Mrna-encapsulated double nanoparticles, method for preparing same, and pharmaceutical use thereof

Abstract

The present invention relates to messenger RNA (mRNA)-encapsulated double nanoparticles, a method for preparing same, and a pharmaceutical use thereof and, more specifically to: mRNA-encapsulated double nanoparticles comprising a first nanoparticles core in which mRNA, which is an active ingredient, is bonded to an anionic polymer surface or is inserted into the anionic polymer surface, and a second nanoparticle shell in which a biocompatible polymer or a polymer conjugate is formed to surround the outside of the core; a composition for mRNA delivery comprising the double nanoparticles; a composition for a vaccine comprising the double nanoparticles, and the like. The double nanoparticles according to the present invention can be used to safely and efficiently deliver mRNA to a target site.

Description

Double nanoparticles encapsulated with mRNA, their preparation method and their pharmaceutical uses

[0001] The present invention relates to mRNA delivery technology, and more particularly, to mRNA-encapsulated double nanoparticles, a method for producing the same, and pharmaceutical uses thereof.

[0002] Existing mRNA delivery vehicles are broadly divided into lipid-based lipid nanoparticles (LNPs) and cationic polymer-based nanoparticles (PNPs).

[0003] LNPs are composed of ionizable lipids and PEGylated lipids and can efficiently encapsulate mRNA. However, there are reports that the PEG contained in the PEGylated lipids may form anti-PEG antibodies in the human body, which may cause side effects such as hypersensitivity reactions, complement activation, anaphylaxis, and urticaria. Furthermore, since various ionizable lipids and lipid composition ratios that make up LNPs are already patented by domestic and foreign companies, it is not easy to develop LNPs for mRNA delivery while avoiding these patents.

[0004] In the case of PNP, polymers with strong cationic charge (e.g., polyethyleneimine, protamine, etc.) are mainly used, but the release of mRNA is limited due to the strong binding force between these and mRNA, resulting in low mRNA expression efficiency. In addition, polymers with large molecular weights, non-degradable properties, and strong cationic charge have the potential to cause toxicity and side effects through non-specific binding with intracellular proteins or important components, which makes it difficult to obtain approval as a pharmaceutical.

[0005] Accordingly, there is a need for an mRNA delivery technology that is safe, has fewer side effects, and can further improve mRNA expression efficiency.

[0006] The purpose of the present invention is to provide mRNA-encapsulated nanoparticles capable of safely and efficiently delivering mRNA and a method for manufacturing the same.

[0007] Another object of the present invention is to provide a composition for mRNA delivery comprising the mRNA-encapsulated nanoparticles.

[0008] Another object of the present invention is to provide a vaccine composition comprising the mRNA-encapsulated nanoparticles.

[0009] To achieve the above purpose, a double nanoparticle encapsulated with mRNA is provided, comprising a first nanoparticle core in which an active ingredient, messenger RNA (mRNA), is bound to a cationic polymer surface or encapsulated therein; and a second nanoparticle shell formed by surrounding the outside of the core with a biocompatible polymer or polymer conjugate.

[0010] The present invention provides a composition for mRNA delivery comprising a double nanoparticle in which the above mRNA is encapsulated.

[0011] The present invention provides a vaccine composition comprising a double nanoparticle in which the above mRNA is encapsulated.

[0012] The present invention provides a method for producing mRNA-encapsulated dual nanoparticles, comprising the steps of: preparing a polymer solution by dissolving a biocompatible polymer or polymer conjugate in an organic solvent; evaporating the solvent of the polymer solution to form a thin film; and adding a mixed solution of a positively charged solution containing a cationic polymer and a negatively charged solution containing mRNA to the formed thin film and crushing the mixed solution to obtain nanoparticles.

[0013] In addition, the present invention provides a method for producing a double nanoparticle encapsulating mRNA, comprising the steps of: mixing a positively charged solution containing a cationic polymer and a negatively charged solution containing mRNA to produce a first nanoparticle in which the mRNA is bound to the surface of the cationic polymer or encapsulated therein; dissolving a biocompatible polymer or polymer conjugate in an organic solvent to produce a second nanoparticle; and mixing the first nanoparticle aqueous solution and the second nanoparticle aqueous solution to obtain a double nanoparticle in the form of a first nanoparticle core-second nanoparticle shell.

[0014] The mRNA-encapsulated double nanoparticle according to the present invention can control mRNA release by binding the active ingredient mRNA to the cationic polymer surface or encapsulating it inside the first nanoparticle core, and the second nanoparticle shell of the biocompatible polymer or polymer conjugate surrounding the first nanoparticle core acts as a nanocarrier, so that the mRNA can be safely and efficiently delivered to the target site, and can be utilized as a fast-acting or sustained mRNA expression carrier.

[0015] The dual nanoparticles according to the present invention exhibit superior gene expression efficiency, immune activation ability, etc. compared to mRNA single preparations and mRNA-encapsulated single nanoparticles, and thus can be utilized as a more effective vaccine for the target disease targeted by the mRNA.

[0016] Figure 1 shows the in vitro gene expression efficiency of mRNA-encapsulated double nanoparticles prepared by the thin film hydration method or the nanoparticle-nanoparticle fusion method in mouse muscle cells (C2C12).

[0017] Figure 2 shows the in vitro gene expression efficiency of mRNA-encapsulated dual nanoparticles prepared by the thin film method or nanoparticle-nanoparticle fusion method in mouse bone marrow-derived dendritic cells (BMDCs).

[0018] Figure 3 shows the in vivo gene expression efficiency by time period after intramuscular administration of mRNA-encapsulated dual nanoparticles prepared by the thin film hydration method in C57BL / 6J mice.

[0019] Figure 4 shows the in vivo gene expression efficiency by time period after intramuscular administration of mRNA-encapsulated dual nanoparticles manufactured by the nanoparticle-nanoparticle fusion method in C57BL / 6J mice.

[0020] Figure 5 shows the results of evaluating the immune activation ability of mRNA-encapsulated dual nanoparticles manufactured by the nanoparticle-nanoparticle fusion method in mouse bone marrow-derived dendritic cells (BMDCs).

[0021] Figure 6 shows the results of an evaluation of the anticancer vaccine efficacy of dual nanoparticles encapsulating mOVA prepared by a nanoparticle-nanoparticle fusion method in a C57BL / 6J mouse cancer model using B16-OVA.

[0022] Figure 7 shows the results of evaluating the distribution of immune cells and cytokine activation in lymphoid tissue and cancer tissue 18 days after administration of dual nanoparticles containing mOVA prepared by the nanoparticle-nanoparticle fusion method in a C57BL / 6J mouse cancer model using B16-OVA.

[0023] Figure 8 shows the results of blood cytokine production on day 18 after administration of dual nanoparticles encapsulating mOVA prepared by the nanoparticle-nanoparticle fusion method in a C57BL / 6J mouse cancer model using B16-OVA.

[0024] Figure 9 shows the results of OVA-specific cytokine production in spleen cells 18 days after administration of dual nanoparticles encapsulating mOVA prepared by the nanoparticle-nanoparticle fusion method in a C57BL / 6J mouse cancer model using B16-OVA.

[0025] Hereinafter, the present invention will be described in detail.

