Application of engineered vesicles modified by connecting adhesion molecules 3 in preparation of medicine for treating bone injury

Abstract

The invention discloses an application of a JAM3 modified engineered vesicle in preparation of a medicine for treating bone injury, the JAM3 modified engineered vesicle is prepared, and the osteogenesis promoting effect of the JAM3 modified engineered vesicle on bone marrow mesenchymal stem cells (BMSCs) is proved through ALP staining, RT-qPCR, WB and other experiments at the cellular level, so that the osteogenesis promoting effect of the JAM3 modified engineered vesicle on the bone marrow mesenchymal stem cells (BMSCs) is improved, and the osteogenesis promoting effect of the JAM3 modified engineered vesicle on the bone marrow mesenchymal stem cells (BMSCs) is improved. Meanwhile, the effect of JAM3 modified engineered vesicles on promoting osteogenesis of bone defect mice is defined through in-vivo experiments. According to the invention, osteogenesis direction differentiation and bone formation capability of BMSC can be promoted, and bone formation capability of bone defect mice can be promoted in vivo; the compound can be used for treating related diseases such as bone injury.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to application of junction adhesion molecule 3 modified engineered vesicles in preparation of a drug for treating bone injury. BACKGROUND

[0002] Bone defects can occur in various bones of the human body due to trauma, infection, tumor or surgery, resulting in loss or discontinuity of bone tissue, and further affecting the appearance and function of the patient, and often accompanied by bone healing disorders. Traditional repair methods include bone grafting, which has problems such as limited donor, infection and immune rejection. Traditional materials include metal scaffolds and growth factors, which have problems of slow healing and possible ectopic ossification, respectively. Therefore, it is urgent to develop a targeted material with biological activity to promote the healing of bone injury.

[0003] Extracellular vesicles are nanoscale membrane vesicles actively secreted by cells, carrying biological active molecules such as proteins, nucleic acids and lipids, and participating in cell-to-cell communication. Its biocompatibility, low immunogenicity and targeted delivery capacity make it have significant potential in disease diagnosis, drug carriers and tissue regeneration. Engineering modification can further optimize its function. Studies have shown that blood-derived vesicles from young mice can improve the mitochondrial function of the aging bone marrow microenvironment, thereby promoting the healing of bone fractures in old mice. Extracellular vesicles with high expression of T cell immunoglobulin mucin molecule 3, which has immunosuppressive function, can target macrophages, thereby improving the microenvironment for bone regeneration.

[0004] The natural vesicles currently relied on in the field of bone regeneration have strong heterogeneity and poor targeting. Although some studies have improved the function of vesicles through engineering modification, there is still a lack of precise design based on specific markers of cell function. SUMMARY

[0005] The application aims at the deficiencies of the prior art, and provides application of junction adhesion molecule 3 modified engineered vesicles in preparation of a drug for treating bone injury. Junction adhesion molecule 3 is identified as a specific marker for promoting osteogenic differentiation of bone marrow mesenchymal stem cells for the first time, and an engineered vesicle is constructed based on this, which significantly improves the efficiency of bone defect repair.

[0006] The application aims at the deficiencies of the prior art, and provides application of junction adhesion molecule 3 modified engineered vesicles in preparation of a drug for treating bone injury. Junction adhesion molecule 3 is identified as a specific marker for promoting osteogenic differentiation of bone marrow mesenchymal stem cells for the first time, and an engineered vesicle is constructed based on this, which significantly improves the efficiency of bone defect repair. In one aspect, the application provides application of junction adhesion molecule 3 modified engineered vesicles in preparation of a drug for treating bone injury.

[0007] In one possible implementation manner, the junction adhesion molecule 3 modified engineered vesicles are prepared by gene overexpression method, specifically as follows. The adhesion molecule 3 overexpression plasmid was co-incubated with cells for 24-72 hours, and the expression of EGFP green fluorescence was observed by fluorescence microscopy to confirm that the plasmid was successfully transfected into the cells. After successful transfection, serum-free cell supernatant was collected, and engineered vesicles modified with linker-adhesion molecule 3 were extracted by ultracentrifugation.

