Preparation method and application of multifunctional layered nanosheet

Abstract

The invention discloses a preparation method and application of a multifunctional layered nanosheet. The biomimetic hydrogel is prepared through a hydrothermal method, folic acid (FA) is modified on the surface of the biomimetic hydrogel, and the biomimetic hydrogel has the capacity of targeted recognition of pro-inflammatory phenotype macrophages, has the biomimetic activity of multiple enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and can effectively remove ROS; meanwhile, a succinate dehydrogenase (SDH) inhibitor dimethylmalonic acid (DMM) is loaded in an intercalation mode of the nano material, SDH activity is inhibited in a slow release mode, ROS outbreak caused by mitochondrial reverse electron transfer (RET) is blocked from the source, then a mitochondrial autophagy pathway mediated by BNIP3-LC3B is started, and dynamic and steady state recovery of mitochondria is achieved. The system finally realizes macrophage mitochondrial function reconstruction, oxidation environment improvement and transfer of healthy mitochondria to osteoblasts, so that the osteogenic differentiation capability is recovered, and the effect of treating osteoporosis is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of nanomaterial preparation, biomedical materials and their application in bone metabolic diseases, and relates to a method for preparing a multifunctional layered nanosheet and its application (a method for preparing a modified magnesium-manganese layered nanosheet and its application in a drug for treating osteoporosis); in particular, it relates to a method for preparing a magnesium-manganese double hydroxide (MgMn-LDH) nanomaterial with multiple enzyme activities that can target pro-inflammatory macrophages and its application in treating osteoporosis). Background Art

[0002] Osteoporosis is a systemic skeletal disease characterized by decreased bone mass and degeneration of bone tissue microarchitecture, which leads to increased bone fragility and increased fracture risk. Under normal physiological conditions, macrophages can maintain bone homeostasis by polarizing into different phenotypes. In the pathological state of osteoporosis, inflammatory macrophages trigger reverse electron transfer in mitochondria due to excessive activity of SDH, thereby increasing the production of reactive oxygen species (ROS) and delivering oxidatively damaged mitochondria to osteocytes. Since osteoporosis itself is also a chronic disease, this long-term mitochondrial transmission gradually affects the function of bone cells, reduces bone formation, and exacerbates the progression of osteoporosis.

[0003] Traditional treatments for osteoporosis mostly focus on improving the osteoblastic capacity of osteoblasts or inhibiting osteoclast activity. However, the issue of macrophages transmitting oxidatively damaged mitochondria to osteoblasts is often overlooked. While some treatments improve oxidative stress in the microenvironment and protect osteoblasts from ROS, due to overactive SDH, abnormal mitochondria are not effectively rescued or cleared, resulting in continued production of substantial ROS. Numerous reports have focused on altering macrophage polarization to treat osteoporosis. However, macrophage polarization is a highly dynamic process, making precise regulation difficult. Furthermore, even if macrophage polarization to the M2 phenotype is successfully induced, this phenotype may be difficult to maintain long-term in the inflammatory microenvironment, resulting in inconsistent therapeutic efficacy. Long-term promotion of M2 polarization may also suppress normal immune function and increase the risk of infection or tumorigenesis. Finally, even if the M1 polarization state is effectively stabilized, reducing the production of damaged mitochondria, the clearance of already severely damaged and difficult-to-repair mitochondria remains ineffective, hindering the generation of healthy mitochondria. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a method for preparing multifunctional layered nanosheets (modified magnesium-manganese layered nanosheets) with high biological antioxidant capacity, enhanced mitochondrial function and osteogenic ability; another purpose of the present invention is to provide an application of multifunctional layered nanosheets in drugs for the treatment of osteoporosis.

