CN113925836A

CN113925836A - RANKL-eliminating cell membrane-coated nano bait, and preparation and application thereof

Info

Publication number: CN113925836A
Application number: CN202111152475.9A
Authority: CN
Inventors: 殷黎晨; 周炀
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2021-09-29
Filing date: 2021-09-29
Publication date: 2022-01-14

Abstract

The application provides a cell membrane coating nano bait for removing a nuclear factor kappa B receptor activator factor ligand (RANKL), and preparation and application thereof. Specifically, the present application provides a nano-bait comprising: (a) a nano-core; and (b) an osteoclast precursor cell membrane coating the nanocore. The nano bait may be RAW-PLGA, wherein RAW cell membrane is the cell membrane of osteoclast precursor cells, and PLGA is the nano core. The application also provides a preparation method and application of the nano bait, in particular to application in treating diseases related to osteoclast excess or hyperfunction (such as osteoporosis). The nano bait in the application can effectively remove highly expressed RANKL in osteoporosis, can escape from macrophage capture, has long-term blood circulation after systemic administration, and has great potential in clinical treatment of osteoporosis.

Description

RANKL-eliminating cell membrane-coated nano bait, and preparation and application thereof

Technical Field

The present application belongs to the field of biomaterials and medicine. In particular, the present application relates to cell membrane coating techniques, and more particularly to osteoclast membrane coated nanocomplexes, which are effective in binding and clearing highly expressed RANKL.

Background

Osteoporosis is a progressive skeletal disease characterized by a decrease in bone mineral density and quality, a disruption of bone microstructure, and an increase in bone fragility. Postmenopausal osteoporosis caused by estrogen deficiency is the most common type of disorder in women, with approximately 50% of women experiencing at least one fracture after menopause. In menopausal women, estrogen deficiency results in upregulation of RANKL, which further activates the nuclear factor- κ B (NF- κ B) pathway by binding to RANK of 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 signal molecules such as p38, ERK and JNK. Activation of both pathways results in differentiation of monocytes into osteoclasts and initiates transcription programs of genes like Matrix Metalloproteinases (MMPs) and alkaline phosphatase (ALPs), ultimately promoting bone resorption and inducing bone loss.

Clinically, Osteoprotegerin (OPG) and antibodies such as Denosumab (Denosumab) have been widely used to clear RANKL to inhibit postmenopausal osteoporosis. However, existing RANKL inhibitors (mainly protein drugs) are often of limited utility due to their short blood circulation time, non-ideal biodistribution, complex manufacturing process, and antibody resistance.

Osteoclasts (OCs) are the main functional cells for bone resorption and play an important role in bone development, growth, repair and reconstruction. Osteoclasts, which originate from the blood-based monocyte-macrophage system, are specialized terminally differentiated cells that can be fused by their mononuclear precursor cells in a variety of ways to form large multinucleated cells. The osteoclast isolation culture method starts from the 80 th of the 20 th century and reaches 7 months of 2018, and mainly comprises the following steps: bone marrow mechanical separation method, bone marrow cell induction method, spleen stem cell induction method, blood mononuclear cell induction method, mouse RAW264.7 cell line induction method and bone giant cell tumor separation method.

Osteoclasts are known for their bone resorption function. And as one of the bone tissue components, functions as bone resorption (bone resorption). Osteoclasts correspond functionally to osteoblasts (also known as bone-forming cells). The two are synergistic and play an important role in the development and formation of bones. In addition, osteoclasts have other biological effects. Such as hematopoietic regulation, bone formation regulation, regulation of angiogenesis in bone, and hormonal action of osteocalcin. Osteoclast dysfunction can cause bone resorption abnormality, and if the osteoclast dysfunction is enhanced, bone degenerative diseases such as osteoporosis, cancer bone metastasis, arthritis and the like can be caused; if the function of the bone is impaired or declined, the bone may cause osteopetrosis, incompetence of compact bone formation, osteitis deformans, massive osteolysis and the like.

The research of the osteoclast signal path provides a new direction for developing a new medicine for targeting and blocking the differentiation and the function of the osteoclast: RANKL is involved in the process of osteoclast formation and differentiation in diseases such as myeloma and osteocarcinoma, and by blocking the RANK/RANKL pathway, osteoclast differentiation can be prevented, bone resorption can be prevented, bone density can be maintained, and fracture risk can be reduced.

Therefore, there is a great need in the art to develop a new safer and more effective drug for preventing or treating diseases associated with the osteoclast RANKL pathway, in particular osteoporosis (e.g. postmenopausal osteoporosis).

Disclosure of Invention

The application provides a nano-composite which can effectively combine and eliminate high-expression RANKL, can escape from macrophage capture, has long-term blood circulation after systemic administration, and has great potential in clinical treatment of RANKL overexpression related diseases (particularly osteoporosis).

In a first aspect of the present application, there is provided a nano-bait comprising: (a) a nano-core; and (b) an osteoclast precursor cell membrane coating the nanocore.

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 (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), Polycaprolactone (PCL), polylysine, polyglutamic acid, poly n-butyl cyanoacrylate (PBCA), chitosan, gelatin; inorganic nanoparticles such as gold, silicon, iron or copper.

In some embodiments, the nanocore has one or more characteristics selected from the group consisting of: is negatively charged; the particle size is 50-200 nm.

In some embodiments, the osteoclast precursor cell is derived from: bone marrow hematopoietic stem cells, spleen stem cells, bone marrow or blood mononuclear cells, giant cell tumors of bone, myeloid dendritic cells, osteoclast-like cells. In some embodiments, the osteoclast precursor cell is selected from: RAW264.7 cells, human bone marrow or peripheral blood mononuclear/macrophage, ANA-1 cells, J774A.1 cells, THP-1 cells. In some embodiments, the osteoclast precursor cell is selected from: natural osteoclast precursor cell, osteoclast precursor cell formed by inducing differentiation, and osteoclast precursor cell engineered by genetic engineering. In some embodiments, the osteoclast precursor cell is derived from a human, a non-human primate (e.g., orangutan, ape, monkey), a rodent (e.g., rat, mouse, guinea pig, hamster, rabbit), an artiodactyl (e.g., sheep, cow, pig, camel, alpaca), a marmot (e.g., horse). In some embodiments, the osteoclast precursor cell is derived from: the subject to be administered is autologous, allogeneic to the subject, or a different species from the subject.

In some embodiments, the cell membrane of the osteoclast precursor cell expresses a macrophage specific surface marker, e.g., one or more molecular markers selected from the group consisting of: RANK, MAC-1, macrosialoprotein, IFN-gamma R, TNF-alpha R, and IL-6R.

