lipid nanoparticles

Abstract

To provide a lipid nanoparticle being a gene delivery carrier that can be delivered to a spleen dendritic cell highly efficiently.SOLUTION: A lipid nanoparticle contains pH-sensitive cationic lipid and polyalkylene glycol modified lipid, where the ratio of the content of the pH-sensitive cationic lipid to the total amount of lipids constituting the lipid nanoparticles is 10 to 25 mol%, the ratio of the content of the polyalkylene glycol modified lipid to the total amount of lipids constituting the lipid nanoparticles is 0.5 to 1.75 mol%, and a number average particle diameter is 150 nm or more.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to lipid nanoparticles that are useful as gene delivery carriers that can deliver genes to splenic dendritic cells with high efficiency. [Background technology]

[0002] mRNA medicine is one of the modalities attracting attention for its clinical application as a gene therapy. mRNA medicine is a next-generation drug that expresses proteins desirable for disease treatment by artificially synthesizing mRNA, a biological component, and administering it to the body. mRNA has attracted attention as a pharmaceutical molecule with potential applications in a wide range of disease areas because it does not pose the risk of insertional mutations into the genome, its sequence can be instantly designed once the gene is identified, and it can theoretically express any protein. Therefore, mRNA, particularly as a vaccine, is expected to contribute significantly to personalized cancer treatment and rapid responses to viral mutations and pandemics in infectious diseases (Non-Patent Document 1). In the development of a vaccine against SARS-CoV-2, Moderna shipped the first batch of an investigational drug (mRNA-loaded lipid nanoparticle formulation) just 42 days after the publication of the viral genome DNA sequence, demonstrating the potential of mRNA vaccines as a rapid response technology for infectious disease control.

[0003] Vaccines are generally required to have immunostimulatory effects. Meanwhile, single-stranded RNA is recognized by Toll-like receptor 7 / 8 (TLR7 / 8), which is primarily localized in endosomes, and induces type I interferon responses. In other words, mRNA has strong immunogenicity mediated by the innate immune system. Therefore, in mRNA vaccines, mRNA plays both the role of target protein expression and immunostimulatory effects (Non-Patent Documents 2 and 3).

[0004] Lipid nanoparticles (LNPs) are used as carriers for encapsulating nucleic acids such as mRNA and small interfering RNA (siRNA) and delivering them to target cells. For example, lipid nanoparticles containing pH-sensitive cationic lipids as constituent lipids have been reported as lipid nanoparticles that are electrically neutral at physiological pH but change to cationic in a weakly acidic pH environment (Patent Document 1). Lipid nanoparticles that are positively charged in blood interact nonspecifically with plasma proteins and are rapidly eliminated from the blood by the reticuloendothelial system. In contrast, lipid nanoparticles containing pH-sensitive cationic lipids as constituent lipids are uncharged under neutral conditions in blood but are positively charged under the weakly acidic conditions in endosomes, allowing for efficient endosomal escape.

[0005] BioNTech has reported RNA-lipoplex (RNA-LPX) as a systemically administered mRNA vaccine (Non-Patent Document 2). RNA-LPX is prepared by mixing liposomes with mRNA, which have a lipid composition of 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 1:1 (molar ratio), prepared by simple hydration or ethanol dilution, so that the N / P ratio, which is the ratio of the number of quaternary ammonium groups in DOTMA to the number of phosphate groups in the mRNA, is 1.3 / 2. RNA-LPX exhibits extremely high spleen selectivity as measured by gene expression, with particularly high gene expression in splenic dendritic cells. As of February 2021, RNA-LPX is currently undergoing phase I clinical trials for use in melanoma, prostate cancer, human papillomavirus type 16-positive head and neck cancer, triple-negative breast cancer, and ovarian cancer (Non-Patent Document 3). Additionally, Tozinameran, the world's first mRNA drug approved for emergency use by the U.S. Food and Drug Administration (FDA) on December 11, 2020, as a preventive vaccine for COVID-19 (Corona virus disease-19), is an LNP-type formulation (Non-Patent Document 4). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2018 / 230710 [Patent Document 2] International Publication No. 2018 / 190423 [Non-patent literature]

[0007] [Non-Patent Document 1] Pardi et al., NATURE REVIEWS DRUG DISCOVERY, 2018, vol. 17, p.261-279. [Non-patent document 2] Kranz et al., Nature, 2016, vol.534, p.396-401. [Non-patent document 3] Sahin et al., Nature, 2020, vol.585, p.107-112. [Non-patent document 4] Polack et al., The New ENGLAND JOURNAL of MEDICINE, 2020, vol. 383(27), p.2603-2615. [Non-patent document 5] Sato et al., Journal of Controlled Release, 2019, vol.295, p.140-152. [Non-patent document 6] Stroock et al., Science, 2002, vol.295, p.647-651. [Non-Patent Document 7] Hajj et al., Nano Letters, 2020, vol.20, p. 5167-5175. Summary of the Invention [Problem to be solved by the invention]

[0008] RNA-LPX is a pioneering systemic mRNA cancer vaccine. However, there are still few reports of systems that enable efficient nucleic acid delivery to cells other than hepatic parenchymal cells, such as immune cells, and there is room for improvement in efficiency.

[0009] An object of the present invention is to provide lipid nanoparticles that serve as gene delivery carriers that can deliver genes to splenic dendritic cells with high efficiency. [Means for solving the problem]

[0010] The present inventors discovered that the efficiency of gene expression in splenic dendritic cells can be improved by incorporating a pH-sensitive cationic lipid and a polyalkylene glycol-modified lipid into the constituent lipids of lipid nanoparticles that encapsulate nucleic acids for gene expression, such as siRNA, and by adjusting the size of the lipid nanoparticles to a number-average particle diameter of 150 nm or more, and thus completed the present invention.

[0011] That is, the present invention provides the following lipid nanoparticles and the like. [1] Contains a pH-sensitive cationic lipid and a polyalkylene glycol-modified lipid, The content ratio of the pH-sensitive cationic lipid to the total amount of lipids constituting the lipid nanoparticles is 40 to 70 mol%, The content ratio of the polyalkylene glycol-modified lipid to the total amount of lipids constituting the lipid nanoparticles is 0.5 to 1.75 mol%, Lipid nanoparticles having a number-average particle diameter of 150 nm or more. [2] The lipid nanoparticles according to [1], having a number-average particle diameter of 600 nm or less. [3] The pH-sensitive cationic lipid is represented by the following general formula (I-1):

[0012] [ka]

[0013] [In formula (I-1), R 11 and R 12 are each independently a linear C 10-14 Alkyl group, linear C with one or two unsaturated bonds 10-20 Alkenyl group, or -CH(R 15 )(R 16 )(R 15 and R 16 are each independently a linear C 5-10 n1 and n2 each independently represent an integer of 6 to 10; p1 and q1 each represent an integer of 0 or greater that satisfies p1+q1=3 to 8; r1 represents 0 or 1; R 13 and R14 are each independently a linear C 1-3 Alkyl or aryl C 1-3 is an alkyl group, or R 13 and R 14 are linked to each other, and the nitrogen atom is a pyrrolidine ring, a piperidine ring, a morpholine ring, or a C 1-3 forming a piperazine ring optionally substituted with an alkyl group. The lipid nanoparticles according to [1] or [2], which are represented by the formula: [4] The lipid nanoparticles according to any one of [1] to [3] above, further comprising one or more selected from the group consisting of sterols and phospholipids. [5] The lipid nanoparticles according to any one of [1] to [4] above, which contain a nucleic acid. [6] The lipid nanoparticle of [5], wherein the nucleic acid is siRNA. [7] The lipid nanoparticle of [5], wherein the nucleic acid is mRNA or plasmid DNA. [8] The lipid nanoparticle of any one of [5] to [7] above, wherein the nucleic acid is a gene to be expressed in splenic dendritic cells. [9] A pharmaceutical composition containing the lipid nanoparticles according to any one of [1] to [8] above as an active ingredient.

[10] The pharmaceutical composition according to [9] above, which is used in cancer vaccine therapy.

[11] The pharmaceutical composition according to [9] above, which is used for immunostimulation.

[12] A method for expressing a foreign gene, comprising administering to a subject animal (excluding humans) lipid nanoparticles according to any one of [5] to [8] above, which encapsulate a foreign gene to be expressed in splenic dendritic cells, and expressing the foreign gene in the splenic dendritic cells of the subject animal.

[13] A method for producing the lipid nanoparticles according to any one of [5] to [8] above using a flow channel structure, A formation step of forming lipid nanoparticles in the flow channel structure from a lipid solution in which all lipid components constituting the lipid nanoparticles are dissolved in ethanol and an aqueous solution containing the nucleic acid; A dialysis step of dialysis the solution containing lipid nanoparticles obtained in the formation step in a buffer solution of pH 6.8 to 7.6; and the flow path structure includes a first inlet path for introducing a first fluid and a second inlet path for introducing a second fluid, which are independent of each other and have a certain length, and which join together to form a single dilution flow path; the dilution flow path has a flow path portion that is two-dimensionally curved in at least a part thereof, When the axial direction or extension direction of the dilution flow path upstream of the bent flow path portion is defined as the X direction, the width direction of the dilution flow path perpendicularly intersecting the X direction is defined as the Y direction, and the flow path width of the dilution flow path upstream of the bent flow path portion is defined as y0, the bent flow path portion is formed by at least two or more structures that protrude alternately from both side wall surfaces of the dilution flow path opposing each other in the Y direction toward the flow path center in approximately the Y direction (approximately the +Y direction, approximately the -Y direction), have constant heights h1, h2... of at least 1 / 2y0 but less than 1y0, and have constant widths x1, x2... in the X direction, and that regulate the flow path width of the dilution flow path, and are provided at constant intervals d1, d2... The lipid solution is introduced through the first inlet channel, and the aqueous solution containing the nucleic acid is introduced through the second inlet channel, A method for producing lipid nanoparticles, wherein the aqueous solution containing the nucleic acid has a sodium chloride concentration of 280 mM or more and is acidic.

[14] The method for producing lipid nanoparticles according to

[13] , wherein the aqueous solution containing the nucleic acid has a sodium chloride concentration of 500 mM or less.

[15] A kit for use in the method for producing lipid nanoparticles according to

[13] or

[14] , A dried lipid composition containing all lipid components constituting the lipid nanoparticles, and A dried product containing sodium chloride that can be dissolved in water to prepare an aqueous solution with a sodium chloride concentration of 280 mM or more. A kit for producing lipid nanoparticles, comprising:

[16] The kit for producing lipid nanoparticles according to

[15] , further comprising one or more selected from the group consisting of ethanol, phosphate saline, and nucleic acids. [Effects of the Invention]

[0014] The lipid nanoparticles according to the present invention can highly express encapsulated genes in splenic dendritic cells, making them useful as splenic dendritic cell-specific gene delivery carriers for cancer vaccine therapy and gene therapy. [Brief explanation of the drawings]

[0015] [Figure 1] 1 is a diagram schematically illustrating the structure of one embodiment of a flow channel structure used in the production of lipid nanoparticles according to the present invention. [Figure 2] This figure shows the interaction profile of factors that significantly affect the size (ζ average particle diameter) of lipid nanoparticles, obtained by statistically analyzing the measurement results of the physical properties of lipid nanoparticles in test plots A-1 to A-14 in Example 1. [Figure 3] FIG. 1 shows the results of measuring the Nluc expression levels (per mg of protein) in the liver and spleen of mice administered lipid nanoparticles of test groups A-1 to A-14 in Example 1. [Figure 4] FIG. 1 shows the results of measuring the amount of lipid nanoparticles taken up into cells in dendritic cells, B cells, and macrophages in the spleens of mice administered lipid nanoparticles of test groups A-1 to A-14 in Example 1. [Figure 5] FIG. 1 shows the results of measuring the expression levels of Nluc in dendritic cells, B cells, and macrophages in the spleens of mice administered lipid nanoparticles of test groups A-1 to A-14 in Example 1. [Figure 6] FIG. 1 shows the results of measuring the amount of lipid nanoparticles taken up into cells in dendritic cells, B cells, and macrophages in the spleens of mice administered lipid nanoparticles of test groups B-1 to B-8 in Example 2. [Figure 7] FIG. 1 shows the results of measuring the expression levels of Nluc in dendritic cells, B cells, and macrophages in the spleens of mice administered lipid nanoparticles of test groups B-1 to B-8 in Example 2. [Figure 8]FIG. 1 shows the results of measuring the expression level of Nluc in dendritic cells in the spleen of mice administered lipid nanoparticles of Test Groups A-1 to A-14 and Test Groups B-1 to B-8 in Examples 1 and 2. [Figure 9] FIG. 1 shows the relationship between the amount of lipid nanoparticles taken up by splenic dendritic cells and the expression level of IA / IE in mice administered lipid nanoparticles from test groups A-1 to A-14 and test groups B-1 to B-8 in Examples 1 and 2 (A), and the relationship between the expression level of Nluc gene in splenic dendritic cells and the expression level of IA / IE (B). [Figure 10] This figure shows the relationship between the amount of lipid nanoparticles taken up by splenic dendritic cells and the expression level of IA / IE in mice administered lipid nanoparticles from test groups A-11, A-15, and A1-16 in Example 3 (A), and the relationship between the expression level of Nluc gene in splenic dendritic cells and the expression level of IA / IE (B). [Figure 11] This figure shows the changes over time in the ζ-average particle size (nm) (A) and RNA encapsulation rate (%) (B) when lipid nanoparticles from test groups A-11, A-15, and A1-16 were stored at 4°C in Example 3. [Figure 12] FIG. 1 shows the results of measuring the tumor volume over time in mice subcutaneously implanted with E.G7-OVA cells after pre-administration of OVA-mRNA-loaded lipid nanoparticles (mOVA-A11-LNP) in Example 4. [Figure 13] FIG. 10 shows the results of measuring the tumor volume over time in mice that were subcutaneously transplanted with E.G7-OVA cells and then administered with mOVA-A11-LNP in Example 4. [Figure 14] FIG. 1 shows the results of measuring the tumor volume over time in mice that underwent a second E.G7-OVA cell transplant in Example 4. [Figure 15] FIG. 10 shows the results of measuring the tumor volume over time in mice that were subcutaneously transplanted with E.G7-OVA cells and then administered with mOVA-A11-LNP in Example 4. [Figure 16]This is a diagram showing the results of measuring the Nluc expression levels in the liver, spleen, lung, kidney, and inguinal lymph nodes of mice administered with each lipid nanoparticle in Example 5. [Figure 17] This is a diagram showing the measurement results of the percentage (%) of EGFP-positive cells (A), the average fluorescence intensity of EGFP in splenic dendritic cells (B), and the expression level of I-A / I-E (C) in the whole splenic dendritic cells of mice administered with each lipid nanoparticle in Example 5. [Figure 18] This is a diagram showing the results of measuring the tumor volume of mice administered with each lipid nanoparticle over time after subcutaneous transplantation of E.G7-OVA cells in Example 6.