[0026]

[0027] To circumvent patents on lipid nanoparticles and develop a biodegradable, low-toxic mRNA delivery system, the inventors selected a biocompatible polymer instead of lipids as the mRNA delivery system. Furthermore, to avoid anti-PEG antibody-induced immune responses, they designed an amphiphilic polymer using a biocompatible small molecule instead of PEG, and fabricated self-assembled nanoparticles thereof to enhance colloidal stability. The resulting nanoparticles demonstrated controllable mRNA release and excellent gene expression efficiency, thereby completing the present invention.

[0028]

[0029] The present invention provides a double nanoparticle encapsulating mRNA.

[0030] More specifically, the mRNA-encapsulated double nanoparticle according to the present invention may include a first nanoparticle core in which the active ingredient, messenger RNA (mRNA), is bound to the surface of a cationic polymer or encapsulated therein; and a second nanoparticle shell formed by surrounding the outside of the core with a biocompatible polymer or polymer conjugate.

[0031] In the first nanoparticle core, the mRNA (messenger RNA) serves to contain and transmit genetic information capable of synthesizing a protein, and some sequences may be chemically modified or the ends may be modified to increase the stability or activity thereof, but the present invention is not limited thereto.

[0032] The cationic polymer may be at least one selected from the group consisting of branched polyethylenimine (bPEI), reducible polyethylenimine (RPC), α-poly-L-lysine (PLL), epsilon-poly-lysine (ε-poly-L-lysine; EPL), and reducible ε-poly-L-lysine-Nanogel (REPL-NG), and the number average molecular weight of each polymer may be in the range of 1 to 100 kDa, preferably 1 to 50 kDa, but is not limited thereto.

[0033] The first nanoparticle may be formed in a form in which the mRNA is bound to the surface of the cationic polymer or encapsulated inside the cationic polymer due to electrostatic attraction between the cationic polymer and the mRNA, and more specifically, the mRNA may be formed in a form in which all or part of the mRNA is exposed on the surface of the first nanoparticle or in a form in which all or part of the mRNA is encapsulated inside the first nanoparticle, but is not limited thereto.

[0034] The mRNA may be included in an amount of 0.01 to 30 parts by weight, preferably 1 to 20 parts by weight, based on 100 parts by weight of the total nanoparticles, but is not limited thereto.

[0035] Depending on the type of cationic polymer applied to the first nanoparticle, the release rate of mRNA can be controlled by the decomposition rate and the difference in the strength of interaction between the polymer and mRNA, so the first nanoparticle can function as a nanocontroller.

[0036]

[0037] In the second nanoparticle shell, the biocompatible polymer or polymer conjugate may include a degradable or non-degradable polymer or polymer conjugate, and preferably, glycocholic acid-poly(epsilon-caprolactone) polymer conjugate [Glycocholic acid-Poly(ε-caprolactone) conjugate; GCAPCL-P], dexamethasone-poly(epsilon-caprolactone) polymer conjugate [Dexamethasone-Poly(ε-caprolactone) conjugate; DexPCL-P], choline-poly(epsilon-caprolactone) polymer conjugate [Choline-Poly(ε-caprolactone) conjugate; CholPCL-P], triphenylphosphonium-poly(epsilon-caprolactone) polymer conjugate [Triphenylphosphonium-Poly(ε-caprolactone) conjugate; TPCL-P], betaine-poly(ε-caprolactone) conjugate [Betaine-Poly(ε-caprolactone) conjugate; BPCL-P], carnitine-poly(ε-caprolactone) conjugate [Carnitine-Poly(ε-caprolactone) conjugate; CPCL-P], succinate-poly(ε-caprolactone) conjugate [succinate-Poly(ε-caprolactone) conjugate; snPCL-P], cytidine triphosphate-poly(ε-caprolactone) conjugate [CTP-Poly(ε-caprolactone) conjugate; CTPCL-P], poly(epsilon-caprolactone) diol [Poly(ε-caprolactone) diol; PCLdiOH], poly(epsilon-caprolactone) dicarboxyl [Poly(ε-caprolactone) dicarboxyl; It may be at least one selected from the group consisting of, but is not limited to, poly(lactic acid); PLA), poly(lactic acid-glycolic acid) [Poly(Lactide-glycolid); PLGA].

[0038] The above second nanoparticles can be manufactured by, but are not limited to, a thin film hydration method, a nanoprecipitation method, or a nanoparticle-nanoparticle fusion method.

[0039] The second nanoparticle may function as a nanocarrier capable of delivering mRNA, or the first nanoparticle in which the mRNA is bound to the cationic polymer surface or encapsulated therein, to a target site.

[0040] The above-mentioned double nanoparticles of the first nanoparticle core and the second nanoparticle shell can be formed with an average particle diameter of 20 to 700 nm and can have a zeta potential of -90 to 50 mV.

[0041]

[0042] The present invention provides a composition for mRNA delivery comprising a double nanoparticle in which the above mRNA is encapsulated.

[0043] By encapsulating the mRNA in the dual nanoparticles according to the present invention, the mRNA can be delivered to the target site more safely and effectively, and the mRNA release rate, etc. can be controlled, and gene expression efficiency can be improved. In addition, immune activation can be enhanced by activating TNF-α and increasing IL-2 production.

[0044] According to one embodiment of the present invention, it can be confirmed that the double nanoparticles encapsulated with the mRNA have a gene expression efficiency that is improved by 1 to 80 times or more within 3 to 24 hours after administration to muscle tissue.

[0045] The above composition may be a pharmaceutical composition or health food composition for delivering mRNA for preventing, improving, or treating cancer.

[0046] The above cancer disease may be any one selected from the group consisting of colorectal cancer, breast cancer, triple-negative breast cancer (TNBC), cervical cancer, lung cancer, pancreatic cancer, colon cancer, bone cancer, skin cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma, but is not limited thereto.

[0047]

[0048] In this specification, "pharmaceutical composition" means a composition administered for a specific purpose, and for the purposes of the present invention, it means administered to prevent or treat a disease targeted by mRNA, for example, cancer or at least one symptom thereof.

[0049] The pharmaceutical composition according to the present invention can be prepared according to conventional methods in the pharmaceutical field. The pharmaceutical composition can be combined with an appropriate pharmaceutically acceptable carrier depending on the formulation, and, if necessary, can be prepared by further including excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. The appropriate carrier, etc., can be selected differently depending on the dosage form and formulation, as long as it does not inhibit the activity and properties of the mRNA according to the present invention.

[0050] The pharmaceutical composition according to the present invention can be applied in any dosage form, and more specifically, it can be formulated and used as an oral dosage form, an external preparation, a suppository, and a parenteral dosage form of a sterile injection solution according to a conventional method.

[0051] In the pharmaceutical composition according to the present invention, the pharmaceutical composition can be administered in a pharmaceutically effective amount.

[0052] As used herein, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing adverse effects.

[0053] The effective dosage level of the pharmaceutical composition may vary depending on the intended use, the patient's age, sex, weight, and health condition, the type and severity of the disease, the activity and sensitivity of the drug, the method of administration, the time of administration, the route of administration, and the excretion rate, the duration of treatment, the drugs used in combination or concurrently, and other factors well known in the medical field. For example, although not fixed, it may generally be administered once or several times daily at a dosage of 0.001 to 1000 mg / kg, preferably 0.01 to 100 mg / kg. The above dosage does not limit the scope of the present invention in any way.