In some embodiments, the cell membrane of the osteoclast precursor cell has the following characteristics: the native structural integrity (e.g., primary, secondary, tertiary, or quaternary structural integrity) or activity (e.g., binding activity, receptor activity, signaling pathway activity) inherent to the cell membrane is maintained or retained.

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

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

In some embodiments, the morphology of the nano-bait is spherical, cubic, conical, cylindrical, prismatic, pyramidal, or other regular or irregular shape; the size range is 1 nanometer to 10 micrometers or any value or range of values therebetween.

In some embodiments, the nano-bait has one or more of the following characteristics selected from the group consisting of: (1) has the ability to specifically bind to and clear RANKL; (2) decreased macrophage endocytosis compared to unloaded nanonuclei; (3) have an extended in vivo half-life (e.g., a circulation half-life of more than 10 hours) compared to unloaded nanonuclei; (4) reduce mRNA and protein levels of gene-related factors of the RANKL/RANK/OPG system (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).

In some aspects of the present application, there is provided a method of making a nano-bait of the present application, the method comprising: (A) providing a nanocore; (B) providing a cell membrane of an osteoclast precursor cell; (C) and (c) wrapping the cell membrane on the nano-core to form the nano-bait.

In some embodiments, step (a) is performed by one or more methods selected from the group consisting of: nano-precipitation, emulsion solvent evaporation, ionic gel, direct dissolution, dialysis, emulsification, media milling, high pressure homogenization, supercritical fluid, quasi-emulsion solvent diffusion, and solid reversed phase micelle solution.

In some embodiments, step (B) is performed by lysis and component separation of osteoclast precursor cells, e.g., the lysis comprises: ultrasonic lysis, enzymatic lysis, chemical lysis, homogenate lysis and/or hypotonic swelling lysis; the separation comprises: centrifugation (e.g., stepwise), precipitation, filtration, magnetic beads, chromatographic separation.

In some embodiments, step (C) comprises applying an external force to encapsulate the cell membrane onto the nanocore, for example using sonic (e.g., ultrasound), mechanical co-extrusion, electroporation, or heating.

In some embodiments, the method comprises: osteoclast precursor cells are cracked by ultrasonic, and cell membranes are obtained by step-by-step centrifugation; and carrying out ultrasonic treatment on the obtained cell membrane and the nano nucleus together to obtain the nano bait. In some embodiments, the osteoclast precursor cell is a RAW264.7 cell, the nanocore is PLGA, and the mass ratio of the cell membrane to the nanocore is 4: 1; and/or the collective sonication is 100W for 2 minutes.

In some aspects of the present application, there is provided a product comprising a nano-bait of the present application or component (a) and component (b) for forming a nano-bait; and, optionally, a pharmaceutically or physiologically acceptable carrier.

In some aspects of the present application, there is provided the use of the inventive nano-bait, or the compositions of components (a) and (b) thereof, for the preparation of a product for the prevention and/or treatment of diseases associated with osteoclastogenesis hyperactivity or hyperfunction (e.g. RANKL hyperactivity).

In some aspects of the present application, there is provided a method of preventing and/or treating a disease associated with osteoclastogenesis hyperactivity or hyperfunction (e.g., RANKL hyperactivity), the method comprising administering to a subject in need thereof a prophylactically and/or therapeutically effective amount of a nanobait of the present application or a composition of, or a product comprising, components (a) and (b) thereof.

In some aspects of the present application, there is provided a nano-bait of the present application or a composition of components (a) and (b) thereof or a product comprising the same for use in the prevention and/or treatment of a disease associated with osteoclast overabundance or hyperactivity (e.g. RANKL hyperactivity).

In some embodiments, the disease associated with osteoclastogenesis hyperactivity or hyperfunction (e.g., RANKL hyperactivity) is selected from the group consisting of: degenerative or lost bone conditions, such as osteoporosis, particularly osteoporosis due to altered levels of estrogen (e.g. menopause), periodontitis, periapical periodontitis, peri-implantitis; bone metastasis from cancer (e.g., breast, prostate, lung); arthritis (e.g., chronic arthritis); osteitis deformans; myeloma (e.g., multiple myeloma); osteocarcinoma large cell carcinoma.

Any combination of the above-described solutions and features may be made by those skilled in the art without departing from the inventive concept and scope of the present application. Other aspects of the present application will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

The present application is further described with reference to the accompanying drawings, wherein the showings are for the purpose of illustrating embodiments of the application only and not for the purpose of limiting the scope of the application.

FIG. 1: the preparation process of RAW-PLGA nano bait is shown in figure 1A; and RAW-PLGA morphology under transmission electron microscopy (fig. 1B).

FIG. 2: hydrated particle size and potential of PLGA, RAW vesicles and RAW-PLGA.

FIG. 3: characteristic protein bands of RAW264.7 membrane and RAW-PLGA under Western blot analysis.

FIG. 4: particle size of RAW vesicles and RAW-PLGA at different time points in PBS containing 10% fetal bovine serum.

FIG. 5: CLSM images and flow charts of RAW264.7 cells after co-incubation with PLGA or RAW-PLGA.

FIG. 6: cell survival after co-incubation of RAW264.7 cells with RAW-PLGA, and secreted amounts of TNF- α and IL-6.

FIG. 7: relative RANKL concentration after PLGA or RAW-PLGA action (reflecting RANKL relative removal).

FIG. 8:^Cy3RANKL and^Cy5fluorescence emission spectra of RAW-PLGA mixed solutions.

FIG. 9: extracellular RANKL levels after co-incubation of RAW264.7 cells with RANKL and RAW-PLGA.

FIG. 10: CLSM images after co-incubation of RAW264.7 cells with RAW-PLGA or RANKL antibodies.

FIG. 11: expression of relative levels of c-Fos mRNA following co-incubation of bone marrow derived mononuclear macrophages with RAW-PLGA and RANKL.

FIG. 12: levels of p-ERK, p-p38, and p-JNK proteins after co-incubation of bone marrow derived mononuclear macrophages with RAW-PLGA and RANKL.

FIG. 13: TRAP stained images after co-incubation of bone marrow derived mononuclear macrophages with RAW-PLGA and RANKL.

FIG. 14: pharmacokinetics of PLGA, RAW vesicles, RBC-PLGA and RAW-PLGA following intravenous injection.

FIG. 15: RANKL protein levels in OVX mice treated with PBS, PLGA, RAW-PLGA, respectively.

FIG. 16: osteoporosis-related mRNA levels in OVX mice after RAW-PLGA treatment.

FIG. 17: TRACP-5b, BGP, BAP protein levels in OVX mice after RAW-PLGA treatment.

