NMN-loaded nanoparticles on stem cell exosomes stimulated by alendronate sodium and their applications

Abstract

This invention relates to the fields of regenerative medicine and healthcare, specifically to alendronate sodium-stimulated stem cell exosomes loaded with NMN nanoparticles and their applications. The nanoparticles of this invention are prepared by first seeding mouse bone marrow mesenchymal stem cells (MBMSCs) into HDF using human dermal fibroblasts (HDF) as the feeder cells, followed by pretreatment of HDF-MBSCs with ALN to obtain their exosomes (AHM-E). Finally, the purified exosomes are loaded with β-nicotinamide mononucleotide (NMN) to prepare the AHM-E@NMN nanoparticles. The AHM-E@NMN nanoparticles of this invention possess the basic characteristics of exosomes, enabling them to promote osteoblast differentiation in vitro and inhibit OVX-induced bone loss in mice with osteoporosis in vivo, demonstrating a good anti-osteoporosis effect and showing promise for development and use in clinical treatment of osteoporosis drugs.

Description

Technical Field

[0001] This invention relates to the fields of regenerative medicine and healthcare, specifically to nanoparticles loaded with NMN on stem cell exosomes stimulated by sodium alendronate and their applications. Background Technology

[0002] As human society gradually enters an aging phase, the health of the elderly is receiving increasing attention. Osteoporosis, a common metabolic chronic bone disease in the elderly, is primarily associated with abnormal bone loss, impaired bone function, and an increased risk of fractures. This disease seriously endangers the health of the elderly, making it urgent to alleviate the harm caused by osteoporosis. Osteoporosis occurs in both sexes, but women are more prone to severe osteoporotic complications (approximately 80%). Several genetic and environmental factors associated with osteoporosis include aging, menopause (women), and low testosterone levels (men). The consequences of this disease are always associated with loss of independence, high morbidity and economic costs, and even excess mortality, and it can occur at any age and in any racial or ethnic group. Although osteoporosis can be partially prevented and treated today, its pathophysiology is not fully understood. The key to this most common metabolic bone disease is restoring and maintaining the balance between bone formation and resorption.

[0003] Currently, various pharmacological approaches, including hormone replacement therapy (HRT), selective androgen receptor modulators (SARMs), bisphosphonates (BPHs), and calcitonin, are used to treat osteoporosis. Among these drugs, BPHs are one of the most commonly prescribed anti-fracture agents. BPHs are inorganic pyrophosphate analogs that inhibit bone resorption by inducing osteoclast apoptosis, thereby preventing age-related bone loss and deterioration of bone microstructure. Nitrogen-containing BPHs (such as alendronate, risedronate, ibandronate, and zoledronic acid) have the most potent anti-bone resorption properties and are the most commonly used drugs for treating osteoporosis. Among different drugs, alendronate sodium (ALN) is one of the best and most widely studied BPHs for treating osteoporosis.

[0004] As is well known, ALN can prevent hydroxyapatite loss, inhibit osteoclast-mediated bone resorption, and promote osteoblast differentiation, thereby regulating the body's bone homeostasis. This drug is primarily used to treat osteoporosis, bone metastases, and Paget's disease. Recently, it has been proposed as an adjunct analgesic in combination with opioids for cancer pain and as a bone antitumor agent. However, in addition to its low bioavailability (<1% when administered orally), its systemic application can cause numerous side effects, such as fever, esophageal erosion, ulcers, and gastrointestinal problems. Therefore, reducing the toxic side effects of ALN and finding new treatments for osteoporosis are crucial. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned technical problems by providing a sodium alendronate-stimulated stem cell exosome loaded with NMN nanoparticles. These nanoparticles are prepared by first seeding mouse bone marrow mesenchymal stem cells (MBMSCs) into HDF using human dermal fibroblasts (HDF) as feeder cells, followed by pretreatment of HDF-MBSCs with ALN to obtain their exosomes (AHM-E). Finally, the purified AHM-E is loaded with β-nicotinamide mononucleotide (NMN) to prepare the nanoparticles AHM-E@NMN.