[0005] Technical solution: The preparation method of the multifunctional layered nanosheets described in the present invention is prepared by a hydrothermal method, and the preparation steps are as follows:

[0006] (1) Add sodium hydroxide to the prepared ultrapure water and stir at room temperature of 25° until dissolved; then add magnesium nitrate and manganese nitrate to another ultrapure water to dissolve them and pour them into a three-necked flask; dropwise add the dissolved sodium hydroxide into the three-necked flask and stir to obtain a mixed solution;

[0007] (2) After the stirring is completed, the obtained mixed solution is transferred to a reactor for reaction and then cooled to obtain a solution;

[0008] After cooling naturally, the obtained solution is centrifuged to collect the precipitate, which is washed with ethanol and ultrapure water respectively, and the washed precipitate is dissolved with ultrapure water to obtain a mixed solution;

[0009] Subsequently, the prepared 3-aminopropyltrimethoxysilane (APTMS) was mixed with toluene and methanol and then added dropwise to the mixed solution, and stirred for reaction to prepare an LDH solution;

[0010] (3) dissolving the prepared folic acid (FA) in deionized water and then adding it dropwise to the LDH solution prepared in step (2), stirring continuously at room temperature, so that FA is coupled to the LDH surface via an amide bond;

[0011] (4) After the reaction is completed, the precipitate is collected by centrifugation, washed with deionized water, and then dispersed in deionized water for later use to obtain a mixed solution containing FA;

[0012] (5) adding the prepared formamide solution to the FA mixture and stirring it to allow it to insert into and expand the LDH layer gap structure to obtain an expanded FA mixture;

[0013] Then, the pre-prepared dimethylmalonic acid (DMM) solution was slowly added dropwise to the expanded FA mixture and stirred continuously to promote DMM intercalation into the LDH interlayer structure;

[0014] (6) After the reaction is completed, the precipitate is collected by centrifugation and washed with deionized water to obtain the final product, multifunctional layered nanosheets, namely magnesium manganese layered double hydroxide nanosheets MgMn-LDH / DMM@FA.

[0015] Furthermore, the molar ratio of magnesium nitrate to manganese nitrate added to ultrapure water in step (1) is: 3:1;

[0016] The dissolved sodium hydroxide was added dropwise into the three-necked flask at a rate of 60-80 ml / h, nitrogen was passed through the entire process, and the mixture was stirred at 800-1500 rpm for 60-180 min.

[0017] Furthermore, the reaction conditions of the mixed solution in the reaction vessel in step (2) are: reaction at 100-150°C for 16-24 hours;

[0018] The solution is centrifuged at 8000-12000 rpm for 5-15 minutes; the solution is washed 3-4 times with ethanol and ultrapure water respectively; and the precipitate is dissolved in ultrapure water to 10-20 mL to prepare a mixed solution.

[0019] The volume ratio of toluene, methanol and APTMS added to the mixed solution is: 40:4:3; preferably, 20 mL of toluene, 2 mL of methanol and 1.5 mL of 3-aminopropyltrimethoxysilane (APTMS) are added to the mixed solution;

[0020] The reaction mixture was stirred at room temperature under nitrogen for 6 hours to achieve amino functional group modification. After the reaction, the precipitate was collected by centrifugation at 10,000 rpm for 12 minutes to obtain an APTMS-modified LDH solution.

[0021] Furthermore, in step (3), the prepared FA is added dropwise to the APTMS-modified LDH solution and stirred at room temperature of 25°C for 12 hours; preferably, 10 mg of folic acid (FA) is dissolved in 5 mL of deionized water and then added dropwise to 20 mL of the APTMS-modified LDH solution.

[0022] Furthermore, after the reaction in step (4) is completed, the precipitate is collected by centrifugation at 10,000 rpm for 5-15 minutes, washed three times with deionized water, and dispersed in deionized water (preferably 10 mL) to prepare a mixed solution containing FA.

[0023] Furthermore, in step (5), the prepared 20 mL formamide solution is added to the FA mixture for stirring at 800-1500 rpm for 2 hours at 40° C. to expand the LDH interlayer spacing to prepare an expanded FA mixture.

[0024] Furthermore, in step (5), the pre-prepared 20 ml dimethylmalonic acid (DMM) solution is slowly added dropwise to the expanded FA mixture and stirred continuously at 800-1500 rpm for 12 hours at room temperature to promote the entry of DMM into the interlayer structure.

