Application of myeloid-derived suppressor cell-derived vesicles in preparation of medicine for relieving osteoporosis

Abstract

The invention relates to the field of biological medicine, in particular to application of myeloid-derived suppressor cell-derived vesicles in preparation of a medicine for relieving osteoporosis. The bone resorption-resistant drug is entrapped by the myeloid-derived suppressor cell-derived vesicles and is delivered to the myeloid-derived suppressor cells, so that the targeting property of the bone resorption-resistant drug is improved, and bone resorption is inhibited by inhibiting the activity of osteoclasts and the absorption effect of the osteoclasts on bone trabecula; the proportion of abnormal myeloid-derived suppressor cell subgroups in bone marrow can be reduced, formation and activity of osteoclasts are indirectly inhibited, the bone resorption process is further slowed down, and the curative effect of the anti-bone resorption medicine on osteoporosis can be improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of vesicles derived from myeloid-derived suppressor cells in the preparation of drugs for alleviating osteoporosis. Background Art

[0002] Osteoporosis is a common bone metabolic disease in middle-aged and elderly people, especially postmenopausal women. It is characterized by decreased bone mass, destruction of bone microarchitecture, and increased bone brittleness, which significantly increase the risk of fractures. Current treatments for osteoporosis mainly rely on anti-resorptive drugs, such as zoledronic acid (ZOL), which can induce apoptosis in osteoclasts by inhibiting the activity of farnesyl pyrophosphate synthase (FPPS). However, systemic administration of ZOL is prone to non-targeted distribution, and long-term use may cause side effects such as jaw necrosis, nephrotoxicity, and excessive inhibition of systemic bone remodeling, limiting its safety and compliance. Therefore, it is necessary to develop a drug carrier suitable for ZOL with high targeting.

[0003] In recent years, cell-derived nanovesicles (CNVs) have become a hot topic in drug delivery research due to their excellent biocompatibility and targeted delivery potential. Vesicles derived from immune cells, in particular, may retain specific marker molecules from the parent cell membrane, enabling active targeting to homologous cells or the lesion microenvironment. Combining the properties of ZOL with the physiological changes associated with osteoporosis, it is promising to develop CNVs capable of delivering ZOL for the treatment of osteoporosis. Summary of the Invention

[0004] The object of the present invention is to provide a CNVs capable of delivering ZOL to treat osteoporosis.

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

[0006] Application of myeloid-derived suppressor cell-derived vesicles in the preparation of drugs for alleviating osteoporosis.

[0007] Optionally, the myeloid-derived suppressor cell-derived vesicles contain anti-bone resorption drugs.

[0008] Optionally, the anti-bone resorption drug is zoledronic acid.

[0009] Optionally, the myeloid-derived suppressor cell-derived vesicles contain a zoledronic acid solution, and the concentration of the zoledronic acid solution is any value between 2 μg / ml and 8 μg / ml.

[0010] Optionally, the myeloid-derived suppressor cell-derived vesicles are administered by injection.

[0011] Optionally, the dosage of the myeloid-derived suppressor cell-derived vesicles is any value between 0.05 mg / kg and 0.15 mg / kg.

[0012] Optionally, the myeloid-derived suppressor cell-derived vesicles are targeted for delivery to myeloid-derived suppressor cells.

[0013] Optionally, the myeloid-derived suppressor cell-derived vesicles inhibit the activity of osteoclasts and their absorption of trabecular bone, thereby promoting bone formation and inhibiting bone resorption.

[0014] Optionally, the myeloid-derived suppressor cell-derived vesicles reduce the proportion of abnormal myeloid-derived suppressor cell subpopulations in the bone marrow, inhibit the expression and secretion of immunoglobulin IgG, inhibit Fc receptor-dependent osteoclast activation, indirectly inhibit the formation and activity of osteoclasts, and slow down the bone resorption process.

[0015] The beneficial effects of the present invention are that anti-bone resorption drugs are encapsulated in myeloid-derived suppressor cell-derived vesicles and delivered to myeloid-derived suppressor cells, thereby helping to improve the targeting of anti-bone resorption drugs and their efficacy in treating osteoporosis.

