A nanocellular drug with targeted sustained-release function and its application in the treatment of osteoporosis

Abstract

The present invention discloses a nanocellular drug with targeted sustained-release function and its application in the treatment of osteoporosis, including using PLGA to coat NO donors to extend the half-life of NO donors. Secondly, extract the human umbilical vein endothelial cell membrane, use it to coat PLGA-SN, improve the targeting ability of the drug, and couple it to ADSC through bioorthogonal click chemistry to form an ADSC-SNNP nanocellular drug with osteoporosis targeting and sustained-release effects. Then co-induce and culture it with OC precursor cells to clarify its effects on OC differentiation and bone resorption. At the same time, co-culture it with osteoblastic precursor cells MC3T3-E1 to clarify its effects on osteoblast differentiation and bone formation. Then inject it into rats with osteoporosis model to detect its therapeutic effect on osteoporosis, and clarify its molecular mechanism of action by detecting key protein pathways.

Description

Technical Field

[0001] The present invention belongs to the field of stem cell therapy and biomedicine technology, and specifically relates to a nano cell drug with targeted sustained release function and its application in the treatment of osteoporosis. Background Art

[0002] The incidence of osteoporosis in my country continues to rise, and it has become a major health problem that seriously affects the quality of life of the elderly. The release of multiple reports and policies not only reflects the country's high attention to osteoporosis treatment research, but also shows the urgency of research.

[0003] Osteoclasts (OC) and osteoblasts (OB) play a decisive role in bone remodeling, and the imbalance of their number and activity is the core mechanism of osteoporosis. Mesenchymal stem cells such as adipose mesenchymal stem cells (ADSC) have certain homing characteristics and can target osteoporotic sites, affecting osteoblast differentiation and bone formation function as well as osteoclast differentiation and bone resorption function during bone remodeling. Although ADSC has certain potential to repair osteoporosis, its homing efficiency is still insufficient to meet treatment needs.

[0004] Nitric oxide (NO) plays an important role in bone remodeling, but its short half-life, inability to bind specifically, and inability to target limit its application in osteoporosis treatment. The half-life of NO donor (Spermine NONOate, SN) is only 39 minutes, which means that it can only diffuse locally in the tissue and is difficult to reach the osteoporosis lesion. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a nanocellular drug with targeted sustained-release function.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions: comprising adipose-derived mesenchymal stem cells, a donor, a nanocarrier, a donor nanodrug, and a targeting molecule bound to the surface of the carrier;

[0009] The donor is a donor capable of providing carbon monoxide, including a nitric oxide-spermine complex; the nanocarrier includes a polylactic acid-glycolic acid copolymer and a liposome; the donor nanomedicine is a nanocarrier-coated donor; and the targeting molecule is CXCR4.

[0010] As a preferred embodiment of the preparation method of the present invention, the targeting molecule CXCR4 is the only receptor of SDF-1.

[0011] As a preferred solution of the preparation method of the present invention, the drug loading of the donor nano-drug is 9.10-10.62%.

[0012] As a preferred solution of the preparation method of the present invention, the encapsulation rate of the donor nano-drug is 59.8% to 69.8%.

[0013] As a preferred solution of the preparation method of the present invention, the NO release rate of the donor nano-drug at pH 4.5 is 82.56-86.30%.

[0014] As a preferred embodiment of the preparation method of the present invention, the donor nano-drug is in the shape of a sphere with a diameter of 200 to 300 nm.

[0015] As a preferred embodiment of the preparation method of the present invention, the drug loading of a single cell of the nanocellular drug is 0.0875-0.09 ng / cell.

[0016] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a nanocellular drug with targeted sustained-release function.

[0017] As a preferred embodiment of the preparation method of the present invention, a nitric oxide-spermine complex is used as a nitric oxide donor; the nitric oxide donor is coated with a polylactic acid-glycolic acid copolymer nanocarrier to form a donor nanomedicine; the nanomedicine is then coated with a human umbilical vein endothelial cell membrane that highly expresses CXCR4; and finally, the nanomedicine is coupled to adipose mesenchymal stem cells through bio-click chemistry to obtain a nanocellular drug.

[0018] Another object of the present invention is to overcome the deficiencies in the prior art and provide a nanocellular drug with targeted sustained-release function for use in the treatment of osteoporosis.

[0019] As a preferred embodiment of the preparation method of the present invention, the nanocellular drug, after being injected into the body, mainly accumulates in lesion sites such as bones, and the bone lesion sites highly express SDF-1.

[0020] Beneficial effects of the present invention:

[0021] (1) The present invention first encapsulates SN with PLGA to form SN@PLGA nanoparticles, and then encapsulates the surface of SN@PLGA nanoparticles with human umbilical vein endothelial cell membrane (CM) with high expression of CXCR4 to form SNNP nanoparticles. The particle size, surface charge, etc. were detected using a Malvern particle size analyzer. Through condition exploration, nanoparticles with a particle size of about 200-300nm were successfully prepared, and the cell membrane coating was determined using Western blot and Coomassie blue. The surface morphology of the nanoparticles was characterized using TEM and SEM. Then, through metabolic sugar engineering, Ac4ManNAz was co-incubated with ADSC to obtain engineered adipose mesenchymal stem cells of ADSC-N3, and the above results were verified by flow cytometry and immunofluorescence technology. Finally, the SNNP nanoparticles with DBCO inserted on the surface were coupled to ADSC-N3 using bioorthogonal chemistry and copper-free click reaction. Flow cytometry and laser confocal microscopy verified that the SNNP nanoparticles and ADSC had been successfully coupled. This indicates that ADSC-SNNP drugs have been successfully constructed.

[0022] (2) Based on the successful preparation of ADSC-SNNP, the present invention uses Transwell experiments to show that compared with ADSC, the targeted transmembrane transport ability of ADSC-SNNP is significantly enhanced; TRAP staining results show that the ADSC-SNNP drug can inhibit osteoclast differentiation and maturation, while ALP staining and Ars staining show that the drug can promote osteoblast differentiation, maturation and mineralization, and the effect of ADSC-SNNP drug on osteoclasts and osteoblasts is stronger than that of ADSC and SNNP alone.

