Mucosal adhesive PLGA nanoparticles

Abstract

The present invention relates to a non-injectable drug delivery system using mucoadhesive nanoparticles, and to mucoadhesive nanoparticles and a method for producing the same, which prevent deformation of the nanoparticles due to mucosal moisture, etc., and strengthen mucoadhesiveness to prevent loss by binding a mucoadhesive polymer to the surface of the nanoparticles. The present invention also relates to a composition for maturing antigen-presenting cells, a composition for treating infectious diseases, and a composition for treating cancer, which comprise the mucoadhesive nanoparticles. The present invention also relates to mucoadhesive nanoparticles loaded with immune active substances (such as antigens and adjuvants) for cancer treatment and immunotherapy based on antigen-presenting cells including dendritic cells (DCs), and a composition for immune anti-cancer treatment comprising the same.

Description

Technical Field

[0001] The present invention relates to a non-injectable drug delivery system using mucoadhesive nanoparticles. Specifically, the present invention relates to mucoadhesive-PLGA nanoparticles having a mucoadhesive polymer bound to the surface and a method for producing the same, and to a composition for inducing maturation of antigen-presenting cells, a composition for treating infectious diseases, and a composition for treating cancer, each containing the mucoadhesive-PLGA nanoparticles.

Background Art

[0002] Immunotherapy is a method of treating cancer using a patient's own immune system and is one of the currently mainly used cancer treatment methods together with surgery, chemotherapy, radiotherapy, etc. Among such cancer treatment methods, immunotherapy is considered to have the fewest side effects, be the safest and most effective.

[0003] Among the immune cancer drugs used in immunotherapy, methods based on dendritic cells (DC) in particular are being actively studied. Dendritic cells are representative antigen-presenting cells and play a role in enhancing anti-cancer immunity by presenting tumor-specific antigens and assisting the tumor-specific activation of cytotoxic T cells (CD8+ T cells). In order to achieve the effective therapeutic efficacy of such dendritic cell-based immunotherapy, antigen delivery is the first core step. For this purpose, syringe-based administration methods are generally utilized, but syringe-based administration has problems such as not only side effects such as vascular weakness, vasoconstriction, and vascular occlusion, but also frequent occurrence of serious symptoms such as needle phobia and injection refusal reactions. Therefore, there is an actual need for alternative injection routes and delivery methods that can effectively deliver drugs without using a syringe.

[0004] In this regard, research is underway on non-injectable drug delivery therapies that use a method of drug delivery via the mucous membrane by spraying into the oral or nasal cavity. However, there is a problem in that the mucosal adhesion of drug-loaded nanoparticles is low, and they disappear without adhering to the mucous membrane and are delivered to the digestive tract, etc. As related prior art, Korean Published Patent No. 10-2021-0057072 and Korean Published Patent No. 10-2021-0124277 disclose drug delivery methods such as spraying or inhaling drugs loaded onto nanoparticles. However, the aforementioned prior art is mainly characterized by techniques for forming drug formulations into dosage forms in order to load drugs onto nanoparticles, or by techniques for delivering nanoparticles into the body through the lungs by nasal inhalation, and does not disclose anything about improving the mucosal adhesion of nanoparticles. Furthermore, many of the prior arts for cancer treatments using nanoparticles are mainly characterized by the direct delivery of chemical drugs corresponding to anticancer drugs to target sites (cancer cells, tumors) loaded onto nanoparticles, and have limitations that make them unsuitable for use as antigen-presenting cell-based immunotherapy including dendritic cells. [Overview of the project] [Problems that the invention aims to solve]

[0005] The present invention aims to provide a non-injectable drug delivery system using mucosal-adhesive nanoparticles, developed to solve the aforementioned problems of the conventional methods, which prevents deformation of the nanoparticles due to mucosal moisture and other factors, and strengthens mucosal adhesion to prevent their disappearance, by bonding a mucosal-adhesive polymer to the surface of the nanoparticles, and provides mucosal-adhesive nanoparticles and a method for producing the same. The present invention also aims to provide a composition for maturing antigen-presenting cells, a composition for treating infectious diseases, and a composition for treating cancer, which include the mucosal-adhesive nanoparticles. Furthermore, the present invention aims to provide mucosal-adhesive nanoparticles loaded with immunoactive substances (antigens and adjuvants, etc.) for antigen-presenting cell-based cancer immunotherapy, including dendritic cells (DCs), and an immunotherapy composition for cancer treatment containing the same.

[0006] Specifically, one objective of the present invention is to provide mucoadhesive PLGA nanoparticles in which a mucoadhesive polymer is bonded to the surface of PLGA (Poly(D,L-lactide-co-glycolide)) nanoparticles, and a method for producing the same.

[0007] Furthermore, one objective of the present invention is to provide a composition for inducing the maturation of antigen-presenting cells (APCs), comprising the aforementioned mucosal adhesive-PLGA nanoparticles as an active ingredient.

[0008] Furthermore, one objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of infectious diseases, comprising the aforementioned mucosal adhesive-PLGA nanoparticles as an active ingredient.

[0009] Furthermore, one objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer, comprising the aforementioned mucosal adhesive-PLGA nanoparticles as an active ingredient.

[0010] The object of the present invention is not limited to the foregoing, but is provided for all cases in which appropriate effects can be obtained by utilizing the present invention. [Means for solving the problem]

[0011] This invention provides mucoadhesive PLGA nanoparticles in which a mucoadhesive polymer is bonded to the surface of PLGA (Poly(D,L-lactide-co-glycolide)) nanoparticles.

[0012] Furthermore, the present invention provides mucosal adhesive-PLGA nanoparticles in which the mucosal adhesive polymer is selected from the group consisting of catechol (CAT), carrageenan, gelatin, pectin, and polyethylene glycol (PEG).

[0013] Furthermore, the present invention provides mucosal adhesive-PLGA nanoparticles in which the mucosal adhesive polymer is catechol (CAT).

[0014] Furthermore, the present invention provides mucosal adhesive-PLGA nanoparticles containing an antigen inside the nanoparticles.

[0015] Furthermore, the present invention provides mucosal adhesive-PLGA nanoparticles in which the antigen is selected from the group consisting of peptides, siRNA, and mRNA.

[0016] Furthermore, the present invention provides mucosal adhesive PLGA nanoparticles that further contain an adjuvant inside the nanoparticles.

[0017] Furthermore, the present invention provides a composition for inducing the maturation of antigen-presenting cells (APCs), comprising the aforementioned mucosal adhesive-PLGA nanoparticles as an active ingredient.

[0018] Furthermore, the present invention provides that the antigen-presenting cells are dendritic cells, and provides a composition for inducing the maturation of antigen-presenting cells.

[0019] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of infectious diseases, comprising the aforementioned mucosal adhesive-PLGA nanoparticles as an active ingredient.

[0020] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of infectious diseases, wherein the pharmaceutical composition is for oral spraying.

[0021] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising the aforementioned mucosal adhesive-PLGA nanoparticles as an active ingredient.

[0022] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, wherein the pharmaceutical composition induces the maturation of antigen-presenting cells (APCs).