[0016] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 TRAP staining analysis of MDSCs-induced osteoclast formation before and after OVX;

[0018] Figure 2 Volcano plot of differentially expressed genes in MDSCs transcriptome sequencing before and after OVX;

[0019] Figure 3 qRT-PCR quantitative analysis of MDSCs before and after OVX;

[0020] Figure 4 This is a quantitative analysis of IgG expression and secretion before and after OVX;

[0021] Figure 5 This is the result of the IgG activation MDSCs experiment;

[0022] Figure 6 This is a transmission electron microscopy image of the MDSC-NVS@ZOL shown in Example 1 of the present invention;

[0023] Figure 7 This is a statistical diagram of the particle size of MDSC-NVS@ZOL shown in Example 1 of the present invention;

[0024] Figure 8 This is a statistical diagram of the zeta potential of MDSC-NVS@ZOL shown in Example 1 of the present invention;

[0025] Figure 9 This is a fluorescence labeling distribution diagram of MDSCs co-incubated with MDSC-NVS@ZOL as shown in Example 1 of the present invention;

[0026] Figure 10 This is a flow cytometry analysis diagram of MDSC-NVS@ZOL co-incubated with MDSCs and BMSCs as shown in Example 1 of the present invention;

[0027] Figure 11 The Micro-CT reconstructed image and quantitative analysis diagram shown in Example 1 of the present invention;

[0028] Figure 12 This is a diagram of histological staining and quantitative analysis shown in Example 1 of the present invention;

[0029] Figure 13 This is a diagram showing the detection and analysis of the bone marrow sample and serum sample of the mouse shown in Example 1 of the present invention;

[0030] Figure 14 This is a graph analyzing the levels of bone formation markers in the serum samples of mice shown in Example 1 of the present invention. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0032] In the description of the present invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and therefore should not be construed as limitations on the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0033] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on the specific circumstances.

[0034] In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0035] The inventors constructed an ovariectomy (OVX) model to simulate osteoporosis and studied the changes in various immune cells in mice before and after the model was constructed. They found that the OVX model construction would lead to an abnormal increase in the proportion of myeloid-derived suppressor cells (MDSCs) in the bone marrow. The inventors studied the expression of CD11b at different time points after OVX or sham surgery. + Gr1 + The changing trends of the MDSCs ratio and trabecular bone volume fraction (BV / TV) showed that the two were significantly negatively correlated in the early stage, suggesting that the increase in the MDSCs ratio occurred synchronously with bone loss.

[0036] MDSCs isolated from OVX mice were labeled as OVX-MDSCs group cell samples, and MDSCs isolated from sham-operated mice were labeled as H-MDSCs group cell samples. Further research was conducted on the two groups of cell samples to verify whether the changes in MDSCs are involved in the regulation of bone resorption.

[0037] Receptor Activator for Nuclear Factor-κB Ligand (RANKL) is a key factor in promoting the final differentiation, maturation and functional activity of osteoclasts. M-CSF protein induces RANK expression on the cell membrane of osteoclast precursors, thereby causing these cells to bind to RANKL and produce an effect, ultimately inducing osteoclast differentiation. M-CSF or M-CSF + RANKL was added to two groups of cell samples, and TRAP was detected by tartrate-resistant acid phosphatase (TRAP) staining after incubation. + For the content of multinucleated osteoclasts, see Figure 1 It can be seen that the OVX-MDSCs group samples are more likely to form TRAP under the induction of M-CSF+RANKL than the H-MDSC group samples. + Multinucleated osteoclasts.

[0038] The mechanism of the above conclusions was studied through transcriptome sequencing. Figure 2 , it can be seen that in addition to the upregulation of the osteoclast activation pathway, the B cell receptor (BCR) signaling pathway was significantly upregulated in OVX-MDSCs. The mRNA expression levels of related genes, including the BCR signaling hub Blnk, the key regulatory gene for VDJ rearrangement, the B cell development core transcription factor (Pax5), and the recombination activation gene Rag1 involved in antibody gene rearrangement, were significantly increased. Figure 3 qRT-PCR was used to detect the expression of Blnk, Pax5, and Rag1 in the two groups of cell samples. The results showed that the expression of Pax5, Blnk, and Rag1 in the OV-MDSCs group was increased compared with that in H-MDSCs. Pax5 can promote B cell antibody production by regulating the expression of immunoglobulin heavy chain (IgH) genes, while the synergistic activation of Blnk and Rag1 may enhance the sensitivity of the BCR signaling pathway. Since the expression of all three is upregulated, it can be speculated that the abnormal activation of the BCR signaling pathway in the OVX-MDSCs group may lead to increased IgG expression on the surface of OVX-MDSCs by promoting gene rearrangement, thereby mediating Fc receptor (FcR)-dependent osteoclast activation.

