Micelles composed of amphiphilic peptides and antigen carrier nanoparticles using same

Abstract

The present invention relates to nanoparticles comprising an amphiphilic peptide that forms a micelle structure by self-assembly and a targeting peptide (preferably a water-soluble antigen peptide) that is electrically bound to the surface of the amphiphilic peptide, and a method for preparing the same. The targeting peptide is electrically bound to the surface of the amphiphilic peptide micelle structure to form particles, so that the targeting peptide can be effectively presented to antigen presenting cells, and the weight ratio of the amphiphilic peptide to the targeting peptide is adjusted to adjust the size of the nanoparticles so as to endocytose the cells, so that immunity caused by cytotoxic T cells can be induced. The nanoparticles of the present invention exhibit an effect as a vaccine using only an antigenic determinant at a more accurate site, thereby minimizing side effects. Therefore, the present invention can exhibit excellent antigen-specific antibodies and cellular immunotherapy effects, and can be used in the fields of various vaccine preparation and the like.

Description

Technical Field

[0001] The present invention provides a micellar structure composed of an amphiphilic peptide, and provides nanoparticles in which a targeting peptide is bound to the surface of the micellar structure. According to the present invention, the nanoparticles are used as antigen carriers to have an effect as a vaccine.

[0002] This invention was derived with the support of the following national research and development projects.

[0003] [Project unique number] 1711163105

[0004] [Project number]2021R1A2C1013022

[0005] [Department Name] Ministry of Science, Technology, ICT and Communications

[0006] [Project Management (Professional) Organization Name] Korea Research Foundation

[0007] [Research Project Name] Personal Basic Research (Ministry of Science, Technology and ICT)

[0008] [Research Topic Name] Particle antigenization of water-soluble incomplete antigens using amphiphilic peptides

[0009] [Project implementing organization name] Hanyang University Background Art

[0010] The process of antibody formation, which plays an important role in the body's immune function, can be roughly divided into two types according to the type of antigen. Particulate antigens are recognized by pattern recognition receptors present in macrophages or dendritic cells, etc., invade cells through phagocytosis, are decomposed by proteasomes, and presented on MHC class II molecules. Conversely, water-soluble antigens are recognized by cells with receptors for them, enter cells through receptor-mediated endocytosis, are decomposed, and presented on MHC class II molecules.

[0011] Generally, it is known that antibodies against particulate antigens are easy to produce. In each tissue, for defense, uniformly distributed dendritic cells or macrophages react nonspecifically to particulate antigens to produce antibodies against them. On the contrary, water-soluble antigens are mainly recognized by B cells with specific receptors for them and cells differentiated from B cells. However, B cells are not evenly distributed in all tissues and are mainly located inside the lymph nodes, so they must be accompanied by a corresponding migration process (CurrOpin Immunol, 65, 1-6, 2020), and must have receptors that can bind to antigens. In addition, since B cells have B cell receptors with different characteristics in each cell, not only antibodies to water-soluble antigens are generated, but also the persistence of the generated antibodies is significantly lower than that of particulate antigens.

[0012] On the other hand, when using a partial amino acid sequence to prepare an antibody in the form of a hapten, or using methods such as gene truncation to make a recombinant protein exposed to the outside of the cell for use as an antigen, in addition to the problem of making an antibody with problems in accessibility, it will also cause structural changes in the degree of forming a chemical bond (covalent bond) with the hapten and the carrier protein, and there are side effects that may occur due to the production of antibodies against this part or the production of antibodies against the giant carrier protein (Sci. Adv. 6, eaaax2285, 2020). In order to solve the problem that the antibody generation rate of water-soluble antigens in vivo is significantly lower than that of particulate antigens, attempts have been made to develop various adjuvants such as nanoparticles, but due to low efficacy, low stability, unacceptable drug resistance, difficulty in preparation and toxicity, no alternative has been successfully developed so far (Hum. Vacc. Imm, 10: 9, 2761-2774, 2014).

[0013] When preparing vaccines against nanoparticles, like bacteria or fungi, antibody production capacity is not a big consideration in most cases. Conventional vaccines that weaken pathogens (attenuated vaccines) or kill them (inactivated vaccines) are widely used with high antibody production capacity. In these cases, there are issues with the toxicity or safety of the vaccine itself compared to the antibody production capacity.

[0014] In the case of vaccines against viruses, there are many viruses that cannot be attenuated or inactivated themselves, and in the case of vaccines against cancer, it is impossible to attenuate or inactivate them at all because cancer cell-specific antigens and non-specific antigens exist at the same time. In order to solve these problems, subunit vaccines or recombinant vaccines have been developed as second-generation vaccines, but due to the limitations of water-soluble antigens as mentioned above, there is a problem that antibodies cannot be effectively formed.

[0015] On the other hand, the basic concept of the third-generation vaccine is that when a platform as a delivery system is secured, as long as the target is determined, the (encoded) deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) that can produce its target molecule can be made into a module to use the vaccine, so a new vaccine can be developed in a very short time. Since then, the reality is that many researchers are developing safer and more stable carrier platforms.

[0016] However, messenger RNA (mRNA) vaccines need to produce proteins in the body, so the length cannot be less than a certain level (too small will lead to reduced protein expression). Therefore, it has the limitation of being difficult to accurately target.

[0017] As an example of vaccine development that requires precise targeting, consider the dengue vaccine. The number of patients with dengue virus disease continues to increase, and the number of critically ill patients is also increasing. The need to develop a vaccine is also increasing. Multiple research entities are developing it, and the first licensed product appeared in 2018. However, after clinical use, side effects are large and its use is restricted.

[0018] It is known that there are four types of viruses that cause dengue fever so far. Antibodies are mainly formed against the E protein, which is a surface antigen. The amino acid sequence of the E protein shows high similarity in the four viruses. More than 52% of the amino acid sequences are exactly the same, and more than 80% similarity is observed overall. In other words, the sequence similarity of the E protein, which is the surface protein of the four dengue viruses, is more than 80%. The biggest problem with dengue fever vaccines is that when the correct antibodies are made and neutralizing antibodies are produced, they will have a preventive effect, but if they are not correctly bound in a divalent manner but weakly bound to similar antigens, it is possible that infection is more likely to occur due to antibody-dependent enhancement.

