Lipid nanoparticles for nucleic acid delivery

Abstract

To provide novel lipid nanoparticles that can more effectively exert the function of the encapsulated nucleic acids within cells compared to conventional lipid nanoparticles. [Solution] Lipid nanoparticles comprising nucleic acid, cKK-E12, DOPC, sterol, and DMG-PEG5000, wherein the molar ratio of cKK-E12 to the DOPC content is 0.2 to 6.0, and the content ratio of cKK-E12 to the total amount of constituent lipids in the lipid nanoparticles is 20 to 40 mol% and DOPC is 5 to 25 mol%.

Description

Technical Field

[0001] The present invention relates to lipid nanoparticles, and more particularly to lipid nanoparticles for nucleic acid delivery, containing a cationic lipid and DOPC in a specific ratio.

Background Art

[0002] In recent years, RNA has attracted attention as one of the modalities in gene therapy. RNA has a low risk of integration into the genome and is safer compared to DNA. In addition, once the sequence is determined, RNA is easy to design, can target any gene, or can express any protein in vivo. Therefore, RNA can lead to safe and versatile drug discovery.

[0003] RNAs such as mRNA and siRNA (small interfering RNA) have immunogenicity, are easily enzymatically degraded, and are not taken up by cells, so simply administering them in vivo cannot exert the desired therapeutic effect. Therefore, as a means for efficiently delivering RNA into target tissues or target cells, a means of using lipid nanoparticles (Lipid Nano Particle, LNP) as a carrier has been studied.

[0004] The LNP used in gene therapy and the like is a complex of a nucleic acid as an active ingredient and a lipid that protects the nucleic acid. Examples of the nucleic acid constituting the LNP include nucleic acids such as mRNA, siRNA, antisense oligonucleotide (Antisense Oligonucleotide, ASO), and DNA. As a result of the delivery of the nucleic acid into the target tissue or target cell, the desired biological activity is exerted. In addition, LNP is basically composed of four lipid components: a cationic lipid, a phospholipid, cholesterol, and a PEG lipid.

[0005] Cationic lipids are one of the main components of LNPs, exhibiting charge neutrality at physiological pH and protonating in acidic regions. Cationic lipids are classified into unsaturated, multi-tailed, and biodegradable types based on their structural characteristics. These structural characteristics contribute to the efficiency of intracellular release of nucleic acids (the payload) and the reduction of LNP cytotoxicity. Phospholipids, also known as structural helper lipids in LNPs, contribute to the effectiveness of LNP formulations by promoting membrane fusion between LNPs and cells and facilitating endosomal escape. Cholesterol, like phospholipids, is also a structural helper lipid for LNPs and contributes to promoting the binding of LNPs to cells. PEG lipids play a role in extending the circulating half-life of LNPs in vivo and also affect the size (particle diameter) of LNPs. Numerous improved and derivative products of these four types of lipids have been created to date.

[0006] Various LNPs have been developed by using diverse lipid molecules in various combinations and ratios. For example, Patent Documents 1 and 2 disclose LNPs characterized by containing a specific combination of lipids in a specific ratio. However, the LNPs developed so far are not sufficient in terms of safety and therapeutic efficacy, and there remains a strong need for the development of novel LNPs that are safer and have higher therapeutic efficacy. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent No. 11191849 [Patent Document 2] U.S. Patent No. 11684577 [Non-patent literature]

[0008] [Non-Patent Document 1] Lee et al., International Journal of Molecular Sciences, 2011, vol.12(5), p.3263-3287. [Overview of the project] [Problems that the invention aims to solve]

[0009] The present invention aims to provide a novel LNP that can effectively exert the function of the encapsulated nucleic acid within the cell. [Means for solving the problem]

[0010] As a result of diligent research into the above-mentioned problems, the inventors of this invention discovered that LNPs containing a cationic lipid having a specific structure and DOPC, a type of phospholipid, in a specific ratio exhibit high cellular activity, that is, the encapsulated nucleic acid exerts high activity within the cell. Based on this finding, the inventors furthered their research and completed the present invention. In other words, the present invention is as follows:

[0011] [1] Lipid nanoparticles, It contains nucleic acids, cKK-E12, and DOPC. Lipid nanoparticles in which the molar ratio of cKK-E12 to DOPC content is 0.2 to 15.0. [2] Lipid nanoparticles according to [1], wherein the molar ratio of cKK-E12 to DOPC content is 0.8 to 8.0. [3] The lipid nanoparticles according to [1] or [2], wherein the content ratio of cKK-E12 to the total amount of constituent lipids in the lipid nanoparticles is 10 to 45 mol% and DOPC is 5 to 30 mol%. [4] Lipid nanoparticles according to any of the above [1] to [3], wherein the content ratio of cKK-E12 to the total amount of constituent lipids in the lipid nanoparticles is 20 to 40 mol% and DOPC is 5 to 25 mol%. [5] Lipid nanoparticles according to any of the above [1] to [4], wherein the content ratio of cKK-E12 to the total amount of constituent lipids in the lipid nanoparticles is 20 to 35 mol% and DOPC is 5 to 25 mol%. [6] Lipid nanoparticles according to any of [1] to [5] above, wherein the content ratio of cKK-E12 to the total amount of constituent lipids in the lipid nanoparticles is 20 to 30 mol% and DOPC is 10 to 25 mol%. [7] The lipid nanoparticles further comprise sterols and PEG lipids, Lipid nanoparticles according to any of the [1] to [6] above, wherein the content ratio of sterols to the total amount of constituent lipids in the lipid nanoparticles is 20 to 85 mol% and PEG lipids is 0.5 to 5 mol%. [8] A lipid nanoparticle of any of the above [1] to [7], wherein the nucleic acid is mRNA or siRNA. [9] Lipid nanoparticles of any of the above [1] to [8], wherein the average particle size of the lipid nanoparticles is 30 to 250 nm.

