Aendronate sodium stimulated stem cell exosome-loaded NMN nanoparticles and application thereof

Abstract

The invention relates to the field of regenerative medicine and medical health, in particular to nano particles of NMN loaded on stem cell exosome stimulated by alendronate sodium and application of the nano particles. According to the nanoparticles, firstly, human dermal fibroblasts (HDF) are used as feeder layer cells, mouse bone marrow mesenchymal stem cells (MBMSC) are inoculated to the HDF, then ALN is used for pretreating the HDF-MBSC, an exosome (AHM-E) of the HDF-MBSC is obtained, and finally the purified exosome is loaded with beta-nicotinamide mononucleotide (NMN), so that the nanoparticles AHM-E-NMN are prepared. The AHM-E-coated NMN nanoparticles have basic characteristics of exosomes, can promote osteoblast differentiation in vitro and inhibit bone loss of OVX-induced osteoporosis mice in vivo, reflect a good anti-osteoporosis effect, and are expected to be applied to development and use of medicines for clinically treating osteoporosis.

Description

Technical Field

[0001] The present invention relates to the field of regenerative medicine and medical health, and in particular to alendronate-stimulated stem cell exosomes loaded with NMN nanoparticles and applications thereof. Background Art

[0002] At present, human society is gradually entering an aging structural state, and the physical health of the elderly has attracted much attention. Osteoporosis is a metabolic chronic bone disease common in the elderly population, which is mainly related to abnormal bone mass reduction, bone tissue dysfunction and increased fracture risk. This disease seriously endangers the physical health of the elderly. Therefore, it is urgent to alleviate the harm caused by osteoporosis to the elderly. Osteoporosis occurs in both sexes, but women are more likely to develop serious osteoporosis complications (about 80%). Several genetic and environmental factors related to 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, which 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 yet fully understood. The key to this most common metabolic bone disease is to restore and maintain the balance between bone formation and resorption.

[0003] Currently, a variety of pharmacological approaches, including hormone replacement therapy (HRT), selective androgen receptor modulators (SARMs), bisphosphonates (BPH), and calcitonin, have been used to treat osteoporosis. Among the various drugs, BPH is one of the most commonly prescribed anti-fracture drugs. BPH is an analog of inorganic pyrophosphate, which inhibits bone resorption by inducing osteoclast apoptosis, thereby preventing age-related bone loss and deterioration of bone microarchitecture. Nitrogen-containing BPH (such as alendronate, risedronate, ibandronate, and zoledronic acid) have the most effective anti-resorptive properties and are the most commonly used drugs for the treatment of osteoporosis. Among the different drugs, alendronate sodium (ALN) is one of the best and most widely studied BPH for the treatment of osteoporosis.

[0004] It is well known that ALN can prevent hydroxyapatite loss, inhibit osteoclast-mediated bone resorption, promote osteoblast differentiation and bone formation, and thus regulate the body's bone homeostasis. The drug is mainly used to treat osteoporosis, bone metastasis and Paget's disease. Recently, it has been proposed as an adjuvant analgesic in combination with opioids to treat cancer pain symptoms and as a bone anti-tumor agent. However, in addition to its low bioavailability (<1% when administered orally), its systemic application can cause many side effects, such as fever, esophageal erosion, ulcers and gastrointestinal problems. Therefore, it is crucial to reduce the toxic side effects of ALN and find new treatments for osteoporosis. Summary of the invention

[0005] The purpose of the present invention is to solve the above technical problems and provide a nanoparticle of NMN loaded with stem cell exosomes stimulated by sodium alendronate. The nanoparticle first uses human dermal fibroblasts (HDF) as feeder cells, inoculates mouse bone marrow mesenchymal stem cells (MBMSC) into HDF, then pretreats HDF-MBSC with ALN and obtains its exosomes (AHM-E), and finally loads the purified AHM-E with β-nicotinamide mononucleotide (NMN) to prepare nanoparticles AHM-E@NMN.

