Novel theragnostic nanocomposite comprising NMDA receptor antibodies and fluorescent compounds, and uses thereof

Abstract

A nanocomposite according to the present invention, comprising mesoporous silica nanoparticles (MSNs), fluorescent compounds, and NMDA receptor marker antibodies, is a theragnostic nanocomposite material which enables early diagnosis of inflammatory diseases and simultaneous treatment thereof, by having the NMDA receptor marker antibodies that bind to NMDA receptors when inflammatory macrophages are activated at an inflammatory site. The nanocomposite can be widely used for the diagnosis and treatment of inflammatory diseases and monitoring of the treatment process.

Description

Novel theranostic nanocomposite comprising NMDA receptor antibody and fluorescent substance and use thereof

[0001] The present invention relates to a theranostic nanocomposite based on mesoporous silica nanoparticles (MSN) that can simultaneously perform early diagnosis and treatment of inflammatory diseases.

[0002] The inflammatory response is a biological process that defends the body against external pathogens, and is characterized by fever, redness, swelling, pain, and loss of function. The inflammatory response induced by macrophages is a representative innate immune response and is initiated by the binding of pathogen-associated molecular patterns (PAMPs) to pattern recognition receptors (PRRs) expressed on the surface of macrophages. Lipopolysaccharides (LPS) are substances that induce macrophage-mediated inflammatory responses. They bind to receptors expressed on the surface of macrophages and induce inflammatory responses through the secretion of inflammatory cytokines such as tumor necrosis factor (TNF)-α and interferon (IFN)-γ.

[0003] The NMDA (N-methyl-D-aspartate) receptor, a neuroreceptor that regulates neuronal signaling in inflammatory macrophages, is a tetramer composed of protein subunits GluN1, GluN2, and GluN3. It has a protein domain on the outside of the postsynaptic membrane that can bind ligands such as glutamate and glycine, a transmembrane domain that crosses the postsynaptic membrane, and a cytoplasmic domain on the inside of the postsynaptic membrane.

[0004] Porous silica nanoparticles are materials capable of delivering DNA and chemicals into animal cells or skin. Their large surface area, ease of processing, and ability to maintain a consistent shape make them a widely used drug delivery material. Porous silica nanoparticles are classified into micro, meso, and macro categories based on pore size.

[0005] Mesoporous silica nanoparticles (MSN) are silica materials with regularly arranged mesopores of extremely uniform size, ranging from 2 to 50 nm. MSNs must be uniform in size to ensure consistent and controlled interactions with cells. Based on these properties, silica nanoparticles are being utilized as carriers in drug delivery systems (DDS).

[0006] Theragnosis is a concept that enables early diagnosis and simultaneous treatment of diseases, enabling real-time evaluation of treatment effects. Theragnosis requires non-invasive imaging modalities such as optical imaging, magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography (PET). Optical imaging relies on fluorescence or bioluminescence, and the fluorescent material used for optical imaging utilizes light in the near-infrared region, with a wavelength of 650 to 900 nm.

[0007] Against this backdrop, the inventors of the present invention have confirmed that inflammatory diseases can be diagnosed and treated simultaneously when NMDA receptors associated with the activity of inflammatory macrophages are labeled on nanoparticles, thereby completing the present invention.

[0008] [Prior Art Literature]

[0009] [Patent Document]

[0010] Republic of Korea Patent Publication No. 10-2015-0079436 (July 8, 2015)

[0011] The purpose of the present invention is to provide a theranostic nanocomposite capable of diagnosing inflammatory diseases at an early stage and simultaneously treating them.

[0012] To achieve the above object, the present invention provides a nanocomposite comprising mesoporous silica nanoparticles (MSN); a fluorescent material bound to the surface or pores of the nanoparticles; and an NMDA receptor labeling antibody bound to the surface of the nanoparticles.

[0013] The present invention also provides a contrast agent comprising the nanocomposite as an active ingredient.

[0014] The present invention also provides a composition for treating and diagnosing inflammatory diseases, which comprises the nanocomposite as an active ingredient and has an anti-inflammatory drug loaded inside the nanocomposite.