[10] Any of the lipid nanoparticles from [1] to [9] above, wherein the PDI of the lipid nanoparticle is 0.3 or less.

[11] A method for producing any of the lipid nanoparticles described in [1] to

[10] above, comprising the following steps: A step to prepare a lipid solution containing cKK-E12, DOPC, sterols, and PEG lipids. A step of preparing a nucleic acid solution containing nucleic acids, and A step of mixing the lipid solution and the nucleic acid solution.

[12] A pharmaceutical composition comprising any of the lipid nanoparticles described in [1] to

[10] above. [Effects of the Invention]

[0012] According to the present invention, it is possible to provide a novel LNP that can more effectively exert the function of the encapsulated nucleic acid within the cell. [Modes for carrying out the invention]

[0013] The present invention will be described in detail below.

[0014] <Lipid Nanoparticle> The lipid nanoparticles of this embodiment (hereinafter sometimes referred to as "the lipid nanoparticles of the present invention" or "the LNP of the present invention", etc.) contain nucleic acid, cKK-E12, and DOPC, and the molar ratio of cKK-E12 to the content of DOPC is 0.2 to 15. cKK-E12 (3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione, Cas number: 1432494-65-9) is a kind of cationic lipid. By using a combination of DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, Cas number: 4235-95-4) and a specific cationic lipid as the lipids constituting the LNP, an LNP excellent in cell activity can be obtained. The reason why the LNP of the present invention is excellent in cell activity is not clear, but it is presumed that one of the reasons is that the particle size distribution is sharp. LNP with extremely large particle diameters or, conversely, extremely small particle diameters are difficult to be taken up by target cells. It is presumed that the LNP of the present invention has a narrow particle size distribution, few particles of a size that are difficult to be taken up by target cells, and a high uptake efficiency into target cells.

[0015] Hereinafter, "cKK-E12" may be referred to as "cationic lipid A".

[0016] In the present specification, the cellular activity of the LNP means the strength of the physiological function exerted when the nucleic acid encapsulated in the LNP is introduced into cells. For example, when the nucleic acid contained in the LNP is mRNA, the cellular activity of the LNP is the strength of the expression of the protein encoded by the mRNA in the cells, and the higher the expression level of the protein, the stronger the cellular activity is evaluated. When the nucleic acid contained in the LNP is a functional nucleic acid for RNA interference such as siRNA, the cellular activity of the LNP is the strength of the suppression of the expression of the gene targeted by the siRNA, and the lower the expression level of the protein encoded by the gene, the stronger the cellular activity is evaluated. The cellular activity of the LNP is affected by various factors such as the stability until the LNP reaches the target cells, the uptake efficiency of the LNP into the target cells, and the ease of release of the nucleic acid from the LNP in the target cells (endosomal escape efficiency).

[0017] The LNP of the present invention contains a nucleic acid. The nucleic acid contained in the LNP of the present invention may be DNA, RNA, or a chimeric nucleic acid of DNA and RNA. Examples of DNA include genomic DNA, cDNA, plasmid DNA, antisense oligonucleotide (ASO), and the like. Examples of RNA include mRNA, siRNA, miRNA, antisense RNA, and the like. The nucleic acid contained in the LNP of the present invention may be single-stranded or double-stranded. It may also be linear or circular.

[0018] The nucleic acid contained in the LNP of the present invention may be a nucleic acid composed of natural nucleobases, an artificial nucleic acid, or a nucleic acid containing natural nucleobases and artificial nucleobases. Examples of artificial nucleic acids include peptide nucleic acid (PNA), LNA (Locked Nucleic Acid), alkynyl nucleic acid, and the like. For example, the nucleic acid contained in the LNP of the present invention may be unmodified, or may be modified or altered at any part of the nucleic acid by a method known per se for the purpose of nucleic acid stabilization or the like.

[0019] In one embodiment, the nucleic acid contained in the LNP of the present invention may be contained in a vector. The term "vector" as used in this specification encompasses all types of vectors. Examples include plasmid vectors, cosmid vectors, artificial chromosome vectors (such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs)), phage vectors, or viral vectors (such as adenovirus vectors or baculovirus vectors), and any other vector known to those skilled in the art.

[0020] The molar ratio of cationic lipid A and DOPC constituting the LNP of the present invention is typically 0.2 to 15.0 ([Ratio of cationic lipid A content to total constituent lipids of LNP (mol%)] / [Ratio of DOPC content to total constituent lipids of LNP (mol%)]) (hereinafter sometimes referred to as the "cationic lipid / DOPC ratio").

