Osteoclast targeted fusion probiotic external vesicle as well as preparation method and application thereof

Abstract

The invention discloses an osteoclast targeted fusion probiotic external vesicle as well as a preparation method and application thereof. The method comprises the following steps: constructing a pClyA-DC-STAMP recombinant plasmid by adopting a one-step cloning kit; then, transforming into EcN, and culturing a secondary pre-culture by adopting a two-step temperature method, so as to promote the efficient expression of the EcN and to intersperse the outer vesicles of the DC-STAMP protein on the surface of a secretory membrane; finally, separation and purification are carried out, and the engineering probiotic outer vesicle BEV-DCS is prepared and obtained. The BEV-DCS can efficiently load the functional polypeptide capable of inhibiting osteoclast differentiation, the in-vivo stability of the BEV-DCS is improved, the intracellular delivery efficiency of the precursor osteoclast of the BEV-DCS is enhanced, and then the osteoclast differentiation inhibiting effect of the BEV-DCS is improved. In an ovariectomy mouse osteoporosis model, the obvious bone targeting ability and osteoporosis treatment effect are shown, and the obvious toxic and side effects are avoided. The invention provides a safe and efficient osteoclast targeted delivery drug carrier.

Description

Technical Field

[0001] The invention relates to an osteoclast-targeted fusion probiotic exosome and a preparation method and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] Osteoporosis (OP) is a common bone disease in the elderly, and its main clinical manifestations include decreased bone density, destruction of bone microstructure, and weakened bone strength. The occurrence of osteoporosis is mainly due to the imbalance of bone formation and bone resorption regulated by osteoblasts and osteoclasts. In response to this mechanism, the drugs currently used in clinical practice mainly include parathyroid hormone and its analog teriparatide that promote bone formation, as well as bisphosphonates and calcitonin that inhibit osteoclast function. However, these traditional therapeutic drugs often have problems with poor therapeutic effects and significant toxic side effects. Therefore, it is still urgent to develop new therapeutic drugs to treat OP more safely and effectively.

[0003] Given the widespread use of teriparatide and calcitonin, functional peptide drugs are being developed in large quantities for safer and more effective prevention and treatment of OP. These functional peptides can precisely regulate signaling pathways associated with osteoclast or osteoblast differentiation, thereby specifically regulating bone formation and resorption. FRATtide (FRAT) is a protein derived from the interaction with glycogen synthase kinase-3β (GSK3β) that can inhibit the phosphorylation of GSK3β. Our previous studies have shown that FRAT can reduce bone loss in ovariectomized mice by inhibiting NFATc1-mediated osteoclast differentiation. However, the low proteolytic stability and cell targeting permeability of FRAT severely limit its clinical application. Therefore, the use of nano-delivery carriers to protect FRAT from proteolysis and enhance the targeting permeability of FRAT osteoclast precursors is expected to promote its clinical translational application in the treatment of osteoporosis.

[0004] Bacterial extracellular vesicles (BEVs), especially those secreted by probiotics, have attracted increasing attention as a new generation of nano-delivery carriers. Similar to extracellular vesicles (EVs) from mammalian cells, the nanoscale structure of BEVs endows them with stable drug loading capacity, efficient cell penetration ability, and good biosafety. Mature bacterial culture technology and bacterial gene editing methods have further promoted the simple, large-scale and customized production of BEVs. In the latest study, BEVs from Escherichia coli Nissle 1917 (EcN) were successively used as carriers to target the delivery of their physically encapsulated SOST siRNA and biologically expressed BMP-2 to the bone microenvironment for regulating bone metabolism, thereby alleviating ovariectomy-induced OP. However, BEVs with the ability to target and penetrate osteoclast precursors have not yet been developed. Summary of the invention

[0005] The purpose of the present invention is to address the problems of poor in vivo enzymatic stability and low cell targeting penetration of existing osteoporosis therapeutic polypeptide drugs. The present invention provides an osteoclast-targeted fusion probiotic exosome and a preparation method and application thereof, which is expected to be used as a safe and efficient osteoclast-targeted drug delivery carrier for the prevention and treatment of osteoporosis-related diseases.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a probiotic extracellular vesicle BEV-DCS for osteoclast targeting fusion, wherein the probiotic is Escherichia coli Nissle 1917 (EcN), and the probiotic extracellular vesicle membrane surface expresses an osteoclast targeting fusion protein DC-STAMP;

[0008] The BEV-DCS is obtained by constructing a recombinant plasmid expressing the osteoclast targeting fusion protein DC-STAMP, transforming the recombinant plasmid into competent cells of EcN to obtain a recombinant Escherichia coli strain, culturing the recombinant Escherichia coli strain and inducing the expression of DC-STAMP protein, and then extracting bacterial extracellular vesicles.