[0039] See Figure 4 The expression of IgG on the cell membrane of the two groups of MDSCs was detected by flow cytometry. + Gr1 + Characterization of MDSCs showed that IgG in the bone marrow of mice after OVX + The proportion of MDSCs was significantly higher than that without OVX. Since IgG is a secretory antibody, the IgG content in the serum of mice after OVX or sham operation was also detected by ELISA. It was found that the serum IgG level of OVX mice was significantly increased, which was consistent with the bone marrow IgG. + The increased proportion of MDSCs corresponds to the phenotype, which verifies the above inference. The number of replicates in this experiment was 3. * indicates P < 0.05, and ** indicates P < 0.01.

[0040] To investigate the role of IgG / FcR signaling in MDSCs osteoclast activation, MDSCs in the bone marrow of healthy mice were activated with IgG, and then treated with FcR-specific inhibitors, namely Anti-CD16 / 32 neutralizing antibodies, followed by RANKL-induced osteoclast activation. Figure 5TRAP staining revealed that inhibitor-treated cells significantly decreased the number of multinucleated osteoclasts compared to the untreated group, with reduced cell size and decreased nuclear fusion. qRT-PCR results showed that FcR signaling blockade significantly downregulated the mRNA expression levels of key regulatory genes for osteoclast activation, such as Nfatc1 and Osca. This experiment was replicated three times within each group. ** indicates P < 0.01, and *** indicates P < 0.0001.

[0041] Based on the above experimental results, the inventors attempted to encapsulate ZOL in myeloid-derived suppressor cell-derived vesicles (MDSC-NVs) and verify its therapeutic effect on osteoporosis.

[0042] This application claims the use of myeloid-derived suppressor cell-derived vesicles in the preparation of a drug for alleviating osteoporosis. By encapsulating an anti-bone resorption drug in myeloid-derived suppressor cell-derived vesicles and delivering it to the myeloid-derived suppressor cells, the drug's targeting and efficacy for osteoporosis are enhanced.

[0043] In some embodiments, the myeloid-derived suppressor cell-derived vesicles are loaded with an anti-resorptive drug.

[0044] In some embodiments, the antiresorptive drug is zoledronic acid

[0045] In some embodiments, the myeloid-derived suppressor cell-derived vesicles are loaded with a zoledronic acid solution, and the concentration of the zoledronic acid solution is any value between 2 μg / ml and 8 μg / ml, for example, any value between 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml and 8 μg / ml.

[0046] In some embodiments, myeloid-derived suppressor cell-derived vesicles are administered by injection.

[0047] In some embodiments, the dosage of myeloid-derived suppressor cell-derived vesicles is any value between 0.05 mg / kg and 0.15 mg / kg, for example, any value between 0.05 mg / kg, 0.07 mg / kg, 0.09 mg / kg, 0.11 mg / kg, 0.13 mg / kg, and 0.15 mg / kg.

[0048] In some embodiments, myeloid-derived suppressor cell-derived vesicles are targeted for delivery to myeloid-derived suppressor cells.

[0049] In some embodiments, myeloid-derived suppressor cell-derived vesicles inhibit the activity of osteoclasts and their resorption of trabecular bone, thereby promoting bone formation and inhibiting bone resorption.

[0050] In some embodiments, myeloid-derived suppressor cell-derived vesicles reduce the proportion of abnormal myeloid-derived suppressor cell subpopulations in the bone marrow, inhibit the expression and secretion of immunoglobulin IgG, and suppress Fc receptor-dependent osteoclast activation, indirectly inhibiting the formation and activity of osteoclasts and slowing bone resorption. See the following examples for details.