[0021] The LNP of the present invention exhibits superior cell activity by containing cationic lipid A and DOPC in the ratios within the above range. The lower limit of the cationic lipid / DOPC ratio of the LNP of the present invention may be 0.2 or higher, 0.3 or higher, 0.4 or higher, 0.5 or higher, 0.6 or higher, 0.7 or higher, 0.8 or higher, 0.9 or higher, 1.0 or higher, 1.5 or higher, 2.0 or higher, 2.5 or higher, 3.0 or higher, 3.5 or higher, 4.0 or higher, 4.5 or higher, 5.0 or higher, 7.0 or higher, 9.0 or higher, 11.0 or higher, or 13.0 or higher. Preferably, it is 0.3 or higher, more preferably 0.4 or higher, even more preferably 0.5 or higher, and even more preferably 0.6 or higher. Furthermore, the upper limit of the cationic lipid / DOPC ratio of the LNP of the present invention may be 15.0 or less, 14.0 or less, 13.0 or less, 12.0 or less, 11.0 or less, 10.0 or less, 9.0 or less, 8.0 or less, 7.0 or less, 6.0 or less, 5.0 or less, 4.5 or less, 4.0 or less, 3.5 or less, 3.0 or less, 2.5 or less, 2.0 or less, 1.5 or less, 1.0 or less, 0.9 or less, 0.7 or less, or 0.5 or less. Preferably, it is 14.0 or less, more preferably 13.0 or less, and even more preferably 12.0 or less. The cationic lipid / DOPC ratio of the LNP of the present invention is preferably 0.2 to 15.0, more preferably 0.8 to 8.0, even more preferably 0.8 to 6.0, and even more preferably 0.8 to 3.0.

[0022] The cKK-E12 content ratio in the LNP of the present invention is not particularly limited, as long as the cationic lipid / DOPC ratio is within the range of 0.2 to 15.0. From the viewpoint of obtaining higher cell activity, the cKK-E12 content ratio relative to the total amount of constituent lipids in the LNP of the present invention is preferably 10 to 45 mol%, more preferably 20 to 40 mol%, even more preferably 20 to 35 mol%, and particularly preferably 20 to 30 mol%.

[0023] The DOPC content ratio in the LNP of the present invention is not particularly limited, as long as the cationic lipid / DOPC ratio is within the range of 0.2 to 15.0. From the viewpoint of obtaining higher cell activity, the DOPC content ratio to the total amount of constituent lipids in the LNP of the present invention is preferably 5 to 30 mol%, more preferably 5 to 25 mol%, and even more preferably 10 to 25 mol%.

[0024] In the LNP of the present invention, the content ratio of cKK-E12 to DOPC relative to the total amount of constituent lipids is preferably 20-40 mol% for cKK-E12 and 5-25 mol% for DOPC, more preferably 20-35 mol% for cKK-E12 and 5-25 mol% for DOPC, and even more preferably 20-30 mol% for cKK-E12 and 10-25 mol% for DOPC. By having the cKK-E12 and DOPC content ratio within the above range, the cellular activity exhibited by the LNP can be further enhanced. In particular, when the cKK-E12 content is 20-30 mol% and the DOPC content is 10-25 mol%, excellent cellular activity can be achieved regardless of the composition of constituent lipids other than cKK-E12 and DOPC.

[0025] Furthermore, in one embodiment, the LNP of the present invention may contain lipids other than cationic lipids and DOPC as constituent lipids of the LNP. Examples of such other lipids include PEG lipids and sterols.

[0026] In one embodiment, the LNP of the present invention may further contain a PEG lipid (also referred to as a "PEGylated lipid"). In this specification, "PEG lipid" means any lipid modified with a PEG (polyethylene glycol) group. Examples of PEG lipids that may be included in the LNP of the present invention include 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-5000 (DMG-PEG5000), 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), 1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG2000), and CREMOPHOR EL. Examples include, but are not limited to, EL, polyoxyethylene sorbitan monooleate, etc. In one preferred embodiment, the PEG lipids may be DMG-PEG5000, DMG-PEG2000, and ALC-0159.

[0027] In one embodiment, the LNP of the present invention may contain sterols (also referred to as "steroid alcohols"). In this specification, "sterol" means a subgroup of steroids that can be produced by plants, animals, or fungi. Examples of sterols that may be included in the LNP of the present invention include, but are not limited to, cholesterol, ergosterol, campesterol, oxysterol, antrosterol, desmosterol, nicasterol, sitosterol, and stigmasterol. In one preferred embodiment, the sterol may be cholesterol.