[0008] In one possible implementation, the engineered vesicles modified with the connecting and adhering molecule 3 are prepared by a vesicle surface modification method, specifically: 0.1–100 μg of the binding adhesion molecule 3 protein was inoculated with 5–20 mg of DSPE-PEG5000-NHS in 2 ml of PBS overnight at 2–8°C. The PBS concentration was 5–20 mM and the pH was 7.2–7.6. Unbound DSPE-PEG5000-NHS was removed by centrifugation, filtration, and washing. The mixture was washed several times with 5-20 mM PBS. The resulting JAM3-PEG-DSPE was bound to the exovesicles at 2-8°C for 12-48 hours. Unbound JAM3-DSPE-NHS and free linker 3 were concentrated and washed several times with 5-20 mM PBS buffer at 3000-6000 g in a centrifugal filter at 2-8°C. The mixture was then resuspended in 5-20 mM PBS to form JAM3. + External vesicle solution; The obtained JAM3 + The outer vesicle solution was centrifuged at 8000-100000g for 2-2.5 hours at 2-8 °C, washed with 5-20mM PBS buffer, and resuspended in 5-20mM PBS buffer.

[0009] In one possible implementation, the sources of vesicles include, but are not limited to, animal and plant cells, tissues, body fluids, exocrine secretions and their products; the methods for obtaining vesicles include, but are not limited to, differential centrifugation, density gradient centrifugation, polymer precipitation, and magnetic bead enrichment.

[0010] In one possible implementation, the site of the bone injury includes the craniofacial bones, spine, and limb bones; the type of injury includes traumatic and pathological.

[0011] On the other hand, this application provides a medicament for treating bone injuries, comprising engineered vesicles modified with adhesion molecule 3.

[0012] In one possible implementation, the dosage form of the drug includes injections, tablets, capsules, powders, and hydrogels.

[0013] In one possible implementation, the solvent for the injection is a phosphate buffer solution or physiological saline.

[0014] The technical solutions provided by the embodiments of this application may include the following beneficial effects: As can be seen from the above embodiments, this application of the present invention demonstrates the osteogenic effect of JAM3-modified engineered vesicles on bone marrow mesenchymal stem cells (BMSCs) by preparing junctional adhesion molecule 3 (JAM3) modified engineered vesicles at the cellular level through experiments such as ALP staining, RT-qPCR, and WB; at the same time, it clarifies the osteogenic effect of JAM3-modified engineered vesicles on bone defects in mice through in vivo experiments.

[0015] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0016] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0017] Figure 1 A flowchart for preparing JAM3-modified engineered vesicles using gene overexpression.

[0018] Figure 2 A flowchart illustrating the preparation of JAM3-modified engineered vesicles using a vesicle surface modification method.

[0019] Figure 3 This is a schematic diagram characterizing engineered vesicles modified with adhesion molecule 3, where A represents TEM observations of Ctrl-EVs and JAM3. + Morphological characteristics of EVs (scale bar: 100 nm), B represents NTA detection of Ctrl-EVs and JAM3. + Particle size distribution of EVs, C represents WB detection of Ctrl-EVs and JAM3. + The expression of signature proteins in EVs.

[0020] Figure 4 Figure 4 shows the experimental results of the osteogenic effect of engineered vesicles modified with adhesion molecule 3 on BMSCs in Example 4 (*). P <0.05.** P <0.01. **** P<0.0001), where A is a representative image of COL1A1 immunofluorescence staining (scale bar: 100 μm), B is the quantitative analysis of COL1A1 immunofluorescence staining, C is a representative image of ALP staining (scale bar: 200 μm), D is the quantitative analysis of ALP staining, E is a representative image of ARS staining (scale bar: 200 μm), F is the quantitative analysis of ARS staining, and G is JAM3. + Osteogenesis differentiation-related genes after co-culturing EVs and BMSCs ( Bmp2, Ocn, Osterix and Ruxn2 mRNA expression levels (n=3).

[0021] Figure 5 The figure shows the experimental results of the bone repair effect of JAM3 modified engineered vesicles on mice with jaw defects in Example 5 (**P<0.01. ****P<0.0001), where A is a representative image of micro CT (scale bar: 200 μm), B is the quantitative results of bone mineral density (BMD) and bone volume fraction (BV / TV), C is a representative image of MASSON staining (scale bar: 200 μm), and D is the quantitative percentage of new bone formation area (n=5). Detailed Implementation

[0022] The present invention will now be described in detail with reference to the accompanying drawings. Unless otherwise specified, the features of the following embodiments and implementations can be combined with each other.

[0023] It should be noted that all animal experiments involved in the specific implementation of this application follow the "Regulations on the Management of Laboratory Animals" and the relevant norms of the Institute of Laboratory Animal Ethics (IACUC) to ensure that the animal euthanasia process is humane and legal, and has been approved by the Animal Experiment Ethics Committee of Zhejiang University. The ethics number is: ZJU20250160.

