Osteogenesis-like cell membrane coated nanoparticles for targeted delivery of curcumin as well as preparation method and application of osteogenesis-like cell membrane coated nanoparticles

Abstract

The invention relates to the technical field of biology, and discloses osteoblast-like cell membrane coated nanoparticles for targeted delivery of curcumin as well as a preparation method and application of the osteoblast-like cell membrane coated nanoparticles. The osteoblast-like cell membrane coated nanoparticles are prepared from curcumin, a polylactic acid-glycolic acid copolymer and an MC3T3-E1 cell membrane, wherein the polylactic acid-glycolic acid copolymer wraps the curcumin to form nano-particles, and the MC3T3-E1 cell membrane wraps the nano-particles to form the osteoblast-like cell membrane coated nano-particles. The osteoblast-like cell membrane coated nanoparticles have bone tissue targeting property, can improve the stability and bioavailability of curcumin while reducing side effects, can synchronously inhibit bone resorption and promote bone formation, and provides a new strategy for effective treatment of osteoporosis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to osteoblast-like cell membrane-coated nanoparticles for targeted delivery of curcumin, as well as a preparation method and application thereof. Background Art

[0002] Osteoporosis is a systemic metabolic bone disease characterized by decreased bone mass and deterioration of bone microarchitecture. In severe cases, it can significantly increase the risk of hip and vertebral fractures. Currently, over 200 million people worldwide suffer from osteoporosis, posing a serious health threat to the elderly. Postmenopausal osteoporosis (PMOP) is the most common disease, caused by estrogen deficiency. Approximately 50% of women experience at least one fracture after menopause. The World Health Organization (WHO) has listed PMOP as one of the three major health threats to the elderly, alongside diabetes and cardiovascular disease. Therefore, finding effective prevention and treatment methods has become a pressing medical research priority.

[0003] The pathogenesis of osteoporosis is complex, and estrogen deficiency is one of the main causes. It not only affects the balance of bone metabolism, but also plays an important role in the occurrence and development of the disease by activating the inflammatory response. Studies have shown that estrogen deficiency can activate monocytes and lymphocytes, and increase the levels of inflammatory factors such as TNF-α and IL-6. As a multifunctional cytokine, TNF-α not only participates in the inflammatory immune response, but also regulates bone metabolism. Excessive TNF-α plays a key role in the development of osteoporosis by promoting bone resorption and inhibiting bone formation. It can enhance RANKL-induced osteoclast formation through multiple pathways, while inducing osteoblast apoptosis, downregulating RUNX2 and OSX transcription, impairing osteoblast differentiation and inhibiting mineralization. Therefore, TNF-α is not only a proinflammatory factor of the osteoporotic inflammatory response, but also a key driver of bone metabolism imbalance.

[0004] Curcumin, a natural polyphenolic compound, has been widely used in the treatment of osteoporosis due to its anti-inflammatory, antioxidant, and osteogenic properties. However, its poor water solubility, chemical stability, and photostability limit its application. Polymer nanoparticles are often used as drug carriers due to their good biocompatibility. PLGA nanoparticles, in particular, can enhance curcumin's bioavailability and achieve sustained drug release by encapsulating it. However, in the pathological microenvironment of osteoporosis, an ideal drug carrier must not only achieve sustained drug release through controlled degradation but also possess active targeting capabilities to achieve selective accumulation at bone lesions, thereby enhancing therapeutic precision.

[0005] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0006] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide osteoblast-like cell membrane-coated nanoparticles for targeted delivery of curcumin and a preparation method and application thereof, aiming to solve the problem that existing curcumin drug carriers lack the ability to actively target bone lesions.

[0007] The technical solutions of the present invention are as follows:

[0008] In a first aspect, an osteoblast-like cell membrane-coated nanoparticle for targeted delivery of curcumin is provided, comprising: curcumin, poly(lactic-co-glycolic acid) copolymer, and MC3T3-E1 cell membrane;

[0009] The polylactic acid-glycolic acid copolymer wraps the curcumin to form nanoparticles, and the MC3T3-E1 cell membrane wraps the nanoparticles to form osteoblast-like cell membrane-coated nanoparticles.

[0010] In a preferred technical solution, the mass ratio of the membrane protein on the MC3T3-E1 cell membrane, the poly(lactic-co-glycolic acid) copolymer, and curcumin is (5-15):(5-15):1.

[0011] In a preferred technical solution, the polylactic acid-glycolic acid copolymer and / or the MC3T3-E1 cell membrane are modified with a fluorescent probe.

[0012] In a further preferred technical solution, the fluorescent probe is selected from one of DiO, DiD, DiI, and DiR.