[0006] The nanoparticle AHM-E@NMN provided by the present invention, wherein HDF provides good growth factors for MBMSCs to promote their proliferation, and HDF-MBMSCs treated with ALN can secrete exosomes containing small molecules such as osteoblast-related protein factors, which can promote osteoblast differentiation, avoiding the toxic side effects of direct administration of ALN to the body; and NMN is an important NAD + The intermediate can promote the intracellular NAD level, thereby reducing cell senescence and promoting osteoblast differentiation, thereby promoting the bone healing ability of osteoporotic mice. At present, there is no report on AHM-E loaded NMN nanoparticles obtained by ALN pretreatment of MBMSCs, which is of great significance for reducing the toxic side effects of ALN and preparing nanoparticles for the treatment of osteoporosis.

[0007] The present invention also provides a method for preparing alendronate-stimulated stem cell exosomes loaded with NMN nanoparticles, which specifically comprises 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 HDFs, and the HDFs are feeder cells of MBMSCs;

[0011] The exosomes are obtained by extracting exosomes AHM-E from cell culture fluid after stem cells are stimulated by alendronate sodium ALN;

[0012] S3: Loading NMN into AHM-E exosomes: Dissolve NMN in PBS solution, then mix the exosome AHM-E obtained in S2 and the NMN solution evenly, add them into the electroporation cup for electroporation, collect the AHM-E@NMN supernatant after the end, add 0.5mL exosome preservation solution, and store it for later use.

[0013] Furthermore, the method for obtaining HDF described in S1 is: take adult skin tissue, cut it into pieces and put it into a culture dish, add 0.1% dispase II solution, seal it with a sealing film, and digest it in a 4°C refrigerator overnight. On the next day, use tweezers to take the dermis and cut it into pieces, transfer it to a centrifuge tube, add a mixed solution containing 0.1% collagen type I enzyme, 0.1% collagen type II enzyme, and 0.1% collagen type IV enzyme, and digest it in a 37°C water bath for 2 hours, filter it with a 100μm mesh, take the filtrate, centrifuge it at 300×g for 5 minutes, resuspend the precipitate with high-glucose DMEM complete culture medium containing 10% FBS, 1% penicillin and streptomycin, inoculate it into a T25 cell culture flask, and change the medium every other day.

[0014] Further, the method for obtaining MBMSCs described in S1: bone marrow cells from the femur and tibia of mice were taken, filtered using a 70 μM cell filter, the filtrate was collected by centrifugation at 300×g for 10 min, and suspended in MBMSC culture medium, the cells were inoculated into a T25 cell culture flask and incubated in a humidified incubator at 37°C with 5% CO2. After 1 day, non-adherent cells were removed by washing twice with DPBS, and 5 mL of MBMSC culture medium was added, and the cells could be subcultured when they reached a confluence of about 80% within a week.

[0015] Further, the method of stimulating stem cells to produce exosomes by alendronate sodium ALN described in S2 is: P9 generation HDFs passivated with 10 μg / mL mitomycin C were cultured at a rate of 2×10 4 Cells / cm 2 The cells were seeded in a six-well plate at a density of 5×10 4 Cells / cm 2 MBMSCs were inoculated on HDF at a cell density of 100 μL and the medium was changed every day; after 5 days of culture, the MBMSC medium containing ALN was replaced and cultured for 1 day; the supernatant of the HDF-MBMSC cells treated with ALN was collected, centrifuged at 700×g for 10 min to remove cell debris, and then centrifuged at 9000×g for 30 min at 4°C, and the supernatant was collected again, and exosomes were isolated by ExoEasy Maxi kit, and 100 μL PBS was added to resuspend the precipitate, and the exosome concentration was detected by BCA protein quantification method, and the exosomes were divided into 100 μL per tube at a concentration of 2 g / mL and frozen at -80°C for later use, namely AHM-E;

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

[0017] Furthermore, the electroporation conditions in S3 are: electric field intensity 1 KV / cm, electroporation temperature 37° C., and electroporation time 5 min.

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

[0019] The present invention also provides the use of nanoparticles AHM-E@NMN in preparing a drug for treating osteoporosis.

[0020] Furthermore, the osteoporosis treatment drug is a drug loaded with NMN in the exosomes of MBMSCs stimulated by ALN with HDF as feeder cells.