[0023] (3) The present invention first verified the strengthening effect of SNNP on the targeting homing ability of ADSC in an in vivo experiment. The results of in vivo imaging of the drug showed that after the ADSC-SNNP drug was injected into the body, it mainly accumulated in the lesion sites such as bones. Subsequently, through HE, Micro-CT scanning, immunofluorescence technology and other detection methods performed on the bone site, the results showed that the drug effectively promoted the recovery of the number and area of ​​trabeculae in the osteoporotic lesion site, indicating that the ADSC-SNNP drug can alleviate and reverse osteoporosis. Subsequently, HE staining of the main organs was performed, and the results showed that there was no obvious pathological damage to important organs such as the liver and kidneys. Finally, the cell drug was determined to have good biosafety through blood routine, blood biochemistry and other tests. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0025] Figure 1 This is a flow chart for the preparation of ADSC-SNNPs in Example 1 of the present invention.

[0026] Figure 2 The isolation, culture, flow cytometry and three-lineage differentiation identification diagram of ADSCs of the present invention (wherein, Figure 2 A is the cell morphology diagram, Figure 2 B is the detection of surface markers and identification of multi-lineage differentiation potential. Figure 2 C is the result of staining after induction of tri-lineage differentiation).

[0027] Figure 3 The particle size, surface charge and release detection diagram of the SNNP nanoparticles of the present invention at different pH values ​​(wherein, Figure 3 A is the particle size diagram of SNNP nanoparticles, Figure 3 B is the surface charge diagram, Figure 3 (C) is a graph showing the sustained release effect of nanoparticles at different pH values.

[0028] Figure 4 The coating detection and morphology observation diagram of the NO donor nanoparticles of the present invention (wherein, Figure 4 A is a picture of CM coating on the surface of SNNP detected by protein immunoblotting. Figure 4 B is the TEM image of PLGA, SN@PLGA, CM, and SN@PLGA@CM. Figure 4 C is the SEM image of SN@PLGA at different pH).

[0029] Figure 5 This is a verification diagram for the preparation of ADSC-SNNP of the present invention (wherein, Figure 5 A is the flow cytometry image of ADSC-SNNP coupling. Figure 5 B is the flow cytometry fluorescence intensity statistical diagram. Figure 5 C is the laser confocal detection image of ADSC-SNNP coupling).

[0030] Figure 6 This is a diagram of detecting the transmembrane transport ability of cells using the Transwell method of the present invention.

[0031] Figure 7 This is a diagram of the effect of ADSC-SNNP on MC3T3-E1 osteogenic differentiation detected by fluorescence quantitative PCR of the present invention (wherein, Figure 7A is the change of ADSC gene ALPL transcription level detected by fluorescence quantitative PCR. Figure 7 B is the change of transcription level of ADSC gene BGLAP detected by fluorescence quantitative PCR. Figure 7 C is the change of ADSC gene COL1A1 transcription level detected by fluorescence quantitative PCR. Figure 7 D is the change of Runx2 transcription level detected by fluorescence quantitative PCR in ADSCs).

[0032] Figure 8 TRAP staining was used to detect the inhibition of osteoclast differentiation by ADSC-SNNP.

[0033] Fig. 9 ALP and Ars staining images after ADSC-SNNP intervention (wherein, Fig. 9 A is ALP staining of MC3T3-E1 after 7 days of osteogenic induction. Fig. 9 B: Ars staining of MC3T3-E1 after 21 days of osteogenic induction).

[0034] Fig.10 This is a thermal image of the main organs and hind limbs of osteoporosis model rats after injection of fluorescently labeled stem cell drugs (including Fig.10 A is the fluorescence intensity of the main organs and hind limbs of rats observed by in vivo imaging. Fig.10 (B) Statistical graph of fluorescence intensity of major organs and hind limbs).

[0035] Fig.11 This is a HE staining image of the femur section of the osteoporosis model rat of the present invention.

[0036] Fig.12 This is a Micro-CT image of the distal femur of the present invention (wherein, Fig.12 A is the three-dimensional reconstructed z-axis section of the distal femur of the osteoporosis model rat. Fig.12 B is a two-dimensional cross-sectional view of the distal femur of osteoporosis model rats).

[0037] Fig.13 This is a serum ELISA test chart of bone formation related cytokines of the present invention (wherein, Fig.13 A is Bmp2 serum ELISA test; Fig.13 B is RUNX2 serum ELISA test; Fig.13 C is the ALP serum ELISA test diagram).

[0038] Fig.14 For the bone resorption related cytokine serum ELISA test of the present invention (wherein, Fig.14 A is OPG serum ELISA test; Fig.14 B is RANKL serum ELISA test; Fig.14 C is OPG / RANKL data statistics; Fig.14 D is TRAP serum ELISA test).

[0039] Fig.15 This is the IF detection of OCN protein in femoral slices of the present invention.

[0040] Fig.16 This is the IF detection of TRAP protein in femoral slices of the present invention.

[0041] Fig.17 The hemolytic statistics and body weight change trend diagram of the drug of the present invention ( Fig.17 A is a statistical graph of hemolytic detection data of ADSC-SNNP drugs; Fig.17 (B) is a graph showing the body weight change trend of osteoporosis model rats during treatment.

[0042] Fig.18 This is the blood biochemical diagram of ALT (alanine aminotransferase), AST (aspartate aminotransferase), CRE (creatinine), and UREA (urea) of the present invention.

[0043] Fig.19 The blood routine items of the present invention ( Fig.19 A stands for WBC (white blood cell count), Neu (neutrophil count), Lym (lymphocyte count), Mon (monocyte count), and RBC (red blood cell count); Fig.19 B is PLT (platelet count), Eos (eosinophil count), MCV (mean corpuscular volume), HCT (hematocrit); Fig.19 C is MPV (mean platelet volume), PCT (platelet packed volume), PDW (platelet volume distribution width), MCH (mean corpuscular hemoglobin content), RDW-CV (red blood cell volume distribution width-coefficient of variation ratio)).

[0044] Fig. 20 This is HE staining of the viscera of the osteoporosis model rats of the present invention. DETAILED DESCRIPTION

[0045] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0046] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0047] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0048] Unless otherwise specified, the reagents and consumables used in the embodiments of the present invention are commercially available.

[0049] The gene sequence of CXCR4 can be downloaded from the NCBI database (https: / / wwwncbi.nlmnih.gov / ) as shown in SEQ ID NO.1, Gene ID: 7852.

