Mucoadhesive-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 thereof and a method for preparing 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 of which contains the mucoadhesive-PLGA nanoparticles. [Background technology]

[0002] Immunotherapy is a method of treating cancer using the patient's own immune system, and is one of the main cancer treatment methods currently used, along with surgery, chemotherapy, radiation therapy, etc. Among these cancer treatment methods, immunotherapy is considered to have the fewest side effects, is safe, and is highly effective.

[0003] Among the immunotherapy anticancer agents, methods based on dendritic cells (DCs) are being actively researched. Dendritic cells are representative antigen-presenting cells that play a role in enhancing anticancer immunity by presenting tumor-specific antigens and assisting tumor-specific activation of cytotoxic T cells (CD8+ T cells). To achieve effective therapeutic efficacy of such dendritic cell-based immunotherapy, antigen delivery is the first and most important step. For this reason, syringe-based administration methods are generally used, but syringe-based administration has problems such as side effects such as vasoweakness, vasoconstriction, and vasoblockage, as well as frequent occurrence of serious symptoms such as needle phobia and injection rejection. Therefore, there is a need for alternative injection routes and delivery methods that can effectively deliver drugs without using syringes.

[0004] In this regard, research is being conducted on non-injectable drug delivery therapeutic agents that use a drug delivery method via mucosa by spraying into the oral or nasal cavity, but there is a problem that the drug-loaded nanoparticles have low mucosal adhesion and disappear without adhering to the mucosa, and are delivered to the digestive tract, etc. As related prior art, Korean Patent Publication No. 10-2021-0057072 and Korean Patent Publication No. 10-2021-0124277 disclose drug delivery methods such as loading drugs onto nanoparticles and spraying or inhaling them, but the main features of the prior art are a technology for formulating a drug formulation to load drugs onto nanoparticles or a technology for delivering nanoparticles into the body through the lungs by inhaling the nanoparticles into the nasal cavity, and there is no disclosure at all regarding improving the mucosal adhesion of nanoparticles. In addition, many of the prior art technologies for cancer therapeutic agents using nanoparticles have a main technical feature of loading chemical drugs corresponding to anticancer drugs onto nanoparticles and delivering them directly to the target location (cancer cells, tumors), which is not suitable for use as an antigen-presenting cell-based immunotherapy including dendritic cells. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention is a non-injectable drug delivery system using mucoadhesive nanoparticles, which has been developed to solve the above-mentioned problems, and the main object of the present invention is to provide a mucoadhesive nanoparticle and a method for producing the same, which prevents deformation of the nanoparticles due to mucosal moisture, etc., and strengthens the mucoadhesive force to prevent disappearance by binding a mucoadhesive polymer to the surface of the nanoparticle. Another object of the present invention is to provide a composition for maturing antigen-presenting cells, a composition for treating infectious diseases, and a composition for treating cancer, which include the mucoadhesive nanoparticles. Another object of the present invention is to provide a mucoadhesive nanoparticle 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 including the same.

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

[0007] Another object of the present invention is to provide a composition for inducing maturation of antigen-presenting cells (APCs), comprising the mucoadhesive-PLGA nanoparticles as an active ingredient.

[0008] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating infectious diseases, comprising the mucoadhesive-PLGA nanoparticles as an active ingredient.

[0009] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the mucoadhesive-PLGA nanoparticles as an active ingredient.

[0010] The object of the present invention is not limited to the above description, but is provided for the purpose of all cases in which the present invention can be utilized to obtain suitable effects. [Means for solving the problem]

[0011] The present invention provides mucoadhesive-PLGA (Poly(D,L-lactide-co-glycolide)) nanoparticles having a mucoadhesive polymer bound to the surface of the PLGA nanoparticles.

[0012] The present invention also provides mucoadhesive-PLGA nanoparticles, wherein the mucoadhesive polymer is selected from the group consisting of catechol (CAT), carrageenan, gelatin, pectin and polyethylene glycol (PEG).

[0013] The present invention also provides mucoadhesive-PLGA nanoparticles, wherein the mucoadhesive polymer is catechol (CAT).

[0014] The present invention also provides mucoadhesive-PLGA nanoparticles, which contain an antigen inside the nanoparticles.

[0015] The present invention also provides mucoadhesive-PLGA nanoparticles, wherein the antigen is selected from the group consisting of peptides, siRNAs and mRNAs.

[0016] The present invention also provides mucoadhesive-PLGA nanoparticles further comprising an adjuvant inside the nanoparticles.

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

[0018] The present invention also provides a composition for inducing maturation of antigen-presenting cells, wherein the antigen-presenting cells are dendritic cells.

[0019] The present invention also provides a pharmaceutical composition for preventing or treating an infectious disease, comprising the mucoadhesive-PLGA nanoparticles as an active ingredient.

[0020] The present invention also provides a pharmaceutical composition for preventing or treating an infectious disease, wherein the pharmaceutical composition is for spraying into the oral cavity.

[0021] The present invention also provides a pharmaceutical composition for preventing or treating cancer, comprising the mucoadhesive-PLGA nanoparticles as an active ingredient.

[0022] The present invention also provides a pharmaceutical composition for preventing or treating cancer, which induces 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 spraying into the oral cavity.

