Lipid nanoparticles for preventing influenza virus infection and preparation method therefor

Abstract

The present invention relates to a lipid nanoparticle and a method for preparing same, wherein melittin, a peptide capable of directly penetrating cell membranes and facilitating mucus layer penetration, is conjugated with a biocompatible lipid to form a melittin-lipid conjugate and is used in combination with an ionizable lipid, a helper lipid, cholesterol, and a lipid-PEG to prepare the lipid nanoparticle, whereby the lipid nanoparticle exhibits high stability and low toxicity while effectively delivering influenza A virus mRNA via intranasal administration to induce a prophylactic vaccine effect.

Description

Lipid nanoparticles for the prevention of influenza virus infection and a method for manufacturing the same

[0001] The present invention relates to lipid nanoparticles for the prevention of influenza virus infection and a method for manufacturing the same.

[0002] Nucleic acid-loaded lipid nanoparticle formulations used for the prevention or treatment of specific diseases (such as influenza) are receiving widespread attention due to their simple manufacturing process, high cell delivery efficiency, biocompatibility, and adaptive immune system activity. As described in the patent document below, conventional lipid nanoparticles generally consist of ionizable lipids, helper lipids, cholesterol, and lipid-PEG as constituent components.

[0003] <Patent Literature>

[0004] Published Patent: No. 10-2014-0097276 (Published Aug. 06, 2014) "Method for producing lipid nanoparticles for drug delivery"

[0005] Conventional lipid nanoparticles are known to be delivered into cells primarily through the process of endocytosis. However, there is a problem with these lipid nanoparticles being unable to effectively penetrate the mucus layer. The mucus layer has high viscoelasticity, making it easy for particles with surface charges or large sizes to attach to the mucus and restrict their movement. In particular, most lipid nanoparticles are removed or degraded after attaching within the mucus and fail to reach the target site. To address this, it is necessary to design lipid nanoparticles capable of penetrating the mucus layer; however, there are limitations in that the materials used for this purpose may reduce the cell delivery capacity of the lipid nanoparticles or cause side effects such as toxicity in the body.

[0006] The present invention has been devised to solve the above-mentioned problems,

[0007] The present invention aims to provide lipid nanoparticles and a method for producing the same, which induce a preventive vaccine effect by effectively delivering influenza A virus mRNA via nasal administration while exhibiting high stability and low toxicity, by forming a melittin-lipid conjugate by binding melittin, a peptide capable of directly penetrating a cell membrane and assisting in penetrating a mucus layer, to a biocompatible lipid, and further utilizing the melittin-lipid conjugate as well as ionized lipids, helper lipids, cholesterol, and lipid-PEG.

[0008] The present invention is implemented by an embodiment having the following configuration to achieve the aforementioned objective.

[0009] According to one embodiment of the present invention, lipid nanoparticles for preventing influenza virus infection according to the present invention comprise a melittin-lipid conjugate, wherein the melittin-lipid conjugate is formed by the binding of melittin and lipid, and the nucleic acid is characterized as being mRNA comprising an antigen-coding base sequence for inducing antibodies specific to the influenza virus.

[0010] According to another embodiment of the present invention, lipid nanoparticles for preventing influenza virus infection according to the present invention are characterized by further comprising a cation-carrying lipid, a neutral lipid, cholesterol, and a conjugate of polyethylene glycol and lipid.

[0011] According to another embodiment of the present invention, in the lipid nanoparticles for preventing influenza virus infection according to the present invention, the melittin-lipid conjugate is characterized by being formed by attaching a lipid head to the C-terminus of melittin.

[0012] According to another embodiment of the present invention, lipid nanoparticles for preventing influenza virus infection according to the present invention are characterized by penetrating a mucus layer to deliver the mRNA, thereby forming antibodies specific to the influenza virus in the body and defending against infection by the influenza virus.

[0013] According to another embodiment of the present invention, lipid nanoparticles for preventing influenza virus infection according to the present invention are characterized by being administered nasally.

[0014] According to another embodiment of the present invention, the surface of the lipid nanoparticles for preventing influenza virus infection according to the present invention is characterized by the presence of polyethylene glycol and melittin together.

[0015] According to another embodiment of the present invention, in the lipid nanoparticles for preventing influenza virus infection according to the present invention, the charge ratio of the phosphate group of the mRNA and the nitrogen group of the cationic lipid is 1:5 to 7, the cationic lipid is used in an amount of 40 to 60 mol% relative to the total moles of molecules constituting the lipid nanoparticles, the neutral lipid is used in an amount of 8 to 12 mol% relative to the total moles of molecules constituting the lipid nanoparticles, the cholesterol is used in an amount of 36 to 40 mol% relative to the total moles of molecules constituting the lipid nanoparticles, the polyethylene glycol and lipid conjugate is used in an amount of 1.3 to 1.7% relative to the total moles of molecules constituting the lipid nanoparticles, and the melittin-lipid conjugate is used in an amount of 0.3 to 0.7 mol% relative to the total moles of molecules constituting the lipid nanoparticles.