[0019] Therefore, in order to prepare a multivalent vaccine with minimal side effects, it is necessary to form a vaccine with only antigenic determinants at more accurate locations. However, existing nucleic acid vaccine platforms cannot meet these requirements. Therefore, the inventors have completed the present invention in order to provide an antigen that targets only the modified portion of a marker of a virus or tumor marker. Summary of the invention

[0020] Technical problem to be solved by the invention

[0021] In one aspect, nanoparticles are provided, comprising an amphiphilic peptide that forms a micellar structure by self-assembly and a targeting peptide (eg, a water-soluble antigen peptide) that is electrically bound to the surface of the amphiphilic peptide.

[0022] On the other hand, a method for preparing nanoparticles is provided, which includes: mixing amphiphilic peptides in an aqueous solution to form a micelle structure; and mixing a targeting peptide (eg, a water-soluble antigen peptide) to electrically bind to the surface of the formed micelle structure.

[0023] Still another aspect of the present invention provides multivalent vaccine nanoparticles that utilize only antigenic determinants at more accurate locations to minimize side effects.

[0024] Means for solving technical problems

[0025] In order to improve the limitations of the above-mentioned prior art, the present inventors have developed a method in which self-granulated micelles of various amphipathic peptides with a small molecular weight of about 10 or less amino acids are formed, and haptenic peptides are bound to the micelle surface by electrical binding so as to be presented to antigen-presenting cells.

[0026] Hereinafter, the present invention will be described in detail.

[0027] The terms used in the present invention are only used to illustrate specific examples and are not intended to limit the present invention. Unless otherwise defined, all terms used herein (including technical terms or scientific terms) have the same meaning as those commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0028] The present invention provides nanoparticles, wherein the nanoparticles include a micellar structure formed by self-assembly (refer to Figure 1 ) an amphiphilic peptide and a targeting peptide electrically bound to the surface of the amphiphilic peptide (or a micellar structure composed of the amphiphilic peptide).

[0029] In the present specification, the term "amphiphilic peptide" refers to a peptide containing both polar hydrophilic amino acids and nonpolar hydrophobic amino acids. Since the amphiphilic peptide has the amino acid composition, it can interact with both hydrophilic molecules and hydrophobic molecules.

[0030] In one embodiment of the present invention, the amphiphilic peptide may contain 1 to 10 (or 1 to 15) amino acids. In one embodiment of the present invention, the amphiphilic peptide is a peptide represented by the following formula I.

[0031] CnXm Formula I

[0032] wherein C is a charged amino acid, X is a hydrophobic amino acid which is the same or different amino acid, n=an integer from 1 to 5, and m=an integer from 3 to 10.

[0033] That is, the nanoparticles of the present invention may contain micelles formed by amphiphilic peptides represented by Formula I: CnXm(I) (wherein C is a charged amino acid, X is a hydrophobic amino acid that is the same or different amino acid, n=an integer from 1 to 5, and m=an integer from 3 to 10). In one embodiment, the number of the charged amino acids may be 2 to 5, and the number of the hydrophobic amino acids may be 4 to 8.

[0034] In one embodiment of the present invention, as hydrophilic amino acids, the amphiphilic peptide may include amino acids with positive charge, such as arginine (R), histidine (H) and lysine (K), etc., or amino acids with negative charge, such as aspartic acid (D), glutamic acid (E), etc. In one embodiment, the hydrophilic amino acids can make the micelle surface positively charged or negatively charged by being located on the micelle surface.

[0035] In one embodiment of the present invention, as the hydrophobic amino acid, the amphiphilic peptide may include one or more selected from the group consisting of valine, phenylalanine, tryptophan, isoleucine, leucine, proline, methionine and alanine.

[0036] In one embodiment of the present invention, the amphipathic peptide may include arginine (R) as a hydrophilic amino acid and valine (V) as a hydrophobic amino acid.

[0037] In one embodiment of the present invention, the amphiphilic peptide can be R3V6 (RRRVVVVVV, sequence number 1) consisting of the amino acid sequence of sequence number 1 comprising three arginines as positively charged hydrophilic amino acids and six valines as hydrophobic amino acids, R4V7 (RRRRVVVV VVV, sequence number 2) consisting of the amino acid sequence of sequence number 2 comprising four arginines as positively charged hydrophilic amino acids and seven valines as hydrophobic amino acids, E3V6 (EEEVVVVVV, sequence number 3) consisting of the amino acid sequence of sequence number 3 comprising three glutamic acids as negatively charged hydrophilic amino acids and six valines as hydrophobic amino acids, or E4V7 (EEEEVVVVVVVV, sequence number 4) consisting of the amino acid sequence of sequence number 4 comprising four glutamic acids as negatively charged hydrophilic amino acids and seven valines as hydrophobic amino acids.

[0038] In one embodiment of the present invention, the "targeting peptide" is a peptide that is combined with an amphiphilic peptide (electrical). The "targeting peptide" of the present invention is preferably an "antigenic peptide", for example, it can be a "hapten peptide".

[0039] In the present invention, the term "hapten peptide" refers to a small molecule substance (e.g., a small molecule peptide, a small molecule toxin, etc.) that can be electrically bound to the surface of the micelle composed of the amphiphilic peptide. The molecular weight and size of the hapten are small, so it cannot cause the body to produce antibodies for immune response alone, but it can act as a specific antigen in vitro that reacts with the corresponding antibody to form a precipitate of antigen-antibody reaction. In addition, when the hapten is combined with a carrier of a sufficiently large size, antibodies can be produced, such as a size that can induce antibodies such as carrier proteins.

[0040] In one embodiment of the present invention, the "hapten peptide" consists of 6 to 15 amino acids or 8 to 12 amino acids, preferably, consists of a sequence of about 10 amino acids.

[0041] As the whole of the "targeting peptide" of the present invention, it is preferred to have a "positive charge" or a "negative charge" and have a charge opposite to the surface charge of the micellar structure. The targeting peptide of the present invention has, for example, a charge of +1, +2, +3, -1, -2, or -3.

[0042] In one embodiment of the present invention, the targeting peptide can bind to the surface of the amphipathic peptide. Specifically, the targeting peptide can be electrically bound to the amphipathic peptide with opposite polarity (see Figure 2 and Figure 3 ).

[0043] In a preferred embodiment of the present invention, the "hapten peptide" may consist of the amino acid sequence of SEQ ID NOs. 5 to 11.