[0025] The present invention also provides a method for preparing mucoadhesive-PLGA nanoparticles having mucoadhesive polymers bound to the surface of the PLGA nanoparticles, the method comprising the steps of: 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, with the mixture, a compound in which PVA-NH2, in which an amino group has been introduced into polyvinyl alcohol (PVA), and a mucoadhesive polymer, in which a carboxyl group has been introduced, are chemically bound to each other.

[0026] The present invention also provides a method for preparing CAT-PLGA nanoparticles having catechol (CAT) bound to the surface of PLGA nanoparticles, the method comprising the steps of: 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, in which an amino group has been introduced into PVA, and 3,4-dihyroxyhydrocinnamic acid (CAT-COOH) are chemically bound to each other.

[0027] As one embodiment of the present invention, there is provided a mucoadhesive-PLGA (Poly(D,L-lactide-co-glycolide)) nanoparticle having a mucoadhesive polymer bound to the surface of the PLGA nanoparticle.

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

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

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

[0031] [ka]

[0032] In the present invention, the catechol is bound to the surface of the PLGA nanoparticles through chemical modification, and can increase the adhesion, adsorption, fixation and deposition ability of the nanoparticles to mucosa through physical entanglement, hydrogen bond, hydrophobic bond or ionic interaction, etc. Also, it is biodegradable and non-toxic and can be used in vivo without side effects, and can be fixed to functional amine groups, thiol groups or imidazole groups in the mucosa layer through chemical interaction in mucosa 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 ratio of lactic acid:glycolic acid (x:y) of the PLGA is not particularly limited, and may be used in a ratio that can be used when preparing PLGA nanoparticles. Non-limiting examples of the ratio of lactic acid:glycolic acid include 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, or 10:90, more specifically, 30:70 to 70:30, and even more specifically, about 40:60 to 60:40. In one embodiment of the present invention, a PLGA having a ratio of lactic acid:glycolic acid of about 50:50 is used, and when used within the above range, the in vivo degradation rate of the PLGA nanoparticles can be more appropriately adjusted, and the release rate of the drug or the like loaded in the nanoparticles can be more appropriately adjusted, and therefore, there is an advantage that it is more suitable for preparing PLGA nanoparticles for in vivo drug delivery.

[0037] The mucoadhesive-PLGA nanoparticles of the present invention have a mucoadhesive polymer bound to the surface of the nanoparticle, which prevents deformation of the nanoparticles due to moisture in the mucosa and prevents loss of the drug loaded in the nanoparticles, and also strengthens the adhesiveness of the nanoparticles to the mucosa, allowing the nanoparticles to effectively adhere, fix and deposit on the mucosa, thereby preventing loss of the drug and effectively delivering it to cells.

[0038] In the present invention, the mucosa refers to a mucosa to which the mucoadhesive-PLGA nanoparticles can come into contact and adhere, particularly a biological mucosa, and non-limiting examples thereof include oral mucosa and nasal mucosa.

[0039] In the present invention, the mucoadhesive-PLGA nanoparticles may contain an antigen therein.

[0040] In the present invention, the term "antigen" is a general term for all substances that enter the body and cause an immune response, and generally refers to all substances that are recognized as foreign substances in the body, induce an immune reactive state, and react with immune cells or antibodies of a subject that has been sensitized thereto. In the present invention, the term "antigen" may be used interchangeably with the term "immunogen," and includes a molecule that contains one or more epitopes that can stimulate the host immune system to cause a secretory, humoral, or cellular immune response specific to the antigen. In the present invention, non-limiting examples of the antigen may include peptides (including polypeptides and proteins), siRNAs, or mRNAs.

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

[0042] In the present invention, the mucoadhesive-PLGA nanoparticles may contain an adjuvant therein.

[0043] In the present invention, the term "adjuvant" means a substance that has a positive effect on the action of other substances pharmacologically or immunologically, in the sense of supporting or assisting, and is also called an immune enhancer or immune promoter.

[0044] In the present invention, the type of the adjuvant is not particularly limited, and any adjuvant used in the present technical field or related fields may be used. Non-limiting examples of the adjuvant may include TLR3 adjuvants (Poly I:C, Poly I:CLC (hiltonol), PolyI: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 that it can be loaded with an antigen and delivered to the living body with excellent efficiency in the mucoadhesive-PLGA nanoparticles of the present invention, and can be effectively used in cancer treatment immunotherapy.

[0045] In another embodiment of the present invention, there is provided a composition for inducing maturation of antigen-presenting cells (APCs), comprising the mucoadhesive-PLGA nanoparticles of the present invention as an active ingredient.

[0046] In the present invention, "antigen-presenting cells (APCs)" refer to cells that introduce protein antigens into cells (endocytosis), and then present antigen-derived peptide fragments together with major histocompatibility complex (MHC) molecules to T cells to activate them. Representative antigen-presenting cells include dendritic cells (DCs), B cells, macrophages, etc., which correspond to the main immune cells responsible for cellular immunity in the body. When an antigen enters the body, these cells introduce it into the cells and then make it appear on the surface, thereby playing the role of antigen-expressing cells that allow other cells of the immune system (such as T cells) to recognize the antigen.