[0016] According to another embodiment of the present invention, a composition for preventing influenza virus infection according to the present invention is characterized by comprising lipid nanoparticles according to any one of claims 1 to 7.

[0017] According to another embodiment of the present invention, a method for manufacturing lipid nanoparticles for preventing influenza virus infection according to the present invention comprises: a conjugate formation step of forming a melittin-lipid conjugate by combining melittin and lipids; a lipid solution preparation step of preparing a lipid solution containing ionized lipids, helper lipids, cholesterol, and lipid-PEG; a melittin solution preparation step of preparing a melittin solution containing the melittin-lipid conjugate; a nucleic acid solution preparation step of preparing a nucleic acid solution containing nucleic acids; and a particle formation step of forming lipid nanoparticles by mixing and reacting a melittin solution with a mixture formed by mixing the lipid solution and the nucleic acid solution, wherein the nucleic acid is an mRNA containing an antigen-coding base sequence for inducing antibodies specific to the influenza virus.

[0018] According to another embodiment of the present invention, a method for preparing lipid nanoparticles for preventing influenza virus infection according to the present invention comprises, in the conjugate formation step, a reactive group introduction step of introducing a reactive group to the C-terminus of melittin, a functional group introduction step of introducing a functional group to the head portion of the lipid, and a binding step of reacting the reactive group of melittin with the functional group of the lipid to bind the lipid head to the C-terminus of melittin; in the particle formation step, the charge ratio of the phosphate group of the mRNA and the nitrogen group of the cationic lipid is 1:5 to 7, the cationic lipid is used in an amount of 40 to 60 mol% relative to the total moles of molecules constituting the lipid nanoparticle, the neutral lipid is used in an amount of 8 to 12 mol% relative to the total moles of molecules constituting the lipid nanoparticle, the cholesterol is used in an amount of 36 to 40 mol% relative to the total moles of molecules constituting the lipid nanoparticle, and the conjugate of the polyethylene glycol and the lipid is of the molecules constituting the lipid nanoparticle It is characterized by being used at 1.3 to 1.7% of the total moles, and the melittin-lipid conjugate is used at 0.3 to 0.7 mol% of the total moles of molecules constituting the lipid nanoparticles.

[0019] The present invention can achieve the following effects through the previously described embodiment.

[0020] The present invention forms a melittin-lipid conjugate by combining melittin, a peptide capable of directly penetrating cell membranes and assisting in the penetration of mucus layers, with a biocompatible lipid, and manufactures lipid nanoparticles by further utilizing the melittin-lipid conjugate as well as ionized lipids, helper lipids, cholesterol, and lipid-PEG, thereby providing a preventive vaccine effect by effectively delivering influenza A virus mRNA via nasal administration while exhibiting high stability and low toxicity.

[0021] FIG. 1 is a schematic diagram of lipid nanoparticles according to one embodiment of the present invention.

[0022] FIG. 2 is a graph showing the distribution of hydrodynamic diameters of lipid nanoparticles through a dynamic light scattering technique according to one embodiment of the present invention.

[0023] FIG. 3 is a graph showing the results of an in vitro experiment to evaluate the mRNA delivery efficacy of lipid nanoparticles according to one embodiment of the present invention.

[0024] FIG. 4 is a graph showing the results of an in vitro experiment to evaluate the efficacy of intracellular introduction of lipid nanoparticles according to one embodiment of the present invention.

[0025] FIG. 5 is a graph showing experimental results for evaluating the mucus layer penetration and cell delivery ability of lipid nanoparticles according to one embodiment of the present invention.

[0026] Fig. 6 shows the coating showing the results of quantitatively analyzing the image of Fig. 5.

[0027] FIGS. 7 and 8 are graphs showing the results of an in vivo experiment to evaluate the efficacy of an influenza preventive vaccine of lipid nanoparticles according to one embodiment of the present invention.

[0028] Hereinafter, lipid nanoparticles for the prevention of influenza virus infection according to the present invention and a method for manufacturing the same will be described with reference to the attached drawings. Unless otherwise specifically defined, all terms in this specification have the same general meaning as understood by a person skilled in the art to which the present invention pertains, and if there is a conflict with the meaning of a term used in this specification, the definition used in this specification shall prevail. Furthermore, detailed descriptions of known functions and configurations that may unnecessarily obscure the essence of the present invention are omitted. Throughout the specification, when a part is described as “comprising” a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components.