[0013] In a second aspect, a method for preparing osteoblast-like cell membrane-coated nanoparticles as described in the first aspect is provided, comprising the steps of:

[0014] dissolving curcumin and poly(lactic acid-co-glycolic acid) in an organic solvent to obtain a mixed solution;

[0015] adding water dropwise into the mixed solution, controlling the volume ratio of the water to the mixed solution to be 1:(2-10), to obtain nanoparticles;

[0016] The nanoparticles and MC3T3-E1 cell membranes are mixed, ultrasonicated, and extruded through a polycarbonate membrane with a pore size of 100-300 nm to obtain osteoblast-like cell membrane-coated nanoparticles.

[0017] According to a preferred technical solution, in the mixed solution, the concentration of curcumin is 0.5-2 mg / mL, and the concentration of the polylactic acid-glycolic acid copolymer is 5-15 mg / mL.

[0018] In a preferred technical solution, the organic solvent is selected from one or more of dimethyl sulfoxide, ethyl acetate, methanol, and dichloromethane.

[0019] In a preferred technical solution, the specific conditions of the ultrasound include: a frequency of 30-50 kHz, a power of 50-200 W, and a time of 1-10 min.

[0020] In a preferred technical solution, the step of extruding through a polycarbonate membrane with a pore size of 100-300 nm is repeated at least three times.

[0021] In a third aspect, a use of the osteoblast-like cell membrane-coated nanoparticles described in the first aspect in the preparation of a drug for treating osteoporosis is provided.

[0022] Beneficial Effects: The present invention provides osteoblast-like cell membrane-coated nanoparticles for targeted delivery of curcumin. Compared with traditional treatments, the osteoblast-like cell membrane-coated nanoparticles of the present invention are bone-tissue targeted, offering high delivery efficiency, excellent therapeutic efficacy, and low toxic side effects. They not only achieve targeted delivery of curcumin to the osteoporotic bone microenvironment but also help neutralize the inflammatory factor TNF-α, thereby reducing its harmful effects on bone metabolism. Therefore, the osteoblast-like cell membrane-coated nanoparticles of the present invention can be used as a curcumin delivery vehicle, providing a new strategy for the effective treatment of osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The present invention is a schematic diagram of the preparation process of osteoblast-like cell membrane-coated nanoparticles and the mechanism of action of the targeted delivery of curcumin for the treatment of postmenopausal osteoporosis.

[0024] Figure 2 : are related results of the preparation and characterization of osteoblast-like cell membrane-coated nanoparticles in the examples; wherein, A is a Venn diagram of the overlap between PMOP and bone-related cell membrane genes; B is a typical transmission electron microscopy (TEM) image of CNPs and OCNPs; C and D are the particle size and Zeta potential of OM, CNPs and OCNPs (n=3); E is a confocal microscopy (CLSM) image of OCNPs in MC3T3-E1 cells, showing the co-localization of cell nuclei (blue), OM (green) and PLGA (red), scale bar = 30 μm; F is a Western blot analysis of CXCR4 and TNFR on the surface of OM, OCNPs and CNPs; G is the particle size change of CNPs and OCNPs in H2O within 7 days (n=3); H is the in vitro curcumin release curve of CNPs and OCNPs (n=3).

[0025] Figure 3 : are the results of the in vitro biosafety assessment and cellular uptake study of osteoblast-like cell membrane-coated nanoparticles in the embodiment; wherein, A and B are the cell activities of MC3T3-E1 and RAW264.7 cells after incubation with different concentrations of NPs and ONPs for 24 hours (n=3); C is the image of the hemolysis test results of CNPs and OCNPs; D is the absorbance of blood samples after incubation with CNPs and OCNPs at a wavelength of 540 nm (n=3); E is the serum protein adsorption analysis after incubation with CNPs and OCNPs for 0.5, 1, and 2 hours (n=3); F and G are the results of the hemolysis test of RAW264.7 cells andDiD NPs and O DiD Confocal microscopy images (scale bar = 30 μm) of NPs incubated for 4 hours, and flow cytometry analysis results; H and I are the results of MC3T3-E1 cells pretreated with TNF-α. DiD NPs and O DiD Confocal images of NPs uptake (scale bar = 20 μm) and flow cytometry analysis results; J is the proportion of MC3T3-E1 cells that did not uptake DiD in the TNF-α pretreatment group (n = 3).

[0026] Figure 4 : are related result graphs of the TNF-α neutralization effect of osteoblast-like cell membrane-coated nanoparticles in the embodiment; wherein, A is a schematic diagram of the mechanism of action of ONPs in neutralizing TNF-α; B is the concentration determination of TNF-α after treatment with NPs, RNPs, OM and ONPs at an initial concentration of 500 pg / L (n=3); C is the residual TNF-α level after treatment with different concentrations of ONPs (n=3); D and E are cell viability and apoptosis of MC3T3-E1 cells after treatment with PBS, TNF-α / NPs and TNF-α / ONPs (scale bar=50 μm, n=3); F and G are ALP staining and quantitative analysis of MC3T3-E1 cells after 7 days of differentiation (scale bar=200 μm, n=3); H and I are ARS staining and quantitative analysis of MC3T3-E1 cells after 14 days of differentiation (scale bar=200 μm, n=3).