[0051] Example 1:

[0052] The method for preparing myeloid-derived suppressor cell-derived vesicles as shown in a preferred embodiment of the present application includes:

[0053] S1. Collect MDSCs from healthy mice after sorting, wash them three times with phosphate buffered saline (PBS), and then resuspend them in PBS to a concentration of approximately 1×10 7 / mL, and then ZOL was added to make the concentration of 5μg / ml to obtain a cell suspension.

[0054] S2. Slowly squeeze the cell suspension through a 1.0 μm polycarbonate filter to remove the supernatant. Repeat 13 times to obtain the cell membrane solution. The filter should be fixed vertically to the end of the syringe and the operation should be carried out on ice.

[0055] S3. Replace the 0.4 μm polycarbonate filter membrane, squeeze the cell membrane liquid to obtain the supernatant, repeat 15 times, then replace the 0.2 μm polycarbonate filter membrane to squeeze the supernatant, repeat 15 times to obtain a vesicle suspension.

[0056] S4. Centrifuge at 1500 g for 5 minutes, collect the supernatant, and then centrifuge at 15,000 g for 1 hour. The resulting precipitate is MDSC-NVs loaded with ZOL, i.e., MDSC-NVS@ZOL. Resuspend in PBS and store at -80°C.

[0057] In step S1, high-purity MDSCs are obtained by flow cytometry sorting, and multiple washing steps are performed to reduce the interference of free impurities such as bovine serum albumin on subsequent vesicle purification.

[0058] In step S2, cells are mechanically disrupted by extrusion through a filter membrane to form cell membrane fragments. Multiple extrusions are performed to ensure complete cell lysis and to preliminarily homogenize the membrane fragments.

[0059] In step S3, the pore size of the filter is gradually reduced to remove large particle debris, such as organelles and unlysed cells, while retaining uniform small vesicles. Multiple extrusions through the same pore size filter help improve vesicle uniformity and avoid excessive aggregation of membrane fragments.

[0060] In step S4, unbroken cells, cell nuclei, mitochondria and other large particles are removed by low-speed centrifugation, and then vesicles are enriched by high-speed centrifugation.

[0061] Take 10 μL of MDSC-NVS@ZOL suspension of appropriate concentration and drop it onto the copper mesh surface. After standing for 5 minutes for adsorption, remove the excess liquid with filter paper. Add 2% phosphotungstic acid and stain for 1 minute. After blotting, dry at room temperature. Use transmission electron microscopy (TEM) to observe the morphology of MDSC-NVS@ZOL. Figure 6 , it can be seen that MDSC-NVS@ZOL is in a complete spherical state and is filled with drug solution.

[0062] The particle size of the vesicles was measured by dynamic light scattering (DLS), with the detection temperature set at 25°C and the measurement angle at 90°. The results were repeated three times and the data were analyzed to obtain the particle size distribution curve and polydispersity index (PDI). The same instrument was used, switched to the Zeta potential mode, and the sample was injected and the data were automatically measured and analyzed again. All data in this example are expressed as mean ± standard deviation (mean ± SD) within the group. Statistical analysis was performed using GraphPad Prism 9.0.0 software. Please refer to Figure 7 and Figure 8 The average hydrodynamic diameter of MDSC-NVS@ZOL was 104.73 ± 8 nm, and the vesicles were uniform in size and well dispersed. The surface charge of MDSC-NVS@ZOL was -7.06 ± 2.1 mV. This negative charge facilitates vesicle stability in physiological environments and reduces nonspecific adsorption. In other words, the MDSC-NVS@ZOL prepared in this example exhibited intact morphology, uniform particle size, and stable structure.

[0063] 500 μL of vesicle suspension was placed in a 1.5 mL EP tube. After centrifugation at 8000 rpm for 10 minutes, the prepared DiO staining solution was added. The cells were stained in the dark at 37°C for 15 minutes. After washing twice with PBS, the MDSC-NVS@ZOL was fluorescently labeled with DiO. The labeled MDSC-NVS@ZOL was then co-incubated with red fluorescent phalloidin to specifically label actin filaments (F-actin) and MDSCs whose nuclei were labeled with DAPI. MDSCs double-labeled using the same method served as a blank control. Observation and photography were performed using an inverted fluorescence microscope. See [see figure 1] for details. Figure 9 , it can be seen that MDSCs can successfully take up MDSC-NVs@ZOL, and the vesicles can co-localize with the cell membrane and endocytic vesicles.