[0029]

[0030] Lipid nanoparticles according to one embodiment of the present invention are prepared by reacting ionized lipids, helper lipids, cholesterol, lipid-PEG, and melittin-lipid conjugates, thereby improving the delivery efficiency of nucleic acids such as mRNA, siRNA, and pDNA carried thereon. For example, the mRNA carried thereon may be an mRNA containing an antigen-coding base sequence for inducing antibodies (proteins) specific to the influenza virus (hereinafter referred to as 'IV mRNA'). The IV mRNA may utilize known technology, and as an example, HAss-Fe mRNA in which a stabilized H1 HA stem region is fused with H. pylori-derived ferritin may be used. The lipid nanoparticles of the present invention, which carry IV mRNA thereon, effectively penetrate the mucus layer to effectively deliver the IV mRNA, thereby enabling the formation of antibodies specific to the influenza virus in the body and defense against infection by the influenza virus. In other words, since the present invention aims to enable the effective delivery of a drug (IV mRNA) by allowing lipid nanoparticles loaded with the drug (IV mRNA) to effectively penetrate the mucus layer, thereby enabling nasal administration of the drug (IV mRNA) rather than the IV mRNA itself, a detailed description of the IV mRNA is omitted. That is, although an example regarding the preventive effect against Type A influenza virus infection is described below, it becomes possible to prevent infection by other types of influenza viruses by varying the IV mRNA loaded therein.

[0031] The above ionizable lipid may be a lipid that carries a positive charge depending on the pH of the solution, and the ionizable lipid is used such that the charge ratio of the phosphate group of the nucleic acid supported thereon to the nitrogen group of the ionizable lipid is 1:5-7, and it is preferable to use it in an amount of 40 to 60 mol% relative to the total moles of molecules constituting the lipid nanoparticles. For example, SM-102, which carries a positive charge in acidic solutions, may be used as the ionizable lipid. In the following examples, lipid nanoparticles were prepared using SM-102 as the ionizable lipid, but this is merely an example and the ionizable lipid is not limited to SM-102. Since the ionizable lipid is a known component constituting conventional lipid nanoparticles, various positively charged lipids used in conventional lipid nanoparticles may be used.

[0032] The helper lipid mentioned above may be a neutral lipid, and it is preferable to use it in an amount of 8 to 12 mol% relative to the total moles of molecules constituting the lipid nanoparticles. For example, DSPC may be used as the helper lipid. In the following examples, lipid nanoparticles were prepared using DSPC as the helper lipid, but this is merely an example and the helper lipid is not limited to DSPC. Since the helper lipid is a known component constituting conventional lipid nanoparticles, various neutral lipids used in conventional lipid nanoparticles may be used.

[0033] It is preferable to use the cholesterol in an amount of 36-40 mol% relative to the total moles of molecules constituting the lipid nanoparticles. Since the cholesterol is a known component constituting conventional lipid nanoparticles, various cholesterol derivatives used in conventional lipid nanoparticles may also be used.

[0034] The above lipid-PEG is a conjugate of polyethylene glycol and lipid, formed by bonding polyethylene glycol to the head portion of the lipid, and is preferably used in an amount of 1.3-1.7 mol% relative to the total moles of molecules constituting the lipid nanoparticle. For example, DMG-PEG may be used as the lipid-PEG. In the following examples, lipid nanoparticles were prepared using DMG-PEG as the lipid-PEG, but this is merely an example and the lipid-PEG is not limited to DMG-PEG. Since the lipid-PEG is a known component constituting conventional lipid nanoparticles, various lipid-PEGs used in conventional lipid nanoparticles may be used. For example, when preparing the lipid-PEG, neutral lipids such as phosphedidylethanolamine, phosphedidylethanolamine derivatives, ceramide, and DMG may be used as the lipid.

[0035] The above-mentioned melittin-lipid conjugate (lipid-MEL) is a conjugate of melittin and lipid, formed by binding melittin to the head portion of the lipid, and is preferably used in an amount of 0.3 to 0.7 mol% relative to the total moles of molecules constituting the lipid nanoparticle. The above-mentioned melittin-lipid conjugate can form a melittin-lipid conjugate by chemically reacting the reactive group of melittin with the functional group of the lipid. The reactive group can be introduced into the melittin, or the amine group present in the melittin can be used as the reactive group. The functional group introduced into the lipid can be a known compound, and the functional group can be introduced into the head portion of the lipid by a known method. For example, NHS (N-hydroxysuccinimide) that reacts with the amine group and NHS-amine, and SH groups (thiol groups) for disulfide bonding can be introduced into the lipid head by a known method and used as functional groups. For example, as a first example, an NHS can be introduced into the lipid head and the NHS of the lipid can be reacted with the amine group of melittin to bind the lipid to melittin; as a second example, a substance having an SH group at the N- or C-terminus of melittin (e.g., cysteine) can be introduced (to form CYS-MEL), a maleimide reactive group can be introduced into the lipid head (to form a lipid-linker), and when the CYS-MEL and the lipid-linker are reacted, the lipid head is bound to the N- or C-terminus of melittin through a maleimide-thiol group binding reaction. In the following examples, DOPE was used as the lipid to prepare the melittin-lipid conjugate, but this is merely an example and the lipid used to prepare the melittin-lipid conjugate is not limited to DOPE. Since the melittin-lipid conjugate is intended to partially replace lipid-PEG, a known component constituting conventional lipid nanoparticles, it can be prepared using a lipid (e.g., a neutral lipid) used in the preparation of lipid-PEG.