[0024] This application provides the use of engineered vesicles modified with adhesion molecule 3 in the preparation of drugs for treating bone injuries.

[0025] It should be noted that the bone injuries mainly include the craniofacial bones, spine, and limb bones; the injury types are mainly traumatic and pathological, such as fractures and osteonecrosis.

[0026] Methods for preparing JAM3-modified engineered vesicles include, but are not limited to, gene overexpression or vesicle surface modification.

[0027] The gene overexpression method specifically involves: co-incubating the JAM3 overexpression plasmid with cells for 24-72 hours, and observing the expression of EGFP green fluorescence using a fluorescence microscope to confirm successful transfection into the cells; after successful transfection, collecting the serum-free cell supernatant and extracting JAM3-modified engineered vesicles using ultracentrifugation. The obtained JAM3-modified engineered vesicles can be stored at 2–8°C for no more than one week; if storage for more than one week is required, it is advisable to store them at -80°C or under liquid nitrogen conditions to maintain their biological activity.

[0028] The vesicle surface modification method specifically involves: binding JAM3 protein (0.1–100 μg, preferably 1–10 μg) with N-hydroxysuccinimide-activated polyethylene glycol-linked 1,2-distearate-sn-glycerol-3-phosphate ethanolamine (DSPE-PEG-NHS) (5–20 mg) overnight in 2 ml PBS (5–20 mM, pH 7.2–7.6) at 2–8°C. Unbound DSPE-PEG5000-NHS is removed by centrifugation and washing using an Amicon centrifugal filtration device (Millipore, UFC810096, 10 kDa molecular weight cutoff), followed by three washes with 10 mM PBS (4°C, 3000–6000 g, 10 minutes each). Then, the resulting JAM3-PEG-DSPE is bound to the outer vesicles at 2–8°C for 12–48 hours. Unbound JAM3-DSPE-NHS and free JAM3 were concentrated and washed 2-3 times with PBS buffer (5-20 mM, pH 7.2-7.6, 3000-6000 g, 10-15 min each time) in an Amicon centrifuge filter (Millipore, UFC810096, 100 kDa molecular weight cutoff) at 2-8°C. The mixture was then resuspended in PBS buffer (5-20 mM, pH 7.2-7.6) to form JAM3. + Exovesicle solution. For further purification, the obtained JAM3+ exovesicle solution was ultracentrifuged at 8000-100000g for 2-2.5 hours at 2-8 °C, then washed once with PBS buffer (5-20mM, pH 7.2-7.6) and resuspended in PBS buffer (5-20mM, pH 7.2-7.6).

[0029] The following detailed description is provided in conjunction with specific examples.

[0030] Example 1: Preparation of JAM3-modified engineered vesicles by gene overexpression like Figure 1As shown, the JAM3 overexpression plasmid was co-incubated with cells for 48 hours, and the expression of EGFP green fluorescence was then observed using a fluorescence microscope to confirm successful transfection into the cells. After successful transfection, serum-free cell supernatant was collected, and JAM3 overexpression vesicles were extracted using ultracentrifugation or size exclusion chromatography, ultrafiltration, immunoaffinity capture, and other methods. The JAM3-modified engineered vesicles were stored at 4°C for up to one week, and at -80°C for more than one week to ensure their biological activity.

[0031] Example 2: Preparation of JAM3-modified engineered vesicles by vesicle surface modification method like Figure 2 As shown, JAM3 (100 μg) was bound to DSPE-PEG5000-NHS (10 mg) overnight in 2 ml PBS (10 mM, pH 7.4) at 4°C. Unbound DSPE-PEG5000-NHS was removed by centrifugation and washing with an Amicon centrifuge filter (Millipore, UFC810096, 10 kDa molecular weight cutoff), followed by three washes with 10 mM PBS (4°C, 4500 g, 10 min each). The resulting JAM3-PEG-DSPE was then bound to extravesicles (e.g., erythrocyte extravesicles, 2, erythrocyte granules) at 4°C for 24 h. Unbound JAM3-DSPE-NHS and free JAM3 were concentrated and washed three times with PBS buffer (10 mM, pH 7.4, 4500 g, 10 min each time) in an Amicon centrifuge filter (Millipore, UFC810096, 100 kDa molecular weight cutoff) at 4 °C. For further purification, the resulting JAM3+ exovesicle solution was ultracentrifuged at 100,000 g for 2 h at 4 °C, washed once with PBS buffer (10 mM, pH 7.4), and resuspended in 2 ml of PBS buffer (10 mM, pH 7.4).