FIG. 18: TRAP stained images of OVX mice femurs after RAW-PLGA treatment and osteoclast counts.

FIG. 19: typical micro-computer tomography (μ CT) images of OVX mouse femoral microstructure after PLGA or RAW-PLGA treatment and CT quantified bone density (BMD), bone body score (BV/TV), trabecular number (Tb.N; 1/mm) and trabecular gap (Tb.Sp; mm).

FIG. 20: representative hematological parameters (FIG. 20A) and biochemical parameters (FIG. 20B) of C57/BL6 mice after PBS or RAW-PLGA treatment.

FIG. 21: h & E stained images of the major organs of C57/BL6 mice after PBS or RAW-PLGA treatment.

In each figure, "+" indicates p <0.05, "+" indicates p <0.01, and "+" indicates p < 0.001.

Detailed Description

Provided herein is a cell membrane-coated nanobelt capable of specifically clearing RANKL, thereby preventing RANKL from binding RANK of osteoclast precursor cells and initiating differentiation of osteoclasts. Particularly, the RAW-PLGA nano bait has longer circulation time in vivo, and overcomes the defects of short blood circulation time of protein drugs (such as antibody drugs) used in the prior art and the like.

In some embodiments of the present application, osteoclast precursor cells are first lysed by ultrasound and RAW cell membranes are obtained by stepwise centrifugation; and carrying out ultrasonic treatment on the RAW cell membrane and the PLGA nano-core for 2 minutes (100W) to obtain the RAW-PLGA nano-decoy. Relevant experiments show that the nano bait has high-efficiency and stable RANKL clearing capacity, can avoid being endocytosed by macrophages and can prevent premature clearing. In an Ovariectomy (OVX) mouse model, RAW-PLGA nano bait is injected into a mouse body in a tail vein injection mode, so that the abnormally increased RANKL is successfully reduced, and the physiological index and the bone index of the OVX mouse are obviously improved.

All numerical ranges provided herein are intended to expressly include all numbers between the end points of the ranges and numerical ranges there between. The features mentioned in the present application or the features mentioned in the embodiments can be combined. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

As used herein, "comprising," having, "or" including "includes" comprising, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …", and "consisting of … …" are subordinate concepts of "comprising", "having", or "including". For example, "substantially" may include 90% or more of all that is referred to.

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

The recitation of a range of values is to be considered as having all the possible subranges explicitly disclosed as well as individual values within that range. For example, a range description such as 1 to 6 should be considered to have the explicitly 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.

Nano bait

As used herein, the terms "nano-bait", "cell membrane coated/encapsulated nano-bait/material", used interchangeably, refer to an artificially synthesized nano-material comprising a nano-core and a cell membrane encapsulated outside it, which may have the function of camouflaging biomimetics to trap and remove undesirable factors or components within the system.

As used herein, the term "osteoclast precursor cell" refers to a cell produced from hematopoietic stem cells in bone marrow, and mainly includes monocytes and macrophages. The osteoclast precursor cells herein may be genetically engineered to overexpress on their surface agents that help to increase osteoclast affinity and/or RANKL clearance, such as, for example, overexpressing RANK, etc.

As used herein, the term "cell membrane" refers to a naturally occurring biological membrane obtained from an osteoclast precursor cell or organelle thereof, or a modified, altered membrane having biological activity of all or part of an osteoclast precursor cell. The cell membrane for use herein may be a cell membrane obtained from an osteoclast precursor cell herein, which may be isolated, have a portion of the components (e.g., lipids, sugar chains) removed, and/or have a portion of the components added (e.g., overexpressed RANK, other cell surface antigen).

As used herein, the term "nanocore" refers to any nanoparticle having a nanoscale size that can be used to support a cell membrane of the present application. Materials that can be used to prepare the nano-bait nano-core of the present application include, but are not limited to, polymeric nanomaterials or inorganic nanomaterials, such as polylactic acid-co-glycolic acid (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), Polycaprolactone (PCL), polylysine, polyglutamic acid, poly n-butyl cyanoacrylate (PBCA), chitosan, gelatin; gold, silicon, iron, copper, etc.

The nano-baits of the present application can have various suitable shapes, such as spherical, cubic, conical, cylindrical, prismatic, pyramidal, or other regular or irregular shapes, depending on the materials or methods used. The size of the nano-baits of the present application can range from 1 nanometer to 10 microns or any value or range of values therebetween, such as 10 nanometers to 5 microns, 500 nanometers to 1 micron, and the like.

Preparation method of nanometer bait

Also provided herein is a method of making a nano-bait of the present application, the method comprising:

(A) providing a nanocore;

(B) providing a cell membrane of an osteoclast precursor cell;

(C) and (c) wrapping the cell membrane on the nano-core to form the nano-bait.

The nanonuclei of the present application can be prepared from raw materials (e.g., using a nanoprecipitation process) using various methods known in the art, or can be purchased directly from various suppliers. The nanonuclei may have an opposite potential to the cell membrane to form a charge attraction that further stabilizes the nanobaits.

Osteoclast precursor cell membranes can be obtained by cell lysis and isolation, for example, the lysis includes: ultrasonic lysis, enzymatic lysis, chemical lysis, homogenate lysis and/or hypotonic swelling lysis; the separation comprises: centrifugation (e.g., stepwise), precipitation, filtration, magnetic beads, chromatographic separation. Cells may be harvested, cultured, engineered, etc. prior to obtaining the cell membrane to obtain osteoclast precursor cells in the desired number and function.

The cell membrane should have some structural integrity and retain the desired functionality and be capable of partially or completely encapsulating the nanocore. Preferably, the cell membrane is capable of completely encapsulating the nanocore to increase stability of the nanocoalures. In some embodiments, the cell membrane has a size greater than or equal to the surface area of the nanocore. In some embodiments, functional molecules such as functional epitopes, receptors, etc. on the surface of the osteoclast precursor cell membrane are retained on the cell membrane.

The coating of the nanocore by the cell membrane can be realized by applying an external force. For example, encapsulation can be achieved using acoustic (e.g., ultrasonic), mechanical (e.g., mechanical co-extrusion), electrical (e.g., electroporation), thermal (e.g., thermal), and the like. In some embodiments, the methods of the present application may comprise: osteoclast precursor cells are cracked by ultrasonic, and cell membranes are obtained by step-by-step centrifugation; and carrying out ultrasonic treatment on the obtained cell membrane and the nano nucleus together to obtain the nano bait.

