stem cells need to be gene-edited to obtain bone marrow mesenchymal stem cells that overexpress RANK and CXCR4. The step of obtaining the bone marrow mesenchymal stem cells that overexpress RANK and CXCR4 in the present invention includes: obtaining the coding sequence (CDS) and amino acid sequence of Tnfrsf11a (encoding Rank protein) and Cxcr4 genes, then constructing a Tnfrsf11a overexpression lentiviral vector and transfecting bone marrow mesenchymal stem cells, screening RANK-positive cells, and then transfecting the screened positive cells with a CXCR4 overexpression lentiviral vector, and after 72 hours of proliferation, RANK and CXCR4 double-positive cells are screened out to obtain bone marrow mesenchymal stem cells that overexpress RANK and CXCR4.

[0038] In the present invention, the pore sizes of the membranes used for extrusion are 4-6 μm, 0.5-1.5 μm, preferably 5 μm, 1 μm. The membrane of the present invention is a polycarbonate membrane.

[0039] In the present invention, the first centrifugation parameter in step (1) is: 0.5×10 5 -1.5×10 5 g, 5-10 min; the parameters for the second centrifugation are: molecular weight cut-off 80-120 kDa, 900-1100 g, 8-12 min. The first centrifugation parameter in step (1) of the present invention is preferably 1.0×10 5 g, 8 min, and the parameters for the second centrifugation were: molecular weight cutoff 100 kDa, 1000 g, 10 min.

[0040] In the present invention, the amount of bone marrow mesenchymal stem cell nanovesicles used in step (2) is 10 9 -10 11 Particles, preferably 10 10 particles; the amount of melatonin used is 490-510 mg, preferably 500 mg.

[0041] In the present invention, the ultrasonic parameters in step (2) are: amplitude 18-22%, 28-32s on / off cycle 2.5-3.5min, repeated 5-7 times, with a cooling interval of 1.5-2.5min between each cycle; preferably, amplitude 20%, 30s on / off cycle 3min, repeated 6 times, with a cooling interval of 2min between each cycle.

[0042] In the present invention, the centrifugation parameters in step (2) and step (3) are: first centrifuge at 1100-1300 rpm for 10 min, then centrifuge at 1×10 4 -2×10 4g for 25-35 min; preferably, centrifuge at 1200 rpm for 8-12 min and then at 1.2×10 4 The mixture was centrifuged at 400 g for 30 min. The purpose of the first low-speed centrifugation in step (2) of the present invention is to remove unencapsulated Mel, and the purpose of the second high-speed centrifugation is to further purify the RCNVs precipitate loaded with Mel. In step (3) of the present invention, the purpose of the first low-speed centrifugation is to remove unreacted DSPE-PEG-NHS-(AspSerSer)6, and the purpose of the second high-speed centrifugation is to further purify the Mel@OST-RCNVs precipitate.

[0043] In the present invention, the concentration of the bone marrow mesenchymal stem cell nanovesicles loaded with melatonin in step (3) is 10 11 -10 13 Particles / mL, preferably 10 12 The concentration of the DSPE-PEG-NHS-coupled (AspSerSer)6 is 8-12 μM, preferably 10 μM. The DSPE-PEG-NHS-coupled (AspSerSer)6 solution of the present invention is prepared according to conventional methods and is prepared by the Orthopedic Laboratory of Shanghai Sixth Hospital.

[0044] The present invention also provides the use of the bone-targeted nanovesicle or the bone-targeted nanovesicle obtained by the preparation method in the preparation of a drug for preventing and / or treating osteoporosis.

[0045] In the present invention, the Mel@RCNVs are dispersed in the bone microenvironment (BME) and are designed to play an anti-osteoclastogenic effect by removing RANKL; the Mel@OST-RCNVs target the bone formation surface, bind to the RANKL of osteoblasts and activate the RANKL reverse signal to promote osteogenesis. The present invention prepares nanovesicles. In the RANK-RANKL signal, the RANKL of osteoblasts binds to the RANK of osteoclasts to trigger osteoclast formation; in the RANKL reverse signal, the osteoclast-derived vesicles expressing RANK bind to the RANKL on the surface of osteoblasts and activate the transcription of osteogenesis-related factors. The nanovesicles prepared by the present invention mimic the osteoclast-derived vesicle RANK, and promote osteogenesis through the RANKL reverse signal, restoring the osteoblast / osteoclast balance and the macrophage efferocytosis. The present invention proposes a promising bone-targeted nanovesicle for treating senile osteoporosis by restoring the osteoblast / osteoclast balance and regulating the efferocytosis function mediated by the circadian clock of macrophages.

[0046] In the present invention, unless otherwise specified, all components, reagents or culture media are commercially available products well known to those skilled in the art.

[0047] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] Example 1

[0049] 1. Cell culture

[0050] Isolation and culture of bone marrow mesenchymal stem cells (BMSCs): C57BL / 6J (male, 6 weeks old) were sacrificed and sterilized with 75% alcohol. The femur and tibia were dissected and cut at the end. The bone marrow was rinsed with PBS, and the rinsed bone marrow cells were collected and resuspended with mouse bone marrow mesenchymal stem cell culture medium (MUXMX-90011, OriCell, CyagenBioscience, Guangzhou, China). When the cells reached 80% confluence, they were separated using Solase cell dissociation buffer (RP01021, Xiu Ren Biotechnology Co., Ltd.). When cultured to the third generation, they were used for subsequent experiments. Positive surface markers [CD90 (bsm-30163A-FITC, Bioss, China), CD105 (A25377, ABclonal Technology, Wuhan, China), and CD73 (E-AB-F1089D, ElabScience, Wuhan, China)] and negative markers of BMSCs [CD34 (A23108, ABclonal Technology) and CD45 (E-AB-F1136UF, ElabScience)] were used for flow cytometry (FCM) (FongCyte TM , Beijing Charon Biotechnology Co., Ltd.) cell identification.