[0036] As previously described, conventional lipid nanoparticles carrying nucleic acids consist of ionized lipids, helper lipids, cholesterol, and lipid-PEG as components. However, in the present invention, a melittin-lipid conjugate is additionally used during the preparation of lipid nanoparticles. During the lipid nanoparticle preparation process, ionized lipids that carry a positive charge at low pH and mRNA that carries a negative charge bind through electrostatic attraction to form a reverse micelle structure. Subsequently, during the solvent exchange process from an acidic solvent containing ethanol to neutral PBS via an ultracentrifugation filter, the ionized lipids, helper lipids, cholesterol, lipid-PEG, and melittin-lipid conjugate, which were present as single molecules in the solution and have become neutral due to the increase in pH and decrease in the ethanol ratio, surround the reverse micelle to stabilize the reverse micelle structure, thereby forming lipid nanoparticles. At this time, PEG and melittin coexist on the surface of the lipid nanoparticles, and due to the shielding effect of the PEG present on the surface, the entire melittin is not exposed to the outside.

[0037]

[0038] A method for manufacturing lipid nanoparticles according to another embodiment of the present invention comprises: a conjugate formation step of forming a melittin-lipid conjugate by combining melittin and lipids; a lipid solution preparation step of preparing a lipid solution containing ionized lipids, helper lipids, cholesterol, and lipid-PEG; a melittin solution preparation step of preparing a melittin solution containing the melittin-lipid conjugate; a nucleic acid solution preparation step of preparing a nucleic acid solution containing nucleic acids; and a particle formation step of forming lipid nanoparticles by mixing and reacting a melittin solution with a mixture formed by mixing the lipid solution and the nucleic acid solution. The nucleic acid may be IV mRNA.

[0039] The above conjugate formation step is a step of forming a melittin-lipid conjugate by combining melittin and lipids, and may include, for example, a reactive group introduction step of introducing a reactive group to the N- or C-terminus of melittin, a functional group introduction step of introducing a functional group to the head portion of the lipid, and a binding step of reacting the reactive group of the melittin with the functional group of the lipid to cause the lipid head to bind to the N- or C-terminus of the melittin.

[0040] The above lipid solution preparation step is a step of preparing a lipid solution comprising ionized lipids, helper lipids, cholesterol, and lipid-PEG, for example, the lipid solution can be prepared by mixing ionized lipids, helper lipids, cholesterol, and lipid-PEG in a solution containing ethanol.

[0041] The above-mentioned melittin solution preparation step is a step of preparing a melittin solution containing a melittin-lipid conjugate, for example, the melittin solution can be prepared by mixing the melittin-lipid conjugate with a solution containing water or ethanol.

[0042] The above nucleic acid solution preparation step is a step of preparing a nucleic acid solution containing nucleic acid, for example, the nucleic acid solution can be prepared by mixing nucleic acid and an acidic solvent.

[0043] The particle formation step described above is a step of forming lipid nanoparticles by mixing a melittin solution into a mixture formed by mixing the lipid solution and the nucleic acid solution and reacting the mixture. For example, lipid nanoparticles can be formed by mixing a melittin solution into a mixture formed by mixing the lipid solution and the nucleic acid solution, then exchanging the solvent to raise the pH and removing ethanol. In the particle formation step, ionized lipids, helper lipids, cholesterol, lipid-PEG, and melittin-lipid conjugates are mixed in a molar ratio of 50:10:38:1.5:0.5, and nucleic acids and ionized lipids may be used such that the charge ratio of the phosphate group of the nucleic acid to the nitrogen group of the ionized lipid is 1:6.

[0044]

[0045] Another embodiment of the present invention relates to a composition for preventing influenza virus infection comprising the lipid nanoparticles.

[0046]

[0047] Another embodiment of the present invention relates to a composition for preventing influenza virus infection comprising the lipid nanoparticles.

[0048]

[0049] The present invention will be described in more detail below through examples. However, these examples are intended only to further explain the invention and do not limit the scope of the invention.