Medicament, pharmaceutical composition or kit

The present application also provides a medicament, pharmaceutical composition or kit comprising an effective amount of the nano-bait or composition of components (a) and (b) of the present application, and a pharmaceutically acceptable carrier. As used herein, the terms "active material" or "active material of the present application" are used interchangeably to refer to a nano-bait or a composition of components (a) and (b). The composition of the components (a) and (b) can comprise the components (a) and (b) and an optional carrier which are stored independently, and the components (a) and (b) can be mixed with the optional carrier and prepared into the nano bait medicament for prevention and/or treatment before use.

In some embodiments, the medicament may be for the prevention and/or treatment of a disease associated with overactivation of osteoclasts. For example, the active substances, products comprising said active substances, of the present application can be used for the prevention and/or treatment of bone degenerative or bone loss diseases caused by overactivation of osteoclasts, such as osteoporosis, in particular osteoporosis caused by altered estrogen levels (e.g. menopause), periodontitis, periapical periodontitis, periimplantitis; bone metastases of cancers (e.g., breast, prostate, lung), arthritis (e.g., chronic arthritis), osteitis deformans, myeloma (e.g., multiple myeloma), large cell bone cancer.

As used herein, the terms "comprising" or "including" include "comprising," consisting essentially of … …, "and" consisting of … …. As used herein, the term "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. As used herein, the term "effective amount" refers to an amount that produces a function or activity in and is acceptable to humans and/or animals.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences, Mack pub.Co., N.J.1991.

Pharmaceutically acceptable carriers in the compositions may comprise liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting or emulsifying agents, flavoring agents, pH buffering substances and the like may also be present in these carriers. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8.

As used herein, the term "unit dosage form" refers to a dosage form that is formulated for single administration of the compositions of the present application for ease of administration, including, but not limited to, various solid dosage forms (e.g., tablets), liquid dosage forms, capsules, sustained release formulations.

It will be appreciated that the effective dose of the active substance used may vary with the severity of the subject to be administered or treated. The specific condition is determined according to the individual condition of the subject (e.g., the subject's weight, age, physical condition, desired effect), and is within the judgment of a skilled physician.

The composition of the present application may be in solid form (e.g., granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets) or liquid form (e.g., oral liquid) or other suitable forms. The administration route can be as follows: intravenous injection, intraperitoneal injection, intralesional injection, oral administration, local administration, intramuscular injection, intradermal injection, rectal administration, inhalation and the like.

In addition, other active substances for ameliorating and treating diseases associated with osteoclast excess or hyperactivity may be contained in the composition of the present application. For example, the additional active substance is selected from the group consisting of: osteoclast inhibitors, antibiotics, antitumor agents, anti-inflammatory agents and the like are commonly used in clinic.

The nano-baits of the present application can also be combined with other drugs and therapeutic approaches such as chemotherapy, radiation therapy, phototherapy, cryotherapy, surgery, cell therapy, transplantation, and the like.

Specific examples

Some specific embodiments of the present application are provided in this section, it being understood that these examples are not intended to limit the scope of the application, but are merely to aid in understanding the application.

In some embodiments of the present application, there is provided a cell membrane-coated nanobait having a structure of RAW-PLGA, wherein RAW is a cell membrane of an osteoclast precursor cell and PLGA is a nanonucleus.

In some embodiments of the present application, the RAW-PLGA cell membrane-coated nano-bait may be prepared by a method comprising disrupting osteoclast precursor cells by ultrasound and obtaining RAW cell membranes by stepwise centrifugation; and carrying out ultrasonic treatment on the RAW cell membrane and the PLGA nano-core for 2 minutes (100W) to obtain the RAW-PLGA nano-decoy.

In some embodiments of the present application, the cell membrane of the osteoclast precursor cell is separated by ultrasound and centrifugation. Specifically, RAW264.7 cells were suspended in a medium containing 20mM Tris-HCl (pH 7.5), 10mM KCl, 75mM sucrose, 2mM MgCl₂And protease/phosphatase inhibitor in homogenization buffer. The suspension was disrupted with a JY92-IIN homogenizer (75W), and then the supernatant was collected by centrifugation at 20000g for 25 minutes, and the cell membrane was collected by centrifugation at 100000g for 35 minutes. The protein content of the collected cell membranes was determined using the BCA kit. The membrane containing about 5mg of membrane protein may be from 3X 10⁷Extracted from each RAW264.7 cell.

In some embodiments of the present application, the PLGA nanocore is prepared by acetone evaporation. Specifically, 1mL of acetone with PLGA (10mg/mL) dissolved therein was added dropwise to 2mL of deionized water, and the mixture was stirred in the open air until the acetone was completely evaporated.

In some embodiments of the present application, the RAW-PLGA nano-bait is prepared by an ultrasonic method. Specifically, RAW cell membranes and PLGA nanonuclei were sonicated for 2 minutes in a mass ratio of 1:4 using a bath sonicator (Fisher Scientific FS30D, 100W). As one advantage, the RAW-PLGA nano-bait of the present application is composed of FDA-approved PLGA and cell membrane of biological origin, and has excellent biocompatibility and safety.

The application further discloses application of the cell membrane coated nano bait in preparation of a medicine for resisting postmenopausal osteoporosis.

As a specific example, the method for preparing RAW-PLGA nano-bait according to the present application is shown in fig. 1A. Specific preparation methods are exemplified by:

(1) RAW264.7 cells were suspended in a medium containing 20mM Tris-HCl (pH 7.5), 10mM KCl, 75mM sucrose, 2mM MgCl₂And a tablet of protease/phosphatase inhibitor in homogenization buffer. The suspension was disrupted with a JY92-IIN homogenizer (75W), and then the supernatant was collected by centrifugation at 20000g for 25 minutes, and the cell membrane was collected by centrifugation at 100000g for 35 minutes. The protein content of the collected cell membranes was determined using the BCA kit. The membrane containing about 5mg of membrane protein may be from 3X 10⁷Extracted from RAW264.7 cells.

(2) 1mL of acetone dissolved with PLGA (10mg/mL) was added dropwise to 2mL of deionized water, and the mixture was stirred in the open air until the acetone was completely evaporated, yielding PLGA nanonuclei.

(3) The RAW cell membrane and the PLGA nano-core are treated by ultrasonic treatment for 2 minutes by a bath ultrasonic instrument (Fisher Scientific FS30D, 100W) according to the mass ratio of 1:4 to obtain the RAW-PLGA nano-decoy.

Advantages of the present application

The application at least comprises the following advantages:

1. the nano bait coated by the cell membrane inherits a surface receptor of an osteoclast precursor cell, and directly clears RANKL through the recognition effect of the receptor, so that potential adverse reactions are avoided.

2. The introduction of the nanocore (e.g., PLGA) limits the flow of membrane components, greatly improving the serum stability of the nanocastray.