[0009] Furthermore, the recombinant plasmid expresses a gene fragment having a sequence as shown in SEQ ID NO:1.

[0010] In a second aspect, the present invention provides a method for preparing the osteoclast-targeted fusion probiotic exosomes as described above, comprising the following steps:

[0011] Step 1. Synthesis of recombinant plasmid: using pET28a as a cloning plasmid, the DC-STAMP gene and the ClyA gene are connected by molecular cloning technology to obtain a DC-STAMP-ClyA connection fragment; the pET28a plasmid is digested with a restriction endonuclease to obtain a linearized pET28a plasmid, and the DC-STAMP-ClyA connection fragment is connected to the linearized pET28a plasmid by a ligase to complete the construction of the pClyA-DC-STAMP recombinant plasmid;

[0012] Step 2. Preparation and cultivation of engineered probiotics: preparing probiotic Escherichia coli competent cells, transferring the recombinant plasmid constructed in step 1 into the competent cells by electroporation to complete the construction of the probiotic recombinant strain, culturing the probiotic recombinant strain and inducing DC-STAMP protein expression to obtain a probiotic bacterial liquid;

[0013] Step 3. Extraction of engineered probiotic extracellular vesicles: First, the probiotic bacterial liquid obtained in step 2 is collected by centrifugation to obtain the probiotic fermentation liquid expressing DC-STAMP, and then the bacterial extracellular vesicles are extracted by ultracentrifugation to obtain osteoclast-targeted fusion probiotic extracellular vesicles BEV-DCS, wherein the osteoclast-targeted fusion protein DC-STAMP is expressed on the membrane surface of the probiotic extracellular vesicles.

[0014] In the present invention, the DC-STAMP gene in step 1 may also be replaced by other related genes expressed on the membrane of osteoclast precursor cells, such as one or more selected from CD47, CD36, CX-43 and c-Fms.

[0015] In the present invention, the probiotic EcN in step 2 may also be replaced by other Gram-negative probiotics that are easily genetically modified, such as at least one of Akkermansia muciniphila (Akk) and Lactobacillus reuteri (L.reuteri).

[0016] Furthermore, the culturing in step 2 comprises: firstly, the probiotics are cultured in a 37°C shaker for 12 to 24 hours, and then cultured in a 37°C shaker until the OD 600 The inducing DC-STAMP protein expression comprises: adding isopropyl-β-D-thiogalactoside (IPTG) at 16°C to induce DC-STAMP protein expression.

[0017] Furthermore, the ultracentrifugation method for extracting bacterial extracellular vesicles in step 3 includes: centrifuging the probiotic fermentation broth at 10000-20000g to remove bacterial precipitates to obtain a supernatant, filtering the supernatant and then centrifuging twice at 1×10 5 ~1.5×10 5 The extracellular vesicles expressing DC-STAMP protein on the membrane were collected by ultracentrifugation

[0018] In a third aspect, the present invention provides the use of the osteoclast-targeted fusion probiotic extracellular vesicle BEV-DCS as described above in loading drugs that inhibit osteoclast differentiation and preparing drugs that inhibit osteoclast differentiation by targeted delivery.

[0019] Furthermore, the drug for inhibiting osteoclast differentiation includes the functional polypeptide FRATtide (FRAT) for inhibiting osteoclast differentiation.

[0020] In a fourth aspect, the present invention provides a method for loading the osteoclast-targeted fusion probiotic extracellular vesicle BEV-DCS as described above with an inhibitory functional polypeptide FRATtide, comprising: first incubating the functional polypeptide FRATtide and the osteoclast-targeted fusion probiotic extracellular vesicle BEV-DCS in a solution, and then introducing the functional polypeptide FRATtide into the probiotic extracellular vesicle by electroporation, thereby completing the loading of the functional polypeptide.