[0015] The present invention also provides a method for preparing a nanocomposite, comprising the steps of: (a) preparing a first nanoparticle by conjugating PEG-maleimide to a mesoporous silica nanoparticle; (b) preparing a second nanoparticle by conjugating a fluorescent material to the first nanoparticle; and (c) conjugating an NMDA receptor-labeled antibody and a hydrophilic biocompatible polymer to the second nanoparticle.

[0016] The present invention also provides a method for preventing or treating an inflammatory disease, comprising administering to a subject a pharmaceutically effective amount of the composition.

[0017] The present invention also provides a method for diagnosing an inflammatory disease, comprising the steps of: administering the composition to a subject in a pharmaceutically effective amount; and detecting a fluorescent signal from the composition.

[0018] The nanocomposite comprising mesoporous silica nanoparticles (MSN), a fluorescent material, and an NMDA receptor-labeled antibody according to the present invention enables direct observation of macrophages involved in the early stages of an inflammatory response, thereby facilitating the early diagnosis of inflammatory diseases and effective treatment simultaneously. Furthermore, it is non-toxic, highly biocompatible, and allows for real-time monitoring of inflammatory substances, enabling the tracking of inflammatory substances and the assessment of therapeutic efficacy of drugs.

[0019] Figure 1 is a schematic diagram showing the mechanism of NMDA receptors, the in vivo distribution and therapeutic effect of a nanocomplex containing an NMDA receptor according to the present invention.

[0020] FIG. 2A is a schematic diagram of a manufacturing process of a nanocomposite according to the present invention, FIG. 2B shows the results of a fluorescence analysis image confirming whether a fluorescent dye is loaded into the nanocomposite, FIG. 2C shows the results of observing the size of the nanocomposite using a transmission electron microscope (TEM), and FIG. 2D shows the results of a wavelength analysis confirming whether a fluorescent dye is loaded into the nanocomposite.

[0021] Figure 3 shows the results of confirming the synthesis process of each nanocomposite using Fourier transform infrared spectroscopy (FT-IR).

[0022] Figure 4A shows the results of confirming the pore structure and size of each nanocomposite using a nitrogen gas adsorption method, and Figure 4B shows the results in a table.

[0023] Figure 5 shows the results of confirming the presence or absence of synthesis for each nanocomposite using zeta potential analysis.

[0024] Figure 6A shows the results of evaluating the toxicity of the nanocomplex confirmed through CCK-8 analysis, and Figure 6B shows the results of confirming the binding of the nanocomplex to bone marrow-derived macrophages (BMDM) through FACS analysis.

[0025] Fig. 7A is a schematic diagram of the experimental design to confirm whether the nanocomplexes label inflammation depending on whether they contain NMDA receptors, Fig. 7B is the experimental results confirming whether labeling occurs at the site of inflammation after nanocomplex injection through a chronic inflammation animal model experiment, Fig. 7C is the experimental results confirming the labeling efficiency at the site of inflammation 24 hours after nanocomplex injection through a chronic inflammation animal model experiment, Figs. 7D and 7E are the results confirming the labeling of inflammatory macrophages depending on whether they contain NMDA receptors through image analysis figures, and Fig. 7F is a bar graph showing the experimental results of Fig. 7C.

[0026] Figure 8A shows the results of an experiment in which the labeling efficiency at the site of inflammation was confirmed through a chronic inflammation animal model experiment depending on whether the anti-inflammatory agent dexamethasone (DEX) was treated 24 hours after the injection of the nanocomposite, and Figures 8B and 8C show the results of confirming the labeling of inflammatory macrophages through image analysis figures depending on whether the NMDA receptor was included and whether the dexamethasone was treated.

[0027] Figure 9A shows the results of analyzing the size of the sole and the fluorescence image of the inflamed area by time period after injection of carrageenan (CG), an inflammatory substance, and PBS to determine the process of inflammation induction and treatment. Figures 9B and 9C show the results of analyzing the image signal to determine the effect of reducing inflammation when the inflammatory substance was injected, and the results of analyzing the size of the sole. Figures 9D and 9E show the results of analyzing the image signal to determine the effect of reducing inflammation when the inflammatory substance was injected, and the results of analyzing the size of the sole.