[0006] The nanoparticles AHM-E@NMN provided by this invention contain HDF, which provides excellent growth factors for MBMSCs, promoting their proliferation. HDF-MBMSCs treated with ALN can then secrete exosomes containing small molecules such as osteoblast-related protein factors, promoting osteoblast differentiation and avoiding the toxic side effects of direct ALN administration. NMN is an important NAD+... + The intermediate can enhance bone healing in mice with osteoporosis by promoting intracellular NAD levels, thereby reducing cellular senescence and promoting osteoblast differentiation. Currently, there are no reports of AHM-E-loaded NMN nanoparticles obtained by pretreating MBMSCs with ALN, which is of great significance for the preparation of nanoparticles to reduce the toxic side effects of ALN and treat osteoporosis.

[0007] This invention also provides a method for preparing NMN-loaded nanoparticles from stem cell exosomes stimulated by sodium alendronate, specifically comprising the following steps:

[0008] S1: Obtain human dermal fibroblasts (HDF) and mouse bone marrow mesenchymal stem cells (MBMSC);

[0009] S2: Alendronate sodium (ALN) stimulates stem cells to produce exosomes;

[0010] The stem cells are MBMSCs seeded on HDF, where HDF is a feeder layer of MBMSCs;

[0011] The exosomes were obtained by extracting exosomes AHM-E from the cell culture medium after alendronate sodium (ALN) stimulation of stem cells.

[0012] S3: Loading NMN into AHM-E exosomes: Dissolve NMN in PBS solution, then mix the exosomes AHM-E obtained in S2 with the NMN solution, add to an electroporation vessel for electroporation, collect the AHM-E@NMN supernatant after electroporation, add 0.5 mL of exosome preservation solution, and store for later use.

[0013] Further, the method for obtaining HDF described in S1 is as follows: adult skin tissue is minced and placed in a culture dish, 0.1% dispersant enzyme II solution is added, the dish is sealed with sealing film, and digested overnight at 4°C. The next day, dermis is minced with tweezers, transferred to a centrifuge tube, and a mixed solution containing 0.1% collagen type I enzyme, 0.1% collagen type II enzyme, and 0.1% collagen type IV enzyme is added. The mixture is digested in a 37°C water bath for 2 hours, filtered through a 100μm mesh sieve, and the filtrate is centrifuged at 300×g for 5 minutes. The precipitate is resuspended in high-glucose DMEM complete medium containing 10% FBS, 1% penicillin, and streptomycin, and then inoculated into T25 cell culture flasks. The medium is changed every other day.

[0014] Further, the method for obtaining MBMSCs described in S1 is as follows: Bone marrow cells from the femur and tibia of mice are collected, filtered using a 70 μM cell filter, and the filtrate is collected by centrifugation at 300 × g for 10 min. The cells are then resuspended in MBMSC medium, seeded into T25 cell culture flasks, and incubated in a humidified incubator at 37°C with 5% CO2. After one day, the cells are washed twice with DPBS to remove non-adherent cells, and 5 mL of MBMSC medium is added. Once the cells reach approximately 80% confluence within one week, they can be passaged.

[0015] Furthermore, the method described in S2 for stimulating stem cells to produce exosomes using alendronate sodium (ALN) is as follows: P9 generation HDF, which has been passivated by 10 μg / mL mitomycin C, is processed at a rate of 2 × 10⁻⁶... 4 cells / cm 2 Cells were seeded at a density of 5 × 10⁶ cells per well in six-well plates and cultured overnight. On the second day, the culture medium was changed to MBMSC medium, and after 1 hour, the cells were seeded at a density of 5 × 10⁶ cells per well. 4 cells / cm 2 MBMSCs were seeded at the appropriate cell density on HDF and the medium was changed daily. After 5 days of culture, the medium was replaced with MBMSC medium containing ALN and cultured for another day. The supernatant of the HDF-MBMSC cell culture after ALN treatment was collected, centrifuged at 700×g for 10 min to remove cell debris, and then centrifuged at 9000×g for 30 min at 4℃. The supernatant was collected again, and exosomes were isolated using the ExoEasy Maxi kit. The precipitate was resuspended in 100 μL of PBS, and the exosome concentration was detected by BCA protein quantification. The exosomes were aliquoted into 100 μL tubes at a concentration of 2 g / mL and stored at -80℃ for later use, i.e., AHM-E.