[0021] Furthermore, the specific steps of the method include: first preparing a FRAT solution with a concentration of 50 to 2000 μg / mL and a 8 ~10 11 particles / mL of a probiotic extracellular vesicle solution; then the FRAT solution and the probiotic extracellular vesicle solution are mixed in a volume ratio of (0.1-5):1 and incubated for 1-5 hours; then the incubated mixed solution is electroporated under the conditions of a voltage of 100V, a resistance of 200Ω and a capacitance of 100μF; after the electroporation, the mixture is allowed to stand at 30-45°C for 30-120 minutes; finally, the mixed solution is ultracentrifuged to remove residual FRAT molecules to obtain BEV-DCS loaded with a functional polypeptide FRAT, namely FRAT@BEV-DCS.

[0022] In some embodiments of the present invention, the concentration of the FRAT solution is preferably 2000 μg / mL, and the concentration of the engineered exosomes is preferably 1×10 11 particles / mL; the volume ratio of the FRAT to the engineered exosome solution is preferably 1:1, and the mixed incubation time is preferably 3 hours; the stationary temperature after electroporation is preferably 37°C, and the stationary time is preferably 60 minutes.

[0023] In a fifth aspect, the present invention provides a probiotic exosome for targeted osteoclast fusion as described in the first aspect or a probiotic exosome for targeted osteoclast fusion obtained by the method described in the second aspect or the fourth aspect, and its use in the preparation of drugs and preparations for inhibiting osteoclast differentiation.

[0024] The present invention endows probiotic exosomes with better targeting ability of osteoclast precursor cells through biosynthesis, and further loads osteoclast inhibitory polypeptide, thereby improving the stability of functional polypeptide and the ability to penetrate cell membrane. Such modified exosomes can be used to prepare therapeutic preparations for bone diseases targeting overactive osteoclasts, and to explore the pathogenesis and treatment of orthopedic diseases.

[0025] In a sixth aspect, the present invention provides an application of the osteoclast-targeted probiotic exosomes as described in the first aspect or the osteoclast-targeted probiotic exosomes prepared by the method described in the second aspect or the fourth aspect in the preparation of drugs and preparations for preventing and treating osteoporosis-related diseases.

[0026] Furthermore, the disease range includes any skeletal system disease caused by osteoporosis, osteoporotic fractures or bone loss.

[0027] Furthermore, the dosage form of the drug and preparation can be a solution, suspension, syrup or emulsion in an oil medium or an aqueous medium, or an extract, powder, granule, tablet, capsule, spray, ointment or gel.

[0028] Compared with existing delivery carriers, the osteoclast-targeted probiotic fusion exosomes provided by the present invention have the following beneficial effects:

[0029] (1) The preparation method of the probiotic exosomes BEV-DCS for osteoclast targeted fusion of the present invention is simple and easy, and the exosomes have a high yield, high purity, low cost, and can be customized; the exosomes can be efficiently loaded with functional polypeptides that inhibit osteoclast differentiation, and after loading, the functional polypeptides can be improved in vivo enzymatic stability and enhanced intracellular penetration of osteoclast precursors;

[0030] (2) The osteoclast-targeted probiotic extracellular vesicle FRAT@BEV-DCS described in the present invention can effectively inhibit osteoclast differentiation after being loaded with the osteoclast inhibitory peptide FRAT; it demonstrated significant bone targeting ability and osteoporosis treatment effect in the osteoporosis model of ovariectomized mice without obvious toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the pClyA-DC-STAMP recombinant plasmid constructed in Example 1 of the present invention;

[0032] Figure 2 The transmission electron microscope image (A) and particle size distribution diagram (B) of BEV-DCS prepared in Example 2 of the present invention;

[0033] Figure 3 This is a diagram showing the encapsulation efficiency of BEV-DCS loaded with functional polypeptides in Example 3 of the present invention;

[0034] Figure 4 This is a diagram showing the enzymatic stability of the BEV-DCS protective functional polypeptide in Example 4 of the present invention;

[0035] Figure 5 This is a diagram showing the intracellular penetration effect of the BEV-DCS functional enhancement polypeptide in Example 5 of the present invention;

[0036] Figure 6 This is a diagram showing the effect of the BEV-DCS improved functional polypeptide in Example 6 of the present invention on inhibiting osteoclast differentiation, wherein A is the quantitative statistical result of the number of osteoclasts, and B is the quantitative statistical result of the area of ​​osteoclasts;