[0051] Isolation and culture of macrophages (BMDMs): Mouse bones were obtained and bone marrow cells were washed as above. These cells were collected and cultured in 6-well plates (CLPT006, Parker Gent Bioscience Co., Ltd., Suzhou, China) using modified Eagle's medium α (MEMα) (PM150421, Procell Life Science & Technology, Wuhan, China) containing 10% FBS overnight. After 24 h, adherent cells were discarded and floating cells were collected to continue cell culture. At this stage, macrophage colony stimulating factor (M-CSF) (30 ng / ml, CB34, Novotin, Shanghai, China) was supplemented in the culture medium to stimulate macrophage colony formation.

[0052] 2. Gene Editing of Bone Marrow Mesenchymal Stem Cells

[0053] The coding sequence (CDS, NCBI No. 7852) and amino acid sequence of Tnfrsf11a (encoding Rank protein NCBI No. 8792) and Cxcr4 genes were obtained from NCBI. Then, a Tnfrsf11a overexpression lentiviral vector was constructed and transfected into the bone marrow mesenchymal stem cells obtained from the above culture. RANK-positive cells were sorted using fluorescence-activated cell sorting (FACS). These cells were then transfected with a CXCR4 overexpression lentiviral vector. After 72 hours of proliferation, these cells were sorted using FACS to obtain double-positive cells for subsequent research. The gene editing of the above-mentioned bone marrow mesenchymal stem cells was entrusted to Shanghai Zhichuang Haotian Biotechnology Co., Ltd.

[0054] 3. Preparation and characterization of bone marrow mesenchymal stem cell nanovesicles (RCNVs)

[0055] 1×10 6 The above gene-edited MSCs were dispersed in PBS and extruded through 5 μm and 1 μm pore size polycarbonate membranes using a micro extruder (Avanti PolarLipids, Birmingham, Alabama). The extruded samples were then placed on a 1 × 10 5 The precipitate was collected by centrifugation at 1000 g for 8 min. The precipitate was resuspended and further purified by centrifugation at 1000 g for 10 min using a centrifugal filter (Millipore) with a molecular weight cutoff of 100 kDa. The total protein concentration of the purified RCNVs was detected using a BCA protein assay kit. The particle size and zeta potential of RCNVs were determined using a Zetasizer Nano ZS90 (Malvern). The morphology of RCNVs was observed using a transmission electron microscope (JEM-1200EX, JEOL Co., Ltd.).

[0056] 4. Melatonin (Mel) loading

[0057] RCNVs (10 10 The mixture was then incubated at 37°C for 1 h to promote membrane recovery. The unencapsulated Mel was then removed by centrifugation at 1200 rpm for 10 min. The mixture was then centrifuged at 1.2 × 10 4 The Mel-loaded RCNVs precipitate was further purified by centrifugation at 400 × g for 30 min, and the precipitate was resuspended in ultrapure water to obtain melatonin-loaded bone marrow mesenchymal stem cell nanovesicles (Mel@RCNVs).

[0058] 5. Preparation and characterization of Mel@OST-RCNVs

[0059] Mel@RCNVs(10 12 The cells were incubated with DSPE-PEG-NHS-conjugated (AspSerSer)6 (10 μM, prepared by the Shanghai Sixth Hospital Orthopedics Laboratory according to conventional methods) in ultrapure water at 4°C overnight. Unreacted DSPE-PEG-NHS-(AspSerSer)6 was removed by centrifugation at 1200 rpm for 10 min. 4 After centrifugation at 400 × g for 30 min, the Mel@OST-RCNVs pellet was washed and resuspended in ultrapure water to obtain Mel@OST-RCNVs. To verify the successful modification, the (AspSerSer)6 peptide was labeled with Cy3 orange-red fluorescence, and the Cy3-labeled Mel@OST-RCNVs were captured by NHS-magnetic beads (1 μM, BMB-1-N, Biotyscience, Beijing, China) incubated with RANKL and then observed under an IX71 Olympus microscope (Olympus, Tokyo, Japan).

[0060] 6. Combination preparation of Mel@RCNVs and Mel@OST-RCNVs: The content of active drug carrier Mel@RCNVs) accounts for about 25% of the total weight, and the excipient or carrier material (such as Mel@OST-RCNVs) is supplemented to 100%.

[0061] Test Example 1

[0062] 1. Experimental methods

[0063] (1) Melatonin release assay

[0064] In vitro melatonin release assay was performed using PBS (pH = 7.4). Equal amounts of samples (Mel@RCNVs or Mel@OST-RCNVs) were placed in dialysis bags at 37°C. At a series of predetermined time points, 1 mL of solution outside the dialysis bag was taken out of the release system. The concentration of melatonin was determined using an ultraviolet spectrophotometer (UV-2000, LANGUAGES, FRANKVILI, WI).

[0065] (2) Uptake of NVs by BMDMs.

[0066] BMDMs were cultured in special culture dishes for CLSM (2 × 10 4Different NVs (labeled with DiR) were incubated with BMDMs for 4 h, then washed with PBS for 3 min and 3 times, fixed with 4% paraformaldehyde (PFA) for 10 min, incubated with 4',6-diamidino-2-phenylindole (DAPI) and phalloidin-FITC, and then observed with a Leica TCSNT1 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany).