[0028] Figure 10A shows the results of confirming the inflammation induction and treatment process by time zone analysis of the size of the sole and fluorescent image of the inflamed area after injection of LPS and PBS, which are inflammatory substances; Figures 10B and 10C show the results of confirming the inflammation reduction effect by image signal analysis and the results of the sole size analysis when the inflammatory substance was injected; Figures 10D and 10E show the results of confirming the effect by image signal analysis and the results of the sole size analysis when the inflammatory substance was not injected.

[0029] Figures 11a to 11c show the results of confirming the presence or absence of inflammation in each organ using optical imaging equipment after injecting TP, a chronic inflammatory substance, and CG and LPS, acute inflammatory substances, into muscles and the soles of the feet.

[0030] Hereinafter, the present invention will be described in detail.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0032] When the present invention is said to “include” a certain component or a certain step, this does not mean that other components or other steps are excluded, but rather that other components or other steps may be further included, unless specifically stated otherwise.

[0033] In the present invention, the term "inflammatory disease" is a general term for diseases in which inflammation is the main lesion, and the inflammatory disease is not particularly limited thereto as long as the symptoms thereof can be alleviated, relieved, improved or treated by the nanocomposite of the present invention, or a contrast agent or composition containing the nanocomposite as an active ingredient.

[0034] In the present invention, the term “prevention” means any act of inhibiting or delaying the onset of an inflammatory disease by administering a nanocomposite according to the present invention, a contrast agent or composition containing the nanocomposite as an active ingredient.

[0035] In the present invention, the term "treatment" means any act in which the symptoms of a subject suspected of having or developing an inflammatory disease are improved or beneficially changed by administering the nanocomposite, a contrast agent or composition containing the nanocomposite as an active ingredient.

[0036] In the present invention, the term "subject" may mean a mammal suffering from an inflammatory disease or at risk of such a disease by administering (applying) the nanocomposite according to the present invention, a contrast agent or composition containing the nanocomposite as an active ingredient, and preferably means a human.

[0037] The present invention provides a nanocomposite comprising mesoporous silica nanoparticles (MSN); a fluorescent material bound to the surface or pores of the nanoparticles; and an NMDA receptor labeling antibody bound to the surface of the nanoparticles.

[0038] The above mesoporous silica nanoparticles are nanoparticles with regularly arranged pores of 2 to 50 nm in size, and are nanomaterials widely used in the fields of catalysts, adsorbents, polymer binders, optical devices, bio-imaging materials, drug delivery agents, and biomedicine.

[0039] The above nanocomposite may additionally include a hydrophilic biocompatible polymer coated on the surface of the nanoparticle.

[0040] The hydrophilic biocompatible polymers include polyethylene glycol (PEG), poly(carboxybetaine) (pCB), poly-(sulphobetaine) (pSB), phosphobetaine polymers, methoxy polyethylene glycol (mPEG), poly(ethylene glycol) monomethyl ether, phosphatidylcholines (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), monodisperse PEG, branched PEG, and multi-arm polyethylene glycol. It may be at least one selected from the group consisting of PEG), preferably methoxy polyethylene glycol (mPEG), but is not limited thereto.

[0041] The above biocompatible polymer can play a role in improving blood circulation.

[0042] The average pore size of the above nanocomposite may be 2 to 50 nm, preferably 2 to 10 nm, but is not limited thereto.

[0043] The fluorescent material may be at least one selected from the group consisting of DID (1,1'-Dioctadecyl-3,3,3',3'-tetramethlindodicarbocyanine), ICG (indocyanine green), methylene blue, IRDye800CW, Cy5.5 (Sulfo-Cyanine5.5), and Flamma 675, and is preferably DID (1,1-Dioctadecyl-3,3,3,3-tetramethlindodicarbocyanine), but is not limited thereto.

[0044] The above fluorescent material is a near-infrared fluorescent material, and can absorb light in the wavelength range of 700 to 900 nm and emit fluorescence, but is not limited thereto.