[0025] Furthermore, after the reaction in step (6) is completed, the precipitate is collected by centrifugation at 10,000 rpm for 5-15 minutes, washed three times with deionized water, and vacuum dried to obtain the final product, functional layered nanosheets, i.e., magnesium manganese layered double hydroxide nanosheets MgMn-LDH / DMM@FA.

[0026] Furthermore, the multifunctional layered nanosheets prepared in step (6) have a particle size of 150 nm and a hexagonal layered structure, with good dispersibility and high specific surface area; the modified magnesium manganese layered nanosheets, surface-modified with folic acid (FA) as a targeting agent, can selectively target pro-inflammatory macrophages expressing folic acid receptors, and achieve targeted delivery in the osteoporotic inflammatory bone marrow microenvironment; the nanosheets have superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) biomimetic enzyme activities, which can scavenge various ROS including superoxide anions, hydrogen peroxide, and hydroxyl free radicals, and improve the oxidative stress state of the bone marrow; the intercalated loaded dimethylmalonic acid (DMM) ) as a succinate dehydrogenase (SDH) inhibitor, can effectively block mitochondrial reverse electron transfer (RET) in pro-inflammatory macrophages, reduce ROS burst, and improve mitochondrial damage from the source; the nanosystem can activate the BNIP3-LC3B-mediated mitochondrial autophagy pathway, clear severely damaged mitochondria, and restore the dynamic balance of macrophage mitochondria; the repaired macrophages can transport healthy mitochondria to adjacent bone-forming cells, alleviate osteoblast aging, and improve their osteogenic differentiation ability; in the ovariectomy model, it can significantly increase the bone volume fraction (BV / TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th), improve bone microstructure, and show a good anti-osteoporosis treatment effect.

[0027] Furthermore, the prepared multifunctional layered nanosheets are used in drugs for the treatment of osteoporosis; the multifunctional layered nanosheets target pro-inflammatory macrophages, clear a large amount of reactive oxygen species (ROS) in the bone marrow microenvironment, and regulate the dynamic homeostasis of mitochondria, thereby significantly reducing the inflammatory stress state in the bone marrow, promoting the generation and transfer of healthy mitochondria, and thereby restoring the function and homeostasis of osteoblasts, ultimately achieving bone mass increase and reconstruction of trabecular structure in osteoporosis.

[0028] Beneficial Effects: Compared with the prior art, the present invention features: the nanosheet possesses highly efficient biological antioxidant capacity and can simultaneously mimic the functions of three natural enzymes: superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), comprehensively clearing excess ROS in pro-inflammatory macrophages and alleviating oxidative stress. Simultaneously, the nanosheet inhibits succinate dehydrogenase (SDH) activity by slowly releasing dimethyl malonate (DMM), blocking the ROS burst induced by mitochondrial reverse electron transfer (RET) at the source, activating the BNIP3–LC3B-mediated mitophagy pathway, clearing damaged mitochondria and promoting mitochondrial renewal, thereby restoring the metabolic state and immune homeostasis of pro-inflammatory macrophages. The repaired macrophages can transfer healthy mitochondria to surrounding osteoblasts, enhancing their mitochondrial function and bone formation, breaking the imbalance in the original bone marrow inflammatory environment and ultimately achieving the effect of promoting bone regeneration in an osteoporosis model. Animal experiments have confirmed that this material can significantly increase the number and thickness of trabeculae and improve bone microstructure. It is a precise intervention method for osteoporosis with multiple synergistic mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the appearance of the layered nanosheets of the present invention (transmission electron microscopy and elemental analysis);

[0030] Figure 2 is a graph of the macrophage targeting effect of the pro-inflammatory phenotype of the lamellar nanosheets of the present invention (flow cytometry);

[0031] Figure 3 Schematic diagram of the in vitro SOD, CAT, and GPx nanozyme effects of the layered nanosheets of the present invention;

[0032] Figure 4 Schematic diagram of the scavenging ability and biocompatibility of the DHE fluorescent probe for ROS in the environment in the present invention;

[0033] Figure 5 This is a schematic diagram of evaluating the mitochondrial rescue effect of the present invention by observing the mitochondrial morphology in macrophages using a mitochondrial mitosox fluorescent probe and a color-transmitted electron microscope;

[0034] Figure 6 This is a schematic diagram of collecting mitochondria from macrophages and co-incubating them with osteoblasts to analyze ALP, ARS staining, and osteoblast marker gene qPCR in the present invention, which observes the promoting effect of layered nanosheets on osteogenic differentiation after rescuing mitochondrial damage;

[0035] Figure 7 Schematic diagram of the therapeutic evaluation of the layered nanosheets of the present invention on femoral bone loss in ovariectomized mice. DETAILED DESCRIPTION

[0036] The present invention is further described below with reference to the accompanying drawings and specific embodiments.