[0047] Among the antigen-presenting cells, dendritic cells are known to be present mainly in tissues that come into contact with the external environment, such as the skin, nose, lungs, stomach, or intestinal lining, and cells in the skin in particular are called Langerhans cells. Dendritic cells can be found in an immature state in the blood, and when activated, they are known to migrate to lymphatic organs and interact with T cells and B cells to initiate immune responses. Dendritic cells are also known to extend protrusions called dendrites at a certain developmental stage.

[0048] Dendritic cells are derived from hemopoietic bone marrow progenitor cells, which initially transform into immature dendritic cells (DCs) that exhibit high endocytic activity and T-cell activation. Immature dendritic cells continually phagocytose pathogens such as viruses and bacteria in the environment through pattern recognition receptors (PRRs) such as toll-like receptors (TLRs). TLRs recognize specific chemical signatures found on a subset of pathogens, and immature dendritic cells phagocytose the cell membrane from living autologous cells through a process called nibbling. When they come into contact with existing antigens, they are activated into mature dendritic cells and migrate to lymph nodes. Immature dendritic cells phagocytose pathogens and break down their own proteins into small fragments. When they mature, these fragments are presented on the cell surface using MHC molecules and increase cell surface receptors that act as co-receptors in T-cell activation, such as CD(Cluster of Differentiation)80, CD86, and CD40. By presenting antigens derived from pathogens together with non-antigen specific costimulatory signals, they can activate helper T-cells, killer T-cells, as well as B cells.

[0049] The cytokines produced by dendritic cells vary depending on the cell type. Lymphocytic dendritic cells can produce a large amount of type-1 IFN (Interferon), which attracts more activated macrophages to enable phagocytosis. It is known that lymphocytic dendritic cells are involved in central and peripheral immune regulation, while myeloid dendritic cells are involved in immune induction against foreign antigens and infections. Therefore, if dendritic cells do not function normally, autoimmune diseases such as diabetes, rheumatoid arthritis, and allergic hypersensitivity reactions may appear, or normal immune responses may not occur against infectious diseases or the development of cancer. As mentioned above, dendritic cells play an important role in enhancing the immune function of the body itself, and inducing the maturation of dendritic cells is an important issue in cell immunotherapy using dendritic cells.

[0050] The composition for inducing maturation of antigen-presenting cells, which contains the mucoadhesive-PLGA nanoparticles of the present invention as an active ingredient, effectively delivers immune activators (e.g., antigens and adjuvants) contained in the nanoparticles to induce maturation of immature antigen-presenting cells (e.g., dendritic cells). Thus, when antigen-presenting cells mature, the expression of surface protein display factors increases, which induces the activity of T cells, thereby increasing the immune response.

[0051] Specifically, mature antigen-presenting cells express MHC type I and type II antigens at higher levels than immature antigen-presenting cells, and can regulate CD (Cluster of Differentiation) 80+, CD83+, and CD86+. More MHC expression induces increased antigen density on the surface of antigen-presenting cells, and upregulation of costimulatory molecules CD80 and CD86 can enhance T cell activation signals through costimulatory molecular counterparts such as CD28 on T cells.

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

[0053] In the present invention, the type of the antigen-presenting cells is not particularly limited, and non-limiting examples thereof may be dendritic cells, Langerhans cells, macrophages, mononuclear cells, B cells, etc. In one embodiment of the present invention, dendritic cells are targeted, and as one of the most powerful antigen-presenting cells that act as a bridge between the innate and adaptive immune systems, they have the advantage of exerting superior immune activity.

[0054] In another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating an infectious disease, comprising the mucoadhesive-PLGA nanoparticles as an active ingredient.

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

[0056] In another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, comprising the mucoadhesive-PLGA nanoparticles as an active ingredient.

[0057] In the present invention, the type of cancer (specific disease name or disease site, etc.) is not particularly limited, and may include all cancers whose 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 preventing or treating cancer, which contains the mucoadhesive-PLGA nanoparticles of the present invention as an active ingredient, can treat not only cancers at local sites where it is directly contacted, but also cancers and tumors occurring in various organs, tissues, cells, etc. in the body through the systemic circulation of T 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 prepared and used as a formulation for oral spray, it is particularly effective in treating oral cancer or head and neck cancer.

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

[0059] The pharmaceutical composition of the present invention can effectively induce maturation of antigen-presenting cells, and can also promote activation of T cells, e.g., cytotoxic CD8+ T cells, and may be utilized in various ways as an effective immunotherapy-based pharmaceutical composition.

[0060] The pharmaceutical composition of the present invention may be prepared in any dosage form that can be introduced into the body and may be administered by any administration method. The pharmaceutical composition of the present invention has been developed as a nanoparticle with excellent mucoadhesion, adsorption, fixation and deposition ability in order to solve the problems of the conventional injection-based administration method, and therefore may be formulated to be easily administered to mucosa, especially oral or nasal mucosa. It may also be prepared as a spray dosage form for oral spray so that the drug can be delivered and absorbed more easily and effectively. The method of preparing the spray dosage form is not particularly limited and may be performed by a method used in the art or related fields.