[0028] When the LNP of the present invention contains cationic lipid A, DOPC, sterols, and PEG lipids, the content ratio of each lipid component to the total amount of constituent lipids in the LNP may be as follows: The lower limit of the content ratio of cationic lipid A may be 5 mol% or more, 6 mol% or more, 7 mol% or more, 8 mol% or more, 9 mol% or more, 10 mol% or more, 15 mol% or more, 20 mol% or more, 25 mol% or more, 30 mol% or more, 35 mol% or more, 40 mol% or more, or 50 mol% or more. The upper limit of cationic lipid A may be 65 mol% or less, 60 mol% or less, 55 mol% or less, 50 mol% or less, 45 mol% or less, 40 mol% or less, 35 mol% or less, 30 mol% or less, 25 mol% or less, 20 mol% or less, 15 mol% or less, or 10 mol% or less. The content ratio of cationic lipid A is preferably 5 to 65 mol%, more preferably 10 to 65 mol%, even more preferably 10 to 60 mol%, even more preferably 10 to 45 mol%, and particularly preferably 20 to 40 mol%. The lower limit of the DOPC content ratio may be 5 mol% or more, 6 mol% or more, 7 mol% or more, 8 mol% or more, 9 mol% or more, 10 mol% or more, 15 mol% or more, 20 mol% or more, or 25 mol% or more. The upper limit of the DOPC content ratio may be 30 mol% or less, 25 mol% or less, 20 mol% or less, 15 mol% or less, or 10 mol% or less. The DOPC content ratio is preferably 5 to 30 mol%, more preferably 5 to 25 mol%. The lower limit of the sterol content ratio may be 15 mol% or more, 20 mol% or more, 25 mol% or more, 30 mol% or more, 35 mol% or more, or 40 mol% or more. Furthermore, the upper limit of the sterol content ratio may be 50 mol% or less, 45 mol% or less, 40 mol% or less, 35 mol% or less, 30 mol% or less, 25 mol% or less, or 20 mol% or less. The sterol content ratio is preferably 15 to 50 mol%, more preferably 20 to 50 mol%, also preferably 20 to 85 mol%, and even more preferably 30 to 75 mol%. The PEG lipid content ratio is preferably 0.5 to 5 mol%, more preferably 0.5 to 3 mol%, even more preferably 0.5 to 2 mol%, also preferably 1.0 to 5.0 mol%, more preferably 2.0 to 5.0 mol%, and even more preferably 2.0 to 4.0 mol%.

[0029] When the LNP of the present invention contains cationic lipid A, DOPC, sterols, and PEG lipids, higher cell activity can be obtained. Therefore, the content ratio of cationic lipid A to the total amount of constituent lipids in the LNP is preferably 25-30 mol%, DOPC is 10-15 mol%, and PEG lipid is 2-5%. It is also preferable that the content ratio of cationic lipid A is 20-25 mol%, DOPC is 20-25 mol%, and PEG lipid is 2-4 mol%.

[0030] The ratio of lipids to nucleic acids in the LNP of the present invention is not particularly limited as long as the desired effects of the present invention are obtained. For example, the N / P ratio (total number of positively charged ionizable lipid amine groups (N) to total number of negatively charged nucleic acid phosphate groups (P)) is usually 2 to 48, preferably 4 to 32, more preferably 4 to 12, also preferably 2 to 10, and more preferably 2 to 6, but is not limited to these values. The N / P ratio in the LNP of the present invention may preferably be 4 to 8, more preferably 5 to 7 (for example, 6).

[0031] Furthermore, in one embodiment, the LNP of the present invention may contain components other than nucleic acids and lipids. Examples of such components include, but are not limited to, surfactants, hyaluronic acid, or derivatives thereof.

[0032] In one embodiment, the LNP of the present invention may contain a surfactant. Examples of surfactants that may be included in the LNP of the present invention include, but are not limited to, polyoxyethylene sorbitan monooleate (e.g., polysorbate 80), polyoxyethylene polyoxypropylene glycol (e.g., Pluronic® F68), sorbitan fatty acid esters (e.g., sorbitan monolaurate, sorbitan monooleate), polyoxyethylene derivatives (e.g., polyoxyethylene hydrogenated castor oil 60, polyoxyethylene lauryl alcohol), glycerin fatty acid esters, or polyethylene glycol alkyl ethers. In one preferred embodiment, the surfactant included in the LNP of the present invention is polyoxyethylene polyoxypropylene glycol, glycerin fatty acid ester, or polyethylene glycol alkyl ether.

[0033] In one embodiment, the LNP of the present invention may further contain hyaluronic acid or a derivative thereof in addition to nucleic acids and lipids. Examples of hyaluronic acid derivatives include compounds obtained by dehydrating and condensing hyaluronic acid on the hydroxyl group of fatty acid glyceryl.

[0034] In one embodiment, the average particle diameter of the LNP of the present invention may have average particle diameters of about 30 nm to about 250 nm, about 30 nm to about 200 nm, about 30 nm to about 170 nm, about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, and about 70 nm to about 80 nm. In a preferred embodiment, the average particle diameter of the LNP of the present invention may be about 30 nm to about 200 nm.

[0035] Where used herein, the term "approximately" refers to a value similar to the reference value stated when applied to the value of interest. The term "approximately" means a range of values ​​that fall within ±10% of the reference value.

[0036] In one embodiment, the lower limit of the polydispersity index (PDI) of the LNP of the present invention may be 0.001 or higher, but is not limited thereto. The upper limit of the PDI of the LNP of the present invention is preferably 0.35 or lower, more preferably 0.30 or lower, even more preferably 0.20 or lower, even more preferably 0.15 or lower, and also preferably 0.12 or lower.

[0037] In the present invention and this specification, the average particle size of LNP is measured by dynamic light scattering and is a volume-based D 50 This refers to the particle size (nm). The PDI of LNPs is calculated from the particle size distribution measured by dynamic light scattering.