[0037] As shown in the figure, the modified magnesium-manganese layered nanosheets described in the present invention are obtained by dissolving sodium hydroxide, manganese nitrate, and magnesium nitrate in ultrapure water, reacting them through a hydrothermal synthesis method, and then modifying them with folic acid and dimethyl malonate; their overall particle size is about 150nm, and they have a lamellar structure with good stability, and have targeting of pro-inflammatory phenotype macrophages and efficient nanozyme activity.

[0038] The preparation method comprises the following steps:

[0039] Step (1): Mg(NO3)2·6H2O (3 mmol) and Mn(NO3)2·6H2O (1 mmol) were dissolved in 5 mL of deionized water, respectively, and after complete dissolution, the mixture was poured into a three-necked flask. Separately, NaOH (6.8 mmol) was dissolved in 40 mL of deionized water and added dropwise to the three-necked flask at a rate of 80 mL / h. The entire reaction process was carried out at room temperature under nitrogen protection with a stirring rate of 1000 rpm for 60 min to obtain a mixed suspension.

[0040] Step (2): centrifuge the mixture at 10,000 rpm for 10 min, discard the supernatant, collect the precipitate, and wash it twice with deionized water to remove residual ions;

[0041] Step (3): resuspend the washed precipitate in 40 mL of deionized water, transfer it to a high-pressure hydrothermal reactor, react at 100° C. for 16 hours, and cool naturally after the reaction;

[0042] Step (4): The reaction product was centrifuged at 10,000 rpm for 10 min to collect the precipitate, which was then washed three times with ethanol and three times with deionized water, and finally dissolved in 10 mL of ddH2O for later use;

[0043] Step (5): Add 20 mL of toluene, 2 mL of methanol, and 1.5 mL of 3-aminopropyltrimethoxysilane (APTMS) to the obtained LDH dispersion, and stir at room temperature under nitrogen protection for 6 hours to introduce amino functional groups;

[0044] Step (6): After the reaction is completed, the precipitate is collected by centrifugation at 10,000 rpm for 12 minutes, the supernatant is discarded, and the APTMS-modified LDH product is obtained after washing;

[0045] Step (7): 10 mg of folic acid (FA) was weighed and dissolved in 5 mL of deionized water, and then added dropwise to 20 mL of the LDH solution obtained in step (6), and stirred at 4°C for 12 hours to form an amide bond;

[0046] Step (8): After the reaction, the precipitate was collected by centrifugation at 10,000 rpm for 10 min, washed three times with deionized water, and dispersed in 10 mL of deionized water for later use;

[0047] Step (9): Add 20 mL of formamide to the above solution and stir at 40°C for 2 hours to allow the formamide molecules to intercalate and expand the interstitial structure of the LDH layer;

[0048] Step (10): Slowly add 20 mL of pre-prepared dimethylmalonic acid (DMM) solution dropwise to the mixture after the formamide expansion, and continue stirring at room temperature for 12 hours to promote the insertion of DMM into the LDH interlayer structure;

[0049] Step (11): After the reaction is completed, the mixture is centrifuged at 10,000 rpm for 10 minutes to collect the precipitate, which is then washed three times with deionized water to obtain MgMn-LDH / DMM@FA nanosheets, which are the final product.

[0050] The present invention provides an application of a multifunctional layered nanosheet in the preparation of a drug for treating osteoporosis.