[0067] (3) CCK-8 assay

[0068] The cytotoxicity of NVs was evaluated using a commercial CCK-8 kit (C0005, TargetMol, Boston, USA) using mouse BMDMs and BMSCs. These cells were incubated with different concentrations of Mel@RCNVs or Mel@OST-RCNVs for 24 h. Cell viability was then measured following the manufacturer’s protocol.

[0069] (4) In vitro RANKL neutralization

[0070] RANKL (200 pg / ml) (PRP1114, Abbkine, Wuhan, China) solution was prepared and mixed with RCNVs, Mel@RCNVs and Mel@OST-RCNVs (1.0×10 10 After incubation at 37°C for 2 h, the mixture was incubated at 1.2×10 4 The supernatant was collected and the concentration of residual RANKL was determined using an enzyme-linked immunosorbent assay kit (Shanghai ZCIBIO Technology, Shanghai, China).

[0071] (5) Anti-osteoclastogenesis effect in vitro

[0072] BMDMs were plated at 2.0 × 10 per well. 5 Cells were seeded at a density of 10 cells / mL in a 24-well plate (801006, NEST Biotechnology, Wuxi, China) overnight. The culture medium components were as follows: MEMα, 12% fetal bovine serum (FBS) (UR50100, Umibio Co., Ltd., Shanghai, China), macrophage colony stimulating factor (M-CSF) (30 ng / ml), and RANKL (50 ng / ml). Then, PBS, RCNVs, and Mel@RCNVs (1.0 × 10 cells / well) were added to each well, respectively. 9 After 5 days, the cells were treated with 4% PFA for 10 min, then rinsed three times with PBS and stained with tartrate-resistant acid phosphatase (TRAP) staining solution (Sigma Aldrich).

[0073] (6) Polarization of bone marrow-derived macrophages

[0074] BMDMs were cultured in glass bottom culture dishes (6 × 10 4 To induce M1 polarization, MEMα medium containing 12% FBS, lipopolysaccharide (LPS) (500 ng / mL) and interferon-γ (20 ng / mL) was used to induce M1 polarization for 48 h. To study the effects of Mel@RCNVs and Mel@OST-RCNVs, Mel@RCNVs and Mel@OST-RCNVs were supplemented to the culture system during the induction process.

[0075] (7) Immunofluorescence staining

[0076] Cells cultured in glass-bottomed culture dishes (1050000, SAINING) for confocal microscopy were treated with 4% PFA for 10 min, then rinsed three times with PBS and permeabilized with 0.2% TritonX-100 for 5 min. For BMDMs and osteoblasts that required phalloidin staining, they were incubated with 1.0% bovine serum albumin (BSA) solution containing phalloidin-FITC (YEASEN Biotechnology Co., Ltd., Shanghai, China) for 30 min; after washing again, the cells were incubated with primary antibodies overnight. For BMSCs overexpressing RANK and CXCR4, they were directly incubated with primary antibodies overnight, then rinsed three times with PBS for 3 min each time, and then incubated with fluorescent dye-conjugated secondary antibodies for 1 h. The cells were then washed again and stained with DAPI. Among them, the primary antibodies used included Runx2 (RM3794, Biodragon, Suzhou, China), Osterix (abs120764, Absin, Shanghai, China), CXCR4 (E1A064G-1, EnoGene, Nanjing, China), and RANK (ATA36616, AtaGenix Laboratory Co., Ltd., Wuhan, China).

[0077] (8) Osteogenesis Induction

[0078] Osteoblasts MC3T3-E1 (CM-0378, Purcell) were thawed from a special freezing medium (AC05L053, Life-iLab, Shanghai, China) and cultured overnight in MEMα and 15% fetal bovine serum (FBS) (Inner Mongolia Wanrui Biotechnology Co., Ltd.). When good proliferation was restored, they were re-seeded into 12-well plates (803012, CELLPROBIO, Suzhou, China) (4 × 10 cells per well). 4cells) and cultured again overnight. When 70-80% attachment was reached, they were cultured for osteogenic induction in commercial mouse osteogenic differentiation medium (ODM) (Orisai). The culture medium was changed every 3 days. To study the effects of Mel@RCNVs or Mel@OST-RCNVs, Mel@RCNVs or Mel@OST-RCNVs were supplemented to the culture system during the induction process, respectively. The formation of calcium nodules was evaluated using Alizarin Red S staining solution (G1450, Solarbio, Beijing, China) according to the manufacturer's protocol. Alkaline phosphatase (ALP) activity, which reflects osteogenic activity, was detected using an alkaline phosphatase activity assay kit (BC2145, Solarbio) according to the manufacturer's protocol.

[0079] (9) Biodistribution of nanovesicles (NVs)

[0080] Each NV was stained with lipophilic DiR or DiO fluorescent dye (KGE2604-10, Jiyuan Biotechnology, Nanjing, China). Each mouse was injected via the tail vein with 50 μL of 1.0 × 10 10 At 4h and 8h after injection, the main organs of DiR-injected mice were collected and imaged using the AniView100 multimodal in vivo animal imaging system (Guangzhou Bio-Thera Biotechnology Co., Ltd.).

[0081] (10) Senile osteoporosis (SOP) model and nanovesicle drug delivery

[0082] Male C57 / BL6 mice aged 56 weeks were housed in specific pathogen-free cages. Mel@RCNVs, Mel@OST-RCNVs, and the combination of Mel@RCNVs and Mel@OST-RCNVs (Nano-Cocktail) were intravenously injected once a week for 8 weeks. The dose was 1.0×10 10 NVs. Male C57 / BL6 mice aged 56 weeks injected with an equal amount of PBS were used as the control group. Young mice aged 12 weeks injected with an equal amount of PBS were used as the reference normal group.

