Cell membrane-coated nanotraps that deplete rankl, their manufacture and use

Abstract

The present application provides a cell membrane-coated nanolure for clearing receptor activator of nuclear factor kappa B ligand (RANKL), preparation and application thereof. Specifically, the present application provides a nanolure, which comprises: (a) a nanocore; and (b) an osteoclast precursor cell membrane coated on the nanocore. The nanolure can be RAW-PLGA, wherein the RAW cell membrane is the cell membrane of an osteoclast precursor cell, and the PLGA is the nanocore. The present application also provides a preparation method of the nanolure and application thereof, especially in the treatment of diseases related to excessive or hyperfunction of osteoclasts (such as osteoporosis). The nanolure in the present application can effectively clear the high expression of RANKL in osteoporosis, can escape from macrophage capture, and has a long blood circulation after systemic administration, and has great potential in the clinical treatment of osteoporosis.

Description

Technical Field

[0001] This application belongs to the fields of biomaterials and medicine. Specifically, this application relates to cell membrane coating technology, and more specifically to a nanocomposite coated on an osteoclast membrane, which can effectively bind to and scavenge highly expressed RANKL. Background Technology

[0002] Osteoporosis is a progressive skeletal disease characterized by decreased bone mineral density and quality, disruption of bone microstructure, and increased bone fragility. In women, postmenopausal osteoporosis caused by estrogen deficiency is the most common type, with approximately 50% of women experiencing at least one fracture after menopause. In postmenopausal women, estrogen deficiency leads to the upregulation of RANKL, which further activates the nuclear factor-κB (NF-κB) pathway by binding to RANK on osteoclast precursor cells, thereby upregulating c-Fos gene expression. Furthermore, RANKL activates the mitogen-activated protein kinase (MAPK) pathway by initiating phosphorylation of a series of signaling molecules such as p38, ERK, and JNK. Activation of both pathways leads to the differentiation of monocytes into osteoclasts and initiates the transcriptional program of genes such as matrix metalloproteinases (MMPs) and alkaline phosphatases (ALPs), ultimately promoting bone resorption and inducing bone loss.

[0003] In clinical practice, osteoprotegerin (OPG) and antibodies such as denosumab have been widely used to clear RANKL to inhibit postmenopausal osteoporosis. However, existing RANKL inhibitors (mainly protein drugs) often have limited applications due to their short blood circulation time, poor biodistribution, complex manufacturing processes, and antibody resistance.

[0004] Osteoclasts (OCs) are the main functional cells responsible for bone resorption and play a crucial role in bone development, growth, repair, and remodeling. Originating from the bloodline mononuclear-macrophage system, osteoclasts are specialized terminally differentiated cells that can fuse from their mononuclear precursor cells through various pathways to form large multinucleated cells. The isolation and culture of osteoclasts began in the 1980s. As of July 2018, the main methods for culturing osteoclasts included: mechanical bone marrow isolation, bone marrow cell induction, spleen stem cell induction, blood mononuclear cell induction, mouse RAW 264.7 cell line induction, and giant cell tumor isolation.

[0005] Osteoclasts are known for their bone resorption function. As a component of bone tissue, they perform bone resorption. Osteoclasts function in contrast to osteoblasts (also known as bone-forming cells). Together, they play a crucial role in bone development and formation. In addition, osteoclasts have other biological functions, such as regulating hematopoiesis, bone formation, and intraosseous angiogenesis, and acting as a hormone in osteocalcin. Abnormal osteoclast function leads to abnormal bone resorption. Hyperfunction can cause degenerative bone diseases such as osteoporosis, bone metastases from cancer, and arthritis; while dysfunction or decline can cause osteosclerosis, osteogenesis imperfecta, osteodegenerative osteomyelitis, and massive osteolysis.

[0006] Research on osteoclast signaling pathways has provided a new direction for developing new drugs that target and block osteoclast differentiation and function: RANKL is involved in the formation and differentiation of osteoclasts in diseases such as myeloma and large cell carcinoma of bone. By blocking the RANK / RANKL pathway, the differentiation process of osteoclasts can be stopped, bone resorption can be prevented, bone density can be maintained, and the risk of fracture can be reduced.

[0007] Therefore, there is an urgent need in this field to develop a new, safer, and more effective drug for the prevention or treatment of diseases related to the osteoclast RANKL pathway, especially osteoporosis (e.g., postmenopausal osteoporosis). Summary of the Invention

[0008] This application provides a nanocomposite that can effectively bind to and scavenge overexpressed RANKL while escaping from macrophage capture and exhibiting prolonged blood circulation after systemic administration. It has great potential in the clinical treatment of RANKL overexpression-related diseases (especially osteoporosis).

[0009] In a first aspect of this application, a nanodecoy is provided, comprising: (a) a nanonucleus; and (b) an osteoclast precursor cell membrane encapsulating the nanonucleus.

[0010] In some embodiments, the nanocore is made of one or more materials selected from the group consisting of: polymeric nanoparticles such as polylactic-co-glycolic acid copolymer (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polylysine, polyglutamic acid, polybutyl cyanoacrylate (PBCA), chitosan, and gelatin; and inorganic nanoparticles such as gold, silicon, iron, or copper.

[0011] In some embodiments, the nanonuclei have one or more characteristics selected from the group consisting of: being negatively charged; and having a particle size of 50-200 nm.

[0012] In some embodiments, the osteoclast precursor cells are derived from: bone marrow hematopoietic stem cells, spleen stem cells, bone marrow or blood mononuclear cells, giant cell tumors of bone, myeloid dendritic cells, and osteoclast-like cells. In some embodiments, the osteoclast precursor cells are selected from: RAW 264.7 cells, human bone marrow or peripheral blood mononuclear / macrophages, ANA-1 cells, J774A.1 cells, and THP-1 cells. In some embodiments, the osteoclast precursor cells are selected from: naturally occurring osteoclast precursor cells, induced osteoclast precursor cells, and genetically engineered osteoclast precursor cells. In some embodiments, the osteoclast precursor cells are derived from humans, non-human primates (e.g., chimpanzees, apes, monkeys), rodents (e.g., rats, mice, guinea pigs, hamsters, rabbits), even-toed ungulates (e.g., sheep, cattle, pigs, camels, alpacas), and perissodactyls (e.g., horses). In some embodiments, the osteoclast precursor cells are derived from: the recipient organism itself, an allogeneic organism of the recipient organism, or a different species.

[0013] In some embodiments, the cell membrane of osteoclast precursor cells expresses macrophage-specific surface markers, such as one or more molecular markers selected from the group consisting of RANK, MAC-1, macrophage sialic acid protein, IFN-γR, TNF-αR, and IL-6R.

[0014] In some embodiments, the cell membrane of osteoclast precursor cells has the following characteristics: maintaining or preserving the original structural integrity (e.g., primary, secondary, tertiary, or quaternary structural integrity) or activity (e.g., binding activity, receptor activity, signal transduction pathway activity) of the cell membrane.

[0015] In some embodiments, the mass ratio of cell membrane to nanonucleus is 1:100 to 1:0.1, or 1:80 to 1:20, such as 1:64 to 1:4.