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

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

[0063] As another embodiment of the present invention, a) mixing an aqueous solution containing an antigen and an adjuvant with an organic solution containing PLGA (Poly(D,L-lactide-co-glycolide)); b) providing a method for producing CAT-PLGA nanoparticles having catechol (CAT) bound to the surface of PLGA nanoparticles, the method including a step of mixing, with the mixture, PVA-NH2, in which an amino group has been introduced into polyvinyl alcohol (PVA), and a compound (PVA-CAT), in which 3,4-dihyroxyhydrocinnamic acid (CAT-COOH) is chemically bound 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 antigen and adjuvant are dissolved in the primary water phase, and PLGA is dissolved in the oil phase, which is an organic solvent, and then mixed to form a primary emulsion. The primary emulsion is then mixed with another secondary water phase, a polyvinyl alcohol-catechol (PVA-CAT) aqueous solution, to form a secondary emulsion, and the oil in the secondary emulsion is evaporated to produce the nanoparticles.

[0065] In the case of PLGA nanoparticles prepared using general PVA as the secondary aqueous phase during preparation of an emulsion formulation of PLGA nanoparticles, there are problems such as the nanoparticles being deformed by mucosal moisture, causing the drug loaded therein to disappear, and the adhesion to the mucosa is very weak, causing the nanoparticles to be delivered to the digestive tract without being fixed to the mucosa. In contrast, the nanoparticles of the present invention use a PVA-mucoadhesive polymer (e.g., PVA-CAT) in which a mucoadhesive polymer (e.g., CAT) is chemically bonded to PVA as the secondary aqueous phase to prepare mucoadhesive-PLGA nanoparticles having a mucoadhesive polymer bonded to the surface, thereby preventing deformation of the nanoparticles due to mucosal moisture, preventing the drug loaded therein to disappear, and strengthening the adhesion of the nanoparticles to the mucosa, allowing the nanoparticles to effectively adhere, fix and deposit on the mucosa, thereby preventing the drug from disappearing and effectively delivering it to cells.

[0066] In one embodiment of the present invention, the chemical reaction formula for the method and process for preparing 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 producing the PVA-CAT is as follows.

[0069] PVA was modified using carbonyldiimidazole (CDI) and ethylenediamine (EDA) to prepare PVA-NH2. Specifically, DMSO (60 mL) containing CDI (74 mg) was added to PVA (400 mg) and 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, and the CDI-PVA was dried in a vacuum oven. Next, CDI-PVA (400 mg) and EDA (4 g) were dissolved in DMSO (100 mL), 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 the 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 the mixture was further stirred at 24°C for 24 hours. Then, 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, a method for preparing the CAT-PLGA nanoparticles of the present invention using the PVA-CAT is as follows.

[0071] 1 mg of E7 as an antigen and 2 mg of poly I:C as an adjuvant 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 for 5 seconds with 3-second intervals). The primary emulsion was sonicated with the secondary water 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, the CAT-PLGA(E7+poly I:C)-NPs were centrifuged at 15,800×g for 30 minutes to prepare CAT-PLGA(E7+poly I:C)-NPs.

[0072] Terms not specifically defined in the present invention are to be interpreted as having the meaning normally used in the art. Furthermore, the term "or" described in the present invention may be interpreted as including "and" unless otherwise specified.

[0073] The scope of the present invention is not limited by the specific description disclosed in the present invention, and each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all possible combinations of various elements disclosed in the present invention are considered to belong to the scope of the present invention. In addition, a person having ordinary skill in the art can recognize or ascertain many equivalents to specific examples of the present invention through routine experimentation, and such equivalents are considered to belong to the scope of the present invention. Effect of the Invention

[0074] The present invention relates to a non-injectable drug delivery system using mucoadhesive nanoparticles. The present invention has an advantage that the nanoparticles are prevented from being deformed by mucosal moisture, etc., and the drug loaded on the nanoparticles is prevented from being lost by binding a mucoadhesive polymer to the surface of the nanoparticles, and the nanoparticles are effectively adhered, fixed and deposited on the mucosa by strengthening the adhesion of the nanoparticles to the mucosa, thereby preventing the drug from being lost and effectively delivered to cells. In addition, the present invention has an advantage that when an antigen is loaded inside the nanoparticles, it can be effectively delivered in vivo or intracellularly, and can be used in various ways as a composition for maturing antigen-presenting cells, a composition for treating infectious diseases and a composition for treating cancer. In addition, the present invention has an advantage that the nanoparticles can be loaded with immune active substances (such as antigens and adjuvants) for antigen-presenting cell-based cancer treatment immunotherapy, including dendritic cells (DCs), and can be used for cancer treatment immunotherapy by activating antigen-specific cytotoxic T cells (CD8+ T cells). [Brief description of the drawings]