[0038] <Method for producing lipid nanoparticles> The method for producing lipid nanoparticles of this embodiment (hereinafter sometimes referred to as "the production method of the present invention") is a method for producing LNPs of the present invention that include at least cationic lipid A, DOPC, sterol, and PEG lipid as constituent lipids, and comprises the following steps. A step of preparing a lipid solution containing cationic lipid A, DOPC, sterols and PEG lipids, A step of preparing a nucleic acid solution containing nucleic acids, and, A step of mixing the lipid solution and the nucleic acid solution.

[0039] The cationic lipids A, DOPC, sterols, and PEG lipids used are those incorporated into the LNP of the present invention as described above.

[0040] In the production method of the present invention, first, a lipid solution containing cationic lipid A, DOPC, sterols, and PEG lipids is prepared. Specifically, cationic lipid A, DOPC, sterols, and PEG lipids are added to an organic solvent in a desired molar ratio. The organic solvent is not particularly limited as long as it is capable of dissolving all of cationic lipid A, DOPC, sterols, and PEG lipids. Examples of such organic solvents include alcoholic solvents such as methanol, ethanol, propanol, isopropanol, and butanol; ester-based solvents such as ethyl acetate and butyl acetate; ketone-based solvents such as acetone, methyl ethyl ketone, and methyl isobutyl ketone; and chlorine-based solvents such as chloroform. Alternatively, a mixed solvent of two or more of these organic solvents, or a mixed solvent of one or more of these organic solvents with water, may also be used. In the production method of the present invention, from the standpoint of relatively high safety for living organisms, an alcoholic solvent or a mixed solvent of water and an alcoholic solvent is preferred, ethanol or a mixed solvent of ethanol and water is more preferred, and ethanol is particularly preferred.

[0041] Furthermore, the nucleic acid solution in the production method of the present invention can be prepared by mixing a nucleic acid such as DNA or RNA with an aqueous solvent capable of dissolving the nucleic acid. The aqueous solvent is not particularly limited as long as it can dissolve the nucleic acid, and any solvent may be used. Examples of aqueous solvents include water and buffer solutions (e.g., citrate buffer solution, acetate buffer solution, etc.), but are not limited to these. In one preferred embodiment, the aqueous solvent is an acetate buffer solution.

[0042] In one embodiment, the pH of the aqueous solvent used in the production method of the present invention is usually 3.0 to 10.0, preferably 3.0 to 8.0, more preferably 3.0 to 7.0, and even more preferably 3.5 to 5.5.

[0043] In the manufacturing method of the present invention, the mixing of the lipid solution and the nucleic acid solution may be carried out by any means as long as the lipid solution and the nucleic acid solution are uniformly mixed. Examples of means for mixing the lipid solution and the nucleic acid solution include, but are not limited to, pipetting and microfluidic mixing devices. In a preferred embodiment, a microfluidic mixing device is used in the manufacturing method of the present invention.

[0044] In one embodiment, the lipid solution and the nucleic acid solution are mixed using a microfluidic mixing apparatus. By using a microfluidic mixing apparatus, the lipid solution and the nucleic acid solution can be mixed quickly and thoroughly. As the microfluidic mixing apparatus used in the manufacturing method of the present invention, for example, a microfluidic mixing apparatus outlined in Non-Patent Literature 1 can be used. A suitable microfluidic mixing apparatus used in the manufacturing method of the present invention is any apparatus that can mix the lipid solution and the nucleic acid solution and thereby produce the desired LNP. One example is, but is not limited to, NanoAssemblr (manufactured by Precision Nanosystems).

[0045] In microfluidic mixing, rapid and sufficient mixing is typically achieved by bringing multiple sample solutions into contact at different flow rates, thereby enhancing the diffusion effect between the flows of the sample solutions. The flow rate ratio between the two solutions can affect the size of the LNPs prepared. In one embodiment, in the mixing of a lipid solution and a nucleic acid solution, the flow rate ratio of the lipid solution to the nucleic acid solution ([flow rate of lipid solution (mL / min)]:[flow rate of nucleic acid solution (mL / min)]) is typically 1:1 to 10:1, preferably 2:1 to 3:1, but 1:10 to 1:1 is also preferred, and 1:2 to 1:3 is more preferred, but is not limited to these values.

[0046] In one aspect, the mixture of the lipid solution and the nucleic acid solution mixed by the microfluidic mixing device may be subjected to downsizing and purification as necessary. Such downsizing and purification may be carried out using methods known per se, such as ultrafiltration and filtration using a filter.

[0047] <Pharmaceutical composition containing LNP> The present invention also provides a pharmaceutical composition containing the LNP of the present invention (hereinafter sometimes referred to as "the pharmaceutical composition of the present invention").