[0016] Furthermore, the ALN concentration in the MBMSC medium containing ALN is 2.5-10 μM.

[0017] Furthermore, the electro-spinning conditions described in S3 are: electric field strength 1KV / cm, electro-spinning temperature 37℃, and electro-spinning time 5min.

[0018] Furthermore, in S3, AHM-E and NMN are loaded in a mass ratio of 5:1.

[0019] This invention also provides the application of AHM-E@NMN nanoparticles in the preparation of drugs for treating osteoporosis.

[0020] Furthermore, the osteoporosis treatment drug is an NMN-loaded exosome of MBMSCs with HDF as the feeder layer cells, stimulated by ALN.

[0021] Furthermore, the osteoporosis treatment drug is a drug that promotes osteoblast differentiation.

[0022] Furthermore, the osteoporosis treatment drug is a drug that promotes bone healing in OVX-induced osteoporotic mice.

[0023] Furthermore, the osteoporosis treatment drug is a drug that inhibits osteoclast differentiation induced by OVX in osteoporotic mice.

[0024] The advantages of this invention are:

[0025] 1. This invention is the first to culture MBMSCs using HDF as a feeder cell layer and collect exosomes secreted by HDF-MBMSCs under ALN stimulation under these culture conditions. NMN is loaded onto these exosomes using electroporation, thus constructing a nanoparticle containing NMN-loaded on alendronate-stimulated stem cell exosomes. The AHM-E@NMN nanoparticles of this invention have been experimentally verified to have good anti-osteoporosis properties and biosafety.

[0026] 2. The AHM-E@NMN nanoparticles of this invention are prepared by loading NMN onto AHM-E. The AHM-E is extracted from HDF-MBMSCs pretreated with ALN. When used in combination with NMN, it can promote osteoblast differentiation and bone healing in OVX-induced osteoporotic mice, inhibit osteoclast growth in OVX-induced osteoporotic mice in vivo, and cause no significant gastric damage to OVX-induced mice. Therefore, AHM-E@NMN nanoparticles are expected to be applied in the clinical treatment of osteoporosis. Attached Figure Description

[0027] Figure 1 This is a graph showing the effect of different concentrations of ALN on the proliferation of MBMSC cells.

[0028] Figure 2 The following are the characterization data of the nanoparticles of this invention: Figure 2 -A is a scanning electron microscope image of AHM-E and AHM-E@NMN nanoparticles. Figure 2 -B is the Western blotting diagram of AHM-E and AHM-E@NMN nanoparticles.

[0029] Figure 3 This is a graph showing the effects of different nanoparticles on osteoblast proliferation.

[0030] Figure 4 This is a graph showing the effect of AHM-E@NMN on osteoblast differentiation: Figure 4 -A shows ALP and ARS staining results after osteoblasts were treated with AHM-E and AHM-E@NMN nanoparticles. Figure 4 -B is a statistical graph showing the quantitative analysis of ALP staining in osteoblasts treated with AHM-E and AHM-E@NMN nanoparticles. Figure 4 -C is a statistical graph of ARS staining quantitative analysis of osteoblasts after treatment with AHM-E and AHM-E@NMN nanoparticles. Scale bar: 100μm.

[0031] Figure 5 This is an in vivo evaluation of the effect of AHM-E@NMN on bone healing in osteoporotic mice: Figure 5 - Micro-CT analysis of mouse tibial specimens from each group (A) Figure 5 -B Quantitative analysis of bone volume / tissue volume (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) of the femur in four groups of mice based on Micro-CT.

[0032] Figure 6 This is a diagram showing the inhibition of osteoclast differentiation by AHM-E@NMN; Figure 6 -A shows the H&E and TRAP staining images of mouse tibia specimens from each group. Figure 6 -B is a quantitative analysis graph based on TRAP staining of bone volume / tissue volume (BV / TV), erosion surface area (ES / BS), and number of osteoclasts per tissue area (Oc.S / BS).