[0045] In the case of GFP, a fluorescent material used in the past, there was a disadvantage that the 500 nm wavelength range in which GFP emits light overlapped with the wavelength range of biological substances in the body, making it difficult to distinguish the fluorescence of GFP alone. Proteins in the body, such as hemoglobin in the blood, absorb light in the visible wavelength range, and water and fat absorb light in the infrared wavelength range. Considering this, it can be seen that the influence of spontaneous luminescence can be minimized in the near-infrared region of 700 to 900 nm. Since the near-infrared wavelength is a long wavelength, it is harmless to the human body, has good tissue penetration, and the signal-to-background ratio is optimized to minimize background fluorescence, so visualization can be maximized even when the fluorescence signal is weak, and it is widely used in near-infrared imaging.

[0046] The above fluorescent material can be linked to the surface or pores of the nanoparticles through electromagnetic and amine bonds.

[0047] The above NMDA receptor labeling antibody can be linked to the surface of the nanoparticle via a maleimide bond.

[0048] The above hydrophilic biocompatible polymer can be coated on the surface of nanoparticles through an amine bond.

[0049] The size of the above nanocomposite may be 50 to 200 nm, preferably 100 to 150 nm, but is not limited thereto.

[0050] The present invention also provides a contrast agent comprising the nanocomposite as an active ingredient.

[0051] The present invention also provides a composition for treating and diagnosing inflammatory diseases, which comprises the nanocomposite as an active ingredient and has dexamethasone, an anti-inflammatory drug, loaded inside the nanocomposite.

[0052] The above inflammatory disease may be at least one selected from the group consisting of inflammatory bowel disease, atopic dermatitis, edema, dermatitis, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, atherosclerosis, pharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, inflammatory spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis of the shoulder joint, tendonitis, tenosynovitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, and multiple sclerosis, but is not limited thereto.

[0053] The composition may be a pharmaceutical composition, and the pharmaceutical composition may be administered to a mammal, including a human, by any method. For example, the composition may be administered orally or parenterally, and parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topically, sublingually, or rectally, or may be applied to the skin.

[0054] The pharmaceutical composition may be formulated as a preparation for oral administration or parenteral administration according to the route of administration as described above. When formulated, it may be prepared using one or more buffers (e.g., saline or PBS), carbohydrates (e.g., glucose, mannose, sucrose, or dextran, etc.), antioxidants, bacteriostats, chelating agents (e.g., EDTA or glutathione), fillers, bulking agents, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrating agents, or surfactants, diluents, or excipients.

[0055] Solid preparations for oral administration include tablets, pills, powders, granules, liquids, gels, syrups, slurries, suspensions, capsules, etc., and these solid preparations can be prepared by mixing the pharmaceutical composition of the present invention with at least one excipient, for example, starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethyl-cellulose, or gelatin. For example, tablets or sugar-coated tablets can be obtained by mixing an active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and then processing the mixture into a granule mixture.

[0056] In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to the commonly used simple diluents, such as water or liquid paraffin, various excipients may be included, such as wetting agents, sweeteners, flavoring agents, or preservatives.

[0057] Additionally, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrating agent in some cases, and anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives may be additionally included.

[0058] When administered parenterally, the pharmaceutical composition of the present invention may be formulated in the form of injections, transdermal administration agents, and nasal inhalants together with a suitable parenteral carrier according to methods known in the art. In the case of injections, they must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for injections include, but are not limited to, solvents or dispersion media containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and / or vegetable oils. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. To protect the above injection from microbial contamination, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal, may be additionally included. In addition, the above injection may, in most cases, additionally include an isotonic agent, such as sugar or sodium chloride.

[0059] Transdermal administration agents include ointments, creams, lotions, gels, topical solutions, pastes, liniments, and aerosols. "Transdermal administration" as used herein refers to topically administering a pharmaceutical composition to the skin, thereby delivering an effective amount of the active ingredient contained in the pharmaceutical composition into the skin.

[0060] For inhalation dosage forms, the active ingredient used according to the present invention may conveniently be delivered in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant, such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or another suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. For example, gelatin capsules and cartridges for use in inhalers or insufflators may be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in the well-known prescription book of all pharmaceutical chemistry (Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour).