[0016] In some embodiments, the nanonucleus is a PLGA and the cell membrane is the cell membrane of a mononuclear macrophage (e.g., the RAW264.7 cell line).

[0017] In some embodiments, the nanodecoy is shaped as a sphere, cube, cone, cylinder, prism, pyramid, or other regular or irregular shape; and its size ranges from 1 nanometer to 10 micrometers or any value or range thereof.

[0018] In some embodiments, the nanodecoy has one or more of the following features selected from the group consisting of: (1) having the ability to specifically bind to and scavenge RANKL; (2) having reduced macrophage endocytosis compared to an empty nanonucleus; (3) having a prolonged in vivo half-life (e.g., a circulating half-life exceeding 10 hours) compared to an empty nanonucleus; and (4) reducing the mRNA and protein levels of gene-related factors (e.g., RANKL, RANK, TRAF6), osteoclastogenesis-related factors (NFATc1, c-Fos, ctsK, TRAP, RECK), and / or bone resorption-related factors (MMP-2, MMP-9, MMP-13) of the RANKL / RANK / OPG system.

[0019] In some aspects of this application, a method for preparing the nanodecoy of this application is provided, the method comprising: (A) providing a nanonucleus; (B) providing a cell membrane of osteoclast precursor cells; and (C) encapsulating the cell membrane onto the nanonucleus to form the nanodecoy.

[0020] In some embodiments, step (A) is performed by one or more methods selected from the group consisting of: nanoprecipitation, emulsion solvent evaporation, ion gelation, direct dissolution, dialysis, emulsification, media grinding, high-pressure homogenization, supercritical fluid, emulsion-like solvent diffusion, and solid reverse micelle solution.

[0021] In some embodiments, step (B) is performed by lysis and component separation of osteoclast precursor cells, for example, the lysis includes: ultrasonic lysis, enzymatic lysis, chemical lysis, homogenization lysis and / or hypotonic swelling lysis; the separation includes: centrifugation (e.g., stepwise centrifugation), precipitation, filtration, magnetic beading, chromatography.

[0022] In some embodiments, step (C) includes applying an external force to wrap the cell membrane around the nanonucleus, for example by acoustic methods (e.g., ultrasound), mechanical co-extrusion, electroporation, or heating.

[0023] In some embodiments, the method includes: lysing osteoclast precursor cells by ultrasound and obtaining cell membranes by stepwise centrifugation; co-ultrasounding the obtained cell membranes with nanonuclei to obtain nanodecoys. In some embodiments, the osteoclast precursor cells are RAW 264.7 cells, the nanonuclei are PLGA, the mass ratio of the cell membrane to the nanonuclei is 4:1; and / or the co-ultrasound is at 100W for 2 minutes.

[0024] In some aspects of this application, a product is provided comprising the nanodecoy of this application or components (a) and (b) for forming the nanodecoy; and optionally, a pharmaceutically or physiologically acceptable carrier.

[0025] In some aspects of this application, the use of the nanodecoy of this application or compositions of its components (a) and (b) in the preparation of products for the prevention and / or treatment of diseases related to osteoclast excess or hyperfunction (e.g., RANKL overactivity) is provided.

[0026] In some aspects of this application, a method is provided for the prevention and / or treatment of diseases related to osteoclast excess or hyperfunction (e.g., RANKL overactivity), the method comprising administering to a subject in need a preventive and / or therapeutic effective amount of the nanodecoy of this application or a composition of its components (a) and (b) or a product comprising the former.

[0027] In some aspects of this application, compositions of the nanodecoy of this application or its components (a) and (b) or products comprising the former are provided for the prevention and / or treatment of diseases associated with osteoclast excess or hyperfunction (e.g., RANKL overactivity).

[0028] In some implementations, diseases associated with excessive or hyperactive osteoclasts (e.g., excessive RANKL) are selected from: degenerative or loss-prone bone diseases, such as osteoporosis, especially osteoporosis caused by changes in estrogen levels (e.g., menopause); periodontitis; periapical periodontitis; peri-implantitis; bone metastases from cancers (e.g., breast cancer, prostate cancer, lung cancer); arthritis (e.g., chronic arthritis); osteitis degenerativeis; myeloma (e.g., multiple myeloma); and large cell carcinoma of bone.

[0029] Those skilled in the art can combine the foregoing technical solutions and features in any way without departing from the inventive concept and protection scope of this application. Other aspects of this application will be obvious to those skilled in the art due to the disclosure herein. Attached Figure Description

[0030] The present application will be further described below with reference to the accompanying drawings, which are shown only for illustrating the embodiments of the present application and are not intended to limit the scope of the present application.

[0031] Figure 1 Schematic diagram of RAW-PLGA nanodecoy preparation process ( Figure 1 A); and the morphology of RAW-PLGA under transmission electron microscopy (A ...). Figure 1 B).

[0032] Figure 2 Hydrated particle size and potential of PLGA, RAW vesicles and RAW-PLGA.

[0033] Figure 3 Characteristic protein bands from RAW 264.7 membrane and RAW-PLGA were analyzed by Western blot.

[0034] Figure 4 Particle size of RAW vesicles and RAW-PLGA at different time points in PBS containing 10% fetal bovine serum.

[0035] Figure 5 Images and flow cytometry plots of RAW 264.7 cells after co-incubation with PLGA or RAW-PLGA.

[0036] Figure 6 Cell survival rate of RAW 264.7 cells after co-incubation with RAW-PLGA, and secretion levels of TNF-α and IL-6.

[0037] Figure 7 : Relative RANKL concentration after PLGA or RAW-PLGA treatment (reflecting the relative removal of RANKL).

[0038] Figure 8 : Cy3 RANKL and Cy5 Fluorescence emission spectrum of RAW-PLGA mixed solution.

[0039] Figure 9 Extracellular RANKL levels in RAW 264.7 cells after co-incubation with RANKL and RAW-PLGA.

[0040] Figure 10 CLSM images of RAW 264.7 cells after co-incubation with RAW-PLGA or RANKL antibodies.

[0041] Figure 11 The relative expression level of c-Fos mRNA in bone marrow-derived monocytes and macrophages after co-incubation with RAW-PLGA and RANKL.

[0042] Figure 12 Levels of p-ERK, p-p38, and p-JNK proteins in bone marrow-derived monocytes and macrophages after co-incubation with RAW-PLGA and RANKL.

[0043] Figure 13 TRAP staining images of bone marrow-derived mononuclear macrophages after co-incubation with RAW-PLGA and RANKL.

[0044] Figure 14 Pharmacokinetics of PLGA, RAW vesicles, RBC-PLGA and RAW-PLGA after intravenous injection.

[0045] Figure 15 RankL protein levels in OVX mice after treatment with PBS, PLGA, and RAW-PLGA, respectively.

[0046] Figure 16 Osteoporosis-related mRNA levels in OVX mice after RAW-PLGA treatment.

[0047] Figure 17 Levels of TRACP-5b, BGP, and BAP proteins in OVX mice after RAW-PLGA treatment.

[0048] Figure 18 TRAP staining images and osteoclast counts of the femur of OVX mice after RAW-PLGA treatment.