[0037] Figure 7 This is a statistical diagram of the fluorescence distribution of BEV-DCS loaded with functional polypeptides in Example 7 of the present invention in the main organs and femur of mice, wherein A to F are the fluorescence quantitative statistical results of the heart (A), liver (B), spleen (C), lung (D), kidney (E), and hind limb (F), respectively;

[0038] Figure 8 Micro-CT (A) and effect diagrams of bone volume fraction (B), bone surface area / total volume (C), and trabecular number (D) of ovariectomized osteoporotic mice treated with BEV-DCS loaded with functional polypeptides in Example 8 of the present invention. DETAILED DESCRIPTION

[0039] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0040] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0041] Unless otherwise specified, the EcN strains, materials, reagents, etc. used in the following examples can be obtained through commercial channels.

[0042] Example 1 Preparation of probiotic extracellular vesicles for osteoclast targeted fusion

[0043] (1) Synthesis of recombinant plasmid: Using biological genetic engineering technology, pET28a was used as a cloning plasmid, and the amplified DC-STAMP gene (ID: NC_000081.7) and ClyA (ID: NP_415700.4) gene were connected by molecular cloning technology. The sequence of the connection fragment is shown in SEQ ID NO: 1. The pET28a plasmid was digested with restriction endonucleases, and the DC-STAMP-ClyA connection fragment (SEQ ID NO: 1) was connected to the linearized pET28a plasmid by ligase to complete the construction of the pClyA-DC-STAMP recombinant plasmid. The spectrum of the constructed recombinant plasmid is shown in Figure 1 shown.

[0044] The sequence of the DC-STAMP-ClyA linker fragment (SEQ ID NO: 1) is as follows:

[0045]

[0046] (2) Preparation of engineered probiotics: Prepare probiotic Escherichia coli competent cells, transfer the recombinant plasmid into the competent cells by electroporation, and complete the construction of the EcN-pClyA-DC-STAMP recombinant strain.

[0047] (3) Cultivation of engineered probiotics: Primary cultures were obtained by incubating the strains at 37°C and 220 rpm overnight. The primary cultures were inoculated into fresh medium and cultured for 12 hours to obtain secondary cultures. In order to specifically express DC-STAMP protein in EcN, a two-step temperature protocol was adopted. Specifically, the recombinant strains were introduced into LB fermentation broth and cultured at 37°C with shaking at 160 rpm until they reached OD 600 Subsequently, the culture temperature was lowered to 16°C while maintaining the shaking speed at 160 rpm, and IPTG was introduced for an 18-hour induction phase to promote the expression of DC-STAMP.

[0048] (4) Extraction of engineered probiotic extracellular vesicles: First, remove the bacteria by low-speed centrifugation at 10,000 g for 15 minutes, and then remove the bacterial debris with a 0.22 μm filter to obtain the probiotic fermentation broth with high expression of DC-STAMP. 5 The BEV-DCS were collected by ultracentrifugation at 4000 g for 90 min, resuspended in PBS, and purified by ultracentrifugation again under the same conditions. Once purified, the BEV-DCS were redissolved in PBS and stored at -80 °C for subsequent use.

[0049] Example 2 Method and characterization of osteoclast-targeted probiotic extracellular vesicles loaded with functional polypeptides that inhibit osteoclast differentiation

[0050] (1) Synthesis of osteoclast differentiation inhibitory polypeptide: The sequence of osteoclast inhibitory polypeptide FRAT is Ac-DPHRLLQQLVLSGNLIKEAVRRLHSR-NH 2 (SEQ ID NO: 2), with an amino resin with a capacity of 0.33 mmol / g as a solid support. In the amino acid coupling, standard Fmoc solid phase peptide synthesis was adopted, 1-hydroxybenzotriazole (HOBt) and N,N-diisopropylcarbodiimide (DIC) were used as the condensation system, and 20% piperidine in N,N-dimethylformamide (DMF) solution was used as the Fmoc deprotection reagent. The sequence of coupling, washing, deprotection and additional washing was repeated until the full sequence of amino acids was assembled. Purification was performed by preparative reverse phase high performance liquid chromatography (HPLC). The relative molecular mass of the peptide was verified by electrospray ionization mass spectrometry (ESI-MS), and the purity of the peptide was evaluated by analytical HPLC.