[0048] The compounding amount of the LNP of the present invention contained in the pharmaceutical composition of the present invention is not particularly limited. The lower limit of the compounding amount of the LNP of the present invention contained in the pharmaceutical composition of the present invention is usually 0.01% by mass or more, preferably 0.1% by mass or more, 0.5% by mass or more, 1.0% by mass or more, 1.5% by mass or more, 2.0% by mass or more, 2.5% by mass or more, 3.0% by mass or more, 3.5% by mass or more, 4.0% by mass or more, 4.5% by mass or more, 5.0% by mass or more, 5.5% by mass or more, 6.0% by mass or more, 6.5% by mass or more, 7.0% by mass or more, 7.5% by mass or more, 8.0% by mass or more, 8.5% by mass or more, 9.0% by mass or more or 9.5% by mass or more, but is not limited thereto. Further, the upper limit of the compounding amount is usually 100% by mass or less, preferably 99.9% by mass or less, 99.0% by mass or less, 95.0% by mass or less, 90.0% by mass or less, 85.0% by mass or less, 80.0% by mass or less, 75.0% by mass or less, 70.0% by mass or less, 65.0% by mass or less, 60.0% by mass or less, 55.0% by mass or less, 50.0% by mass or less, 45.0% by mass or less, 40.0% by mass or less, 35.0% by mass or less, 30.0% by mass or less, 25.0% by mass or less, 20.0% by mass or less, 15.0% by mass or less or 10.0% by mass or less, but is not limited thereto.

[0049] The pharmaceutical composition of the present invention may contain components other than the LNP of the present invention. Examples of such components include pharmaceutically acceptable carriers. A pharmaceutically acceptable carrier generally means an inert and non-toxic solid or liquid filler, diluent, or encapsulating material that does not react with the active ingredient. Examples of pharmaceutically acceptable carriers used in the pharmaceutical composition of the present invention include, but are not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and mixtures thereof. Furthermore, in order to bring the physical properties of the pharmaceutical composition of the present invention closer to physiological conditions, the pharmaceutical composition of the present invention may contain pharmaceutically acceptable auxiliary substances. Examples of such auxiliary substances include, but are not limited to, pH adjusters, buffers, isotonic adjusters, wetting agents, etc.

[0050] The pharmaceutical composition of the present invention may be administered orally or parenterally. Parenteral administration may include, but is not limited to, transdermal, subcutaneous, intravenous, intra-arterial, intramuscular, intraperitoneal, vaginal, and intranasal administration. The number of administrations may be a single dose or multiple doses.

[0051] The target population to which the pharmaceutical composition of the present invention can be applied is not particularly limited. Examples of target populations to which the pharmaceutical composition of the present invention can be applied include, but are not limited to, mammals (humans, chimpanzees, dogs, cats, horses, cattle, sheep, goats, rats, mice, rabbits, pigs, etc.). In a preferred embodiment, the target population may be humans. [Examples]

[0052] The present invention will be described in more detail in the following examples, but the present invention is not limited in any way by these examples.

[0053] [Example 1] LNPs containing mRNA were prepared using cKK-E12 as the cationic lipid, DOPC or DSPC (1,2-distearoyl-sn-glycero-3-phosphatidylcholine, Cas number: 816-94-4) as the phospholipid, cholesterol as the sterol, and DMG-PEG5000 or ALC-0159 as the PEG lipid, and their physical properties and cellular activity were investigated. The mRNA used was the mRNA encoding green fluorescent protein GFP (SEQ ID NO: 1).

[0054] (Preparation of LNPs) Cationic lipids, phospholipids, cholesterol, and PEG lipids were mixed in the molar ratios shown in Table 1 to obtain a lipid mixture. This mixture was then dissolved in 99.5% ethanol to obtain a 10 mmol / L lipid solution. Furthermore, mRNA encoding GFP (SEQ ID NO: 1) was dissolved in acetate buffer (25 mM sodium acetate, pH 3.5-5.5) to obtain a nucleic acid solution of 0.167 mg / mL. The obtained lipid solution and nucleic acid solution were mixed at room temperature using a microfluidic mixer (Nanoassemblr, Precision Nanosystems) at a flow rate ratio of 1:2 ([lipid solution flow rate (4 mL / min)]:[nucleic acid solution flow rate (8 mL / min)]) to obtain a dispersion. The obtained dispersion was diluted 20-fold with D-PBS(-) buffer (Fujifilm Wako Pure Chemical Industries, Ltd.) and subjected to centrifugal ultrafiltration using a centrifugal filter unit (Amicon Ultra-4, Millipore). Subsequently, the solution after centrifugal ultrafiltration was further filtered using a 0.22 μm syringe filter (Millipore) to prepare LNPs. The obtained LNPs were stored at 4°C.

[0055] (Measurement of mRNA inclusion rate) A portion of the prepared LNP solution was taken, and the LNPs were dissolved by adding 0.5% Triton X-100. The nucleic acid concentration was then measured using the nucleic acid quantification kit "Quant-iT RiboGreen RNA Assay Kit" (Thermo Fisher Scientific). Additionally, a portion of the prepared LNP solution was taken again, and the nucleic acid concentration was measured in the same manner without adding Triton X-100 to determine the concentration of mRNA not encapsulated in the LNPs. From these measurement results, the mRNA encapsulation rate (%) in the LNPs was calculated.