[0049] Figure 19 Typical microcomputed tomography (μCT) images of femoral microstructure in OVX mice after PLGA or RAW-PLGA treatment, along with CT-quantified bone mineral density (BMD), bone volume fraction (BV / TV), trabecular bone number (Tb.N; 1 / mm), and trabecular bone spacing (Tb.Sp; mm).

[0050] Figure 20 Representative hematological parameters of C57 / BL6 mice after treatment with PBS or RAW-PLGA ( Figure 20 A) and biochemical parameters ( Figure 20 B).

[0051] Figure 21 H&E staining images of major organs of C57 / BL6 mice after treatment with PBS or RAW-PLGA.

[0052] In each figure, "*" indicates p < 0.05, "**" indicates p < 0.01, and "***" indicates p < 0.001. Detailed Implementation

[0053] This application provides a cell membrane-coated nanodecoy that can specifically remove RANKL, thereby preventing RANKL from binding to RANK in osteoclast precursor cells and initiating osteoclast differentiation. In particular, the RAW-PLGA nanodecoy has a long circulation time in vivo, overcoming the shortcomings of short blood circulation time of protein drugs (such as antibody drugs) used in the prior art.

[0054] In some specific embodiments of this application, osteoclast precursor cells are first lysed by ultrasound, and RAW cell membranes are obtained by stepwise centrifugation. Then, the RAW cell membranes and PLGA nanonuclei are sonicated together for 2 minutes (100W) to obtain RAW-PLGA nanodecoys. Related experiments show that this nanodecoy possesses highly efficient and stable RANKL clearance capabilities and can avoid endocytosis by macrophages, preventing premature clearance. In an ovariectomized (OVX) mouse model, RAW-PLGA nanodecoys were injected into mice via tail vein injection, successfully downregulating abnormally elevated RANKL and significantly improving physiological and bone parameters in OVX mice.

[0055] All numerical ranges provided herein are intended to clearly include all values ​​falling between the endpoints of the range and the range of values ​​between them. Features mentioned in this application or in the embodiments may be combined. All features disclosed in this specification may be used in any compositional form, and each feature disclosed in the specification may be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0056] As used herein, “containing,” “having,” or “including” includes “comprising,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.” For example, “substantially” can include 90% or more of what is referred to.

[0057] When used in a series of two or more items, the term "and / or" means that any of the listed items can be used alone or in combination with any one or more of the listed items. For example, the expression "A and / or B" is intended to mean one or both of A and B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B and / or C" is intended to mean A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C.

[0058] The description of a numerical range should be considered to have exactly disclosed all possible subranges as well as individual numerical values ​​within the range. For example, a range description such as 1 to 6 should be considered to have exactly disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within the range, such as 1, 2, 3, 4, 5, and 6.

[0059] Nano decoys

[0060] As used herein, the terms “nanodecoy” and “cell membrane-coated / encapsulated nanodecoy / material” are used interchangeably to refer to artificially synthesized nanomaterials containing a nanonucleus and an outer cell membrane, which can have the function of camouflaging biomimicry to trap and remove harmful factors or components within a system.

[0061] As used herein, the term "osteoclast precursor cells" refers to cells generated from hematopoietic stem cells in the bone marrow, primarily including monocytes and macrophages. The osteoclast precursor cells in this application may be genetically engineered to overexpress substances on their surface that enhance osteoclast affinity and / or RANKL clearance, such as overexpression of RANK.

[0062] As used herein, the term "cell membrane" refers to a naturally occurring biological membrane derived from osteoclast precursor cells or their organelles, or a modified or altered membrane possessing all or part of the biological activity of osteoclast precursor cells. The cell membrane used in this application may be a cell membrane derived from the osteoclast precursor cells of this application, which may be separated, have some components removed (e.g., lipids, glycans) and / or have some components added (e.g., overexpressed RANK, other cell surface antigens).

[0063] As used herein, the term "nanocore" refers to any nanoparticle with a nanoscale size that can be used to support the cell membrane of this application. Materials that can be used to prepare the nanodecoy nanocore of this application include, but are not limited to, polymeric nanomaterials or inorganic nanomaterials, such as polylactic-co-glycolic acid copolymer (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polylysine, polyglutamic acid, polybutyl cyanoacrylate (PBCA), chitosan, gelatin; gold, silicon, iron, copper, etc.

[0064] Depending on the materials or methods used, the nanodecoys of this application can have various suitable shapes, such as spheres, cubes, cones, cylinders, prisms, pyramids, or other regular or irregular shapes. The size of the nanodecoys of this application can be any value or range between 1 nanometer and 10 micrometers, such as 10 nanometers to 5 micrometers, 500 nanometers to 1 micrometer, etc.

[0065] Preparation method of nano decoys

[0066] This application also provides a method for preparing the nano-decoy of this application, the method comprising:

[0067] (A) Provides nano-cores;

[0068] (B) Provides the cell membrane for osteoclast precursor cells;

[0069] (C) The cell membrane is wrapped around the nanonucleus to form the nanodecoy.

[0070] The nanonuclei of this application can be prepared from raw materials using various methods known in the art (e.g., nanoprecipitation), or purchased directly from suppliers. The nanonuclei can have an opposite electrical potential to the cell membrane to form charge attraction, further stabilizing the nanodecoy.

[0071] Osteoclast precursor cell membranes can be obtained through cell lysis and separation, such as lysis including: ultrasonic lysis, enzymatic lysis, chemical lysis, homogenization lysis, and / or hypotonic swelling lysis; separation including: centrifugation (e.g., stepwise centrifugation), precipitation, filtration, magnetic beading, and chromatography. Before obtaining the cell membrane, cells can be collected, cultured, and engineered to obtain osteoclast precursor cells with the desired quantity and function.

[0072] The cell membrane should possess structural integrity and retain the necessary functions, and be able to partially or completely encapsulate the nanonucleus. Preferably, the cell membrane can completely encapsulate the nanonucleus to increase the stability of the nanodecoy. In some embodiments, the cell membrane has a size greater than or equal to the surface area of ​​the nanonucleus. In some embodiments, the cell membrane retains functional epitopes, receptors, and other functional molecules from the osteoclast precursor cell membrane surface.

[0073] The coating of nanonuclei by the cell membrane can be achieved by applying external force. For example, it can be achieved by sound waves (e.g., ultrasound), mechanical force (e.g., mechanical co-extrusion), electrical energy (e.g., electroporation), or thermal energy (heating). In some embodiments, the method of this application may include: lysing osteoclast precursor cells by ultrasound and obtaining cell membranes by stepwise centrifugation; and sonicating the obtained cell membranes and nanonuclei together to obtain nanodecoys.

[0074] Drugs, pharmaceutical compositions or kits

[0075] This application also provides a medicament, pharmaceutical composition, or kit containing an effective amount of the nanodecoy of this application or a composition of components (a) and (b), and a pharmaceutically acceptable carrier. As used herein, the terms "active substance" or "active substance of this application" are used interchangeably and refer to the nanodecoy or a composition of components (a) and (b). The composition of components (a) and (b) may contain separately stored components (a) and (b) and optionally a carrier, which can be mixed with the optional carrier and prepared into a nanodecoy medicament for preventive and / or therapeutic use prior to use.