[0051] Example 1:

[0052] (1) Experimental materials:

[0053] Mouse MC3T3-E1 cells and BMDM cells: (Bone Metabolism Laboratory, Institute of Translational Medicine, a certain university); DHE detection kit (Beyotime, S0063); flow cytometry antibodies CD86 and F4 / 80 (Proteintech, PE-65068, FITC-98236); superoxide dismutase (SOD) test kit (WST-8 method) (Keming Biotechnology, SOD-1-W); total glutathione peroxidase detection kit (Beyotime, S0058); MitoSOX Red probe (MCE, HY-D1055); Calcein / PI cell viability and cytotoxicity assay kit: (Beyotime, C2015M); Cell mitochondria isolation kit (Beyotime, C3601); ALP staining kit: (Beyotime, P0321S); ARS staining kit (Beyotime, C0138); TRIzol reagent (Beyotime, R0016); Reverse transcription reagent (TaKaRa, RR037A);

[0054] MEM-α medium: (Pricella, PM150421); MC3T3-E1 cell osteogenic differentiation medium (OriCell, MUXMT-90021)

[0055] (2) Experimental methods:

[0056] 1. Preparation and morphological observation of layered nanosheets (transmission electron microscopy):

[0057] The layered nanosheets were dissolved in anhydrous ethanol and ultrasonicated until fully dissolved. Then, the morphology and elemental composition were observed using transmission electron microscopy and elemental analysis. Figure 1 The results of a) show that the particle size is about 150 nm and the particle size is uniformly distributed. Figure 1 b) The morphology is also similar to a flower;

[0058] 2. Targeting effect of pro-inflammatory macrophages by layered nanosheets (flow cytometry):

[0059] The layered nanosheets were dissolved in 5 ml of ultrapure water, sonicated until fully dissolved, and labeled with CE6. They were then injected into the femoral medullary cavity of ovariectomized mice, and the cells in the medullary cavity were collected and thoroughly mixed with red blood cell lysis buffer to remove red blood cells. Finally, the proportion of macrophages labeled with CE6 was detected by flow cytometry after labeling with CD86 and F4 / 80 antibodies. Figure 2 a, b); It can be seen that LDH can effectively target pro-inflammatory macrophages through FA modification;

[0060] 3. In vitro nanozyme activity (SOD, CAT, GPx) of layered nanosheets:

[0061] SOD: Prepare the reaction system of xanthine and xanthine oxidase in the kit; add different concentrations of MgMn-LDH and MgMn-LDH / DMM@FA to the reaction system; incubate at 37°C for 20 minutes. Measure the absorbance of the reaction solution at 560 nm; calculate the SOD activity based on the change in absorbance; the degree of absorbance reduction reflects the magnitude of SOD activity ( Figure 3 a);

[0062] CAT: Different concentrations of MgMn-LDH and MgMn-LDH / DMM@FA were added to H2O2 solution and incubated at 37°C for 20 minutes. The dissolved oxygen content in the solution was measured using a dissolved oxygen meter. The amount of O2 produced by H2O2 catalyzed by MgMn-LDH and MgMn-LDH / DMM@FA was calculated to evaluate CAT activity ( Figure 3 b,c);

[0063] GPx: Prepare the reaction system containing GSH and NADPH in the kit; add different concentrations of MgMn-LDH and MgMn-LDH / DMM@FA to the reaction system; incubate at 37°C for a period of time; measure the consumption of NADPH; calculate the GPx activity ( Figure 3 d);

[0064] 4. The scavenging ability and biocompatibility of ROS in the environment through DHE fluorescent probe:

[0065] The layered nanosheets were co-incubated with RAW264.7 cells for 24 h, 48 h, and 72 h, and the cell viability was determined by CCK-8 assay. The results showed that the layered nanosheets ( Figure 4 a) There was no significant difference compared with the control group in the concentration range of 0-40μg / ml; 60μg / ml was selected as the safe biological action concentration of the layered nanosheets; then the cell survival rate of the culture medium under different H2O2 concentrations was screened ( Figure 4 b) As a microenvironment for simulating osteoporosis micro-oxidative stress; finally, culture medium with different H2O2 concentrations was added and incubated with the layered nanosheets. The CCK-8 results showed that compared with other concentrations, 40 μg / ml of the layered nanosheets could significantly rescue the damaged macrophages ( Figure 4 c); Similarly, incubation with lamellar nanosheets in a culture medium with a H2O2 concentration of 100 μM, live-dead staining results showed a rescue effect on macrophages, and DHE staining showed the scavenging effect of lamellar nanosheets on ROS ( Figure 4 d, e);