[0061] The present invention also provides a method for preparing a nanocomposite, comprising the steps of: (a) preparing a first nanoparticle by conjugating PEG-maleimide to a mesoporous silica nanoparticle; (b) preparing a second nanoparticle by conjugating a fluorescent material to the first nanoparticle; and (c) conjugating an NMDA receptor-labeled antibody and a hydrophilic biocompatible polymer to the second nanoparticle.

[0062]

[0063] The present invention also provides a method for preventing or treating an inflammatory disease, comprising administering to a subject a pharmaceutically effective amount of the composition.

[0064]

[0065] The present invention also provides a method for diagnosing an inflammatory disease, comprising the steps of administering the composition to a subject in a pharmaceutically effective amount; and detecting a fluorescent signal from the composition.

[0066]

[0067] The description of the above manufacturing method, prevention or treatment method and diagnosis method, the effects thereof and all related descriptions are the same as those described above, so the description thereof is omitted to avoid excessive complexity of this specification due to duplicate description.

[0068]

[0069] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0070]

[0071] Example 1. Experimental materials and methods

[0072]

[0073] 1-1. Experimental materials

[0074]

[0075] Hexadecyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), and aminopropyltriethoxysilane (APTES) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Methoxy-PEG24-NHS ester (m-PEG24-NHS ester) and maleimide PEG-NHS (Mal-PEG2-NHS ester) were purchased from NANOCS (NY, USA). Phosphate-buffered saline (PBS, pH 7.4) was supplied by BioWorld (Seongnam, Korea). 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DID) was purchased from Thermo Scientific (Rockford, USA). Lipopolysaccharide (LPS) from Escherichia coli O111:B4, dizocilpine (MK-801), glutamate, N-methyl-D-aspartate (NMDA), rapamycin, 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and trifluoperazine (TFP) were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). Rabbit IgG isotype control was purchased from Abcam (Cambridge, United Kingdom). NMDAR1 polyclonal antibody was purchased from Thermo Fisher Scientific Co.(Waltham, MA, USA). CellMask green plasma membrane stain and Hoechst 33342 (trihydrochloride) were purchased from Thermo Fisher Scientific.

[0076]

[0077] 1-2. Nanocomposite manufacturing method

[0078]

[0079] Mal-PEG-MSNs, which are mesoporous silica nanoparticles (MSNs) with carboxyl groups (-COOH) on their surfaces, were labeled with the fluorescent dye DID (1,1-Dioctadecyl-3,3,3,3-tetramethlindodicarbocyanine) in PBS (pH 7.4) at room temperature within 1 hour, thereby generating Mal-DID@MSNs. After that, NMDA receptor-labeled antibody, which is an inflammation marker antibody, and mPEG (methoxy polyethylene glycol) that improves blood flow were mixed in a 5:5 ratio based on molecular weight, and then reacted for 24 hours using dilution water (DW) at room temperature to prepare nanocomposites containing mesoporous silica nanoparticles (MSNs), fluorescent substances, hydrophilic biocompatible polymers, and NMDA receptor-labeled antibodies (NMDAR-PEG-DID@MSNs) and nanocomposites containing IgG-labeled antibodies (IgG-PEG-DID@MSNs). In addition, for chemical analysis, nanocomposites excluding only fluorescent substances (NMDAR-PEG-MSNs and IgG-PEG-MSNs) and nanocomposites excluding fluorescent substances, NMDA receptor-labeled antibodies, and IgG-labeled antibodies (Mal-PEG-MSNs, Mal-MSNs) were prepared using the same method.

[0080]

[0081] Example 2. Structural evaluation of nanocomposites

[0082]

[0083] The manufactured nanocomposite was tested for loading of fluorescent dye using an IVIS (In vivo imaging system) bioimaging system (Fig. 2B). As a result, it was confirmed that the DID fluorescence wavelength signal was synthesized by the IVIS fluorescence imaging system.

[0084] The size of the manufactured nanocomposite was analyzed by TEM (Fig. 2C). As a result, it was confirmed that the synthesized mesoporous silica nanoparticles were synthesized with a size distribution of 100 to 150 nm.

[0085] The shape and dye loading of the manufactured nanocomposite were confirmed by UV-vis absorption spectrum (Fig. 2D). As a result, DID fluorescence confirmed the synthesis of nanoparticles by presenting the same silica wavelength and fluorescence wavelength.