[0056] Specifically, the reagents included in the nucleic acid quantification kit were added to the sample and reacted. Afterward, an excitation wavelength of 485 nm was applied, and the fluorescence intensity at 528 nm was measured. Based on two pre-prepared standard curves, depending on the presence or absence of a surfactant (Triton X-100), the total mRNA concentration in each sample (fluorescence intensity of the sample with surfactant added) and the concentration of mRNA not encapsulated in LNPs (fluorescence intensity of the sample without surfactant added) were calculated from the fluorescence intensity of each sample. Based on these, the mRNA inclusion rate (%) and mRNA yield (%) were calculated using the following formula. "Total mRNA concentration at the start of the reaction" refers to the total mRNA concentration used in the reaction after adding the reagents from the nucleic acid quantification kit.

[0057] [mRNA inclusion rate (%)] = ([Total mRNA concentration in sample (%)] - [Concentration of mRNA not encapsulated in LNP (%)]) / [Total mRNA concentration in sample (%)] × 100 (%) [mRNA yield (%)] = [Total mRNA concentration in sample (%)] / [Total mRNA concentration in preparation (%)] × 100 (%)

[0058] (Measurement of average particle size of LNPs) The average particle size of LNPs was measured by dynamic light scattering. Specifically, diluted samples were added to a measurement cuvette, and a dynamic light scattering instrument (Zetasizer, Malvern Panalytical) was used to irradiate them with laser light (633 nm) and measure the particle size (nm) and PDI.

[0059] (Cell assay) AAVpro 293T cells, pre-cultured using FBS-DMEM (DMEM containing 10% FBS and 1 mM SP), were placed in 24-well plates at a rate of 80,000 cells / cm². 2 The cells were seeded in the specified manner and cultured until 70-80% confluence (37°C, 5% CO2). 24 hours after seeding, the culture medium was changed (FBS-DMEM). Next, the prepared LNP was added to each well to transfect the AAVpro 293T cells, and they were further cultured. 24 hours after transfection, the cells were detached from the wells and collected by adding a trypsin-like enzyme (TrypLE select, Thermo Fisher Scientific).

[0060] (Measurement of cell activity (%)) The collected cells were passed through a strainer, and then the cell count and GFP emission intensity were measured using a FACS Verse flow cytometer (Becton Dickinson). The measurement results were analyzed using the analysis software "FlowJo" to determine the percentage of eGFP-positive cells ([number of GFP-expressing cells] / [total number of cells] × 100%) and MFI (Mean Fluorescence Intensity: average value of GFP emission intensity). The percentage of eGFP-positive cells (%) was defined as cell activity (%). LNPs with cell activity of 1% or more were evaluated as useful carriers, i.e., carriers that can effectively exert the function of the encapsulated nucleic acid within the cell.

[0061] [Table 1]

[0062] Table 1 shows the measurement results for the average particle size (nm), PDI, mRNA inclusion rate (%), and cell activity (%) of each prepared LNP. Comparing test groups 1 and 2, which shared the same cationic lipid, the cell activity of the LNP using DOPC as the phospholipid was clearly higher. These results confirm that when using LNPs as carriers to deliver nucleic acids for protein expression, such as mRNA, to cells, combining DOPC and cKK-E12 as constituent lipids of LNPs significantly improves the efficiency of nucleic acid delivery to cells. In other words, LNPs with DOPC and cKK-E12 as constituent lipids are shown to be very useful as carriers for introducing nucleic acids for protein expression, such as mRNA, into cells.

[0063] [Example 2] LNPs containing mRNA were prepared using cKK-E12 as the cationic lipid, DOPC as the phospholipid, cholesterol as the sterol, and DMG-PEG5000 as the PEG lipid, and their physical properties and cellular activity were investigated. The mRNA used was the mRNA encoding FLuc, a luciferase protein (SEQ ID NO: 2).

[0064] (Preparation of LNPs) Cationic lipids, phospholipids, cholesterol, and PEG lipids were mixed in the molar ratios shown in Tables 2-6 to obtain a lipid mixture. This mixture was then dissolved in 99.5% ethanol to obtain a 10 mmol / L lipid solution. Furthermore, the mRNA encoding FLuc was dissolved in acetate buffer (25 mM sodium acetate, pH 3.5-5.0) to obtain a nucleic acid solution of 0.167 mg / mL. The obtained lipid solution and nucleic acid solution were mixed by pipetting at room temperature in a volume ratio of 1:2 to obtain a dispersion. The obtained dispersion was diluted 20-fold with D-PBS(-) buffer (Fujifilm Wako Pure Chemical Industries, Ltd.) and subjected to centrifugal ultrafiltration using a centrifugal filter unit (Amicon Ultra-4, Millipore). Subsequently, the solution after centrifugal ultrafiltration was further filtered using a 0.22 μm syringe filter (Millipore) to prepare LNPs. The obtained LNPs were stored at 4°C.

[0065] (Measurement of mRNA inclusion rate) A portion of the prepared LNP solution was taken, and the LNPs were dissolved by adding 0.5% Triton X-100. The nucleic acid concentration was then measured using the nucleic acid quantification kit "Quant-iT RiboGreen RNA Assay Kit" (Thermo Fisher Scientific). Additionally, a portion of the prepared LNP solution was taken again, and the nucleic acid concentration was measured in the same manner without adding Triton X-100 to determine the concentration of mRNA not encapsulated in the LNPs. From these measurement results, the mRNA encapsulation rate (%) in the LNPs was calculated.