[0050]

[0051] <Example 1> Preparation of Melittin-Lipid Conjugate

[0052] 1. Melittin (hereinafter referred to as 'C-CYS-MEL'), in which a cysteine ​​amino acid is bound to the C-terminal region, was prepared in an 8 mL vial by dissolving it in water at a concentration of 1 mg / mL. DOPE-maleimide, a lipid with a maleimide reactive group added to the hydrophilic head portion, was dissolved in an ethanol solvent at a concentration of 1 mg / mL to form a DOPE-maleimide solution. The DOPE-maleimide solution was injected into the vial at a rate of 0.2 mL / min so that the molar ratio of DOPE-maleimide to C-CYS-MEL was 1.2:1, and the mixture was stirred and reacted for 24 hours to allow the maleimide-thiol group binding reaction to proceed, thereby forming a melittin-lipid conjugate (lipid-C-MEL). The melittin-lipid conjugate (lipid-C-MEL) was then obtained by performing dialysis and freeze-drying.

[0053] 2. A melittin-lipid conjugate (lipid-N-MEL) was obtained by using the same conditions as in Example 1, except that melittin with a cysteine ​​amino acid bound to the N-terminal region was used instead of C-CYS-MEL.

[0054]

[0055] <Example 2> Preparation of lipid nanoparticles

[0056] 1. Lipid nanoparticles 1

[0057] (1) SM-102 (ionized lipid), DSPC (helping lipid), cholesterol, and DMG-PEG (lipid-PEG) were each dissolved in ethanol to prepare SM-102 solution, DSPC solution, cholesterol solution, and lipid-PEG solution, respectively, and the above melittin-lipid conjugate (lipid-C-MEL) was dissolved in deionized water to prepare lipid-C-MEL solution.

[0058] (2) 10 µg of mRNA was added to deionized water and sodium acetate solution (pH 4.5) was added to prepare the mRNA solution. The mRNA used was Enhanced Green Fluorescence Protein mRNA (EGFP mRNA) purchased from TriLink.

[0059] (3) Calculate the amount of ionized lipid required so that the charge ratio of the phosphate group of the mRNA and the nitrogen group of the ionized lipid is 1:6, and prepare a lipid mixture solution by mixing SM-102 solution, DSPC solution, cholesterol solution, and DMG-PEG solution in a sterile 2 mL vial so that the molar ratio of SM-102 : DSPC : cholesterol : DMG-PEG : lipid-C-MEL is 50 : 10 : 38 : 1.5 : 0.5 and adding ethanol to make the final volume 50 µL, then inject the mRNA solution into the vial containing the lipid mixture solution using a 1 mL insulin syringe, and additionally inject the lipid-C-MEL solution into the vial containing the lipid mixture solution and mRNA solution using a pipette, and then react for 1 hour to form lipid nanoparticles, and then centrifuge the solution containing the lipid nanoparticles using an ultra-centrifugal filter at a rate of 14,200 xg. Centrifuged for 10 minutes, 400 µL of 0.01 M PBS with pH 7 was added to the centrifuge tube, centrifuged again at a speed of 14,200 xg for 10 minutes, and this was repeated once more. Then, an inverted ultracentrifuge filter was inserted into a 1.5 mL microtube, and centrifuged at a speed of 1,000 xg for 3 minutes to collect the particle solution inside the filter into a 1.5 mL centrifuge tube. After measuring the volume of the lipid nanoparticle solution using a pipette, 0.01 M PBS was added so that the final volume was 100 µL based on 10 µg of mRNA, thereby preparing the lipid nanoparticle solution.

[0060] 2. Lipid nanoparticles 2

[0061] A lipid nanoparticle solution was prepared under the same conditions as in Example 2, except that lipid-N-MEL was used instead of lipid-C-MEL.

[0062] 3. Lipid nanoparticles 3

[0063] A conventional lipid nanoparticle solution was prepared by making all other conditions the same as in Example 2, except that lipid-C-MEL was not used, so the molar ratio of lipid-C-MEL was 0% and the molar ratio of cholesterol was 38.5.

[0064] 4. Lipid nanoparticles 4

[0065] A lipid nanoparticle solution was prepared under the same conditions as in Example 2, except that the known IV mRNA HAss-Fe mRNA (Sequence No. 1) was used instead of Enhanced Green Fluorescence Protein mRNA (EGFP mRNA).

[0066] 5. Lipid nanoparticles 5

[0067] A lipid nanoparticle solution was prepared under the same conditions as in Example 2, 4, except that lipid-N-MEL was used instead of lipid-C-MEL.

[0068] 6. Lipid nanoparticles 6

[0069] A conventional lipid nanoparticle solution was prepared by making all other conditions the same as those in Example 2, 4, except that lipid-C-MEL was not used, so the molar ratio of lipid-C-MEL was 0% and the molar ratio of cholesterol was 38.5.