[0083] (11) Enzyme-linked immunosorbent assay (ELISA)

[0084] For ELISA of cell culture supernatants, the supernatants were centrifuged at 300 × g for 10 min and at 1.2 × 10 3The supernatant was collected by centrifugation at 1000 × g for 15 min. The supernatant was then used for ELISA test according to the manufacturer's instructions. For the detection of serum cytokines and bone biochemical markers, blood samples were centrifuged at 1000 g for 30 min to obtain serum, and then ELISA test was performed using ELISA kits according to the manufacturer's instructions. The ELISA kits used in this study were as follows: interleukin-1β (IL-1β, JLW18442-48T, Jianglai Biotechnology, Shanghai, China), mouse type I procollagen N-terminal propeptide (P1NP, JL20174-48T, Jianglai Biotechnology, Shanghai, China), tumor necrosis factor-α (TNF-α, RE1060MF, Red Biotechnology, Wuhan, China), bone alkaline phosphatase (BAP, MU30270; β-cross-linked C-terminal peptide (β-CTX, MU30091; tartrate-resistant acid phosphatase 5b (TRAP5b, MU30923; Bio-Swamp, Wuhan, China).

[0085] (12) Routine blood examination and biochemical test

[0086] For routine blood test, mice were anesthetized with sodium pentobarbital, and whole blood was obtained through the ophthalmic venous plexus. Blood was collected in K2-EDTA blood collection tubes and analyzed using an automatic blood analyzer (Mindray BC5000, Mindray Co., Ltd., Shenzhen, China). For biochemical detection, serum was obtained from whole blood by centrifugation, and serum was analyzed using an automatic chemical analyzer (Ray Du Chemray-240, Ray Du Life and Analytical Science Co., Ltd., Shenzhen, China).

[0087] (13) Quantitative polymerase chain reaction (qPCR)

[0088] Cells were collected and total RNA was extracted using the SteadyPure Universal RNA Extraction Kit (AG21017, Hunan Accurate Biotechnology Co., Ltd., Changsha, China). The collected RNA was treated with a mouse ribonuclease inhibitor (K1046, APExBIO, Houston, USA) to avoid degradation. The mRNA samples were then reverse transcribed using a complementary DNA (cDNA) synthesis kit (Takara Bio, Shiga, Japan). PCR reactions were performed using a BioRad CFX96 instrument (BioRad, California, USA) and a SYBR GreenqPCRMix kit (Starlight, FS-Q1002 Beijing Everlasting Star Biotechnology).

[0089] (14) Western Blotting

[0090] These nanovesicles (NVs) or cells were treated with a protein extraction kit (Beyotime) to obtain total protein samples. A protease and phosphatase inhibitor cocktail (P001, Suzhou New Cell & Molecular Biotech) was used to avoid protein degradation and dephosphorylation. YoungPAGE gels prepared commercially were used. TM Western blot analysis was performed with the help of GenScript (Nanjing GenScript Biotechnology Co., Ltd., GenScript, M01004). The primary antibodies used in this section include: RANK (Nanjing Bioworld Biotechnology Co., Ltd., Bioworld, BZ16158), CXCR4 (Wuhan Abotek Biotechnology Co., Ltd., ABclonal Technology, A1303), Akt (Chengdu Zhengneng Biotechnology Co., Ltd., ZENBIO, 342529), p-Akt (Thr308) (Chengdu Zhengneng Biotechnology Co., Ltd., ZENBIO, 341790), S6K1 (Chengdu Zhengneng Biotechnology Co., Ltd., ZENBIO, 380469), p-S6K1 (Thr389) (Chengdu Zhengneng Biotechnology Co., Ltd., ZENBIO, 310310), PRAS40 (Chengdu Zhengneng Biotechnology Co., Ltd., ZENBIO, R381889) and PRAS40 (Thr246) (Chengdu Zhengneng Biotechnology Co., Ltd., ZENBIO, 370375). Horseradish peroxidase (HRP)-labeled secondary antibody (Wuhan Abbkine Biotechnology Co., Ltd., Abbkine, A21020) and Sparkjade ECL ultrasensitive luminescent liquid (Shandong Sparkjade Biotechnology Co., Ltd., ED0015-A) were used to visualize protein bands.

[0091] (15) Immunofluorescence (IF) staining of bone tissue sections

[0092] Mouse bone samples were fixed with 4% ice-cold paraformaldehyde (PFA) overnight and then rinsed with a commercial 10% ethylenediaminetetraacetic acid (EDTA) decalcification solution for 4 weeks. Then, rinsed overnight with a dehydration solution [2% polyvinyl pyrrolidone (PVP) and 20% sucrose]. After that, the bones were embedded in an embedding gel (2% PVP, 9% gelatin, and 20% sucrose) for quick freezing and then cut into 10 μm thick slices. These slices were incubated overnight with various primary antibodies. The primary antibodies used in this section include: RANK (Wuhan AtaGenix Laboratories Co., Ltd., ATA36616), CXCR4 (Wuhan Aiboteike Biotechnology Co., Ltd., ABclonalTechnology, A1303), and osteocalcin (Hangzhou Hua'an Biotechnology Co., Ltd., HUABIO, ER1919-20). After incubation with the primary antibody, the sections were washed with PBS for 3 minutes each time for a total of 3 times, and then incubated with the fluorescent dye-labeled secondary antibody for 1 hour. After that, the sections were washed again and treated with 4',6-diamidino-2-phenylindole (DAPI). The sections were then observed and photographed under a microscope.