[0050] Example 1

[0051] (1) Cell culture: Human umbilical vein endothelial cell line (HUVEC) was selected, RPMI 1640 medium was added, and 10% fetal bovine serum was added, and cultured in a 37°C constant temperature incubator with a CO2 concentration of 5%. Cell culture and processing were carried out in a sterile environment. The cell density was observed under an optical microscope. 1 mL of 0.25% trypsin was added to each culture dish, and the dish was placed in an incubator for incubation for 2 min. After the cells fell off, 2 mL of culture medium was added to terminate the digestion. The cells were collected by centrifugation at 1500 r / min for 5 min, the supernatant was discarded, and complete culture medium was added to blow the cell pellet to make a cell suspension, which was then transferred to the corresponding culture dish for subculture.

[0052] (2) ALP, Oil Red O, and Alzheimer Blue staining:

[0053] ALP staining: ADSC cells were seeded in a six-well plate. When they grew to 90% confluence, osteogenic induction medium (DMEM medium containing 10% FBS, 1% double antibody, 10 nM dexamethasone, 50 mg / L L-ascorbic acid, and 10 mM sodium β-glycerophosphate) was added. The medium was changed every 3 days and cultured continuously for 7 days. The cells were then treated with 4% paraformaldehyde for 20 minutes, washed with PBS, added with ALP staining solution, incubated at room temperature for 1 hour, and photographed under a microscope.

[0054] Oil red O staining: ADSC cells were seeded in a six-well plate, and when they grew to 90% confluence, adipogenic induction medium A (10% FBS, 1% double antibody, insulin (57μg / mL), IBMX (111μg / mL), dexamethasone (39ng / mL), indomethacin (72μg / mL)) was added. After three days, adipogenic induction medium B (10% FBS, 1% double antibody, insulin (57μg / mL)) was replaced for maintenance. After 24 hours, it was replaced with adipogenic induction solution A for induction, and this was repeated for 3 cycles. When there are more lipid droplets but they are relatively small, adipogenic induction solution B can be used for maintenance for 3-5 days to increase the lipid droplets so that Oil red O staining can be performed.

[0055] Aris blue staining: Before the chondrogenic induction experiment, it is necessary to count the cells after routine digestion. 5 Transfer the cells to a 15mL centrifuge tube and centrifuge at 250g for 4min. Aspirate the supernatant, add 0.5mL culture medium, resuspend the precipitate obtained from the previous centrifugation to wash the mesenchymal stem cells, and centrifuge at 150g for 5min at room temperature. Resuspend the precipitate obtained in the previous step with 0.5mL mesenchymal stem cell chondrogenic induction complete medium. Centrifuge at 150g for 5min at room temperature. Loosen the cap of the centrifuge tube to facilitate gas exchange and place it in an incubator at 37℃, 5% CO2 for culture. When the cell clusters gather, flick the bottom of the centrifuge tube to detach the chondrogenic balls from the bottom of the tube and suspend them in the liquid. Starting from the start of inoculation, replace the cells with fresh chondrogenic induction complete medium every 2-3d. After changing the medium, flick the bottom of the centrifuge tube to detach the chondrogenic balls from the bottom of the tube and suspend them in the liquid. Slightly loosen the cap of the centrifuge tube and place it in an incubator at 37℃, 5% CO2 to continue induction culture. Generally, after 21-28 days of induction, the chondrocytes can be fixed with formalin, embedded in paraffin, and sectioned, and finally stained with Aris blue.

[0056] (3) Cell membrane preparation: Prepare PBS containing 50uM PMSF, scrape the cells with a cell scraper in an ice bath, centrifuge at 700g for 10min, resuspend in PBS, centrifuge at 700g for 5min, then resuspend in 1mL PBS, transfer to a cryopreservation tube, and freeze-thaw repeatedly between liquid nitrogen and a 37°C water bath for 5 times; centrifuge again at 700g to remove nuclear fragments and unbroken cells, then centrifuge at 20,000g in a refrigerated centrifuge at 4°C for 1h.

[0057] (4) Preparation of SN@PLGA nanoparticles: 25 mg of PLGA was dissolved in 2 mL of dichloromethane, and SN was dissolved in PBS at pH 8. The two were mixed and sonicated in an ice bath at 200 W for 3 min. 10 mL of 2% PVA aqueous solution was prepared. The mixed liquid in the first step was added to PVA and sonicated in an ice bath for 15 min at a power of 200 W. The sonicated liquid was collected and magnetically stirred at 600 rpm for 2 h at room temperature to evaporate the dichloromethane. The collected product was centrifuged at 4°C and 12,000 rpm for 30 min and washed 3 times with DDW. After centrifugation, the supernatant was removed from the product and resuspended in PBS. The obtained solution was stored at 4°C.

[0058] (5) Determination of encapsulation efficiency and drug loading: The SN content in the microparticles was determined by microwave digestion of PLGA. A known amount of microparticle suspension was placed in 1M NaOH, the suspension was kept in an ultrasonic bath for 10 min, stirred until the PLGA was completely hydrolyzed, and then the final transparent hydrolyzate was determined by ultraviolet spectrophotometry at a wavelength of 252 nm. The standard solution for the calibration curve was prepared by mixing SN of different concentrations in 1M NaOH. The sample was prepared three times, and the encapsulation efficiency was calculated as follows: EN% = Cf / Ct × 100%. In the formula, Cf is the amount of free drug in the supernatant after decomposition; Ct is the total amount of drug added during the preparation of nanoparticles. Drug loading formula

[0059] The formula is as follows: LE (%) = We / Wm × 100% (1)

[0060] In formula (1), LE represents the percentage of drug loading in the nanoparticles; We represents the amount of drug encapsulated in the nanoparticles; and Wm represents the total weight of the drug-loaded nanoparticles.