Modes for Carrying Out the Invention

[0016] Hereinafter, embodiments of the present invention will be specifically described. In the specification of the present application, "X1 to X2 (X1 and X2 are real numbers satisfying X1 < X2)" means "X1 or more and X2 or less".

[0017] The lipid nanoparticles according to the present invention have a number average particle diameter of 150 nm or more. Immune cells have developed macropinocytosis and phagocytosis pathways that take up relatively large extracellular substances. The lipid nanoparticles according to the present invention are efficiently taken up by splenic dendritic cells because of their large number average particle diameter of 150 nm or more. The number average particle diameter of the lipid nanoparticles according to the present invention is not particularly limited as long as it is 150 nm or more, preferably 150 to 700 nm, more preferably 150 to 600 nm, further preferably 200 to 600 nm, still further preferably 300 to 600 nm, and particularly preferably 400 to 600 nm.

[0018] <0​​​​The polydispersity index (PdI) of the lipid nanoparticles according to the present invention is about 0.01 to 0.7, preferably about 0.01 to 0.5, and more preferably about 0.01 to 0.2. The zeta potential can be in the range of 1 mV to 20 mV, and preferably in the range of 5 mV to 15 mV.

[0020] The lipid nanoparticles according to the present invention contain a pH-sensitive cationic lipid and a polyalkylene glycol-modified lipid. The content of the pH-sensitive cationic lipid relative to the total amount of lipids constituting the lipid nanoparticles is 40 to 70 mol %, and the content of the polyalkylene glycol-modified lipid relative to the total amount of lipids constituting the lipid nanoparticles is 0.5 to 1.75 mol %.

[0021] The lipid nanoparticles of the present invention have excellent blood retention and endosomal escape ability due to the inclusion of a pH-sensitive cationic lipid as a constituent lipid component. The ratio of the content of the pH-sensitive cationic lipid to the total amount of lipids constituting the lipid nanoparticles in the lipid nanoparticles of the present invention ([amount of pH-sensitive cationic lipid (mol)] / ([amount of total lipids constituting the lipid nanoparticles (mol)]) × 100%) is not particularly limited as long as it is 40 to 70 mol%, but is preferably 40 to 65 mol%, and more preferably 40 to 60 mol%.

[0022] The pH-sensitive cationic lipid constituting the lipid nanoparticles of the present invention may be one type only, or may be two or more types. When the pH-sensitive cationic lipid constituting the lipid nanoparticles of the present invention is two or more types, the amount of pH-sensitive cationic lipid means the total amount of lipid molecules corresponding to the pH-sensitive cationic lipid among the lipid molecules constituting the lipid nanoparticles.

[0023] As the pH-sensitive cationic lipid, for example, a lipid represented by the following general formula (I) (hereinafter, sometimes referred to as "pH-sensitive cationic lipid (I)") is preferably used.

[0024] [ka]

[0025] In the general formula (I), a represents an integer of 3 to 5, and is preferably 4. b represents 0 or 1. When b is 0, it means that there is no —O—CO— group and it is a single bond.

[0026] In general formula (I), R 1 and R 2 each independently represents a group represented by the following general formula (A): In general formula (A), q represents an integer of 1 to 9; r represents 0 or 1; s represents an integer of 1 to 3; t represents 0 or 1; u represents an integer of 1 to 8; c represents 0 or 1; and v represents an integer of 4 to 12. However, when b and c are simultaneously 0, this does not include the cases where q is an integer of 3 to 5, r and t are 1, s is 1, and u+v is an integer of 6 to 10.

[0027] [ka]

[0028] As the pH-sensitive cationic lipid (I), R 1 and R 2 It is preferable that the hydrocarbon chain of R is relatively long. Specifically, in the pH-sensitive cationic lipid (I), 1 and R 2 is preferably a group having 20 or more carbon atoms, that is, in general formula (A), q+2r+s+2t+u+c+v is preferably an integer of 19 or more. In particular, for the pH-sensitive cationic lipid (I), q+2r+s+2t+u+c+v is preferably an integer of 19 to 33, more preferably an integer of 19 to 31, even more preferably an integer of 21 to 31, and even more preferably an integer of 21 to 27.

[0029] pH-sensitive cationic lipid (I) 1 and R 2Examples of the aryl group include a group in which, in general formula (A), r is 1, t is 0, q is an integer of 5 to 11, preferably an integer of 6 to 10, s+u is an integer of 5 to 11, preferably an integer of 6 to 10, c is 1, v is an integer of 4 to 12, and q+s+u+v is an integer of 16 or more; a group in which r is 0, t is 1, q+s is an integer of 5 to 11, preferably an integer of 6 to 10, u is an integer of 5 to 8, c is 1, and v is an integer of 4 to 12 and q+s+u+v is an integer of 16 or greater; a group where r and t are 0, q+s+u is an integer of 13 to 23, preferably 15 to 21, c is 1, v is an integer of 4 to 12, and q+s+u+v is an integer of 18 or greater; or a group where r and t are 0, q+s+u is an integer of 13 to 23, preferably 15 to 21, c is 1, v is an integer of 6 to 10, and q+s+u+v is an integer of 18 or greater.

[0030] In pH-sensitive cationic lipids (I), R 1 and R 2 are not limited as long as they are groups represented by general formula (A), and may be different groups from each other, but are preferably the same group.

[0031] In general formula (I), X represents a group represented by the following general formula (B) or a 5- to 7-membered non-aromatic heterocyclic group. The 5- to 7-membered non-aromatic heterocyclic group represented by X is bonded to (O—CO) b- via a carbon atom.

[0032] [ka]

[0033] In general formula (B), d represents an integer of 0 to 3. When d is 0, it means that there is no —(CH2)— group and it is a single bond.

[0034] In general formula (B), R 3 and R 4 are each independently C 1-4 Alkyl group (alkyl group with 1 to 4 carbon atoms) or C 2-4 R represents an alkenyl group (an alkenyl group having 1 to 4 carbon atoms).3 and R 4 C indicates 1-4 Alkyl group or C 2-4 In the alkenyl group, one or two hydrogen atoms may be replaced by a phenyl group.

[0035] C 1-4 Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, and a t-butyl group. 2-4 Examples of the alkenyl group include a vinyl group, a 1-propenyl group, a 2-propenyl group, a 1-methylvinyl group, a 2-methyl-1-propenyl group, a 1-butenyl group, a 2-butenyl group, and a 3-butenyl group.

[0036] In general formula (B), R 3 and R 4 may be bonded to each other to form a 5- to 7-membered non-aromatic heterocycle. 3 and R 4 Examples of the 5- to 7-membered non-aromatic heterocycle formed by bonding R to each other include a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group, and a 1-piperazinyl group. 3 and R 4 In the 5- to 7-membered non-aromatic heterocycle formed by bonding together, one or two hydrogen atoms in the ring are C 1-4 Alkyl group or C 2-4 The two hydrogen atoms in the ring may be substituted with an alkenyl group. 1-4 Alkyl group or C 2-4 When the alkenyl groups are substituted, they may be substituted with the same groups or with different groups.

[0037] In general formula (I), when X is a 5- to 7-membered non-aromatic heterocyclic group, examples of the heteroatom contained in the heterocyclic group include a nitrogen atom, an oxygen atom, and a sulfur atom. The heteroatom constituting the heterocyclic group may be one, or may be two or more heteroatoms that are the same or different. The heterocyclic group may be a saturated heterocyclic ring and may contain one or more double bonds, but the heterocyclic ring is not an aromatic ring.

[0038] The pKa of the pH-sensitive cationic lipid (I) is not particularly limited, but can be selected, for example, from about 4.0 to 9.0, preferably from about 4.5 to 8.5, and more preferably from about 6 to 8. It is preferable to select the type of each substituent so as to give a pKa within this range.

[0039] The pH-sensitive cationic lipid (I) can be easily produced, for example, by the method specifically shown in the Examples of this specification. By referring to this production method and appropriately selecting raw material compounds, reagents, reaction conditions, etc., a person skilled in the art can easily produce any lipid within the scope of general formula (I).

[0040] As the pH-sensitive cationic lipid contained in the lipid nanoparticles according to the present invention, a lipid represented by the following general formula (I-1) (hereinafter sometimes referred to as "pH-sensitive cationic lipid (I-1)") is particularly preferred.

[0041] [ka]

[0042] In general formula (I-1), p1 and q1 represent integers of 0 or greater that satisfy p1+q1=3 to 8, and r1 represents 0 or 1. When r1 is 0, p1 and q1 are preferably integers of 0 or greater that satisfy p1+q1=3 to 5, and particularly preferably integers of 0 or greater that satisfy p1+q1=4. When r1 is 1, p1 is preferably an integer of 3 to 5 and q1 is an integer of 0 to 2, and particularly preferably p1 is 4 and q1 is an integer of 0 to 2.

[0043] In general formula (I-1), n1 and n2 each independently represent an integer of 6 to 10. In the pH-sensitive cationic lipid represented by general formula (I-1), n1 and n2 are preferably 6, 8, or 10, and more preferably 6.

[0044] In general formula (1), R 13 and R 14 are each independently a linear C 1-3 Alkyl or aryl C 1-3 It is an alkyl group. 1-3 Examples of alkyl groups include methyl, ethyl, and n-propyl groups. 1-3 The alkyl group is phenyl C 1-3 An alkyl group is preferred, and a phenylmethyl group (benzyl group) is particularly preferred.

[0045] In general formula (1), R 13 and R 14 are linked together to form a pyrrolidine ring, a piperidine ring, a morpholine ring, or a ring in which the nitrogen atom is C 1-3 A piperazine ring may be formed which may be substituted with an alkyl group. 13 and R 14 The nitrogen atom of the piperazine ring formed by 1-3 Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, and an isopropyl group, and a methyl group is preferred. In the pH-sensitive cationic lipid represented by general formula (I-1), when r1 is 0, R 13 and R 14are both n-propyl groups or are linked to each other to form a pyrrolidine ring, a piperidine ring, a morpholine ring, a piperazine ring, or a piperazine ring in which a nitrogen atom is substituted with a methyl group, and R 13 and R 14 are more preferably both n-propyl groups or are linked to each other to form a morpholine ring.

[0046] The pKa of the pH-sensitive cationic lipid represented by general formula (I-1) is preferably 4.5 to 7.5, more preferably 5.0 to 7.1, and even more preferably 5.5 to 6.5. For example, in general formula (I-1), R 13 and R 14 The pKa of pH-sensitive cationic lipids in which both R are n-propyl groups is about 6.25, and 13 and R 14 The pKa of a pH-sensitive cationic lipid in which the groups are linked to each other to form a morpholine ring is about 5.85 (Non-Patent Document 5).

[0047] In general formula (1), R 11 and R 12 are each independently a linear C 10-14 Alkyl group, linear C with one or two unsaturated bonds 10-20 Alkenyl group, or -CH(R 15 )(R 16 )(R 15 and R 16 are each independently a linear C 5-10 Among the pH-sensitive cationic lipids represented by general formula (1), the scaffolding R 11 and R 12 However, by using a relatively medium-chain alkyl group, alkenyl group, or branched alkyl group, the fluidity of the scaffold is increased, the efficiency of introduction into splenic dendritic cells is improved, and toxicity is also reduced.

[0048] Linear C 10-14Examples of the alkyl group (an alkyl group having 10 to 14 carbon atoms) include an n-decyl group, an n-undecyl group, an n-dodecyl group, an n-tridecyl group, and an n-tetradecyl group. 11 or R 12 is a linear C 10-14 In the case of an alkyl group, the pH-sensitive cationic lipid represented by general formula (I-1) is 11 and R 12 Both are linear C 10-14 Preferably, R is an alkyl group. 11 and R 12 are each independently an n-undecyl group, an n-dodecyl group, or an n-tridecyl group, and R 11 and R 12 More preferably, R are both n-undecyl, n-dodecyl, or n-tridecyl groups, 11 and R 12 Even more preferably, both are n-tridecyl groups.