[0023] The present invention also provides a pharmaceutical composition for preventing or treating cancer, wherein the pharmaceutical composition activates cytotoxic CD8+ T cells.

[0024] The present invention also provides a pharmaceutical composition for preventing or treating cancer, wherein the pharmaceutical composition is for oral spray.

[0025] The present invention also provides a method for producing mucoadhesive-PLGA nanoparticles, wherein a mucoadhesive polymer is bound to the surface of PLGA nanoparticles, the method comprising: a) mixing an aqueous solution containing an antigen and an adjuvant with an organic solution containing PLGA (Poly(D,L-lactide-co-glycolide)); and b) mixing the mixture with a compound in which PVA-NH2 having an amino group introduced into polyvinyl alcohol (PVA) and a mucoadhesive polymer having a carboxyl group introduced therein are chemically bonded to each other.

[0026] The present invention also provides a method for producing CAT-PLGA nanoparticles, wherein catechol (CAT) is bound to the surface of PLGA nanoparticles, the method comprising: a) mixing an aqueous solution containing an antigen and an adjuvant with an organic solution containing PLGA (Poly(D,L-lactide-co-glycolide)); and b) mixing the mixture with a compound (PVA-CAT) in which PVA-NH2 having an amino group introduced into polyvinyl alcohol (PVA) and 3,4-dihydroxyhydrocinnamic acid (CAT-COOH) are chemically bonded to each other.

[0027] As one specific example of the present invention, we provide mucoadhesive-PLGA nanoparticles in which a mucoadhesive polymer is bonded to the surface of PLGA (Poly(D,L-lactide-co-glycolide)) nanoparticles.

[0028] In the present invention, "mucoadhesive polymer" is a polymer that has adhesive properties to mucous membranes, particularly in vivo mucous membranes, such as oral mucosa or nasal mucosa, and can be applied to living organisms. It may include any polymer that is mucoadhesive and can be applied to living organisms without limitation. Non-limiting examples of the mucoadhesive polymer include catechol (CAT), carrageenan, gelatin, pectin, or polyethylene glycol (PEG). The mucoadhesive polymer has excellent adhesive properties to mucous membranes, particularly in vivo mucous membranes, such as oral mucosa or nasal mucosa, and has the advantage of being applicable to living organisms without side effects.

[0029] In the present invention, the mucosal adhesive polymer may be catechol (CAT).

[0030] In the present invention, "catechol (CAT)" is a compound represented by the following chemical formula 1, and is also called 1,2-dihydroxybenzene.

[0031] [ka]

[0032] In the present invention, the catechol is bonded to the surface of PLGA nanoparticles through chemical deformation, and can increase the adhesion, adsorption, fixation, and deposition ability of the nanoparticles to mucous membranes through physical entanglement, hydrogen bonding, hydrophobic bonding, or ionic interactions. Furthermore, it is biodegradable and non-toxic and can be used in vivo without side effects, and may be fixed to functional amine groups, thiol groups, or imidazole groups in the mucosal layer through chemical interactions in mucous membranes containing moisture, such as oral mucosa and nasal mucosa.

[0033] In the present invention, "PLGA (Poly(D,L-lactide-co-glycolide))" is a copolymer of Poly(lactic acid)(PLA) and Poly(glycolic acid)(PGA), and is represented by the following chemical formula 2.

[0034] [ka]

[0035] In the above formula 2, x represents the number of lactic acid units, and y represents the number of glycolic acid units.

[0036] In the present invention, the lactic acid:glycolic acid ratio (x:y) of the PLGA is not particularly limited and may be used in any ratio usable during the production of PLGA nanoparticles. Non-limiting examples of the lactic acid:glycolic acid ratio include ratios of 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, or 10:90, and more specifically, 30:70 to 70:30, and even more specifically, around 40:60 to 60:40. In one embodiment of the present invention, when a PLGA with a lactic acid:glycolic acid ratio of around 50:50 is used and used within the above range, the biodegradation rate of the PLGA nanoparticles can be more appropriately controlled, and the release rate of drugs and other substances carried within the nanoparticles can be more appropriately controlled, thus having the advantage of being more suitable for the production of PLGA nanoparticles for biodrug delivery.

[0037] The mucosal adhesive-PLGA nanoparticles of the present invention have a mucosal adhesive polymer bound to the surface of the nanoparticles, which prevents deformation of the nanoparticles due to moisture in the mucous membrane, prevents the loss of drugs loaded onto the nanoparticles, and enhances the adhesive force of the nanoparticles to the mucous membrane, so that the nanoparticles adhere, are fixed and deposited on the mucous membrane, thereby preventing the loss of drugs and having the advantage of being effectively delivered to cells.

[0038] In the present invention, the mucous membrane refers to a mucous membrane that can come into contact with and adhere to the mucosal adhesive-PLGA nanoparticles, particularly a biological mucous membrane, and may include, in non-limiting examples, oral mucosa or nasal mucosa.

[0039] In the present invention, the mucosal adhesive-PLGA nanoparticles may contain an antigen internally.

[0040] In the present invention, "antigen" is a general term for all substances that enter a living body and trigger an immune response, and generally refers to all substances that are recognized as external substances in the living body, induce an immunoreactive state, and react with immune cells or antibodies of a target body that has been sensitized to them. In the present invention, the term "antigen" may also be used interchangeably with the term "immunogen," and includes molecules containing one or more epitopes that can promote the host immune system to trigger a secretory, humoral, or cellular immune response specific to that antigen. In the present invention, non-limiting examples of the antigen may include peptides (including polypeptides and proteins), siRNA, or mRNA.

[0041] In one embodiment of the present invention, E7 protein is used as the antigen. In this case, the mucosal adhesive-PLGA nanoparticles of the present invention can be loaded and delivered into the body with excellent efficiency, and are delivered more effectively to antigen-presenting cells. This has the advantage of being able to be effectively utilized in cancer treatment immunotherapy and the like by inducing maturation of antigen-presenting cells and activating T cells.

[0042] In the present invention, the mucosal adhesive-PLGA nanoparticles may contain an adjuvant internally.

[0043] In this invention, "adjuvant" means support or assistance, and refers to a substance that has a beneficial effect on the action of other substances pharmacologically or immunologically, and is also called an immune enhancer or immune stimulant.

[0044] In the present invention, the type of adjuvant is not particularly limited, and adjuvants used in the art or related fields may be used. Non-limiting examples of the adjuvant include TLR3 adjuvants (Poly I:C, Poly I:CLC (hiltonol), Poly I:C12U (ampligen), Poly I:C + CAF01 (CAF05)), TLR7 / TLR8 adjuvants (Imiquimod (R-837), Resiquimod (R-848)), or TLR9 adjuvants (CpG ODN, CpG ODN + MPL / QS21 (AS15)). In one embodiment of the present invention, Poly I:C is used as the adjuvant, and in this case, it has the advantage of being loaded onto the mucosal adhesive-PLGA nanoparticles of the present invention and delivered into the body with excellent efficiency together with the antigen, and can be effectively utilized in cancer treatment immunotherapy and the like.