[0075] [Figure 1] FIG. 1 is a schematic diagram showing the drug delivery and cancer treatment process of the spray-type CAT-PLGA-NP of the present invention. [Diagram 2] FIG. 2 shows a chemical reaction scheme (FIG. 2A) and 1H-NMR analysis results (FIG. 2B) illustrating the PVA-CAT conjugation process by chemical modification in the present invention. [Diagram 3] FIG. 3 shows the results of PVA-CAT conjugate analysis by FT-IR, where the circled number 1 indicates the benzene aromatic ring of CAT-COOH and the circled number 2 indicates the amide I band of PVA-CAT. [Figure 4]FIG. 4 is a schematic diagram, graph, and photograph showing the physicochemical properties of CAT-PLGA-NP. Specifically, FIG. 4A is a schematic diagram showing the manufacturing process of CAT-PLGA(E7+poly I:C)-NP. FIG. 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 into CAT-PLGA-NP was measured by Nanodrop (absorbance: 260 nm) and BCA protein analysis, respectively. FIG. 4C shows the results of measuring the size and zeta potential of CAT-PLGA-NP after spraying, and the loading efficiency of poly I:C and E7 into PLGA-NP and CAT-PLGA-NP after spraying. FIG. 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 of E7 from CAT-PLGA(E7+poly I:C)-NPs at 37°C and 50 RPM. Figure 4F shows the mucin adsorption to CAT PLGA-NPs measured by UV-vis spectrophotometer at 263 nm. The data are expressed as mean ± SD (n=3) (*p<0.001). [Diagram 5] FIG. 5 shows the size distribution of CAT-PLGA-NPs before and after spraying, measured by laser scattering using a particle analyzer. [Figure 6] Figure 6 shows graphs and photographs measuring the binding and intracellular delivery of CAT-PLGA-NP to DCs, and the maturation of DCs induced by CAT-PLGA(E7+poly I:C)-NPs. Specifically, Figure 6A shows the results of measuring the binding of CAT PLGA-NPs to DCs using flow cytometry, and Figure 6B shows the results of measuring the intracellular delivery of CAT-PLGA NPs to DCs using a confocal microscope (magnification: ×200, scale bar: 20 μm). Error bars represent SEM (*p<0.001). [Figure 7]Figure 7 is a graph measuring DC maturation and cytokine production induced by CAT-PLGA(E7+poly I:C)-NP. Specifically, Figure 7A shows the results of measuring the levels of surface markers (CD40, CD80, CD86) by flow cytometry. Figure 7B shows the results of quantifying infectious cytokines and DC activating factors in the culture supernatant of DC cultured with CAT-PLGA(E7+poly I:C)-NP by ELISA. Error bars represent SEM (*p<0.001, **p<0.05). [Figure 8] Figure 8 shows the in-vivo fluorescence images of mice after CAT-PLGA-NP injection monitored using IVIS. Error bars represent SEM (*p<0.01). [Figure 9] FIG. 9 shows the results for adhesion of CAT-PLGA-NPs to the oral mucosa of mice and animals (Murine) (magnification: ×200, scale bar: 100 μm). [Figure 10] FIG. 10 shows the therapeutic efficacy of CAT-PLGA-NPs in the TC-1 tumor tongue model. Specifically, treatment with PLGA(E7+poly I:C)-NPs and CAT-PLGA(E7+poly I:C)-NPs was started 3 weeks after injection of TC-1 tumor cells into C57BL6 mouse tongues. PLGA(E7+poly I:C)-NPs and CAT-PLGA(E7+poly I:C)-NPs were sprayed once a week at a dose of 50 μg E7 and poly I:C. FIG. 10A shows the experimental schedule for treatment, FIG. 10B shows a tongue weight graph, FIG. 10C shows tongue and tumor weight photographs, and FIG. 10D shows a graph showing mouse body weight. Error bars represent SEM (*p<0.001, **p<0.01, ***p<0.05). [Figure 11] Figure 11 shows the results of flow cytometry to measure cytotoxic IFN-γ+CD8+ T cells in tongue, mandibular lymph node and spleen cells. 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 tongue. Specifically, immunohistochemical analysis of cell proliferation marker (Ki67) and measurement of microvessel 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). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0076] The present invention will be described in more detail with reference to the following 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 sense by these examples.

[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-dihyroxyhydrocinnamic acid (CAT-COOH), carbonyldiimidazole (CDI), ethylenediamine (EDA), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride (NHS), methyl methacrylate (MMMA ... Polyinosinic-polycytidylic acid sodium salt (poly I:C) and porcine stomach type II mucin 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 the materials were of analytical grade and were used without further purification. EXAMPLES

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

[0079] In the second step, CAT-COOH was conjugated with PVA-NH2 using NHS and EDC. Specifically, NHS (68 mg) and EDC (108 mg) were added to the 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 further stirred at 24°C for 24 hours. Then, 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 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: Preparation of catechol-conjugated PLGA nanoparticles (CAT-PLGA-NP) PLGA(E7+poly I:C)-NP refers to PLGA nanoparticles containing E7 as an antigen and poly I:C as an adjuvant, and was prepared by 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 for 5 seconds with 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 chloroform was completely evaporated using an evaporator for 10 minutes, the PLGA(E7+poly I:C)-NP was washed three times by centrifugation at 15,800×g for 30 minutes and then stored at 4° C. until use. CAT-PLGA(E7+poly I:C)-NP was prepared in the same manner as the PLGA(E7+poly I:C)-NP, except that 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: Release of E7 from CAT-PLGA(E7+poly I:C)-NPs CAT-PLGA (E7 + poly I:C)-NPs were added to the microtubes and then placed in a water bath for a given time. The microtubes were then centrifuged at 15,800 × g for 60 min, and the supernatant was collected to measure the cumulative release of E7 from CAT-PLGA (E7 + poly I:C)-NPs. The amount of released E7 was measured using a BCA protein assay kit.