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

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

[0023] Furthermore, the drug for treating osteoporosis is a drug that inhibits the differentiation of osteoclasts in OVX-induced osteoporotic mice.

[0024] The present invention has the advantages that:

[0025] 1. In the present invention, MBMSCs were cultured for the first time using HDF as feeder cells, and exosomes secreted by HDF-MBMSCs under the culture condition stimulated by ALN were collected. NMN was loaded by electroporation to construct a nanoparticle of stem cell exosomes loaded with NMN stimulated by sodium alendronate. The AHM-E@NMN nanoparticles of the present invention have been experimentally verified to have good anti-osteoporosis performance and biosafety.

[0026] 2. The AHM-E@NMN nanoparticles of the present invention are prepared by loading NMN with AHM-E. The AHM-E is extracted from HDF-MBMSC pretreated with ALN. When used in combination with NMN, it can promote osteoblast differentiation and bone healing in OVX-induced osteoporosis mice, inhibit osteoclast growth in OVX-induced osteoporosis mice in vivo, and cause no obvious gastric damage to OVX mice. Therefore, AHM-E@NMN nanoparticles are expected to be used in the clinical treatment of osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0028] Figure 2 is the characterization data of the nanoparticles of the present invention: Figure 2 -A is the scanning electron micrograph of AHM-E and AHM-E@NMN nanoparticles, Figure 2 -B is the Western Blotting band image of AHM-E and AHM-E@NMN nanoparticles.

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

[0030] Figure 4 This is the data diagram of the effect of AHM-E@NMN on osteoblast differentiation: Figure 4 -A is the ALP staining and ARS staining images of osteoblasts treated with AHM-E and AHM-E@NMN nanoparticles. Figure 4 -B is a quantitative analysis of ALP staining of osteoblasts treated with AHM-E and AHM-E@NMN nanoparticles. Figure 4 -C is a statistical chart of quantitative analysis of ARS staining after osteoblasts were treated with AHM-E and AHM-E@NMN nanoparticles, scale bar: 100μm.

[0031] Figure 5 This is an in vivo evaluation of the bone healing effect of AHM-E@NMN on osteoporosis mice: Figure 5 -A Micro-CT analysis of tibia specimens of mice in each group, 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 femurs of four groups of mice based on Micro-CT.

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

[0033] Figure 7 This is a data chart showing the effects of ALN and AHM-E@NMN on gastric damage in osteoporosis mice. DETAILED DESCRIPTION

[0034] The technical solutions described in the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described in this specification are only part of the feasible technical solutions of the present invention. Other implementation methods obtained by ordinary technicians in this field based on the embodiments of the present invention without any creative work should be deemed to belong to the scope of protection of the present invention.

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

[0036] (1) Isolation and culture of HDF: Adult skin tissue discarded from surgery was soaked in 75% alcohol in a biosafety cabinet for 2 min for disinfection. The skin tissue was washed at least 3 times in PBS containing penicillin and streptomycin, and then the fat and connective tissue were cut off. The skin was cut into 0.5×0.5 cm pieces and placed in a culture dish. 0.1% dispase II solution was added and sealed with parafilm. The pieces were digested in a refrigerator at 4°C overnight. The next day, the dermis was taken with tweezers and cut into pieces. The pieces were 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 was added. The mixture was digested in a 37°C water bath for 2 h. The tube was filtered with a 100 μm mesh. The filtrate was taken and centrifuged at 300 g for 5 min. The precipitate was resuspended in high-glucose DMEM complete medium containing 10% FBS, 1% penicillin and streptomycin, and inoculated into a T25 cell culture flask. The medium was changed every other day.

[0037] (2) MBMSC isolation and culture: 6-8 week old male C57BL / 6 mice were sacrificed, the femurs and tibias of the mice were dissected, and the bone marrow cells were washed out with DPBS. The bone marrow-derived cells were filtered through a 70 μM cell filter, collected by centrifugation at 300×g for 10 min, and suspended in 1 ml of high-glucose DMEM complete medium (MBMSC medium) containing 20% ​​FBS, 1% penicillin and streptomycin, and 1% glutamine. The cells were inoculated into T25 cell culture flasks and incubated in a humidified incubator at 37°C with 5% CO2. After 1 day, the non-adherent cells were washed twice with DPBS, and 5 ml of fresh MBMSC medium was added. The primary culture reached a confluence of about 80% within one week and could be subcultured.