[0076] In some embodiments, the drug can be used to prevent and / or treat diseases associated with osteoclast overactivation. For example, the active substance of this application, and products containing said active substance, can be used to prevent and / or treat osteoclast overactivation-related bone degeneration or bone loss diseases, such as osteoporosis, especially osteoporosis caused by changes in estrogen levels (e.g., menopause), periodontitis, periapical periodontitis, peri-implantitis; bone metastases from cancers (e.g., breast cancer, prostate cancer, lung cancer), arthritis (e.g., chronic arthritis), osteomyelitis degenerative, myeloma (e.g., multiple myeloma), and large cell carcinoma of bone.

[0077] As used herein, the terms “containing” or “including” include “comprising,” “consistently composed of,” and “composed of.” As used herein, a “pharmaceutically acceptable” ingredient is a substance suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. As used herein, the term “effective amount” refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0078] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0079] Pharmaceutically acceptable carriers in the composition may contain liquids such as water, saline, glycerol, and ethanol. Additionally, these carriers may contain auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffers, etc. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, with a pH usually around 5-8, preferably around 6-8.

[0080] As used herein, the term "unit dosage form" refers to a dosage form prepared for a single dose of the composition of this application for ease of administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules, and sustained-release formulations.

[0081] It should be understood that the effective dose of the active substance used can vary depending on the severity of the condition of the patient being treated. The specific dosage is determined based on the individual patient's circumstances (e.g., weight, age, physical condition, and desired outcome), within the judgment of a skilled physician.

[0082] The compositions of this application may be in solid form (such as granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets) or liquid form (such as oral liquid) or other suitable shapes. Routes of administration may include: intravenous injection, intraperitoneal injection, intralesional injection, oral administration, local administration, intramuscular administration, intradermal administration, rectal administration, inhalation, etc.

[0083] In addition, the compositions of this application may also contain other active substances for improving and treating diseases related to excessive or hyperactive osteoclasts. For example, the other active substances are selected from the group consisting of commonly used osteoclast inhibitors, antibiotics, antitumor agents, anti-inflammatory agents, etc.

[0084] The nanodecoy of this application can also be combined with other drugs and treatments, such as chemotherapy, radiotherapy, phototherapy, cryotherapy, surgery, cell therapy, transplantation, etc.

[0085] Specific examples

[0086] This section provides some specific implementations of this application. It should be understood that these examples are not intended to limit the scope of protection of this application, but only to help understand this application.

[0087] In some specific embodiments of this application, a cell membrane-coated nanodecoy is provided, the structure of which is RAW-PLGA, wherein RAW is the cell membrane of osteoclast precursor cells and PLGA is the nanonucleus.

[0088] In some specific embodiments of this application, the preparation method of RAW-PLGA cell membrane-coated nanodecoy may include lysing osteoclast precursor cells by sonication and obtaining RAW cell membranes by stepwise centrifugation; then sonicating the RAW cell membranes and PLGA nanonuclei together for 2 minutes (100W) to obtain RAW-PLGA nanodecoys.

[0089] In some specific embodiments of this application, the cell membranes of osteoclast precursor cells are separated by sonication and centrifugation. Specifically, RAW 264.7 cells are suspended in a homogenization buffer containing 20 mM Tris·HCl (pH 7.5), 10 mM KCl, 75 mM sucrose, 2 mM MgCl2, and a protease / phosphatase inhibitor. The suspension is disrupted using a JY 92-IIN homogenizer (75W), and the supernatant is collected by centrifugation at 20,000 g for 25 minutes. The cell membrane is then collected by centrifugation at 100,000 g for 35 minutes. The protein content of the collected cell membranes is determined using a BCA kit. A membrane containing approximately 5 mg of membrane protein can be obtained from 3 × 10⁻⁶ cells. 7 Extracted from RAW264.7 cells.

[0090] In some specific embodiments of this application, PLGA nanonuclei are prepared by acetone evaporation. Specifically, 1 mL of acetone containing PLGA (10 mg / mL) is added dropwise to 2 mL of deionized water, and the mixture is then stirred in the open air until the acetone is completely evaporated.

[0091] In some specific embodiments of this application, the RAW-PLGA nanodecoy is prepared by an ultrasonic method. Specifically, RAW cell membranes and PLGA nanonuclei are ultrasonically treated at a 1:4 mass ratio using a bath ultrasonic instrument (Fisher Scientific FS30D, 100W) for 2 minutes. As an advantage, the RAW-PLGA nanodecoy of this application consists of FDA-approved PLGA and biologically derived cell membranes, exhibiting excellent biocompatibility and safety.

[0092] This application further discloses the application of the above-mentioned cell membrane-coated nanodecoys in the preparation of drugs for treating postmenopausal osteoporosis.

[0093] As a specific example, the method for preparing RAW-PLGA nanodecoys in this application is as follows: Figure 1 As shown in Figure A. A specific preparation method is illustrated below:

[0094] (1) RAW 264.7 cells were suspended in homogenization buffer containing 20 mM Tris·HCl (pH 7.5), 10 mM KCl, 75 mM sucrose, 2 mM MgCl2, and one tablet of protease / phosphatase inhibitor. The suspension was disrupted using a JY92-IIN homogenizer (75W), and the supernatant was collected by centrifugation at 20,000 g for 25 minutes. The cell membrane was then collected by centrifugation at 100,000 g for 35 minutes. The protein content of the collected cell membranes was determined using a BCA kit. Membranes containing approximately 5 mg of membrane protein could be obtained from 3 × 10⁻⁶ cells. 7 Extracted from RAW 264.7 cells.

[0095] (2) Add 1 mL of acetone containing PLGA (10 mg / mL) dropwise to 2 mL of deionized water, and then stir the mixture in the open air until the acetone is completely evaporated to obtain PLGA nanonuclei.

[0096] (3) RAW cell membranes and PLGA nanonuclei were ultrasonically treated with a bath ultrasonic instrument (Fisher Scientific FS30D, 100W) for 2 minutes at a mass ratio of 1:4 to obtain RAW-PLGA nanodecoys.

[0097] Advantages of this application

[0098] This application includes at least the following advantages:

[0099] 1. Cell membrane-coated nanodecoys inherit the surface receptors of osteoclast precursor cells and directly clear RANKL through receptor recognition, avoiding potential adverse reactions.

[0100] 2. The introduction of nanonuclei (e.g., PLGA) restricts the flow of membrane components, greatly improving the serum stability of nanodecoys.

[0101] 3. Nanodecoys inherit surface-specific proteins from osteoclast precursor cells, thus avoiding endocytosis by macrophages and prolonging blood circulation.

[0102] 4. Compared with existing osteoporosis treatment drugs, the nano-decoy of this application has advantages such as long circulation, high RANKL neutralization efficiency, and high safety.

[0103] Example

[0104] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present application. Those skilled in the art can make appropriate modifications and variations to the present application, and such modifications and variations are all within the scope of the present application.

[0105] Experimental methods not specifically described in the following examples can be performed using conventional methods in the art, such as those described in *Molecular Cloning: A Laboratory Manual* (3rd edition, New York: Cold Spring Harbor Laboratory Press, 1989), *Animal Cell Culture* (R.Freshney, 1987), or according to the conditions recommended by the supplier. DNA sequencing methods are conventional in the art and can also be provided by commercial companies.