[0045] As another specific example of the present invention, a composition for inducing the maturation of antigen-presenting cells (APCs) is provided, comprising the mucosal adhesive-PLGA nanoparticles of the present invention as an active ingredient.

[0046] In this invention, "antigen-presenting cells (APCs)" refer to cells that, after transferring protein antigens into the cell (endocytosis), present antigen-derived peptide fragments together with major histocompatibility complex (MHC) molecules to T cells and activate them. Representative antigen-presenting cells include dendritic cells (DCs), B cells, and macrophages, which are major immune cells responsible for cellular immunity in the body. These cells play the role of antigen-expressing cells, allowing other cells in the immune system (such as T cells) to recognize antigens by transferring them into the cell and then making them visible on the surface.

[0047] Among the antigen-presenting cells mentioned above, dendritic cells are known to be mainly found in tissues that come into contact with the external environment, such as the skin, nose, lungs, stomach, or intestinal lining, and those found in the skin are specifically called Langerhans cells. Dendritic cells can be found in the blood in an immature state, and when activated, they are known to migrate to lymphoid organs and interact with T cells and B cells to initiate an immune response. Furthermore, dendritic cells are known to extend projections called dendrites at certain developmental stages.

[0048] Dendritic cells originate from hematopoietic bone marrow progenitor cells, which initially differentiate into immature dendritic cells known to exhibit high endocytotic and T-cell activity. Immature dendritic cells constantly phagocytose surrounding pathogens such as viruses and bacteria, a process known to be possible through pattern recognition receptors (PRRs) like TLRs (toll-like receptors). TLRs recognize specific chemical features found on subsets of pathogens, and immature dendritic cells phagocytose the cell membrane of living autologous cells through a process called nibbling. When these cells come into contact with existing antigens, they are activated into mature dendritic cells and migrate to lymph nodes. Immature dendritic cells engulf pathogens and break down their own proteins into small fragments. When they mature, these fragments appear on the cell surface using MHC molecules, and they increase cell surface receptors that act as co-receptors in T-cell activation, such as CD80, CD86, and CD40. By presenting antigens derived from the pathogen along with non-antigen-specific costimulatory signals, they can activate not only helper T-cells and killer T-cells, but also B-cells.

[0049] The cytokines produced by dendritic cells differ depending on the cell type. Lymphoid dendritic cells can produce large amounts of type-1 interferon (IFN), which attracts more activated macrophages and enables phagocytosis. Lymphoid dendritic cells are known to be involved in central and peripheral immune regulation, while myeloid dendritic cells are involved in inducing immunity against exogenous antigens and infections. Therefore, if dendritic cells do not function properly, autoimmune diseases such as diabetes, rheumatoid arthritis, and allergic hypersensitivity reactions may appear, or a normal immune response to infectious diseases and cancer may not occur. As mentioned above, dendritic cells play an important role in enhancing the body's own immune function, and inducing dendritic cell maturation is a crucial challenge in cellular immunotherapy using dendritic cells.

[0050] The antigen-presenting cell maturation induction composition of the present invention, which contains the mucosal adhesive-PLGA nanoparticles as an active ingredient, exhibits the effect of effectively delivering immunoactive substances (e.g., antigens and adjuvants) contained in the nanoparticles to induce maturation of immature antigen-presenting cells (e.g., dendritic cells). As a result, when antigen-presenting cells mature, the expression of surface protein indicators increases, and by inducing T cell activity, the immune response can be increased.

[0051] Specifically, mature antigen-presenting cells express MHC type I and II antigens at higher levels than immature antigen-presenting cells and can regulate CD80+, CD83+, and CD86+. Higher MHC expression induces increased antigen density on the surface of antigen-presenting cells, and the upward regulation of the co-stimulating molecules CD80 and CD86 can enhance T cell activity signals on T cells through co-stimulating molecular counterparts such as CD28.

[0052] The maturation of the antigen-presenting cells can be monitored by methods known in the art, for example, by detecting cell surface markers using analytical methods used in the art, such as flow cytometry or immunohistochemistry. Alternatively, the cells can be monitored through cytokine production analysis (e.g., ELISA or FACS).

[0053] In the present invention, the type of antigen-presenting cell is not particularly limited, and non-limiting examples include dendritic cells, Langerhans cells, macrophages, mononuclear cells, or B cells. In one embodiment of the present invention, dendritic cells are targeted, and these dendritic cells have the advantage of exhibiting superior immune activity as one of the most potent antigen-presenting cells that play a bridge role in the innate and adaptive immune systems.

[0054] As another specific example of the present invention, a pharmaceutical composition for the prevention or treatment of infectious diseases is provided, comprising the mucosal adhesive-PLGA nanoparticles as an active ingredient.

[0055] In the present invention, the type of infectious disease is not particularly limited and refers collectively to diseases caused by infection with various pathogens such as viruses, bacteria, and fungi.

[0056] As another specific example of the present invention, a pharmaceutical composition for the prevention or treatment of cancer is provided, comprising the mucosal adhesive-PLGA nanoparticles as an active ingredient.

[0057] In the present invention, the type of cancer (such as a specific disease name or disease site) is not particularly limited and may include all cancers in which symptoms improve or can be prevented or treated by maturing antigen-presenting cells and activating immune cells such as T cells. The pharmaceutical composition for the prevention or treatment of cancer of the present invention, which contains the mucosal adhesive-PLGA nanoparticles as an active ingredient, can treat not only cancers at the local site to which it is directly applied, but also cancers and tumors that occur in various organs, tissues, and cells in the body through systemic circulation of T cells and other cells activated by the pharmaceutical composition, thereby enabling T cell-mediated anti-cancer immunotherapy for systemic cancers. When the pharmaceutical composition of the present invention is manufactured and used as an oral spray formulation, it is particularly effective in the treatment of oral cancer or head and neck cancer.

[0058] The pharmaceutical composition of the present invention can be further specialized and manufactured and used as a pharmaceutical composition for specific cancer / tumor-specific anti-cancer immunotherapy by loading the mucosal adhesive-PLGA nanoparticles with an antigen or adjuvant specific to the cancer / tumor to be targeted.

[0059] The pharmaceutical compositions of the present invention can effectively induce the maturation of antigen-presenting cells, and can also promote the activation of T cells, such as cytotoxic CD8+ T cells, and may be used in a variety of ways as effective immunotherapy-based pharmaceutical compositions.

[0060] The pharmaceutical compositions of the present invention may be manufactured in any dosage form that can enter the body and may be administered by any method of administration. To address the problems of conventional injection-based administration methods, the pharmaceutical compositions of the present invention have been developed as nanoparticles with particularly excellent mucosal adhesion, adsorption, fixation, and deposition capabilities, and therefore may be formulated for easy administration to mucous membranes, particularly the oral or nasal mucosa. Furthermore, they may be manufactured as an oral spray dosage form for simpler and more effective drug delivery and absorption. The method for manufacturing the spray dosage form is not particularly limited and may be carried out by methods used in the art or related fields.