[0049] Linear C with one or two unsaturated bonds 10-20 The alkenyl group (alkenyl group having 10 to 20 carbon atoms) is a straight-chain C 10-20 Among alkyl groups (alkyl groups with 10 to 20 carbon atoms), it is sufficient if one or two of the saturated bonds between carbon atoms in the alkyl chain are unsaturated bonds, and a straight-chain C 10-20 A group in which one or two of the saturated bonds between carbon atoms near the middle of the alkyl group are unsaturated is preferred, and a straight-chain C 13-18 A group in which one or two of the saturated bonds between carbon atoms near the middle of the alkyl group (an alkyl group having 13 to 18 carbon atoms) are unsaturated is more preferred. Examples of alkyl groups having 10 to 20 carbon atoms include an n-decyl group, an n-undecyl group, an n-dodecyl group, an n-tridecyl group, an n-tetradecyl group, an n-pentadecyl group, an n-hexadecyl group, an n-heptadecyl group, an n-octadecyl group, an n-nonadecyl group, and an n-eicosyl group. A linear C 13-18The alkyl group in which one or two of the saturated bonds between carbon atoms near the middle thereof are unsaturated is more preferably an n-tridecyl group, an n-tetradecyl group, an n-pentadecyl group, an n-hexadecyl group, an n-heptadecyl group, or an n-oxadecyl group in which one or two of the saturated bonds between carbon atoms near the middle thereof are unsaturated, and more preferably a 5-tridecenyl group, a 6-tridecenyl group, a 7-tridecenyl group, an 8-tridecenyl group, a 9-tridecenyl group, a 5-tetradecenyl group, a 6-tetradecenyl group, a 7-tetradecenyl group, an 8-tetradecenyl group, a 9-tetradecenyl group, a 6-pentadecenyl group, a 7-pentadecenyl group, an 8-pentadecenyl group, a 9-pentadecenyl group, a 10-pentadecyl group, a 6-hexadecenyl group, a octadecenyl group, 7-hexadecenyl group, 8-hexadecenyl group, 9-hexadecenyl group, 10-hexadecyl group, 6-heptadecenyl group, 7-heptadecenyl group, 8-heptadecenyl group, 9-heptadecenyl group, 10-heptadecenyl group, 11-heptadecenyl group, 12-heptadecenyl group, 9,12-heptadecenyl group, 7-octadecenyl group, 8 1-octadecenyl group, 9-octadecenyl group, 10-octadecenyl group, 11-octadecenyl group, 7-nonadecenyl group, 8-nonadecenyl group, 9-nonadecenyl group, 10-nonadecenyl group, 11-nonadecenyl group, 8-eicosenyl group, 9-eicosenyl group, 10-eicosenyl group, 11-eicosenyl group, and 12-eicosenyl group.

[0050] R 11 or R 12 is a linear C 10-20 In the case of an alkenyl group, the pH-sensitive cationic lipid represented by general formula (I-1) is 11 and R 12 Both are linear C 10-20 Preferably, R is an alkenyl group. 11 and R 12 are each independently a linear C 13-18 It is more preferable that one or two of the saturated bonds between carbon atoms near the middle of the alkyl group are unsaturated bonds, and R 11 and R 12are each independently a 6-hexadecenyl group, a 7-hexadecenyl group, an 8-hexadecenyl group, a 9-hexadecenyl group, a 10-hexadecyl group, a 6-heptadecenyl group, a 7-heptadecenyl group, an 8-heptadecenyl group, a 9-heptadecenyl group, a 10-heptadecenyl group, an 11-heptadecenyl group, a 12-heptadecenyl group, a 9,12-heptadecenyl group, a 7-octadecenyl group, an 8-octadecenyl group, a 9-octadecenyl group, a 10-octadecenyl group, or an 11-octadecenyl group, and R 11 and R 12 are each independently a 6-heptadecenyl group, a 7-heptadecenyl group, an 8-heptadecenyl group, a 9-heptadecenyl group, a 10-heptadecenyl group, an 11-heptadecenyl group, a 12-heptadecenyl group, or a 9,12-heptadecenyl group, and R 11 and R 12 It is particularly preferred that both are 8-heptadecenyl groups.

[0051] -CH(R 15 )(R 16 )(R 15 and R 16 are each independently a linear C 5-10 Examples of alkyl groups include -CH(-CH 11 )(-C7H 15 ), -CH(-CH 13 )(-C8H 17 ), -CH(-CH 15 )(-C9H 19 ), -CH(-CH 17 )(-C 10 H 21 ) etc. R 11 or R 12 -CH(R 15 )(R 16 ), the pH-sensitive cationic lipid represented by general formula (I-1) is R 11 and R 12 Both are -CH(R 15 )(R 16 ), and R 11 and R 12 are each independently -CH(-CH11 )(-C7H 15 ), -CH(-CH 13 )(-C8H 17 ), -CH(-CH 15 )(-C9H 19 ), or -CH(-CH 17 )(-C 10 H 21 ), and R 11 and R 12 Both are -CH(-CH 13 )(-C8H 17 ) is more preferable.

[0052] The pH-sensitive cationic lipid represented by general formula (I-1) includes R 11 and R 12 However, each independently has a linear C 13-18 It is a group in which one or two saturated bonds between carbon atoms near the middle of the alkyl group are unsaturated, or R 11 and R 12 are each independently an n-undecyl group, an n-dodecyl group, or an n-tridecyl group, and p is 3 to 5, and R 11 and R 12 However, each independently has a linear C 13-18 More preferred are compounds in which one or two of the saturated bonds between carbon atoms near the middle of the alkyl group are unsaturated, and p is 3 to 5. 11 and R 12 are each independently a 6-heptadecenyl group, a 7-heptadecenyl group, an 8-heptadecenyl group, a 9-heptadecenyl group, a 10-heptadecenyl group, an 11-heptadecenyl group, a 12-heptadecenyl group, or a 9,12-heptadecenyl group, and p is 3 to 5, and more preferably a compound in which R 11 and R 12 are both 8-heptadecenyl groups and p is 3 to 5, and 11 and R 12 are both 8-heptadecenyl groups, and R 13 and R 14Particularly preferred are compounds in which both are methyl groups and p is 4.

[0053] The pH-sensitive cationic lipid represented by general formula (I-1) includes R 11 and R 12 are each independently -CH(R 15 )(R 16 ) and p is 3 to 5, and R 11 and R 12 are each independently -CH(-CH 11 )(-C7H 15 ), -CH(-CH 13 )(-C8H 17 ), -CH(-CH 15 )(-C9H 19 ), or -CH(-CH 17 )(-C 10 H 21 ) and p is 3 to 5, and R 11 and R 12 Both are -CH(-CH 13 )(-C8H 17 ) and p is 3 to 5, and R 11 and R 12 Both are -CH(-CH 13 )(-C8H 17 ) and p is 4 are even more preferred.

[0054] The pH-sensitive cationic lipid represented by general formula (I-1) can be easily produced, for example, by the method specifically shown in the Examples of this specification. By referring to this production method and appropriately selecting raw material compounds, reagents, reaction conditions, etc., a person skilled in the art can easily produce any lipid within the scope of general formula (I-1).

[0055] The lipid nanoparticles according to the present invention have a polyalkylene glycol-modified lipid as a constituent lipid component, and thus the surface of the lipid nanoparticles is modified with polyalkylene glycol. Surface modification with polyalkylene glycol, a hydrophilic polymer, can increase the stability of the lipid nanoparticles, such as their blood retention. However, if the density of polyalkylene glycol on the lipid surface is too high, the interfacial stabilization ability is high, and the particle size of the lipid nanoparticles is likely to be small. The lipid nanoparticles according to the present invention have a high stability and a relatively large number-average particle size of 150 nm or more, because the ratio of the content of polyalkylene glycol-modified lipid to the total amount of lipids constituting the lipid nanoparticles ([amount of polyalkylene glycol-modified lipid (mol)] / ([amount of total lipids constituting the lipid nanoparticles (mol)]) × 100%) is 0.5 to 1.75 mol %.

[0056] The content ratio of polyalkylene glycol-modified lipids in the lipid nanoparticles according to the present invention relative to the total amount of lipids constituting the lipid nanoparticles is not particularly limited as long as it is 0.5 to 1.75 mol%, and is preferably 0.5 to 1.6 mol%, and more preferably 0.5 to 1.5 mol%. The polyalkylene glycol-modified lipids constituting the lipid nanoparticles according to the present invention may be of only one type, or may be of two or more types. When two or more types of polyalkylene glycol-modified lipids are constituting the lipid nanoparticles according to the present invention, the amount of polyalkylene glycol-modified lipid means the total amount of lipid molecules corresponding to polyalkylene glycol-modified lipids among the lipid molecules constituting the lipid nanoparticles.

[0057] Examples of polyalkylene glycols that can be used include polyethylene glycol, polypropylene glycol, polytetramethylene glycol, polyhexamethylene glycol, etc. The molecular weight of the polyalkylene glycol is, for example, about 300 to 10,000, preferably about 500 to 10,000, and more preferably about 1,000 to 5,000.

[0058] For example, stearylated polyethylene glycol (e.g., PEG45 stearate (STR-PEG45)) can be used to modify lipids with polyethylene glycol. Other examples include N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-5000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-750]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and N-[carbonyl-methoxypolyethylene glycol Polyethylene glycol derivatives such as 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2k-DSG), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2k-DMG), and the like can also be used, but polyalkylene glycol-modified lipids are not limited to these.

[0059] Among the constituent lipids of the lipid nanoparticles of the present invention, lipids other than the pH-sensitive cationic lipid and the polyalkylene glycol-modified lipid can be lipids generally used in forming liposomes. Examples of such lipids include phospholipids, sterols, glycolipids, saturated or unsaturated fatty acids, etc. These can be used alone or in combination of two or more.

[0060] Examples of phospholipids include glycerophospholipids such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, phosphalidylcholine, cardiolipin, plasmalogen, ceramide phosphorylglycerol phosphate, and phosphatidic acid; and sphingophospholipids such as sphingomyelin, ceramide phosphorylglycerol, and ceramide phosphorylethanolamine. Phospholipids derived from natural products such as egg yolk lecithin and soybean lecithin can also be used. The fatty acid residues in the glycerophospholipids and sphingophospholipids are not particularly limited, but examples include saturated or unsaturated fatty acid residues having 12 to 24 carbon atoms, with saturated or unsaturated fatty acid residues having 14 to 20 carbon atoms being preferred. Specific examples include acyl groups derived from fatty acids such as lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, lignoceric acid, etc. When these glycerolipids or sphingolipids have two or more fatty acid residues, all of the fatty acid residues may be the same group or may be different groups.

[0061] Examples of sterols include animal-derived sterols such as cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol, and dihydrocholesterol; plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, campesterol, and brassicasterol; and microbial-derived sterols such as zymosterol and ergosterol. Examples of glycolipids include glyceroglycolipids such as sulfoxyribosylglyceride, diglycosyldiglyceride, digalactosyldiglyceride, galactosyldiglyceride, and glycosyldiglyceride; and sphingoglycolipids such as galactosylcerebroside, lactosylcerebroside, and ganglioside. Examples of saturated or unsaturated fatty acids include saturated or unsaturated fatty acids having 12 to 20 carbon atoms such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid.

[0062] The constituent lipids of the lipid nanoparticles according to the present invention preferably contain a neutral lipid in addition to the pH-sensitive cationic lipid and the polyalkylene glycol-modified lipid, more preferably one or more selected from the group consisting of phospholipids and sterols, and even more preferably phospholipids and cholesterol. When the lipid nanoparticles according to the present invention contain both phospholipids and sterols as constituent lipids, the ratio of the phospholipid content to the total lipid content constituting the lipid nanoparticles ([amount of phospholipid (mol)] / ([amount of total lipids constituting the lipid nanoparticles (mol)]) × 100%) is preferably 5 to 40 mol%, more preferably 5 to 30 mol%, even more preferably 5 to 20 mol%, and even more preferably 5 to 15 mol%. When the lipid nanoparticles according to the present invention contain both phospholipids and sterols as constituent lipids, the ratio of the sterol content to the total amount of lipids constituting the lipid nanoparticles ([amount of sterol (mol)] / ([amount of total lipids constituting the lipid nanoparticles (mol)]) × 100%) is preferably 5 to 40 mol%, more preferably 10 to 40 mol%, even more preferably 15 to 40 mol%, and even more preferably 20 to 40 mol%.

[0063] The lipid nanoparticles according to the present invention can be subjected to appropriate surface modification, if necessary. The blood retention of the lipid nanoparticles according to the present invention can be improved by modifying the surface with a hydrophilic polymer, etc. In some cases, surface modification can be achieved by using lipids modified with these modifying groups as constituent lipids of the lipid nanoparticles.

[0064] In producing the lipid nanoparticles according to the present invention, lipid derivatives that can be used to enhance blood retention include, for example, glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivatives, glutamic acid derivatives, polyglycerin phospholipid derivatives, etc. Furthermore, hydrophilic polymers that can be used to enhance blood retention include, in addition to polyalkylene glycol, dextran, pullulan, ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, carrageenan, etc. for surface modification.