[0106] Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to one skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be used in the methods of this application. The preferred methods and materials described herein are for illustrative purposes only.

[0107] All data are expressed as mean ± standard deviation, and statistical analysis was performed using Student's t-test. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 and a p-value < 0.001 were considered highly statistically significant.

[0108] Example 1: Preparation and Characterization of RAW-PLGA Nanodecoys

[0109] according to Figure 1 The cell membrane-coated nanocomposite—RAW-PLGA nanodecoy—of this application was prepared using the process described in section A. The specific preparation steps are as follows:

[0110] (1) Preparation of membrane material: Mouse monocyte / macrophage-like cells RAW 264.7 (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number SCSP-5036, culture medium was DMEM containing 10% FBS, 37℃, 5% CO2) were suspended in homogenization buffer containing 20 mM Tris·HCl (pH 7.5), 10 mM KCl, 75 mM sucrose, 2 mM MgCl2, and protease / phosphatase inhibitor (purchased from Pierce, catalog number A32953, each tablet dissolved in 10 mL of solution). The cells in the suspension were lysed using a JY 92-IIN homogenizer (75W), and the supernatant was collected by centrifugation at 20000g for 25 minutes. The cell membrane was then collected by centrifugation at 100000g for 35 minutes. The protein content of the collected cell membranes was determined using a BCA kit. The protein content was approximately 3 × 10⁻⁶. 7 Membrane material containing approximately 5 mg of membrane protein was extracted from one RAW264.7 cell.

[0111] (2) Preparation of nanonuclei: 1 mL of acetone containing polylactic acid-glycolic acid copolymer (PLGA) (PLGA was purchased from Sigma-Aldrich, catalog number 719900, acid-capped, lactide:glycolide 50:50, molecular weight 38000-54000) 10 mg / mL was added dropwise to 2 mL of deionized water. The mixture was then stirred in the open air until the acetone was completely evaporated to obtain PLGA nanonuclei.

[0112] (3) RAW cell membranes and PLGA nanonuclei were ultrasonically treated in deionized water at a preset mass ratio (1:64 to 2:1) for 2 minutes using a bath ultrasonic instrument (Fisher Scientific FS30D, 100W) to obtain RAW-PLGA nanodecoys.

[0113] The results showed that uniform nanodecoys were obtained when the RAW:PLGA mass ratio was 1:64–2:1, while the nanodecoys possessed complete coatings and suitable particle sizes at a mass ratio of 1:16–1:4. The prepared nanodecoys could be stored at room temperature for one month without precipitation or particle size changes.

[0114] The obtained cell membrane-coated nanocomposite was characterized as follows:

[0115] After staining with uranyl acetate (0.2 wt%), the morphology of RAW-PLGA nanodecoys was observed using transmission electron microscopy (TEM, TECNAI G2, FEI, US). See details... Figure 1 .like Figure 1 As shown, the nanodecoy has a spherical structure and a clearly identifiable membrane structure.

[0116] The hydrodynamic dimensions and zeta potential of the nanodecoy were determined using a Zetasizer Nano ZS90 (Malvern Instruments, Ltd., UK). See details... Figure 2 .like Figure 2 As shown, the hydrodynamic size of the nanodecoy is approximately 109.5 nm, and the zeta potential is approximately -14.5 mV.

[0117] Macrophage-specific surface markers on RAW vesicles and RAW-PLGA nanodecoys were examined by Western blot: integrative proteins: MAC-1 and macrosialin; cytokine-binding receptors: IFN-γR, TNF-αR, IL-6R, and RANK. Primary antibodies against MAC-1, macrosialin, IFN-γR, TNF-αR, IL-6R, RANK, and GAPDH were used at a concentration of 1:1000, and HRP-labeled secondary antibodies were used at a concentration of 1:500. See details... Figure 3 .like Figure 3 As shown, the RAW cell membrane encapsulated on the nanonucleus expresses essentially the same surface markers as the natural cell membrane, suggesting that it may have similar membrane functions as the natural cell.

[0118] Serum stability of the nanodecoys was assessed by measuring their particle size in deionized water or DMEM containing 10% FBS. See details... Figure 4 .like Figure 4 As shown, the particle size of RAW vesicles changed significantly over time, while the particle size of RAW-PLGA remained essentially constant. This result indicates that the rigid PLGA nanonucleus restricts the flow of the membrane components surrounding it, thus improving the stability of the entire RAW-PLGA in serum.

[0119] In summary, the method of this application yielded a stable nanodecoy comprising a PLGA nanocore and a RAW (roughly encapsulated) osteoclast precursor cell membrane, which can provide surface proteins similar to those of the corresponding natural cells, such as various integrins and cytokine binding receptors.

[0120] Example 2: Cellular experiments with RAW-PLGA nanodecoys: endocytosis, biocompatibility, and immunostimulation.

[0121] Laser confocal microscopy was used to study the endocytosis of nanodecoys. RAW 264.7 cells were cultured at 4 × 10⁶ cells per dish. 4 Cells were seeded in a number of culture dishes (Ф=20mm) and incubated for 24 hours (medium: DMEM containing 10% FBS, 37℃, 5% CO2). After staining the cell membranes with DiI (purchased from Beyotime Biotechnology, 5μg / mL), the cells were then... DiD PLGA (with DiD (purchased from Beyotime Biotechnology) added to a PLGA-acetone solution at a PLGA / DiD mass ratio of 1000:1, followed by centrifugation to remove excess DiD after acetone evaporation (20000g, 15 minutes)) or RAW- DiD PLGA (using RAW cell membranes and) DiD PLGA nanonucleus preparation method is the same as that for RAW-PLGA. DiD The final concentration of PLGA component in the culture medium was 100 μg. DiD Cells were incubated at 37°C for 4 hours with PLGA / mL. After washing three times with cold PBS, cells were observed using CLSM (Leica, TCS SP5, Germany). See details... Figure 5 Confocal experimental results show that RAW- DiD The intracellular fluorescence of PLGA was significantly weaker than that of other PLGA cells. DiD PLGA and flow cytometry results also showed that RAW- 264.7 cells were endocytinated with RAW- DiD The level of PLGA nanodecoys is higher than that of endocytosis DiD The PLGA level was approximately 20 times lower. These results indicate that the cell membrane encapsulation significantly reduces the proportion of nanodecoys endocytosed by macrophages. Therefore, the RAW-PLGA of this application can effectively escape macrophage capture, avoiding premature clearance and thus exerting a more sustained effect in vivo.

[0122] Further investigation was conducted into the biocompatibility and immunostimulatory activity of RAW-PLGA. RAW 264.7 cells were cultured at a density of 1 × 10⁶ cells per well. 4 Cells were seeded in 96-well plates and cultured for 24 hours. Then, the cells were incubated with RAW-PLGA nanodecoys (5-160 μg PLGA / mL) at 37°C for 24 hours. Cell viability was assessed using the MTT assay, and the concentrations of TNF-α and IL-6 in the supernatant were quantified using an ELISA kit (purchased from Sanjia Biochemical Supplies). See details... Figure 6RAW-PLGA nanodecoys do not affect cell viability at concentrations ranging from 5-160 μg PLGA / mL, nor do they stimulate the production of TNF-α and IL-6. Therefore, the RAW-PLGA of this application possesses high biocompatibility and low immunostimulatory activity.