[0093] (16) Tartrate-resistant acid phosphatase (TRAP) staining of bone tissue sections

[0094] Mouse bone samples were collected and fixed and decalcified according to the above method. The samples were then sliced ​​into 10 μm slices with the help of Wuhan Pinnofei Biotechnology Co., Ltd. Afterwards, TRAP staining was performed on the slices using a TRAP staining kit (Beijing Solarbio Technology Co., Ltd., Solarbio, G1492). The nuclei were stained with 1% methyl green staining solution (Beijing Solarbio Technology Co., Ltd., Solarbio, G1652). The positive staining areas were analyzed by ImageJ software (National Institutes of Health, NIH, Bethesda, Maryland).

[0095] (17) Microcomputed tomography (μCT) analysis

[0096] The dissected femur was fixed with 4% paraformaldehyde overnight and then scanned using a μCT device (Bruker, Skyscan 1276). The region of interest for trabecular bone analysis was set around the growth plate, approximately 0.15 mm distal to the growth plate to 0.4 mm proximal. CT-Vox software was used for three-dimensional reconstruction of the image, and CT-An software was used to obtain tissue morphometric parameters.

[0097] (18) Statistical analysis

[0098] Statistical analysis was performed using GraphPadPrism 8.0. Statistical comparisons between two groups were performed using Student's t test. Statistical comparisons between three or more groups were performed using one-way ANOVA. Numerical data were expressed as mean ± standard deviation (Mean ± SD). In this study, statistical differences were defined as: *P < 0.05, **P < 0.01, ***P < 0.001, and #P < 0.0001.

[0099] 2. Results Analysis

[0100] (1) Preparation and characterization results of Mel@RCNVs and Mel@OST-RCNVs

[0101] The preparation process of Mel@RCNVs and Mel@OST-RCNVs is shown in the figure. Figure 2 As shown in A. IF staining confirmed that after gene editing of BMSCs, RANK and CXCR4 were overexpressed in BMSCs via lentiviral vector ( Figure 2 B). IF was then used to confirm the retention of RANK and CXCR4 in RCNVs ( Figure 2 C) Since no permeabilization was performed, the binding of antibodies further demonstrated that these antigenic proteins are located on the outer surface of RCNVs.

[0102] In addition, the amount of RANK and CXCR4 on control NVs (NVs prepared from unedited BMSCs using the same method) and RCNVs was detected by ELISA, and it was determined that the amount of CXCR4 and RANK on each RCNV was 52 and 77, respectively, while less than 1 CXCR4 or RANK protein was detected on each control NV ( Figure 2 D) and then measured by dynamic light scattering (DLS) 6 The average yield of RCNVs at the single-cell level was 24213 particles per cell ( Figure 2 E) Using a protein quantification kit, the protein 6 The average protein content of RCNVs prepared from 10 cells was 70.12 μg, or 2.90 × 10 -6 ng protein. Transmission electron microscopy (TEM) images showed that RCNVs were spherical ( Figure 2 F).

[0103] Furthermore, Mel was loaded into RCNVs using ultrasound with an encapsulation efficiency of 23.19%. To verify the successful modification of NVs, the carboxyl terminus of the peptide was labeled with a Cy3 fluorophore. RANKL-binding immunomagnetic beads were used to capture the modified NVs. Strong orange-red fluorescence ( Figure 2 G), indicating that (AspSerSer)6 was successfully anchored on these NVs. DLS measurements showed that these NVs had similar size distributions, with a peak diameter of approximately 110 nm ( Figure 3 ).

[0104] Subsequently, the release characteristics of Mel@RCNVs and Mel@OST-RCNVs were studied in vitro in 50% fetal bovine serum (FBS) at 37°C, which simulates body fluid. Figure 2 As shown in Figure 3, Mel showed a slow release, reaching a plateau of approximately 90% at 48 h. This release profile can avoid undesired release in the circulation before achieving bone-specific accumulation. After Mel loading and (AspSerSer)6 peptide modification, Western blot analysis confirmed that all of these NVs retained abundant RANK and CXCR4 proteins ( Figure 2 I), indicating that these nanomanipulations had no destructive effect on these proteins.

[0105] In addition, Mel@RCNVs and Mel@OST-RCNVs were incubated in 50% FBS and their diameter and ζ potential changes were monitored. Both NVs showed stable size and ζ potential within a week without obvious changes ( Figure 2 J). Moreover, cell-derived NVs generally have a good safety profile. Mel@RCNVs and Mel@OST-RCNVs are not cytotoxic even at very high concentrations ( Figure 4 ) and negligible hemolysis ( Figure 5 ).

[0106] (2) Results of bone targeting ability determination of nanovesicles (NVs)

[0107] The bone-targeting properties of the nanovesicles were further investigated using biophotonic imaging and tissue sections. Nanovesicles prepared from gene-edited BMSCs or pristine BMSCs were stained with DiR, a lipophilic fluorescent dye, and administered intravenously ( Figure 6 A). Then, the main organs were collected for biodistribution assay after 4h and 8h, such as Figure 6As shown in B, the fluorescence intensity of Mel@RCNVs and Mel@OST-RCNVs groups in bones was higher than that of the control group, and there was no statistical difference in fluorescence intensity between Mel@RCNVs and Mel@OST-RCNVs. In addition, the fluorescence intensity of Mel@RCNVs and Mel@OST-RCNVs in other organs was lower than that of the control group ( Figure 6 C).

[0108] The histological distribution of Mel@OST-RCNVs in bone tissue was further observed. First, the osteoblast surface was labeled with xylenol orange (XO, a red fluorescent dye that specifically binds to the newly mineralized part of the bone) and the NVs were labeled with the green fluorescent DiO dye. A clear colocalization was observed in the Mel@OST-RCNVs group, while this red-green colocalization was hardly observed in the Mel@RCNVs and control groups ( Figure 6 D). This result suggests that Mel@OST-RCNVs are distributed in bone-forming areas. Then, the cellular distribution of these NVs was investigated; the colocalization of Col1a1 (a marker for early osteoblasts) and NVs was evaluated in order to determine whether the NVs were expressed in the bone. + Distribution in osteoblasts. Col1a1 is a collagen component in the bone matrix, so cortical and trabecular bones are Col1a1 positive and can be identified under a microscope. + Obvious colocalization was observed in the cells, while this colocalization was hardly observed in Mel@RCNVs and the control group ( Figure 6 E, 7F).