3. The nanometer bait can avoid the endocytosis of macrophage by inheriting the surface specificity protein of osteoclast precursor cell, thereby prolonging blood circulation.

4. Compared with the existing osteoporosis treatment medicines, the nano bait has the advantages of long circulation, high RANKL neutralization efficiency, high safety and the like.

Examples

The present application is further illustrated with reference to specific examples. It should be understood that these examples 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, which are within the scope of the present application.

The experimental procedures, for which specific conditions are not indicated in the following examples, can be carried out by methods conventional in the art, for example, with reference to the molecular cloning, A Laboratory Manual (third edition, New York, Cold Spring Harbor Laboratory Press, New York: Cold Spring Harbor Laboratory Press, 1989), Animal Cell Culture (Animal Cell Culture, R.I. Freshney, 1987) or according to the conditions suggested by the supplier. Methods for sequencing DNA are conventional in the art and tests are also available from commercial companies.

Unless otherwise indicated, percentages and parts are by weight. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present application. The preferred embodiments and materials described herein are intended to be exemplary only.

All data are expressed as mean ± standard deviation and were statistically analyzed using Student's t-test. Differences between the two groups were judged significant at p <0.05, and were very significant at p <0.01 and p < 0.001.

EXAMPLE I preparation and characterization of RAW-PLGA Nanodexide

The cell membrane-coated nanocomposite, RAW-PLGA decoy, of the present application was prepared according to the procedure described in fig. 1A. The preparation method comprises the following specific steps:

(1) preparation of membrane material: mouse monocyte/macrophage-like cells RAW264.7 (purchased from cell bank of Chinese academy of sciences, catalog number SCSP-5036, culture medium DMEM containing 10% FBS, 37 ℃, 5% CO)₂) Suspended in a medium containing 20mM Tris-HCl (pH 7.5), 10mM KCl, 75mM sucrose, 2mM MgCl₂And protease/phosphatase inhibitors (purchased from Pierce, cat. No. A32953, each tablet dissolved in 10mL of solution). Cells in the suspension were disrupted with a JY92-IIN homogenizer (75W), and then the supernatant was collected by centrifugation at 20000g for 25 minutes, and the cell membrane was collected by centrifugation at 100000g for 35 minutes. The protein content of the collected cell membranes was determined using the BCA kit. May be from about 3 × 10⁷The membrane material containing about 5mg of membrane protein was extracted from each RAW264.7 cell.

(2) Preparing a nano core: 1mL of acetone dissolved with poly (lactic-co-glycolic acid) -PLGA (PLGA was purchased from Sigma-Aldrich, catalog number 719900, acid-capped, lactide: glycolide 50:50, molecular weight 38000-54000)10mg/mL was added dropwise to 2mL of deionized water, and the mixture was stirred in the open air until the acetone was completely evaporated to obtain PLGA nanonuclei.

(3) And (2) ultrasonically treating the RAW cell membrane and the PLGA nano nucleus for 2 minutes in deionized water by using a bath ultrasonic instrument (Fisher Scientific FS30D, 100W) according to a preset mass ratio (1: 64-2: 1) to obtain the RAW-PLGA nano decoy.

The results show that, at RAW: the PLGA mass ratio is 1:64-2:1, uniform nanometer bait can be obtained, and the nanometer bait has complete coating and proper grain size at the mass ratio of 1:16-1: 4. The obtained nano-bait can be stored at room temperature for one month after preparation without precipitation or particle size change.

The resulting cell membrane-coated nanocomplexes were characterized as follows:

after staining with uranyl acetate (0.2 wt%), the morphology of the RAW-PLGA nano-bait was observed using a transmission electron microscope (TEM, TECNAI G2, FEI, US). See in particular fig. 1. As shown in fig. 1, the nano-bait is in a spherical structure and has a distinct and clear membrane structure.

The hydrodynamic size and zeta potential of the nanoscopic bait were determined using a Zetasizer Nano ZS90(Malvern Instruments, ltd., UK). See in particular fig. 2. As shown in FIG. 2, the nano-bait has a hydrodynamic size of about 109.5nm and a zeta potential of about-14.5 mV.

Macrophage specific surface markers on RAW vesicles and RAW-PLGA nano-baits were examined by Western blot: integrin: MAC-1 and Macrosialon (Macrosialin); cytokine binding receptors: IFN- γ R, TNF- α R, IL-6R and RANK). The concentration of MAC-1, macrosialoprotein, IFN-gamma R, TNF-alpha R, IL-6R, RANK and GAPDH primary antibody is 1:1000, and the concentration of HRP-labeled secondary antibody is 1: 500. See in particular fig. 3. As shown in fig. 3, RAW cell membranes wrapped on the nanonucleus have substantially the same surface marker expression as natural cell membranes, suggesting that they may have similar membrane function as natural cells.

Serum stability of the nano-baits was evaluated by measuring the particle size of the nano-baits in deionized water or DMEM containing 10% FBS. See in particular fig. 4. As shown in fig. 4, the particle size of RAW vesicles changed significantly with time, while the particle size of RAW-PLGA remained essentially unchanged. The results indicate that the rigid PLGA nanocore limits the flow of membrane components wrapped outside it, allowing for improved stability of the whole RAW-PLGA in serum.

In conclusion, the stable nanotopodium bait comprising a PLGA nanonucleus and an osteoclast precursor cell membrane RAW effectively wrapping the nanonucleus, which is capable of providing surface proteins similar to those of corresponding native cells, such as various integrins and cytokine binding receptors, on the surface, is prepared by the method of the present application.

Example two, cell experiments with RAW-PLGA nano-decoys: endocytosis, biocompatibility and immunostimulatory

The endocytosis of the nanocoalts was studied using laser confocal experiments. RAW264.7 cells were plated 4 times per dish×10⁴The number of cells was seeded on a cell culture dish (. PHI.: 20mm) and cultured for 24 hours (the medium was DMEM containing 10% FBS, 37 ℃, 5% CO)₂). After staining the cell membranes with DiI (purchased from Biyunnan biosome, 5. mu.g/mL), the cells were incubated with^DiDPLGA (DiD (purchased from Muyunnan biosciences) was added to a PLGA acetone solution at a PLGA/DiD mass ratio of 1000:1, and after acetone was volatilized, excess DiD (20000g, 15 minutes) or RAW-^DiDPLGA (Using RAW cell membranes and^DiDPLGA nanometer core is prepared in the same method as RAW-PLGA: (^DiDThe final concentration of PLGA component in the medium was 100. mu.g^DiDPLGA/mL) was incubated at 37 ℃ for 4 hours. After three washes with cold PBS, cells were visualized by CLSM (Leica, TCS SP5, Germany). See in particular fig. 5. The results of confocal experiments show that RAW-^DiDThe intracellular fluorescence of PLGA was significantly weaker than that of^DiDPLGA, flow cytometry results also showed RAW-containing protein endocytosed by RAW264.7 cells^DiDThe level ratio of PLGA nano bait is endocytosed^DiDPLGA levels were about 20 times lower. The results show that the proportion of the nanometer bait endocytosed by macrophages is greatly reduced due to the wrapping of the cell membrane. Thus, the RAW-PLGA of the present application can effectively escape macrophage capture, avoiding premature clearance, and thus can more permanently function in vivo.