[0123] Example 3: In vitro binding and scavenging effects of RAW-PLGA nanodecoy on RANKL

[0124] RANKL (200 pg / mL, Sigma-Aldrich) was mixed with PLGA or RAW-PLGA (1 mg PLGA / mL) in deionized water and incubated at 37°C for 2 hours. Then, it was centrifuged at 16100 g for 10 minutes. The concentration of RANKL in the supernatant was quantified using an ELISA kit. See details... Figure 7 The results showed that RAW-PLGA bound approximately 53% of RANKL, while PLGA had almost no binding effect.

[0125] RANKL binding was further detected by FRET. RBC-PLGA was used as a negative control. Red blood cells collected from whole blood of female C57 / BL6 mice were resuspended in 0.25×PBS and centrifuged at 800g for 5 minutes. This process was repeated until hemoglobin was completely removed, and the red blood cell membranes were collected. The protein content of the collected cell membranes was determined using a BCA kit. RAW cell membranes or red blood cell membranes were mixed with Cy5-NHS (purchased from Macklin) at a mass ratio of 1000:1, and then prepared according to the RAW-PLGA preparation procedure. Cy5 RBC-PLGA and Cy5 RAW-PLGA.

[0126] Will be used as FRET pairs Cy3 RANKL (200 pg / mL) and red blood cell membrane-bound Cy5 RBC-PLGA or Cy5 RAW-PLGA (1 mg PLGA / mL) was mixed and incubated as described above. The temperature was maintained at 565-750 nm (λ). ex Fluorescence emission spectra were collected in the range of 550 nm. See details... Figure 8 The results showed that energy transfer occurred in the RAW-PLGA group, but not in the RBC-PLGA group. This result further demonstrates the effective and specific binding of RAW-PLGA nanodecoys to RANKL.

[0127] Further investigation was conducted to assess real-time RANKL clearance in the presence of cells. RAW 264.7 cells were cultured at a density of 1 × 10⁶ cells per well. 4Cells were seeded in 96-well plates and cultured for 24 hours. Then, the cells were incubated at 37°C with RANKL (100 ng / mL) and RAW-PLGA nanobait (100 μg PLGA / mL). RANKL clearance efficiency was assessed by measuring the RANKL concentration in the cell culture medium at predetermined time intervals. See details... Figure 9 The addition of RAW-PLGA resulted in a significant decrease in RANKL levels. These results collectively demonstrate that RAW-PLGA has a strong and specific scavenging effect on RANKL.

[0128] Example 4: Effects of RAW-PLGA nanodecoys on osteoclast precursor cells

[0129] To demonstrate that the nanodecoy blocked the binding between RANKL and RAW 264.7 cell membranes, RAW 264.7 cells were arranged at a ratio of 4 × 10⁶ cells per dish. 4 Cells were seeded in a number of culture dishes (Ф=20mm) and incubated for 24 hours. Cells were stained with DiI (5μg / mL, a cell membrane dye) and then... FITC Cells were cultured in fresh RANKL medium (100 ng / mL). After incubation for 4 hours with RANKL antibody (0.05 mg / mL, Abcam) or RAW-PLGA (100 μg PLGA / mL, based on PLGA), cells were washed three times with cold PBS, and nuclei were stained with Hoechst 33258 (5 μg / mL) and observed using CLSM. See details... Figure 10 The results showed that the proportion of RANKL binding to RAW 264.7 cells was significantly reduced after the addition of RAW-PLGA.

[0130] Further investigation was conducted into the blocking effect of RAW-PLGA on the NF-κB pathway. Mouse bone marrow mononuclear macrophages were isolated from mouse bone marrow and divided into groups of 1 × 10⁻⁶ cells per well. 4 Cells were seeded in 96-well plates and cultured for 24 hours. They were then cultured in fresh medium containing M-CSF (30 ng / mL) and RANKL (100 ng / mL) and incubated with RAW-PLGA (100 μg PLGA / mL) for 24 hours. c-Fos mRNA levels were quantified using real-time PCR. See details... Figure 11 After RAW-PLGA treatment, the abnormally elevated levels of the osteoclast-associated transcription factor c-Fos were reduced by 81%.

[0131] Simultaneously, the blocking effect of RAW-PLGA on the MAPK pathway was investigated. Mouse bone marrow mononuclear macrophages were isolated and treated as described above. The levels of p-ERK, p-p38, and p-JNK proteins were quantified using ELISA. See details... Figure 12 After RAW-PLGA treatment, the levels of p-ERK, p-p38, and p-JNK were significantly lower than those in the RANKL treatment group and essentially returned to normal levels.

[0132] Subsequently, the inhibitory effect of RAW-PLGA on osteoclast differentiation was investigated holistically. Mouse bone marrow mononuclear macrophages (BMMs) were cultured in fresh medium containing M-CSF (30 ng / mL) and RANKL (100 ng / mL) and incubated with RAW-PLGA nanobait (100 μg PLGA / mL) for 96 hours. Cells were fixed with 4% formalin buffer for 10 minutes, washed three times with PBS, stained with a TRAP staining kit, and imaged under an inverted microscope (Leica DM4000, Solms, Germany). See details... Figure 13 The results showed that BMM differentiated into large multinucleated osteoclasts after induction. Figure 13 (See middle image). After RAW-PLGA treatment, the number and diameter of osteoclasts were significantly reduced, and their morphology was similar to that of the uninduced control cells. Figure 13 (Left image vs. Right image)

[0133] The above results collectively demonstrate that RAW-PLGA has a strong inhibitory effect on osteoclast differentiation.

[0134] Example 5: In vivo half-life study of RAW-PLGA nanodecoys

[0135] To demonstrate that RAW-PLGA has a long in vivo circulation time, the pharmacokinetics of RAW-PLGA after intravenous injection were studied.

[0136] Female C57 / BL6 mice (6-8 weeks old, 18-20g, purchased from Shanghai Slack Laboratory Animal Co., Ltd., housed in a clean room at a rate of four per cage, with free access to water, a 12:12 hour light-dark cycle, and a temperature of 25±1℃. The animal experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Soochow University) were administered 10mg of [a certain medication / method]. DiD PLGA / kg or 2.5mg DiD RAW vesicles / kg dose intravenously DiD PLGA DiD RAW vesicles (prepared by mixing RAW cell membranes and DiD at a mass ratio of 1000:1, followed by sonication for 2 minutes), RBC- DiD PLGA or RAW DiD PLGA. Blood was collected at predetermined time points and analyzed by spectrophotometry (λ). ex =644nm, λ em=663nm) to determine plasma DiD PLGA or DiD The content of RAW. Calculate the cycle half-life (t). 1 / 2 See details. Figure 14 RAW-PLGA displays are superior to PLGA (t) 1 / 2 =4.08 hours) and RAW vesicles (t 1 / 2 =3.11 hours) significantly prolonged blood circulation time, its t 1 / 2 It takes 13.26 hours.