[0051] (2) Loading of osteoclast inhibitory peptide FRAT: FRAT was mixed with PBS at a volume ratio of 1:1 with a concentration of 1.0×10 11 The probiotic extracellular vesicles of particles / mL were combined with the engineered probiotic extracellular vesicles. The mixture was placed in an electroporation tube and incubated for 3 hours. An electroporator was used for electrotransformation, and the electroporation conditions were set to 100V, 200Ω and 100μF. After the perforation was completed, it was allowed to stand at 37°C for 30 minutes. After standing, the unloaded polypeptide drug was removed by ultrafiltration centrifugation to obtain the FRAT@BEV-DCS.

[0052] (3) Characterization of osteoclast-targeted probiotic extracellular vesicles loaded with functional peptides that inhibit osteoclast differentiation

[0053] Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to characterize the morphology and particle size distribution of probiotic extracellular vesicles. Figure 2 As shown, BEV-DCS exhibits a typical bilayer membrane structure with an average diameter of about 150 nm.

[0054] Example 3 Encapsulation efficiency of osteoclast-targeted probiotic extracellular vesicles loaded with functional polypeptides that inhibit osteoclast differentiation

[0055] A series of FRAT solutions with a concentration gradient of 100, 200, 400, 800 and 1000 μg / mL were prepared with 1.0×10 11 The FRAT concentration in the engineered probiotic exosomes was determined by HPLC to determine the encapsulation rate. Figure 3 As shown, when the concentration of FRAT solution was 1000 μg / mL, an encapsulation efficiency of up to 20.4% could be achieved, proving that the electroporation method can efficiently load peptide drugs into engineered probiotic extracellular vesicles.

[0056] Example 4 Characterization of the enzymatic stability of osteoclast-targeted probiotic extracellular vesicles to protect the functional polypeptides loaded to inhibit osteoclast differentiation

[0057] The chymotrypsin solution was mixed with FRAT and FRAT@BEV-DCS at 37°C. At different time intervals, 50 μL of the mixture was added to 100 μL of acetonitrile to terminate the enzymatic hydrolysis. After centrifugation at 10000 g for 10 minutes, the supernatant was collected and analyzed by HPLC. Figure 4As shown in the figure, free FRAT degraded rapidly, and only 18.52% of FRAT was intact after 48 hours. However, the content of FRAT in FRAT@BEV-DCS could still reach 87.9% after 48 hours under the protection of probiotic extracellular vesicles, indicating that BEV-DCS nanocarriers effectively protected the loaded FRAT from protease degradation.

[0058] Example 5 Characterization of the effect of osteoclast-targeted fusion of probiotic extracellular vesicles on improving the intracellular penetration of functional peptides loaded to inhibit osteoclast differentiation

[0059] The FITC-labeled FRAT peptide was synthesized by solid phase synthesis, and the fluorescently labeled FRAT peptide was electroporated into BEV-DCS. FITC-FRAT and FITC-FRAT@BEV-DCS were incubated with osteoclast precursor cells for 6 hours. After rinsing with PBS three times, they were incubated with the blue fluorescent nuclear dye Hoechst at 37°C for 30 minutes. The internalization analysis fluorescence signals of FRAT and FRAT@BEV-DCS were observed by confocal laser scanning microscopy (CLSM). Figure 5 As shown, the FITC fluorescence signal enriched in the FRAT@BEV-DCS group was significantly stronger than that in the FRAT group, indicating that the BEV-DCS nanocarrier effectively enhanced the intracellular penetration efficiency of FRAT molecules in precursor osteoclasts.

[0060] Example 6 Characterization of the inhibitory effect of BEV-DCS loaded with functional peptides on osteoclast differentiation

[0061] Osteoclast formation was quantitatively detected by tartrate-resistant acid phosphatase (TRAP) staining. BMMs were seeded into 96-well plates at a density of 8,000 cells per well. After 24 h of incubation, M-CSF at a concentration of 30 ng / ml, RANKL at 50 ng / mL, and FRAT and FRAT@BEV-DCS were added for incubation. Fresh culture medium was replaced every 2 days. After successful osteoclast induction, the cells were fixed in 4% paraformaldehyde at room temperature and stained with a TRAP staining kit. Positive multinuclear osteoclasts were counted under an optical microscope, and the percentage of osteoclasts in each well was measured using ImageJ software. According to the statistical graph of TRAP staining results, multinuclear osteoclasts were formed after stimulation in the control group, while the number of differentiated osteoclasts decreased after the addition of FRAT and FRAT@BEV-DCS. Compared with FRAT, FRAT@BEV-DCS had a significant inhibitory effect on the differentiation of BMMs into osteoclasts, and the number and area of ​​quantifiable osteoclasts were reduced by 1.9 times and 3.3 times, respectively ( Figure 6 A and 6B), confirming that FRAT@BEV-DCS has a stronger inhibitory effect on osteoclastogenesis.