[0054] The pharmaceutical composition according to the present invention can be administered to any animal that can develop a disease targeted by the mRNA, and the animal can include, for example, humans and primates, as well as livestock such as cows, pigs, horses, and dogs.

[0055] The pharmaceutical composition according to the present invention can be administered via an appropriate route of administration depending on the formulation form, and can be administered via various routes, either oral or parenteral, as long as it can reach the target tissue. The method of administration is not particularly limited, and can be administered by conventional methods such as oral, rectal, intravenous, intramuscular, or skin application, respiratory inhalation, intrauterine epidural, or intracerebroventricular injection.

[0056] The pharmaceutical composition according to the present invention can be used alone for the prevention or treatment of a disease targeted by the mRNA, or can be used in combination with surgery or other drug treatments.

[0057]

[0058] In this specification, "health food" includes health functional foods manufactured and processed using raw materials or ingredients with functionality useful to the human body, and refers to foods with high medical and healthcare effects that are processed so that, in addition to providing nutrition, the mRNA for the purpose of the present invention efficiently exhibits biological regulation functions such as prevention of diseases targeted by the mRNA, for example, cancer, biological defense, immunity, and recovery.

[0059] The health food according to the present invention can be manufactured in the form of powder, granules, tablets, capsules, syrup, or beverage. There is no limitation on the form the health food can take, and it can be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods.

[0060] The above health foods may include all foods in the conventional sense. For example, they may include beverages and various drinks, fruits and their processed foods (canned fruit, jam, etc.), fish, meats and their processed foods (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.), and all functional foods in the conventional sense. They may also include foods used as animal feed.

[0061] The health food composition according to the present invention can be manufactured by further including food additives (food additives) commonly used in the art and other appropriate auxiliary ingredients that are acceptable in terms of food science. Unless otherwise specified, the suitability as a food additive can be determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety. Items listed in the above 'Food Additives Codex' include, for example, chemical synthetics such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, sucrose pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents for noodles, preservative preparations, and tar color preparations.

[0062] The above other auxiliary ingredients may additionally contain, for example, flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, coloring agents, pectic acid, alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents, etc. In particular, as the natural carbohydrates, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol, and erythritol can be used, and as the sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame can be used.

[0063] The effective dose of the dual nanoparticles contained in the health food according to the present invention can be appropriately adjusted depending on the purpose of use, such as prevention or improvement of a target disease.

[0064] The above health food composition has the advantage of being made from food and having no side effects that may occur with long-term use of general medicines, and is highly portable, so it can be taken as a supplement for preventing or improving the target disease.

[0065]

[0066] The present invention provides a vaccine composition comprising a double nanoparticle in which the above mRNA is encapsulated.

[0067] The above composition can be used as a pharmaceutical composition for a vaccine for a disease targeted by the above mRNA, and for example, it can be an anticancer vaccine composition for cancer, but is not limited thereto.

[0068] According to another embodiment of the present invention, in a cancer model formed of melanoma cells producing OVA, in which the ovalbumin (OVA)-expressing mRNA or the double nanoparticles encapsulating the mRNA were administered into the muscle, it was confirmed that when the double nanoparticles encapsulating the mRNA were administered, the tumor volume and tumor weight could be significantly reduced compared to when only the mRNA was administered.

[0069] In addition, it can be confirmed that the vaccine including the double nanoparticles encapsulating the mRNA increases the production of immune cells such as dendritic cells, cytotoxic T cells, and OVA-specific cytotoxic T cells that help in anticancer action, and interferon gamma (IFN-γ), a key regulator of the immune response, and increases the production of OVA-specific cytokines (TNF-α, IL-6, IL-12, IFN-γ), thereby activating the immune response.

[0070] Accordingly, the double nanoparticles encapsulated with the above mRNA can be utilized as an effective anticancer vaccine composition, but are not limited thereto.

[0071]

[0072] The present invention provides a method for producing a double nanoparticle encapsulated with the above mRNA.

[0073] More specifically, the method for producing a double nanoparticle encapsulating mRNA according to the present invention may include the steps of: preparing a polymer solution by dissolving a biocompatible polymer or polymer conjugate in an organic solvent; evaporating the solvent of the polymer solution to form a thin film; and adding a mixed solution of a positively charged solution containing a cationic polymer and a negatively charged solution containing mRNA to the formed thin film and crushing the mixed solution to obtain nanoparticles.

[0074] In the step of preparing the polymer solution, the biocompatible polymer or polymer conjugate is selected from the group consisting of glycocholic acid-poly(ε-caprolactone) conjugate [Glycocholic acid-Poly(ε-caprolactone) conjugate; GCAPCL-P], dexamethasone-poly(ε-caprolactone) conjugate [DexPCL-P], choline-poly(ε-caprolactone) conjugate [CholPCL-P], and triphenylphosphonium-poly(ε-caprolactone) conjugate [Triphenylphosphonium-Poly(ε-caprolactone) conjugate; TPCL-P], betaine-poly(ε-caprolactone) conjugate [Betaine-Poly(ε-caprolactone) conjugate; BPCL-P], carnitine-poly(ε-caprolactone) conjugate [Carnitine-Poly(ε-caprolactone) conjugate; CPCL-P], succinate-poly(ε-caprolactone) conjugate [succinate-Poly(ε-caprolactone) conjugate; snPCL-P], cytidine triphosphate-poly(ε-caprolactone) conjugate [CTP-Poly(ε-caprolactone) conjugate; CTPCL-P], poly(ε-caprolactone) diol [Poly(ε-caprolactone) diol; PCL diOH ] Poly(ε-caprolactone) dicarboxyl [Poly(ε-caprolactone) dicarboxyl; PCL diCOOH ], polylactic acid [Poly(Lactic acid); PLA], and poly(lactic acid-glycolic acid) [Poly(Lactide-glycolid); PLGA].

[0075] The organic solvent may be at least one selected from the group consisting of ethanol, methanol, chloroform, tetrahydrofuran, dichloromethane, and dimethyl sulfoxide.

[0076] The step of forming the above thin film can be performed by evaporating the solvent from the polymer solution using a rotary evaporator under reduced pressure.

[0077] The step of obtaining the above nanoparticles can be performed by adding a mixed solution of a positively charged solution containing a cationic polymer and a negatively charged solution containing mRNA to the formed thin film and using an ultrasonic disruptor.

[0078] The cationic polymer may be at least one selected from the group consisting of branched polyethylenimine (bPEI), reducible polyethylenimine (RPC), α-polylysine (α-poly-L-lysine; PLL), epsilon-polylysine (ε-poly-L-lysine; EPL), and reducible ε-poly-L-lysine-Nanogel (REPL-NG).

[0079] This allows for obtaining nanoparticles of uniform size, and preferably, a first nanoparticle core in which mRNA is bound to the surface of a cationic polymer or encapsulated inside the mixed solution can be formed, and then a second nanoparticle shell surrounding the outside of the first nanoparticle core with a biocompatible polymer or polymer conjugate can be formed, thereby obtaining a core-shell type double nanoparticle.