[0086]

[0087] Example 3. Evaluation of the Synthesis Process of Nanocomposites

[0088]

[0089] The synthesis process of NMDAR-PEG-DID@MSNs, IgG-PEG-DID@MSNs, NMDAR-PEG-MSNs, IgG-PEG-MSNs, Mal-PEG-MSNs, Mal-MSNs nanocomposites, and MSNs nanoparticles was analyzed by Fourier-transform infrared spectroscopy (FTIR) (Fig. 3).

[0090] 1800 ~ 1600 cm, the stretching vibration peak of the carboxyl group (-COOH), which is the reactive group of MSNs -1 and 3000 ~ 2700 cm -1 , 800 ~ 500 cm, which is the stretching vibration peak of the amine group (NH2).-1 and 3500 ~ 3100 cm -1 And the stretching vibration peak of maleimide is 1750 ~ 1650 cm -1 The synthesis of nanomedicines was confirmed through peak generation and reduction of functional groups such as .

[0091]

[0092] Example 4. Confirmation of the pore structure and size of the nanocomposite.

[0093]

[0094] The pore structure and size of each nanocomposite were confirmed by nitrogen gas adsorption method (Fig. 4a and Fig. 4b). The synthesis of MSN was confirmed by changes in BET (Brunauer-Emmett-Teller) surface area, pore volume, and pore size according to the synthesis process. The analysis of surface area, pore volume, and pore size through nitrogen analysis confirmed that the surface area volume, pore volume, and pore size within the nanoparticles changed according to each synthesis process, confirming that the structure of the nanoparticles changed according to the synthesis.

[0095]

[0096] Example 5. Reaction analysis by manufacturing process of nanocomposites

[0097]

[0098] Zeta potential, conductivity, and standard deviation were measured for the surface changes by introducing functional groups such as amine (NH2) and carboxyl (COOH) groups. Further verification was performed using zeta potential analysis after each step. MSNs (-16.2 mV), Mal-MSNs (18.4 mV), Mal-PEG-MSNs (3.64 mV), IgGPEG-MSNs (23.4 mV), NMDAR-PEG- MSNs (24.6 mV), IgG-PEG-DID@MSNs (23.4 mV), and NMDAR-PEGDID@MSNs (23.1 mV) were identified. The above results confirmed the reaction between carboxyl groups and amines, and at the same time, confirmed that the synthesized materials for each reaction process were successfully labeled (Fig. 5).

[0099]

[0100] Example 6. Evaluation of cell stability and efficacy

[0101]

[0102] To evaluate the toxicity of NMDAR-PEG-DID@MSNs, 2 x 10 bone marrow-derived macrophages (BMDMs) were injected at 0, 1, 2, 4, and 8 mg / mL. 4 After the nanoparticles were placed into cells and cultured in a CO2 incubator at 37 °C for 24 hours, the nanoparticles were removed and cytotoxicity was confirmed through a CCK-8 assay. The experimental results confirmed that the cells were stable without statistical significance (Fig. 6A).

[0103] To confirm the functional binding of NMDAR-PEG-DID@MSNs to the cell membrane of LPS-stimulated BMDM, we performed fluorescence-activated cell sorter (FACS) analysis to visualize the cell surface binding of imaging probes (Fig. 6B). Intact and LPS-stimulated BMDM were cultured with NMDAR-PEG-DID@MSNs (or IgG-PEG-DID@MSNs) and M1 macrophage-specific antibodies, such as CD11b and F4 / 80 Ab. FACS analysis revealed that NMDAR-PEG-DID@MSNs, but not IgG-PEG-DID@MSNs, shifted the basal expression of NMDAR1 in intact BMDM, but the activity of CD11 and F4 / 80. These results suggest a close correlation between NMDA receptors and macrophages.

[0104]

[0105] Example 7. Confirmation of the diagnosis and therapeutic effect of inflammation after inducing chronic inflammation.