[0044] In one embodiment of the present invention, the hapten may be a peptide prepared based on a partial amino acid sequence of DDOST protein (dolichyl-diphosphooligosaccharide-protein glycosyltransferase 48k Da subunit precursorglycosyltransferase, NP_005207), for example, the hapten peptide of the present invention may be composed of any one of sequence numbers 5 to 7. Sequence number 5 (SDLGQHTLIV) of the present invention represents charge-1 in a neutral solution, sequence number 6 (LEDTLSSE) represents charge-2 in a neutral solution, and sequence number 7 (P GSQRYSQTGN) represents charge+1 in a neutral solution. However, as a compound that can only be electrically bound to the surface of a carrier peptide by carrying a charge, any "hapten" that forms an antibody against it when chemically bound to KLH protein or albumin (albumin) protein and injected into a living body can be used as the hapten peptide proposed in the present invention.

[0045] In one embodiment of the present invention, the size of the nanoparticles can be adjusted according to the weight ratio of the amphiphilic peptide and the targeting peptide used as the carrier. For example, the size of the nanoparticles can be adjusted by adjusting the weight ratio (w / w) of the amphiphilic peptide and the targeting peptide to 1:5 to 5:1, 1:4 to 4:1 or 1:3 to 3:1.

[0046] In one embodiment of the present invention, the nanoparticles can contain an amphipathic peptide and a hapten at a weight ratio (w / w) of 1:3 to 3:1. Specifically, when the amphipathic peptide and the hapten peptide are mixed at a weight ratio of 3:1 and 1:3, the particle size is analyzed by Zetasizer, and the results confirm that the particle size can be adjusted to 60-150 nm and 300-800 nm, respectively. According to the size adjustment of the nanoparticles as described above, antibody immunity or cellular immunity can be induced.

[0047] In one embodiment of the present invention, the size of the nanoparticles is 50nm to 150nm, 50nm to 300nm, 50nm to 700nm, 150nm to 400nm or 100nm to 300nm, preferably, has a size of 150nm to 350nm.

[0048] In one embodiment of the present invention, when the amphiphilic peptide and the hapten peptide are contained in a weight ratio of 1:3, 3:1, the size of the nanoparticles is 60 to 150 nm, and can be 300 nm to 800 nm. In this case, it is confirmed that the phagocytosis of the nanoparticles by macrophages increases (see Figure 1 ), presented to the surface by antigen presenting cells (APC), can more effectively increase the production of immunoglobulins.

[0049] In one embodiment of the present invention, when the amphiphilic peptide and the hapten peptide are contained in a weight ratio of 3:1, 1:3, the size of the nanoparticle is 60-150 nm, and can be 300 nm to 800 nm. In this case, the phagocytic effect of the nanoparticle on macrophages is relatively small (see Figure 1 ), the results showed that the formation of immunoglobulins decreased. On the contrary, it was confirmed that the cytotoxic T lymphocytes specific to the hapten peptide increased compared with the helper T cells or B cells specific to the hapten peptide.

[0050] In addition, the present invention provides a method for preparing nanoparticles, the method comprising: a step of mixing an amphiphilic peptide in an aqueous solution to form a micelle structure; and a step of mixing a targeting peptide to electrically bind to the surface of the formed micelle structure.

[0051] In the method for preparing nanoparticles of one embodiment of the present invention, the amphiphilic peptide may be a peptide represented by the following formula I:

[0052] CnXm Formula I

[0053] wherein C is a charged amino acid, X is a hydrophobic amino acid which is a different amino acid, n=an integer from 1 to 5, and m=an integer from 3 to 10.

[0054] One embodiment of the present invention is characterized in that the step of forming the micelle structure can be performed in a glucose solution with a concentration of 5% to 20%. Specifically, when the amphiphilic peptide and the hapten peptide are mixed in a glucose solution with a concentration of 5% to 20%, a more stable micelle structure can be formed.

[0055] In one embodiment of the present invention, the step of forming the micelle structure can be performed by further including cholesterol in a weight ratio of 1:5 to 5:1 compared to the amphiphilic peptide. Specifically, the amphiphilic peptide and cholesterol are adjusted to a mass ratio of 1:5, 1:3, 1:1, etc., and after mixing, the mixture is allowed to stand at room temperature for 2 hours to produce particles with relatively small size variation.

[0056] In one embodiment of the present invention, the method for preparing nanoparticles is characterized in that the amphiphilic peptide and the targeting peptide are mixed in a weight ratio of 1:5 to 5:1 to form the targeting peptide into particles.

[0057] In one embodiment of the present invention, in the step of forming particles, the antigenic peptide may be a hapten peptide.

[0058] In addition, the present invention provides a vaccine composition comprising the nanoparticles. The vaccine composition can be used as an immunotherapeutic agent.

[0059] The vaccine composition of the present invention may target dengue virus infection, fever with thrombocytopenia syndrome or influenza virus infection, but is not limited thereto.

[0060] In one embodiment of the present invention, the vaccine can be used for the prevention or treatment of diseases using immune cells (B cells or T cells).

[0061] In the present specification, the term "prevention" may refer to any action of inhibiting an individual disease or delaying the onset of the disease by administering the pharmaceutical composition according to an aspect.

[0062] In the present specification, the term "treatment" may refer to any action to ameliorate or improve disease symptoms by administering the pharmaceutical composition according to an aspect.

[0063] In addition, the present invention provides a pharmaceutical composition for preventing or treating immune diseases, comprising the nanoparticles.

[0064] According to one embodiment of the present invention, the immune disease can be directed against diseases caused by pathogens such as viruses and microorganisms or all diseases caused by pathogenic substances. Specifically, the pathogen itself or a part thereof or the pathogenic substance can be applied as a hapten. The pharmaceutical composition may also include a pharmaceutically acceptable carrier or diluent. The pharmaceutically acceptable carrier or diluent may be known in the art. The carrier or diluent may be lactose, glucose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water (e.g., saline and sterile water), syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, intravenous infusion, buffer, maltodextrin solution, glycerol, ethanol, dextran, albumin or any combination thereof. The pharmaceutical composition may also include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent or a preservative.