[0070]

[0071] <Example 3> Confirmation of Formation of Melittin-Conjugated Lipid Nanoparticles

[0072] 1. For the lipid nanoparticles (LNP-C-MEL) prepared in Example 2-1, the lipid nanoparticles (LNP-N-MEL) prepared in Example 2-2, and the conventional lipid nanoparticles (LNP) prepared in Example 2-3, the results were measured using dynamic light scattering with a particle size analyzer and are shown in Figure 2, and the Z-average value of the hydrodynamic diameter and the polydispersity index (PI) values ​​are shown in Table 1.

[0073] 2. Looking at Figure 2 and Table 1, it can be seen that the hydrodynamic diameter of LNP-C-MEL and LNP-N-MEL increases compared to the existing LNP, indicating that lipid nanoparticles bound to melittin are formed in Examples 1 and 2 of Example 2.

[0074] 3. As a result of conducting the same experiment as in Example 3, 1 on the lipid nanoparticles prepared in Examples 4 to 6 of Example 2, it was confirmed that the diameter of the lipid nanoparticles prepared in Examples 4 and 5 of Example 2 was larger than the diameter of the existing lipid nanoparticles prepared in Example 6 of Example 2.

[0075] Z-Average(nm) / PILNP124.3 / 0.07488LNP-C-MEL156.4 / 0.07271LNP-N-MEL216.1 / 0.1759

[0076]

[0077] <Example 4> Evaluation of mRNA delivery efficacy of melittin-conjugated lipid nanoparticles

[0078] 1. Approximately 200,000 Calu-3 cells were seeded in a 12-well plate, and each group was treated for 3 hours with the respective lipid nanoparticle solutions prepared in Examples 1, 2, and 3 of Example 2, at a concentration of 0.5 μg of EGFP mRNA. Afterward, the fluorescence intensity of the expressed Enhanced Green Fluorescence protein was measured for each group using a flow cytometer, and the results are shown in Figure 3. Meanwhile, in Figures 3 and 4, CTRL indicates the case where no lipid nanoparticles were used; the lipid nanoparticles prepared in Example 2, 1 are labeled LNP-C-MEL, the lipid nanoparticles prepared in Example 2, 2 are labeled LNP-N-MEL, and the conventional lipid nanoparticles prepared in Example 2, 3 are labeled LNP.

[0079] 2. As shown in Figure 3, it can be seen that LNP-N-MEL has a genetic material expression level approximately 4.18 times higher than that of conventional lipid nanoparticles (LNP), LNP-C-MEL has a genetic material expression level approximately 5.86 times higher than that of conventional lipid nanoparticles (LNP), and LNP-C-MEL has a genetic material expression efficiency approximately 1.4 times higher than that of LNP-N-MEL.

[0080]

[0081] <Example 5> Evaluation of Cell Uptake Efficiency of Melittin-Conjugated Lipid Nanoparticles

[0082] 1. Approximately 200,000 Calu-3 cells were seeded in a 12-well plate, and for each group, 0.5 μg of EGFP mRNA was used to treat each lipid nanoparticle solution prepared in Examples 1, 2, and 3 of 2 (provided that fluorescently stained lipid nanoparticles were used, and that fluorescently stained lipid nanoparticles could be obtained by using a DSPC solution in which 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine (DID) was additionally mixed during the preparation of the lipid nanoparticles) for 3 hours. Then, the amount of DID fluorescence contained in the cells of each group was measured using a flow cytometer, and the results are shown in Figure 4.

[0083] 2. Looking at Figure 4, lipid-C-MEL showed 17.63 times higher intracellular incorporation compared to conventional lipid nanoparticles (LNP), lipid-N-MEL showed 3.03 times higher intracellular incorporation compared to standard lipid nanoparticles (LNP), and lipid-C-MEL showed 5.82 times higher intracellular incorporation compared to lipid-N-MEL.

[0084]

[0085] <Example 6> Evaluation of the permeability of a mucus layer formed by conjugated interface culture of melittin-bound lipid nanoparticles