[0061] (6) Drug release detection: The nanoparticle suspension was added to 5 mL of PBS solution with different pH values, namely pH 4.5, pH 7.4 and pH 8, and shaken at 120 rpm at 37°C. 500 μL of samples were collected at predetermined time intervals, supplemented with the same volume of PBS, and centrifuged at 12,000 g for 10 min. The supernatant was then directly analyzed by a spectrophotometer at a wavelength of 252 nm to determine the undecomposed SN. The precipitate was digested with 1 M NaOH to determine the residual SN in the microparticles. All samples were prepared in triplicate for analysis. The apparent NO release (%) was calculated by the following formula (2):

[0062] NO release amount (%) = 100 - (% unreleased SN + % undecomposed SN in supernatant) (2)

[0063] (7) Preparation of SNNPs: First, the cell membrane was filtered through a 0.45 μm filter and resuspended in PBS, pH 7.4, and the protein concentration of the cell membrane was measured. Then, the cell membrane and PLGA (25 mg / mL) were added to a centrifuge tube at a mass ratio of 1:5, and the tube was sonicated in a water bath at 80% power for 10 min, followed by an ice bath. The tube was then centrifuged at 4°C, 8000 rpm, for 20 min to separate the nanoparticles and unbound cell membranes.

[0064] (8) The prepared nanoparticles are tested for particle size and zeta potential using a Malvern particle size analyzer.

[0065] (9) Protein immunoblotting: Extract HUVEC and SNNP proteins using RIPA lysis buffer supplemented with a protease inhibitor cocktail. Determine protein concentration using the BCA method and perform quantitative processing. Prepare samples according to 20 μg protein content per sample, add 5× loading buffer, boil for 10 min to denature the protein, and load directly on electrophoresis or place in a -20°C refrigerator for later use. Use sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins, add protein markers on both sides of the sample to mark the molecular weight, and facilitate the indication of the target protein location. Prepare separation gels with an acrylamide concentration of 8-12% and a concentration gel with a concentration of 5% according to the molecular weight of the protein.

[0066] The electrophoresis conditions were set to 70V (1h) + 90V (2h), and the transfer conditions were set to 200mA (1.5-3h), which can be adjusted according to the molecular weight of the protein. After the transfer, the target protein was transferred to the polyvinylidene fluoride (PVDF) membrane, blocked with 5% bovine serum albumin or skim milk powder at room temperature for 1.5h, washed three times with TBST and incubated with the primary antibody at 4°C for 12h, washed three times with TBST after the primary antibody incubation, and then selected the appropriate secondary antibody according to the source of the primary antibody and incubated at room temperature for 1.5h. The ultrasensitive ECL luminescent liquid A liquid and B liquid were mixed in a 1:1 ratio to incubate the PVDF membrane, remove bubbles, use a developer to develop and pay attention to the storage of the original data. ImageJ was used to analyze the protein expression level.

[0067] Preparation of electrophoresis solution: weigh 3.03g Tris, 14.4g Glycine, 1g SDS, add DDW to dissolve and make up to 1L.

[0068] Preparation of transfer solution: weigh 5.8g Tris, 2.9g Glycine, 0.37g SDS, 200mL methanol, add DDW to dissolve and make up to 1L.

[0069] Preparation of TBST solution: accurately weigh 24.2g Tris and 87.6g NaCl, dissolve in deionized water and dilute to 1L to prepare 10×TBS stock solution, store at 4°C. Prepare 1×TBS before use, add 900mL deionized water to dilute 100mL 10×TBS stock solution to prepare 1L 1×TBS working solution, add 1mL Tween-20 and mix well before use.

[0070] (10) The prepared nanoparticles were sent for transmission electron microscopy and scanning electron microscopy observation.

[0071] (11) Flow cytometry: ADSCs were seeded in a six-well plate and grown to about 90%. The cells were digested with trypsin and digested with serum medium. The cells were collected by centrifugation at 1000 r / min and resuspended in 4°C precooled PBS. The cells were collected by centrifugation at 1000 r / min and resuspended in precooled PBS again. The cells were then incubated with fluorescent primary antibodies at 4°C for 1 h and then tested on a flow cytometer.

[0072] (12) Immunofluorescence staining: ADSC cells were seeded in a six-well plate and digested with trypsin until the cells grew to about 90%. The digestion was terminated by medium with serum, and the cells were collected by centrifugation at 1000 r / min. The cells were resuspended in 4°C precooled PBS, and the cells were collected by centrifugation at 1000 r / min. The cells were resuspended again in precooled PBS, and the cell suspension was dropped on a slide and observed after sealing.

[0073] (13) Preparation of ADSC-SNNPs: First, Ac4ManNAz was added to ADSCs in advance and co-cultured for 24 h. Then, the prepared cell membrane was resuspended in PBS and incubated with DSPE-PEG-DBCO (40 μg / mL) at 37°C for 30 min. The engineered cell membrane was then wrapped onto the surface of the nanoparticles and washed twice with PBS and collected by centrifugation. Subsequently, SNNPs were added to the culture dish of engineered ADSCs and incubated at 37°C for 15 min. After trypsin digestion, the ADSC cells with surface-anchored SNNPs were collected by centrifugation at 800 g for 5 min, washed with PBS, and resuspended in PBS for in vitro or in vivo studies.

[0074] (14) Determination of the amount of bio-orthogonal nanoparticles loaded in a single cell: ADSC cells were plated in a six-well plate and Ac4ManNAz was added to co-culture for 24 h. SNNPs were then prepared and resuspended in 1 mL of basal medium. Then, the cells were added to the six-well plate of ADSC cells and incubated at 37°C for 15 min. The supernatant was collected, and 50 μL of the supernatant was added to 50 μL of 1 M NaOH solution. The SN content in the supernatant was calculated by microwave digestion (the amount of cell-bound nanoparticles on the well plate / the number of cells = the amount of bio-orthogonal nanoparticles loaded in a single cell ng / cell). Figure 1The preparation flow chart of ADSC-SNNPs.

[0075] Isolation, culture and identification of ADSCs:

[0076] Figure 2 A shows that the obtained ADSCs have characteristic cell morphology, and the results of flow cytometry are as follows Figure 2 B shows that the third generation of cultured ADSCs uniformly expressed CD29 and CD90, with positive rates of 98.9% and 99.1%, respectively. Figure 2 C shows the staining results of ADSCs after tri-lineage differentiation induction, which show that they have the potential to differentiate into osteoblasts, adipocytes and chondrocytes, indicating that the isolated ADSCs have high purity, good activity and intact functions.