[0027] Figure 5 : are the results of the blood circulation and targeting ability of DiD-labeled ONPs in osteoporosis (OVX) mice in the examples; wherein, A and B are the fluorescence images and quantitative analysis of DiD-labeled ONPs in whole blood collected at different time points after intravenous injection, n = 3; C and D are the biodistribution of NPs and ONPs in OVX mice; E is a confocal laser scanning microscopy (CLSM) image of accumulated NPs and ONPs in bone tissue sections, scale bar = 30 μm; F is the quantitative analysis of fluorescence intensity in femur and tibia, n = 3.

[0028] Figure 6 Figures 3 and 4 are related results of the anti-osteoporosis effect of OCNPs on OVX mice in the examples; wherein, A is a schematic diagram of the mouse model; B is a representative micro-CT image of the proximal femur; CF is a quantitative analysis of bone mineral density (BMD), trabecular volume fraction (Th.BV / TV), trabecular number (Tb.N) and trabecular separation (Th.Sp) in the sham group, saline group, curcumin group, CNPs group and OCNPs group, n=5.

[0029] Figure 7: These are the relevant result graphs of OCNPs treatment promoting bone formation and inhibiting bone resorption in the embodiment; wherein, A is the quantitative analysis of TNF-α concentration in serum, n=5; B and C are the quantitative analysis of serum TRAP-5b and OCN concentrations, n=5; D is a representative image of H&E staining, scale bar=200 μm; E is a representative image of TRAP staining, osteoclasts are stained red, scale bar=50 μm; F is a representative image of BMP-2 immunofluorescence staining, BMP-2 is labeled in red, cell nuclei are labeled in blue, scale bar=20 μm; G is the quantitative analysis of the number of osteoclasts shown by TRAP staining, n=5; H is the quantitative analysis of the number of BMP-2 positive cells, n=5.

[0030] Figure 8 : are relevant result graphs of the in vivo biocompatibility evaluation of osteoblast-like cell membrane-coated nanoparticles in the examples; wherein, AD are the hematological parameters of mice in the sham operation group, normal saline group, curcumin group, CNPs group, and OCNPs group; EH are the test results of biochemical markers related to liver and kidney function (n=5); I is the H&E staining image of the main organs of osteoporosis mice with different treatment regimens, scale bar = 100 μm. DETAILED DESCRIPTION

[0031] The present invention provides osteoblast-like cell membrane-coated nanoparticles for targeted delivery of curcumin, as well as a preparation method and application thereof. To make the objectives, technical solutions and effects of the present invention clearer and more specific, the present invention is further described in detail below.

[0032] Cell membrane-coated nanoparticles have shown significant advantages in nanomedicine: the cell membrane coating provides "camouflage" for the nanoparticles, avoiding clearance by the mononuclear phagocytic system, prolonging circulation time, reducing immunogenicity and maximizing biocompatibility; specific cell membrane-coated nanoparticles have targeting capabilities, and achieve specific tissue accumulation with the help of targeting molecules on the membrane surface, such as macrophage membrane-coated nanoparticles targeting atherosclerotic plaques through integrin α4 / β1, and platelet membrane-coated nanoparticles can cross the blood-brain barrier to target damaged cerebral blood vessels; in addition to targeting, some membrane-coated nanoparticles can also specifically bind to and neutralize pathological molecules such as cytokines and toxins through membrane surface receptors, such as lipopolysaccharide-treated macrophage membrane-coated nanoparticles neutralizing pro-inflammatory factors through specific receptor affinity. In recent years, cell membrane-coated nanoparticles have demonstrated promising results in the treatment of osteoporosis. Studies have demonstrated that membrane-coated nanoparticles derived from microvascular endothelial cells overexpressing the CXCR4 receptor can be delivered to osteoporotic sites in response to a gradient of SDF-1 concentrations. Other researchers have developed membrane-coated nanoparticles that can scavenge RANKL and TNF-α, achieving the dual effects of inhibiting bone resorption and promoting bone formation. Therefore, optimizing screening strategies for cell membrane-coated nanoparticles in osteoporosis treatment to achieve a synergistic effect of targeted drug delivery and cytokine neutralization will provide new insights into improving efficacy.

[0033] Based on this, an embodiment of the present invention provides an osteoblast-like cell membrane-coated nanoparticle, comprising: curcumin, poly(lactic-co-glycolic acid) copolymer, and MC3T3-E1 cell membrane;

[0034] The polylactic acid-glycolic acid copolymer wraps the curcumin to form nanoparticles, and the MC3T3-E1 cell membrane wraps the nanoparticles to form osteoblast-like cell membrane-coated nanoparticles.