[0064] After DiO fluorescently labeled MDSC-NVS@ZOL was co-incubated with MDSCs and bone marrow mesenchymal stem cells (BMSCs) for 24 hours, the proportion of DiO positive cells in MDSCs and BMSCs was quantitatively analyzed by flow cytometry. Figure 10 , it can be seen that the proportion of DiO-positive cells in MDSCs is 15.3%, while the proportion of DiO-positive cells in BMSCs is only 3.8%, which shows that the uptake capacity of MDSCs for self-derived vesicles is much higher than that of BMSCs, and the targeting is significant, which verifies that MDSC-NVs@ZOL achieves specific targeting of homologous MDSCs through the mother cell membrane molecules retained on the surface. Therefore, MDSC-NVs as a delivery platform can not only protect the drug structure, but also achieve local enrichment, reduce non-targeted toxic and side effects, and improve treatment safety and compliance. In this embodiment, the comparison between the two groups adopts the unpaired t-test, and the comparison of more than two groups of data adopts one-way analysis of variance (ANOVA). The statistical significance level is set at P<0.05, where * represents P<0.05, ** represents P<0.01, *** represents P<0.001, and ns represents P>0.05.

[0065] The ability of MDSC-NVS@ZOL to inhibit osteoporosis was validated through animal experiments. Twenty-four six-week-old female C57BL / 6 mice were randomly divided into a sham group, an OVX+PBS group, an OVX+ZOL group, and an OVX+MDSC-NVs@ZOL group, with six mice in each group. The sham group underwent a sham surgery, while the other three groups underwent ovariectomy (OVX) models. The model construction steps included anesthetizing mice with an intraperitoneal injection of 50 mg / kg of 2% sodium pentobarbital. Once fully anesthetized, the mice were mounted on a mouse board and surgical procedures were performed under sterile conditions. The laparotomy was followed by bilateral ovarian incision, ovarian ligation, and ovariectomy, followed by suturing of the abdominal cavity. For sham surgery, only the laparotomy was performed, the ovaries were located, and then repositioned, followed by suturing of the abdominal cavity. Throughout the surgical procedure, strict animal ethics guidelines were adhered to, with careful attention paid to animal protection and minimizing bleeding and tissue damage. Mice were treated with penicillin sodium at a dose of 100 U / g for three consecutive days after surgery to prevent infection. Starting four weeks after surgery, treatment fluid was injected into the tail vein of the mice once a week for a total of four injections. The sham and OVX+PBS groups received PBS as the treatment fluid, the OVX+ZOL group received ZOL in PBS at a dose of 0.1 mg / kg, and the OVX+MDSC-NVs@ZOL group received MDSC-NVs@ZOL resuspended in PBS at a dose of 0.1 mg / kg. Eight weeks after surgery, all mice were sacrificed, and femurs, bone marrow cells, and serum were collected for subsequent experiments. Serum samples were obtained from mice by ex vivo sampling. After anesthesia, orbital blood was collected from each group of mice using heparin-rinsed forceps. The collected blood samples were allowed to stand at 4°C for 12 hours and then centrifuged at 1500 g for 20 minutes. The supernatant was then collected for later use to obtain serum samples.