[0065] The pharmaceutical composition can be formulated using pharmaceutically acceptable carriers and / or excipients according to methods known to those of ordinary skill in the art, and can be prepared in unit volume form or built into a large-capacity container. At this time, the dosage form can be a solution, suspension, syrup or emulsion in an oil or mercury solvent, or in the form of an extract, powder, powder, granule, tablet or capsule, and can further include a dispersant or stabilizer. The aqueous solvent may include physiological saline or phosphate buffered saline (PBS). The pharmaceutical composition according to one embodiment can be formulated in an oral or parenteral form, preferably in a parenteral form. For intramuscular, intraperitoneal, subcutaneous and intravenous administration forms, a sterile solution of the active ingredient is usually prepared, and a buffer capable of appropriately adjusting the pH of the solution may be included. For intravenous administration, an isotonic agent may be included to impart isotonicity to the preparation, etc.

[0066] The dosage (effective dose) of the pharmaceutical composition according to one embodiment can be variously prescribed according to factors such as formulation method, administration mode, patient age, weight, sex, symptom, diet, administration time, administration route, excretion rate and reaction sensitivity. As long as it is a person of ordinary skill in the art, these factors can be considered to appropriately adjust the dosage. The number of administrations can be once a day or more than twice within the range of clinically acceptable side effects. For the administration site, it can be administered at 1 or more than 2 places, daily or at intervals of 2 to 5 days, and the total number of days of administration can reach 1 to 30 days for each treatment. If necessary, the same treatment can be repeated after an appropriate time. For animals other than humans, the same dosage as humans can be administered per kilogram (kg), or, for example, the volume ratio (e.g., average value) of the organs (such as the heart, etc.) of the target animal and humans can be used to convert the above dosage to administer. Possible routes of administration may include parenteral (e.g., subcutaneous, intramuscular, intraarterial, intraperitoneal, intradural or intravenous), local (including percutaneous), and injection, or insertion of an implant device or substance. Examples of animals to be treated according to one embodiment include humans and other target mammals, specifically humans, monkeys, mice, rats, rabbits, sheep, cows, dogs, horses, pigs, and the like.

[0067] In addition, the present invention provides a health functional food for preventing or improving immune diseases, comprising the nanoparticles as an effective ingredient.

[0068] The nanoparticles, immune diseases and prevention are as described above.

[0069] In the present specification, "improvement" may refer to all actions of suppressing an immune disease in an individual or delaying the onset of the disease by administering the composition according to one aspect.

[0070] The health functional food defined in the present invention can be a health functional food that has fully established its functionality and safety for the newly defined human body through the law on health functional food revised in 2008, and is included in the relevant provisions on the identification of functional ingredients for health functional food stipulated in Announcement No. 2008-72 of the Ministry of Food and Drug Safety of Korea.

[0071] When the composition of the present invention is included in the health function food for use, the composition can be added, it can also be used with other health function foods or health function food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use. Usually, the active ingredient of the composition according to the present invention can be included with 0.01% by weight to 15% by weight of the total food weight, preferably, it can be included with 0.2% by weight to 10% by weight, when prepared into a beverage, based on 100mL, it can be included with 0.1g to 30g, preferably, it can be included with a ratio of 0.2g to 5g, and the beverage can also be composed of all natural ingredients. However, when taking it for the purpose of regulating health and hygiene for a long time, the amount can be below the range, and since there is no problem in terms of safety, the active ingredient can also be used in an amount above the range.

[0072] The health functional food composition according to the present invention can be formulated into the dosage form of conventional health functional food known in the relevant technical field. The health functional food can be made into, for example, powders, granules, tablets, pills, capsules, suspensions, emulsions, syrups, impregnation agents, liquids, extracts, chewing gums, teas, jellies or beverages, etc., preferably, can be formulated into beverage forms. As the acceptable carrier or additive on the food science, any carrier or additive that can be used in the technical field that is known to be used for preparing pre-prepared dosage forms can be used. And, food used as animal feed can be included.

[0073] According to the purpose or preference, the health functional food may include nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectin acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerol, alcohol, carbonating agents used in carbonated beverages, etc. In addition, it may include pulp used to prepare natural fruit juices, fruit juice drinks and vegetable drinks. In addition, the health functional food composition may further include food additives. Whether it is appropriate as a "food additive" can be determined according to the general rules and general test methods of the food additive code approved by the Korean Ministry of Food and Drug Safety, etc., according to the relevant specifications and standards of the item, unless otherwise specified.

[0074] In addition, the present invention provides use of the nanoparticles prepared for medicine for the prevention or treatment of immune diseases.

[0075] Effects of the Invention

[0076] According to the present invention, when an amphiphilic peptide that forms a micellar structure by self-assembly and a targeting peptide (e.g., a water-soluble antigen peptide) that is electrically bound to the surface of the amphiphilic peptide are mixed in a specified weight ratio, the size of the formed nanoparticles can be adjusted and, based on their size, presented to helper T lymphocytes to induce the production of antibodies or activate cytotoxic T lymphocytes and cellular immunity, thereby showing a targeting peptide-specific immunotherapy effect.

[0077] In the present invention, nanoparticles can induce immune responses of antigen presenting cells, B cells and helper T cells without chemical deformation to generate antibodies in vivo, and can induce the generation of targeted peptide-specific cytotoxic T lymphocytes. Therefore, they can be used not only in fields such as vaccine preparation, but also in cellular immunity that has not yet been put into practical application, and thus can be used for the prevention and treatment of various diseases such as various viral diseases and cancers.

[0078] The nanoparticles of the present invention can be used as multivalent vaccines with minimal side effects by using hapten peptides as targeting peptides, and can only configure antigenic determinants at more accurate locations than the prior art. That is, the nanoparticles (vaccine platform) of the present invention can provide antigens that only target modified portions of viruses or tumor markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 This is a schematic diagram for confirming the micelle formation conditions for all possible amphipathic peptides to ensure that the nanoparticles (vaccine platform) of the present invention are used as the object. The nanoparticles (peptide platform) of the present invention refer to amphipathic peptides that form micelles by self-assembly in an aqueous solution. In addition, when the formed micelles are decomposed in antigen presenting cells and presented on MHC class 2 molecules, antibodies will also be generated against the micelle-forming peptides. Therefore, in order to suppress the formation of antibodies to this as much as possible, it is composed of only two kinds of amino acids that form bipolarity.

[0080] Figure 2 It is shown that the nanoparticles ( Figure 2 Schematic diagram of the preparation steps of modular antigens. According to the present invention, a targeting peptide (hapten peptide) is bound to the surface of micelles formed by amphiphilic peptides to form nanoparticles.

[0081] Figure 3 Schematic diagram of the formation of nanoparticles (peptide vaccine platform) according to the present invention. The nanoparticles of the present invention are formed by granulation after the targeting peptide (preferably, hapten peptide) is added to the amphiphilic peptide.