[0035] Specifically, the preparation process of the osteoblast-like cell membrane-coated nanoparticles and the mechanism of action of the targeted delivery of curcumin for the treatment of postmenopausal osteoporosis are as follows: Figure 1 As shown. The present invention screened and identified optimized bone-related cell membranes and developed osteoblast-like cell (MC3T3-E1) membrane-coated nanoparticles as a new carrier for the treatment of PMOP. Previous studies have found that MC3T3-E1 cells express the chemokine receptor CXCR4 and can respond to the key chemokine SDF-1 for bone remodeling. This characteristic helps to target the delivery of curcumin to osteoporotic sites where SDF-1 is highly expressed; at the same time, the MC3T3-E1 cell membrane expresses TNF receptors (TNFRs), which can specifically bind to TNF-α and reduce the level of free TNF-α in vivo and in vitro. Therefore, osteoblast-like cell membrane-coated nanoparticles can not only achieve targeted delivery of curcumin to the osteoporotic bone microenvironment, but also help to neutralize the inflammatory factor TNF-α to reduce its harm to bone metabolism. By targeting osteoporotic sites and alleviating the effects of oxidative stress and inflammation on bone metabolism, osteoblast-like cell membrane-coated nanoparticles as curcumin delivery carriers provide a new strategy for the effective treatment of osteoporosis.

[0036] In one embodiment, the mass ratio of the membrane protein, poly(lactic-co-glycolic acid) and curcumin on the MC3T3-E1 cell membrane is (5-15):(5-15):1.

[0037] In one embodiment, the poly(lactic-co-glycolic acid) and / or the MC3T3-E1 cell membrane are modified with a fluorescent probe.

[0038] In a more specific embodiment, the fluorescent probe is selected from one of DiO, DiD, DiI, and DiR.

[0039] In one embodiment, the molecular weight of the poly(lactic acid-co-glycolic acid) is 50,000-150,000.

[0040] The present invention provides a method for preparing osteoblast-like cell membrane-coated nanoparticles as described above, comprising the steps of:

[0041] dissolving curcumin and poly(lactic acid-co-glycolic acid) in an organic solvent to obtain a mixed solution;

[0042] adding water dropwise into the mixed solution, controlling the volume ratio of the water to the mixed solution to be 1:(2-10), to obtain nanoparticles;

[0043] The nanoparticles and MC3T3-E1 cell membranes are mixed, ultrasonicated, and extruded through a polycarbonate membrane with a pore size of 100-300 nm to obtain osteoblast-like cell membrane-coated nanoparticles.

[0044] In one embodiment, the volume ratio of water to the mixed solution is 1:(2-6); preferably, the volume ratio of water to the mixed solution is 1:4.

[0045] In one embodiment, in the mixed solution, the concentration of curcumin is 0.5-2 mg / mL, and the concentration of poly(lactic-co-glycolic acid) is 5-15 mg / mL.

[0046] In one embodiment, the organic solvent is selected from one or more of dimethyl sulfoxide, ethyl acetate, methanol, and dichloromethane.

[0047] In one embodiment, the specific conditions of the ultrasound include: a frequency of 30-50 kHz, a power of 50-200 W, and a time of 1-10 min.

[0048] In one embodiment, the step of extruding through a polycarbonate membrane with a pore size of 100-300 nm is repeated at least three times; preferably, the step of extruding through a polycarbonate membrane with a pore size of 100-300 nm is repeated ten times.

[0049] In a specific embodiment, the preparation method comprises the steps of:

[0050] dissolving curcumin and poly(lactic acid-co-glycolic acid) in an organic solvent to obtain a mixed solution;

[0051] adding water dropwise into the mixed solution, controlling the volume ratio of the water to the mixed solution to be 1:(2-10), to obtain a solution containing nanoparticles;

[0052] dialyzing the solution containing the nanoparticles to remove the curcumin and the organic solvent to obtain the nanoparticles;

[0053] The nanoparticles and MC3T3-E1 cell membranes are mixed, ultrasonicated, and extruded through a polycarbonate membrane with a pore size of 100-300 nm to obtain osteoblast-like cell membrane-coated nanoparticles.

[0054] In one embodiment, the method for preparing the MC3T3-E1 cell membrane comprises the steps of:

[0055] MC3T3-E1 cells were cultured in MEMα medium containing fetal bovine serum and penicillin-streptomycin to a cell density of 85-95%. The medium was removed, and the cells were washed with phosphate buffered saline. The cells were trypsinized, and the cell suspension was collected and centrifuged to obtain a cell pellet.

[0056] adding a hypotonic lysis solution containing phenylmethylsulfonyl fluoride to the cell pellet, performing hypotonic lysis and disruption, and collecting the supernatant by first centrifugation;

[0057] The supernatant was then centrifuged a second time to collect the precipitate, thereby obtaining the MC3T3-E1 cell membrane.

[0058] In a more specific embodiment, in the MEMα culture medium containing fetal bovine serum and penicillin-streptomycin, the volume percentage of the fetal bovine serum is 10%, and the volume percentage of the penicillin-streptomycin is 1%.