[0080]

[0081] Another method for producing a double nanoparticle encapsulating mRNA according to the present invention may include the steps of mixing a positively charged solution containing a cationic polymer and a negatively charged solution containing mRNA to produce a first nanoparticle in which the mRNA is bound to the surface of the cationic polymer or encapsulated therein; dissolving a biocompatible polymer or polymer conjugate in an organic solvent to produce a second nanoparticle; and mixing the first nanoparticle aqueous solution and the second nanoparticle aqueous solution to obtain a double nanoparticle in the form of a first nanoparticle core-second nanoparticle shell.

[0082] The step of manufacturing the second nanoparticles can be performed by dissolving the biocompatible polymer or polymer conjugate in an organic solvent, evaporating the solvent to form a thin film, and then using an ultrasonic disruptor to obtain nanoparticles of uniform size.

[0083] Alternatively, the step of manufacturing the second nanoparticles may be performed by dissolving the biocompatible polymer or polymer conjugate in an organic solvent, adding purified water while stirring to form the biocompatible polymer or polymer conjugate into self-assembled nanoparticles, and dialyzing the nanoparticle dispersion containing the self-assembled nanoparticles into an aqueous phase to remove the organic solvent.

[0084] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0085]

[0086] <Example 1> Preparation of nanocarrier nanoparticles using thin film hydration and evaluation of physicochemical properties

[0087] Nanoparticles were prepared by dissolving polymers in volatile solvents [e.g., ethanol (E), methanol (M), chloroform (C), tetrahydrofuran (T), dichloromethane (D)] and then using the thin film hydration method (U), and were designated by the code names UE, UM, UC, UT, and UD, respectively.

[0088] The polymers are glycocholic acid-poly(ε-caprolactone) conjugate [Glycocholic acid-Poly(ε-caprolactone) conjugate; GCAPCL-P], dexamethasone-poly(ε-caprolactone) conjugate [DexPCL-P], choline-poly(ε-caprolactone) conjugate [CholPCL-P], triphenylphosphonium-poly(ε-caprolactone) conjugate [Triphenylphosphonium-Poly(ε-caprolactone) conjugate; TPCL-P], betaine-poly(ε-caprolactone) conjugate [Betaine-Poly(ε-caprolactone) conjugate; BPCL-P], carnitine-poly(ε-caprolactone) conjugate [Carnitine-Poly(ε-caprolactone) conjugate; CPCL-P], succinate-poly(ε-caprolactone) conjugate [succinate-Poly(ε-caprolactone) conjugate; snPCL-P], cytidine triphosphate-poly(ε-caprolactone) conjugate [CTP-Poly(ε-caprolactone) conjugate; CTPCL-P], poly(ε-caprolactone) diol [Poly(ε-caprolactone) diol; PCL diOH] Poly(ε-caprolactone) dicarboxyl [Poly(ε-caprolactone) dicarboxyl; PCL diCOOH ], polylactic acid [Poly(Lactic acid); PLA], poly(lactic acid-glycolic acid) [Poly(Lactide-glycolid); PLGA], etc. can be used, and these can be used alone or in combination.

[0089] For example, GCAPCL-NP:UE, DexPCL-NP:UE, CholPCL-NP:UE, SnPCL-NP:UE, and PCL prepared as polymer solutions diOH -GCAPCL-P, DexPCL-P, CholPCL-P, snPCL-P and PCL for the manufacture of NP:UE diOH 2 mg was dissolved in 1 mL of ethanol to prepare 2 mg / mL. To prepare TPCL-NP:UC, BPCL-NP:UC, 2 mg of TPCL-P, BPCL-P were dissolved in 1 mL of chloroform to prepare 2 mg / mL. CPCL-NP:UM, CTPCL-NP:UM, PCL diCOOH -CPCL-P, CTPCL-P, PCL for manufacturing NP:UM diCOOH 2 mg was dissolved in 1 mL of methanol to prepare 2 mg / mL.

[0090] The prepared polymer solution was transferred to a glass vial, and the organic solvent was evaporated using a rotary evaporator under reduced pressure to form a thin film. After that, purified water was added to the glass vial where the thin film was formed to prepare a polymer concentration of 1 mg / mL, and then a nanocarrier of uniform size was prepared using an ultrasonicator, and the particle size and zeta potential were measured (Table 1).

[0091] Table 1 below shows the particle size and zeta potential of nanocarriers manufactured by the thin film hydration method.

[0092] Particle size (nm)Zeta potential (mV)GCAPCL-NP:UE48.3 ± 13.9-39.4 ± 1.4DexPCL-NP:UE35.9 ± 10.8-31.2 ± 0.5CholPCL-NP:UE58.4 ± 16.254.5 ± 0.4snPCL-NP:UE54.2 ± 2.4-41.5 ± 2.0TPCL-NP:UC35.1 ± 2.660.9 ± 2.2BPCL-NP:UC75.7 ± 8.570.3 ± 5.1CPCL-NP:UM62.4 ± 7.250.2 ± 2.3CTPCL-NP:UM73.9 ± 21.0-60.1 ± 11.7PCL diOH -NP:UE94.9 ± 25.3-24.1 ± 0.8PCL diCOOH -NP:UM70.2 ± 19.1-24.1 ± 2.0PLA-NP:UE58.7 ± 17.1-37.6 ± 2.4PLGA-NP:UE65.3 ± 17.3-13.3 ± 1.5

[0093]

[0094] <Example 2> Preparation of nanocarrier nanoparticles using nanoprecipitation and evaluation of physicochemical properties

[0095] After dissolving the polymer in an organic solvent miscible with water [e.g., DMSO (dimethyl sulfoxide; S), ethanol (E)], purified water was added while stirring to form self-assembled nanoparticles of the polymer conjugate, which were indicated using the code names NS and NE, respectively.

[0096] The polymer may be GCAPCL-P, DexPCL-P, CholPCL-P, TPCL-P, BPCL-P, CPCL-P, snPCL-P, CTPCL-P, folic acid-poly(ε-caprolactone) conjugate [Folate-Poly(ε-caprolactone) conjugate; FPCL-P], glycocholic acid-polylactic acid conjugate (GCAPLA-P), glycocholic acid-poly(lactic-co-glycolic acid) conjugate [Glycocholic acid-Poly(lactic-co-glycolic acid) conjugate; GCAPLGA-P], etc., and these may be used alone or in combination.

[0097] After dissolving the polymer in an organic solvent at a concentration of 20 mg / mL, purified water was added while stirring to prepare a nanoparticle dispersion so that the final concentration of the polymer conjugate was 1 mg / mL. The dispersion containing the polymer nanoparticles was dialyzed against an aqueous solution for 12 h using a dialysis membrane with a MWCO of 1000 daltons to remove the organic solvent. The purified nanocarrier was obtained, and its particle size and zeta potential were measured (Table 2).

[0098] Table 2 below shows the particle size and zeta potential of nanocarriers manufactured by the nanoprecipitation method.