[0032] Example 3: Identification of JAM3-modified engineered vesicles The morphological characteristics of the JAM3-modified engineered vesicles prepared in Examples 1 and 2 were observed using transmission electron microscopy (TEM). The particle size of the JAM3-modified engineered vesicles prepared in Examples 1 and 2 was analyzed using nanoparticle tracking analysis (NTA). TEM observation showed that their surface morphology was a typical biconcave disk shape, with particle radii between 50-200 nm, which meets the radius requirements for external vesicles. This indicates that the extracted vesicles are external vesicles. Furthermore, the expression levels of marker proteins of the JAM3-modified engineered vesicles were detected using Western blotting (WB).

[0033] Figure 3 This section demonstrates JAM3-modified engineered vesicles constructed via gene overexpression. First, we co-cultured cells with either a control plasmid or a JAM3 overexpression plasmid, then collected cells (Ctrl-Cell and JAM3+Cell) and their supernatants. Control vesicles (Ctrl-EVs) and JAM3-modified engineered vesicles (JAM3+Cell) were extracted from the supernatants. + EVs). Figure 3 As shown in Figure A, two types of vesicles, Ctrl-EVs and JAM3, were detected by TEM. + EVs all exhibit typical cup-shaped or spherical structures with a diameter of approximately 150 nm. Figure 3 The B in the text shows that NTA technology is used to process Ctrl-EVs and JAM3. + Quantitative analysis was performed on the particle size distribution of EVs. The results showed that the mode of Ctrl-EVs was 142 nm, while that of JAM3... + The mode of the particle size of EVs is 138 nm. Figure 3 The C in the figure shows that donor cells (Ctrl-Cell and JAM3) were analyzed using the Western blot method. + Cell) and Ctrl-EVs, JAM3 + EVs were analyzed for classic exosome marker proteins, and the results showed that both types of vesicles highly expressed classic exosome marker proteins (such as Annexin V, EPCAM, and Flotillin-1), while GM130 expression was not detected, indicating that the extracted exosomes had high purity. Meanwhile, JAM3-modified engineered vesicles highly expressed JAM3 protein, further validating the successful enrichment of JAM3 in the prepared JAM3-modified engineered vesicles.

[0034] Example 4: Osteogenesis effect of JAM3-modified engineered vesicles on BMSCs S1. Isolation of mouse bone marrow mesenchymal stromal cells (BMSCs) C57BL6 cells were euthanized at 6-10 weeks using the carbon dioxide inhalation method. Under aseptic conditions, the femur and tibia were removed, and the attached muscle tissue was cleaned repeatedly with PBS and α-MEM culture medium. The ends of the bones were cut off, and the bone marrow was flushed out with α-MEM complete culture medium containing 10% fetal bovine serum and penicillin-streptomycin antibiotics until the bone turned white. The medium was changed every 2-3 days thereafter, and the cells were ready for use after approximately 10 days.

[0035] S2. Osteogenesis of BMSCs by JAM3-Modified Engineered Vesicles The experiment was set up in two groups: (1) JAM3-modified engineered vesicles: 6×10 4 BMSCs were seeded at a density of vesicles / mL, and JAM3-modified engineered vesicles were added every other day to a final concentration of 25 μg / mL. Thereafter, vesicles of the same concentration were added every 3 days when the medium was changed, and the culture was continued for 7 days. (2) Control group: BMSCs of the same density were inoculated and an equal volume of control group vesicles were added every other day. The medium change cycle was the same as that of the experimental group.