[0061] As another specific example of the present invention, a) A step of mixing an aqueous solution containing an antigen and an adjuvant with an organic solution containing PLGA (Poly(D,L-lactide-co-glycolide)), and b) A method for producing mucoadhesive-PLGA nanoparticles in which a mucoadhesive polymer is bonded to the surface of PLGA nanoparticles, comprising the step of mixing the aforementioned mixture with a compound in which polyvinyl alcohol (PVA) has an amino group introduced into it (PVA-NH2) and a mucoadhesive polymer with a carboxyl group (-COOH) introduced into it are chemically bonded to each other.

[0062] The mucosal adhesive PLGA nanoparticles of the present invention are in the form of a water-in-oil-in-water (w / o / w) secondary emulsion. The nanoparticles can be produced by first dissolving an antigen and an adjuvant in a primary aqueous phase and PLGA in an organic solvent oil phase, then mixing them to form a primary emulsion. A secondary emulsion is then formed by mixing the primary emulsion with an aqueous solution of polyvinyl alcohol and a compound to which a mucosal adhesive polymer is bound, which is another secondary aqueous phase. Finally, the oil in the formed secondary emulsion is evaporated.

[0063] As another specific example of the present invention, a) A step of mixing an aqueous solution containing an antigen and an adjuvant with an organic solution containing PLGA (Poly(D,L-lactide-co-glycolide)), and b) A method for producing CAT-PLGA nanoparticles in which catechol (CAT) is bonded to the surface of PLGA nanoparticles is provided, comprising the step of mixing the aforementioned mixture with a compound (PVA-CAT) in which polyvinyl alcohol (PVA) to which an amino group has been introduced and 3,4-dihydroxyhydrocinnamic acid (CAT-COOH) are chemically bonded to each other.

[0064] The CAT-PLGA nanoparticles of the present invention are in the form of a water-in-oil-in-water (w / o / w) secondary emulsion. The nanoparticles can be produced by first dissolving an antigen and an adjuvant in a primary aqueous phase and PLGA in an organic solvent oil phase, then mixing them to form a primary emulsion. A secondary emulsion is then formed by mixing another secondary aqueous phase, a polyvinyl alcohol-catechol (PVA-CAT) aqueous solution, with the primary emulsion, and finally evaporating the oil from the formed secondary emulsion.

[0065] When manufacturing PLGA nanoparticles in emulsion formulations, if PLGA nanoparticles are produced using PVA as the secondary aqueous phase, the nanoparticles may deform due to mucosal moisture, causing the drug loaded inside to disappear. Furthermore, the adhesion to the mucous membrane is very weak, resulting in problems such as the nanoparticles not being fixed to the mucous membrane and being delivered to the digestive tract, etc. In contrast, the nanoparticles of the present invention use a PVA-mucosal adhesive polymer (e.g., PVA-CAT), which is a PVA-mucosal adhesive polymer (e.g., CAT) chemically bonded to PVA as the secondary aqueous phase, to produce mucosal adhesive-PLGA nanoparticles with the mucosal adhesive polymer bonded to the surface. This prevents deformation of the nanoparticles due to mucosal moisture, prevents the disappearance of the drug loaded onto the nanoparticles, and strengthens the adhesion of the nanoparticles to the mucous membrane, allowing the nanoparticles to effectively adhere, fix, and deposit on the mucous membrane, thereby preventing drug loss and effectively delivering the drug to cells.

[0066] In one embodiment of the present invention, the chemical reaction equation for the method and process for producing PVA-CAT, in which CAT and PVA are chemically bonded, is as follows.

[0067] [ka]

[0068] According to one embodiment of the present invention, the method for manufacturing the PVA-CAT is as follows.

[0069] PVA-NH2 was prepared by modifying PVA using carbonyldiimidazole (CDI) and ethylenediamine (EDA). Specifically, DMSO (60 mL) containing CDI (74 mg) was added to PVA (400 mg), and the mixture was stirred at 24°C for 4 hours. Next, the mixture was precipitated in butanol to remove unreacted reagents, and CDI-activated PVA (CDI-PVA) was obtained. The CDI-PVA was then dried in a vacuum oven. Next, CDI-PVA (400 mg) and EDA (4 g) were dissolved in 100 mL of DMSO, stirred at 50°C for 48 hours, and then dried in a vacuum oven. In the second step, CAT-COOH was conjugated with PVA-NH2 using N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride (EDC). Specifically, NHS (68 mg) and EDC (108 mg) were added to a CAT-COOH solution (37.3 mg / mL), and the mixture was stirred at 24°C for 4 hours. Next, PVA-NH2 (400 mg) was added to the mixture, and it was stirred again at 24°C for 24 hours. After that, the CAT-labeled PVA (PVA-CAT) was separated using a dialysis membrane (cut off Mw000), and then freeze-dried to obtain PVA-CAT.

[0070] According to one embodiment of the present invention, the method for producing CAT-PLGA nanoparticles of the present invention using the PVA-CAT is as follows.

[0071] 1 mg of E7 as the antigen and 2 mg of poly I:C as the adjuvant were dissolved in 200 μL of deionized water. This mixture was then mixed with 2 mL of chloroform solution containing PLGA (20 mg / ml) using a probe-type sonicator (SONICS, Newtown, CT, USA) at 4°C for 30 seconds (6 pulses of 5 seconds each, with 3-second intervals between pulses). The primary emulsion was sonicated with a secondary aqueous phase (10 mL of 1.0% w / v PVA-CAT) at 4°C for 5 minutes to form a secondary (w / o / w) emulsion. After completely evaporating the chloroform using an evaporator for 10 minutes, CAT-PLGA(E7+poly I:C)-NP was centrifuged at 15,800 × g for 30 minutes to produce CAT-PLGA(E7+poly I:C)-NP.

[0072] Terms not specifically defined in this invention shall be interpreted as having the meanings commonly used in the art. Furthermore, unless otherwise specified, the expression “or” in this invention may be interpreted as including the concept of “and.”

[0073] The scope of the present invention is not limited by the specific descriptions disclosed herein, and each description and embodiment disclosed herein may also apply to each other description and embodiment. That is, all possible combinations of the various elements disclosed herein are to be interpreted as falling within the scope of the present invention. Furthermore, a person with ordinary skill in the art may recognize or confirm numerous equivalents to specific embodiments of the present invention through ordinary experimentation, and such equivalents are to be interpreted as falling within the scope of the present invention. [Effects of the Invention]

[0074] This invention relates to a non-injectable drug delivery system using mucoadhesive nanoparticles. By binding a mucoadhesive polymer to the surface of the nanoparticles, the invention prevents deformation of the nanoparticles due to mucosal moisture, thereby preventing the loss of the drug loaded onto the nanoparticles. Furthermore, it enhances the adhesive force of the nanoparticles to the mucosa, allowing the nanoparticles to effectively adhere, fix, and deposit on the mucosa, thereby preventing drug loss and effectively delivering the drug to cells. In addition, when an antigen is loaded inside the nanoparticles, the invention has the advantage of being able to be effectively delivered in vivo or into cells and used in a variety of applications as a composition for maturing antigen-presenting cells, a composition for treating infectious diseases, and a composition for treating cancer. Furthermore, by loading immunoactive substances (antigens and adjuvants, etc.) for antigen-presenting cell-based cancer immunotherapy, including dendritic cells (DCs), onto the nanoparticles, the invention has the advantage of being able to be used in cancer immunotherapy by activating antigen-specific cytotoxic T cells (CD8+ T cells). [Brief explanation of the drawing]