[0099] Particle size (nm)Zeta potential (mV)GCAPCL-NP:NS121.2 ± 23.9-31.0 ± 1.0GCAPLA-NP:NS78.6 ± 18.9-29.7 ± 2.5GCAPLGA-NP:NS75.9 ± 17.3-34.2 ± 1.6DexPCL-NP:NS112.5 ± 23.6-29.4 ± 1.6snPCL-NP:NS47.5 ± 9.3-47.4 ± 3.5TPCL-NP:NS3.6 ± 3.945.4 ± 13.4BPCL-NP:NS43.7 ± 7.736.7 ± 1.8CPCL-NP:NS47.6 ± 3.935.9 ± 2.5FPCL-NP:NS99.9± 3.0-27.0 ± 1.0

[0100]

[0101] <Example 3> Preparation of mRNA-encapsulating nanocontroller nanoparticles through electrostatic attraction and evaluation of their physicochemical properties.

[0102] A positively charged solution containing cationic polymers such as branched polyethylenimine (bPEI, molecular weight 25 kDa), reducible polyethylenimine (RPC, molecular weight 10 kDa), α-poly-L-lysine (PLL, molecular weight 20 kDa), ε-poly-L-lysine (EPL, molecular weight 4.5 kDa), and reducible ε-poly-L-lysine-Nanogel (REPL-NG) and a negatively charged solution containing messenger ribonucleic acid (mRNA) were prepared, respectively. The two solutions were mixed to form nanocontrollers, and their particle size and zeta potential were measured (Table 3).

[0103] Table 3 below shows the particle size and zeta potential of the nanocontroller encapsulated with mRNA.

[0104] Particle size (nm)Zeta potential (mV)bPEI / mRNA(WR0.13)57.5 ± 15.9-57.9 ± 1.9bPEI25kDa / mRNA(WR0.65)68.9 ± 18.324.6 ± 3.2RPC / mRNA(WR0.2)53.9 ± 15.2-55.9 ± 9.8RPC / mRNA(WR2)74.4 ± 19.544.9 ± 1.6RPC / mRNA(WR5)49.5 ± 13.835.4 ± 1.3PLL / mRNA(WR0.2)66.4 ± 18.8-23.2 ± 2.0PLL / mRNA(WR2)69.9 ± 19.641.1 ± 0.1PLL / mRNA(WR5)67.6 ± 18.940.6 ± 0.5EPL / mRNA(WR0.2)52.5 ± 15.6-43.3 ± 3.9EPL / mRNA(WR2)154.2 ± 44.417.2 ± 0.5EPL / mRNA(WR5)126.5 ± 33.419.2 ± 0.9REPL3-NG / mRNA(WR0.2)75.1 ± 20.5-45.3 ± 4.2REPL3-NG / mRNA(WR2)213.0 ± 48.219.1 ± 0.5REPL3-NG / mRNA(WR5)102.0 ± 19.516.9 ± 0.5REPL5-NG / mRNA(WR2)234.4 ± 45.315.5 ± 1.3REPL5-NG / mRNA(WR5)98.8 ± 18.917.4 ± 3.1REPL10-NG / mRNA(WR2)348.3 ± 69.515.0 ± 0.7REPL10-NG / mRNA(WR5)145.4 ± 36.417.6 ± 1.8

[0105]

[0106] <Example 4> Preparation of mRNA-encapsulated double nanoparticles using thin film hydration and evaluation of their physicochemical properties.

[0107] As in Example 1, the freeze-dried polymer or polymer conjugate was dissolved in a volatile solvent, transferred to a glass vial, and the solvent was evaporated using a rotary evaporator under reduced pressure to form a thin film. 2 mL of the cationic polymer and mRNA nanocontroller aqueous solution prepared in Example 3 were added to the glass vial in which the thin film was formed, and an ultrasonic disruptor was used to prepare mRNA-encapsulated dual nanoparticles of a uniform size of NanoController-in-NanoCarrier type, and the particle size and zeta potential thereof were measured (Table 4).

[0108] Table 4 below shows the particle size and zeta potential of mRNA-encapsulated double nanoparticles manufactured by the thin film hydration method.

[0109] Particle size (nm)Zeta potential (mV)bPEI / mRNA(WR0.13)@CTPCL-NP:U64.2 ± 18.4-84.3 ± 1.3RPC / mRNA(WR0.2)@CTPCL-NP:U68.7 ± 19.5-83.6 ± 2.0PLL / mRNA(WR(0.2)@CTPCL-NP:U63.2 ± 17.5-50.0 ± 2.5bPEI / mRNA(WR0.13)@GCAPCL-NP:U30.8 ± 8.2-64.4 ± 1.2bPEI / mRNA(WR0.65)@TPCL-NP:U67.4 ± 18.853.7 ± 1.1bPEI / mRNA(WR0.65)@CPCL-NP:U585.5 ± 82.918.9 ± 0.3

[0110]

[0111] <Example 5> Preparation of mRNA-encapsulated double nanoparticles using nanoparticle-nanoparticle fusion and evaluation of their physicochemical properties.

[0112] Dual nanoparticles manufactured by the nanoparticle-nanoparticle fusion method (F) were indicated using the code name F. Specifically, after manufacturing a nanocarrier in Example 1 or Example 2 and manufacturing a nanocontroller in Example 3, the nanocarrier aqueous solution and the nanocontroller aqueous solution were mixed to manufacture mRNA-encapsulated nanocontroller-in-nanocarrier type dual nanoparticles, and the particle size and zeta potential thereof were measured (Table 5).

[0113] Table 5 below shows the particle size and zeta potential of mRNA-encapsulated dual nanoparticles manufactured by the nanoparticle-nanoparticle fusion method.

[0114] Particle size (nm)Zeta potential (mV)bPEI25kDa / mRNA(WR0.13)@CTPCL-NP:F71.7 ± 19.7-68.8 ± 1.2RPC / mRNA(WR0.2)@CTPCL-NP:F65.3 ± 18.6-50.6 ± 1.7PLL / mRNA(WR0.2)@CTPCL-NP:F62.8 ± 17.5-50.5 ± 2.2EPL / mRNA(WR0.2)@CTPCL-NP:F69.4 ± 19.5-37.6 ± 0.5REPL3-NG / mRNA(WR0.2)@CTPCL-NP:F72.7 ± 20.0-35.9 ± 0.6bPEI / mRNA(WR0.13)@GCAPCL-NP:F39.0 ± 11.4-49.7 ± 0.7bPEI / mRNA(WR0.65)@TPCL-NP:F81.6 ± 21.937.4 ± 1.2bPEI / mRNA(WR0.65)@CPCL-NP:F128.9 ± 34.630.0 ± 0.6

[0115]

[0116] <Example 6> In vitro gene expression efficiency evaluation of mRNA-encapsulated double nanoparticles manufactured by thin film hydration or nanoparticle-nanoparticle fusion method

[0117] bPEI25kDa / mFLUC@CTPCL-NP:U, a dual nanoparticle encapsulating mRNA (hereinafter referred to as 'mFLUC') expressing luciferase (Luc) was manufactured by the thin film hydration method of Example 4, and bPEI25kDa / mFLUC@CTPCL-NP:F, a dual nanoparticle encapsulating mFLUC, was manufactured by the nanoparticle-nanoparticle fusion method of Example 5. Then, the in vitro gene delivery efficiency thereof was determined by comparing and evaluating with the control group such as bPEI25kDa / mFLUC (WR0.13) manufactured in Example 3 or mRNA@CTPCL-NP:U prepared by the method of Example 1.