[0086] 1. The mucus layer penetration efficacy of the lipid nanoparticles (LNP-MEL) prepared in Example 2-5 and the conventional lipid nanoparticles (LNP) prepared in Example 2-6 was evaluated. Specifically, an insert containing a membrane made of a 0.4 μm porous PET permeable membrane was placed in a 24-well plate, and the unit area (cm²) of the porous PET permeable membrane in the space above the insert 2) 5.0x10 per 6Calu-3 cells were seeded, and cell culture solution was filled into the lower space and the cells were allowed to attach for 2 days. After the cells were attached, the cell culture solution in the upper space where the cells were located was removed. After removing the cell culture solution, the cells were cultured exposed to air for 7 days to allow the cells to differentiate and secrete their own mucus to form a mucus layer. The respective lipid nanoparticle solutions prepared in Examples 5 and 6 of Example 2, at a concentration of 0.5 μg based on HAss-Fe mRNA, were applied to the upper part of the cell layer where the mucus layer had formed for 3 hours. After fixation using 4% paraformaldehyde (PFA), the mucus layer was stained with Wheat Germ Agglutinin conjugated with alexa fluor 555, the cell nuclei were stained with DAPI, and the cytoskeleton was stained with Phaalloidin conjugated with alexa fluor 488. The results were analyzed using a fluorescence microscope and are shown in Figure 5, and the results were quantified and shown in Figure 6. In Fig. 5, blue represents the nucleus, green the cytoskeleton, red the mucus layer, and white the lipid nanoparticles.

[0087] 2. As can be seen in Figure 5, when examining the degree of penetration into the red mucus layer and the vertical viewpoint images for each group, it can be confirmed that the fluorescence of the particles in LNP-MEL is evenly distributed over a wider area compared to conventional lipid nanoparticles (LNP). Looking at Figure 6, which quantifies this, it can be confirmed that LNP-MEL has 31.57 times more particles that have penetrated the mucus layer compared to conventional lipid nanoparticles (LNP).

[0088] 3. As a result of conducting the same experiment as in Example 6, 1 on the lipid nanoparticles prepared in Example 2, 4, it was confirmed that the lipid nanoparticles prepared in Example 2, 4 also had significantly greater mucus layer permeability compared to the lipid nanoparticles prepared in Example 2, 6.

[0089]

[0090] <Example 7> Evaluation of Influenza Virus Protective Activity of Melittin-Conjugated Lipid Nanoparticles Upon Nasal Administration

[0091] 1. The immunogenic ability of the lipid nanoparticles (LNP-C-MEL) prepared in Example 2-4, the lipid nanoparticles (LNP-N-MEL) prepared in Example 2-5, and the conventional lipid nanoparticles (LNP) prepared in Example 2-6 was evaluated.

[0092] 2. Specifically, lipid nanoparticle solutions prepared in Examples 4 to 6 of Example 2, each at a rate of 10 μg based on HAss-Fe mRNA, were administered intranasally to Balb / cAnNCrlOri rats a total of three times at 2-week intervals. Serum samples were collected one week after the second and third administrations, and serum IgG titers were measured using enzyme immunoassay; the results are shown in Figure 7. To measure serum IgG titers, 2 μg / ml of recombinant protein hemagglutinin H1N1 (A / California / 07 / 2009) (Sino Biological) was placed in a Maxisorp-coated 96-well immunoplate. -1 The test was performed by coating overnight at 4°C, blocking with 3% skim milk / 0.05% PBST (phosphate buffered saline with 0.05% Tween-20) at room temperature for 2 hours the next day, serially diluting the obtained mouse serum twofold and treating the coated plate at room temperature for 2 hours, treating with HRP conjugated Rabbit anti-Mouse IgG at room temperature for 1 hour, treating with TMB substrate and solution (SeraCare) at room temperature for 15 minutes, stopping the reaction with 1M H3PO4, and measuring the absorbance at a wavelength of 450 nm.

[0093] 2. In addition, lipid nanoparticle solutions prepared in Examples 4 to 6 of Example 2, each containing 10 μg of HAss-Fe mRNA, were administered intranasally to Balb / cAnNCrlOri rats a total of three times at 2-week intervals, and after 2 days, H1N1 (A / Puerto Rico / 8 / 34) was administered intranasally at 5 times the 50% lethal dose (LD50). The survival and changes in body weight of the rats were measured for 2 weeks, and the results are shown in Figure 8.

[0094] 3. As shown in Figure 7, after the second vaccination, LNP-N-MEL showed neutralizing antibody values ​​1.23 times higher than LNP, LNP-C-MEL showed 1.33 times higher than LNP, and LNP-C-MEL showed 1.09 times higher than LNP-N-MEL. However, in the case of the third vaccination, similar neutralizing antibody values ​​were observed in all three groups, so it was possible to confirm that the lipid nanoparticles prepared in Examples 4 and 5 of Example 2 had superior or similar neutralizing antibody efficacy compared to the existing lipid nanoparticles (LNP).

[0095] 4. As shown in Figure 8, the results showed 0% survival when using PBS, the negative control, 60% survival when using LNP or LNP-N-MEL, and 100% survival when using LNP-C-MEL. In addition, regarding body weight loss, the results showed weight loss and death when using PBS, and some mice lost weight and died when using LNP-N-MEL and LNP. However, it was confirmed that there was no significant weight loss when using LNP-C-MEL, indicating that LNP-C-MEL induces a more effective protective immune response compared to the existing substrate nanoparticle (LNP).