[0066] The femurs were decalcified and dehydrated before embedding and sectioning for storage. After removing excess muscle from the femurs, they were fixed in 4% paraformaldehyde for two days and washed three times with PBS. The femurs were immersed in a 14% EDTA solution (pH 7.2-7.4) and placed on a shaker. The solution was changed every two days for 21 days of decalcification. Any residual decalcification solution on the surface was then rinsed with deionized water. Each femur was dehydrated in a gradient of 70%, 80%, 90%, 95%, and 100% ethanol, with each concentration treated for one hour. After dehydration, the femurs were immersed in an environmentally friendly, transparent deparaffinizing solution for 4-6 hours and then placed in a paraffin solution overnight to protect them from moisture. Before embedding, the embedding machine was preheated four hours in advance to melt the paraffin, and the cryogenic freezer was started one hour in advance to precool the paraffin. During embedding, ensure that each femur was placed on the paraffin base at the same angle. After embedding, the embedded femurs were placed in a cryogenic freezer to solidify and sliced at a thickness of 6 μm using a vibratome. The slices were flattened in 45°C hot water, transferred to adhesive slides, and then dried in a 65°C oven for 2 hours to obtain the femoral specimens used in subsequent experiments.

[0067] Bone mineral density (BMD) is a commonly used indicator for measuring bone mass, and is of great significance for early screening of osteoporosis and assessment of fracture risk. Bone volume fraction (Bone Volume / Tissue Volume, BV / TV) and trabecular thickness (Trabecular Thickness, Tb.Th) can measure the integrity of trabecular structure. The number of trabecular bones (Trabecular Number, TB.N) can measure the strength and compressive resistance of bones. Micro-CT was used to scan and reconstruct the cancellous bones of mice in three dimensions, and to scan and reconstruct the femurs of mice in two dimensions. The bone density (BMD), bone volume fraction (BV / TV), trabecular number (TB.N), and trabecular thickness (TB.Th) of each sample were detected and quantitatively analyzed. For reconstruction and analysis results, please see Figure 11It can be seen that OVX caused a significant decrease in the bone density of mice, with a decrease of 25%, and the bone volume fraction (BV / TV) and the number of trabeculae (TB.N) decreased by 28% and 20% respectively. The number of trabeculae (TB.N) also decreased significantly. This indicates that the femur of the mice has experienced trabecular sparseness and increased separation, and significant trabecular structural degeneration has occurred. Traditional ZOL treatment partially improved bone microstructure. In contrast, MDSC-NVs@ZOL treatment had a significantly better inhibitory effect on changes in bone density, bone volume fraction and trabecular thickness than ZOL treatment. Bone density increased by 32% compared with the OVX+PBS group, almost approaching the level of the Sham group, while bone volume fraction (BV / TV) and trabecular number (TB.N) increased by 26% and 18% respectively. This shows that MDSC-NVs@ZOL treatment can alleviate bone loss in OVX mice. This targeted delivery system can effectively improve trabecular structure and reverse skeletal degeneration caused by bone loss.

[0068] See Figure 12 Femoral bone samples from each group of mice were observed and analyzed by histological staining. Femoral samples from each group of mice were stained with H&E, and trabecular area was quantitatively analyzed based on the area of the blue area in the images. Femoral samples from each group of mice were also stained with Masson staining, and relative collagen content was quantitatively analyzed based on the area of the blue area in the images. The H&E and Masson staining images and analysis results showed that compared with the sham group, the number of trabeculae under the distal femoral epiphyseal plate in the OVX+PBS group was significantly reduced. Treatment with MDSC-NVs@ZOL effectively increased trabecular bone formation and significantly increased collagen fibril formation, indicating that the targeted delivery system can effectively reverse bone loss. Femoral samples from each group of mice were then stained with TRAP. Osteoclasts with wine-red cytoplasm and blue nuclei were identified under the microscope and counted to obtain the osteoclast count (Oc.s). The total osteoclast count (Oc.s) was calculated based on the area of the red area in 3-5 randomly selected fields of view and quantitatively analyzed. The activity of osteoclasts is a key factor affecting the degree of bone resorption. According to the experimental results, MDSC-NVs@ZOL intervention significantly reduced the number of osteoclasts attached to trabecular bone, and quantitative results also confirmed that MDSC-NVs@ZOL intervention reduced the number of osteoclasts / bone surface area (Oc.S / BS) by 41%, suggesting that it significantly inhibited the activity of osteoclasts and the resorption of osteoclasts on trabecular bone.