[0084] Example 4: Mucin adsorption by CAT-PLGA-NPs To confirm the mucin adsorption of 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). The mixtures were centrifuged at 15,800×g for 30 min, and the supernatants were collected and the amount of unadsorbed mucin was measured by UV-vis spectrophotometer at 263 nm.

[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 the approved protocol for the proper use and care of the specific pathogen-free housing facility at Konkuk University by the Animal Care Committee of Konkuk University Veterinary Hospital (Ref. No.: KU20214).

[0086] TC-1 cells (expressing HPV type 16 E6 and E7 proteins) were cultured in RPMI 1640 medium supplemented with 0.1% gentamicin and 10% fetal bovine serum (Biowest, Nuaille, France). 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: Conjugation and intracellular delivery of CAT-PLGA-NPs Before confirming the binding and intracellular delivery of CAT-PLGA-NPs, the fluorescent dye Cy5.5 was loaded onto the CAT-PLGA-NPs as a model drug. To measure the binding efficiency of CAT-PLGA-NPs, DCs were cultured with CAT-PLGA(Cy5.5)-NPs for 5 and 30 min at 37°C, respectively. After the culture, DCs were washed with PBS, then stained with FITC-labeled anti-CD11c and analyzed by flow cytometry (BD Facscalibur with CELLQuest software, BD biosciences, Franklin Lakes, NJ, USA). For confocal microscopy, DCs were cultured with CAT-PLGA(Cy5.5)-NPs for 30 min at 37°C. DCs were then fixed with 4% paraformaldehyde (w / v) for 10 min at 24° C. and stained with 1 μM Sytox® green (Life Technologies, Carlsbad, CA, USA) in PBS for 10 min. Intracellular delivery of CAT-PLGA(Cy5.5)-NPs in DCs was observed using a confocal microscope (LSM 710, Carl Zeiss, Oberkochen, Germany).

[0088] Example 7: DC maturation and cytokine production DCs were cultured at 5 × 10 per well. 6The cells were cultured in 6-well plates at a density of 100x ...

[0089] Example 8: Adhesion of CAT-PLGA-NPs to mouse oral mucosa To evaluate the mucoadhesion of CAT-PLGA(Cy5.5)-NPs, CAT-PLGA(Cy5.5)-NPs were sprayed onto the oral mucosa of C57BL / 6 mice. The fluorescent signal of CAT-PLGA(Cy5.5)-NPs 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)-NPs, immunohistochemistry (IHC) analysis was performed using oral mucosa tissues. CAT-PLGA(Cy5.5)-NPs were sprayed onto the oral mucosa tissue layer and incubated for 30 min, after which the mucosa tissues were immediately washed twice with PBS. The tissues were fixed using 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), and tissues were counterstained with H&E (hematoxylin and eosin, Leica biosystems, Buffalo, IL, USA) and analyzed using a microscope (Eclipse NI, Nikon, Tokyo, Japan).

[0090] Example 9: Therapeutic efficacy of CAT-PLGA-NPs To generate tumors, TC-1 cells (4 × 10 in 25 μL HBSS) were cultured in 1000 x 100 cells / mL. 4C57BL / 6 mice were injected with 50 μg of E7 and poly I:C into the tongues of mice (n=5 per group). To evaluate the therapeutic efficacy, vaccination was started 3 days after the mice were injected with tumor cells. Three groups of mice, (1) control group (negative), (2) control group, (3) PLGA(E7+poly I:C)-NPs, and (4) CAT-PLGA(E7+poly I:C)-NPs (50 μg of E7 and poly I:C, respectively), were vaccinated once a week for a total of three times, and tongue weights and mouse weights were recorded. Tongues, mandibular lymph nodes, and spleen cells were also collected from the mice to confirm the activation of cytotoxic CD8+ T cells. Cells isolated from the tissue were resuspended in 1 mL of RPMI 1640 containing 10% FBS, 0.1% gentamicin, and 1.0% β-mercaptoethanol, and cultured with GolgiPlug (BD Biosciences, San Diego, CA, USA) and E7 (1 μg / mL) for 24 h. The cells were then washed and stained with APC-anti-CD8a and FITC-anti-IFN-γ to identify IFN-γ+CD8+ T cells using flow cytometry. H&E analysis, IHC analysis for cell proliferation (anti Ki67, Abcam, Cambridge, UK), microvessel density (MVD, anti-CD31, Abcam Cambridge, UK), apoptotic cell death (TUNEL, Trevigen, Gaithersbug, MD, USA) and CD8+ T cell population (anti-CD8, Biolegend, San Diego, CA, USA) were performed using tongue tissues isolated from mice. Stained tissues were analyzed using bright-field and fluorescent microscopes. Analysis was recorded in random fields of each slide (five random fields at ×400 magnification).