[0036] BMSC osteogenic capacity in all groups was assessed using immunofluorescence, ALP, alkaline phosphatase staining, alizarin red staining, and RT-qPCR. Figure 4 As shown in Figures A and B, the fluorescence intensity of COL1A1 immunofluorescence staining in BMSCs with JAM3-modified engineered vesicles was higher than that in BMSCs with control group vesicles. Figure 4 As shown in C and D, the proportion of ALP-stained areas in BMSCs with JAM3-modified engineered vesicles was higher than that in BMSCs with control group vesicles. Figure 4 As shown in E and F, the absorbance of BMSCs with JAM3-modified engineered vesicles at 562 nm after ARS staining was higher than that of BMSCs with control group vesicles. Figure 4 As shown in G, two groups of BMSCs were collected for RT-qPCR. BMSCs with JAM3-modified engineered vesicles were added. Bmp2 (Bone morphogenetic protein 2) Ocn (Osteocalcin) Osterix (Osteoblast-specific transcription factors) Runx2 The transcriptional level of the key transcription factor for osteoblast differentiation (JAM3) was higher than that of the control group. This indicates that JAM3-modified engineered vesicles can promote osteogenic activity in BMSCs by enhancing the expression of osteogenic-related genes. Example 5: Bone repair effect of JAM3-modified engineered vesicles on jawbone defects in mice Six- to ten-week-old male C57BL6 mice were selected. A "critical bone defect" model of the mandible was created in the bilateral mandibular angle region using a 1.5 mm dental bur. JAM3-modified engineered vesicles were placed at the left defect site, with 100 μg of JAM3-modified engineered vesicles added to each mandibular defect site. An equal amount of control vesicles was added to the right defect site after modeling. Samples were collected after 7 days to observe the promoting effect of JAM3-modified engineered vesicles on bone defect repair. Figure 5 As shown in Figure A, the mice in the JAM3-modified engineered vesicle group exhibited more neobone formation in the microCT reconstruction images of bone defects compared to the control group. Figure 5 As shown in Figure B, the quantitative values ​​of bone mineral density (BMD) and bone volume fraction (BV / TV) at bone defects in mice in the JAM3-modified engineered vesicle group were both higher than those in the control group. Figure 5 As shown in C and D, MASSON staining of bone defects in mice with JAM3-modified engineered vesicles revealed more new bone formation compared to the control group. This indicates that JAM3-modified engineered vesicles can promote bone defect repair in vivo.

[0037] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

[0038] The above embodiments are only used to illustrate the design concept and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. The protection scope of the present invention is not limited to the above embodiments. Therefore, all equivalent changes or modifications made based on the principles and design ideas disclosed in the present invention are within the protection scope of the present invention.

Claims

1. Application of engineered vesicles modified with a linker-adhesion molecule 3 in the preparation of drugs for treating bone injuries.

2. The application according to claim 1, characterized in that, The engineered vesicles modified with the adhesion molecule 3 were prepared via gene overexpression, specifically as follows: The adhesion molecule 3 overexpression plasmid was co-incubated with cells for 24-72 hours, and the expression of EGFP green fluorescence was observed by fluorescence microscopy to confirm that the plasmid was successfully transfected into the cells. After successful transfection, serum-free cell supernatant was collected, and engineered vesicles modified with linker-adhesion molecule 3 were extracted by ultracentrifugation.

3. The application according to claim 1, characterized in that, The engineered vesicles modified with the connecting and adhering molecule 3 were prepared by a vesicle surface modification method, specifically: 0.1–100 μg of the binding adhesion molecule 3 protein was inoculated with 5–20 mg of DSPE-PEG5000-NHS in 2 ml of PBS overnight at 2–8°C. The PBS concentration was 5–20 mM and the pH was 7.2–7.

6. Unbound DSPE-PEG5000-NHS was removed by centrifugation, filtration, and washing. The mixture was washed several times with 5-20 mM PBS. The resulting JAM3-PEG-DSPE was bound to the exovesicles at 2-8°C for 12-48 hours. Unbound JAM3-DSPE-NHS and free linker 3 were concentrated and washed several times with 5-20 mM PBS buffer at 3000-6000 g in a centrifugal filter at 2-8°C. The mixture was then resuspended in 5-20 mM PBS to form JAM3. + External vesicle solution; The obtained JAM3 + The external vesicle solution was ultracentrifuged at 8000-100000g for 2-2.5 hours at 2-8 °C, washed with 5-20mM PBS buffer, and resuspended in 5-20mM PBS buffer.

4. The application according to claim 1, characterized in that, The sources of vesicles include, but are not limited to, animal and plant cells, tissues, body fluids, exocrine secretions and their products; the methods for obtaining vesicles include, but are not limited to, differential centrifugation, density gradient centrifugation, polymer precipitation, and magnetic bead enrichment.

5. The application according to claim 1, characterized in that, The locations of the bone injuries include the craniofacial bones, spine, and limb bones; the types of injuries include traumatic and pathological.

6. A drug for treating bone injuries, characterized in that, This includes engineered vesicles modified with adhesive molecules 3.

7. The drug according to claim 6, characterized in that, The dosage forms of the drug include injections, tablets, capsules, powders, and hydrogels.

8. The medicament according to claim 7, characterized in that, The solvent for the injection is a phosphate buffer solution or physiological saline.

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