[0118] Specifically, 50,000 mouse muscle cells (hereinafter referred to as 'C2C12 cells') or 500,000 mouse bone marrow derived dendritic cells (hereinafter referred to as 'BMDCs') were plated in 12-well or 48-well plates, respectively, and cultured for 24 hours. Then, the prepared bPEI25kDa / mFLUC@CTPCL-NP:U or bPEI25kDa / mFLUC@CTPCL-NP:F solution was transfected into the cultured cells using 5 μg of mRNA per well, and 24 or 48 hours later, the gene expression efficiency of the transfected cells was evaluated using the expression level of luciferase.

[0119] In the experiment using C2C12 cells, the medium was changed to serum-free medium before starting transfection, and then changed back to serum-containing medium 4 hours after transfection. As a result, as shown in Fig. 1, in C2C12 cells, compared to the control group bPEI25kDa / mFLUC (WR0.13), the gene expression efficiency of bPEI25kDa / mFLUC@CTPCL-NP:U or bPEI25kDa / mFLUC@CTPCL-NP:F was 2.2-fold or 2.9-fold higher at 24 hours, and 4.3-fold or 3.3-fold higher at 48 hours, respectively. In addition, as shown in Fig. 2, in BMDCs cells, the gene expression efficiency of bPEI25kDa / mFLUC@CTPCL-NP:U or bPEI25kDa / mFLUC@CTPCL-NP:F was 2.2 times or 4.9 times higher than that of the control group mFLUC@CTPCL-NP:U at 24 hours, and the gene expression efficiency of bPEI25kDa / mFLUC@CTPCL-NP:U or bPEI25kDa / mFLUC@CTPCL-NP:U was similar to that of the other control group bPEI25kDa / mFLUC (WR0.13) at 24 hours, and was 2.2 times higher than that of the other control group bPEI25kDa / mFLUC at 24 hours.

[0120]

[0121] <Example 7> Evaluation of in vivo gene expression efficiency of mRNA-encapsulated double nanoparticles manufactured by thin film hydration method

[0122] To determine the in vivo gene delivery efficiency of the mRNA-encapsulated dual nanoparticles manufactured in Example 4, bPEI25kDa / mFLUC@CTPCL-NP:U, RPC / mFLUC@CTPCL-NP:U, or PLL / bPEI25kDa / mFLUC@CTPCL-NP:U encapsulating mFLUC were prepared so that 5 μg of mRNA was contained in 100 μL of nanoparticle solution and administered into the thigh muscle of C57BL / 6J (7-week-old) mice. 3, 6, 12, and 24 hours after administration, muscle tissues at the administration site were isolated and dissociated, and the in vivo gene expression efficiency was evaluated using the expression level of luciferase in the tissue lysate.

[0123] As a result, as shown in Fig. 3 and Table 6, compared to the control group mFLUC@CTPCL-NP:U in mouse muscle tissue, the gene expression efficiency of bPEI25kDa / mFLUC@CTPCL-NP:U, RPC / mFLUC@CTPCL-NP:U, and PLL / mFLUC@CTPCL-NP:U was 61-fold, 8.2-fold, and 5.9-fold higher at 3 hours, 65.6-fold, 75.7-fold, and 5.6-fold higher at 6 hours, 40.7-fold, 14.1-fold, and 22.6-fold higher at 12 hours, and 27.3-fold, 17.5-fold, and 68.5-fold higher at 24 hours, respectively.

[0124] In addition, it was confirmed that the mRNA expression level reached its maximum at 6 hours after administration for RPC / mFLUC@CTPCL-NP:U, 12 hours after administration for bPEI25kDa / mFLUC@CTPCL-NP:U, and 24 hours after administration for PLL / mFLUC@CTPCL-NP:U. From this, it was confirmed that the mRNA release rate can vary depending on the degradation rate of the cationic polymer used and the difference in the strength of the interaction between the polymer and the gene, and thus the time point at which mRNA expression reaches its maximum can appear differently.

[0125] Table 6 below shows the relative in vivo gene expression efficiency of mRNA-encapsulated dual nanoparticles manufactured by the thin film hydration method.

[0126] Time elapsed after administration (h) 361224mFLUC@CTPCL-NP:U1111bPEI25kDa / mFLUC@CTPCL-NP:U61.065.640.727.3RPC / mFLUC@CTPCL-NP:U8.275.714.117.5PLL / mFLUC@CTPCL-NP:U5.95.622.668.5

[0127]

[0128] <Example 8> Evaluation of in vivo gene expression efficiency of mRNA-encapsulated dual nanoparticles manufactured by nanoparticle-nanoparticle fusion method

[0129] To determine the in vivo gene delivery efficiency of mRNA-encapsulated dual nanoparticles manufactured by the nanoparticle-nanoparticle fusion method of Example 5, bPEI25kDa / mFLUC@CTPCL-NP:F, RPC / mFLUC@CTPCL-NP:F, PLL / mFLUC@CTPCL-NP:F, EPL / mFLUC@CTPCL-NP:F, or REPL3-NG / mRNA@CTPCL-NP:F encapsulated with mFLUC were prepared so that 5 μg of mRNA was contained in 100 μL of nanoparticle solution and administered to the thigh muscles of C57BL / 6J (7-week-old) mice. Muscle tissues at the injection site were isolated and dissociated at 3, 6, 12, and 24 hours after administration, and the in vivo gene expression efficiency was evaluated using the expression level of luciferase in the tissue lysate.

[0130] As a result, as shown in Fig. 4 and Table 7, compared to the control group mFLUC@CTPCL-NP:F in mouse muscle tissue, the gene expression efficiency of bPEI25kDa / mFLUC@CTPCL-NP:F, RPC / mFLUC@CTPCL-NP:F, PLL / mFLUC@CTPCL-NP:F, EPL / mFLUC@CTPCL-NP:F, or REPL3-NG / mFLUC@CTPCL-NP:F was 21.7-fold, 52.4-fold, 17.7-fold, 7.9-fold, and 13-fold at 3 hours after administration, respectively; 17.3-fold, 12.3-fold, 17.1-fold, 0.6-fold, and 1.2-fold at 6 hours, respectively; 2.3-fold, 7.4-fold, 11.4-fold, 1.3-fold, and 1.5-fold at 12 hours, respectively; and 24 hours after administration, respectively. It was confirmed that it was 8.2 times, 2.8 times, 3.5 times, 28.1 times, and 8.3 times higher.

[0131] Table 7 below shows the relative in vivo gene expression efficiency of mRNA-encapsulated dual nanoparticles manufactured by the nanoparticle-nanoparticle fusion method.