[0066] 5. Evaluation of mitochondrial rescue effect by observing mitochondrial morphology in macrophages using mitochondrial mitosox fluorescent probe and color-coded electron microscopy:

[0067] LPS was used to induce the pro-inflammatory phenotype of macrophages. RAW264.7 cells were seeded in 12-well plates and incubated with the layered nanosheets for 72 h. Mitochondria-derived oxidative stress in pro-inflammatory macrophages was then detected using mito SOX. The layered nanosheets effectively reduced the production of ROS in mitochondria. Figure 5 a);

[0068] Female C57 / BL6 mice (8 weeks old, 20 g) were housed in a clean room, with 6 mice per cage. As previously reported, an osteoporosis model was established by ovariectomy (OVX). The mice were then injected with the layered nanosheets through the tail vein. Eight weeks later, the mice were killed, and cells in the femoral bone marrow cavity were collected. After fixation, the mitochondrial morphology of the macrophages was observed under a transmission electron microscope. It can be seen that after ovariectomy, the matrix of the mice became thinner, the electron density decreased, and even vacuolated. The cristae were disordered, broken, or even disappeared. After intervention with the layered nanosheets, the mitochondrial morphology was restored to the level of the blank control group ( Figure 5 b);

[0069] 6. After co-incubation of mitochondria from macrophages with osteoblasts, ALP and ARS staining and qPCR of osteoblast marker genes were performed to observe the effect of mitochondria from macrophages after intervention with the layered nanosheets on osteogenic differentiation;

[0070] RAW264.7 cells were co-incubated with the layered nanosheets for 72 h, and the mitochondria of RAW264.7 cells were isolated using a mitochondrial isolation kit. MC3T3-E1 cells were seeded in a 12-well plate for 24 h, and the mitochondria of RAW264.7 were co-incubated with them. After 7 days, the cells were washed three times with PBS, fixed with paraformaldehyde (4%) solution for 15 minutes, and washed with PBS. Afterwards, the osteogenic activity was detected using an alkaline phosphatase colorimetric kit ( Figure 6 a, b); Similarly, cells were stained with ARS solution 21 days after osteogenic induction ( Figure 6 c, d); The same MC3T3-E1 cells were co-cultured with mitochondria from macrophages treated with different treatments and lamellar nanosheets for 7 days to analyze the expression of osteogenic genes in MC3T3-E1 cells. TRIzol reagent was added to extract RNA from the cells, and vazyme cDNA was obtained using TaKaRa reverse transcription reagent. The obtained cDNA was used for further qPCR detection; RT-PCR was performed using real-time PCR mixture on a light cycler ( Figure 6 e) It can be seen that the layered nanosheets not only alleviate the inhibition of osteoblast differentiation caused by damaged mitochondria;

[0071] 7. Evaluation of the therapeutic effect of layered nanosheets on femoral bone loss in ovariectomized mice:

[0072] Female C57 / BL6 mice (8 weeks old, 20 g) were housed in a clean room, with 6 mice per cage. Ovariectomy (OVX) was performed as previously reported to establish an osteoporosis model. The mice were then injected with the layered nanosheets via the tail vein. Eight weeks later, the mice were sacrificed, and the femurs were collected for bone mass analysis using micro-CT. Figure 7 a, 7b), it can be seen that the layered nanosheets effectively restored the bone mass of ovariectomized mice; the femur was fixed and decalcified for a long time, and paraffin sections were performed after complete decalcification. HE staining was performed, and it can be seen that the trabeculae in the ovariectomized mice were sparse, while the trabeculae in the femurs of mice treated with layered nanosheets could reach the level of normal mice ( Figure 7 c).