[0109] The macrophage phagocytosis of these nanovesicles was further investigated. Macrophages were labeled with CD68, and colocalization was observed when they engulfed DiR-labeled NVs. Only a small amount of red fluorescence was observed in the control group due to the lack of bone targeting ability. In Mel@RCNVs and Mel@OST-RCNVs, red-green colocalization areas were very common, indicating that all NVs in these groups could be cleared by macrophage phagocytosis. Therefore, macrophages are expected to play a therapeutic role after "ingesting" these nanovesicles.

[0110] (3) Anti-osteoclastogenesis effect of NVs in each group in vitro

[0111] Because Mel@RCNVs are dispersed in the bone microenvironment rather than distributed at the bone formation interface, Mel@RCNVs are designed to exert anti-osteoclastogenic effects by scavenging RANKL. First, the RANK-mediated RANKL uptake effect was investigated by quantifying the remaining RANKL by ELISA after 2 h of incubation with different nanovesicles (NVs). Figure 7 A). Figure 7 B and Figure 8 As shown in Figure 3, RCNVs, Mel@RCNVs, and Mel@OST-RCNVs all exhibited significant RANKL clearance effects, and there was no statistical difference in their RANKL clearance effects. Only anti-RANK preincubation could block this effect, indicating that this effect depends on the abundance of RANK on NVs.

[0112] Next, the anti-osteoclastogenic effect was investigated by detecting the expression of osteoclastogenesis-related genes (c-Fos and Nfatc1) and bone resorption-related genes (Mmp9 and Ctsk) during osteoclastogenesis. Figure 7 As shown in Figure CF, Mel@RCNVs and RCNVs significantly reduced the expression of these genes, and there was no statistical difference between Mel@RCNVs and RCNVs. Then, the phosphorylation of ERK, JNK, and p38 was detected by ELISA to study the activation of the mitogen-activated protein kinase (MAPK) pathway, which plays a key role in osteoclast formation, and the results showed that they were all reduced to a low level close to that of bone marrow-derived macrophages not treated with RANKL ( Figure 7 GI). In addition, the osteoclastogenesis of BMDMs was confirmed by tartrate-resistant acid phosphatase (TRAP) staining, and the results showed that Mel@RCNVs or RCNVs significantly inhibited osteoclastogenesis ( Figure 7 J, Figure 7 K), and reduced the fusion index ( Figure 7 L).

[0113] (4) Verification results of Mel@OST-RCNVs promoting osteogenesis via RANKL reverse signaling in vitro

[0114] When osteoblast precursor cells were induced to differentiate into osteoblasts, they were incubated with different nanovesicles, and the expressions of key osteogenic transcription factors Runx2 and Osterix were determined by immunofluorescence staining on days 3 and 7. Fig. 9 A). The results showed that Mel@OST-RCNVs promoted the expression of Runx2 and Osterix compared with the control group; there was no statistical difference between Mel@OST-RCNVs and Mel@RCNVs or Mel@RCNVs and RCNVs groups ( Fig. 9 B, C), indicating that the effect of melatonin on osteogenic differentiation is negligible; it may be that the activation of osteogenic transcription factors by RANKL reverse signaling is strong enough to make the effect of melatonin unclear. In addition to protein expression, ARS staining was also used to confirm the osteogenic effect. The results ( Fig. 9 D, E) also showed that the RCNV vesicle component of Mel@OST-RCNVs increased the mineralization of osteoblasts.

[0115] Subsequently, we further confirmed whether the RANK protein of Mel@OST-RCNVs plays a core role in activating osteogenesis. Soluble RANKL (sRANKL) was used to deplete the binding ability of Mel@OST-RCNVs with osteoblast RANKL by competition. Then, the transcription of osteogenesis-related genes including Runx2, Osterix, collagen type I α1 (Col1a1) and alkaline phosphatase (Alp) was detected. Fig. 9 As shown in FI, Mel@OST-RCNVs significantly promoted the transcription of these genes, while sRANKL supplementation significantly inhibited the promoting effect of Mel@OST-RCNVs. In addition, the activation of the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin complex 1 (mTORC1) pathway by Mel@OST-RCNVs and the elimination of RANK masking mediated by sRANKL pretreatment were also examined; Fig. 9 As shown in Figure J, stimulation with Mel@RCNVs or Mel@OST-RCNVs resulted in activation and phosphorylation of Akt, S6K, and PRAS40, representing key nodes of the PI3K-Akt-mTORC1 pathway. Mel@OST-RCNVs or Mel@OST-RCNVs occupied with RANK clearly showed significantly weaker activation. These results further support the conclusion that Mel@OST-RCNVs promote osteogenesis by activating RANKL reverse signaling in osteoblasts.