[0038] Example 2: MBMSC cell proliferation experiment

[0039] MBMSC cells were cultured at 5×10 3 The cells were seeded in a 96-well plate at a density of 100 cells / well. After the cells adhered, 100 μL of MBMSC culture 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 make the final concentration of 5 mg / mL and incubated for another 4 h. The absorbance at 560 nm was measured with an ELISA reader and the statistical data was plotted.

[0040] Experimental results: In order to observe the effect of different concentrations of ALN on BMSC cell proliferation, the present invention treated MBMSC cells with different concentrations of ALN. The MTT results are as follows Figure 1The results showed that different concentrations of ALN promoted the growth of MBMSC cells first and then inhibited them. At a concentration of 5 μM ALN, the proliferation of MBMSC reached a peak, and at a concentration of 20 μM ALN, MBMSC was significantly inhibited. Therefore, the present invention selected 5 μM ALN to treat MBMSC cells for exosome preparation.

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

[0042] (1) ALN stimulation of MBMSCs cultured on HDFs as feeder cells: P9 HDFs inactivated with 10 μg / mL mitomycin C were cultured at 2×10 4 Cells / cm 2 The cells were seeded in a six-well plate at a density of 5×10 4 Cells / cm 2 MBMSCs were seeded on HDFs at a cell density of 1.5 mL and the medium was changed daily. After 5 days of culture, 1.5 mL of MBMSC medium containing 5 μM ALN was replaced and culture was continued for 1 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, and then centrifuged at 9000 × g for 30 min at 4°C. The supernatant was collected again and exosomes were isolated 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 divided into 100 μL per tube at a concentration of 2 g / mL and frozen at -80°C for later use, i.e. AHM-E.

[0044] (3) AHM-E and NMN were loaded at a mass ratio of 5:1: 20 mg of NMN powder was dissolved in 1 mL of sterile PBS solution, 50 μl of previously aliquoted AHM-E was added, and the mixture was mixed with a pipette. The mixed solution was transferred to an electroporation cup for electroporation. The electric field strength was set to 1 KV / cm, the temperature was 37°C, and the electroporation was performed for 5 min. After the electroporation, the solution was collected and centrifuged at 4°C and 700 × g for 5 min to remove the unbound NMN and other impurities. Finally, the supernatant was collected as the AHM-E@NMN solution, and 0.5 mL of exosome preservation solution was added. At this time, the concentration of AHM-E@NMN was 80 mg / mL. After mixing evenly, 50 μl of AHM-E@NMN solution was aliquoted into each tube and stored at -80°C for later use.

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

[0046] The present embodiment is different from the embodiment 1 in that the step (1) of replacing 1.5 mL of MBMSC culture medium with 5 μM ALN and continuing to culture for 1 day is replaced by replacing 1.5 mL of MBMSC culture medium and continuing to culture for 1 day.

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

[0048] The difference between this embodiment and comparative example 1 is that the MBMSC cells are not cultured on HDF cells, but are 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, and observed and photographed using a transmission electron microscope;

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

[0052] Experimental results: The morphology of the samples observed by transmission electron microscopy is as follows Figure 2 -A, AHM-E varies in size, most of which are elliptical with a diameter of about 70-110nm. The membrane structure with a white transparent aperture on the outside is the phospholipid bilayer, which is consistent with the structure of exosomes; AHM-E@NMN is significantly larger than AHM-E, with a circular structure of about 160nm, and AHM-E presents a complete membrane structure, indicating that NMN was successfully loaded onto AHM-E by electroporation. Western Blotting experimental results are shown in Figure 2 As shown in Figure 5-B, the surfaces of AHM-E and AHM-E@NMN both express exosome marker proteins CD63, CD81, and CD9, indicating that the AHM-E@NMN prepared by the present invention can display exosome characteristics.