Claims

1. A method for preparing multifunctional layered nanosheets, characterized in that: The preparation steps are as follows: (1) Add sodium hydroxide to the prepared ultrapure water and stir at room temperature of 25° until dissolved; then add magnesium nitrate and manganese nitrate to another ultrapure water to dissolve them and pour them into a three-necked flask; dropwise add the dissolved sodium hydroxide into the three-necked flask and stir to obtain a mixed solution; (2) After the stirring is completed, the obtained mixed solution is transferred to a reactor for reaction and then cooled to obtain a solution; After cooling naturally, the obtained solution is centrifuged to collect the precipitate, which is washed with ethanol and ultrapure water respectively, and the washed precipitate is dissolved with ultrapure water to obtain a mixed solution; Subsequently, the prepared APTMS was mixed with toluene and methanol and then added dropwise to the mixed solution, and stirred for reaction to prepare an LDH solution; (3) Dissolve the prepared FA in deionized water and add it dropwise to the LDH solution prepared in step (2), stirring continuously at room temperature; (4) After the reaction is completed, the precipitate is collected by centrifugation, washed with deionized water, and then dispersed in deionized water for later use to obtain a mixed solution containing FA; (5) Add the prepared formamide solution to the FA mixture and stir to obtain the expanded FA mixture, then add the pre-prepared DMM solution dropwise to the expanded FA mixture and continue stirring; (6) After the reaction is completed, the precipitate is collected by centrifugation and washed with deionized water to obtain the final product, multifunctional layered nanosheets, namely magnesium manganese layered double hydroxide nanosheets MgMn-LDH / DMM@FA.

2. The method for preparing the multifunctional layered nanosheet according to claim 1, wherein: The molar ratio of magnesium nitrate to manganese nitrate added to ultrapure water in step (1) is 3:1; The dissolved sodium hydroxide was added dropwise into the three-necked flask at a rate of 60-80 ml / h, nitrogen was passed through the entire process, and the mixture was stirred at 800-1500 rpm for 60-180 min.

3. The method for preparing the multifunctional layered nanosheet according to claim 1, wherein: The reaction conditions of the mixed solution in the reaction vessel in step (2) are: reaction at 100-150° C. for 16-24 hours; The solution is centrifuged at 8000-12000 rpm for 5-15 minutes; the solution is washed 3-4 times with ethanol and ultrapure water respectively; and the precipitate is dissolved in ultrapure water to 10-20 mL to prepare a mixed solution. The volume ratio of toluene, methanol and APTMS added to the mixed solution is 40:4:

3. The mixture was stirred at room temperature under nitrogen for 6 hours. After the reaction was completed, the precipitate was collected by centrifugation at 10,000 rpm for 12 minutes to prepare an LDH solution.

4. The method for preparing the multifunctional layered nanosheet according to claim 1, wherein: In the step (3), the prepared FA is added dropwise to the LDH solution and stirred at room temperature of 25° C. for 12 hours.

5. The method for preparing the multifunctional layered nanosheet according to claim 1, wherein: After the reaction in step (4) is completed, the precipitate is collected by centrifugation at 10,000 rpm for 5-15 minutes, washed three times with deionized water, and dispersed in deionized water.

6. The method for preparing the multifunctional layered nanosheet according to claim 1, wherein: In step (5), the prepared formamide solution is added to the FA-containing mixed solution and stirred at 800-1500 rpm for 2 hours at 40°C.

7. The method for preparing the multifunctional layered nanosheet according to claim 1, wherein: In step (5), the pre-prepared DMM solution is added dropwise to the expanded FA mixture and the stirring is continued under the following conditions: stirring is continued at 800-1500 rpm at room temperature for 12 hours.

8. The method for preparing the multifunctional layered nanosheet according to claim 1, wherein: After the reaction in step (6) is completed, the precipitate is collected by centrifugation at 10,000 rpm for 5-15 minutes, washed three times with deionized water, and dried in vacuo.

9. The method for preparing the multifunctional layered nanosheet according to claim 1, wherein: The multifunctional layered nanosheets prepared in step (6) have a particle size of 150 nm and present a hexagonal layered structure.

10. Use of the multifunctional layered nanosheets prepared by the method according to any one of claims 1 to 9 in drugs for treating osteoporosis.

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