[0106]

[0107] 7-1. Confirmation of labeling after nanocomposite injection

[0108]

[0109] Turpentine (TP), a chronic inflammation-inducing substance, was injected into the right thigh of mice, and the same amount of PBS was injected into the left thigh as a negative control. After 1, 3, 5, and 24 hours of nanocomposite (NMDAR-PEG-DID@MSNs) injection, the presence of labeling in chronic inflammation sites was confirmed in real time using 5 mice each (Figs. 7a to 7c).

[0110] The labeling of the nanocomplex (NMDAR-PEG-DID@MSNs) at the inflamed site was confirmed 1, 3, 5, and 24 hours after injection. The experimental results confirmed that the nanocomplex was highly efficiently labeled in the right thigh of TP-injected mice. This confirmed that NMDA antibodies were labeled in activated macrophages in the inflamed area (Fig. 7a).

[0111] 24 hours after TP and PBS injection, mice were dissected and the degree of labeling in each organ was confirmed through ex-vivo imaging (Fig. 7b). Results confirmed that nanoparticles labeled activated macrophages at the site of inflammation.

[0112] The presence of macrophage labeling and inflammation-inducing processes of nanocomplexes containing NMDA receptors (NMDAR-PEG-DID@MSNs) and nanocomplexes not containing NMDA receptors (IgG-PEG-DID@MSNs) were confirmed (Fig. 7c).

[0113]

[0114] 7-2. Confirmation of labeling and therapeutic effects after combined injection of nanocomposite and therapeutic agent

[0115]

[0116] Turpentine (TP), a chronic inflammatory agent, was injected into the right thigh of mice, and PBS was injected into the left thigh as a negative control. Afterwards, the nanocomplex capable of only detecting inflammation (NMDAR-PEG-DID@MSNs (DEX -)) and the nanocomplex capable of both detecting inflammation and treating inflammation (NMDAR-PEG-DID@MSNs (DEX +)) were injected into 5 mice each to simultaneously confirm the efficacy of inflammation diagnosis and treatment.

[0117] In the group of mice injected with TP, the inflammation-inducing process and therapeutic efficacy were evaluated by time point. In the group co-injected with DEX (NMDAR-PEG-DID@MSNs (DEX +)), an inflammation-reducing effect was observed as image signals (Fig. 8A). These results were confirmed through image signal analysis, and the inflammation-suppressing effect was significantly observed in the DEX + group (Fig. 8B). In the left thigh control group injected with PBS, it was confirmed that both nanomedicines (NMDAR-PEG-DID@MSNs (DEX -) and NMDAR-PEG-DID@MSNs (DEX +)) showed minimal image signals (Fig. 8C).

[0118] Through the above results, it was confirmed that the novel theranostic nano-medicine capable of detecting and treating inflammation developed in the present invention can be applied simultaneously to the diagnosis and treatment of inflammation.

[0119]

[0120] Example 8. Confirmation of the diagnosis and therapeutic effect of inflammation after induction of acute inflammation.

[0121]

[0122] Carrageenan, an acute inflammation-inducing substance, was injected into the right foot of mice, and the same amount of PBS was injected into the left foot as a negative control. After injection, the presence of labeling in acute inflammation sites in real time was confirmed 1, 3, 5, and 24 hours after injection of the nanocomplex capable of only labeling inflammation (NMDAR-PEG-DID@MSNs (DEX -)) and the nanocomplex capable of both labeling and treating inflammation (NMDAR-PEGDID@MSNs (DEX +)) using 5 mice each.

[0123] The labeling of nanocomplexes (NMDAR-PEG-DID@MSNs) at the site of inflammation was confirmed by image signal, signal analysis, and footpad size analysis at 1, 3, 5, and 24 hours after injection (Figs. 9A to 9E).

[0124] LPS, an acute inflammation-inducing substance, was injected into the right paw of mice, and the same amount of PBS was injected into the left paw as a negative control. Then, 1 h, 3 h, 5 h, and 24 h after injection of the nanocomplex capable of only labeling inflammation (NMDAR-PEG-DID@MSNs (DEX -)) and the nanocomplex capable of both labeling and treating inflammation (NMDAR-PEGDID@MSNs (DEX +)), the labeling of the nanocomplex (NMDAR-PEG-DID@MSNs) at the site of inflammation was confirmed by image signal, signal analysis, and paw size analysis for each of 5 mice (Figs. 10A to 10E).