[0065] Furthermore, to promote nuclear translocation of the lipid nanoparticles according to the present invention, the surface of the lipid nanoparticles can be modified with an oligosaccharide compound of three or more sugars. The type of oligosaccharide compound of three or more sugars is not particularly limited, but for example, an oligosaccharide compound having about 3 to 10 sugar units bonded thereto can be used, and preferably an oligosaccharide compound having about 3 to 6 sugar units bonded thereto can be used. Among these, oligosaccharide compounds that are preferably glucose trimers or hexamers can be used, and more preferably, oligosaccharide compounds that are glucose trimers or tetramers can be used. More specifically, isomaltotriose, isopanose, maltotriose, maltotetraose, maltopentaose, or maltohexaose can be suitably used, and among these, maltotriose, maltotetraose, maltopentaose, or maltohexaose in which glucose is α1-4-linked is more preferred. Maltotriose or maltotetraose is particularly preferred, and maltotriose is the most preferred. The amount of the lipid nanoparticle surface modified with the oligosaccharide compound is not particularly limited, but is, for example, about 1 to 30 mol %, preferably about 2 to 20 mol %, and more preferably about 5 to 10 mol % relative to the total lipid amount.

[0066] The method for surface-modifying lipid nanoparticles with oligosaccharide compounds is not particularly limited, but for example, liposomes in which the surface of lipid nanoparticles is modified with monosaccharides such as galactose or mannose (WO 2007 / 102481) are known, and the surface modification method described in this publication can be adopted. The disclosure of the above publication is incorporated herein by reference in its entirety.

[0067] Furthermore, the lipid nanoparticles of the present invention can be imparted with one or more of the following functions: temperature sensitivity, membrane permeability, gene expression, and pH sensitivity. By appropriately imparting these functions, the retention of the lipid nanoparticles in blood can be improved, and the lipid nanoparticles can be efficiently released from endosomes after endocytosis in target cells, allowing the encapsulated nucleic acid to be more efficiently expressed in splenic dendritic cells.

[0068] The lipid nanoparticles of the present invention may contain one or more substances selected from the group consisting of antioxidants such as tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene, charged substances, and membrane polypeptides. Examples of charged substances that impart a positive charge include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine. Examples of charged substances that impart a negative charge include dicetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Examples of membrane polypeptides include surface membrane polypeptides and integral membrane polypeptides. The amounts of these substances to be incorporated are not particularly limited and can be selected appropriately depending on the purpose.

[0069] The form of the lipid nanoparticles according to the present invention is not particularly limited, but examples thereof include unilamellar liposomes, multilayer liposomes, spherical micelles, and amorphous layered structures dispersed in an aqueous solvent.

[0070] The lipid nanoparticles according to the present invention preferably contain a target component to be delivered into target cells within the lipid membrane-covered particles. The component contained within the lipid nanoparticles according to the present invention is not particularly limited as long as it has a size that allows it to be contained. The lipid nanoparticles according to the present invention can contain any substance, such as nucleic acids, sugars, peptides, low-molecular-weight compounds, and metal compounds.

[0071] Nucleic acids are preferred as components to be encapsulated in the lipid nanoparticles of the present invention. The nucleic acid may be DNA, RNA, or an analog or derivative thereof (e.g., peptide nucleic acid (PNA), phosphorothioate DNA, etc.). The nucleic acid to be encapsulated in the lipid nanoparticles of the present invention may be a single-stranded nucleic acid or a double-stranded nucleic acid, and may be linear or cyclic. The nucleic acid to be encapsulated in the lipid nanoparticles of the present invention is preferably mRNA or plasmid DNA (pDNA), as these have particularly good transfer efficiency into splenic dendritic cells.

[0072] The nucleic acid to be encapsulated in the lipid nanoparticles of the present invention preferably contains a foreign gene for expression in the target cell, and more preferably is a nucleic acid that functions to express the foreign gene in the cell by being incorporated into the cell. The foreign gene may be a gene that is originally contained in the genomic DNA of the target cell (preferably a splenic dendritic cell), or it may be a gene that is not contained in the genomic DNA. Examples of such nucleic acids include gene expression vectors containing a nucleic acid consisting of a base sequence encoding the target gene to be expressed. The gene expression vector may exist as an extrachromosomal gene in the cell into which it is introduced, or it may be incorporated into the genomic DNA by homologous recombination.

[0073] The gene expression vector to be encapsulated in the lipid nanoparticles of the present invention is not particularly limited, and vectors commonly used in gene therapy, etc. can be used. The gene expression vector to be encapsulated in the lipid nanoparticles of the present invention is preferably a nucleic acid vector such as a plasmid vector. The plasmid vector may remain circular, or may be encapsulated in the lipid nanoparticles of the present invention after being pre-cleaved into a linear form. The gene expression vector can be designed by standard methods using commonly used molecular biology tools based on the base sequence information of the gene to be expressed, and can be produced by various known methods.

[0074] The nucleic acid to be encapsulated in the lipid nanoparticles of the present invention is also preferably mRNA encoding a peptide or protein to be translated and expressed in target cells. The peptide or protein encoded by the mRNA may be a protein or partial protein encoded by a gene originally contained in the genomic DNA of the target cells (preferably splenic dendritic cells), or a protein or partial protein not contained in the genomic DNA of the target cells. The structure of the mRNA is not particularly limited as long as it is translatable in the target cells into which it is introduced, but it preferably has a structure similar to that of natural mRNA. Examples of such structures include a 5' cap structure, a 3' poly(A) structure, a 5' untranslated region, and a 3' untranslated region. The mRNA to be encapsulated in the lipid nanoparticles of the present invention can be designed by conventional methods using commonly used molecular biology tools based on the amino acid sequence information of the peptide or the nucleotide sequence encoding the peptide or the like to be expressed in target cells, and can be produced by various known methods.

[0075] The nucleic acid encapsulated in the lipid nanoparticles according to the present invention is preferably a functional nucleic acid that controls the expression of a target gene present in a target cell. Examples of such functional nucleic acids include antisense oligonucleotides, antisense DNA, antisense RNA, siRNA, microRNA, and mRNA. Alternatively, the functional nucleic acid may be pDNA, which serves as an siRNA expression vector that expresses siRNA in cells. The siRNA expression vector can be prepared from a commercially available siRNA expression vector, which may also be modified as appropriate.

[0076] The method for producing lipid nanoparticles according to the present invention is not particularly limited, and any method available to those skilled in the art can be employed. For example, all lipid components are dissolved in an organic solvent such as chloroform, and a lipid membrane is formed by drying under reduced pressure using an evaporator or spray drying using a spray dryer. An aqueous solvent containing the components to be encapsulated in the lipid nanoparticles, such as nucleic acids, is then added to the dried mixture, and the mixture is emulsified using an emulsifier such as a homogenizer, an ultrasonic emulsifier, or a high-pressure injection emulsifier. Liposomes can also be produced by well-known methods for producing liposomes, such as reverse-phase evaporation. To control the size of lipid nanoparticles, extrusion filtration can be performed under high pressure using a membrane filter with a uniform pore size.

[0077] The composition of the aqueous solvent (dispersion medium) is not particularly limited, and examples thereof include buffer solutions such as phosphate buffer, citrate buffer, and phosphate-buffered saline, physiological saline, cell culture media, etc. These aqueous solvents (dispersion media) can stably disperse lipid nanoparticles, but may also contain sugars (aqueous solutions) such as monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose, disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose, trisaccharides such as raffinose and melezinose, polysaccharides such as cyclodextrin, sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, and maltitol, and polyhydric alcohols (aqueous solutions) such as glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ethers, diethylene glycol monoalkyl ethers, and 1,3-butylene glycol. To stably store lipid nanoparticles dispersed in this aqueous solvent for a long period of time, it is desirable to eliminate electrolytes from the aqueous solvent as much as possible from the viewpoint of physical stability such as suppressing aggregation. Also, from the viewpoint of chemical stability of lipids, it is desirable to set the pH of the aqueous solvent to a weakly acidic to near-neutral range (about pH 3.0 to 8.0) and / or remove dissolved oxygen by nitrogen bubbling or the like.

[0078] The lipid nanoparticles according to the present invention can be produced by an alcohol dilution method using a flow channel. The flow channel used for production may be a microflow channel with a built-in three-dimensional micromixer that can achieve instantaneous mixing of two liquids, but because this allows for the formation of a nano-sized lipid particle formation system with high particle size control, it is preferable to use a simple two-dimensional flow channel structure, as described in Patent Document 2, in which baffles (baffle plates) of a fixed width relative to the width of the flow channel are arranged alternately on both sides of the micro-sized flow channel through which the raw material solution flows.

[0079] Specifically, it is preferable to use a flow path structure as shown in Fig. 1 (hereinafter sometimes referred to as the "flow path structure of the present invention"). On the upstream side (left side of the drawing), a first inlet path 10 for introducing a first fluid and a second inlet path 20 for introducing a second fluid, which are independent from each other, each have a certain length and join together to form a single dilution flow path 30 toward the downstream side. The dilution flow path 30 has a flow path portion 50 that is two-dimensionally curved in at least a part thereof, and the curved flow path portion 50 is formed by at least two or more structural elements 40 that protrude alternately from both side wall surfaces of the dilution flow path opposing each other in the Y direction toward the flow path center in approximately the Y direction (approximately the +Y direction, approximately the −Y direction), have constant heights h1, h2... of 1 / 2y0 or more and less than 1y0, and have constant widths x1, x2... in the X direction, and that regulate the flow path width of the dilution flow path, being defined as the X direction, the Y direction, and the flow path width of the dilution flow path upstream of the curved flow path portion 50, respectively. That is, in the region where the structural element 40 is present, the flow path width y1, y2... of the dilution flow path is restricted to 1 / 2y0 or less, particularly 1 / 2y0 or less and 1 / 40y0 or more, over a certain length x1, x2... in the X direction.

[0080] While the flow channel structure of the present invention conceptually has a shape similar to that of a micro-sized flow channel, with approximately rectangular baffles alternately arranged on both sides, as illustrated in FIG. 1 and described above, in practice, it is not limited to a structure formed by disposing separate baffles on the flow channel. That is, as long as a flow channel of a similar shape is formed corresponding to the flow channel formed by disposing such baffles, the structure of the structural element 40 is not particularly limited. To form the aforementioned structural element 40, the wall surfaces of the flow channel structure may be integrally formed while being bent into a predetermined shape (while maintaining a substantially constant thickness), thereby forming a two-dimensional flow channel shape that bends and contracts as defined above. Naturally, such embodiments are included in the flow channel structure of the present invention. Such two-dimensional flow channels can be relatively easily formed by, for example, injection molding, cast molding, or molding using a three-dimensional printer using thermoplastic resins, thermosetting resins, ultraviolet-curable resins, metals, or glassy materials.

[0081] The channel width y0 of the dilution channel 30 after the first inlet channel 10 and the second inlet channel 20 join depends to some extent on the particle size of the nano-sized lipid particles to be formed, but is typically about 20 to 1000 μm, more preferably about 100 to 200 μm. To obtain lipid particles of the desired nano size, specifically, for example, within a particle size range of about 10 to 100 nm, it is somewhat necessary to dilute the lipid solution with a diluent medium with a channel width y0 within the above range.

[0082] Furthermore, the height h1, h2... (length in the Y direction) of each structural element 40 is ½y0 or more and less than 1y0, preferably ½y0 or more and 39 / 40y0 or less, more preferably ½y0 or more and ¾y0 or less, of the flow path width y0 of the dilution flow path 30 on the upstream side. The presence of each structural element 40 reduces the flow path width y1, y2... from the flow path width y0 of the dilution flow path 30 on the upstream side to a width less than ½y0 but greater than 0. Note that the heights h1, h2... of the structural elements 40 provided in the curved flow path portion 50 do not necessarily have to be identical and may be different as long as the above-mentioned predetermined conditions are satisfied. The flow path widths y1, y2... formed thereby may also be different. For example, the widths h1, h2... of each structural element 40 may gradually increase in the downstream direction, narrowing the flow path widths y1, y2.... The heights h1, h2... (lengths in the Y direction) of each structural element 40 are predetermined, and the flow path widths y1, y2... at the locations where these elements exist are narrowed to widths less than 1 / 2y0, thereby improving the efficiency of molecular diffusion.

[0083] Although it is not particularly limited and depends on other conditions such as the size of the lipid particles to be obtained, the number of structural elements 40, the width (length in the X direction) x1, x2... of each mixer structural element 40, and the interval d1, d2... between adjacent structural elements 40, specifically, for example, if the flow path width y0 of the upstream dilution flow path is 200 μm, it is desirable that the heights h1, h2... of each structural element 40 be 100 μm to less than 200 μm. Therefore, the flow path width y1, y2... at the position where each structural element 40 is present is less than 1 / 2y0 and greater than 0, that is, approximately less than 100 μm.

[0084] The widths (lengths in the X direction) x1, x2... of each structural element 40 are influenced by other conditions, such as the size of the lipid particles to be obtained, the number of structural elements 40, the heights h1, h2... (lengths in the Y direction) of each structural element 40, and the spacing d1, d2... between adjacent structural elements 40, but are preferably set to approximately 1 / 10y0 or more and 5y0 or less of the channel width y0 of the upstream dilution channel. Specifically, for example, if the channel width y0 of the upstream dilution channel is 20 to 1000 μm, typically 200 μm, then the widths x1, x2... of each structural element 40 are desirably approximately 20 μm to 1000 μm. The widths x1, x2... of each structural element 40 do not necessarily have to be identical and may be different as long as the above-mentioned predetermined conditions are met. For example, the widths x1, x2... may gradually increase in the downstream direction.