[0077] Characterization of SNNP nanoparticles:

[0078] The size and surface charge of the synthesized SNNP nanoparticles were tested. Figure 3 As shown in A, the Malvern particle size analyzer results showed that the particle size of the synthesized PLGA was about 227±12.9 nm, the particle size of SN@PLGA was about 244.3±8.2 nm, the particle size of CM was about 372.3±26.1 nm, and the particle size of SN@PLGA@CM (SNNP) was about 335.9±9.1 nm. Figure 3 B shows that the zeta potentials of PLGA, SN@PLGA, CM, and SN@PLGA@CM are -2.72±0.34, -2.75±0.37, -8.89±1.07, and -6.41±1.07, respectively. The microwave digestion method was used to detect the sustained release effect of nanoparticles at different pH values. The results showed that ( Figure 3 C), the release rate of the nanoparticles in the acidic environment of pH 4.5 was higher than that of the other two groups, and 85% of NO could be released within 12 hours, while the release in the alkaline environment was slow, and only 45% of NO was released within 12 hours. This shows that the constructed nanoparticle system has a strong sustained release ability, and most of the SN drugs can be retained before reaching the acidic osteoporotic lesions, and the nanoparticles that reach the lesions can be quickly released into the microenvironment.

[0079] Then the coating and morphology of SNNP nanoparticles were observed. Due to the high expression of CXCR4 protein on the surface of HUVEC, Figure 4 The cell membrane (CM) shown in A was successfully coated on the surface of PLGA nanoparticles. Figure 4B shows that PLGA presents a uniform spherical structure with a particle size of about 100 nm under transmission electron microscopy, SN@PLGA presents a uniform spherical structure with a particle size of about 100 nm under transmission electron microscopy, CM presents an uneven hollow spherical structure with a particle size of about 150 nm under transmission electron microscopy, and SN@PLGA@CM presents a uniform spherical structure with a particle size of about 200 nm under transmission electron microscopy. Figure 4 C is a scanning electron microscope image of SN@PLGA nanoparticles taken after being placed in different pH environments for 3 hours. The results in the figure show that in an acidic environment of pH 4.5, PLGA nanoparticles cannot maintain a complete spherical membrane structure, and compared with PLGA nanoparticles in pH = 7, there is a more serious structural damage, while PLGA nanoparticles in pH = 8 maintain a relatively complete membrane structure.

[0080] The present invention achieves the purpose of coupling the nanoparticles to the surface of ADSC-N3 by inserting DBCO into the CM on the surface of the SNNP nanoparticles. Figure 5 As shown in Figure A, through flow cytometry, the present invention found that ADSC cells incubated with Ac4ManNAz can undergo copper-free click reaction, i.e., SPAAC reaction, with SNNP nanoparticles with DBCO inserted on their surfaces, while ordinary ADSC and SNNP with DBCO inserted, and ADSC-N3 and SNNP without DBCO inserted, cannot couple. Figure 5 In B, the statistical results also showed that there were significant differences between the ADSC-N3 and DBCO-SNNP groups and other experimental groups. Figure 5 In C, the coupling between ADSC and SNNP was detected by laser confocal microscope. The results in the figure show that the fluorescent nanoparticles are mainly coupled to the cell membrane surface of stem cells, which is consistent with the results of flow cytometry. At the same time, the drug loading of a single cell was detected to be 0.0875ng / cell.

[0081] Example 2

[0082] (1) ADSCs were seeded in a 6-well plate, incubated overnight with SNNPs, then washed with PBS, and RNA was extracted with Trizol, which was then reverse transcribed using a kit. According to the experimental protocol, 2 μL of cDNA was added to each well, followed by the target gene primer sequence and SYBR fluorescent dye, and enzyme-free water was added to make up to 20 μL of the system. The CT value of the sample was detected using a fluorescent quantitative PCR instrument according to the set program. The relative mRNA level was then calculated according to the formula.

[0083] Table 1 Primer sequences used for fluorescence quantitative PCR

[0084]

[0085]

[0086] (2) Transwell cell targeted migration assay:

[0087] The prepared cell suspension was added to the upper chamber, and 500 μL of chemokine solution was added to the lower chamber. The control group used normal serum-containing culture medium without chemokines. The co-culture system was placed in a humidified cell culture incubator for 24 hours, and crystal violet staining was performed after the cells spread and attached to the surface of the lower chamber. The images were observed and photographed under a microscope, and three areas were randomly selected for cell counting, and statistical data were analyzed.

[0088] Effect of ADSC-SNNP in the treatment of osteoporosis:

[0089] In view of the low expression of CXCR4 on the surface of ADSCs and the high expression of SDF-1 in osteoporotic lesions, SNNPs were coated with cell membranes that highly expressed CXCR4 to enhance the targeted homing effect of ADSCs. Figure 6 The results showed that the transmembrane transport efficiency of the ADSC-SNNP complex in PBS was not affected by SNNP coupling; however, after adding SDF-1, the ADSC-SNNP group showed a significantly enhanced targeted transmembrane transport ability compared with the ADSC group alone. This result fully proves that HUVEC cell membrane coated SNNP effectively improves the homing efficiency of ADSC to osteoporotic lesions.

[0090] Example 3

[0091] (1) Fluorescence quantitative PCR:

[0092] MC3T3-E1 was inoculated in a Transwell plate, and the corresponding drugs were added for incubation for 7 days, followed by PBS washing, and Trizol was added to extract RNA, and then reverse transcription was performed using a kit. According to the experimental plan, 2 μL of cDNA was added to each well, and the target gene primer sequence and SYBR fluorescent dye were added, and enzyme-free water was added to make up to 20 μL of the system. The CT value of the sample was detected using a fluorescent quantitative PCR instrument according to the set program. The relative mRNA level was then calculated according to the formula.

[0093] Table 2 Primer sequences used for fluorescence quantitative PCR

[0094]

[0095]

[0096] (2) Raw264.7 osteoclast differentiation induction and TRAP staining:

[0097] Raw264.7 cells were revived and available after 8 passages. They were inoculated in a 12-well plate at a cell number of 6000 / well, and osteoclast differentiation induction medium was added. The culture was continued for three days, and the medium was changed once a day. TRAP staining can be performed on the fifth day from the date of inoculation. During the induction process, two parallel experimental groups with different pH values ​​were set at the same time, and the pH value of the culture medium in one group was adjusted to 6.8. The specific steps of TRAP staining are to first prepare the fixative and staining solution according to the instructions of the kit, preheat both in a 37°C water bath, pour out the cell culture solution in the cell plate to be stained, wash with preheated PBS 3 times, add the fixative for 1 minute, pour out the fixative, wash 3 times with PBS, add the staining solution, incubate at 37°C for 1 hour, then pour out the staining solution, wash with PBS, and observe and shoot under a microscope.