[0075] [Figure 1] Figure 1 is a schematic diagram illustrating the drug delivery and cancer treatment process of the spray-type CAT-PLGA-NP of the present invention. [Figure 2] Figure 2 shows the chemical reaction equation (Figure 2A) and the 1H-NMR analysis results (Figure 2B) illustrating the PVA-CAT conjugation process by chemical modification in the present invention. [Figure 3] Figure 3 shows the results of PVA-CAT junction analysis by FT-IR, where circled number 1 indicates the benzene aromatic ring of CAT-COOH, and circled number 2 indicates the amide I band of PVA-CAT. [Figure 4]Figure 4 shows schematic diagrams, graphs, and photographs illustrating the physicochemical properties of CAT-PLGA-NP. Specifically, Figure 4A is a schematic diagram showing the manufacturing process of CAT-PLGA(E7+poly I:C)-NP. Figure 4B shows the results of measuring the size and zeta potential of CAT-PLGA NP by laser light scattering using a particle size analyzer. The loading efficiency of poly I:C and E7 onto CAT-PLGA-NP was measured by Nanodrop (absorbance: 260 nm) and BCA protein analysis, respectively. Figure 4C shows the measurement results of the size and zeta potential of CAT-PLGA-NP after spraying, and the loading efficiency of poly I:C and E7 onto PLGA-NP and CAT-PLGA-NP after spraying. Figure 4D shows the results of measuring the morphology of CAT-PLGA-NP by FE-SEM (scale bar: 500 nm). Figure 4E shows the cumulative release measurement results of E7 in CAT-PLGA(E7+poly I:C)-NP under conditions of 37°C and 50 RPM. Figure 4F shows the results of mucin adsorption to CAT PLGA-NP measured at 263 nm using a UV-vis spectrophotometer. The above data are expressed as mean ± SD (n=3) (*p<0.001). [Figure 5] Figure 5 shows the size distribution of CAT-PLGA-NPs before and after spraying, as measured by laser scattering using a particle size analyzer. [Figure 6] Figure 6 shows graphs and photographs of CAT-PLGA-NP binding to DCs and intracellular delivery, as well as CAT-PLGA(E7+poly I:C)-NP-induced DC maturation. Specifically, Figure 6A shows the results of measuring CAT-PLGA-NP binding to DCs using flow cytometry, and Figure 6B shows the results of measuring intracellular delivery of CAT-PLGA-NP to DCs using confocal microscopy (magnification: ×200, scale bar: 20 μm). Error bars represent SEM results (*p<0.001). [Figure 7]Figure 7 is a graph showing the maturation and cytokine production of dendritic cells (DCs) induced by CAT-PLGA(E7+poly I:C)-NP. Specifically, Figure 7A shows the results of flow cytometry measurements of surface marker levels (CD40, CD80, CD86). Figure 7B shows the results of quantification of infectious cytokines and DC activators in the culture supernatant of DCs cultured with CAT-PLGA(E7+poly I:C)-NP using ELISA. Error bars represent SEM results (*p<0.001, **p<0.05). [Figure 8] Figure 8 shows the in-vivo fluorescence images of mice after CAT-PLGA-NP spraying, monitored using IVIS. Error bars represent SEM (*p<0.01). [Figure 9] Figure 9 shows the results regarding the adhesion of CAT-PLGA-NP to the oral mucosa of mice and animals (mucin) (magnification: ×200, scale bar: 100 μm). [Figure 10] Figure 10 shows the therapeutic efficacy of CAT-PLGA-NP in a TC-1 tumor tongue model. Specifically, treatment with PLGA(E7+poly I:C)-NP and CAT-PLGA(E7+poly I:C)-NP was initiated 3 weeks after injecting TC-1 tumor cells into the tongues of C57BL6 mice. PLGA(E7+poly I:C)-NP and CAT-PLGA(E7+poly I:C)-NP were sprayed once a week at a dose of 50 μg E7 and poly I:C. Figure 10A is the experimental schedule for treatment, Figure 10B is a graph of tongue weight, Figure 10C is a photograph of tongue and tumor weight, and Figure 10D is a graph showing the body weight of the mice. Error bars represent SEM (*p<0.001, **p<0.01, ***p<0.05). [Figure 11] Figure 11 shows the results of measuring cytotoxic IFN-γ+CD8+ T cells in the tongue, mandibular lymph nodes, and spleen cells using flow cytometry. Error bars represent SEM (*p<0.001, **p<0.01, ***p<0.05). [Figure 12] Figure 12 shows the results of H&E staining and immunohistochemical analysis of the tongue. Specifically, immunohistochemical analysis of the cell proliferation marker (Ki67) and measurement of microvascular density (MVD, CD31), TUNEL, and CD8+ T cell infiltration were performed using mouse tongue tissue (H&E scale bar: 500 μm, (scale bar: 25 μm)). Error bars represent SEM (*p<0.001, **p<0.01, ***p<0.05). [Modes for carrying out the invention]

[0076] The present invention will be described in more detail below through specific examples. However, these examples are merely illustrative for explaining the present invention, and should not be construed as limiting the scope of the present invention in any way.

[0077] [material] Poly(d,l-lactide-co-glycolide), PLGA, Resomer® RG502H, 50:50 monomer ratio, MW 10-12 kDa, polyvinyl alcohol (PVA, MW 9-10 kDa), 3,4-dihydroxyhydrocinnamic acid (CAT-COOH), carbonyldiimidazole (CDI), ethylenediamine (EDA), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride Hydrochloride (EDC), porcine stomach-derived type II mucin, and polyinosinic-polycytidylic acid sodium salt (poly I:C) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dimethylsulfoxide (DMSO) was purchased from Biosesang (Bundang, Korea). E7 peptide (TNYLFSPNGPIARAW) was purchased from Anygen (Gwangju, Korea). Fetal bovine serum (FBS) was purchased from Welgene (Gyeongsan, Korea). RPMI 1640 medium was purchased from Biowest (Nuaille, France). Hoechst 33342 was purchased from Invitrogen (Carisbad, CA, USA). Cy5.5-NHS was purchased from Lumiprobe (Hunt Valley, MD, USA). FITC-labeled anti-mouse CD11c, PE-labeled anti-mouse CD40, CD80, and CD86 were purchased from Biolegend (San Diego, CA, USA).FITC-labeled anti-mouse IFN-γ, and mouse TNF-α, IL-6, and IL-1β ELISA Ready-SET-Go kits were purchased from eBioscience (San Diego, CA, USA). APC-conjugated anti-CD8a was purchased from Invitrogen (Waltham, MA, USA). All materials were analytical grade and were used without further purification. [Examples]

[0078] Example 1: Chemical deformation of PVA bonded CAT PVA-NH2 was prepared by deforming PVA using CDI and EDA. Specifically, DMSO (60 mL) containing CDI (74 mg) was added to PVA (400 mg), and the mixture was stirred at 24°C for 4 hours. Next, the mixture was precipitated in butanol to remove unreacted reagents, and CDI-activated PVA (CDI-PVA) was obtained. The CDI-PVA was then dried in a vacuum oven. Next, CDI-PVA (400 mg) and EDA (4 g) were dissolved in 100 mL of DMSO, stirred at 50°C for 48 hours, and then dried in a vacuum oven.