[0062] Example 7 Characterization of in vivo targeted distribution of BEV-DCS loaded with functional polypeptides

[0063] 8-week-old female C57BL / 6J mice were injected with Cy5.5-labeled FRAT and FRAT@BEV-DCS at a concentration of 50 μM via the tail vein. Six hours after injection, the osteoporotic mice were humanely killed, and their main organs and hind limbs were taken for in vitro imaging to evaluate the fluorescence distribution of FRAT and FRAT@BEV-DCS in the main organs and femur of the mice. Figure 7 As shown in the figure, compared with FRAT treatment, the fluorescence intensity in the femur of mice treated with FRAT@BEV-DCS increased significantly, and the proportion accumulated in the hindlimbs reached 28.7%, which was significantly higher than 12.5% ​​of FRAT. These results confirmed the in vivo bone targeting of FRAT@BEV-DCS mediated by osteoclast recognition and fusion DC-STAMP protein expressed on its membrane surface.

[0064] Example 8 Evaluation of the effect of BEV-DCS loaded with functional polypeptides in treating osteoporosis

[0065] (1) Establishment of the ovariectomized osteoporosis mouse model and grouping experiments: Animal studies were conducted in accordance with the principles of the National Institutes of Health on the care and use of experimental animals. The experimental protocol was approved by the Ethics Committee of Shanghai University (approval number ECSHU 2024-075). Female C57BL / 6J mice aged 8 weeks were housed in a standardized, controlled environment. Most mice were anesthetized with sodium pentobarbital and bilaterally ovariectomized to induce osteoporosis, and then randomly assigned to three groups: a sham operation group without ovariectomy, a control group without treatment, and two treatment groups receiving FRAT and FRAT@BEV-DCS, respectively. Mice in the treatment group were injected with FRAT or FRAT@BEV-DCS via the tail vein at a dose of 5 mg / kg, once a week for 7 weeks.

[0066] (2) Evaluation of osteoporosis treatment effect: After 7 weeks of treatment, all mice were killed, and one femur was removed and fixed with 4% PFA for micro-computed tomography (Micro-CT). Continuous sections were made from each femur from 0.15 mm below the growth plate to 0.4 mm proximal. The scan data was processed to generate three-dimensional visualization images of the epiphyseal and trabecular regions. These three-dimensional reconstructions were examined and analyzed using Data Viewer software for the bone volume fraction (BV / TV), bone surface area / total volume (BS / TV), and trabecular number (Tb.N) of each group. According to Figure 8 Micro-CT scans shown in A showed obvious trabecular bone loss without any treatment, indicating that the OVX model was successfully established. Figure 8BD analysis showed that after FRAT@BEV-DCS treatment, BV / TV and BS / TV increased significantly, bone loss gradually decreased, and Tb.N also showed an increasing trend. FRAT@BEV-DCS has a very significant therapeutic effect on osteoporosis, as shown by the fact that the trabecular bone mass is almost the same as that of the sham operation group. This indicates that FRAT@BEV-DCS can effectively reduce ovariectomy-induced bone loss.

[0067] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A probiotic extracellular vesicle BEV-DCS for osteoclast targeting fusion, characterized in that: The probiotics are Escherichia coli Nissle 1917 (EcN), and the probiotics outer vesicle membrane surface expresses osteoclast targeting fusion protein DC-STAMP; The BEV-DCS is obtained by constructing a recombinant plasmid expressing the osteoclast targeting fusion protein DC-STAMP, transforming the recombinant plasmid into competent cells of EcN to obtain a recombinant Escherichia coli strain, culturing the recombinant Escherichia coli strain and inducing the expression of DC-STAMP protein, and then extracting bacterial extracellular vesicles.

2. The osteoclast-targeted probiotic extracellular vesicle BEV-DCS according to claim 1, characterized in that: The recombinant plasmid expresses a gene fragment as shown in SEQ ID NO:

1.