[0085] The distances d1, d2... between adjacent structural elements 40 depend on other conditions, such as the size of the lipid particles to be obtained, the number of structural elements 40, the heights h1, h2... (lengths in the Y direction) of each structural element 40, and the widths x1, x2... (lengths in the X direction) of each structural element 40, but are preferably set to approximately 1 / 10y0 or more and 5y0 or less of the channel width y0 of the upstream dilution channel. Specifically, for example, if the channel width y0 of the upstream dilution channel is 20 to 1000 μm, typically 200 μm, the distances d1, d2... between adjacent structural elements 40 are preferably set to approximately 20 μm to 1000 μm. The distances d1, d2... between adjacent structural elements 40 do not necessarily have to be identical and may be different as long as the above-mentioned predetermined conditions are met. For example, the distances d1, d2... may be gradually narrower downstream.

[0086] In the flow channel structure of the present invention, the axial direction or extension direction of the upstream dilution channel is defined as the X direction, and the width direction of the dilution channel perpendicularly intersecting the X direction is defined as the Y direction. As described above, each structural element 40 extends alternately from both side walls toward the center of the channel in the approximate Y direction (approximately +Y direction and approximately −Y direction), and has wall surfaces that are approximately perpendicular to the channel direction (X direction). However, this angle does not necessarily have to be strictly 90°; even a slight inclination can be effective. While not particularly limited, specific examples include angles within the range of approximately 30 to 150°, more preferably 40 to 140°, and particularly preferably 80 to 100°. Furthermore, a certain degree of roundness is also acceptable for the shape of the corners of each structural element 40 on the channel center side. While not particularly limited, for example, a radius of 50 μm or less, more preferably 20 μm or less, may be acceptable. However, in order to obtain more controllable and uniform nano-sized lipid particles, it is desirable to minimize these tolerances. 1, the axial direction of the upstream dilution flow channel in the flow channel structure or the X direction, which is its extension direction, is shown as a straight line for convenience, but this X direction merely indicates the axial direction of the dilution flow channel, and in reality, it is not limited to such a straight line and may be curved, for example, with a certain curvature. In such a case, the Y direction, which is the width direction of the dilution flow channel that intersects perpendicularly with the X direction, refers to the direction perpendicular to the X direction at the unit length.

[0087] Furthermore, since the flow path structure of the present invention is a two-dimensional flow path structure as described above, the size of the flow path in the depth direction (paper thickness direction in Figure 1) is not particularly limited, but it is preferable to set it to, for example, about 10 to 1000 μm, more preferably about 50 to 200 μm.

[0088] The flow channel used when producing lipid nanoparticles according to the present invention by the alcohol dilution method is not particularly limited as long as the dilution flow channel 30 in the flow channel structure shown in Figure 1 is a flow channel that can generate a three-dimensional flow. For example, the flow channel structure of the present invention may be a chaotic micromixer (staggered herringbone mixer) (Non-Patent Document 6) in which a chaotic flow is generated by grooves or minute protrusions formed on the wall surface of the flow channel, instead of the two-dimensionally curved flow channel portion 50 in at least a part of the dilution flow channel 30.

[0089] Dilution in the flow channel structure of the present invention depends on molecular diffusion. The faster the dilution rate of the raw lipid solution, the smaller the size of the lipid particles produced. Therefore, by adjusting the width, length, and arrangement of the structure (baffle), the dilution rate of the raw solution can be controlled, making it possible to form lipid nanoparticles with higher particle size controllability than conventional methods.

[0090] In the flow channel structure of the present invention, as shown in FIG. 1, a first inlet channel 10 for introducing a first fluid and a second inlet channel 20 for introducing a second fluid are joined together to form a single dilution channel, each having a fixed length. A lipid solution in which lipid components are dissolved in ethanol is introduced through the first inlet channel 10, and an aqueous solution is introduced through the second inlet channel 20. The aqueous solution introduced through the second inlet channel 20 contains dissolved water-soluble components to be loaded onto the lipid nanoparticles. For example, by introducing an aqueous solution containing nucleic acid (nucleic acid-containing aqueous solution) through the second inlet channel 20, the lipid solution is diluted with the nucleic acid-containing aqueous solution in the dilution channel, and lipid nanoparticles loaded with nucleic acid are formed in this process (formation process).

[0091] The nucleic acid-containing aqueous solution can be prepared by dissolving the nucleic acid to be loaded onto the lipid nanoparticles in an aqueous solvent. The aqueous solvent is not particularly limited as long as it can stably dissolve the nucleic acid and can stably disperse the produced lipid nanoparticles. Examples of the aqueous solvent include buffer solutions such as acetate buffer, citrate buffer, phosphate buffer, and phosphate-buffered saline, physiological saline, and cell culture media. These aqueous solvents (dispersion media) may further contain monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose; disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose; trisaccharides such as raffinose and melezinose; polysaccharides such as cyclodextrin; sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, and maltitol; and polyhydric alcohols such as glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, and 1,3-butylene glycol.

[0092] When the pH of the nucleic acid-containing aqueous solution is acidic, lipid nanoparticles are formed in an acidic environment. Therefore, the pH-sensitive cationic lipids contained in the lipid nanoparticles are positively charged, and the nucleic acid is efficiently loaded onto the lipid nanoparticles through electrostatic interactions with the negatively charged nucleic acid. Therefore, when forming lipid nanoparticles loaded with nucleic acid, it is preferable to prepare the nucleic acid-containing aqueous solution by dissolving the nucleic acid in an acidic buffer solution. The pH of the acidic buffer solution is preferably in the range of 3.5 to 6.0, more preferably in the range of 3.8 to 5.5, even more preferably in the range of 3.8 to 5.0, and even more preferably in the range of 3.8 to 4.5. Examples of acidic buffer solutions that can be used include acetate buffers and citrate buffers.

[0093] When the pH of the nucleic acid-containing aqueous solution is acidic, the lipid nanoparticles formed in the channel structure are preferably dialyzed with a buffer solution of neutral pH (pH 6.8 to 7.6) such as PBS, which can be administered to living organisms, in the dialysis step, thereby obtaining lipid nanoparticles that can be administered relatively safely to animals.

[0094] The nucleic acid-containing aqueous solution is preferably adjusted so that the sodium chloride (NaCl) concentration is 280 mM or higher. Generally, when a channel structure such as that shown in FIG. 1 is used, the particle diameter of the lipid nanoparticles formed is 100 nm or less, with most of them being around 20 nm. However, when the salt concentration of the nucleic acid-containing aqueous solution is high, the charge repulsion between nucleic acids is alleviated and hydration is inhibited, resulting in destabilization of the interface and an increase in the particle diameter of the lipid nanoparticles formed. Then, by dialyzing these lipid nanoparticles with an increased particle size against a buffer solution exhibiting a neutral pH, the lipid nanoparticles fuse together, resulting in large lipid nanoparticles with a number-average particle diameter of 300 nm or greater. The NaCl concentration of the nucleic acid-containing aqueous solution is not particularly limited as long as it is 280 mM or higher, but is preferably 280 to 500 mM, more preferably 280 to 450 mM, and even more preferably 280 to 420 mM.

[0095] The flow channel structure of the present invention has a plurality of inlet channels that are independent of each other, and these inlet channels each have a certain length and merge to form a single dilution channel, and may have three inlet channels. When the flow channel structure of the present invention has three inlet channels, the first inlet channel, the second inlet channel, and the third inlet channel can each have a certain length and merge to form a single dilution channel so that the first fluid introduced from the first inlet channel comes into contact with the third fluid introduced from the third inlet channel before merging with the second fluid introduced from the second inlet channel. When producing lipid nanoparticles loaded with nucleic acid, a lipid solution in which lipid components are dissolved in ethanol is introduced from the first inlet channel, a nucleic acid-containing aqueous solution is introduced from the second inlet channel, and the aqueous solvent used to prepare the nucleic acid-containing aqueous solution is introduced from the third inlet channel.

[0096] The aqueous dispersion of lipid nanoparticles, which has been dialyzed as necessary, can be stored in a dried state until use. When the aqueous dispersion of lipid nanoparticles is freeze-dried or spray-dried, stability may be improved by using a sugar (aqueous solution) such as monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose, disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose, trisaccharides such as raffinose and melezinose, polysaccharides such as cyclodextrin, and sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, and maltitol. In addition, when freezing the aqueous dispersion, stability may be improved by using a polyhydric alcohol (aqueous solution) such as the above-mentioned sugars, glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, or 1,3-butylene glycol.

[0097] The lipid nanoparticles of the present invention can be more easily produced by providing a kit containing various reagents and the like used in the production of the lipid nanoparticles. For example, when producing lipid nanoparticles loaded with nucleic acid using the channel structure, a kit for producing lipid nanoparticles preferably includes a dried lipid composition containing all the lipid components constituting the lipid nanoparticles and a dried product containing NaCl and intended for dissolving in water to prepare an aqueous solution having a NaCl concentration of 280 mM or higher. The lipid solution to be introduced into the first channel of the channel structure shown in FIG. 1 can be easily prepared by dissolving the dried lipid composition containing all the lipid components constituting the lipid nanoparticles in ethanol. Furthermore, the nucleic acid-containing aqueous solution to be introduced into the second channel of the channel structure shown in FIG. 1 can be easily prepared by mixing the dried product to be dissolved in water to prepare an aqueous solution having a NaCl concentration of 280 mM or higher, water, and the nucleic acid to be loaded onto the lipid nanoparticles. The kit preferably further contains one or more selected from the group consisting of ethanol, phosphate saline, and nucleic acid.

[0098] When lipid nanoparticles according to the present invention encapsulating a gene expression vector are administered to an animal, the gene expression vector encapsulated in the lipid nanoparticles is expressed with high efficiency in splenic dendritic cells. Similarly, when lipid nanoparticles according to the present invention encapsulating mRNA are administered to an animal, the mRNA encapsulated in the lipid nanoparticles is introduced into splenic dendritic cells and translated, resulting in highly efficient expression of the peptides encoded by the mRNA. Furthermore, when lipid nanoparticles according to the present invention encapsulating an siRNA expression vector are administered to an animal, the siRNA expression vector encapsulated in the lipid nanoparticles is expressed with high efficiency in splenic dendritic cells, suppressing the expression of the gene targeted by the expression vector.

[0099] Due to this highly selective gene expression activity in splenic dendritic cells, the lipid nanoparticles of the present invention function as gene expression carriers targeting splenic dendritic cells. By encapsulating a foreign gene or mRNA to be expressed in splenic dendritic cells in the lipid nanoparticles of the present invention and then administering them to a subject animal, the foreign gene and mRNA are expressed in the splenic dendritic cells of the subject animal. Therefore, the lipid nanoparticles of the present invention are useful as an active ingredient in pharmaceutical compositions used in gene therapy, for example, as an active ingredient in pharmaceutical compositions used in gene therapy targeting splenic dendritic cells.

[0100] In addition, the lipid nanoparticles according to the present invention are particularly useful as a nucleic acid vaccine loaded with a nucleic acid encoding an antigen. The nucleic acid vaccine may be a DNA vaccine, but is preferably an mRNA vaccine. Since mRNA vaccines are sufficient for introduction into the cytoplasm, they have higher versatility than DNA vaccines that require introduction into the cell nucleus. When the lipid nanoparticles according to the present invention are mRNA vaccines, when the lipid nanoparticles are administered to an animal, the mRNA loaded on the lipid nanoparticles is translated in the spleen dendritic cells of the animal, and an antigen protein is expressed. The immune system is activated by the expressed antigen protein. In addition, since mRNA itself has strong immunogenicity, mRNA vaccines can be expected to acquire immunity against antigen proteins without using an immunostimulant such as an adjuvant in combination. That is, the lipid nanoparticles according to the present invention are useful as vaccines used in cancer vaccine therapy and infectious disease treatment.

[0101] The animal to which the lipid nanoparticles according to the present invention are administered is not particularly limited and may be a human or an animal other than a human. Examples of non-human animals include mammals such as cows, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters, guinea pigs, and birds such as chickens, quails, and ducks. In addition, the administration route when administering the lipid nanoparticles according to the present invention to an animal is not particularly limited, but parenteral administration such as intravenous administration, enteral administration, intramuscular administration, subcutaneous administration, transdermal administration, nasal administration, and pulmonary administration is preferred.

Examples

[0102] Next, examples will be shown to explain the present invention in more detail, but the present invention is not limited to the following examples.

[0103] <Production of lipid nanoparticles loaded with RNA> In the following experiments, lipid nanoparticles loaded with nucleic acids were produced using a lipid nanoparticle production apparatus (「iLiNP」, manufactured by Lylac Pharma) including a flow channel structure as shown in FIG. 1.

[0104] The nucleic acid-containing aqueous solution was prepared by dissolving the nucleic acid to be loaded onto lipid nanoparticles at 0.142 mg RNA / mL in acetate buffer (25 mM, pH 4.0) with NaCl added as needed. The nucleic acid loaded onto lipid nanoparticles was a mixture of mRNA encoding nanoluciferase (NLuc) (NLuc-mRNA) and siRNA targeting human polo-like kinase 1 (hPLK1) (double-stranded RNA:hPLK1-siRNA) with no mouse homology (NLuc-mRNA:hPLK1-siRNA) at a weight ratio of 1:19, unless otherwise noted. The hPLK1-siRNA used was a 2'-OMe-modified form that has been shown not to induce type I IFN responses. The reason for loading the siRNA mixture onto lipid nanoparticles, rather than mRNA alone, was to increase the amount of lipid nanoparticles administered to animals without changing the mRNA dose, thereby improving the sensitivity of the lipid nanoparticle uptake efficiency measurement.