[0079] In the second stage, CAT-COOH was conjugated with PVA-NH2 using NHS and EDC. Specifically, NHS (68 mg) and EDC (108 mg) were added to a CAT-COOH solution (37.3 mg / mL), and the mixture was stirred at 24°C for 4 hours. Next, PVA-NH2 (400 mg) was added to the mixture and stirred further at 24°C for 24 hours. After that, the CAT-labeled PVA (PVA-CAT) was separated using a dialysis membrane (cut off Mw000), and then freeze-dried to obtain PVA-CAT.

[0080] The formation of PVA-CAT as described above was confirmed by 1H-NMR (500 MHz, HRMAS-FT NMR, Billerica, MA, USA) and Fourier transform infrared (FT-IR) (Nicolet 5700, Thermo, Waltham, MA, USA).

[0081] Example 2: Production of catechol-bound PLGA nanoparticles (CAT-PLGA-NP) PLGA(E7+poly I:C)-NP refers to PLGA nanoparticles containing E7 as the antigen and poly I:C as the adjuvant, and was manufactured using a w / o / w (water-in-oil-in-water) evaporation method. Specifically, 1 mg of E7 and 2 mg of poly I:C were dissolved in 200 μL of deionized water, and then mixed with 2 mL of chloroform solution containing PLGA (20 mg / ml) using a probe-type sonicator (SONICS, Newtown, CT, USA) at 4°C for 30 seconds (6 pulses of 5 seconds each at 3-second intervals). The primary emulsion was sonicated with a secondary water phase (10 mL of 1.0% w / v PVA) at 4°C for 5 minutes to form a secondary (w / o / w) emulsion. After completely evaporating the chloroform using an evaporator for 10 minutes, the PLGA(E7+poly I:C)-NP was centrifuged at 15,800 × g for 30 minutes and washed three times, then stored at 4°C until use. The production of CAT-PLGA(E7+poly I:C)-NP was the same as the production procedure for PLGA(E7+poly I:C)-NP described above, but PVA-CAT was used as the secondary aqueous phase.

[0082] The size and zeta potential of CAT-PLGA(E7+poly I:C)-NPs were measured by dynamic light scattering using an electrophoretic light scattering photometer (SZ-100, HORIBA, Kyoto, Japan). The loading efficiency of E7 was measured using a BCA protein analysis kit (Pierce Biotechnology, Rockford, IL, USA), and poly I:C was measured at 260 nm using a NanoDrop1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The morphology of PLGA-NPs and CAT PLGA-NPs before and after spraying was confirmed using a field emission scanning electron microscope (FE-SEM) (SU8000, HITACHI, Tokyo, Japan).

[0083] Example 3: Emission of E7 from CAT-PLGA(E7+poly I:C)-NP CAT-PLGA (E7 + poly I:C)-NP was added to microcentrifuge tubes and placed in a water bath for a specified time. Next, the microcentrifuge tubes were centrifuged at 15,800 × g for 60 minutes, and the supernatant was collected to measure the cumulative release of E7 with CAT-PLGA(E7+poly I:C)-NP. The amount of released E7 was measured using a BCA protein analysis kit.

[0084] Example 4: Mucin adsorption by CAT-PLGA-NP To confirm mucin adsorption by CAT-PLGA-NP, mucin and CAT-PLGA-NP mixtures were prepared at various mixing ratios (mucin:NP w / w, 1:0.25, 1:1, 1:4). After centrifuging the mixtures at 15,800 × g for 30 minutes, the supernatant was collected and the amount of unadsorbed mucin was measured at 263 nm using a UV-vis spectrophotometer.

[0085] Example 5: Preparation of mice and cell lines Female C57BL / 6 mice (5-6 weeks old) were purchased from ORIENT (Gapyeong, Korea). All mice were maintained in accordance with protocols approved by the Animal Management Committee of Konkuk University Veterinary Hospital (Ref. No.: KU20214) for the proper use and management of specific pathogen-free housing facilities at Konkuk University.

[0086] TC-1 cells (expressing HPV type 16 HPV E6 and E7 proteins) were cultured in RPMI 1640 medium supplemented with 0.1% gentamicin and 10% fetal bovine serum (Biowest, Nuaille, France). Dendritic cells (DCs) were obtained from the bone marrow of C57BL / 6 mice and cultured in RPMI 1640 medium supplemented with 0.1% gentamicin, 10% FBS, and 20 ng / mL mouse recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0087] Example 6: Binding and intracellular delivery of CAT-PLGA-NP Before confirming the binding and intracellular delivery of CAT-PLGA-NP, the fluorescent dye Cy5.5 was loaded onto CAT-PLGA-NP as a model drug. To measure the binding efficiency of CAT-PLGA-NP, DCs were cultured with CAT-PLGA(Cy5.5)-NP at 37°C for 5 minutes and 30 minutes, respectively. After culturing, DCs were washed with PBS, stained with FITC-labeled anti-CD11c, and analyzed using flow cytometry (BD Facscalibur with CELLQuest software, BD biosciences, Franklin Lakes, NJ, USA). For confocal microscopy, DCs were cultured with CAT-PLGA(Cy5.5)-NP at 37°C for 30 minutes. Subsequently, the DCs were fixed with 4% paraformaldehyde (w / v) at 24°C for 10 minutes and stained with 1 μM Sytox® green (Life Technologies, Carlsbad, CA, USA) in PBS for 10 minutes. Intracellular delivery of CAT-PLGA(Cy5.5)-NP in DCs was observed using a confocal microscope (LSM 710, Carl Zeiss, Oberkochen, Germany).

[0088] Example 7: DC maturation and cytokine production DC 5 x 10 per well 6Cells were cultured in 6-well plates at the specified cell density. As a single control group for DCs, poly I:C (50 μg), CAT-PLGA-NP, PLGA(E7+poly I:C)-NP (50 μg E7 and poly I:C, respectively), and CAT-PLGA(E7+poly I:C)-NPs (50 μg E7 and poly I:C, respectively) were cultured for 30 minutes, after which the medium containing PLGA-NP was removed. DCs were then cultured for another 24 hours and stained with FITC-anti-CD11c, PE-anti-CD40, PE-anti-CD80, and PE-anti-CD86. DC maturation was measured using flow cytometry, and cytokines (TNF-α, IL-6, and IL-1β) secreted from DCs were identified using a cytokine-specific ELISA kit (eBioscience, San Diego, CA, USA).