The biocompatibility and immunostimulatory properties of RAW-PLGA were further investigated. RAW264.7 cells were plated at 1X 10 per well⁴Numbers of cells were seeded in 96-well plates and cultured for 24 hours, and then cells were incubated with RAW-PLGA nano-bait (5-160. mu.g PLGA/mL) for 24 hours at 37 ℃. Cell viability was determined by the MTT method and TNF-. alpha.and IL-6 concentrations in the supernatants were quantified using an ELISA kit (purchased from Sanjia Biochemical Supplies). See in particular fig. 6. The RAW-PLGA nano bait does not affect cell survival rate in the concentration range of 5-160 mug PLGA/mL, and does not stimulate the production of TNF-alpha and IL-6. Thus, the RAW-PLGA of the present application has high biocompatibility and low immunostimulatory function.

Example III in vitro binding and scavenging action of RAW-PLGA Nannoceptory on RANKL

RANKL (purchased from Sigma-Aldrich, 200pg/mL) was mixed with PLGA or RAW-PLGA (1mg PLGA/mL) in deionized water and incubated at 37 ℃ for 2 hours. Then centrifuged at 16100g for 10 min. RANKL concentration in the supernatant was quantified using an ELISA kit. See in particular fig. 7. The results show that: RAW-PLGA bound about 53% RANKL, while PLGA had little binding effect.

RANKL binding was further detected by FRET. RBC-PLGA was used as a negative control. Erythrocytes collected from whole blood of female C57/BL6 mice were resuspended in 0.25 XPBS and centrifuged at 800g for 5 minutes. This procedure was repeated until hemoglobin was completely removed, and the red cell membranes were collected, and the protein content of the collected cell membranes was measured using the BCA kit. Mixing RAW cell membrane or erythrocyte membrane with Cy5-NHS (from Macklin) at a mass ratio of 1000:1, and preparing according to the preparation process of RAW-PLGA^Cy5RBC-PLGA and^Cy5RAW-PLGA。

to be a FRET pair^Cy3RANKL (200pg/mL) and erythrocyte membrane-encapsulated^Cy5RBC-PLGA or^Cy5RAW-PLGA (1mg PLGA/mL) was mixed and incubated as described above. At 565-_ex550nm) was collected. See in particular fig. 8. The results showed that energy transfer occurred in the RAW-PLGA group, while it did not. This result again demonstrates the efficient specific binding of RAW-PLGA nano-decoys to RANKL.

The real-time RANKL clearance in the presence of cells was further examined. RAW264.7 cells were plated at 1X 10 per well⁴Numbers of cells were seeded in 96-well plates and cultured for 24 hours, then cells were incubated with RANKL (100ng/mL) and RAW-PLGA nano-decoy (100 μ g PLGA/mL) at 37 ℃. RANKL clearance efficiency was evaluated by measuring RANKL concentration in the cell culture medium at predetermined time intervals. See in particular fig. 9. RANKL showed a significant downward trend after the addition of RAW-PLGA. The results jointly show that RAW-PLGA has strong specific clearing effect on RANKL.

EXAMPLE four Effect of RAW-PLGA Nannoceptor on osteoclast precursor cells

In order to show that the nano bait blocks the combination between RANKL and RAW264.7 cell membranes, the RAW264.7 cells are arranged at 4X 10 cells per dish⁴The number of the seeds is inoculated on a cell culture dish(20 mm) and cultured for 24 hours. Cells were stained with DiI (5. mu.g/mL, cell membrane stain) and then incubated with^FITCRANKL (100ng/mL) in fresh medium. After 4 hours incubation with RANKL antibody (0.05mg/mL from Abcam) or RAW-PLGA (100. mu.g PLGA/mL as PLGA), cells were washed three times with cold PBS, stained for nuclei with Hoechst 33258 (5. mu.g/mL) and visualized by CLSM. See in particular fig. 10. The results show that the RANKL ratio bound to RAW264.7 cells is greatly reduced after the addition of RAW-PLGA.

The blocking effect of RAW-PLGA on NF-kB pathway was further investigated. Separating mouse bone marrow mononuclear macrophage from mouse bone marrow at a ratio of 1 × 10 per well⁴The number of cells was seeded on a 96-well plate and cultured for 24 hours. The cells were then cultured in fresh medium containing M-CSF (30ng/mL) and RANKL (100ng/mL) and incubated with RAW-PLGA (100. mu.g PLGA/mL) for 24 hours. Real-time PCR was used to quantify c-Fos mRNA levels. See in particular fig. 11. Abnormally elevated levels of osteoclast-associated transcription factor c-Fos were reduced by 81% following RAW-PLGA treatment.

And simultaneously, the blocking effect of RAW-PLGA on MAPK pathway is researched. Mouse bone marrow mononuclear macrophages were isolated and treated as described above. p-ERK, p-p38 and p-JNK protein levels were quantified using ELISA. See in particular fig. 12. After the treatment of RAW-PLGA, the levels of p-ERK, p-p38 and p-JNK are all obviously reduced compared with the RANKL treatment group, and the levels are basically restored to normal levels.

Thereafter, the inhibitory effect of RAW-PLGA on osteoclast differentiation was studied in bulk. Mouse bone marrow mononuclear macrophages (BMM) were cultured in fresh medium containing M-CSF (30ng/mL) and RANKL (100ng/mL) and incubated with RAW-PLGA nano-bait (100 μ g PLGA/mL) for 96 hours. Cells were fixed with 4% formalin buffer for 10 min, washed three times with PBS, stained using TRAP staining kit, and imaged under an inverted microscope (Leica DM4000, Solms, Germany). See in particular fig. 13. The results show that: BMM was induced to differentiate into large multinucleated osteoclasts (fig. 13, panel), whereas upon treatment with RAW-PLGA, osteoclasts were dramatically reduced in number and diameter, with morphology close to that of the uninduced control cells (fig. 13, left panel vs. right panel).

The results jointly indicate that the RAW-PLGA has strong inhibiting effect on the differentiation of osteoclast.