[0069] Bone marrow and serum samples were collected from each group of mice. The proportion of IgG-positive MDSCs in the mouse bone marrow samples was detected by flow cytometry and quantitatively analyzed. The IgG content in the mouse serum samples was then determined using an ELISA kit and a standard curve method. The test results are shown in the table. Figure 13 , it can be seen that OVX will cause the proportion of IgG-positive MDSCs in the mouse bone marrow to increase significantly. Traditional ZOL treatment is difficult to effectively inhibit this change. After MDSC-NVs@ZOL treatment, the proportion of IgG-positive MDSCs decreased significantly, with a decrease of 47%, and even recovered to a level close to that of the Sham group. The changes in the IgG content in mouse serum are consistent with the results obtained by flow cytometry. According to the research results of the present invention, IgG-positive MDSCs can promote osteoclast activation and participate in the occurrence and development of osteoporosis. Combined with the results obtained in this experiment, it is further verified that MDSC-NVs@ZOL treatment can inhibit the weakening of immunosuppressive function caused by OVX, inhibit abnormal MDSCs subpopulations, improve the local microenvironment of the bone marrow, and curb bone loss from the source.

[0070] The levels of bone formation markers PINP and CTX-Ⅰ in mouse serum samples were detected by ELISA kits and quantitatively analyzed. Figure 14 , it can be seen that in the OVX+PBS group of mice, the level of CTX-1, a bone resorption marker, in the serum was significantly increased, while the bone formation marker PINP was significantly decreased. Traditional ZOL treatment can only reduce the content of CTX-1 to a certain extent. After MDSC-NVs@ZOL treatment, the PINP level increased by about 34%, returning to a level close to that of the Sham group, indicating that bone formation was improved. At the same time, the CTX-1 level decreased by about 30%, which was significantly better than ZOL treatment, indicating that bone resorption was inhibited and the treatment had a good therapeutic effect. Therefore, the delivery of ZOL through MDSC-NVs has a positive effect on promoting bone formation and reducing the secretion of autoantibodies, and effectively inhibits bone resorption.

[0071] The present invention has the beneficial effect of providing a delivery system for anti-bone resorption drugs. By encapsulating the anti-bone resorption drugs in myeloid-derived suppressor cell-derived vesicles and delivering them to the myeloid-derived suppressor cells, the system helps improve the targeting of the anti-bone resorption drugs. Furthermore, the system inhibits the activity of osteoclasts and their resorption of trabecular bone, thereby promoting bone formation and inhibiting bone resorption. Furthermore, the system can reduce the proportion of abnormal myeloid-derived suppressor cell subpopulations in the bone marrow, indirectly inhibiting the formation and activity of osteoclasts and further slowing the bone resorption process. This system helps improve the efficacy of anti-bone resorption drugs for osteoporosis.

[0072] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0073] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. Application of myeloid-derived suppressor cell-derived vesicles in the preparation of drugs for alleviating osteoporosis.

2. The use according to claim 1, characterized in that The myeloid-derived suppressor cell-derived vesicles contain anti-bone resorption drugs.

3. The use according to claim 2, characterized in that The anti-bone resorption drug is zoledronic acid.

4. The use according to claim 3, characterized in that The myeloid-derived suppressor cell-derived vesicles contain a zoledronic acid solution, and the concentration of the zoledronic acid solution is any value between 2 μg / ml and 8 μg / ml.

5. The use according to claim 4, characterized in that The myeloid-derived suppressor cell-derived vesicles are administered by injection.

6. The use according to claim 5, characterized in that The dosage of the myeloid-derived suppressor cell-derived vesicles is any value between 0.05 mg / kg and 0.15 mg / kg.

7. The use according to claim 1, wherein The myeloid-derived suppressor cell-derived vesicles are delivered to myeloid-derived suppressor cells in a targeted manner.

8. The use according to claim 1, wherein The myeloid-derived suppressor cell-derived vesicles inhibit the activity of osteoclasts and their absorption effect on trabecular bone, promote bone formation, and inhibit bone absorption.

9. The use according to claim 1, wherein The myeloid-derived suppressor cell-derived vesicles reduce the proportion of abnormal myeloid-derived suppressor cell subpopulations in the bone marrow, inhibit the expression and secretion of immunoglobulin IgG, inhibit Fc receptor-dependent osteoclast activation, indirectly inhibit the formation and activity of osteoclasts, and slow down the bone resorption process.

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