[0098] (3) Osteogenesis induction and ALP staining of MC3T3-E1 cell line:

[0099] MC3T3-E1 cells were seeded in Transwell 24-well plates. When they grew to 90% confluence, osteogenic induction medium was added. Intervention cells and nanoparticles were also added to the upper chamber. The medium was changed every 3 days and cultured continuously for 7 days. The cells were then fixed with 4% paraformaldehyde for 20 minutes, washed with PBS, and ALP staining solution was added. The cells were incubated at room temperature for 1 hour and photographed under a microscope.

[0100] Osteogenesis induction and Ars staining of MC3T3-E1 cell line:

[0101] After 21 days of osteogenic induction culture of MC3T3-E1 cells, the cells were fixed with 4% paraformaldehyde for 20 minutes, washed with PBS, added with Ars staining solution, incubated at 37°C for 1 hour, and observed under a microscope.

[0102] Effects of ADSC-SNNP on osteoclast differentiation and maturation:

[0103] A positive group, an ADSC-SNNP group, and a negative group were set up. Among them, both the positive group and the ADSC-SNNP group were added with osteoclast induction medium, while the negative group used normal medium. Considering the acidic characteristics of the pathological microenvironment of osteoporosis, we also specifically verified the effect of ADSC-SNNP on osteoclast differentiation under acidic conditions. TRAP staining results showed ( Figure 8 ), no osteoclasts were generated in the negative group and TRAP was not expressed; while multinucleated TRAP-positive osteoclasts were generated in the positive group. It is worth noting that although the ADSC-SNNP group highly expressed TRAP, multinucleated osteoclasts were not generated. This indicates that ADSC-SNNP blocked the fusion of osteoclast precursor cells and significantly inhibited the differentiation and maturation of osteoclasts, regardless of normal pH or acidic pH environment.

[0104] Fluorescence quantitative PCR detection of the effect of ADSC-SNNP on osteogenic differentiation of MC3T3-E1:

[0105] MC3T3-E1 is an osteoblast precursor cell that can be induced to differentiate into osteoblasts. We studied the effect of ADSC-SNNP on osteoblast differentiation through in vitro experiments. Fluorescence quantitative results showed that ( Figure 7 ), Figure 7 A is the change diagram of ADSC gene ALPL expression detected by fluorescence quantitative PCR. Figure 7 B is the change diagram of ADSC gene BGLAP expression detected by fluorescence quantitative PCR. Figure 7 C is the change diagram of ADSC gene COL1A1 expression detected by fluorescence quantitative PCR. Figure 7 D is a graph showing changes in the expression of the ADSC gene Runx2 detected by fluorescence quantitative PCR. Compared with other groups, the expression of osteogenic-related genes ALPL, BGLAP, COL1A1, and Runx2 were significantly upregulated after ADSC-SNNP treatment, indicating that ADSC-SNNP significantly promoted the transcription level of MC3T3-E1 osteogenic differentiation-related genes.

[0106] To verify the effect of ADSC-SNNP on osteoblast differentiation and bone formation, we co-cultured ADSC-SNNP with MC3T3-E1 using Transwell upper and lower chambers. ALP staining results showed ( Fig. 9 A), compared with other groups, the ADSC-SNNP group showed a more significant effect in promoting osteogenic differentiation of MC3T3-E1 cells. To further verify the effect of ADSC-SNNP on bone formation, we performed Ars staining. The results showed ( Fig. 9 B), ADSC-SNNP group produced more mineral accumulation compared with other groups. This shows that ADSC-SNNP can promote osteoblast differentiation and bone formation.

[0107] Example 3

[0108] ADSC-SNNP clinical treatment effect and safety testing:

[0109] (1) Animal experiment: 8-week-old Wistar rats were housed in an animal room with a constant temperature (24±5℃) and a 12-h day-night cycle, with free access to food and water. Rats that had undergone ovariectomy and were fed for 3 months were randomly divided into 6 groups: sham group; saline group; SN group; SNNP group; ADSC group; and ADSC-SNNP group, with 6 rats in each group.

[0110] The osteoporosis model rats were treated. The sham operation group was the rats whose abdominal cavity was only opened but whose ovaries were not removed. The saline group was injected with saline through the tail vein, the SN group was injected with SN solution through the tail vein, the SNNP group was injected with SNNP nanoparticle solution through the tail vein, the ADSC group was injected with ADSC cell suspension through the tail vein, and the ADSC-SNNP group was injected with ADSC-SNNP cell suspension through the tail vein. The treatment was once a week for 8 consecutive weeks.

[0111] After treatment, rats were fasted for 12 hours and killed by cervical dislocation after anesthesia. Blood, serum, heart, liver, spleen, lung, kidney and other visceral tissues as well as femur and tibia were collected. Blood and serum were quickly tested for blood routine and blood biochemistry by instruments, and the remaining serum was frozen in a -80℃ refrigerator for subsequent ELISA testing. Visceral tissues and bones were fixed with 4% paraformaldehyde for making sections and subsequent morphological observations.

[0112] The body weight of each rat was recorded every week during the treatment, and statistics and analysis were performed after the treatment.

[0113] (2) Evaluation of ADSC-SNNP drug hemosolubility:

[0114] Whole blood treated with heparin from healthy Wistar rats was collected and centrifuged at 1600 r / min for 5 min. The supernatant was removed and the red blood cells were washed three times. The purified red blood cells were dispersed in PBS to prepare a 20% volume concentration red blood cell solution. 100 μL of the red blood cell solution was added to 900 μL of ADSC-SNNP suspension (the final concentration of SN was 0.765, 1.913, 3.825, 7.65, 15.3, 22.95, 30.6, and 38.25 μg / mL, respectively). DDW and PBS were used as positive and negative controls for incubation with red blood cells, respectively. The culture medium was incubated in a shaker at 37°C for 4 h. After the incubation, it was centrifuged at 1600 r / min for 5 min to remove unbroken red blood cells. The sample was taken and 100 μL of the supernatant was transferred to a 96-well plate. The absorbance of the supernatant was measured at a wavelength of 540 nm. The hemolysis rate was calculated according to the following formula (3):

[0115] Hemolysis rate (%) = (sample absorbance - negative control absorbance) / (positive control absorbance - negative control absorbance) × 100 Formula (3)

[0116] (3) Near-infrared imaging of drug distribution in vivo: DiR-stained ADSCs and ADSC-SNNP drugs were prepared and injected into rats via the tail vein. Samples were collected 6 hours later and observed by instruments.