[0125] The results confirmed that the signal from the nanoparticles labeled with the anti-inflammatory drug gradually decreased. Furthermore, a decrease in the size of the footpads was also observed. Based on these results, we confirmed that the drug's efficacy led to a decrease in activated macrophages.

[0126]

[0127] Example 9. Confirmation of the therapeutic effects by organ after inducing chronic and acute inflammation.

[0128]

[0129] TP, a chronic inflammation-inducing substance, and CG and LPS, acute inflammation-inducing substances, were injected into the muscles and footpads of mice, respectively, and 24 hours later, ex vivo, the presence or absence of inflammation was examined using optical imaging equipment. Experiments were conducted using a nanocomposite capable of only labeling inflammation (NMDAR-PEG-DID@MSNs (DEX -)) and a nanocomposite capable of labeling and treating inflammation (NMDAR-PEG-DID@MSNs (DEX +)).

[0130] As confirmed by ex vivo imaging and post-dissection imaging, chronic and acute inflammation were confirmed to be associated with a gradual decrease in nanoparticle signal from anti-inflammatory drug-labeled nanoparticles (Figures 11a to 11c). Furthermore, a decrease in footpad size was observed. Based on these results, we confirmed that the drug efficacy resulted in a decrease in activated macrophages.

Claims

1. Mesoporous silica nanoparticles (MSN); A fluorescent material bound to the surface or pores of the nanoparticles; and NMDA receptor labeling antibody bound to the surface of the above nanoparticles; Nanocomposite comprising:

2. A nanocomposite according to claim 1, further comprising a hydrophilic biocompatible polymer coated on the surface of the nanoparticle.

3. In the second paragraph, the hydrophilic biocompatible polymer is at least one selected from the group consisting of polyethylene glycol (PEG), polycarboxybetaine (pCB), polysulfobetaine (pSB), phosphobetaine polymer, methoxy polyethylene glycol (mPEG), polyethylene glycol monomethyl ether, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), monodisperse polyethylene glycol (monodisperse PEG), branched polyethylene glycol (branched PEG), and multi-arm polyethylene glycol (multi-arm PEG).

4. A nanocomposite according to claim 1, wherein the fluorescent material is at least one selected from the group consisting of DID, ICG, methylene blue, IRDye800CW, Cy5.5, and Flamma 675.

5. A nanocomposite according to claim 1, wherein the fluorescent material is connected to the surface or pores of the nanoparticles through electromagnetic coupling.

6. A nanocomplex according to claim 1, wherein the NMDA receptor-labeled antibody is linked to the surface of the nanoparticle via a maleimide bond.

7. A nanocomposite in the second paragraph, wherein the hydrophilic biocompatible polymer is coated on the surface of the nanoparticle through an amine bond.

8. A nanocomposite according to claim 1, wherein the size of the nanocomposite is 50 to 200 nm.

9. A contrast agent comprising the nanocomposite of paragraph 1 as an active ingredient.

10. A composition for treating and diagnosing inflammatory diseases, comprising the nanocomposite of paragraph 1 as an active ingredient and an anti-inflammatory drug loaded inside the nanocomposite.

11. A composition for treating and diagnosing an inflammatory disease, wherein the inflammatory disease is at least one selected from the group consisting of inflammatory bowel disease, atopic dermatitis, edema, dermatitis, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, atherosclerosis, pharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, inflammatory spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis of the shoulder joint, tendinitis, tenosynovitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, and multiple sclerosis. 12.(a) A step of manufacturing a first nanoparticle by binding PEG-maleimide to mesoporous silica nanoparticles; (b) a step of manufacturing a second nanoparticle by binding a fluorescent material to the first nanoparticle; and (c) a step of binding an NMDA receptor-labeled antibody and a hydrophilic biocompatible polymer to the second nanoparticle; A method for producing a nanocomposite, comprising:

13. A method for preventing or treating an inflammatory disease, comprising administering to a subject a pharmaceutically effective amount of the composition of Article 10.

14. A method for diagnosing an inflammatory disease, comprising: administering to a subject a pharmaceutically effective amount of the composition of claim 10; and detecting a fluorescent signal from the composition.

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