[0053] Example 5: Osteoblast proliferation assay

[0054] Mouse embryonic osteoblast precursor cells (MC3T3-E1 cells) were cultured at 2×10 3The cells were seeded in a 96-well plate at a density of 100 cells / well. After the cells adhered to the wall, 100 μL of the corresponding drugs were added to each well according to the groups: Control group (PBS solution), AHM-E group (12 mg / mL), AHM-E@NMN group (12 mg / mL), HM-E@NMN group (12 mg / mL), ME@NMN group (12 mg / mL). After 1, 3, 5, and 7 days, the culture medium containing 10% CCK-8 was replaced and incubated for another 4 hours. The A value at 450 nm was measured by an enzyme reader, and the statistical data was plotted.

[0055] Experimental results: In order to detect the effect of AHM-E@NMN on osteoblast proliferation, the present invention set different nanoparticles to treat MC3T3-E1 cells. The CCK-8 results are as follows Figure 3 As shown, compared with the Control group, the proliferation rate of osteoblasts in the four groups treated with nanoparticles was significantly accelerated. The above results indicate that AHM-E, AHM-E@NMN, HM-E@NMN and ME@NMN treatment 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 promotes osteoblast proliferation more significantly than the other three groups, while the HM-E@NMN group promotes osteoblast proliferation more significantly. This indicates that exosomes combined with NMN can promote osteoblast differentiation, but the exosomes secreted by MBMSC 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 MC3T3-E1 cell density was adjusted to 1×10 5 Cells / well were seeded in 6-well plates. After the cells adhered to the wall, the corresponding drugs were added according to the grouping: Control group (PBS solution), AHM-E group (12 mg / mL), AHM-E@NMN group (12 mg / mL). The culture medium used was osteogenic induction medium. After culturing for 21 days, the cell samples were collected and fixed with 4% paraformaldehyde for 20 minutes. After washing with PBS, the cells were incubated with 1 mL of ALP staining solution at 37°C for 1 hour, and the results were imaged under a microscope. ImageJ software was used to quantify the ALP staining intensity.

[0058] (2) Alizarin red (ARS) staining: The density of MC3T3-E1 cells was adjusted to 1×10 5Cells / well were seeded in 6-well plates. After the cells adhered to the wall, the corresponding drugs were added according to the grouping: Control group (PBS solution), AHM-E group (12 mg / mL), AHM-E@NMN group (12 mg / mL), and the culture medium used was osteogenic induction medium. After culturing for 21 days, the cells in each group were fixed with 4% paraformaldehyde for 10 minutes, washed with PBS three times, stained with Alizarin Red for 30 minutes, washed with distilled water, and observed and photographed. Image J software was used to quantify the ARS staining intensity.

[0059] Experimental results: ALP activity and calcification level evaluation are the hallmark indicators of osteoblast differentiation. First, observe the ALP staining results. Figure 4 -A, compared with the control group, the gray-black particles of MC3T3-E1 treated with AHM-E and AHM-E@NMN were more, and the AHM-E@NMN group had the most. Combined with the quantitative analysis results Figure 4 -B shows that the ALP activity in the AHM-E@NMN group was the highest. Secondly, the degree of osteoblast mineralization was observed by ARS staining. The staining results are shown in Figure 4 -A shows that compared with the Control group, the red area in the AHM-E group and the AHM-E@NMN group was wider and there were more calcified nodules. The quantitative analysis of ARS staining was as follows Figure 4 -C shows that the AHM-E@NMN group has the highest osteoblast calcification level. Combined with the ALP staining and ARS staining results, it shows that AHM-E@NMN can effectively promote the ALP activity of osteoblasts and increase 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 divided into sham operation group (Sham group), ovariectomy group (OVX group), AHM-E group and AHM-E@NMN group in groups of 6. The fat tissue around the ovaries was removed in the Sham group, and the other groups were removed to establish osteoporosis models. Eight weeks after surgery, the Sham group and OVX group were gavaged with normal saline, the AHM-E group was gavaged at a dose of 30 mg (AHM-E) / kg (mouse weight), and the AHM-E@NMN group was gavaged at a dose of 30 mg (AHM-E@NMN) / kg (mouse weight), once every other day. After 12 weeks of continuous administration, the mice were killed, and the femurs and tibias of the mice were collected for CT scanning, H&E and TRAP staining.