[0033] Figure 7 This is a graph showing the effects of ALN and AHM-E@NMN on gastric damage in osteoporotic mice. Detailed Implementation

[0034] The technical solutions described in this invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only some feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.

[0035] Example 1: Isolation and culture of human skin fibroblasts (HDF) and mouse bone marrow mesenchymal stem cells (MBMSCs)

[0036] (1) Isolation and culture of HDF: In a biosafety cabinet, surgically discarded adult skin tissue was soaked in 75% alcohol for 2 min for disinfection. The skin tissue was washed at least 3 times in PBS containing penicillin and streptomycin. Then, fat and connective tissue were removed, and the skin was cut into 0.5×0.5cm pieces. The pieces were placed in a culture dish, 0.1% dispersant enzyme II solution was added, the dish was sealed with sealing film, and the skin was digested overnight at 4°C. The next day, the dermis was cut into small pieces with tweezers and transferred to a centrifuge tube. A mixed solution containing 0.1% collagen type I enzyme, 0.1% collagen type II enzyme, and 0.1% collagen type IV enzyme was added and digested in a 37°C water bath for 2 h. The mixture was filtered through a 100μm mesh sieve, and the filtrate was centrifuged at 300g for 5 min. The precipitate was resuspended in high-glucose DMEM complete medium containing 10% FBS, 1% penicillin and streptomycin, and inoculated into T25 cell culture flasks. The medium was changed every other day.

[0037] (2) MBMSC isolation and culture: Male C57BL / 6 mice aged 6-8 weeks were sacrificed. The femur and tibia of the mice were dissected, and bone marrow cells were washed with DPBS. The bone marrow-derived cells were filtered through a 70 μM cell filter, centrifuged at 300 × g for 10 min, and resuspended in 1 ml of high-glucose DMEM complete medium (MBMSC medium) containing 20% ​​FBS, 1% penicillin and streptomycin, and 1% glutamine. The cells were seeded into T25 cell culture flasks and incubated in a humidified incubator at 37°C with 5% CO2. After 1 day, the cells were washed twice with DPBS to remove non-adherent cells, and 5 ml of fresh MBMSC medium was added. The primary culture reached approximately 80% confluence within one week and could be passaged.

[0038] Example 2: MBMSC cell proliferation experiment

[0039] MBMSC cells were loaded at 5 × 10 3 Cells were seeded at a density of 100 cells / well in a 96-well plate. After the cells adhered, 100 μL of MBMSC medium containing different concentrations of ALN (0, 2.5, 5, 10, 20 μM) was added to each well. One day later, MTT solution was added to bring the final concentration to 5 mg / mL and the cells were incubated for another 4 hours. The absorbance at 560 nm was measured using a microplate reader and the data were plotted.

[0040] Experimental Results: To observe the effect of different concentrations of ALN on the proliferation of BMSC cells, this invention treated MBMSC cells with different concentrations of ALN. MTT results are as follows: Figure 1The results showed that different concentrations of ALN initially promoted the growth of MBMSCs, then inhibited it. At a concentration of 5 μM ALN, the effect of promoting MBMSC proliferation reached its peak, while at a concentration of 20 μM ALN, it had a significant inhibitory effect on MBMSCs. Therefore, this invention selected 5 μM ALN to treat MBMSCs for exosome preparation.

[0041] Example 3: Preparation of AHM-E@NMN nanoparticles

[0042] (1) ALN stimulation culture of MBMSCs with HDF as feeder cells: P9 generation HDF cells that had been inactivated by 10 μg / mL mitomycin C were cultured at a rate of 2 × 10⁻⁶ 4 cells / cm 2 Cells were seeded at a density of [specific value] in six-well plates and cultured overnight. On the second day, the culture medium was changed to MBMSC medium, and after 1 hour, [cells were seeded at a density of] 5 × 10⁶ cells / well. 4 cells / cm 2 MBMSCs were seeded at the appropriate cell density on HDF and the medium was changed daily. After 5 days of culture, the medium was replaced with 1.5 mL of MBMSC medium containing 5 μM ALN and cultured for another day.