[0089] Example 8: Adhesion of CAT-PLGA-NP to mouse oral mucosa To evaluate the mucosal adhesion of CAT-PLGA(Cy5.5)-NP, CAT-PLGA(Cy5.5)-NP was sprayed onto the oral mucosa of C57BL / 6 mice. The fluorescence signal of CAT-PLGA(Cy5.5)-NP was monitored using an in vivo imaging system (IVIS, excitation: 630 nm, emission: 710 nm). For further evaluation of the adhesive effect of CAT-PLGA(Cy5.5)-NP, immunohistochemical (IHC) analysis was performed using oral mucosal tissue. CAT-PLGA(Cy5.5)-NP was sprayed onto a layer of oral mucosal tissue, incubated for 30 minutes, and then immediately washed twice with PBS. The tissue was fixed using an optimal cutting temperature compound (OCT) (Tissue Tek, Torrance, CA, USA). For IHC analysis, tissue slides were stained with Hoechst 33342 and analyzed using a fluorescence microscope (BX61-32FDIC, Olympus, Tokyo, Japan). Additionally, tissues were stained with H&E (hematoxylin and eosin, Leica Biosystems, Buffalo, IL, USA) as a control and analyzed using a microscope (Eclipse NI, Nikon, Tokyo, Japan).

[0090] Example 9: Therapeutic efficacy of CAT-PLGA-NP To produce tumors, TC-1 cells (4 × 10⁶ cells in 25 μL HBSS) 4The tumor cells were injected into the tongues of C57BL / 6 mice (n=5 per group). To evaluate the therapeutic effect, vaccination was started 3 days after the injection of tumor cells into the mice. Three groups of mice, (1) control group (negative), (2) control group, (3) PLGA(E7+poly I:C)-NP, and (4) CAT-PLGA(E7+poly I:C)-NP (50 μg each of E7 and poly I:C), were vaccinated once a week for a total of three doses, and tongue weight and mouse weight were recorded. In addition, to confirm the activation of cytotoxic CD8+ T cells, tongue, mandibular lymph node, and spleen cells were collected from the mice. Cells isolated from the aforementioned tissue were resuspended in 1 mL of RPMI 1640 containing 10% FBS, 0.1% enthemicin, and 1.0% β-mercaptoethanol, and cultured for 24 hours with GolgiPlug (BD Biosciences, San Diego, CA, USA) and E7 (1 μg / mL). Next, the cells were washed, stained with APC-anti-CD8a and FITC-anti-IFN-γ, and identified as IFN-γ+CD8+ T cells using flow cytometry. IHC analyses for H&E analysis, cell proliferation (anti-Ki67, Abcam, Cambridge, UK), microvascular density (MVD, anti-CD31, Abcam Cambridge, UK), cell autodestruction (TUNEL, Trevigen, Gaitersbug, MD, USA), and CD8+ T cell population (anti-CD8, Biolegend, San Diego, CA, USA) were performed using tongue tissue isolated from mice. Stained tissues were analyzed using bright-field and fluorescence microscopy. The analysis was recorded in arbitrary fields on each slide (five arbitrary fields at ×400 magnification).

[0091] [Statistical analysis] Differences in continuous variables were analyzed using Student's t-test to compare two groups, and differences between multiple groups were compared using analysis of variance (ANOVA). In this example, all p-values ​​< 0.05 were considered statistically significant.

[0092] [Experimental Results] Experimental Result 1: Induction of dendritic cell (DC)-based anti-cancer immune response in oral cancer by tumor-specific antigen delivery using sprayable CAT-PLGA-NP. The inventors designed and manufactured CAT-PLGA-NP, a spray-type mucosal immobilization-deposition nanoparticle delivery system, which effectively induces a DC-based immune response by easily and readily delivering tumor-specific antigens to DCs in oral cancer patients and others who experience injection rejection.

[0093] Experimental Result 2: Chemical Deformation of PVA-bonded CAT Before manufacturing CAT-PLGA-NP, CAT-COOH and PVA-NH2 were joined by chemical deformation (Figure 2A). The joining of PVA-CAT was performed by (1) the OH (aromatic C-OH) of CAT-COOH, (2) the CH2 (methylene) of CAT-COOH, and (3) the CH2 (methylene) of PVA. 1 The spectra were measured by 1H-NMR (Figure 2B). PVA-CAT was also confirmed using FT-IR (Figure 3). The 1470 cm⁻¹ values ​​for CAT-COOH and PVA-CAT were also measured. -1 ~1622cm -1 The absorption peak between these points is attributed to the aromatic ring C=C vibrational band (benzene aromatic ring). In particular, the stretching of the amide I band at 1643 cm² is due to the C=O vibrational coupling in PVA-CAT. -1 The peaks indicate that CAT and PVA were well coupled with the COOH of CAT and the NH2 of PVA.

[0094] Experimental Result 3: Physical Properties of CAT-PLGA-NP CAT-PLGA(E7+polyI:C)-NPs were fabricated as shown in Figure 4A. The size of PLGA-NPs and CAT-PLGA-NPs was approximately 200 nm (Figure 4B). The zeta potential was approximately -70 mV, and the loading efficiencies of poly I:C and E7 were confirmed to be 35% (poly I:C) and 70% (E7), respectively (Figure 4B). Furthermore, the sprayed PLGA-NPs and CAT-PLGA-NPs showed no difference compared to the "pre-spray" condition (Figure 4C), and there was no difference in size distribution (Figure 5). The morphology of PLGA-NPs and CAT-PLGA-NPs was measured by FE-SEM (Figure 4D). The shape of CAT-PLGA-NPs before and after spraying was spherical, and there was no change in shape after spraying. These results suggest that CAT-PLGA-NPs are suitable as a spray-type formulation. Next, the release of E7 from CAT-PLGA-NPs was confirmed (Figure 4E). The cumulative emission patterns of E7 from PLGA-NP and CAT-PLGA-NP were similar, with burst emissions observed within 8 hours.

[0095] Mucin is an important glycoprotein in mucous membranes and plays a role in mucosal structure. Therefore, we confirmed the adsorption effect of CAT-PLGA-NP. Compared to CAT-unlabeled PLGA-NP, mucin was adsorbed much more effectively onto the surface of CAT-PLGA-NP when the amount of CAT-PLGA-NP increased (Figure 4F).

[0096] Experimental result 4: Binding and intracellular delivery of CAT-PLGA-NP The binding of CAT-PLGA(Cy5.5)-NP to DCs was measured using flow cytometry, confirming that CAT-PLGA(Cy5.5)-NP exhibited higher binding efficiency compared to PLGA(Cy5.5)-NP (Figure 6A). Furthermore, intracellular delivery of CAT-PLGA(Cy5.5)-NP was measured using confocal microscopy. The relative fluorescence intensity of CAT-PLGA(Cy5.5)-NP was confirmed to be higher than that of PLGA(Cy5.5)-NP (Figure 6B). These results suggest that CAT labeling makes CAT-PLGA(Cy5.5)-NP more suitable than PLGA(Cy5.5)-NP for binding to DCs and intracellular delivery.