3. A method for preparing the osteoclast-targeted fusion probiotic extracellular vesicle BEV-DCS according to claim 1 or 2, characterized in that: The following steps are involved: Step 1. Synthesis of recombinant plasmid: using pET28a as a cloning plasmid, the DC-STAMP gene and the ClyA gene are connected by molecular cloning technology to obtain a DC-STAMP-ClyA connection fragment; the pET28a plasmid is digested with a restriction endonuclease to obtain a linearized pET28a plasmid, and the DC-STAMP-ClyA connection fragment is connected to the linearized pET28a plasmid by a ligase to complete the construction of the pClyA-DC-STAMP recombinant plasmid; Step 2. Preparation and cultivation of engineered probiotics: preparing probiotic Escherichia coli competent cells, transferring the recombinant plasmid constructed in step 1 into the competent cells by electroporation to complete the construction of the probiotic recombinant strain, culturing the probiotic recombinant strain and inducing DC-STAMP protein expression to obtain a probiotic bacterial liquid; Step 3. Extraction of engineered probiotic extracellular vesicles: First, the probiotic bacterial liquid obtained in step 2 is collected by centrifugation to obtain the probiotic fermentation liquid expressing DC-STAMP, and then the bacterial extracellular vesicles are extracted by ultracentrifugation to obtain osteoclast-targeted fusion probiotic extracellular vesicles, wherein the osteoclast-targeted fusion protein DC-STAMP is expressed on the membrane surface of the probiotic extracellular vesicles.

4. The preparation method according to claim 3, characterized in that: The culturing in step 2 includes: firstly, growing the probiotics in a primary culture in a 37°C shaker for 12 to 24 hours, and then growing them in a secondary culture at 37°C until the OD 600 The inducing DC-STAMP protein expression comprises: adding IPTG at 16°C to induce DC-STAMP protein expression.

5. The preparation method according to claim 3, characterized in that: The ultracentrifugation method for extracting bacterial extracellular vesicles in step 3 comprises: centrifuging the probiotic fermentation broth at 10,000 to 20,000 g to remove bacterial precipitates to obtain a supernatant, filtering the supernatant and then centrifuging twice at 1×10 5 ~1.5×10 5 The extracellular vesicles expressing DC-STAMP protein on the membrane were collected by ultracentrifugation.

6. Use of the osteoclast-targeted fusion probiotic extracellular vesicle BEV-DCS according to claim 1 or 2 in loading drugs that inhibit osteoclast differentiation and preparing drugs that inhibit osteoclast differentiation by targeted delivery.

7. The use according to claim 6, characterized in that: The drug for inhibiting osteoclast differentiation includes the functional polypeptide FRATtide (FRAT) for inhibiting osteoclast differentiation.

8. A method for loading the osteoclast-targeted fusion probiotic extracellular vesicle BEV-DCS with the inhibitory functional polypeptide FRATtide according to claim 1 or 2, characterized in that: include: First, the functional polypeptide FRATtide and the osteoclast-targeted fused probiotic extracellular vesicles BEV-DCS are incubated in a solution, and then the functional polypeptide FRATtide is introduced into the probiotic extracellular vesicles by electroporation, thereby completing the loading of the functional polypeptide.

9. The method according to claim 8, characterized in that The specific steps of the method include: first preparing a FRAT solution with a concentration of 50 to 2000 μg / mL and a 1×10 8 ~10 11 particles / mL of a probiotic extracellular vesicle solution; then the FRAT solution and the probiotic extracellular vesicle solution are mixed and incubated for 1 to 5 hours at a volume ratio of (0.1 to 5):1; then the mixed solution after incubation is electroporated and transfected under the conditions of a voltage of 100V, a resistance of 200Ω and a capacitance of 100μF; after the electroporation, the mixture is allowed to stand at 30 to 45°C for 30 to 120 minutes; finally, the mixed solution is ultracentrifuged to remove residual FRAT molecules, and BEV-DCS loaded with functional polypeptide FRAT is obtained.

10. Use of the osteoclast-targeted probiotic exosomes of claim 1 or 2, or the osteoclast-targeted probiotic exosomes prepared by any of the methods of claims 3-5 or 8-9 in the preparation of drugs and preparations for inhibiting osteoclast differentiation.

11. Use of the osteoclast-targeted probiotic exosomes of claim 1 or 2, or the osteoclast-targeted probiotic exosomes prepared by any of the methods of claims 3-5 or 8-9 in the preparation of drugs and preparations for preventing and treating osteoporosis-related diseases.

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