[0043] (2) Preparation of AHM-E: The supernatant of MBMSC cell culture after ALN treatment was collected and centrifuged at 700×g for 10 min to remove cell debris. Then, it was centrifuged at 9000×g for 30 min at 4℃ and the supernatant was collected again. Exosomes were obtained by separation using the ExoEasyMaxi kit. The precipitate was resuspended in 100 μL PBS and the exosome concentration was detected by BCA protein quantification method. The exosomes were aliquoted into 100 μl tubes at a concentration of 2 g / mL and stored at -80℃ for later use, which is AHM-E.

[0044] (3) Load AHM-E and NMN in a mass ratio of 5:1: Dissolve 20 mg of NMN powder in 1 mL of sterile PBS solution until fully dissolved, add 50 μl of previously dispensed AHM-E, mix with a pipette, transfer the mixed solution to an electroporation cup, set the electric field strength to 1 KV / cm, the temperature to 37℃, electroporate for 5 min, collect the solution at 4℃, centrifuge at 700×g for 5 min to remove unbound NMN and other impurities, and finally collect the supernatant as AHM-E@NMN solution, and add 0.5 mL of exosome preservation solution. At this time, the concentration of AHM-E@NMN is 80 mg / mL. After mixing evenly, dispense 50 μl of AHM-E@NMN solution into each tube and store at -80℃ for later use.

[0045] Comparative Example 1: Preparation of HM-E@NMN Nanoparticles

[0046] The difference between this embodiment and Example 1 is that the MBMSC medium containing 1.5 mL of 5 μM ALN in step (1) is replaced with 1.5 mL of MBMSC medium and cultured for 1 day.

[0047] Comparative Example 2: Preparation of ME@NMN Nanoparticles

[0048] The difference between this embodiment and Comparative Example 1 is that the MBMSC cells were not cultured on HDF cells, but were directly seeded into a six-well plate.

[0049] Example 4: Characterization of AHM-E@NMN

[0050] (1) Transmission electron microscopy observation of AHM-E and AHM-E@NMN: AHM-E and AHM-E@NMN prepared in Example 3 were diluted 500 times and transported on ice. They were observed and photographed using a transmission electron microscope.

[0051] (2) Western Blotting to identify the marker proteins of AHM-E and AHM-E@NMN: Total protein was extracted from AHM-E and AHM-E@NMN prepared in Example 3, and the exosome marker proteins were identified using Western Blotting technology.

[0052] Experimental results: Transmission electron microscopy revealed the following sample morphology as follows: Figure 2 As shown in Figure -A, AHM-Es vary in size, with most exhibiting an elliptical shape of approximately 70-110 nm in diameter. The outer membrane structure, displaying a white transparent halo, is a phospholipid bilayer, consistent with the structure of exosomes. In contrast, AHM-E@NMN is significantly larger than AHM-E, exhibiting a circular structure of approximately 160 nm. Furthermore, AHM-E displays a complete membrane structure, indicating that the electroporation method successfully loaded NMN onto AHM-E. Western blotting results are shown below. Figure 2 As shown in Figure B, both AHM-E and AHM-E@NMN express exosome marker proteins CD63, CD81, and CD9 on their surfaces, indicating that the AHM-E@NMN prepared in this invention can exhibit exosome characteristics.

[0053] Example 5: Osteoblast proliferation experiment

[0054] Mouse embryonic osteoblast precursor cells (MC3T3-E1 cells) were used at a rate of 2×10⁻⁶. 3Cells were seeded at a density of 100 μL / well in 96-well plates. After cell adhesion, 100 μL of the corresponding drug was added to each well according to the group: Control group (PBS solution), AHM-E group (12 mg / mL), AHM-E@NMN group (12 mg / mL), HM-E@NMN group (12 mg / mL), and ME@NMN group (12 mg / mL). After 1, 3, 5, and 7 days, the medium was replaced with 10% CCK-8 and incubated for another 4 hours. The A value at 450 nm was measured with a microplate reader and the data were plotted.