[0082] Figure 4The images (left) and electron microscope images (right) are used to measure the particle sizes of the amphiphilic peptide (R3V6) and R3V6 (or R3L6)-hapten peptide according to one embodiment of the present invention. Figure 4 As shown, micelles composed only of amphiphilic peptides formed spherical particles having a size between 100 and 300 nm, and when the hapten peptide was electrically linked to the amphiphilic peptide, particles having a size between 150 and 350 nm were formed.

[0083] Figure 5 These are images of nanoparticles internalized by macrophages, taken by confocal fluorescence microscopy according to their size. It can be confirmed that the uptake of macrophage lines varies depending on the size of the nanoparticles produced, and modular vaccines with an average size of about 200 to 300 nm are easily absorbed.

[0084] Figure 6 This is a graph showing the expression of target protein-specific IgG and IgM antibodies in the blood of mice administered with nanoparticles made according to the present invention. When nanoparticles of 200-300 nm in size were treated (●), the immunogenicity was greater than when nanoparticles of 30-50 nm in size were administered (■).

[0085] Figure 7 : is a comparison diagram of the nanoparticles according to the present invention and antibodies formed by the KLH conjugate method. In order to compare the amount of production, the case of inducing antibodies by binding with keyhole limpet hemocyanin, which is most commonly used when inducing antibodies with water-soluble incomplete antigens, was compared together. The results confirmed that no antibodies were produced when only water-soluble incomplete antigens were injected ( Figure 7 The upper end of the detection combination is lane 1), and antibodies are also generated when combined with keyhole limpet hemocyanin to induce antibodies ( Figure 7 When the nanoparticles of the present invention are used to induce antibodies, a large amount of antibodies can be induced compared to the conventional keyhole limpet hemocyanin method ( Figure 7 Lanes 3 and 4 in the upper end detection combination).

[0086] Figure 8The images of nanoparticles internalized by macrophages were taken using a confocal fluorescence microscope. Macrophages were incubated with nanoparticles for 2 hours (nanoparticle concentration: 40ug / ml). Hapten is represented by red fluorescence. Nucleus is stained with blue for control. (A) Amphipathic peptide: hapten peptide 3:1 (B) Amphipathic peptide: hapten peptide = 1:3.

[0087] Fig. 9 This is a titration curve of the hapten peptide, and is a graph that confirms the specific IgG response. BALB / c mice were immunized with 3:1 (w / w) nanoparticles or 1:3 (w / w) nanoparticles supplemented with carrier protein, hapten, and adjuvant on days 0 and 21, respectively, and blood was collected twice a week for enzyme-linked immunosorbent assay (ELISA) analysis. Fig. 9 Mesoparticles are amphipathic peptides.)

[0088] Fig.10 : is a graph confirming the effect of nanoparticles on antigen-specific IgG response. BALB / c mice were immunized with emulsified 3:1 (w / w) or 1:3 (w / w) nanoparticles on days 0 and 21. Mice were sacrificed on day 35 and whole blood was collected. The serum was analyzed by dot blot for whole protein containing hapten as part. ( Fig.10 Mesoparticles are amphipathic peptides.)

[0089] Fig.11 647 positive channel is used for the detection of hapten-specific B cells. And, hapten-specific B cells are a subphylum. Fig.11 A is the result of amphipathic peptide (Carrier), and the subphylum group showed 15.1%. Fig.11 The B is the result of hapten peptide, and the subphylum group showed 14.3%. Fig.11 C is the result of (amphipathic peptide:hapten peptide) 3:1 (w / w) nanoparticles, and the subphylum group showed 33.6%. Fig.11 The D is the result of 1:3 (w / w) nanoparticles, and the subphylum group showed 23.5%.

[0090] Fig.12 The results of flow cytometry analysis of hapten-specific cytotoxic T cell populations. Flow cytometry was performed on spleen cells harvested from immunized mice. The Alexa 647 positive channel was used for the detection of hapten-specific cytotoxic T cells. Also, hapten- and CD8-specific T cells are subphyla. Fig.12A is the result of carrier analysis, and the subphylum group showed 0.78%. Fig.12 B is the result of hapten analysis, and the subphylum group showed 0.67%. Fig.12 The C is 3:1 (w / w) nanoparticle analysis results, the subphylum group showed 1%. Fig.12 The D was 1:3 (w / w) nanoparticle analysis results, the subphylum group showed 2.1%. DETAILED DESCRIPTION

[0091] Hereinafter, the present invention will be described in further detail by way of examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples and experimental examples.

[0092] Embodiment and Experimental Examples

[0093] Experimental Example 1. Materials and Methods

[0094] 1.1 Preparation of nanoparticles (microparticle soluble hapten) using amphiphilic peptides

[0095] Recombinant amphiphilic peptides and hapten peptides were produced and provided by ANYGEN, Gwangju, South Korea. To prepare nanoparticles (peptide-soluble hapten) using amphiphilic peptides, amphiphilic peptides (peptides of sequence numbers 1 to 4, respectively) were dissolved in distilled water at a concentration of 4 mg / ml. Also, hapten peptides (peptides of sequence numbers 5 to 11, respectively) were dissolved in distilled water at a concentration of 0.5 mg / ml. Next, a fixed amount of hapten (50 ug) was mixed with two different amounts of amphiphilic peptides at a ratio of 3:1 and 1:3 (w / w), and incubated at room temperature (25°C) for 30 minutes.

[0096] 1.2 Characteristics of nanoparticle size

[0097] To characterize the size of the amphiphilic peptides and nanoparticles, the diameters of micelles and nanoparticles formed by the amphiphilic peptides (carriers) were determined using a Zetasizer Nano ZS system (Malvern Instruments, Malvern, UK). After decomposition, the micelle stock solution was placed in a cuvette. After incubation for 30 minutes, the nanoparticles were diluted to a final volume of 1 ml. Next, the average size was analyzed using Dispersion Technology software 4.3. The nanoparticles were diluted with ultrapure water (refractive index 1.33, viscosity 0.89) and used for analysis at room temperature. Observations are shown as triplicate averages + / - standard deviations.