[0137] The results suggest that the RAW-PLGA nanodecoy of this application has a long circulating half-life in vivo, and can exert its therapeutic effect more effectively compared with drugs with shorter circulating half-lives (such as protein drugs).

[0138] Example 6: In vivo anti-osteoporosis effect of RAW-PLGA nanodecoys

[0139] The effect of RAW-PLGA on osteoporosis-related biochemical indicators was assessed in vivo. Ovariectomized (OVX) mouse models were established by bilateral ovariectomy in female C57 / BL6 mice. For the next 60 days, PLGA or RAW-PLGA was administered intravenously every 3 days at a dose of 10 mg PLGA / kg, with an equal volume of PBS injected as a negative control. On day 60, mice were sacrificed, peripheral blood was collected, and serum was extracted by centrifugation at 500g for 10 minutes at 4°C. Serum RANKL levels were quantified using an ELISA kit. See details below. Figure 15 The serum RANKL concentration in RAW-PLGA-treated OVX mice was reduced by 57% compared to the OVX group (p<0.0001), almost returning to normal levels.

[0140] Further investigation was conducted to detect the mRNA levels of RANKL (RANKL / RANK / OPG system genes: RANKL, RANK, TRAF6), osteoclastogenesis-related key factors (osteoclast-related transcription factors: NFATC1, c-Fos; and osteoclast-specific genes: ctSK, TRAP, RECK), and bone resorption-related key factors (MMP-2, MMP-9, and MMP-13) in femoral tissue. Femoral tissue was frozen in liquid nitrogen and homogenized with Trizol reagent to isolate total RNA, and real-time PCR was used to detect the corresponding mRNA levels. The primer pairs (forward primer + reverse primer) used for real-time PCR of RANK, c-Fos, RANKL, TRAF6, NFATC1, ctSK, TRAP, RECK, MMP-2, MMP-9, MMP-13, and GAPDH are shown in SEQ ID NO:1-24 of the sequence listing.

[0141] For details of the results, please refer to [link / details]. Figure 16 Following RAW-PLGA treatment, key factors of the RANKL / RANK / OPG system (RANKL, RANK, and TRAF6) returned to normal levels, indicating that the entire system had been rebalanced. Simultaneously, RAW-PLGA not only restored abnormally upregulated osteoclast-related transcription factors (NFATc1 and c-Fos) to normal levels but also suppressed the expression levels of osteoclast-specific genes (ctsK, TRAP, and RECK), demonstrating its strong ability to inhibit osteoclast differentiation. Furthermore, bone resorption-related genes (MMP-2, MMP-9, and MMP-13) largely returned to normal levels, which will further benefit osteoporosis prevention and bone formation.

[0142] Simultaneously, bone resorption and bone formation during postmenopausal osteoporosis were assessed by detecting biochemical markers of bone turnover. Serum levels of TRACP-5b, BGP, and BAP were quantified using an ELISA kit. See details... Figure 17 After RAW-PLGA processing, the levels of these metrics almost returned to normal.

[0143] Example 7: Histological Effects of RAW-PLGA Nanoparticle Decoy Therapy on Bone Tissue

[0144] Histological analysis was performed on the femurs of RAW-PLGA-treated OVX mice. On day 60 after bilateral ovariectomy (administration regimen as in Example 6), mice were sacrificed, femoral tissue was harvested, fixed in 10% formalin buffer, and then incubated in decalcification solution (14% EDTA) at room temperature for 1 month to decalcify. The femurs were then embedded in paraffin and sectioned at a thickness of 8 μm. Staining was performed using a TRAP staining kit, and the number of osteoclasts (N.Oc / BS) on each bone surface was counted. See details... Figure 18 RAW-PLGA effectively reduced the erosion of bone trabeculae by osteoclasts, while reducing the number of osteoclasts by 61%, almost restoring it to normal levels.

[0145] Example 8: Imaging Effects of RAW-PLGA Nanoparticle Decoy Therapy on Bone Tissue

[0146] The restoration of femoral integrity was further analyzed using micro-CT. On day 60 after bilateral oophorectomy (drug administration regimen as in Example 6), right femoral specimens from OVX mice were scanned using micro-CT (Skyscan 1176). High-resolution scans (9–20 mm) were obtained (resolution: 8.8 mm, source voltage: 50 kV, source current: 500 mA, rotation step: 0.7 U). The dataset was reconstructed using CT analyzer software (Skyscan) to obtain 3D images of the femoral tissue and to measure morphological parameters. Bone erosion from the micro-CT scans was calculated based on the reconstructed data and an internally written Fiji script. The program determined the bone surface and internal bone spaces and filled the pores on the bone surface. Regions of interest (ROIs) in the trabecular bone were selected for analysis of the following morphological parameters: (1) bone mineral density (BMD), (2) bone volume density ratio (BV / TV), (3) number of trabeculae (Tb.N), and (4) intertrabecular spacing (Tb.Sp). See details. Figure 19 The results showed that RAW-PLGA significantly inhibited oophorectomy-induced bone loss and alleviated the osteopenia phenotype in trabeculae. Furthermore, after RAW-PLGA treatment, BMD, BV / TV, Tb.N, and Tb.Sp returned to normal levels.

[0147] The above experimental evidence strongly demonstrates the great potential of RAW-PLGA nanodecoys for treating postmenopausal osteoporosis.

[0148] Example 9: In vivo biocompatibility of RAW-PLGA nanodecoys

[0149] Female C57 / BL6 mice were intravenously injected with either PBS (200 μL) or RAW-PLGA nanocomposite (10 mg PLGA / kg, 200 μL), following the same administration regimen as in Example 6. Blood and major organs (heart, liver, spleen, lung, and kidney) were collected. Hematological assessments were performed using a Cobas 501 automated hematology analyzer (Roche, USA). Serum biochemical parameters were measured using a BC-5380 automated hematology analyzer (Mindray, China). See details... Figure 20 Major organs were fixed in 10% formalin, embedded in paraffin, and sectioned at a thickness of 8 μm. The sections were stained with H&E and observed using a whole-body transmission optical microscope. See details... Figure 21 .

[0150] The results showed that mice treated with RAW-PLGA nanodecoys exhibited no abnormalities in representative hematological and biochemical parameters. Furthermore, no necrosis, inflammation, edema, or other pathological symptoms were detected in H&E-stained cross-sections of major organs. These results indicate that the RAW-PLGA nanodecoys, after systemic administration, possess good biocompatibility and high safety.