[0055] Experimental Results: To investigate the effect of AHM-E@NMN on osteoblast proliferation, this invention involved treating MC3T3-E1 cells with different nanoparticles. CCK-8 results are as follows... Figure 3 As shown, compared with the Control group, the proliferation rate of osteoblasts treated with the four nanoparticle groups was significantly accelerated. The above results indicate that treatment with AHM-E, AHM-E@NMN, HM-E@NMN, and ME@NMN can effectively promote osteoblast proliferation. Among them, the AHM-E, HM-E@NMN, and ME@NMN groups have similar abilities to promote osteoblast proliferation, while the AHM-E@NMN group has a more significant effect on promoting osteoblast proliferation than the other three groups. This indicates that exosomes combined with NMN can promote osteoblast differentiation, but the exosomes secreted by MBMSCs cultured on HDF after ALN pretreatment combined with NMN have a stronger ability to promote osteoblast differentiation.

[0056] Example 6: Evaluation of the effect of AHM-E@NMN on osteoblast differentiation

[0057] (1) Alkaline phosphatase (ALP) activity assay: The density of MC3T3-E1 cells was adjusted to 1×10⁻⁶ cells. 5 Cells were seeded per well in 6-well plates. After cell adhesion, the appropriate drugs were added according to the groups: Control group (PBS solution), AHM-E group (12 mg / mL), and AHM-E@NMN group (12 mg / mL). All culture media used were osteogenic induction media. After culturing for 21 days, cell samples were collected, and cells were fixed with 4% paraformaldehyde for 20 min. After washing with PBS, the cells were incubated with 1 mL of ALP staining solution at 37°C for 1 h, and the results were imaged under a microscope. The ALP staining intensity was quantified using ImageJ software.

[0058] (2) Alizarin Red (ARS) staining: Adjust the MC3T3-E1 cell density to 1×10⁻⁶. 5Cells were seeded per well in 6-well plates. After cell adhesion, the appropriate drugs were added according to the groups: Control group (PBS solution), AHM-E group (12 mg / mL), and AHM-E@NMN group (12 mg / mL). All culture media used were osteogenic induction media. After culturing for 21 days, cells in each group were fixed with 4% paraformaldehyde for 10 min, washed three times with PBS, stained with alizarin red for 30 min, washed with distilled water, and observed and photographed. ImageJ software was used to quantify the ARS staining intensity.

[0059] Experimental Results: ALP activity and calcification levels are key indicators of osteoblast differentiation. First, observe the ALP staining results as follows: Figure 4 -A, compared with the control group, the MC3T3-E1 gray-black particles treated with AHM-E and AHM-E@NMN were more abundant, with the AHM-E@NMN group showing the highest concentration, combined with quantitative analysis results. Figure 4 -B indicates that the AHM-E@NMN group had the highest ALP activity. Secondly, the degree of osteoblast mineralization was observed by ARS staining, and the staining results are as follows: Figure 4 As shown in Figure A, compared to the Control group, the AHM-E group and the AHM-E@NMN group had a wider red area and more calcified nodules. Quantitative analysis of ARS staining was performed as follows: Figure 4 -C indicates that AHM-E@NMN resulted in the highest level of osteoblast calcification. Combined with ALP and ARS staining results, this demonstrates that AHM-E@NMN can effectively promote ALP activity in osteoblasts and enhance their calcification level.

[0060] Example 7: In vivo evaluation of the effect of AHM-E@NMN on bone healing in osteoporotic mice

[0061] Twenty-four animals were randomly assigned to groups of six: sham-operated group (Sham group), ovariectomized group (OVX group), AHM-E group, and AHM-E@NMN group. In the Sham group, periovarian adipose tissue was removed, while in the other groups, the ovaries were removed to establish an osteoporosis model. Eight weeks after surgery, the Sham and OVX groups were administered saline via gavage, the AHM-E group received 30 mg (AHM-E) / kg (mouse weight) via gavage, and the AHM-E@NMN group received 30 mg (AHM-E@NMN) / kg (mouse weight) via gavage. Administration was repeated every other day for 12 weeks. Mice were then sacrificed, and femurs and tibias were collected for CT scans, H&E, and TRAP staining.