[0062] Experimental results: The potential therapeutic effect of AHM-E@NMN in the OVX-induced osteoporosis mouse model was further explored. After the administration, the distal end of the left femur was scanned by Micro-CT and two-dimensionally reconstructed. The results are as follows: Figure 5 -A shows that compared with the Sham group, the femoral cortical bone of the OVX group rats became thinner and the trabecular structure became sparse; compared with the OVX group, the AHM-E group and the AHM-E@NMN group had thicker cortical bone and a tighter trabecular structure, and the effect of the AHM-E@NMN group was the most significant. At the same time, Micro-CT scanning calculated the parameters related to the microstructure of the femur, and quantitatively analyzed the bone volume / tissue volume (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). The statistical results are shown in Figure 5 -B. Compared with the Sham group, the BV / TV, Tb.N and Tb.Th of the OVX group were significantly reduced, and were inhibited in the AHM-E group and the AHM-E@NMN group, and the AHM-E@NMN group had a more obvious effect in inhibiting the decline of each index. At the same time, compared with the Sham group, AHM-E and AHM-E@NMN treatment also inhibited the increase of Tb.Sp in the OVX group. The experimental results show that AHM-E and AHM-E@NMN have therapeutic effects on OVX-induced osteoporosis, among which AHM-E@NMN has a better therapeutic effect.

[0063] H&E staining was used to observe the changes in the femoral structure of mice in each group. Figure 6 -A, Sham group, the femoral tissue structure is dense, the trabeculae are tightly connected, arranged regularly, rarely broken, and the size of the bone marrow cavity is normal. Compared with the Sham group, the femoral tissue separation degree of the Ovx group increased, the trabeculae were severely broken, the bone marrow cavity was significantly enlarged, and the number of cavities increased. Compared with the OVX group, the femoral tissue trabeculae of the mice in the AHM-E group and the AHM-E@NMN group increased significantly, the number of connections increased, the gaps between the trabeculae decreased, the bone marrow cavity became smaller, and the structure became denser. In addition, the AHM-E@NMN group had a better repair effect on the bone marrow cavity.

[0064] The present invention also performs TRAP staining on bone tissue sections, and quantitatively analyzes the bone volume / tissue volume (BV / TV), erosion surface (ES / BS) and number of osteoclasts per tissue area (Oc.S / BS) of TRAP staining. Figure 6 As shown in Figure 2A, compared with the Sham group, the number of osteoclasts in the OVX group increased (the area of ​​the red area is proportional to the number of osteoclasts). After treatment with AHM-E and AHM-E@NMN, the number of osteoclasts decreased, and the AHM-E@NMN group had the least osteoclasts, which is consistent with the quantitative analysis data. Figure 6 -B data results are consistent, indicating that AHM-E@NMN treatment inhibits the increase in the number of osteoclasts 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 lesions in osteoporotic mice

[0066] The animal model was treated as in Example 7, and the experimental groups were divided into: the OVX group was gavaged with normal saline, the ALN group was gavaged at a dose of 30 mg (ALN) / kg (mouse weight), and the AHM-E@NMN group was gavaged at a dose of 30 mg (AHM-E@NMN) / kg (mouse weight). After the administration, the mice were killed and the stomach samples were collected for H&E staining.

[0067] Experimental results: The gastric tissue pathological results of each group of mice are as follows Figure 7 As shown, the gastric mucosa of the Control group was 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 obvious epithelial cell loss and edema as in the ALN group. This result indicates that AHM-E@NMN reduced the damage to the stomach compared with ALN.