[0059] In a more specific embodiment, the concentration of phenylmethylsulfonyl fluoride in the hypotonic lysis solution is 1-3 mM.

[0060] In a more specific embodiment, the specific conditions of the first centrifugation are: rotation speed 1000-2000 rpm, time 5-15 min.

[0061] In a more specific embodiment, the specific conditions of the second centrifugation are: rotation speed 10000-20000 rpm, time 20-40 min.

[0062] In a more specific embodiment, said disruption is achieved by repeated freezing and thawing.

[0063] An embodiment of the present invention provides a use of the osteoblast-like cell membrane-coated nanoparticles described above in the preparation of a drug for treating osteoporosis.

[0064] The present invention will be further described below with reference to specific examples.

[0065] Example

[0066] 1. Materials and Methods

[0067] 1. Materials

[0068] Curcumin was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; poly(lactic-co-glycolic acid) (PLGA) was purchased from Dalian Meilun Biotechnology Co., Ltd.; DiD, DiO, BCIP / NBT alkaline phosphatase detection kits, and TUNEL cell apoptosis detection kits were purchased from Beyotime Biotechnology Co., Ltd.; BCA protein quantification kit was purchased from Thermo Fisher Scientific; CCK-8 kit was purchased from Dojindo Chemical Research Institute, Japan; mouse TNF-α recombinant protein was purchased from PeproTech; all ELISA kits were purchased from Jiangsu Enzyme Immunoassay Co., Ltd.; TNFR antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd.; CXCR4 antibody was purchased from Santa Cruz Biotechnology, USA; osteogenic differentiation induction kit and MC3T3-E1 cells were purchased from Wuhan Punosai Life Science Co., Ltd.

[0069] 2. Cell membrane screening

[0070] PMOP-related genes and membrane protein genes of bone-related cells (BMSCs, osteoblasts, osteoclasts, and osteocytes) were retrieved from the GeneCards database (https: / / www.genecards.org / ). Venn diagrams of PMOP and membrane protein genes were constructed using the Xiantao Academic Platform (https: / / www.xiantao.love / products).

[0071] 3. Osteoblastic membrane (OM) extraction

[0072] MC3T3-E1 cells were seeded in 10 cm culture dishes and cultured to a cell density of 90% in MEMα medium (Gibco, Catalog No. C12571500BT) supplemented with 10% (v / v) fetal bovine serum (Gibco, Catalog No. 10099-141) and 1% (v / v) anti-antibody (Gibco, Catalog No. 15140163). The medium was aspirated, and the cells were washed three times with phosphate-buffered saline (PBS). The cells were then digested with 1 ml of 0.25% (w / v) trypsin and harvested to obtain a cell suspension. The cell suspension was placed in hypotonic lysis buffer (Biyuntian, Catalog No. P0033) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and subjected to 4-5 freeze-thaw cycles. The cells were centrifuged at 1500 rpm for 10 min, and the supernatant was purified by centrifugation at 14000 rpm for 30 min (4°C) to obtain MC3T3-E1 cell membranes, i.e., OM vesicles. Membrane protein concentration was determined by the BCA assay.

[0073] 4. Preparation of osteoblast-like cell membrane-coated nanoparticles

[0074] Curcumin-loaded PLGA nanoparticles (CNPs) were prepared by a modified nanoprecipitation method: 1 mg of curcumin was mixed with 10 mg of PLGA

[0075] (molecular weight 90000) was dissolved in 1 mL DMSO, 4 mL deionized water was slowly added dropwise, and dialyzed (molecular weight cut-off (MWCO)

[0076] 3.5KDa) to remove free curcumin and DMSO, and obtain CNPs, which were stored at 4°C. To facilitate evaluation and fluorescence tracing, the same method was used to prepare PLGA nanoparticles (NPs) without curcumin encapsulation and DiD-labeled PLGA nanoparticles ( DiD NPs).

[0077] Curcumin-loaded osteoblast-like membrane-coated nanoparticles, i.e., OM-coated CNPs (OCNPs), were prepared by direct extrusion: OM vesicles and CNPs were mixed at a ratio of OM vesicle membrane protein:CNPs = 1:1 (w / w), sonicated for 3 min (FS30D, 42 kHz, 100 W), and extruded 10 times through a 200 nm polycarbonate membrane to obtain OCNPs. For ease of evaluation and fluorescence tracing, uncurcumin-loaded osteoblast-like membrane-coated nanoparticles (ONPs) were prepared from OM and NPs using the same method. OM and CNPs were labeled with DiO. DiD NPs were used to prepare dual fluorescent labeled osteoblast-like cell membrane-coated nanoparticles (O DiD NPs).

[0078] 5. Nanoparticle Characterization

[0079] The hydrodynamic diameter and zeta potential of CNPs, OM vesicles, and OCNPs were measured using a zeta potential analyzer. A 1 mg / mL sample was dripped onto a copper grid and negatively stained with 1% (w / v) phosphotungstic acid. The morphology was observed using a transmission electron microscope (JEM-1400). The samples were stored at 37°C, and particle size changes were measured at 1, 3, 5, and 7 days to assess stability.