[0062] Experimental Results: The potential therapeutic effect of AHM-E@NMN in an OVX-induced osteoporosis mouse model was further explored. After drug administration, a Micro-CT scan of the distal left femur was performed, and two-dimensional reconstruction was carried out. The results are as follows: Figure 5 As shown in Figure A, compared with the Sham group, the femoral cortex of rats in the OVX group was thinner and the trabecular structure was sparser; compared with the OVX group, the AHM-E group and the AHM-E@NMN group showed thicker cortex and denser trabecular structure, with the AHM-E@NMN group showing the most significant effect. Simultaneously, Micro-CT scans were used to calculate relevant parameters of femoral microstructure, and quantitative analysis was performed on bone volume / tissue volume (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). The statistical results are shown below. Figure 5 Compared to the Sham group, the OVX group showed significantly reduced BV / TV, Tb.N, and Tb.Th, which were inhibited in both the AHM-E and AHM-E@NMN groups, with the AHM-E@NMN group showing a more significant inhibitory effect on these parameters. Simultaneously, compared to the Sham group, AHM-E and AHM-E@NMN treatments also inhibited the increase in Tb.Sp in the OVX group. These results indicate that AHM-E and AHM-E@NMN have therapeutic effects on OVX-induced osteoporosis, with AHM-E@NMN showing better therapeutic efficacy.

[0063] H&E staining was used to observe changes in the femoral structure of mice in each group. The staining results are as follows: Figure 6 In the Sham group (A), the femoral tissue structure was dense, with tightly connected and regularly arranged trabeculae, few fractures, and a normal medullary cavity size. Compared to the Sham group, the Ovx group showed increased femoral tissue separation, severe trabecular fractures, a significantly enlarged medullary cavity, and increased cavities. Compared to the OVX group, the AHM-E and AHM-E@NMN groups showed significantly increased trabeculae and connections in the femoral tissue, reduced gaps between trabeculae, a smaller medullary cavity, and a denser structure. Furthermore, the AHM-E@NMN group demonstrated better repair of the medullary cavity.

[0064] This invention also involves TRAP staining of bone tissue sections and quantitative analysis of the bone volume / tissue volume (BV / TV), erosion surface area (ES / BS), and osteoclast count per tissue region (Oc.S / BS) based on TRAP staining. The TRAP staining results are as follows: Figure 6 As shown in Figure A, compared to the Sham group, the OVX group showed an increase in osteoclast count (the area of ​​the red region is proportional to the number of osteoclasts). After treatment with AHM-E and AHM-E@NMN, the number of osteoclasts decreased, with the AHM-E@NMN group showing the fewest osteoclasts. This aligns with the quantitative analysis data... Figure 6 The data from -B are consistent, indicating that AHM-E@NMN treatment inhibits the increase in osteoclast numbers induced by OVX. Animal experimental data show that AHM-E@NMN inhibits OVX-induced osteoporosis in vivo.

[0065] Example 8: Evaluation of the effects of ALN and AHM-E@NMN on gastric injury in osteoporotic mice

[0066] Animal model treatment was as described in Example 7. Experimental groups: OVX group was administered physiological saline by gavage, ALN group was administered 30 mg (ALN) / kg (mouse weight) by gavage, and AHM-E@NMN group was administered 30 mg (AHM-E@NMN) / kg (mouse weight) by gavage. After administration, the mice were sacrificed and gastric samples were collected for H&E staining.

[0067] Experimental results: The pathological results of gastric tissue in each group of mice are as follows: Figure 7 As shown, the gastric mucosa in the Control group remained intact. After ALN treatment, the ALN group showed gastric epithelial cell loss (indicated by arrows) and edema (indicated by pentagons), while the AHM-E@NMN group did not show the same significant epithelial cell loss and edema as the ALN group. This result indicates that AHM-E@NMN reduces damage to the stomach compared to ALN.

[0068] In summary, the AHM-E@NMN nanoparticles of this invention possess the basic characteristics of exosomes, enabling them to promote osteoblast differentiation in vitro and inhibit OVX-induced bone loss in mice with osteoporosis in vivo. Furthermore, compared to ALN, they reduce gastric damage, demonstrating their good anti-osteoporosis efficacy and safety, and are expected to be applied to the development and use of drugs for the clinical treatment of osteoporosis.