[0117] (4) Conduct biochemical and blood routine tests on liver and kidney function.

[0118] (5) Preparation of tissue sections: fixation (4% paraformaldehyde, 24h), trim tissue blocks, fix again (4% paraformaldehyde, 4h), rinse with water for 12h, and then dehydrate: put the tissue blocks into different concentrations of alcohol in sequence, first 70%, 12h, then 80%, 2h, then 90%, 2h, then 95% I, 40min, then 95% II, 30min, then 100% I, 30min, then 100% II, 30min, then transparent xylene I, 15min, then xylene II, 10min. Then wax immersion (paraffin I, 2h, then paraffin II, 1h), embedding, sectioning (5μm), drying and storage.

[0119] (6) HE staining: bake the slides (60°C, 30 min), then dewax: xylene I (5 min), xylene II (5 min), then hydrate: immerse the slides in alcohol gradient, 100% alcohol I, 5 min, then 100% alcohol II, 5 min, then 90% alcohol, 5 min, then 80% alcohol, 5 min, then hematoxylin, 10 min, then rinse with water, 1 s, then 1% hydrochloric acid alcohol, 5 s, then 45°C water bath, 3 min, then eosin, 1 min, then 80% alcohol, 5 min, then 90% alcohol, 5 min, then 100% alcohol, 5 min. Seal the slides with neutral resin. After the slides are dried, observe the pathological changes of the tissue under a microscope.

[0120] (7) TRAP staining: Prepare the fixative and staining solutions, preheat them in a 37°C water bath, and prepare the hydrated slides. After adding the fixative for 1 min, pour out the fixative, wash three times with PBS, add the staining solution, incubate at 37°C for 1 h, pour out the staining solution, wash with PBS, and then observe and photograph under a microscope.

[0121] (8) Immunofluorescence staining: bake the slides (60°C, 30 min), then dewax in xylene I (5 min), xylene II (5 min), hydration: immerse the sections in alcohol gradient 100% alcohol I for 5 min, then 100% alcohol II for 5 min, then 90% alcohol for 5 min, then 80% alcohol for 5 min, then 70% alcohol for 5 min, then DDW for 3 min, then PBS for 3 min, circle the target tissue with a histochemical pen, cover the tissue with pepsin antigen retrieval solution, 37°C for 20 min, then PBS for 3 min, blocking solution (1 g BSA dissolved in 100 mL PBS containing 0.5% TritonX-100), room temperature for 1 h, then primary antibody (4°C, 12 h), PBS rinse (5 min / time, 3 times), fluorescent secondary antibody (37°C, 1 h), light-protected PBS rinse (5 min / time, 3 times), dry, seal the slides with anti-fluorescence quencher, and observe under a confocal microscope.

[0122] (9) ELISA: The OD value of each well was measured at a wavelength of 450 nm, and the obtained OD value was used to obtain the corresponding sample concentration value.

[0123] (10) Micro-CT: Examination of femurs fixed in 4% paraformaldehyde.

[0124] Bone-targeted testing of ADSC-SNNP drugs:

[0125] Animal experiment results show that Fig.10 ), in the ADSC-SNNP group, the drug was mainly concentrated in the hind limb area, indicating that it has a strong targeting ability. And compared with the simple ADSC group, the targeting ability of ADSC-SNNP was significantly improved. Fig.10 A is the fluorescence intensity diagram of the main organs and hind limbs observed by the small animal living imaging instrument. Fig.10 B is the statistical diagram of fluorescence intensity of major organs and hind limbs.

[0126] Since the stem cell-nanoparticle drug was injected into the osteoporosis model rats via tail vein injection, a hemolytic test was performed before treatment, e.g. Fig.17 As shown in A, compared with the Water group, different concentrations of the drug had no lytic effect on red blood cells, indicating that the drug had preliminary biosafety. Subsequently, during the 8-week treatment, each osteoporosis model rat was weighed every week before drug administration. Fig.17 As shown in B, the weight data are statistically plotted as a line graph to clearly show the average weight change trend of rats in each group during the treatment period. The results in the graph show that the drug does not cause a sudden increase or decrease in the weight of osteoporosis model rats, indicating that it has a certain degree of biosafety.

[0127] like Fig.18 As shown in A, after the treatment, the rats were killed and serum was taken for blood biochemical testing. The results showed that the average values ​​of biochemical indicators of liver and kidney function of rats in each group were within the normal range. It can be seen that the stem cell-nanoparticle drug has no significant harm to liver and kidney function and has a certain degree of biosafety.

[0128] Fig.19((A) The indicators in the figure are WBC (white blood cell count), Neu (neutrophil count), Lym (lymphocyte count), Mon (monocyte count), and RBC (red blood cell count); (B) The indicators in the figure are PLT (platelet count), Eos (eosinophil count), MCV (mean corpuscular volume), and HCT (hematocrit); (C) The indicators in the figure are MPV (mean platelet volume), PCT (hematocrit), PDW (platelet volume distribution width), MCH (mean corpuscular hemoglobin content), and RDW-CV (red blood cell volume distribution width-coefficient of variation ratio)) The results showed that the average values ​​of various blood routine indicators of rats in each group were within the normal range, indicating that the stem cell-nanoparticle drug has good biosafety.

[0129] like Fig. 20 As shown in the figure, the HE results of the heart, liver, spleen, lung, kidney and other organs of each group of rats showed that compared with the sham operation group, surgery and stem cell-nanoparticle drug treatment did not cause pathological changes in various organs, indicating that the drug has good biosafety.

[0130] HE staining of femoral sections to observe the therapeutic effect of ADSC-SNNP:

[0131] HE staining results ( Fig.11 ) showed that compared with the osteoporotic saline group, the ADSC-SNNP group showed a very significant recovery effect in the number and area of ​​trabeculae. In addition, compared with the SN group, SNNP group and ADSC group, the ADSC-SNNP group showed a larger number of trabeculae and a larger trabecular area, further confirming its superiority in promoting bone formation.