[0080] 6. Membrane protein detection

[0081] The membrane protein composition of OM vesicles and OCNPs was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): 10% (w / v) polyacrylamide gel electrophoresis (75V, 0.5h→140V, 1h), followed by Coomassie Brilliant Blue staining for band visualization. CXCR4 and TNFR expression was detected by Western blot: Proteins were separated by 10% (w / v) SDS-PAGE, transferred to membranes, and incubated with primary antibodies against CXCR4 (1:1000) and TNFR (1:25000) followed by HRP-conjugated secondary antibodies, followed by chemiluminescence visualization.

[0082] 7. Drug loading and in vitro release

[0083] A standard curve of curcumin (0.5-32 μg / mL) was established. After freeze-drying the CNPs, they were dissolved in DMSO and the drug loading (LE) and encapsulation efficiency (EE) were measured using a UV spectrophotometer.

[0084] LE (%) = (mass of curcumin in nanoparticles / total mass of nanoparticles) × 100%;

[0085] EE (%) = (mass of curcumin in nanoparticles / dosage) × 100%;

[0086] The in vitro release was investigated by dialysis method: a dialysis bag containing 1 mL of CNPs / OCNPs solution was placed in PBS containing 0.3% (w / v) Tween 80 (oscillation at 37°C), and samples were taken at regular intervals to determine the cumulative release amount.

[0087] 8. Colocalization studies

[0088] Confocal laser scanning microscopy (CLSM) was used to directly observe the fluorescence distribution of the samples. DiD NPs were co-incubated for 4 h, fixed with 4% (w / v) paraformaldehyde, and cellular uptake was observed by CLSM after DAPI nuclear staining.

[0089] 9. Cytotoxicity Assessment

[0090] The survival rates of MC3T3-E1 and RAW264.7 cells were assessed by CCK-8 assay after treatment with different concentrations of NPs / ONPs (10-200 μg / mL) for 24 hours. Cell viability was observed under a fluorescence microscope after treatment with a live-dead staining kit (Calcein-AM / PI) for 24 hours.

[0091] 10. Blood compatibility

[0092] Hemolysis assay: 1 mg / mL CNPs / OCNPs were incubated with diluted whole blood (1:1.25) for 1 hour, followed by centrifugation and measurement of absorbance at 540 nm. Serum protein adsorption assay: CNPs / OCNPs were incubated with mouse serum (0.5-2 hours), followed by centrifugation and determination of residual protein in the supernatant using the BCA assay.

[0093] 11. Cellular Uptake

[0094] CLSM and flow cytometry were used to evaluate the effect of RAW264.7 cells on DiD NPs / O DiD Phagocytosis of NPs. MC3T3-E1 cells were pretreated with TNF-α (50 ng / mL) for 24 h to induce inflammation, and flow cytometry analysis was performed. DiD NPs / O DiD NPs uptake efficiency.

[0095] 12. TNF-α scavenging ability

[0096] The residual TNF-α concentration of NPs, erythrocyte membrane-coated nanoparticles (RNPs), OM vesicles, and ONPs after incubation with TNF-α (500 pg / mL) for 2 h was measured by ELISA. The clearance effect of different concentrations of ONPs (1-2 mg / mL) was also investigated.

[0097] 13. Promote bone function

[0098] CCK-8 and TUNEL assays were used to assess the rescue effects of ONPs on TNF-α (50 ng / mL)-induced proliferation inhibition and apoptosis in MC3T3-E1 cells. Alkaline phosphatase (ALP) staining and quantitative analysis were used to assess the osteogenic differentiation effects of ONPs in the presence of TNF-α (10 ng / mL). Alizarin red staining was used to quantitatively analyze the improvement in mineralized nodule formation by ONPs.

[0099] 14. In vivo pharmacokinetics

[0100] Tail vein injection DiD NPs / O DiD NPs (2 mg / mL), tail vein blood was collected at 1-48 h, and the fluorescence intensity was quantified by IVIS system after EDTA-K2 anticoagulation to analyze the pharmacokinetic characteristics.

[0101] 15. Bone targeting assessment

[0102] Tail vein injection of osteoporotic mice DiD NPs / O DiD After sacrifice, femurs, tibias, and major organs were harvested for IVIS imaging and quantitative analysis. Decalcified bone tissue sections were stained with DAPI and then analyzed by CLSM to observe the distribution of nanoparticles.

[0103] 16. Animal Experimentation

[0104] Twelve-week-old female C57BL / 6 mice were randomly divided into five groups: sham-operated, saline-treated, curcumin-treated, CNPs-treated, and OCNPs-treated. All mice, except the sham-operated group, underwent bilateral ovariectomy. The mice were injected with 100 μg of the corresponding formulations via the tail vein every three days for eight weeks after surgery. The sham-operated and saline-treated groups received an equal volume of saline.