[0069] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A nanoparticle containing NMN loaded onto alendronate sodium-stimulated stem cell exosomes, characterized in that, The nanoparticles were prepared by first culturing MBMSCs on HDF, then pretreating HDF-MBMSCs with ALN and separating and purifying the exosomes AHM-E secreted by ALN@HDF-MBMSCs, and finally loading the purified AHM-E with NMN to obtain the nanoparticles AHM-E@NMN. In the nanoparticles AHM-E@NMN, the mass ratio of AHM-E to NMN is 5:

1.

2. A method for preparing the nanoparticles according to claim 1, characterized in that, The preparation method includes the following steps: S1: Obtain human dermal fibroblasts (HDF) and mouse bone marrow mesenchymal stem cells (MBMSC); S2: Alendronate sodium (ALN) stimulates stem cells to produce exosomes; The stem cells are MBMSCs seeded on HDF, where HDF is a feeder layer of MBMSCs; The exosomes were AHM-E, which were extracted from the cell culture medium after stem cells were stimulated with alendronate sodium (ALN). S3: Loading NMN into AHM-E exosomes: Dissolve NMN in PBS solution, then mix the AHM-E exosomes obtained in S2 with the NMN solution, add to an electroporation vessel, perform electroporation, collect the AHM-E@NMN supernatant after electroporation, add 0.5 mL of exosome preservation solution, and the AHM-E@NMN is stored for later use.

3. The method for preparing nanoparticles according to claim 2, characterized in that: The method for obtaining MBMSCs described in S1 is as follows: Bone marrow cells from the femur and tibia of mice were filtered using a 70 μM cell filter. The filtrate was collected and centrifuged at 300 × g for 10 minutes, then resuspended in MBMSC medium. The cells were seeded into T25 cell culture flasks and incubated in a humidified incubator at 37°C with 5% CO2. After 1 day, the cells were washed twice with DPBS to remove non-adherent cells, and 5 mL of MBMSC medium was added. The cells were passaged after reaching approximately 80% confluence within one week.

4. The method for preparing nanoparticles according to claim 2, characterized in that: The method for stimulating stem cells to produce exosomes using S2 alendronate sodium (ALN) is as follows: P9 generation HDF, which has been deactivated by 10 μg / mL mitomycin C, is administered at a rate of 2 × 10⁻⁶. 4 cells / cm 2 Cells were seeded at a density of 5 × 10⁻⁶ cells in six-well plates and cultured overnight. The culture medium was then replaced with MBMSC medium on the second day, and after 1 hour, the cells were seeded at a density of 5 × 10⁻⁶ cells / well. 4 cells / cm 2 MBMSCs were seeded at the appropriate cell density on HDF medium, with the medium changed daily. After 5 days of culture, the medium was replaced with MBMSC medium containing ALN and cultured for another day. The supernatant of the HDF-MBMSC cell culture after ALN treatment was collected, centrifuged at 700×g for 10 min to remove cell debris, and then centrifuged at 9000×g for 30 min at 4℃. The supernatant was collected again, and exosomes were isolated using the ExoEasy Maxi kit. The precipitate was resuspended in 100 μL of PBS, and the exosome concentration was detected by BCA protein quantification. The exosomes were aliquoted into 100 μL tubes at a concentration of 2 g / mL and stored at -80℃ for later use, i.e., AHM-E.

5. The method for preparing nanoparticles according to claim 4, characterized in that: The ALN concentration in the MBMSC medium containing ALN is 2.5-10 μM.

6. The method for preparing nanoparticles according to claim 2, characterized in that: S3 is loaded with AHM-E and NMN at a mass ratio of 5:

1.

7. The method for preparing nanoparticles according to claim 2, characterized in that: The electro-rotation conditions described in S3 are: electric field strength of 1KV / cm, temperature of 37℃, and electro-rotation time of 5 minutes.

8. The use of the nanoparticles of claim 1 in the preparation of a drug for treating osteoporosis.

9. The application according to claim 8, characterized in that, The drug for treating osteoporosis is a drug that promotes osteoblast differentiation or promotes bone healing in OVX-induced osteoporotic mice, or a drug that inhibits osteoclast differentiation in OVX-induced osteoporotic mice.

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