EXAMPLE V in vivo half-Life Studies of RAW-PLGA Nanodebait

In order to demonstrate that RAW-PLGA has a longer circulation time in vivo, the pharmacokinetics of RAW-PLGA following intravenous injection was studied.

Female C57/BL6 mice (6-8 weeks, 18-20g, purchased from Shanghai Leike laboratory animals, Inc., four mice/cage were housed in a clean room, water ad libitum, 12:12 hours light and dark cycle, temperature 25. + -. 1 ℃ C. animal laboratory protocol reviewed and approved by the institutional animal Care and use Committee of Suzhou university) at 10mg^DiDPLGA/kg or 2.5mg^DiDDose of RAW vesicles/kg for intravenous injection^DiDPLGA、^DiDRAW vesicles (prepared by mixing RAW cell membranes and DiD at a mass ratio of 1000:1 followed by sonication for 2 minutes), RBC-^DiDPLGA or RAW-^DiDPLGA. Blood was collected at predetermined time points and analyzed by spectrofluorimetry (lambda)_ex＝644nm，λ_em663nm) in plasma^DiDPLGA or^DiDContent of RAW. Calculating the circulating half-life (t)_1/2). See in particular fig. 14. RAW-PLGA shows a ratio PLGA (t)_1/24.08 hr) and RAW vesicles (t)_1/23.11 hours) significantly prolonged blood circulation time, t_1/2It was 13.26 hours.

The results suggest that the RAW-PLGA nano-bait of the present application has a longer circulating half-life in vivo, and can exert its therapeutic effect more effectively than drugs (e.g., proteinaceous drugs) having a shorter circulating half-life.

EXAMPLE VI in vivo anti-osteoporosis Effect of RAW-PLGA Nanodebait

The improvement of the biochemical indexes related to osteoporosis by RAW-PLGA is detected in vivo. A bilateral ovariectomy was performed on female C57/BL6 mice to establish an OVX mouse (ovariectomized mice) model. In the next 60 days, PLGA or RAW-PLGA was injected intravenously every 3 days at a dose of 10mg PLGA/kg and an equal volume of PBS was injected as a negative control. On day 60, mice were sacrificed and peripheral blood was collected and centrifuged at 500g for 10 minutes at 4 ℃ to extract serum. RANKL levels in serum were quantified using an ELISA kit. See in particular fig. 15. Serum RANKL concentrations in RAW-PLGA treated OVX mice were reduced by 57% (p <0.0001) compared to the OVX group, returning almost to normal levels.

The femoral tissues were further examined for mRNA levels associated with RANKL (RANKL/RANK/OPG system genes: RANKL, RANK, TRAF6), key factors associated with osteoclastogenesis (osteoclast-associated transcription factors: NFATc1, c-Fos; and osteoclast-specific genes: ctSK, TRAP, RECK) and key factors associated with bone resorption (MMP-2, MMP-9 and MMP-13). Femoral bone tissue was frozen in liquid nitrogen and homogenized with Trizol reagent to isolate total RNA and the corresponding mRNA levels were detected using real-time PCR. The sequences of 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 respectively shown as SEQ ID NO:1-24 in the sequence table.

The results are shown in particular in FIG. 16. After RAW-PLGA treatment, key factors of the RANKL/RANK/OPG system (RANKL, RANK and TRAF6) decreased to normal levels, indicating that the entire system had rebalanced. Meanwhile, RAW-PLGA not only restored abnormally up-regulated osteoclast-associated transcription factors (NFATc1 and c-Fos) to normal levels, but also suppressed the expression levels of osteoclast-specific genes (ctsK, TRAP and RECK), indicating that it has a strong ability to suppress osteoclast differentiation. While bone resorption related genes (MMP-2, MMP-9 and MMP-13) are substantially restored to normal levels, which would further benefit anti-osteoporosis as well as bone formation.

Bone resorption and bone formation during postmenopausal osteoporosis were also assessed by detecting biochemical markers of bone turnover. TRACP-5b, BGP and BAP levels in serum were quantified using an ELISA kit. See in particular fig. 17. The levels of these indicators also returned almost to normal levels after the RAW-PLGA treatment.

EXAMPLE seventhly, histological effects of RAW-PLGA NanoPredecoy treatment on bone tissue

The femurs of OVX mice treated with RAW-PLGA were subjected to histological analysis. On day 60 after bilateral ovariectomy (dosing schedule same as example six), mice were sacrificed, femoral tissues were harvested, fixed in 10% formalin buffer, and then incubated in decalcifying solution (14% EDTA) at room temperature for 1 month for decalcification. Then, the femur was embedded in paraffin and cross-sectioned at a thickness of 8 μm. The number of osteoclasts per bone surface (n.oc/BS) was calculated using a TRAP staining kit for staining. See in particular fig. 18. RAW-PLGA effectively reduced the extent of osteoclast erosion to trabeculae, and reduced the osteoclast number by 61%, almost returning to normal level.

EXAMPLE eight imaging Effect of RAW-PLGA NanoPredecoy treatment on bone tissue

The recovery of femoral integrity was further analyzed by micro CT. On day 60 after bilateral ovariectomy (dosing schedule same as example six), right femoral specimens of OVX mice were scanned using micro-CT (Skyscan 1176). A high-resolution scan (9-20mm) was obtained (resolution: 8.8mm, source voltage: 50kV, source current: 500mA, rotation step: 0.7U). The data set was reconstructed using CT analyzer software (Skyscan) to obtain 3D images of the femoral tissue and to measure morphometric parameters. Bone erosion for micro-CT scans was calculated based on the reconstructed data and internally written Fiji scripts. The procedure defines the bone surface and the bone interior space and fills the pores of the bone surface. A region of interest (ROI) in the trabecular bone is selected for analysis of the following morphometric parameters, including: (1) bone Mineral Density (BMD), (2) bone volume density ratio (BV/TV), (3) trabecular number (tb.n) and (4) trabecular spacing (tb.sp). See in particular fig. 19. The results show that RAW-PLGA very significantly inhibited ovariectomy-induced bone loss and reduced osteopenic phenotype in the trabeculae. In addition, BMD, BV/TV, Tb.N and Tb.Sp returned to normal levels after treatment with RAW-PLGA.

The experimental evidence strongly proves the great potential of the RAW-PLGA nano bait for postmenopausal osteoporosis.

EXAMPLE nine in vivo biocompatibility of RAW-PLGA Nanocolures

PBS (200. mu.L) or RAW-PLGA nanocomplexes (10mg PLGA/kg, 200. mu.L) were injected intravenously into female C57/BL6 mice, following the same dosing schedule as in example six. Blood and major organs (heart, liver, spleen, lung and kidney) were collected. Hematological evaluations were performed on a Cobas501 automated hematology analyzer (Roche, USA). Serum biochemical parameter levels were determined using a BC-5380 automated chemical analyzer (Mindray, China). See in particular fig. 20. Major organs were fixed in 10% formalin, embedded in paraffin, cross-sectioned at a thickness of 8 μm, stained with H & E, and observed by whole body fluoroscopy optical microscopy. See in particular fig. 21.