[0105] 17. Micro-CT analysis

[0106] The femurs were fixed with 4% (w / v) paraformaldehyde for 24 h, and scanned with VNC-102 micro-CT (90 kV). Three-dimensional reconstruction was performed to analyze parameters such as BMD, Tb.BV / TV, Tb.N, and Tb.Sp.

[0107] 18. Anti-osteoporosis effect

[0108] After 8 weeks of treatment, peripheral blood was collected to measure serum TNF-α, TRACP-5b, and OCN levels. Femoral decalcification and paraffin sectioning were performed, and TRAP staining was used to assess osteoclast activity, and BMP-2 expression was assessed by immunofluorescence.

[0109] 19. In vivo biocompatibility

[0110] Serum ALT, AST, BUN, and CREA levels were measured using an automated biochemical analyzer, and RBC, PLT, WBC, and HGB indices were measured using a hematology analyzer. Pathological changes in major organs were observed using H&E staining.

[0111] 20. Statistical Analysis

[0112] Data are expressed as mean ± SD, and one-way ANOVA (multiple groups) or t-test (two groups) were performed using GraphPad Prism 8.0. The significance thresholds were set at p < 0.05, p < 0.01, p < 0.001, ****p < 0.0001.

[0113] 2. Results and Discussion

[0114] 1. Screening, preparation and characterization of OCNPs

[0115] In recent years, cell membrane functionalized nanoparticles have been widely used as drug delivery carriers for the treatment of bone diseases. However, the ideal cell membrane for the treatment of postmenopausal osteoporosis (PMOP) is still under screening research. The biological functions of bone-related cells and the targeting of cell membranes are key considerations in the screening process. A search of the GeneCards database revealed that there were 1508 PMOP-related genes, 18922 osteoblast membrane-related genes, 10530 BMSCs membrane-related genes, 2349 osteoclast membrane-related genes, and 540 bone cell membrane-related genes. The data were imported into the Xiantao Academic Platform for analysis, and the intersection genes obtained were 1413, 1340, 784, and 306, respectively ( Figure 2 Further analysis showed that the proportion of common genes between different cell membranes and PMOP to the total number of disease-related genes was as follows: osteoblast membrane 93.70%, BMSCs membrane 88.86%, osteoclast membrane 51.99%, and osteocyte membrane 20.29%, indicating that osteoblast membrane is the most relevant candidate membrane material for PMOP treatment ( Figure 2 Middle A).

[0116] The drug loading (LE) of CNPs prepared by nanoprecipitation method was 5.6±0.12%, and the encapsulation efficiency (EE) was 57.46±0.42%. Transmission electron microscopy (TEM) showed that both CNPs and OCNPs formed regular spherical structures, and a clear osteoblast membrane coating was visible on the surface of OCNPs ( Figure 2 The average particle size of CNPs was 130.2 nm ( Figure 2 C), and increased to 155.2 nm after coating; Zeta potential analysis showed that OM, CNPs and OCNPs were all negatively charged, and membrane coating made the potential of OCNPs close to that of CNPs ( Figure 2 Middle D).

[0117] Confocal microscopy (CLSM) showed that OCNPs were enriched around the nucleus of MC3T3-E1 cells ( Figure 2 Middle E). Western blot confirmed that OCNPs retained CXCR4 and TNFR proteins ( Figure 2 F). Stability experiments showed that the particle size of OCNPs remained stable within 7 days, while the particle size of CNPs increased due to aggregation ( Figure 2 Release experiments showed that the cumulative release rates of CNPs and OCNPs within 72 h were 51.23±1.89% and 48.99±1.13%, respectively, confirming that they can improve the solubility of curcumin and achieve sustained release ( Figure 2 Middle H).

[0118] 2. In vitro cytotoxicity and hemocompatibility

[0119] The toxicity experiments of NPs and ONPs on MC3T3-E1 and RAW 264.7 cells showed that there was no significant change in cell viability after treatment with 10-200 μg / mL concentration for 24 h ( Figure 3 A and B). Hemolysis experiments showed that the hemolysis rates of 1 mg / mL CNPs and OCNPs were <5% ( Figure 3 C and D), and there was no difference in serum protein adsorption ( Figure 3 E), confirming its good blood compatibility.

[0120] 3. In vitro immune escape and targeted delivery

[0121] Macrophage phagocytosis experiments showed that the phagocytic rate of ONPs (59.15±1.58%) was significantly lower than that of NPs (91.02±0.65%) ( Figure 3 F and G), indicating that it has immune escape ability. Under inflammatory stimulation, the uptake rate of ONPs by MC3T3-E1 cells increased by 87.44% compared with NPs ( Figure 3 HJ), confirming its targeted delivery advantage.