[0098] 1.3 Cellular absorption of nanoparticles

[0099] Macrophages, RAW264.7 were purchased from ATCC, Virginia, USA. To detect the uptake of nanoparticles by cells, the nanoparticles were labeled with Alexa 647. After labeling, the nanoparticles were incubated with the carrier and the nanoparticles were prepared at 40ug / ml. Cells were seeded on 8 slides at 1×10 4 , and set for 1 day. Two different ratios of 1:3 or 3:1 of nanoparticles were treated in cells for 2 hours. Cells were fixed with 4% for 10 minutes. Cells were blocked with 1% bovine serum albumin for 30 minutes. In the absence of antibody treatment, DAPI negative staining was performed for 1 minute. After mounting the coverslip, the cells were observed with a confocal fluorescence microscope SP5TCS (Leica Biosystems, USA).

[0100] 1.4 Immunization of mice

[0101] Female BALB / C mice were purchased from National Biotech, Seoul, Korea, 6 weeks old and housed under pathogen-free conditions. This study was performed in accordance with the strict guidelines for the care and use of experimental animals of the National Institutes of Health, Korea. The protocol was approved by the Animal Care Committee of Hanyang University Animal Hospital. For immunization, the nanoparticles were emulsified with aluminum hydroxide (20 mg / 100 uL, Sigma-Aldrich, St Louis, MO, USA). Mice were injected with 150 ug of carrier dissolved in 150 ul of phosphate-buffered saline (PBS). Mice were injected with 50 ug of hapten dissolved in 150 ul of PBS. Mice were injected with 3:1 (w / w) nanoparticles (150 ug carrier and 50 ug hapten) combined with 150 ul of PBS and 40 mg of aluminum hydroxide. Mice were injected with 1:3 (w / w) nanoparticles (16.67 ug carrier and 50 ug hapten) combined with 150 ul of PBS and 40 mg of aluminum hydroxide. All groups were injected by intraperitoneal route and injected again on day 21. Blood was collected twice a week from the jugular vein and centrifuged at 2000g for 20 minutes. Serum was taken from the centrifuged blood and frozen at -20°C until analyzed by ELSIA. Mice were sacrificed on day 35. Whole blood was collected and centrifuged at 2000g for 20 minutes. Serum was taken from the centrifuged blood Dot blot and frozen at -20°C until analyzed by dot blot. Spleens were collected for splenocytes for flow cytometry.

[0102] 1.6 Antibody detection by dot blot

[0103] In order to determine the specific antibodies of nanoparticles (or hapten peptides), the serial dilutions of the recombinant protein were inoculated onto polyvinylidene fluoride (PVDF) membranes at a concentration of 1.2ug / ul to 12ng / ul. The membrane was blocked with 2.5% skim milk in PBS (blocking buffer) at room temperature for 2 hours. 20uL of serum was diluted with blocking buffer to a final volume of 3ml and treated on the membrane at room temperature for 2 hours. After serum treatment, the wells were incubated with horseradish peroxidase-coupled anti-mouse IgG antibodies at room temperature for 1 hour. Next, the membrane was treated with enhanced chemiluminescence solution (ECL) and directly analyzed with a chemiluminescence imaging system (model: chemiscope 3400, China Qinxiang Scientific Instruments (C LINX, china)). The points were visualized within 10 seconds. In each step, the membrane was washed 3 times with washing buffer (PBS with Tween).

[0104] 1.7 Harvesting of splenocytes

[0105] Spleens were collected from mice sacrificed on day 35 and stored in culture medium RPMI 1640 (Corning, Kennebunk ME, USA) with 10% fetal bovine serum (FBS) without antibiotics. A 70um cell strainer was placed in a 50mL conical tube and washed with 5mL of RPMI 1640. The spleen was cut with scissors, placed on a cell strainer and gently crushed. The strainer was washed again with 5mL of RPMI 1640. After the suspended cells were transferred to a 15mL conical tube, the sample was stored on ice. The suspended cells were centrifuged at 550g for 5 minutes and the supernatant was discarded. 1mL of ACK (ammonium-chloride-potassium) lysis buffer (Thermofisher Scientific, Massachusetts, USA) was treated in a 15mL conical tube and incubated at room temperature for 5 minutes. Add 4 mL of RPMI 1640 to the conical tube and centrifuge the suspended cells at 550 g for 5 minutes. Repeat this step until the red spots disappear from the cell pellet. Finally, resuspend the cell pellet in 5 mL of RPMI 1640 and use it directly for flow cytometry.

[0106] 1.8 Flow cytometry of viable cells

[0107] To confirm the nanoparticle (or hapten peptide)-specific lymphocyte population, flow cytometry of viable cells was performed. To detect hapten-specific B and T lymphocytes, DDOST recombinant protein containing part of the hapten peptide was labeled with alexa 647 (ab269823, Abcam, Cambridge, UK). The collected splenocytes were centrifuged at 550g for 5 minutes and the supernatant was discarded. The cell pellet was resuspended in cold Dulbecco's phosphate buffered saline (DPBS). This step was repeated 3 times for rinsing. After rinsing, the cells were filtered through a 40um cell strainer and counted. 2×10 6 The cells were transferred to a 1.75 ml E-tube and treated with FcR blocking reagent (for mouse) at 4°C for 10 minutes.

[0108] To characterize B lymphocytes, Alexa 488 conjugated anti-mouse CD19 antibody (ab270176, Abcam, Cambridge, UK) and Alexa 647 labeled recombinant protein were treated with FcR blocking reagent (130-092-575, Miltenybiotec, Bergisch Gladbach, Germany) at 4°C for 1 h. The treated cells were washed three times with flow cytometry fluorescence sorting buffer (FACS buffer). After washing, the cells were resuspended in 1 mL of cold DPBS. PI was added to the cells to detect viable cells within 10 min, and the cells were analyzed using FACS.

[0109] To characterize T lymphocytes, FITC-conjugated anti-mouse CD8 antibody (ab237367, Abcam, Cambridge, UK) and Alexa 647-labeled recombinant protein were treated with FcR blocking reagent at 4 °C for 1 hour. The treated cells were rinsed 3 times with FACS buffer. After rinsing, the cells were resuspended in 1 mL of cold DPBS. PI was added to the cells, surviving cells were detected within 10 minutes, and the cells were immediately analyzed with FACS conto II (BD Biosciences, SanDiego, USA). In all experiments for recovering cells, centrifugation was performed at 300g for 3 minutes.