[0116] (5) Verification of Mel@RCNVs / Mel@OST-RCNVs restoration of circadian rhythm and cell efferocytosis function

[0117] BMDMs from young mice (Young), BMDMs from old mice (Old), BMDMs from young mice treated with small interfering RNA of the Bmal1 gene (Young+siBmal1), and BMDMs from old mice treated with Mel (Old+Mel) were taken. Primary BMDMs from young and old mice and drug-treated BMDMs were synchronized with dexamethasone, and the time of circadian rhythm was calculated from the start of dexamethasone synchronization. qPCR was used to measure the periodic oscillation of circadian rhythm-related genes, including brain and muscle ARNT-like 1 (Bmal1), circadian motor output cycle failure (Clock), cryptochrome 1 (Cry1), and cyclin 1 (Per1). In BMDMs of young mice, the expression of Bmal1, Clock, and Cry1 peaked at 12h and returned to the baseline at 24h, showing a regular circadian rhythm. The expression of Per1 decreased slightly at 6h, then increased to a peak at 18h, and then returned to the baseline at 30h, also forming a regular circadian rhythm. Since Bmal1 plays a central role in the circadian clock, its silencing leads to bone loss. Small interfering RNA was used to interfere with the expression of the Bmal1 gene. After treatment with siRNA (siBmal1) of Bmal1, the expression of the Bmal1 gene was maintained at a low level. Due to the cross-regulation of other circadian genes, the regular rhythms and fluctuations of Clock, Cry1, and Per1 were also weakened, which was more similar to the gene expression in BMDMs of aged mice. However, the treatment of aged BMDMs with melatonin (Mel), a drug that regulates circadian rhythms, restored the rhythms and fluctuations of these genes ( Fig.10 A).

[0118] Then, the function of efferocytosis was investigated by incubating BMDMs with apoptotic osteoblasts ( Fig.10 B) If Fig.10 C and Fig.10 As shown in D, young BMDMs exhibited strong efferocytosis, and most of them were able to phagocytose cell debris, while aged BMDMs exhibited impaired efferocytosis; young BMDMs treated with siBmal1 also exhibited impaired efferocytosis, while aged BMDMs treated with Mel restored efferocytosis. These results indicate that the loss of efferocytosis in BMDMs is mainly due to circadian rhythm disturbance.

[0119] Here, in vivo experiments demonstrated that both Mel@RCNVs and Mel@OST-RCNVs could be phagocytosed by bone marrow-derived macrophages (BMDMs). Therefore, we investigated whether Mel@RCNVs and Mel@OST-RCNVs could restore circadian rhythm and efferocytosis in vitro. Fig.11As shown in A, Mel@RCNVs and Mel@OST-RCNVs treatment reestablished the 24h circadian rhythm and the fluctuation of circadian genes. In addition, like the pharmacological intervention of melatonin (Mel), Mel@RCNVs and Mel@OST-RCNVs treatment also restored the efferocytosis function of BMDMs ( Fig.11 B and Fig.11 C).

[0120] (6) Anti-osteoporosis effect of NVs in each group on senile osteoporosis (SOP) mice

[0121] Aged mice were used as SOP models to study the efficacy of NVs in each group. Aged mice were intravenously injected with NVs in each group once a week for 8 weeks ( Fig.12 A). After administration, the mechanical strength of the tibia was first tested by a three-point bending test. The results showed that the maximum load and stiffness of the tibia were greatly enhanced after administration of NVs in each group ( Fig.12 B, C). Microcomputed tomography (μCT) was also used to assess bone mass and microstructure. Fig.12 As shown in D, age-related bone loss was alleviated after treatment with these nanovesicles (NVs). Although the bone mass of each group after NVs administration was lower than that of young mice, it still showed considerable bone mass. After NVs treatment, key bone morphometric parameters such as bone mineral density (BMD) and trabecular bone volume / tissue volume (Tb.BV / TV) were significantly improved in each group ( Fig.12 E, F). Other parameters, such as trabecular number (Tb.N) and trabecular spacing (Tb.Sp), were also close to the levels of young mice ( Fig.12 G, H). As two key components, both Mel@RCNVs and Mel@OST-RCNVs showed significant restoration of bone mass, and the combined effect of Mel@RCNVs and Mel@OST-RCNVs was more significant.

[0122] In addition, plasma concentrations of RANKL, osteoprotegerin (OPG), tumor necrosis factor-α (TNF-α), and other bone turnover biochemical markers such as serum band 5 tartrate-resistant acid phosphatase (TRAP5b), type I collagen carboxyl terminal peptide (β-CTX), type I procollagen N-terminal propeptide (P1NP), and bone-specific alkaline phosphatase (BAP) were measured. Due to the RANKL scavenging effect, all nanovesicles (NVs) administration naturally reduces RANKL ( Fig.12 I), while OPG, a decoy receptor for RANKL, showed no statistical difference. Due to enhanced efferocytosis and clearance of dead cells, the combination of Mel@RCNVs and Mel@OST-RCNVs reduced the pro-inflammatory TNF-α ( Fig.13TRAP5b and β-CTX represent bone resorption activity, which increases due to aging-related bone loss, but were significantly decreased after administration of all NVs ( Fig.12 J, K). P1NP and BAP, two major bone formation markers, were also improved after treatment. The combination of Mel@RCNVs and Mel@OST-RCNVs showed the best effect in both bone formation and bone resorption ( Fig.12 L, M). The above results demonstrate that nanovesicles have a satisfactory effect on bone mechanical properties, bone mass and microstructure.