[0132] Time elapsed after administration (h) 36 12 2 4 mFLUC@CTPCL-NP: F1111 bPEI 25 kDa / mFLUC@CTPCL-NP: F21.7 17.32.38.2 RPC / mFLUC@CTPCL-NP: F52.4 12.37.42.8 PLL / mFLUC@CTPCL-NP: F17.7 17.111.43.5 EPL / mFLUC@CTPCL-NP: F7.9 0.61.328.1 REPL3-NG / mFLUC@CTPCL-NP: F13.01.21.58.3

[0133]

[0134] <Example 9> Evaluation of the immune activation ability of mRNA-encapsulated dual nanoparticles manufactured by nanoparticle-nanoparticle fusion method

[0135] To determine the immunoactivation ability of mRNA-encapsulated dual nanoparticles manufactured by the nanoparticle-nanoparticle fusion method of Example 5, mRNA-encapsulated bPEI25kDa / mFLUC@CTPCL-NP:F, RPC / mFLUC@CTPCL-NP:F, PLL / mFLUC@CTPCL-NP:F, EPL / mFLUC@CTPCL-NP:F, or REPL3-NG / mRNA@CTPCL-NP:F were treated to BMDCs. Specifically, 100,000 BMDCs were plated in a 96-well plate and treated with the prepared bPEI25kDa / mFLUC@CTPCL-NP:F, RPC / mFLUC@CTPCL-NP:F, PLL / mFLUC@CTPCL-NP:F, EPL / mFLUC@CTPCL-NP:F, or REPL3-NG / mRNA@CTPCL-NP:F solution. After 24 hours, the inflammatory cytokines secreted in the supernatant, tumor necrosis factor-α (TNF-α) and interleukin-12 (IL-12), were quantitatively analyzed using an ELISA assay.

[0136] As a result, as shown in Fig. 5, it was confirmed that RPC / mRNA@CTPCL-NP:F activated TNF-α, and PLL / mRNA@CTPCL-NP:F and REPL3-NG / mRNA@CTPCL-NP:F increased the production of IL-12. From this, it was confirmed that RPC / mRNA@CTPCL-NP:F, PLL / mRNA@CTPCL-NP:F, and REPL3-NG / mRNA@CTPCL-NP:F had immune activation ability.

[0137]

[0138] <Example 10> Evaluation of the anticancer vaccine efficacy of mOVA-encapsulated dual nanoparticles manufactured by nanoparticle-nanoparticle fusion: Confirmation of changes in tumor volume and tumor weight

[0139] To evaluate the anticancer vaccine efficacy of dual nanoparticles loaded with ovalbumin (OVA)-expressing mRNA (hereinafter referred to as 'mOVA') prepared by the nanoparticle-nanoparticle fusion method of Example 5, RPC / mOVA@CTPCL-NP:F or PLL / mOVA@CTPCL-NP:F solutions loaded with mOVA were administered intramuscularly to C57BL / 6J (7-week-old) mice at 5-day intervals for a total of 4 times, and 7 days after the start of administration, mouse melanoma cells (hereinafter referred to as 'B16-OVA') producing 1,000,000 OVA per mouse were subcutaneously administered to form a mouse cancer model.

[0140] The results of the anticancer vaccine efficacy of RPC / mOVA@CTPCL-NP:F or PLL / mOVA@CTPCL-NP:F are shown in Fig. 6. As a result, on the 18th day after administration, compared to the mRNA control group, the tumor volume of the RPC / mOVA@CTPCL-NP:F or PLL / mOVA@CTPCL-NP:F administration group was 1.5 times or 2 times smaller, and the tumor weight was 1.6 times or 1.8 times smaller, respectively. In addition, on the 23rd day, compared to the mRNA control group, the tumor volume of the RPC / mOVA@CTPCL-NP:F or PLL / mOVA@CTPCL-NP:F administration group was 2 times or 1.3 times smaller, and the tumor weight was 1.7 times or 1.1 times smaller, respectively. From this, it was confirmed that RPC / mOVA@CTPCL-NP:F and PLL / mOVA@CTPCL-NP:F had anticancer vaccine efficacy. Additionally, it was confirmed that the nanoparticles themselves were not toxic as the body weight of the mice did not decrease during the experiment.

[0141]

[0142] <Example 11> Evaluation of the anticancer vaccine efficacy of mOVA-encapsulated dual nanoparticles manufactured by nanoparticle-nanoparticle fusion: Confirmation of changes in immune cell distribution and cytokine production in lymphoid tissue and cancer tissue 18 days after administration

[0143] In order to confirm the changes in immune cell distribution and cytokine production by RPC / mOVA@CTPCL-NP:F or PLL / mOVA@CTPCL-NP:F in the mice tested in Example 10, the mice were euthanized on the 18th day after administration, lymphoid tissues and cancer tissues were separated, and the tissues were ground into single cells. Then, the distribution of each immune cell (dendritic cells, cytotoxic T cells, and OVA tetramer-specific T cells) and interferon-gamma (IFN-γ), a key regulator of the immune response within T cells, were confirmed through flow cytometry by fluorescent staining using an antibody that labels immune cells or cytokines, and the immune cell markers are shown in Table 8.

[0144] As a result, as shown in Fig. 7, the ratios of MHCI-labeled dendritic cells and cytotoxic T cells in the lymphatic tissues of mice administered RPC / mOVA@CTPCL-NP:F were increased by 1.9 times and 1.3 times, respectively, compared to the lymphatic tissues of mice administered PBS as a control group, and the amount of IFN-γ production in cytotoxic T cells was increased by approximately 1.1 times. In cancer tissues, the ratios of MHCI-labeled dendritic cells and OVA-specific cytotoxic T cells were increased by 1.3 times and 3.2 times, respectively.

[0145] In addition, in the lymphoid tissues of mice administered PLL / mOVA@CTPCL-NP:F, the proportions of MHCI-labeled dendritic cells and cytotoxic T cells increased by 2.3 times and 1.1 times, respectively, compared to the lymphoid tissues of mice administered PBS as a control group. In the cancer tissues, the proportions of cytotoxic T cells and OVA-specific cytotoxic T cells increased by 1.3 times and 1.5 times, respectively, and IFN-γ in cytotoxic T cells increased by approximately 1.5 times. These results confirmed that RPC / mRNA@CTPCL-NP:F and PLL / mRNA@CTPCL-NP;F increased the production of dendritic cells, cytotoxic T cell population, and IFN-γ, which are helpful for anticancer action.

[0146] Table 8 below shows immune cell markers.

[0147] MHC I, an immune cell marker + Dendritic cellsMHC I, CD11cCytotoxic T cellsCD3, CD8OVA-specific cytotoxic T cellsCD3, CD8, SIINFEKL

[0148]

[0149] <Example 12> Evaluation of the anticancer vaccine efficacy of mOVA-encapsulated dual nanoparticles manufactured by nanoparticle-nanoparticle fusion: Confirmation of changes in cytokine production in blood and spleen cells 18 days after administration

[0150] In order to confirm the changes in blood cytokines and the production of OVA-specific cytokines in spleen cells by RPC / mOVA@CTPCL-NP:F or PLL / mOVA@CTPCL-NP:F in the mice tested in Example 10, the mice were anesthetized 18 days after administration, blood was collected from the heart, plasma was separated, and cytokines related to the inflammatory response in the blood (TNF-α, IL-6, IL-12, IFN-γ) were quantitatively analyzed using an ELISA experiment.

[0151] In addition, mice were euthanized, spleens were isolated, and the tissues were ground into single cells. 50,000 cells were plated on a 48-well plate and treated with OVA at a concentration of 10 μg / mL. After 24 hours, the production of OVA-specific cytokines (TNF-α, IL-6, IL-12, IFN-γ) in the cell culture medium was analyzed using an ELISA test.