[0110] Experimental Example 2. Results

[0111] 2.1 Nanoparticle size determination

[0112] First, in this example, the sizes of the nanoparticles formed by the amphipathic peptide (R3V6) and the amphipathic peptide (R3V6)-hapten peptide were measured and verified by electron microscopy ( Figure 4 ). See Figure 4 Based on this alone, it can be confirmed that the amphiphilic peptide forms micelles (spherical particles) with a size of 50 to 300 nm (preferably 100 to 300 nm). When the hapten peptide is electrically connected to the micelle structure, it can be confirmed that particles with a size between 60 to 150 nm and 300 to 800 nm are formed according to the ratio.

[0113] In addition, in order to characterize the size of the carrier and nanoparticles, the diameters of the micelles and nanoparticles formed by the amphiphilic peptides were determined. More specifically, a fixed amount of the hapten peptide was mixed with two different amounts of the amphiphilic peptides at a weight ratio of 3:1 and 1:3. The size of the micelles prepared according to the present invention was 100-300, the size of the 3:1 (w / w) nanoparticles was 60-150 nm and the size of the 1:3 (w / w) nanoparticles was 300-800 nm (Table 1).

[0114] Table 1

[0115]

[0116] 2.2 Cellular uptake of nanoparticles

[0117] Macrophages are one of the antigen presenting cells, so when the nanoparticles according to the present invention are used as antigens, cellular uptake must occur.

[0118] In this example, the hapten peptide was labeled with the fluorescent material alexa 647 and mixed with the amphiphilic peptide (R3V6) by adjusting the ratio, and the nanoparticles with average sizes of 30-50nm and 200-300nm were treated into the macrophage cell line. Figure 5 It can be confirmed that nanoparticles with a size of 200 to 300 nm easily enter macrophages, while the entry ratio of nanoparticles with a size of 30 to 50 nm is significantly reduced. That is, it can be seen that when the nanoparticles prepared according to the present invention are formed into a size of 150 to 350 nm, the effect as a vaccine is improved.

[0119] In addition, the hapten peptide was mixed at a ratio of 3:1 ( Figure 8 left) and 1:3( Figure 8 After mixing with two different amphiphilic peptides in a 1:1 weight ratio, each of the prepared nanoparticles was treated with a macrophage cell line. After incubation for 2 hours, it was confirmed that the nanoparticles were taken up into the macrophages ( Figure 8 ).

[0120] 2.3 Detection of Nanoparticle (or Hapten Peptide) Specific Antibodies

[0121] 2.3.1 Increase of Nanoparticle (or Hapten Peptide)-Specific IgG Antibodies

[0122] In order to detect the increase of nanoparticle (or hapten peptide) specific IgG antibody, blood was collected from mice twice a week and ELISA was performed. IgM increased only in the group injected with mixed amphipathic peptide (micelle structure) and hapten peptide, existed at the highest amount in about 7 days, and then declined rapidly. In the figure, although a complex graph is shown, no increase in antibody expression of any IgM and IgG was observed in the group injected with only amphipathic peptide or hapten peptide.

[0123] Specifically, the nanoparticles (200-300 in size) prepared according to the present invention are Figure 6 Medium ▲, ●), 30-50nm sized nanoparticles ( Figure 6 ◆, ■) were injected into mice, and 15 days later, the same amount was injected into mice again, and the expression of target peptide (hapten peptide)-specific IgG and IgM antibodies in the mouse blood was confirmed ( Figure 6 ). See Figure 6 It was confirmed that IgM and IgG specific to the hapten peptide were induced in the same manner as in a typical vaccine.

[0124] In addition, the expression of IgG and IgM antibodies according to the mixing ratio of the amphipathic peptide and the hapten peptide was confirmed as follows: Fig. 9 As shown. For IgG, no significant increase was shown after the first vaccination, but after the second booster injection, the optical density (OD) increased in the group where the amphipathic peptide and hapten peptide were mixed at a ratio of 1:3 (w / w). On the other hand, in the group treated with the amphipathic peptide and hapten peptide mixed at a ratio of 3:1 (w / w), the increase in OD was significantly lower than that of the 1:3 group (see Fig. 9 ).

[0125] 2.3.2 Comparison of modular vaccines and antibodies formed by the KLH conjugate approach

[0126] When dot blot analysis of the first targeted protein was performed using antiserum isolated from blood 33 days after vaccine injection, similar to the ELISA data, good results were confirmed when 200-300 nm nanoparticles were administered.

[0127] On the other hand, in order to compare the amount of antibody production, the case of inducing antibodies by binding to keyhole limpet hemocyanin, which is the most commonly used when inducing antibodies with water-soluble incomplete antigens, was compared. The results showed that no antibodies were produced when only the hapten peptide was injected ( Figure 7Lane 1 in the upper detection combination), while antibodies were generated in the case of induction of antibodies by binding to keyhole limpet hemocyanin ( Figure 7 When the nanoparticles of the present invention are used to induce antibodies, it is confirmed that the same or even greater amount of antibodies can be induced compared to the conventional keyhole limpet hemocyanin method ( Figure 7 Lanes 3 and 4 in the upper end detection combination).

[0128] In addition, antibodies confirmed by western blotting were pre-reacted with water-soluble incomplete antigens ( Figure 7 The lower end detection combination) bands all disappeared, thus confirming that the generated antibodies were all specifically generated by water-soluble incomplete antigens. After the experiment, the spleen of the animal used was removed to separate splenocytes, and B cells and T cells were analyzed by flow cytometry.

[0129] 2.3.3 Detection of nanoparticle-specific IgG antibodies

[0130] To confirm the nanoparticle (or hapten peptide)-specific IgG antibodies, we collected whole blood from mice sacrificed on day 35 and performed a dot blot assay. The 1:3 (w / w) nanoparticle spot was confirmed as the most reliable spot, as in the previous ELISA results, and it was confirmed that this diluted as the concentration of the recombinant protein that was highly concentrated and inoculated became dilute. However, no concentrated spots were found in the group with the amphiphilic peptide micelle structure or the hapten peptide alone. On the other hand, in the group where the amphiphilic peptide and hapten were mixed in a 3:1 (w / w) ratio, very light and clear spots were confirmed ( Fig.10 ), as shown in the previous ELISA experimental results, it can be confirmed that the group in which the amphipathic peptide and the hapten were mixed at a ratio of 3:1 (w / w) had a lower antibody production rate than the group in which the amphipathic peptide and the hapten were mixed at a ratio of 1:3 (w / w).