[0097] Experimental Result 5: CAT-PLGA-NP-induced DC maturation and cytokine production Cell surface markers of dendritic cells (DCs) were measured using flow cytometry. DCs treated with CAT-PLGA(E7+poly I:C)-NP showed significantly increased levels of CD40, CD80, and CD86 compared to other groups (Figure 7A). Significant differences were observed, particularly between the control group, the poly I:C group, and the CAT-PLGA-NP group. Furthermore, infectious cytokines (TNF-α, IL-6, IL-1β) were significantly increased compared to other groups (Figure 7B).

[0098] Experimental result 6: Adhesion of CAT-PLGA-NP to mouse oral mucosa To confirm the adhesion of CAT-PLGA-NP to the oral mucosa, CAT-PLGA(Cy5.5)-NP was sprayed onto the oral mucosa of C57BL / 6 mice, and the fluorescence intensity was measured using IVIS. The fluorescence intensity of CAT-PLGA(Cy5.5)-NP in the mucosa was maintained for 4 hours, longer than that of PLGA(Cy5.5)-NP (Figure 8). Furthermore, after separating the oral mucosa from C57BL / 6 mice, CAT-PLGA(Cy5.5)-NP was sprayed onto the mucosa. The adhesive properties of CAT-PLGA(Cy5.5)-NP were confirmed using a fluorescence microscope, and the adhesive effect of CAT-PLGA(Cy5.5)-NP was superior to that of PLGA(Cy5.5)-NP (Figure 9).

[0099] Experimental result 7: Therapeutic efficacy of CAT-PLGA-NP TC-1 cells expressing HPV 16 E6 and E7 proteins are commonly used as tumor models for cancer immunotherapy. Therefore, a TC-1 tongue tumor model was developed to measure the therapeutic efficacy of CAT-PLGA-NP. To produce the mouse tongue tumor model, TC-1 cells (4 × 10⁶) were used. 4 Cells ( / mouse) were injected into the tongues of C57BL / 6 mice (n=5 per group). Vaccination was started 3 days after injecting tumor cells into the C57BL / 6 mice. (1) control group (negative), (2) control group, (3) PLGA(E7+poly I:C)-NP, and (4) CAT-PLGA(E7+poly I:C)-NP (Figure 10A). The CAT-PLGA(E7+poly I:C)-NP group showed significant suppression of tumor growth compared to the control group (56%, p<0.001) and PLGA(E7+poly I:C)-NP (30%, p<0.01) (Figures 10B and C). However, the CAT-PLGA(E7+poly I:C)-NP group showed only small differences in tongue weight and tumor weight compared to the control group (negative) (Figures 10B and C).

[0100] Based on the results described above, the body weight of the vaccinated group was examined (Figure 10D). The CAT-PLGA(E7+poly I:C)-NP group showed consistency, while the control group (p<0.01) and the PLGA(E7+poly I:C)-NP group (p<0.05) showed weight loss. This is because the mice in the control group and the PLGA(E7+poly I:C)-NP group experienced difficulties in food intake due to tumor growth on the tongue (Figure 10D). To evaluate CD8+ T cell activation, IFN-γ+CD8+ T cell populations were identified in the tongue, mandibular lymph nodes, and spleen cells. CAT-PLGA(E7+poly I:C)-NP showed a significant increase in the number of IFN-γ+CD8+ T cells compared to the control group and PLGA(E7+poly I:C). Specifically, this was observed in tongue tissue (p<0.001) compared to the control group and PLGA(E7+poly I:C)-NP (p<0.01), in mandibular lymph nodes (p<0.001) compared to the control group and PLGA(E7+poly I:C)-NP (p<0.05), and in the spleen (p<0.001) compared to the control group and PLGA(E7+poly I:C)-NP (p<0.01) (Figure 11).

[0101] Compared to the control group and PLGA(E7+polyI:C)-NP, H&E staining analysis showed that tumors occupied a larger proportion of the tongue tissue (Figure 12). In addition, tumors were analyzed using IHC analysis for cell proliferation (ki67), microvascular density (MVD, CD31), cell death (TUNEL), and CD8+ T cell markers. Compared to the control group (p<0.01) and PLGA(E7+poly I:C)-NP (p<0.05), CAT-PLGA(E7+poly I:C)-NP showed significantly suppressed cell proliferation, decreased microvascular density, and increased number of cell deaths (Figure 12). Furthermore, the number of CD8+ T cells in the tongue tissue of the CAT-PLGA(E7+poly I:C)-NP group increased compared to the control group (p<0.001) and PLGA(E7+poly I:C)-NP (p<0.01) (Figure 12).

[0102] This specification omits detailed descriptions of matters that can be fully understood and inferred by a person with ordinary skill in the art of the present invention, and allows for a wider variety of modifications to the present invention without changing the technical idea or essential configuration, other than those specifically illustrated herein. Therefore, the present invention may be carried out in ways different from those specifically described and illustrated herein, and this is understandable to a person with ordinary skill in the art of the present invention.

Claims

1. CAT-PLGA nanoparticles that adhere to oral or nasal mucosa, wherein catechol (CAT) is bound to the surface of PLGA (Poly(D,L-lactide-co-glycolide)) nanoparticles, and the interior of the nanoparticles contains an E6 or E7 protein as an antigen and poly I:C as an adjuvant.

2. The oral or nasal mucosal adhesive CAT-PLGA nanoparticles according to Claim 1, wherein the nanoparticles have the efficacy of inducing maturation of antigen-presenting cells (APCs) and activating antigen-specific cytotoxic T cells.

3. The oral or nasal mucosal adhesive CAT-PLGA nanoparticles according to claim 2, wherein the antigen-presenting cells are dendritic cells.

4. The oral or nasal mucosal adhesive CAT-PLGA nanoparticles according to Claim 1, wherein the nanoparticles have the activity to increase one or more pro-inflammatory cytokines selected from the group consisting of TNF-α, IL-6, and IL-1β.

5. The oral or nasal mucosal adhesive CAT-PLGA nanoparticles according to claim 1, wherein the nanoparticles are for oral or nasal spraying.

6. A pharmaceutical composition for the prevention or treatment of oral cancer, comprising oral or nasal mucosal adhesive CAT-PLGA nanoparticles as described in any one of claims 1 to 5 as an active ingredient.

7. The pharmaceutical composition for the prevention or treatment of oral cancer according to claim 6, wherein the pharmaceutical composition is for oral or nasal spray.

8. a) A step of obtaining a mixture by mixing an aqueous solution containing E6 or E7 protein as an antigen and poly I:C as an adjuvant with an organic solution containing PLGA (Poly(D,L-lactide-co-glycolide)), and b) The mixture contains PVA-NH, which is polyvinyl alcohol (PVA) to which an amino group has been introduced. ２ The step of mixing a compound (PVA-CAT) in which 3,4-dihydroxyhydrocinnamic acid (CAT-COOH) is chemically bonded to each other. A method for producing oral or nasal mucosal adhesive CAT-PLGA nanoparticles, wherein catechol (CAT) is bound to the surface of PLGA nanoparticles.

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