The results show that mice treated with RAW-PLGA nano-bait have no abnormalities in representative hematological and biochemical parameters. Furthermore, in H & E stained main organ cross sections, no necrosis, inflammation, edema or other pathological symptoms were detected. These results indicate that the RAW-PLGA nano-bait has good biocompatibility and high safety after systemic administration.

All documents mentioned in this application are incorporated by reference into this application as if each were individually incorporated by reference. Further, it should be understood that various changes or modifications can be made to the present application by those skilled in the art after reading the above teachings of the present application, and these equivalents also fall within the scope of the present application as defined by the appended claims.

Sequence listing

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Claims

1. A nano-bait, comprising:

(a) a nano-core; and

(b) an osteoclast precursor cell membrane coating the nanocore.

2. The nano-bait of claim 1, wherein the nano-core is made of one or more materials selected from the group consisting of: polymeric nanoparticles such as polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), Polycaprolactone (PCL), polylysine, polyglutamic acid, poly n-butyl cyanoacrylate (PBCA), chitosan, gelatin; inorganic nanoparticles such as gold, silicon, iron or copper; and/or

The nanocore has one or more characteristics selected from the group consisting of: is negatively charged; the particle size is 50-200 nm.

3. The nano-bait of claim 1, wherein,

the osteoclast precursor cell is derived from: bone marrow hematopoietic stem cells, spleen stem cells, bone marrow or blood mononuclear cells, giant cell tumors of bone, myeloid dendritic cells, osteoclast-like cells;

for example, the osteoclast precursor cell is selected from: RAW264.7 cells, human bone marrow or peripheral blood mononuclear/macrophage, ANA-1 cells, J774A.1 cells, THP-1 cells; and/or

The osteoclast precursor cell is selected from: natural osteoclast precursor cell, osteoclast precursor cell formed by induced differentiation, osteoclast precursor cell engineered by gene engineering; and/or

The osteoclast precursor cell is derived from a human, a non-human primate (e.g., orangutan, ape, monkey), a rodent (e.g., rat, mouse, guinea pig, hamster, rabbit), an artiodactyl (e.g., sheep, cow, pig, camel, alpaca), an ungulate (e.g., horse); and/or

The osteoclast precursor cell is derived from: the subject to be administered is autologous, allogeneic to the subject, or a different species from the subject.

4. The nanopaste of claim 1, wherein the osteoclast precursor cell membrane expresses macrophage specific surface markers, such as one or more molecular markers selected from the group consisting of: RANK, MAC-1, macrosialoprotein, IFN- γ R, TNF- α R, and IL-6R; and/or

The osteoclast precursor cell has the following characteristics: the native structural integrity (e.g., primary, secondary, tertiary, or quaternary structural integrity) or activity (e.g., binding activity, receptor activity, signaling pathway activity) inherent to the cell membrane is maintained or retained.

5. The nano-bait according to claim 1, wherein the mass ratio of the cell membrane to the nano-core is 1:100 to 1:0.1, or 1:80 to 1:20, 1:64 to 1: 4; and/or

The nanocore is PLGA, and the cell membrane is the cell membrane of mononuclear macrophage (such as RAW264.7 cell line); and/or

The shape of the nano bait is spherical, cubic, conical, cylindrical, prismatic, pyramidal or other regular or irregular shapes; the size range is 1 nanometer to 10 micrometers or any value or range of values therebetween.

6. The nano-bait of any of claims 1 to 5, wherein the nano-bait has one or more of the following characteristics selected from the group consisting of:

(1) has the ability to specifically bind to and clear RANKL;

(2) decreased macrophage endocytosis compared to unloaded nanonuclei;

(3) have an extended in vivo half-life (e.g., a circulation half-life of more than 10 hours) compared to unloaded nanonuclei;

(4) reduce mRNA and protein levels of gene-related factors of the RANKL/RANK/OPG system (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).

7. A method of preparing a nano-bait according to any one of claims 1 to 6, the method comprising:

(A) providing a nanocore;

(B) providing a cell membrane of an osteoclast precursor cell;

(C) wrapping the cell membrane onto the nanocore to form the decoy;

for example, the step (a) is performed by one or more methods selected from the group consisting of: nano-precipitation, emulsion solvent evaporation, ionic gel, direct dissolution, dialysis, emulsification, media milling, high pressure homogenization, supercritical fluid, quasi-emulsion solvent diffusion, solid reversed phase micelle solution; and/or

The step (B) is performed by lysis and component separation of osteoclast precursor cells, for example the lysis comprises: ultrasonic lysis, enzymatic lysis, chemical lysis, homogenate lysis and/or hypotonic swelling lysis; the separation comprises: centrifugation (e.g., stepwise), precipitation, filtration, magnetic beads, chromatographic separation; and/or

Said step (C) comprises applying an external force to encapsulate said cell membrane onto said nanocore, for example using sonic (e.g. ultrasound), mechanical co-extrusion, electroporation, heating; and/or

In one embodiment, the method comprises: osteoclast precursor cells are cracked by ultrasonic, and cell membranes are obtained by step-by-step centrifugation; carrying out ultrasonic treatment on the obtained cell membrane and the nano nucleus together to obtain nano bait;

preferably, the osteoclast precursor cell is a RAW264.7 cell, the nano-core is PLGA, and the mass ratio of the cell membrane to the nano-core is 4: 1; and/or the collective sonication is 100W for 2 minutes.

8. A product, comprising:

a nano-bait according to any one of claims 1 to 6 or components (a) and (b) as defined in any one of claims 1 to 6; and, optionally, a pharmaceutically or physiologically acceptable carrier.

9. Use of a Nanodecoy according to any one of claims 1 to 6, or a composition of components (a) and (b) thereof, for the preparation of a product for the prevention and/or treatment of a disease associated with osteoclastogenesis hyperactivity or hyperfunction (e.g. RANKL hyperactivity).

10. The use according to claim 9, wherein the disease is selected from: degenerative or lost bone conditions, such as osteoporosis, particularly osteoporosis due to altered levels of estrogen (e.g. menopause), periodontitis, periapical periodontitis, peri-implantitis; bone metastasis from cancer (e.g., breast, prostate, lung); arthritis (e.g., chronic arthritis); osteitis deformans; myeloma (e.g., multiple myeloma); osteocarcinoma large cell carcinoma.