[0151] All references to this application are incorporated herein by reference as if each reference were individually incorporated herein by reference. Furthermore, it should be understood that after reading the foregoing teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Suzhou University <120> Cell membrane-coated nanodecoys for RANKL removal, their preparation and application <130> 217779 1CNCN <160> twenty four <170> PatentIn version 3.3 <210> 1 <211> twenty four <212> DNA <213> Artificial sequence <400> 1 agatgtggtc tgcagctctt ccat 24 <210> 2 <211> twenty four <212> DNA <213> Artificial sequence <400> 2 acacacttct tgctgactgg aggt 24 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <400> 3 tttcaacgcg gactacgagg 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <400> 4 gcgcaaaagt cctgtgtgtt 20 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <400> 5 cagaaggaac tgcaacacat 20 <210> 6 <211> twenty one <212> DNA <213> Artificial sequence <400> 6 cagagtgact ttatgggaac c 21 <210> 7 <211> twenty one <212> DNA <213> Artificial sequence <400> 7 ttgcacattc agtgtttttg g 21 <210> 8 <211> 18 <212> DNA <213> Artificial sequence <400> 8 tgcaagtgtc gtgccaag 18 <210> 9 <211> twenty two <212> DNA <213> Artificial sequence <400> 9 ccgttgcttc cagaaaataa ca 22 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <400> 10 tgtgggatgt gaactcggaa 20 <210> 11 <211> twenty one <212> DNA <213> Artificial sequence <400> 11 agcgaacaga ttctcaacag c 21 <210> 12 <211> twenty one <212> DNA <213> Artificial sequence <400> 12 agacagagca aagctcacca t 21 <210> 13 <211> 20 <212> DNA <213> Artificial sequence <400> 13 cgtctctgca cagattgcat 20 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <400> 14 aagcgcaaac ggtagtaagg 20 <210> 15 <211> 20 <212> DNA <213> Artificial sequence <400> 15 ctccagcagt ctcccgtcat 20 <210> 16 <211> twenty one <212> DNA <213> Artificial sequence <400> 16 gttgtgggtg gtcagggtct a 21 <210> 17 <211> twenty three <212> DNA <213> Artificial sequence <400> 17 gcaatacctg aacactttct atg 23 <210> 18 <211> 20 <212> DNA <213> Artificial sequence <400> 18 tctggtcaag gtcacctgtc 20 <210> 19 <211> twenty one <212> DNA <213> Artificial sequence <400> 19 tggcagaggc atacttgtac c 21 <210> 20 <211> 18 <212> DNA <213> Artificial sequence <400> 20 tgtccagctc accagtct 18 <210> twenty one <211> 20 <212> DNA <213> Artificial sequence <400> twenty one ctggaccaaa ctatggtggg 20 <210> twenty two <211> 20 <212> DNA <213> Artificial sequence <400> twenty two ggtccttgga gtgatccaga 20 <210> twenty three <211> 20 <212> DNA <213> Artificial sequence <400> twenty three ttcaccacca tggagaaggc 20 <210> twenty four <211> 20 <212> DNA <213> Artificial sequence <400> twenty four ggcatggact gtggtcatga 20

Claims

1. The application of nanodecoys in the preparation of products for the prevention and / or treatment of osteoporosis, wherein, The nanodecoy includes: (a) A nanocore, said nanocore being made of polylactic-co-glycolic acid copolymer (PLGA) nanoparticles; and (b) The osteoclast precursor cell membrane encapsulating the nanonucleus, wherein the osteoclast precursor cells are RAW 264.7 cells. The size of the nanodecoy ranges from 1 nanometer to 10 micrometers. Furthermore, the cell membrane of the osteoclast precursor cells maintains or retains the integrity of its original primary, secondary, tertiary, or quaternary structures; and maintains or preserves the original binding activity, receptor activity, and signal transduction pathway activity of the cell membrane.

2. The application as described in claim 1, wherein, The osteoporosis mentioned is caused by changes in estrogen levels.

3. The application as described in claim 1, wherein, The osteoporosis mentioned is postmenopausal osteoporosis.

4. The application as described in claim 1, wherein, The nanonucleus has one or more characteristics selected from the group consisting of: being negatively charged; having a particle size of 50-200 nm.

5. The application as described in claim 1, wherein, The osteoclast precursor cells express macrophage-specific surface markers on their cell membranes.

6. The application as described in claim 5, wherein, The macrophage-specific surface markers are one or more molecular markers selected from the group consisting of: RANK, MAC-1, macrophage sialic acid protein, IFN-γR, TNF-αR, and IL-6R.

7. The application as described in claim 1, wherein, The mass ratio of the cell membrane to the nanonucleus is 1:100 to 1:0.

1.

8. The application as described in claim 1, wherein, The mass ratio of the cell membrane to the nanonucleus is 1:80 to 1:

20.

9. The application as described in claim 1, wherein, The mass ratio of the cell membrane to the nanonucleus is 1:64 to 1:

4.

10. The application as described in claim 1, wherein, The nanodecoy can be spherical, cubic, conical, cylindrical, prismatic, pyramidal, or other regular or irregular shapes.

11. The application as described in any one of claims 1 to 10, wherein, The nanodecoy has one or more of the following characteristics selected from the group consisting of: (1) It has the ability to specifically bind to and clear RANKL; (2) Macrophage endocytosis was reduced compared to empty nanonuclei; (3) Compared with empty nanonuclei, it has a longer in vivo half-life; (4) Reduce the mRNA and protein levels of RANKL / RANK / OPG system gene-related factors, osteoclastogenesis-related factors and / or bone resorption-related factors.

12. The application as described in claim 11, wherein, The nanodecoy has a cycling half-life exceeding 10 hours; and / or The gene-related factors of the RANKL / RANK / OPG system are selected from: RANKL, RANK, and TRAF6; Osteoclastogenesis-related factors are selected from: NFATC1, c-Fos, ctsK, TRAP, RECK; and / or, The bone resorption-related factors are selected from MMP-2, MMP-9, and MMP-13.

13. The application as described in claim 1, wherein, The nanodecoy is prepared as follows: (A) Provide a nanocore made of polylactic acid-glycolic acid copolymer (PLGA) nanoparticles; (B) Provides the cell membrane of osteoclast precursor cells, wherein the osteoclast precursor cells are RAW 264.7 cells; (C) The cell membrane is wrapped around the nanonucleus to form the nanodecoy, wherein the size of the nanodecoy is in the range of 1 nanometer to 10 micrometers.

14. The application as described in claim 13, wherein, Step (A) is performed by one or more methods selected from the group consisting of: nanoprecipitation, emulsion solvent evaporation, ionogel method, direct dissolution method, dialysis method, emulsification method, media milling method, high-pressure homogenization method, supercritical fluid method, emulsion-like solvent diffusion method, and solid-state reverse micelle solution method; and / or Step (B) is performed by lysis of osteoclast precursor cells and separation of components; and / or Step (C) includes applying an external force to cause the cell membrane to wrap around the nanonucleus.

15. The application as described in claim 14, wherein, The lysis method is selected from: ultrasonic lysis, enzymatic lysis, chemical lysis, homogenization lysis, and / or hypotonic swelling lysis; The separation process is selected from: centrifugation, precipitation, filtration, magnetic beads, chromatography; and / or Step (C) employs acoustic wave method, mechanical co-extrusion method, electroporation method, and heating method.

16. The application as described in claim 13, wherein, The nanodecoy is prepared as follows: osteoclast precursor cells are lysed by ultrasound, and cell membranes are obtained by stepwise centrifugation; the obtained cell membranes are then sonicated together with nanonuclei to obtain the nanodecoy.

17. The application as described in claim 16, wherein, The common ultrasound was 100 W for 2 minutes.

18. The application as described in claim 1, wherein, The mass ratio of the cell membrane to the nanonucleus is 4:

1.

19. The application as described in claim 1, wherein, The product also includes: a pharmaceutically or physiologically acceptable carrier.

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