[0131] 2.4 Confirmation of Nanoparticle-Specific Lymphocyte Populations

[0132] 2.4.1 Nanoparticle-specific B cell populations

[0133] To confirm the nanoparticle (or hapten peptide) specific B cell population, flow cytometry of viable cells was performed using recombinant proteins labeled with Alexa 647 that partially contained the hapten. After gating on CD19 positive B cells, the histogram was set in the Alexa 647 positive channel and sub-gated with bars ( Fig.11 ).

[0134] Treatment of amphiphilic peptides alone ( Fig.11 , A) group and hapten peptide alone ( Fig.11 , B) The proportion of cells responding to the target protein did not show a significant change, but in the group where the amphipathic peptide:hapten peptide was mixed at a ratio of 3:1 ( Fig.11 , C), showed a greater proportion of 8% of cells responding to the target protein in the group mixed with amphipathic peptide:hapten peptide at 1:3 ( Fig.11 , D), compared with the group treated with amphipathic peptide or hapten peptide, the proportion of cells that showed response to the target protein was larger, about 18%. (See Table 2)

[0135] Table 2

[0136]

[0137] 2.4.2 Hapten-specific cytotoxic T cell population

[0138] To confirm the hapten-specific T cell population, flow cytometry of viable cells was performed using Alexa 647-labeled recombinant proteins that partially contain the hapten. Dot plots are shown, with each axis representing a target-specific channel (X-axis - CD8-specific channel, Y-axis - hapten-specific channel). As in the B cell analysis, amphiphilic peptides were treated alone ( Fig.12 , A) group and hapten peptide alone ( Fig.12 , B) The proportion of cells responding to the target protein did not change significantly, but in the amphipathic peptide:hapten peptide = 1:3 group, it was 0.22% higher than the group injected with amphipathic peptide alone, and 0.33% higher than the group injected with hapten peptide alone ( Fig.12 In contrast, in the group where the amphipathic peptide:hapten peptide = 3:1, the expression of hapten peptide in the amphipathic peptide alone was 1.32% higher than that in the group where the amphipathic peptide was injected alone, and 1.43% higher than that in the group where the hapten peptide was injected alone ( Fig.12 A, B and C), thus confirming that particles with relatively smaller sizes are more likely to increase cytotoxic T cells.

[0139] So far, the preferred embodiments of the present invention have been mainly studied. It should be understood by those skilled in the art that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative perspective rather than a restrictive perspective. The scope of the present invention appears in the scope of the invention claims, rather than the above description, and should be interpreted as all differences within the equivalent scope thereof are included in the present invention.

[0140] Sequence Listing Free Text

[0141] Sequence Number 1

[0142] amphiphilic peptide R3V6

[0143] RRRVVVVVV

[0144] Sequence number 2

[0145] amphiphilic peptide R4V7

[0146] RRRRVVVVVVV

[0147] Sequence number 3

[0148] amphiphilic peptide E3V6

[0149] EEEVVVVVV

[0150] Sequence Number 4

[0151] amphiphilic peptide E4V7

[0152] EEEEVVVVVVV

[0153] Sequence number 5

[0154] Hapten peptide 1

[0155] SDLGQHTLIV

[0156] Sequence Number 6

[0157] hapten peptide 2

[0158] LEDTLSSE

[0159] Sequence Number 7

[0160] Hapten peptide 3

[0161] PGSQRYSQTGN

[0162] Sequence Number 8

[0163] Dengue virus subtype1 hapten peptide

[0164] APTSEIQLTD

[0165] Sequence Number 9

[0166] Dengue virus subtype2 hapten peptide

[0167] SSITEAELTG

[0168] Sequence number 10

[0169] Dengue virus subtype3 hapten peptide

[0170] ASTVEAILPE

[0171] Sequence Number 11

[0172] Dengue virus subtype4 hapten peptide

[0173] SPSVEVKLPD.

Claims

1. A nanoparticle, wherein: The nanoparticles include an amphiphilic peptide that forms a micellar structure through self-assembly and a targeting peptide that is electrically bonded to the surface of the amphiphilic peptide.

2. The nanoparticle according to claim 1, wherein The amphiphilic peptide is a peptide represented by the following formula I: CnXm Formula I Wherein, C is a charged amino acid, X is a hydrophobic amino acid, n=an integer from 1 to 5, and m=an integer from 3 to 10.

3. The nanoparticle according to claim 1, wherein The amphipathic peptide includes arginine R as a hydrophilic amino acid and valine V as a hydrophobic amino acid.

4. The nanoparticle according to claim 1, wherein The amphiphilic peptide is one or more selected from the group consisting of peptides represented by the amino acid sequences of SEQ ID NOs: 1 to 4.

5. The nanoparticle according to claim 1, wherein The targeting peptide has a polarity opposite to the surface polarity of the micelle structure.

6. The nanoparticle according to claim 1, wherein The targeting peptide is an antigenic peptide.

7. The nanoparticle according to claim 6, wherein The antigenic peptide is a haptenic peptide targeting an antigen.

8. The nanoparticle according to claim 1, wherein The targeting peptide is composed of any one of the amino acid sequences from sequence number 5 to sequence number 11.

9. The nanoparticle according to claim 1, wherein The amphiphilic peptide and the targeting peptide are contained in a weight ratio of 1:5 to 5:

1.

10. The nanoparticle according to claim 1, wherein The size of the nanoparticles is 50 nm to 1000 nm.

11. A vaccine composition, wherein: The nanoparticles according to claim 1.

12. The vaccine composition according to claim 11, wherein Used to prevent dengue virus infection, fever with thrombocytopenia syndrome or influenza virus infection.

13. A method for preparing nanoparticles, wherein: include: Step a) mixing an amphiphilic peptide in an aqueous solution to form a micellar structure; as well as Step b) mixing the targeting peptide and electrically bonding it to the surface of the formed micelle structure.

14. The method for preparing nanoparticles according to claim 13, wherein: The amphiphilic peptide is a peptide represented by the following formula I: CnXm Formula I Wherein, C is a charged amino acid, X is a hydrophobic amino acid, n=an integer from 1 to 5, and m=an integer from 3 to 10.

15. The method for preparing nanoparticles according to claim 13, wherein: The amphiphilic peptide and the targeting peptide of step b) are mixed in a weight ratio of 1:5 to 5:

1.

16. The method for preparing nanoparticles according to claim 13, wherein: The targeting peptide is a hapten peptide.

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