[0105] The lipid solution was prepared by dissolving all lipid components constituting the lipid nanoparticles and the lipophilic fluorescent dye DiO in ethanol. DiO was added in an amount of 0.1 mol% based on the total lipid components. By adding DiO to the lipid solution, lipid nanoparticles modified with DiO could be produced.

[0106] An appropriate amount of nucleic acid-containing aqueous solution was dispensed into a 2.5 mL syringe, and an appropriate amount of lipid solution was dispensed into a 1 mL syringe. Each was connected to the lipid nanoparticle production device via a syringe pump, and the acid-containing aqueous solution and lipid solution were introduced into the lipid nanoparticle production device at a flow rate ratio of 3:1 (N / P ratio = 7.4 in the case of the A-11 composition below) and a total flow rate of 500 μL / min.

[0107] The solution discharged from the dilution channel of the lipid nanoparticle production device was transferred to a dialysis membrane (Spectra / Por 4 dialysis membrane, MWCO: 12,000-14,000, Spectrum Laboratories), and the external aqueous phase was replaced with MES buffer (20 mM, pH 6.0) and dialyzed at 4°C for at least 2 hours. The external aqueous phase was then replaced with PBS(-) and dialyzed again at 4°C for at least 2 hours to remove ethanol and exchange the aqueous solvent inside the lipid nanoparticles for PBS. The solution recovered after dialysis was used as the lipid nanoparticle solution (LNP solution).

[0108] <Measurement of mean particle size and zeta potential of lipid nanoparticles> The mean particle size (number average, ζ average) of lipid nanoparticles in PBS(-) and the zeta potential in 10 mM HEPES buffer (pH 7.4) were measured using the dynamic light scattering analyzer "Zetasizer Nano ZS ZEN3600" (Malvern).

[0109] <RNA encapsulation rate in lipid nanoparticles> The RNA encapsulation rate of lipid nanoparticles was quantified by an assay (Ribogreen Assay) using "Quant-iT® RiboGreen® RNA" (Thermo Fisher Scientific), which selectively intercalates with RNA and emits fluorescence.

[0110] A solution (Triton(+)) was prepared by mixing 978.8 μL, 20 μL, and 1.25 μL of HEPES buffer (10 mM, pH 7.4), 10% (w / v) Triton X-100, and RiboGreen, respectively. Another solution (Triton(-)) was prepared by mixing 998.8 μL and 1.25 μL of HEPES buffer (10 mM, pH 7.4) and RiboGreen, respectively. Also, as a calibration curve sample, RNA was diluted with HEPES buffer (10 mM, pH 7.4) to 500 ng RNA / mL to prepare other RNA solutions, and the RNA solutions were appropriately diluted with HEPES buffer to prepare an appropriate dilution series. The LNP solution was diluted with HEPES buffer (10 mM, pH 7.4) to 500 ng / mL.

[0111] The calibration curve sample and the diluted LNP solution were each added to a black 96-well plate for fluorescence measurement at 100 μL / well, and further 100 μL / well of a solution containing RiboGreen (Triton(+) or Triton(-)) was added. After shaking the plate in a shaking incubator ("SI-300", manufactured by AS ONE) at 700 rpm for 30 seconds, it was placed in a microplate reader ("Enspire 2300 Multilabel Reader", manufactured by Perkin Elmer), and the fluorescence intensity of the solution in each well was measured (Excitation / Emission: 500 / 525 nm, Band width: 5 nm, Measurement time: 500 msec / well, Dynamic range: Autorange, Temperature: r.t.). From the obtained fluorescence intensity, the encapsulation efficiency of RNA was calculated according to the following formula. The calibration curve and each LNP sample were measured in duplicate.

[0112] [RNA encapsulation efficiency (%)] = ([RNA concentration in Triton(+)] - [RNA concentration in Triton(-)]) / [RNA concentration in Triton(+)] × 100

[0113] <Preparation of NLuc-mRNA> NLuc-mRNA was prepared by in vitro transcription from pDNA (pUC57-amp-T7-NLuc-IRES-RFP) containing the NLuc gene. First, pDNA (pUC57-amp-T7-NLuc-IRES-RFP) was digested with the restriction enzyme EcoRV. DNA was purified from the digest using the phenol-chloroform method and then dissolved in sterile water to obtain a 1 μg / μL solution of linearized pDNA. This linearized pDNA was then used as a template for transcription and polyadenylation reactions. These reactions were performed using a commercially available kit (mMESSAGE mMACHINETM T7 Ultra Transcription Kit, Invitrogen) according to the protocol provided with the kit. Using this kit, mRNA was synthesized in vitro with a cap analog (m7G nucleotide) added to the 5' end, replacing the 3' OH group with an OCH3 group, and polyadenylation added to the 3' end. The mRNA obtained after the reaction was purified with phenol-chloroform, then precipitated with isopropanol, air-dried, and redissolved in sterile water. The redissolved RNA solution was purified using a commercially available RNA concentration and purification kit. The RNA obtained by in vitro synthesis was subjected to agarose gel electrophoresis, and it was confirmed that the 5'-end cap structure and the 3'-end poly(A) were added.

[0114] <Measurement of Nluc expression levels in the liver and spleen> c57BL / 6N mice were euthanized after tail vein administration of RNA-loaded, DiO-labeled lipid nanoparticles, and their livers and spleens were collected. The livers and spleens were weighed, frozen, and stored at -80°C. A portion of the spleen was removed before freezing for use in splenic immune cell analysis, and the remainder was frozen and stored.

[0115] 1 mL of 1x Passive Lysis Buffer was added to sample tubes containing frozen tissue and zirconia beads (1.4 mm diameter), and the tissue was disrupted using a bead-type cell disrupter at 5,000 rpm at 4°C for 40 seconds. The resulting disrupted tissue was centrifuged at 13,000 rpm at 4°C for 10 minutes, and the supernatant was collected and used as the measurement sample.

[0116] Nluc expression levels were measured using a commercially available luciferase assay system ("Nano-Glo® Luciferase Assay System," manufactured by Promega). First, Nano-Glo Luciferase Assay Substrate and Nano-Glo Luciferase Assay Buffer were mixed at a ratio of 50:1 to obtain Nano-Glo Luciferase Assay Reagent. 25 μL of the measurement sample was mixed with 25 μL of Nano-Glo Luciferase Assay Reagent and incubated for 3 minutes. The luminescence intensity of each sample was then measured using a luminometer ("Luminescencer-PSN AB-2200," manufactured by ATTO). The protein content of each organ relative to the luciferase luminescence intensity was corrected using a BCA (bicinchoninic acid) assay. The BCA assay was performed using a commercially available BCA assay kit (Pierce BCA protein assay, manufactured by Pierce) using BSA (bovine serum albumin) as the protein for preparing a calibration curve.

[0117] <Measurement of intracellular uptake of lipid nanoparticles and Nluc expression levels in splenic immune cells> After a portion of the spleen was minced with scissors, an appropriate amount of Hank's balanced salt solution (-) (HBSS(-)) was added to loosen the cells from the spleen. The resulting suspension was passed through a 40 μm cell strainer and centrifuged at 400 g for 5 minutes at 4°C. After removing the supernatant, the pellet was resuspended in 1 mL of RBC lysis buffer and incubated at room temperature for 3 minutes. The suspension was then diluted 10-fold with HBSS(-) and centrifuged again at 400 g for 5 minutes at 4°C, after which the supernatant was removed. The resulting pellet was resuspended in an appropriate amount of HBSS(-), the number of cells was counted, and the appropriate number of cells was aliquoted into 1.5 mL tubes and centrifuged at 400 g for 5 minutes at 4°C. After removing the supernatant, the pellet was blocked by adding 100 μL / 1.0 × 10 purified anti-mouse CD16 / 32 antibody (prepared at 10 μg / mL in FACS buffer) to the pellet. 6The mixture was added so that the total number of cells was 100, and incubated at 4°C for 10 minutes while tapping every 5 minutes.

[0118] After blocking, the cells were stained with PE-labeled anti-mouse F4 / 80 antibody, APC-labeled anti-mouse CD11c antibody, PerCP / Cyanine5.5-labeled anti-mouse IA / IE antibody, and PE / Cy7-labeled anti-mouse CD19 antibody, and incubated at 4°C for 30 minutes with tapping every 5 minutes. After incubation, the cells were diluted with FACS buffer to 1000 μL per tube and centrifuged at 4°C, 400 g, and 5 minutes. The cells were then washed twice with 1 mL of FACS buffer and used as samples for FACS analysis.

[0119] FACS was performed using a cell sorter ("SH800", Sony Corporation). 6 μL / 1 × 10 propidium iodide (PI) solution was added to the sample. 6 Approximately 15 minutes later, dendritic cells (DC cells), macrophages (Mφ), and B cells were sorted using a cell sorter into 1.5 mL tubes containing 20 μL of 2× Passive Lysis Buffer, each containing 3,000 cells (approximately 20 μL). Based on the expression patterns of cell surface antigens, IA / IE-positive and CD11c-positive cells were sorted as dendritic cells, F4 / 80-positive cells were sorted as macrophages, and CD19-positive cells were sorted as B cells.

[0120] Fluorescence compensation for the cell sorter was performed using a commercially available fluorescence compensation kit (VersaComp Antibody Capture Bead Kit, manufactured by Beckman Coulter). Beads for compensating for fluorescence spillover into the cell sorter were prepared according to the protocol attached to the kit.

[0121] Dendritic cells, B cells, and macrophages fractionated using a cell sorter were measured for the amount of lipid nanoparticle uptake and the amount of Nluc expression in each cell. The amount of lipid nanoparticle uptake was measured as the average value of the DiO fluorescence intensity in each cell. The amount of Nluc expression in each cell was measured as described above using a commercially available luciferase assay system ("Nano-Glo® Luciferase Assay System," manufactured by Promega).

[0122] <Creation of tumor-bearing mice> Tumor-bearing mice were generated by subcutaneously implanting E.G7-OVA cells, a cultured cell line derived from OVA-expressing murine malignant lymphoma, into mice. E.G7-OVA cells were cultured in RPMI-1640 medium at 37°C in a 5% CO2 environment. The number of viable cells was measured using trypan blue staining. E.G7-OVA cells 2 × 10 7 The cell suspension was suspended in cold D-PBS(-) to a concentration of 8 × 10 cells / mL. The cells were stored at 4°C and then injected into a 26G syringe using 8 × 10 5 The cells were subcutaneously transplanted into shaved c57BL / 6N mice (6-week-old, female, provided by slc) at a volume of 40 μL / mouse. The entire process from preparation of the cell suspension to subcutaneous transplantation was completed within 2 hours.

[0123] <Measurement of tumor volume> The tumor volume of the tumor-bearing mice was calculated using a digital caliper every three days from day 6 after E.G7-OVA cell inoculation according to the following formula.

[0124] [Tumor volume (mm 3 )]=[Major diameter (mm 3 )]×[minor diameter (mm 3 )]×[minor diameter (mm 3 )] × 0.52

[0125] Tumor volume 4000mm 3 was the endpoint, and mice beyond this were euthanized by cervical dislocation.

[0126] [Example 1] A definitive screening plan was conducted to clarify the extent of the main effect of each factor during the preparation of nucleic acid-containing lipid nanoparticles on the expression efficiency in splenic dendritic cells. The factors to be investigated were the type and content of pH-sensitive cationic lipid, the content of phospholipid, the content of PEG-lipid, and the NaCl concentration of the nucleic acid-containing aqueous solution, and each level was set.

[0127] The lipid components constituting the lipid nanoparticles were pH-sensitive cationic lipid (CL), phospholipid (PL), cholesterol (chol), and PEG lipid. CL4H6 or CL7H6 was used as the pH-sensitive cationic lipid, and DOPE was used as the phospholipid. PEG lipids were either PEG2k-DMG, which rapidly desorbs in the blood, or PEG2k-DSG, which is less likely to desorb and contributes to blood retention. CL4H6 and CL7H6 were synthesized using the method described in Patent Document 1. Table 1 shows the pH-sensitive cationic lipid content (mol%), phospholipid content (mol%), and PEG lipid content (mol%) relative to the total lipid components for each test group. The cholesterol content (mol%) is the remainder obtained by subtracting the pH-sensitive cationic lipid, phospholipid, and PEG lipid content from 100 mol%.

[0128] [ka]

[0129] The experimental plan was created based on the data analysis software "JMP (registered trademark)" (SAS), and a total of 324 experiments (3 4 ×2 2), 14 types of compositions (A-1 to A-14) were selected. Next, RNA-loaded lipid nanoparticles were prepared using a nucleic acid-containing solution prepared with the lipid composition shown in Table 1 and the NaCl concentration shown in Table 1. The ζ-average particle size (nm), number-average particle size (nm), PdI, and RNA encapsulation rate (%) of each prepared lipid nanoparticle were measured. The lipid nanoparticle samples in each test group were measured in duplicate (n=2). The measurement results are shown in Table 2.

[0130] [Table 1]

[0131] [Table 2]

[0132] As shown in Table 2, lipid nanoparticles with a ζ-average particle size ranging from approximately 80 to 750 nm were prepared. Furthermore, the PdI, an index of lipid nanoparticle uniformity, was 0.2 or less for most lipid nanoparticles, and the RNA encapsulation rate was 70% or higher for all lipid nanoparticles. Statistical analysis was performed to examine the effect of each factor on the ζ-average particle size. It was found that the DOPE ratio, PEG-lipid ratio, and NaCl concentration during lipid nanoparticle preparation had a statistically significant effect on the ζ-average particle size (Table 3, Figure 2). More specifically, it was found that decreasing the DOPE ratio, decreasing the PEG-lipid ratio, and increasing the NaCl concentration during lipid nanoparticle preparation increased the ζ-average particle size of lipid nanoparticles.