[0096]

[0097] Although the applicant has described preferred embodiments of the present invention above, such embodiments are merely examples of implementing the technical concept of the present invention, and any modification or alteration that implements the technical concept of the present invention should be interpreted as falling within the scope of the present invention.

Claims

1. In lipid nanoparticles supporting nucleic acid, The above lipid nanoparticles include a melittin-lipid conjugate, wherein the melittin-lipid conjugate is formed by the binding of melittin and lipids, and Lipid nanoparticles for preventing influenza virus infection, characterized in that the nucleic acid is mRNA containing an antigen-coding base sequence for inducing antibodies specific to the influenza virus.

2. In Paragraph 1, Lipid nanoparticles for preventing influenza virus infection, characterized by further comprising a conjugate of a lipid with a positive charge, a neutral lipid, cholesterol, and polyethylene glycol and lipid.

3. In Paragraph 2, Lipid nanoparticles for preventing influenza virus infection, characterized in that the above-mentioned melittin-lipid conjugate is formed by binding a lipid head to the C-terminus of melittin.

4. In Paragraph 2, The lipid nanoparticles for preventing influenza virus infection described above are characterized by penetrating a mucus layer to deliver the mRNA, thereby forming antibodies specific to the influenza virus in the body and defending against infection by the influenza virus.

5. In Paragraph 4, The above lipid nanoparticles for preventing influenza virus infection are characterized by being administered via the nasal cavity.

6. In Paragraph 2, Lipid nanoparticles for preventing influenza virus infection, characterized by the presence of polyethylene glycol and melittin together on the surface of the lipid nanoparticles for preventing influenza virus infection.

7. In Paragraph 2, Lipid nanoparticles for preventing influenza virus infection, characterized in that the charge ratio of the phosphate group of the mRNA and the nitrogen group of the cationic lipid is 1:5 to 7, the cationic lipid is used in an amount of 40 to 60 mol% relative to the total moles of molecules constituting the lipid nanoparticles, the neutral lipid is used in an amount of 8 to 12 mol% relative to the total moles of molecules constituting the lipid nanoparticles, the cholesterol is used in an amount of 36 to 40 mol% relative to the total moles of molecules constituting the lipid nanoparticles, the polyethylene glycol and lipid conjugate is used in an amount of 1.3 to 1.7% relative to the total moles of molecules constituting the lipid nanoparticles, and the melittin-lipid conjugate is used in an amount of 0.3 to 0.7 mol% relative to the total moles of molecules constituting the lipid nanoparticles.

8. A composition for preventing influenza virus infection characterized by comprising lipid nanoparticles according to any one of claims 1 to 7.

9. A conjugate formation step of forming a melittin-lipid conjugate by combining melittin and lipids; a lipid solution preparation step of preparing a lipid solution containing ionized lipids, helper lipids, cholesterol, and lipid-PEG; a melittin solution preparation step of preparing a melittin solution containing the melittin-lipid conjugate; a nucleic acid solution preparation step of preparing a nucleic acid solution containing nucleic acids; and a particle formation step of forming lipid nanoparticles by mixing and reacting a melittin solution with a mixture formed by mixing the lipid solution and the nucleic acid solution; A method for manufacturing lipid nanoparticles for preventing influenza virus infection, characterized in that the nucleic acid is mRNA containing an antigen-coding base sequence for inducing antibodies specific to the influenza virus.

10. In Paragraph 9, The above conjugate formation step includes a reactive group introduction step for introducing a reactive group to the C-terminus of melittin, a functional group introduction step for introducing a functional group to the head portion of a lipid, and a binding step for reacting the reactive group of melittin with the functional group of the lipid to bind the lipid head to the C-terminus of melittin. A method for manufacturing lipid nanoparticles for preventing influenza virus infection, characterized in that, in the particle formation step, the charge ratio of the phosphate group of the mRNA and the nitrogen group of the cationic lipid is 1:5 to 7, the cationic lipid is used in an amount of 40 to 60 mol% relative to the total moles of molecules constituting the lipid nanoparticles, the neutral lipid is used in an amount of 8 to 12 mol% relative to the total moles of molecules constituting the lipid nanoparticles, the cholesterol is used in an amount of 36 to 40 mol% relative to the total moles of molecules constituting the lipid nanoparticles, the polyethylene glycol and lipid conjugate is used in an amount of 1.3 to 1.7% relative to the total moles of molecules constituting the lipid nanoparticles, and the melittin-lipid conjugate is used in an amount of 0.3 to 0.7 mol% relative to the total moles of molecules constituting the lipid nanoparticles.

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