[0068] In summary, the AHM-E@NMN nanoparticles of the present invention have the basic characteristics of exosomes, can promote osteoblast differentiation in vitro, inhibit bone loss in OVX-induced osteoporosis mice in vivo, and can reduce stomach damage compared to ALN, reflecting its good anti-osteoporosis effect and safety, and is expected to be applied to the development and use of clinical osteoporosis drugs.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the principle of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A nanoparticle containing NMN loaded by stem cell exosomes stimulated by sodium alendronate, characterized in that: The nanoparticles first culture MBMSCs on HDF, then pretreat HDF-MBMSCs with ALN and separate and purify exosomes AHM-E secreted by ALN@HDF-MBMSCs, and finally load the purified AHM-E with NMN to prepare 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 nanoparticle AHM-E@NMN according to claim 1, characterized in that: The preparation method comprises 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 HDFs, and the HDFs are feeder cells of MBMSCs; The exosomes are obtained by extracting exosomes AHM-E from cell culture fluid after stem cells are stimulated by alendronate sodium ALN; S3: Loading NMN into AHM-E exosomes: Dissolve NMN in PBS solution, then mix the exosome AHM-E and NMN solution obtained in S2 evenly, add them into the electroporation cup, and perform electroporation. After the end, collect the AHM-E@NMN supernatant, add 0.5mL of exosome preservation solution, and store AHM-E@NMN for later use.

3. The method for preparing the nanoparticle AHM-E@NMN according to claim 2, characterized in that: S1 The method for obtaining HDF is as follows: take adult skin tissue, cut it into pieces and put it into a culture dish, add 0.1% dispase II solution, seal it with a sealing film, and digest it in a 4°C refrigerator overnight. On the next day, use tweezers to take the dermis and cut it into pieces, transfer it to a centrifuge tube, add a mixed solution containing 0.1% collagen type I enzyme, 0.1% collagen type II enzyme, and 0.1% collagen type IV enzyme, and digest it in a 37°C water bath for 2 hours, filter it with a 100μm mesh, take the filtrate, centrifuge it at 300×g for 5 minutes, resuspend the precipitate with high-glucose DMEM complete culture medium containing 10% FBS, 1% penicillin and streptomycin, inoculate it into a T25 cell culture flask, and change the medium every other day.

4. The method for preparing the nanoparticle AHM-E@NMN according to claim 2, characterized in that: Method for obtaining MBMSC as described in S1: Bone marrow cells from mouse femur and tibia were filtered using a 70 μM cell strainer, the filtrate was collected by centrifugation at 300 × g for 10 minutes, and suspended 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, non-adherent cells were removed by washing twice with DPBS, and 5 mL of MBMSC medium was added. The cells could be subcultured when they reached a confluence of about 80% within a week.

5. The method for preparing the nanoparticle AHM-E@NMN according to claim 2, characterized in that: S2 The method of stimulating stem cells to produce exosomes by alendronate sodium ALN is as follows: P9 generation HDFs passivated with 10 μg / mL mitomycin C were cultured at 2×10 4 Cells / cm 2 The cells were seeded in a six-well plate at a density of 5×10 4 Cells / cm 2 MBMSCs were seeded on HDF at a cell density of 100 μL and the medium was changed every day. After 5 days of culture, the MBMSC medium containing ALN was replaced and cultured for 1 day. The supernatant of the HDF-MBMSC cells treated with ALN was collected and centrifuged at 700×g for 10 min to remove cell debris, and then centrifuged at 9000×g for 30 min at 4°C. The supernatant was collected again and exosomes were isolated using the ExoEasy Maxi kit. 100 μL PBS was added to resuspend the precipitate and the exosome concentration was detected by the BCA protein quantification method. The exosomes were aliquoted into 100 μl tubes at a concentration of 2 g / mL and stored at -80°C for later use, i.e., AHM-E.

6. The method for preparing the nanoparticle AHM-E@NMN according to claim 5, characterized in that: In the MBMSC culture medium containing ALN, the concentration of ALN is 2.5-10 μM.

7. The method for preparing the nanoparticle AHM-E@NMN according to claim 2, characterized in that: S3 AHM-E and NMN were loaded at a mass ratio of 5:

1.

8. The method for preparing the nanoparticle AHM-E@NMN according to claim 2, characterized in that: The electroporation conditions in S3 are as follows: electric field strength of 1 KV / cm, temperature of 37° C., and electroporation for 5 min.

9. Use of the nanoparticle AHM-E@NMN according to claim 1 in preparing a drug for treating osteoporosis.

10. The use according to claim 9, 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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