[0133] [Table 3]

[0134] Furthermore, it was shown that the proportion of PEG lipid and the NaCl concentration during lipid nanoparticle preparation statistically significantly affected the number-average particle size. More specifically, it was found that decreasing the proportion of PEG lipid and increasing the NaCl concentration during lipid nanoparticle preparation increased the number-average particle size of lipid nanoparticles. Furthermore, it was shown that the proportion of pH-sensitive cationic lipid, the proportion of DOPE, and the type of pH-sensitive cationic lipid statistically significantly affected the RNA encapsulation rate. More specifically, it was found that increasing the proportion of pH-sensitive cationic lipid, increasing the proportion of DOPE, and using CL7H6 as the pH-sensitive cationic lipid increased the RNA encapsulation rate of lipid nanoparticles.

[0135] The lipid nanoparticles prepared for each test group were administered at 1.0 mg RNA / kg via the tail vein to c57BL / 6N mice (6-week-old, female, supplied by SLC). 24 hours after administration, the mice were euthanized and Nluc expression levels in the liver and spleen were measured. Furthermore, the intracellular uptake of lipid nanoparticles and Nluc expression levels in dendritic cells, B cells, and macrophages in the collected spleens were measured.

[0136] The results of measuring the Nluc expression level per gram of protein in the liver and spleen of mice administered lipid nanoparticles A-1 to A-14 are shown in Figure 3. Furthermore, the results of measuring the amount of lipid nanoparticle uptake (average value of DiO fluorescence intensity in each cell) in splenic immune cells of mice administered lipid nanoparticles A-1 to A-14 are shown in Figure 4, and the results of measuring the Nluc expression level are shown in Figure 5.

[0137] The Nluc gene expression level in dendritic cells was highest with lipid nanoparticles A-6, A-10, A-11, A-13, and A-14, with A-11 showing the highest gene expression (Figure 5). Meanwhile, the uptake of lipid nanoparticles into cells was greater in macrophages than in dendritic cells, regardless of the lipid nanoparticles used (Figure 4). These results confirmed that the ratio of gene expression to the amount of lipid nanoparticles taken up into cells was higher in dendritic cells than in macrophages. Furthermore, lipid nanoparticles were hardly taken up by B cells (Figure 4), and no gene expression was observed (Figure 5).

[0138] The amount of Nluc expression in the whole spleen was highest for A-11 lipid nanoparticles, followed by A-2 and A-7 lipid nanoparticles (Figure 3). Since A-2 and A-7 lipid nanoparticles showed only slight gene expression in dendritic cells, it was inferred that most of these lipid nanoparticles were taken up by spleen cells other than dendritic cells, such as macrophages. These results indicate that high gene expression in the whole spleen does not necessarily mean that gene expression in dendritic cells will also be high.

[0139] Statistical analysis showed that the pH-sensitive cationic lipid ratio, DOPE ratio, pH-sensitive cationic lipid type, and PEG lipid type all significantly affected Nluc expression in the spleen. Furthermore, lipid nanoparticles with a number-average particle size of 100 nm or greater demonstrated statistically significant higher gene expression in dendritic cells compared with lipid nanoparticles with a number-average particle size of less than 100 nm. Regarding gene expression in splenic dendritic cells (Figure 4), the frequency of occurrence of each level of PEG amount, NaCl concentration, and pH-sensitive cationic lipid type in the lipid nanoparticles in the top five and bottom five test groups suggested that a lower PEG amount, a higher NaCl concentration, and the use of CL4H6 as the pH-sensitive cationic lipid were suitable for enhancing gene expression efficiency in splenic dendritic cells.

[0140] [Example 2] The lipid composition of lipid nanoparticles was reexamined using Taguchi design (partial implementation design), which allows narrowing down the composition by evenly allocating factor values ​​to a set region.

[0141] Based on the results of Example 1, and considering that particle size contributes to gene expression in dendritic cells, the NaCl concentration of the nucleic acid-containing aqueous solution during lipid nanoparticle preparation was set to 300 mM so that the average particle size would be 100 nm or more. Furthermore, considering the main effect of influencing increased gene expression in the spleen, CL4H6 was used as the pH-sensitive cationic lipid. Other factors to be investigated included the CL4H6 content, phospholipid (DOPE) content, type of PEG lipid, and PEG lipid content, and each level was set.

[0142] The experimental plan was created based on the data analysis software "JMP", and a total of 16 (2 4 ), eight compositions (B-1 to B-8) were selected (Table 4). Next, RNA-loaded lipid nanoparticles were prepared using a nucleic acid-containing solution prepared with a NaCl concentration of 300 mM and the lipid composition shown in Table 4. The ζ-average particle size (nm), number-average particle size (nm), PdI, and RNA encapsulation rate (%) of each prepared lipid nanoparticle were measured. The lipid nanoparticle samples in each test group were measured in duplicate (n=2). The measurement results are shown in Table 5.

[0143] [Table 4]

[0144] [Table 5]

[0145] As shown in Table 5, the ζ-average particle size of the lipid nanoparticles in all test groups was in the range of approximately 110 to 420 nm. Furthermore, the PdI and RNA encapsulation rate of all lipid nanoparticles were 0.15 or less and 80% or more, respectively. The results of statistical analysis indicated that the proportion of PEG lipid statistically significantly affected the number-average particle size, and that the proportion of DOPE statistically significantly affected the RNA encapsulation rate. The results of statistical analysis regarding the physical properties of the lipid nanoparticles were generally similar to those of Example 1.

[0146] Next, the lipid nanoparticles of test groups B-1 to B-8 were administered via the tail vein to c57BL / 6N mice, and the Nluc expression levels in the liver and spleen, and the intracellular uptake of lipid nanoparticles and Nluc expression levels in dendritic cells, B cells, and macrophages in the spleen were measured in the same manner as in Example 1. The measurement results for the amount of lipid nanoparticle uptake (average value of DiO fluorescence intensity for each cell) in splenic immune cells of mice administered lipid nanoparticles of B-1 to B-8 are shown in Figure 6, and the measurement results for Nluc expression levels are shown in Figure 7.

[0147] The Nluc gene expression level in dendritic cells was high for lipid nanoparticles B-1, B-3, B-5, and B-7, with B-5 lipid nanoparticles showing the highest gene expression (Figure 7). Meanwhile, the uptake of lipid nanoparticles into cells was higher in macrophages than in dendritic cells, regardless of the lipid nanoparticles used in the test group (Figure 6). These results confirmed that the ratio of gene expression to the amount of lipid nanoparticles taken up into cells was higher in dendritic cells than in macrophages.

[0148] Statistical analysis confirmed a high correlation between the gene expression level in dendritic cells and the particle size of lipid nanoparticles, and this correlation between the two was higher than the experimental results of Example 1. This is presumably because, compared to Example 1, the composition of the lipid nanoparticles was examined within a narrower range in this Example, which resulted in a relatively higher contribution of particle size to gene expression in dendritic cells.

[0149] Figure 8 shows the results of Nluc gene expression levels in splenic dendritic cells for the lipid nanoparticles of test groups A-1 to A-14 and the lipid nanoparticles of test groups B-1 to B-8 in Example 1. Of these lipid nanoparticles, the lipid nanoparticles of A-11 showed the highest Nluc gene expression levels. Furthermore, a high correlation was observed between the particle size of the lipid nanoparticles, their cellular uptake in dendritic cells, and their gene expression in dendritic cells. Therefore, the increase in gene expression in dendritic cells with an increase in particle size of the lipid nanoparticles was thought to be due to an increase in cellular uptake. Furthermore, as in Example 1, a certain correlation was observed between gene expression in the spleen and gene expression in dendritic cells, but it was also shown that lipid nanoparticles that showed high gene expression in the spleen did not necessarily show high gene expression in dendritic cells.

[0150] Furthermore, for these lipid nanoparticles, the relationship between the amount of lipid nanoparticle uptake into dendritic cells (mean DiO fluorescence intensity) and the expression level of IA / IE, a dendritic cell maturation marker, is shown in Figure 9(A), and the relationship between the expression level of Nluc gene in dendritic cells and the expression level of IA / IE is shown in Figure 9(B). As a result, the uptake of lipid nanoparticles into dendritic cells showed a high correlation with the expression level of IA / IE (Figure 9(A)). These results revealed that A-11 lipid nanoparticles are efficiently taken up by splenic dendritic cells, thereby inducing dendritic cell maturation and simultaneously achieving high gene expression. The lipid nanoparticles of the present invention not only efficiently induce gene expression in dendritic cells, but also induce dendritic cell maturation, and are therefore expected to demonstrate high performance as mRNA vaccines.

[0151] [Example 3] In Examples 1 and 2, the reproducibility of A-11 lipid nanoparticles, which showed the highest gene expression in splenic dendritic cells, and the effect of the PEG lipid content were investigated. Specifically, RNA-loaded lipid nanoparticles were prepared using the lipid composition shown in Table 6 in the same manner as in A-11 of Example 1.

[0152] [Table 6]

[0153] The prepared lipid nanoparticles were administered via the tail vein of c57BL / 6N mice in the same manner as in Example 1, and the amount of lipid nanoparticles taken up into cells and the amount of Nluc expression in splenic dendritic cells were measured (n = 2-3). The measurement results of the amount of lipid nanoparticles taken up (average value of DiO fluorescence intensity for each cell) are shown in Figure 10(A), and the measurement results of the amount of Nluc expression are shown in Figure 10(B). As shown in Figure 10, both the amount of lipid nanoparticles taken up into cells and the amount of Nluc expression in splenic dendritic cells decreased depending on the PEG lipid content.

[0154] The prepared lipid nanoparticles were stored at 4°C for 3 weeks, and their physical properties were measured over time. The results are shown in Figure 11. Figure 11(A) shows the measurement results for the ζ-average particle size (nm) of each lipid nanoparticle, and Figure 11(B) shows the measurement results for the RNA encapsulation rate (%) of each lipid nanoparticle. As a result, regardless of the amount of PEG lipid, the RNA encapsulation rate was similar for all lipid nanoparticles, but the ζ-average particle size decreased depending on the amount of PEG lipid. No particular changes in the physical properties were observed for any lipid nanoparticles after storage at 4°C for approximately 3 weeks, confirming their high storage stability.

[0155] [Example 4] The lipid nanoparticles of test group A-11 in Examples 1 and 3 are expected to be applicable as an mRNA vaccine capable of inducing efficient gene expression and maturation in splenic dendritic cells. Therefore, antitumor activity was evaluated using lipid nanoparticles of A-11 loaded with mRNA encoding a tumor-associated antigen.

[0156] Specifically, first, using pDNA containing the ovalbumin (OVA) gene as a template, mRNA encoding OVA (OVA-mRNA) with a cap structure at the 5' end and a poly(A) at the 3' end was prepared by in vitro transcription in the same manner as for NLuc-mRNA. Next, OVA-mRNA-loaded lipid nanoparticles (mOVA-A11-LNP) were prepared in the same manner as in Test Group A-11 in Example 1, except that OVA-mRNA was used instead of NLuc-mRNA.

[0157] The preventive antitumor activity of mOVA-A11-LNP was evaluated as follows. First, mice were intravenously administered mOVA-A11-LNP twice weekly at doses of 0.25 mg (0.25 mg mRNA / kg), 0.5 mg mRNA / kg, or 0.75 mg mRNA / kg (equivalent to the amount of OVA-mRNA per kg of mouse body weight) (n = 3-5 per administration group). Control mice were administered an equal volume of PBS instead of mOVA-A11-LNP. One week later, E.G7-OVA cells were subcutaneously implanted into the right flank, and tumor volume was measured every three days over time. The results of tumor volume measurements are shown in Figure 12. As a result, tumor engraftment was completely prevented in the mOVA-A11-LNP-administered groups at all doses above 0.25 mg mRNA / kg.

[0158] The therapeutic antitumor activity of mOVA-A11-LNP was evaluated as follows. First, E.G7-OVA cells were subcutaneously implanted into the right flank of mice, and tumor volume was measured over time every three days. On days 7, 10, and 14 after implantation, mOVA-A11-LNP was intravenously administered at 0.125 mg mRNA / kg, 0.25 mg mRNA / kg, or 0.5 mg mRNA / kg (n = 3–5 per treatment group). Control mice were administered an equal volume of PBS instead of mOVA-A11-LNP. The results of tumor volume measurements are shown in Figure 13. In the figure, "**" indicates p < 0.01 by nrANOVA followed by SNK test. As a result, a decrease in tumor volume was observed in the mOVA-A11-LNP-treated groups at all doses above 0.125 mg mRNA / kg. Of the 12 mice in the mOVA-A11-LNP administration group, the tumor completely disappeared in one of four mice in the 0.25 mg mRNA / kg administration group and one of three mice in the 0.5 mg mRNA / kg administration group.

[0159] Two mice that had completely disappeared and achieved remission were subcutaneously implanted with E.G7-OVA cells 68 days after the subcutaneous implantation. On days 7 and 10 after implantation, they received the same dose of mOVA-A11-LNP as the first. Figure 14 shows tumor volume measurements for the control PBS-treated group and two mice that achieved remission after the first subcutaneous implantation. As a result, even after reimplantation of E.G7-OVA cells, tumor engraftment was completely suppressed by mOVA-A11-LNP. This is thought to be due to the persistence of memory T cells that had experienced antigen stimulation by OVA expressed in splenic dendritic cells by mOVA-A11-LNP in the mice that received mOVA-A11-LNP, which maintained antitumor activity.