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

[0092] [Experimental Results] Experimental Results 1: Induction of dendritic cell (DC)-based anti-cancer immune responses in oral cancer by tumor-specific antigen delivery using sprayable CAT-PLGA-NPs The present inventors designed and manufactured CAT-PLGA-NP, a spray-type mucosally fixed and deposited nanoparticle delivery vehicle, and effectively induced DC-based immune responses by simply and easily delivering tumor-specific antigens to DCs in oral cancer patients who experience injection rejection.

[0093] Experimental result 2: Chemical modification of PVA-bonded CAT Before preparing CAT-PLGA-NPs, CAT-COOH and PVA-NH2 were conjugated by chemical modification (Figure 2A). The conjugation of PVA-CAT was achieved by the following chemical modification: (1) OH (aromatic C-OH) of CAT-COOH, (2) CH2 (methylene) of CAT-COOH, and (3) CH2 (methylene) of PVA. 1 H-NMR spectrum (Fig. 2B). PVA-CAT was also confirmed using FT-IR (Fig. 3). -1 ~1622cm -1 The absorption peak between 1643 cm and 1703 cm is due to the aromatic ring C=C vibration band (benzene aromatic ring). In particular, in PVA-CAT, the stretching C=O vibration coupling of the amide I band at 1643 cm -1 The peaks indicate that CAT and PVA were well conjugated with the COOH of CAT and the NH2 of PVA.

[0094] Experimental results 3: Physical properties of CAT-PLGA-NPs CAT-PLGA(E7+polyI:C)-NPs were prepared as shown in Figure 4A. The size of PLGA-NPs and CAT-PLGA-NPs was about 200 nm (Figure 4B). The zeta potential was about -70 mV, and the loading efficiency of poly I:C and E7 was confirmed to be 35% (poly I:C) and 70% (E7), respectively (Figure 4B). In addition, the sprayed PLGA-NPs and CAT-PLGA-NPs showed no difference compared to the "before spray" condition (Figure 4C), and did not show any 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 dosage form. Next, the release of E7 from CAT-PLGA-NPs was confirmed (Figure 4E). The cumulative release profiles of E7 from PLGA-NPs and CAT-PLGA-NPs were similar, with a burst release observed within 8 h.

[0095] Mucin is an important glycoprotein in mucosa and plays a role in mucosal structure. Therefore, the adsorption effect of CAT-PLGA-NP was examined. Mucin was adsorbed to the surface of CAT-PLGA-NP with a significantly higher amount of CAT-PLGA-NP than that of CAT-unlabeled PLGA-NP (Figure 4F).

[0096] Experimental result 4: Binding and intracellular delivery of CAT-PLGA-NPs The binding of CAT-PLGA(Cy5.5)-NP to DCs was measured using flow cytometry, and it was confirmed that CAT-PLGA(Cy5.5)-NP showed higher binding efficiency than PLGA(Cy5.5)-NP (Figure 6A). In addition, the intracellular delivery of CAT-PLGA(Cy5.5)-NP was measured using confocal microscopy. It was confirmed that the relative fluorescence intensity of CAT-PLGA(Cy5.5)-NP was higher than that of PLGA(Cy5.5)-NP (Figure 6B). These results suggest that CAT-PLGA(Cy5.5)-NP is more suitable for binding and intracellular delivery to DCs than PLGA(Cy5.5)-NP due to CAT labeling.

[0097] Experimental result 5: DC maturation and cytokine production induced by CAT-PLGA-NPs The cell surface markers of DCs were measured using flow cytometry. DCs treated with CAT-PLGA(E7+poly I:C)-NPs showed a significant increase in CD40, CD80, and CD86 compared to other groups (Figure 7A). In particular, significant differences were observed between the control group, poly I:C group, and CAT-PLGA-NP group. In addition, 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-NPs to mouse oral mucosa To confirm the adhesiveness of CAT-PLGA-NP to the oral mucosa layer, CAT-PLGA(Cy5.5)-NP was sprayed onto the oral mucosa layer of C57BL / 6 mice, and the fluorescence intensity was observed using IVIS. The fluorescence intensity of CAT-PLGA(Cy5.5)-NP in the mucosa layer was maintained for 4 hours, longer than that of PLGA(Cy5.5)-NP (Figure 8). In addition, the oral mucosa layer was separated from C57BL / 6 mice, and CAT-PLGA(Cy5.5)-NP was sprayed onto the mucosa layer. 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-NPs TC-1 cells expressing HPV 16 E6 and E7 proteins are commonly used as a tumor model for cancer immunotherapy. Therefore, a TC-1 tongue tumor model was developed to measure the therapeutic efficacy of CAT-PLGA-NPs. To prepare the mouse tongue tumor model, TC-1 cells (4 × 10 4 C57BL / 6 mice (n=5 per group) were injected with 1000 mg of E7+poly I:C-NPs (cells / mouse) into the tongues of C57BL / 6 mice. Vaccination was started 3 days after tumor cell injection into C57BL / 6 mice. (1) Control group (negative), (2) Control group, (3) PLGA(E7+poly I:C)-NPs, and (4) CAT-PLGA(E7+poly I:C)-NPs (Figure 10A). The CAT-PLGA(E7+poly I:C)-NPs group showed significant inhibition of tumor growth compared to the control group (56%, p<0.001) and PLGA(E7+poly I:C)-NPs (30%, p<0.01) (Figures 10B and C). However, the CAT-PLGA(E7+poly I:C)-NPs group showed small differences in tongue weight and tumor weight compared to the control group (negative) (Figures 10B and C).