[0132] Micro-CT of the distal femur of rats to detect the therapeutic effect of ADSC-SNNP:

[0133] Micro-CT scanning was performed on the distal femur of osteoporosis model rats in each group.

[0134] Through the three-dimensional reconstruction of the femur, a section is selected from the z-axis for observation, such as Fig.12 The number and area of ​​trabeculae in the cancellous bone zone of the ADSC-SNNP group shown in A were significantly restored. Compared with the healthy bone state of the femur of the sham-operated rats, the effect of the ADSC-SNNP group was closest, while other groups, such as the SN group, SNNP group, and ADSC group, did not show such a significant therapeutic effect. Fig.12 B shows a 2D cross-section of the distal femur, where the visual results of trabecular number and area are consistent with the findings in A. Together, these results suggest that the ADSC-SNNP drug has excellent performance in promoting bone remodeling in osteoporotic conditions.

[0135] ELISA detection of osteogenic related cytokines in serum:

[0136] Bmp2, RUNX2 and ALP play a synergistic role in bone formation. As a powerful bone growth factor, Bmp2 promotes osteoblast differentiation and proliferation, laying the foundation for bone formation. RUNX2, as a key transcription factor, regulates the expression of osteoblast differentiation-related genes to ensure the normal progress of bone formation. ALP participates in the bone mineralization process by providing necessary phosphate ions and regulating the concentration of inorganic phosphorus in the extracellular matrix, ensuring the hardness and stability of bones. The three work together to promote healthy bone formation. ELISA serum test results show ( Fig.13 )ADSC-SNNP group Bmp2( Fig.13 A) RUNX2 Fig.13 B) and ALP( Fig.13 C) The expression level was significantly upregulated. This indicates that ADSC-SNNP can significantly upregulate the expression of factors related to bone formation and promote bone repair in the treatment of osteoporosis.

[0137] ELISA detection of bone resorption related cytokines in serum:

[0138] like Fig.14 A and B first detected the two most classic cytokines of the OPG / RANKL axis in serum. It can be seen that after treatment in the ADSC-SNNP group, OPG had the most significant upward trend, while RANKL had an obvious downward trend. The ratio of the two can well reflect the degree of activation of bone resorption. From Figure C, it can be seen that the OPG / RANKL value of the ADSC-SNNP group increased significantly, indicating that the combined treatment of stem cell-nanoparticle drugs significantly reduced bone resorption. Fig.14 D detected the TRAP content in serum. TRAP is a good marker for bone absorption and osteoclast activity. The results in the figure show that the treatment of the ADSC-SNNP group effectively reduced the TRAP content in serum, which indirectly reflected the reduction of osteoclast activity and the inhibition of bone absorption.

[0139] Immunofluorescence technique to detect osteoblast-related proteins:

[0140] OCN in bones was specifically stained by immunofluorescence technique, e.g. Fig.15 As shown in A, it can be seen that the protein positive area and region of the ADSC-SNNP group were significantly higher than those of the normal saline group, SN group, SNNP group, and ADSC group, and were closest to the sham operation group. Therefore, the stem cell-nanoparticle drug has a significant promoting effect on the mineralization of osteoblasts.

[0141] Immunofluorescence detection of osteoclast-related proteins:

[0142] Tartrate resistant acid phosphatase (TRAP), during bone resorption, osteoclasts attach to the surface of the bone, and the secreted acid and protease form a gap between the bone matrix and the osteoclasts. Phosphatanhydrase and H+-ATPase proton pumps form an acidic environment in this gap. TRAP located in the osteoclast microsomes is secreted into this gap through the wavy edge of the osteoclasts, and participates in the degradation of solid calcium phosphate mineralization in the bone matrix together with other enzymes. Therefore, the main positive sites of TRAP protein are concentrated on the surface of the bones. Fig.16 It can be seen that the ADSC-SNNP group has a smaller total positive area than the ADSC group and the SNNP group. At the same time, the number and area of ​​positive proteins are closest to those of the sham operation group, indicating that ADSC-SNNP has a good inhibitory effect on osteoclast activity.

[0143] The present invention first uses PLGA to coat NO donor (PLGA-SN) to extend the half-life of NO donor. Secondly, extract human umbilical vein endothelial cell membrane, use it to coat PLGA-SN, form SNNP nanoparticles, improve the targeting ability of the drug, and couple it to ADSC through bioorthogonal click chemistry to form ADSC-SNNP nanocell drugs with osteoporosis targeting and sustained release. Then it is co-induced and cultured with OC precursor cells to clarify its effects on OC differentiation and bone resorption. At the same time, it is co-cultured with osteoblast-like cells MC3T3-E1 to clarify its effects on osteoblast differentiation and bone formation. Then ADSC-SNNP is injected into rats with osteoporosis model, and its therapeutic effect and biosafety on osteoporosis are detected, and the molecular mechanism of its action is elucidated by detecting key protein pathways.

[0144] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A nanocellular drug with targeted sustained-release function, characterized in that: including, using a nitric oxide-spermine complex as a nitric oxide donor; The nitric oxide donor was coated with a poly(lactic acid-glycolic acid) copolymer nanocarrier to form a donor nanodrug, which was then coated with a human umbilical vein endothelial cell membrane that highly expressed CXCR4. Finally, the nanodrug was coupled to adipose mesenchymal stem cells through bio-click chemistry to obtain a nanocellular drug.

2. The nanocellular drug according to claim 1, characterized in that: The drug loading of the donor nanomedicine is 9.10-10.62%.

3. The nanocellular drug according to claim 1, characterized in that: The encapsulation efficiency of the donor nanomedicine is 59.8% to 69.8%.

4. The nanocellular drug according to claim 1, characterized in that: The NO release rate of the donor nanomedicine at pH 4.5 is 82.56-86.30%.

5. The nanocellular drug according to claim 1, characterized in that: The donor nano drug has a spherical particle size of 200-300 nm.

6. The nanocellular drug according to claim 2, characterized in that: The drug loading capacity of a single cell of the nanocellular drug is 0.0875-0.09 ng / cell.

7. Use of the nanocellular medicine according to claims 1 to 6 in the preparation of a medicine for the treatment of osteoporosis.

8. The use according to claim 7, characterized in that: After being injected into the body, the nanocell drug mainly accumulates in the bone lesion site, and the bone lesion site highly expresses SDF-1.

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