[0160] Furthermore, because no dose-dependent antitumor activity of mOVA-A11-LNP was observed, it was likely that a single dose of 0.125 mg mRNA / kg was saturated for evaluating antitumor activity. Therefore, we investigated the appropriate dose for evaluating the therapeutic antitumor activity of mOVA-A11-LNP. Specifically, mOVA-A11-LNP was administered via the tail vein at single doses of 0.05 mg mRNA / kg, 0.015 mg mRNA / kg, 0.005 mg mRNA / kg, or 0.0015 mg mRNA / kg on days 8 and 11 after subcutaneous implantation of E.G7-OVA cells. The tumor volume measurements for each administration group are shown in Figure 15. As a result, mOVA-A11-LNP exhibited therapeutic antitumor activity at doses of 0.015 mg mRNA / kg or higher.

[0161] [Example 5] To examine the usefulness of the lipid nanoparticles in Test Group A-11 of Example 1, gene expression was compared with two previously reported mRNA delivery systems. One of the two mRNA delivery systems used for comparison was RNA-LPX (Non-Patent Documents 2 and 3, manufactured by BioNTech), which is currently undergoing phase I clinical trials as an mRNA cancer vaccine for melanoma and other diseases. The other was MC3-LNP, an equivalent formulation of Onpattro (manufactured by Alnylam), a lipid nanoparticle for RNA delivery that also has a proven clinical track record. MC3-LNP also demonstrates a certain level of efficiency as an mRNA delivery system, and several examples of its use as a comparative formulation have been reported (Non-Patent Document 7).

[0162] RNA-LPX carrying NLuc-mRNA (mNLuc-RNA-LPX) was prepared as follows. First, DOTMA / DOPE (1:1 molar ratio, total volume 400 μL) dissolved in ethanol to a total lipid concentration of 10 mM was added to a glass test tube, and the solvent was removed using an evaporator. Next, 800 μL of D-PBS(-) was added to the test tube and hydrated for approximately 2 minutes. After stirring with a vortex mixer, the mixture was allowed to stand for approximately 5 minutes. The test tube was sonicated (approximately 30 seconds) using a bath sonicator, and the prepared liposome solution was diluted 5.92-fold with D-PBS(-) (total lipid concentration 0.845 mM). 100 μL of the liposome solution was added to 100 μL of a solution diluted with D-PBS(-) to a concentration of 0.2 mg mRNA / mL while stirring with a vortex mixer (N / P = 1.3 / 2).

[0163] MC3-LNPs loaded with NLuc-mRNA (mNLuc-MC3-LNPs) were prepared in the same manner as in Test Group A-11, except that the lipid composition was MC3 / DSPC / Chol / PEG2k-DMG (50 / 10 / 40 / 1.5 mol%) and no NaCl was added to the nucleic acid-containing aqueous solution.

[0164] The prepared lipid nanoparticles were administered via the tail vein to mice as in Example 1, except that the dose was 0.5 mg mRNA / kg. 24 hours after administration, mice were euthanized, and the liver, spleen, lungs, kidneys, and inguinal lymph nodes were collected and Nluc expression levels were measured. The measurement results are shown in Figure 16. As a result, mNLuc-MC3-LNP showed the highest gene expression in the liver, followed by spleen, inguinal lymph nodes, kidneys, and lungs, with approximately 16-fold higher gene expression in the liver compared to the spleen. This was reasonable, as mNLuc-MC3-LNP is a formulation capable of efficient nucleic acid delivery to hepatic parenchymal cells. mNLuc-RNA-LPX showed the highest gene expression in the spleen, followed by inguinal lymph nodes, lungs, liver, and kidneys, with approximately 270-fold higher gene expression in the spleen compared to the inguinal lymph nodes. This was reasonable, as RNA-LPX is a formulation with extremely high spleen selectivity for gene expression. In contrast, A-11 lipid nanoparticles showed approximately 13-fold and 5-fold higher gene expression in the spleen and approximately 5-fold and 100-fold higher gene expression in the inguinal lymph nodes compared to mNLuc-MC3-LNP and mNLuc-RNA-LPX. These results demonstrate that A-11 lipid nanoparticles are more efficient gene expression carriers in lymphatic tissues than both clinically proven formulations.

[0165] Next, the efficiency of gene expression in splenic dendritic cells of each lipid nanoparticle was compared. First, using pDNA containing the fluorescent protein EGFP gene as a template, mRNA encoding OVA (EGFP-mRNA) with a cap structure at the 5' end and a poly(A) at the 3' end was prepared by in vitro transcription in the same manner as for NLuc-mRNA. Lipid nanoparticles loaded with EGFP-mRNA (mEGFP-A11-LNP) were prepared in the same manner as in experimental group A-11, except that EGFP-mRNA was loaded instead of NLuc-mRNA. In addition, RNA-LPX carrying EGFP-mRNA (mEGFP-RNA-LPX) was prepared in the same manner as mNLuc-RNA-LPX, except that EGFP-mRNA was used instead of NLuc-mRNA, and MC3-LNP carrying EGFP-mRNA (mEGFP-MC3-LNP) was prepared in the same manner as mNLuc-MC3-LNP.

[0166] The prepared lipid nanoparticles were administered to mice via the tail vein in the same manner as in Example 1, except that the dose was 0.5 mg mRNA / kg. 24 hours after administration, the mice were euthanized, their spleens were collected, and splenic dendritic cells were isolated by FACS. Figure 17(A) shows the percentage (%) of EGFP-positive cells relative to the total number of splenic dendritic cells, and Figure 17(B) shows the results of measuring the EGFP mean fluorescence intensity of splenic dendritic cells. The percentage of EGFP-positive cells in the mEGFP-A11-LNP-administered group was approximately 9%, which was approximately 5-fold and approximately 10-fold higher than the mEGFP-MC3-LNP-administered group and the mEGFP-RNA-LPX-administered group, respectively.

[0167] At the same time, the expression levels of IA / IE, a maturation marker for splenic dendritic cells, were measured in the same manner as in Example 1. The measurement results are shown in Figure 17(C). As a result, the mEGFP-A11-LNP-administered group showed significantly higher IA / IE expression levels. These results demonstrate that mEGFP-A11-LNP, compared to both clinically proven formulations, not only exhibits higher gene expression in dendritic cells but also can efficiently induce dendritic cell maturation.

[0168] [Example 6] The antitumor activities of mOVA-A11-LNP prepared in Example 4 were compared with those of RNA-LPX and MC3-LNP.

[0169] RNA-LPX carrying OVA-mRNA (mOVA-RNA-LPX) was prepared in the same manner as for the preparation of mNLuc-RNA-LPX, except that the OVA-mRNA used in Example 4 was used instead of NLuc-mRNA. Similarly, OVA-mRNA-loaded MC3-LNP (mOVA-MC3-LNP) was prepared in the same manner as in the preparation of mNLuc-MC3-LNP, except that OVA-mRNA was loaded instead of NLuc-mRNA.

[0170] Lipid nanoparticles loaded with OVA-mRNA (mOVA-B8-LNP) were also prepared in the same manner as in Test Group B-8 in Example 2, except that OVA-mRNA was loaded instead of NLuc-mRNA. In Example 2, the Nluc expression level of the lipid nanoparticles in Test Group B-8 was equivalent to that of the lipid nanoparticles in Test Group A-11 in the whole spleen, but was lower in splenic dendritic cells.

[0171] The therapeutic antitumor activities of mOVA-A11-LNP, mEGFP-A11-LNP, mOVA-B8-LNP, mOVA-RNA-LPX, and mOVA-MC3-LNP were evaluated using tumor-bearing mice subcutaneously implanted with E.G7-OVA cells. Specifically, E.G7-OVA cells were subcutaneously implanted into the right flank of mice, and tumor volume was measured every three days. Lipid nanoparticles were intravenously administered at 0.03 mg mRNA / kg on days 7 and 10 after implantation (n = 5 per treatment group). Control mice received an equal volume of PBS instead of lipid nanoparticles. The tumor volume measurements are shown in Figure 18. Only the mOVA-A11-LNP treatment group showed a decrease in tumor volume during the treatment period. Since no decrease in tumor volume was observed in the mOVA-RNA-LPX or mOVA-MC3-LNP treatment groups, mOVA-A11-LNP demonstrated superior therapeutic antitumor activity compared with both formulations. Furthermore, no decrease in tumor volume was observed in the mOVA-B8-LNP treatment group, suggesting that gene expression at the immune cell level, rather than overall spleen gene expression, may be an important indicator of antitumor activity. [Explanation of symbols]

[0172] 10...first inlet channel, 20...second inlet channel, 30...dilution channel, 31...junction, 40...structure, 50...bent channel portion.

Claims

1. The composition contains a pH-sensitive cationic lipid, a polyalkylene glycol-modified lipid, and a nucleic acid, The content ratio of the pH-sensitive cationic lipid to the total amount of lipids constituting the lipid nanoparticles is 40 to 70 mol%, The content ratio of the polyalkylene glycol-modified lipid to the total amount of lipids constituting the lipid nanoparticles is 0.5 to 1.75 mol%, The number average particle size is 150 nm or more, The pH-sensitive cationic lipid is represented by the following general formula (I-1): 【Chemistry 1】 [In formula (I-1), R 11 and R 12 each independently represent a linear C 10-14 alkyl group, a linear C 10-20 alkenyl group having one or two unsaturated bonds, or —CH(R 15 )(R 16 ) (R 15 and R 16 each independently represent a linear C 5-10 alkyl group); n1 and n2 each independently represent an integer of 6 to 10; p1 and q1 each represent an integer of 0 or greater that satisfies p1+q1=3 to 8; r1 represents 0 or 1; R 13 and R 14 each independently represent a linear C 1-3 alkyl group or an aryl C 1-3 alkyl group, or R 13 and R 14 are linked together to form a pyrrolidine ring, a piperidine ring, a morpholine ring, or a ring in which the nitrogen atom is a C 1-3 alkyl group] forming a piperazine ring optionally substituted with an alkyl group. is expressed as The nucleic acid is siRNA, mRNA, plasmid DNA, or a gene; Lipid nanoparticles.

2. Nanoparticles described in claim 1 for expressing a target component in splenic dendritic cells.

3. The lipid nanoparticles according to claim 1 or 2, having a number average particle diameter of 600 nm or less.

4. The lipid nanoparticle according to any one of claims 1 to 3, further comprising one or more selected from the group consisting of sterols and phospholipids.

5. A pharmaceutical composition comprising the lipid nanoparticles according to any one of claims 1 to 4 as an active ingredient.

6. The pharmaceutical composition according to claim 5, which is used in cancer vaccine therapy.

7. The pharmaceutical composition according to claim 5, which is used for immunostimulation.

8. A method for expressing a foreign gene, comprising administering the lipid nanoparticles according to any one of claims 1 to 7, which encapsulate a foreign gene to be expressed in splenic dendritic cells, to a subject animal (excluding humans), and expressing the foreign gene in the splenic dendritic cells of the subject animal.

9. A method for producing the lipid nanoparticles according to any one of claims 1 to 4 using a flow channel structure, A formation step of forming lipid nanoparticles in the flow channel structure from a lipid solution in which all lipid components constituting the lipid nanoparticles are dissolved in ethanol and an aqueous solution containing the nucleic acid; a dialysis step of dialysis the solution containing lipid nanoparticles obtained in the formation step in a buffer solution of pH 6.8 to 7.6; and the flow path structure includes a first inlet path for introducing a first fluid and a second inlet path for introducing a second fluid, which are independent of each other and have a certain length, and which join together to form a single dilution flow path; the dilution flow path has a flow path portion that is two-dimensionally curved in at least a part thereof, The bent flow path portion is defined as the axial direction or extension direction of the dilution flow path upstream from this portion as the X direction, and the width direction of the dilution flow path perpendicularly intersecting this X direction as the Y direction. The flow path width of the dilution flow path upstream from this portion is defined as the y direction. ０ In this case, the flow is alternately distributed from both side walls of the dilution flow passages facing each other in the Y direction toward the center of the flow passage in the approximately Y direction (approximately +Y direction, approximately −Y direction) by 1 / 2y ０ More than 1y ０ A constant height h less than １ , h ２ ... and has a constant width x in the X direction １ , x ２ ... and the structure that regulates the flow path width of the dilution flow path is １ , d ２ It is formed by at least two or more The lipid solution is introduced through the first inlet channel, and an aqueous solution containing the nucleic acid is introduced through the second inlet channel, A method for producing lipid nanoparticles, wherein the aqueous solution containing the nucleic acid has a sodium chloride concentration of 280 mM or more and is acidic.

10. The method for producing lipid nanoparticles according to claim 9, wherein the sodium chloride concentration of the aqueous solution containing the nucleic acid is 500 mM or less.

11. A kit for use in the method for producing lipid nanoparticles according to claim 9 or 10, A dried lipid composition containing all lipid components constituting the lipid nanoparticles, and A dried product containing sodium chloride and intended to be dissolved in water to prepare an aqueous solution having a sodium chloride concentration of 280 mM or more. A kit for producing lipid nanoparticles, comprising:

12. The kit for producing lipid nanoparticles according to claim 11, further comprising one or more selected from the group consisting of ethanol, phosphate saline, and nucleic acids.

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