[0152] As a result, as shown in Fig. 8, 18 days after administration, the RPC / mOVA@CTPCL-NP:F administration group showed a 4.8-fold, 52.7-fold, and 4.6-fold increase in IL-6, IL-12, and INF-γ in the plasma compared to the PBS administration group as a control group, and the PLL / mOVA@CTPCL-NP:F administration group showed a 70-fold and 1.6-fold increase in IL-12 and INF-γ, respectively. In addition, as shown in Fig. 9, when cytokines were analyzed after treating splenocytes isolated from mice with OVA 18 days after administration of RPC / mOVA@CTPCL-NP:F or PLL / mOVA@CTPCL-NP:F, it was confirmed that IL-12 increased by approximately 5.7-fold in both administration groups. From these results, it was confirmed that the immune response was activated by RPC / mOVA@CTPCL-NP:F or PLL / mOVA@CTPCL-NP:F, and further that the OVA protein-specific immune response generated by the mRNA delivered by the two types of nanoparticles was activated.

[0153]

[0154] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A first nanoparticle core in which the active ingredient, messenger RNA (mRNA), is bound to the surface of a cationic polymer or encapsulated therein; and An mRNA-encapsulated double nanoparticle comprising a second nanoparticle shell formed by surrounding the outer surface of the core with a biocompatible polymer or polymer conjugate.

2. In paragraph 1, The above first nanoparticle, A dual nanoparticle characterized in that the mRNA is bound to the surface of a cationic polymer or encapsulated therein by electrostatic attraction.

3. In paragraph 1, The above cationic polymer is, A dual nanoparticle characterized by at least one selected from the group consisting of branched polyethylenimine (bPEI), reducible polyethylenimine (RPC), α-poly-L-lysine (PLL), epsilon-poly-lysine (ε-poly-L-lysine; EPL), and reducible ε-poly-L-lysine-Nanogel (REPL-NG).

4. In paragraph 1, The above first nanoparticle, A dual nanoparticle characterized in that the release rate of the mRNA is controlled depending on the type of the cationic polymer.

5. In paragraph 1, The above biocompatible polymer or polymer conjugate is, Glycocholic acid-poly(ε-caprolactone) conjugate [Glycocholic acid-Poly(ε-caprolactone) conjugate; GCAPCL-P], dexamethasone-poly(ε-caprolactone) conjugate [Dexamethasone-Poly(ε-caprolactone) conjugate; DexPCL-P], choline-poly(ε-caprolactone) conjugate [Choline-Poly(ε-caprolactone) conjugate; CholPCL-P], triphenylphosphonium-poly(ε-caprolactone) conjugate [Triphenylphosphonium-Poly(ε-caprolactone) conjugate; TPCL-P], betaine-poly(ε-caprolactone) conjugate [Betaine-Poly(ε-caprolactone) conjugate; BPCL-P], carnitine-poly(ε-caprolactone) conjugate [Carnitine-Poly(ε-caprolactone) conjugate; CPCL-P], succinate-poly(ε-caprolactone) conjugate [succinate-Poly(ε-caprolactone) conjugate; snPCL-P], cytidine triphosphate-poly(ε-caprolactone) conjugate [CTP-Poly(ε-caprolactone) conjugate; CTPCL-P], poly(ε-caprolactone) diol [Poly(ε-caprolactone) diol; PCL diOH ] Poly(ε-caprolactone) dicarboxyl [Poly(ε-caprolactone) dicarboxyl; PCL diCOOH ], polylactic acid [Poly(Lactic acid); PLA], and poly(lactic acid-glycolic acid) [Poly(Lactide-glycolid); PLGA], characterized in that at least one is selected from the group consisting of.

6. In paragraph 1, The above double nanoparticles are, A dual nanoparticle characterized by an average particle diameter of 20 to 700 nm.

7. A composition for mRNA delivery comprising a dual nanoparticle according to any one of claims 1 to 6.

8. In paragraph 7, The above composition, A composition for mRNA delivery characterized by improving gene expression efficiency and immune activation ability.

9. A composition for a vaccine comprising a dual nanoparticle according to any one of claims 1 to 6.

10. A step of preparing a polymer solution by dissolving a biocompatible polymer or polymer conjugate in an organic solvent; A step of forming a thin film by evaporating the solvent of the polymer solution; and A method for producing double nanoparticles encapsulated with mRNA, comprising the step of adding a mixed solution of a positively charged solution containing a cationic polymer and a negatively charged solution containing mRNA to the formed thin film and crushing the mixed solution to obtain nanoparticles.

11. In paragraph 10, The above biocompatible polymer or polymer conjugate is, Glycocholic acid-poly(ε-caprolactone) conjugate [Glycocholic acid-Poly(ε-caprolactone) conjugate; GCAPCL-P], dexamethasone-poly(ε-caprolactone) conjugate [Dexamethasone-Poly(ε-caprolactone) conjugate; DexPCL-P], choline-poly(ε-caprolactone) conjugate [Choline-Poly(ε-caprolactone) conjugate; CholPCL-P], triphenylphosphonium-poly(ε-caprolactone) conjugate [Triphenylphosphonium-Poly(ε-caprolactone) conjugate; TPCL-P], betaine-poly(ε-caprolactone) conjugate [Betaine-Poly(ε-caprolactone) conjugate; BPCL-P], carnitine-poly(ε-caprolactone) conjugate [Carnitine-Poly(ε-caprolactone) conjugate; CPCL-P], succinate-poly(ε-caprolactone) conjugate [succinate-Poly(ε-caprolactone) conjugate; snPCL-P], cytidine triphosphate-poly(ε-caprolactone) conjugate [CTP-Poly(ε-caprolactone) conjugate; CTPCL-P], poly(ε-caprolactone) diol [Poly(ε-caprolactone) diol; PCL diOH ] Poly(ε-caprolactone) dicarboxyl [Poly(ε-caprolactone) dicarboxyl; PCL diCOOH ], polylactic acid [Poly(Lactic acid); PLA], and poly(lactic acid-glycolic acid) [Poly(Lactide-glycolid); PLGA], characterized in that at least one is selected from the group consisting of.

12. In paragraph 10, The above organic solvent is, A manufacturing method characterized in that at least one selected from the group consisting of ethanol, methanol, chloroform, tetrahydrofuran, dichloromethane, and dimethyl sulfoxide.

13. In paragraph 10, The step of obtaining the above nanoparticles comprises: It is performed by manufacturing double nanoparticles of uniform size using an ultrasonic crusher. A manufacturing method, characterized in that the above dual nanoparticle comprises a first nanoparticle core in which mRNA is bound to or encapsulated within a cationic polymer surface and a second nanoparticle shell formed of a biocompatible polymer or polymer conjugate surrounding the outside of the core.

14. A step of mixing a positively charged solution containing a cationic polymer and a negatively charged solution containing mRNA to produce a first nanoparticle in which the mRNA is bound to the surface of the cationic polymer or encapsulated therein; A step of preparing second nanoparticles by dissolving a biocompatible polymer or polymer conjugate in an organic solvent; and A method for producing a double nanoparticle encapsulated with mRNA, comprising the step of mixing the first nanoparticle aqueous solution and the second nanoparticle aqueous solution to obtain a double nanoparticle in the form of a first nanoparticle core-second nanoparticle shell.

15. In paragraph 14, The step of manufacturing the second nanoparticle is as follows: A manufacturing method characterized by performing the method by dissolving a biocompatible polymer or polymer conjugate in an organic solvent, adding purified water while stirring to form self-assembled nanoparticles, and then removing the solvent.

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