[0123] In addition, osteoblast / osteoclast balance and macrophage efferocytosis were investigated by observing bone tissue sections. First, tartrate-resistant acid phosphatase (TRAP) staining was performed to observe the resorption activity of osteoclasts in vivo ( Fig.14 A). Due to aging, bone resorption increases, and after nanovesicle administration, excessive bone resorption was alleviated due to the RANKL neutralization effect of RCNVs. In addition, immunofluorescence (IF) staining of Runx2 and osteocalcin (OCN) in bone tissue was used to observe the differentiation of osteoblasts in vivo; Fig.14 As shown in B and C, Mel@OST-RCNVs treatment significantly increased the expression of Runx2 and osteocalcin. Due to the lack of targeting ability to the bone-forming surface, Mel@RCNVs treatment was not as effective as Mel@OST-RCNVs. Then, the circadian rhythm-related proteins Bmal1 and Clock in macrophages were determined by co-staining with the macrophage marker CD68; Fig.14 As shown in D and E, Mel@RCNVs and Mel@OST-RCNVs significantly promoted the expression of Bmal1 and Clock in macrophages, and the expression of Mer tyrosine kinase (Mertk), a receptor for efferocytosis in macrophages, was also restored with the restoration of circadian rhythm ( Fig.14 F), and CD90-positive cells, which are usually BMSCs, showed decreased Mertk expression ( Fig.14 G). These results suggest that macrophages restore efferocytosis and BMSCs are liberated from non-professional efferocytosis, which is beneficial for maintaining bone homeostasis.

[0124] In addition, the biosafety of bone-targeted nanovesicles was evaluated by blood biochemical analysis and routine blood examination. Analysis of blood biochemical parameters including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), serum creatinine (Scr), and total bilirubin (TBil) showed that the liver and kidney functions of all mice were normal after nanovesicle administration ( Fig.15 Blood routine examination also showed that the nanovesicles have good safety ( Fig.16 ).

[0125] In summary, the experiments show that Mel@RCNVs / Mel@OST-RCNVs nanovesicles restore osteoblast / osteoclast balance and macrophage efferocytosis. Excessive osteoclastogenesis is alleviated by bone-specific RANKL neutralization, and osteogenesis is promoted by RANKL reverse signal activation of osteoblasts. Most importantly, when bone marrow-derived macrophages (BMDMs) phagocytose these nanovesicles (NVs), circadian rhythm and efferocytosis functions are restored, and BMSCs are liberated from non-professional efferocytosis. In summary, the present invention proposes a promising bone-targeted nanovesicle for treating senile osteoporosis by restoring osteoblast / osteoclast balance and regulating the efferocytosis function mediated by the circadian clock of macrophages.

[0126] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A bone-targeted nanovesicle for treating osteoporosis, characterized in that: The bone-targeted nanovesicle is any of the following nanovesicles: (1) Bone marrow mesenchymal stem cell nanovesicles loaded with melatonin; (2) (AspSerSer)6-modified melatonin-loaded bone marrow mesenchymal stem cell nanovesicles; (3) A combination of (1) and (2).

2. The bone-targeted nanovesicle according to claim 1, characterized in that The bone marrow mesenchymal stem cell nanovesicles are obtained by squeezing bone marrow mesenchymal stem cells.

3. The bone-targeted nanovesicle according to claim 1 or 2, characterized in that: The bone marrow mesenchymal stem cells are bone marrow mesenchymal stem cells that overexpress RANK and CXCR4.

4. The method for preparing bone-targeted nanovesicles according to any one of claims 1 to 3, characterized in that: The steps include: (1) dispersing bone marrow mesenchymal stem cells in a PBS solution, then squeezing, collecting the precipitate by a first centrifugation, resuspending the precipitate, centrifuging again, collecting the precipitate, and obtaining bone marrow mesenchymal stem cell nanovesicles; (2) mixing the bone marrow mesenchymal stem cell nanovesicles with melatonin and sonicating the mixture, then incubating the mixture at 36-38° C. for 0.5-1.5 h, and collecting the precipitate by centrifugation to obtain the bone marrow mesenchymal stem cell nanovesicles loaded with melatonin; (3) incubating the bone marrow mesenchymal stem cell nanovesicles loaded with melatonin with (AspSerSer)6 coupled with SPE-PEG-NHS at 3-5° C. overnight, collecting the precipitate by centrifugation, washing and suspending in water to obtain (AspSerSer)6-modified bone marrow mesenchymal stem cell nanovesicles loaded with melatonin; (4) Mixing the bone marrow mesenchymal stem cell nanovesicles loaded with melatonin with the bone marrow mesenchymal stem cell nanovesicles loaded with melatonin modified with (AspSerSer)6 to obtain a nanovesicle composition.

5. The preparation method according to claim 4, characterized in that: The membrane pore sizes used for the extrusion are 4-6 μm and 0.5-1.5 μm respectively.

6. The preparation method according to claim 4, characterized in that: The first centrifugation parameters in step (1) are: 0.5×10 5 -1.5×10 5 g, 5-10 min; the parameters for the second centrifugation are: molecular weight cutoff 80-120 kDa, 900-1100 g, 8-12 min.

7. The preparation method according to claim 4, characterized in that: The amount of bone marrow mesenchymal stem cell nanovesicles used in step (2) is 10 9 -10 11 The amount of melatonin used is 490-510 mg.

8. The preparation method according to claim 4, characterized in that: The ultrasonic parameters in step (2) are: amplitude 18-22%, 28-32s on / off cycle 2.5-3.5min, repeated 5-7 times, with a cooling interval of 1.5-2.5min between each cycle; the centrifugation parameters in steps (2) and (3) are: first centrifugation at 1100-1300rpm for 8-12min, then at 1×10 4 -2×10 4 Centrifuge at 200 g for 25-35 min.

9. The preparation method according to claim 4, characterized in that: The concentration of the bone marrow mesenchymal stem cell nanovesicles loaded with melatonin in step (3) is 10 11 -10 13 particles / mL, and the concentration of the DSPE-PEG-NHS-conjugated (AspSerSer)6 was 8-12 μM.

10. Use of the bone-targeted nanovesicle according to any one of claims 1 to 3 or the bone-targeted nanovesicle obtained by the preparation method according to any one of claims 4 to 9 in preparing a drug for preventing and / or treating osteoporosis.

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