[0100] Based on the results, the body weight of the vaccinated group was confirmed (Figure 10D). The CAT-PLGA(E7+poly I:C)-NP showed consistency, while the control group (p<0.01) and PLGA(E7+poly I:C)-NP (p<0.05) showed weight loss. This was because mice in the control and PLGA(E7+poly I:C)-NP groups experienced difficulty in food intake due to tumor growth in the tongue (Figure 10D). To evaluate CD8+ T cell activation, IFN-γ+ CD8+ T cell populations were confirmed in the tongue, mandibular lymph node, and spleen cells. CAT-PLGA(E7+poly I:C)-NPs 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 in tongue tissue (p<0.001) and PLGA(E7+poly I:C)-NPs (p<0.01), in mandibular lymph nodes (p<0.001) and PLGA(E7+poly I:C)-NPs (p<0.05), and in the spleen (p<0.001) and PLGA(E7+poly I:C)-NPs (p<0.01) (Figure 11).

[0101] The control group and PLGA(E7+polyI:C)-NPs showed a larger proportion of tumor in tongue tissues by H&E staining analysis compared to CAT-PLGA(E7+polyI:C)-NPs (Figure 12). IHC analysis was also used to analyze tumors for markers of cell proliferation (ki67), microvessel density (MVD, CD31), apoptotic death (TUNEL), and CD8+ T cells. CAT-PLGA(E7+poly I:C)-NPs showed a significant inhibition of cell proliferation, a decrease in microvessel density, and an increase in the number of apoptotic cells compared to the control group (p<0.01) and PLGA(E7+poly I:C)-NPs (p<0.05) (Figure 12). In addition, the number of CD8+ T cells in the tongue tissue of the CAT-PLGA(E7+poly I:C)-NP group was increased compared to the control group (p<0.001) and the PLGA(E7+poly I:C)-NP group (p<0.01) (Figure 12).

[0102] In this specification, detailed descriptions of contents that a person having ordinary skill in the technical field of the present invention can fully recognize and infer are omitted, and various modifications are possible within the scope of the technical idea and essential configuration of the present invention other than the specific examples described in this specification. Therefore, the present invention may be carried out in a manner different from that specifically described and exemplified in this specification, and this is a matter that can be understood by a person having ordinary skill in the technical field of the present invention.

Claims

1. Mucoadhesive-PLGA nanoparticles are formed by binding a mucoadhesive polymer to the surface of PLGA (Poly(D,L-lactide-co-glycolide)) nanoparticles.

2. 2. The mucoadhesive-PLGA nanoparticles of claim 1, wherein the mucoadhesive polymer is selected from the group consisting of catechol (CAT), carrageenan, gelatin, pectin, and polyethylene glycol (PEG).

3. The mucoadhesive-PLGA nanoparticles of claim 1, wherein the mucoadhesive polymer is catechol (CAT).

4. The mucoadhesive-PLGA nanoparticles of claim 1, wherein the nanoparticles contain an antigen therein.

5. The mucoadhesive-PLGA nanoparticles of claim 4, wherein the antigen is selected from the group consisting of peptides, siRNA and mRNA.

6. The mucoadhesive-PLGA nanoparticles of claim 4, wherein the nanoparticles further contain an adjuvant therein.

7. A composition for inducing maturation of antigen-presenting cells (APCs), comprising the mucoadhesive-PLGA nanoparticles according to any one of claims 1 to 6 as an active ingredient.

8. The composition for inducing maturation of antigen-presenting cells according to claim 7 , wherein the antigen-presenting cells are dendritic cells.

9. A pharmaceutical composition for the prevention or treatment of infectious diseases, comprising the mucoadhesive-PLGA nanoparticles according to any one of claims 1 to 6 as an active ingredient.

10. The pharmaceutical composition for preventing or treating an infectious disease according to claim 9, wherein the pharmaceutical composition is for oral spray.

11. A pharmaceutical composition for preventing or treating cancer, comprising the mucoadhesive-PLGA nanoparticles according to any one of claims 1 to 6 as an active ingredient.

12. The pharmaceutical composition for preventing or treating cancer according to claim 11 , wherein the pharmaceutical composition is for oral spray.

13. a) mixing an aqueous solution containing an antigen and an adjuvant with an organic solution containing PLGA (Poly(D,L-lactide-co-glycolide)); b) Adding an amino group to polyvinyl alcohol (PVA) to the mixture to prepare PVA-NH ２ and a carboxyl group-introduced mucoadhesive polymer are chemically bonded to each other,

14. a) mixing an aqueous solution containing an antigen and an adjuvant with an organic solution containing PLGA (Poly(D,L-lactide-co-glycolide)); b) Adding an amino group to polyvinyl alcohol (PVA) to the mixture to prepare PVA-NH ２ and 3,4-dihydroxyhydrocinnamic acid (CAT-COOH) are chemically bonded to each other (PVA-CAT), thereby forming a PLGA nanoparticle having catechol (CAT) bonded to the surface thereof.

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