[0066] (Measurement of average particle size of LNPs) The average particle size of LNPs was measured using dynamic light scattering. Specifically, diluted samples were added to a measurement plate, and a dynamic light scattering instrument (DynaPro PlateReader III, Wyatt Technology) was used to irradiate the plate with laser light (633 nm) and measure the particle size (nm) and PDI.

[0067] (Cell assay) AAVpro 293T cells, pre-cultured using FBS-DMEM (DMEM containing 10% FBS and 1 mM SP), were placed in 96-well plates at a rate of 80,000 cells / cm³. 2The cells were seeded and cultured until 70-80% confluence (37°C, 5% CO2). 24 hours after seeding, the prepared LNP was added to each well to transfect AAVpro 293T cells, which were then cultured further. 24 hours after transfection, the Steady-Glo® Luciferase Assay System (Promega) was added, and the luminescence intensity was analyzed using BioTek Synergy Neo2 (Agilent Technologies).

[0068] (Cytotoxicity) AAVpro 293T cells, pre-cultured using FBS-DMEM (DMEM containing 10% FBS and 1 mM SP), were placed in 96-well plates at a rate of 80,000 cells / cm³. 2 The cells were seeded in the specified manner and cultured until 70-80% confluence (37°C, 5% CO2). 24 hours after seeding, the prepared LNPs were added to each well to transfect AAVpro 293T cells, which were then cultured further. 24 hours after transfection, Cell Counting Kit-8 (Dojin Chemical Co., Ltd.) was added, and the cells were cultured for approximately 60 minutes before absorption was analyzed using BioTek Synergy Neo2 (Agilent Technologies). As a control, LNPs prepared in the same manner except without nucleic acid encapsulation were transfected, and absorbance was analyzed in the same way. The relative absorbance (%) of each cell type was defined as the cell viability (%), with the absorbance of the control cells set to 100%.

[0069] [Table 2]

[0070] [Table 3]

[0071] [Table 4]

[0072] [Table 5]

[0073] [Table 6]

[0074] Tables 2-6 show the measurement results for the average particle size (nm), PDI, mRNA inclusion rate (%), luminescence intensity, and cell viability (%) of each prepared LNP. Under the experimental conditions of this experiment, a luminescence intensity of 500,000 or higher is preferable, and 950,000 or higher is more preferable. All LNPs in the test groups showed sufficiently high mRNA inclusion rates, and good cell viability and luminescence intensity. Cells administered with LNPs in test groups with a cationic lipid content of 5-35 mol% and a phospholipid content of 5-30 mol% tended to have sufficiently high cell viability of 90% or higher. In particular, cells administered with LNPs in test groups 8, 10, 12-14, 18, 20-30, 33, and 35-38 showed high luminescence intensity, indicating that these LNPs had excellent cell viability. These results demonstrate that LNPs containing 20-30 mol% cKK-E12 and 10-25 mol% DOPC, with a cationic lipid / DOPC ratio of 0.8-8, exhibit excellent safety and cellular activity, making them highly useful as carriers for introducing nucleic acids for protein expression, such as mRNA, into cells. [Industrial applicability]

[0075] The LNPs of the present invention can more effectively exert the function of the encapsulated nucleic acid within the cell. For this reason, the LNPs of the present invention are particularly useful as carriers for transporting functional nucleic acids such as mRNA and siRNA to target cells, and are especially useful in fields such as nucleic acid drugs.

Claims

1. Lipid nanoparticles, It contains nucleic acids, 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, sterols, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-5000. The molar ratio of 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione to the content of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine is 0.2 to 6.

0. Lipid nanoparticles wherein the content ratio of 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione to the total amount of constituent lipids in the lipid nanoparticles is 20 to 40 mol%, and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine is 5 to 25 mol%.

2. Lipid nanoparticles according to claim 1, wherein the molar ratio of 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione to the content of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine is 0.8 to 6.

0.

3. The lipid nanoparticles according to claim 1, wherein the content ratio of 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione to the total amount of constituent lipids in the lipid nanoparticles is 20 to 35 mol%, and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine is 5 to 25 mol%.

4. The lipid nanoparticles according to claim 1, wherein the content ratio of 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione to the total amount of constituent lipids in the lipid nanoparticles is 20 to 30 mol%, and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine is 10 to 25 mol%.

5. Lipid nanoparticles according to any one of claims 1 to 4, wherein the content ratio of sterols to the total amount of constituent lipids in the lipid nanoparticles is 20 to 85 mol% and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-5000 is 0.5 to 5 mol%.

6. The lipid nanoparticle according to claim 5, wherein the nucleic acid is mRNA or siRNA.

7. The lipid nanoparticles according to claim 5, wherein the average particle size of the lipid nanoparticles is 30 to 250 nm.

8. The lipid nanoparticles according to claim 5, wherein the polydispersity index of the lipid nanoparticles is 0.3 or less.

9. A method for producing lipid nanoparticles according to any one of claims 1 to 4, comprising the following steps: A step of preparing a lipid solution containing 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, sterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-5000, A step of preparing a nucleic acid solution containing nucleic acids, and A step of mixing the lipid solution and the nucleic acid solution.

10. A pharmaceutical composition comprising lipid nanoparticles according to any one of claims 1 to 4.

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