[0122] 4. OCNPs clear TNF-α and promote bone formation

[0123] ONPs clear TNF-α through TNFR on the membrane surface, and the clearance effect is concentration-dependent ( Figure 4In the TNF-α stimulation model, ONPs restored cell viability to 77.61±2.71% (control 56.25±2.50%) ( Figure 4 Middle D), significantly inhibited cell apoptosis ( Figure 4 ALP activity (0.83±0.02 vs. control 1.08±0.07) and mineralization capacity (recovered to 80.31% of the unstimulated group) experiments confirmed that ONPs reversed the inhibition of TNF-α on osteogenic differentiation ( Figure 4 Chinese FI).

[0124] 5. In vivo pharmacokinetics and bone targeting

[0125] The blood circulation time of OCNPs in C57BL / 6 mice was prolonged to 48 h ( Figure 5 A and B). Osteoporosis model showed that ONPs were enriched in the long bones of the lower limbs ( Figure 5 CF), and fluorescence in bone tissue sections confirmed its localization in the trabecular region ( Figure 5 Middle (E), demonstrating CXCR4 / SDF-1 axis-mediated targeting.

[0126] 6. Anti-osteoporosis effect of OCNPs in ovariectomized mice

[0127] Micro-CT showed that the bone mineral density (BMD), bone volume fraction (Tb.BV / TV) and trabecular number (Tb.N) in the OCNPs group increased significantly, and the trabecular separation (Tb.Sp) decreased ( Figure 6 Serological analysis showed that OCNPs reduced the levels of TNF-α and TRACP-5b, and increased osteocalcin ( Figure 7 H&E and TRAP staining showed that bone loss was reduced and osteoclast activity was inhibited ( Figure 7 D, E and G), the up-regulation of BMP-2 expression confirmed its role in promoting bone formation ( Figure 7 F and H).

[0128] 7. Biosafety evaluation

[0129] After 8 weeks of treatment, hematological indicators ( Figure 8 AD) and liver and kidney function ( Figure 8 EH) were normal, and no damage was found in histopathology ( Figure 8 (I) This confirms that the OCNPs system has good security.

[0130] 3. Conclusion

[0131] The osteoblast membrane-coated nanoparticles constructed in this study achieve dual treatment of PMOP through CXCR4 / SDF-1 targeted delivery and TNFR-mediated TNF-α clearance. Its advantages include: (1) targeted delivery reduces side effects; (2) simultaneous inhibition of bone resorption and promotion of bone formation; and (3) nanocarriers improve curcumin stability and bioavailability.

[0132] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. An osteoblast-like cell membrane-coated nanoparticle for targeted delivery of curcumin, characterized in that: include: Curcumin, poly(lactic-co-glycolic acid), and MC3T3-E1 cell membrane; The polylactic acid-glycolic acid copolymer wraps the curcumin to form nanoparticles, and the MC3T3-E1 cell membrane wraps the nanoparticles to form osteoblast-like cell membrane-coated nanoparticles.

2. The osteoblast-like cell membrane-coated nanoparticles according to claim 1, characterized in that The mass ratio of the membrane protein, poly(lactic acid-glycolic acid copolymer) and curcumin on the MC3T3-E1 cell membrane is (5-15):(5-15):

1.

3. The osteoblast-like cell membrane-coated nanoparticles according to claim 1, characterized in that The polylactic acid-glycolic acid copolymer and / or the MC3T3-E1 cell membrane are modified with a fluorescent probe.

4. The osteoblast-like cell membrane-coated nanoparticles according to claim 3, characterized in that The fluorescent probe is selected from one of DiO, DiD, DiI and DiR.

5. A method for preparing osteoblast-like cell membrane-coated nanoparticles according to any one of claims 1 to 4, characterized in that: Including steps: dissolving curcumin and poly(lactic acid-co-glycolic acid) in an organic solvent to obtain a mixed solution; adding water dropwise into the mixed solution, controlling the volume ratio of the water to the mixed solution to be 1:(2-10), to obtain nanoparticles; The nanoparticles and MC3T3-E1 cell membranes are mixed, ultrasonicated, and extruded through a polycarbonate membrane with a pore size of 100-300 nm to obtain osteoblast-like cell membrane-coated nanoparticles.

6. The preparation method according to claim 5, characterized in that In the mixed solution, the concentration of curcumin is 0.5-2 mg / mL, and the concentration of the polylactic acid-glycolic acid copolymer is 5-15 mg / mL.

7. The preparation method according to claim 5, characterized in that The organic solvent is selected from one or more of dimethyl sulfoxide, ethyl acetate, methanol, and dichloromethane.

8. The preparation method according to claim 5, characterized in that The specific conditions of the ultrasound include: a frequency of 30-50 kHz, a power of 50-200 W, and a time of 1-10 min.

9. The preparation method according to claim 5, characterized in that The step of extruding through a polycarbonate membrane having a pore size of 100-300 nm was repeated at least three times.

10. Use of the osteoblast-like cell membrane-coated nanoparticles according to any one of claims 1 to 4 in the preparation of a drug for treating osteoporosis.

Images