Lipid nanoparticle and use thereof

Abstract

A lipid nanoparticle, comprising an ionizable lipid, a steroid compound and a polyethylene glycol-lipid, and not comprising a helper lipid. The ionizable lipid is selected from a compound having a structure represented by formula (I) and / or a pharmaceutically acceptable salt thereof. The three-component lipid nanoparticle not comprising a helper lipid reduces the complexity of the formulation and the difficulty of large-scale production, and further improves the nucleic acid encapsulation efficiency.

Description

Lipid nanoparticles and uses thereof TECHNICAL FIELD

[0001] The present invention belongs to the field of drug delivery, in particular to a lipid nanoparticle for nucleic acid delivery. BACKGROUND

[0002] Lipid nanoparticles (LNP) are the most widely clinically validated nucleic acid delivery vehicles, which can load mRNA, siRNA, DNA, saRNA and other nucleic acid molecules, and have been applied in many fields such as infectious disease prevention, tumor treatment, protein replacement, autoimmune disease, etc. LNP carriers are usually composed of four components, namely auxiliary phospholipid, cholesterol, ionizable lipid and PEG lipid. The ionizable lipid loads the nucleic acid molecule through electrostatic interaction and hydrogen bond, and the auxiliary phospholipid, cholesterol and PEG lipid form the lipid shell. Among them, the auxiliary phospholipid mostly uses DSPC which has strong rigidity and is easy to form a phospholipid film.

[0003] The traditional LNP four components are a classic prescription, which can form a stable mRNA-embedded drug preparation, and has been widely reported and formed a strict patent protection. However, since LNP contains multiple lipids, it is easy to cause complex ratio, cumbersome quality control and other problems in the actual production process; the complex lipid composition also increases the difficulty of impurity control, such as DSPC in the four components which can hydrolyze to form lysophospholipids and may cause potential side reactions; at the same time, DSPC may lead to reduced intracellular transfection efficiency of LNP due to its strong rigidity.

[0004] However, existing studies have shown that the LNP system based on SM102 ionizable lipid has almost no positive charge in a neutral environment, and the binding capacity with negatively charged nucleic acids is greatly reduced, so auxiliary lipids and cholesterol must be used to form a protective shell; at the same time, PEG lipids can help form uniform nanoparticles and prevent the clearance of the monocyte-macrophage system, so they cannot be removed from the prescription. Based on the structural characteristics and functions of the lipids in the existing classic LNP, it is difficult to simplify the lipid components in the prescription. SUMMARY

[0005] In view of the deficiencies in the prior art, the present disclosure replaces the conventional ionizable lipid (such as SM102) with a new ionizable lipid, which can bind to nucleic acid molecules through electrostatic interaction and hydrogen bonding, and can form a three-component lipid nanoparticle (LNP) that does not contain auxiliary phospholipid, thereby reducing the complexity of the prescription and the difficulty of large-scale production. In addition, the present disclosure has surprisingly found that the three-component LNP of the present disclosure further improves the nucleic acid encapsulation rate compared to the four-component LNP based on the conventional ionizable lipid such as SM102.

[0006] According to one aspect of the present disclosure, there is provided a lipid nanoparticle comprising an ionizable lipid, a steroidal compound, and a polyethylene glycol lipid, the ionizable lipid being selected from a compound having a structure shown in Formula (I) and / or a pharmaceutically acceptable salt thereof,

[0007] wherein,

[0008] A1is selected from hydrogen or a residue of a natural amino acid, wherein the -C=0- in the residue of the natural amino acid is bonded to the -NH in Formula (I) to form an amide bond;

[0009] A2is absent, or selected from a hydroxyl group or

[0010] R1, R2, or R3are each independently selected from a residue of a natural fatty acid or a residue of a natural fatty acid ester;

[0011] m is selected from an integer from 0 to 3,

[0012] n, q are each independently selected from an integer from 1 to 3.

[0013] In some embodiments, the lipid nanoparticle does not comprise a helper phospholipid.

[0014] In some embodiments, the lipid component in the lipid nanoparticle consists of the ionizable lipid, the steroidal compound, and the polyethylene glycol lipid.

[0015] In some embodiments, the natural amino acid is selected from a non-polar hydrophobic amino acid, a polar neutral amino acid, an acidic amino acid, or a basic amino acid.

[0016] In some embodiments, the non-polar hydrophobic amino acid is selected from alanine (Ala), valine (Val), leucine (Leu), isoleucine (ile), proline (Pro), methionine (Met), tryptophan (Trp), or phenylalanine (Phe).

[0017] In some embodiments, the polar neutral amino acid is selected from glutamine (Gln), serine (Ser), threonine (Thr), cysteine (Cys), asparagine (Asn), tyrosine (Tyr), or glycine (Gly),

[0018] In some embodiments, the acidic amino acid is selected from aspartic acid (Asp) or glutamic acid (Glu)

[0019] In some embodiments, the basic amino acid is selected from lysine (Lys), arginine (Arg), or histidine (His).

[0020] In some embodiments, A1is selected from H or a group selected from indicates the point of attachment of the group:

[0021] In some embodiments, R1, R2, or R3is each independently selected from a residue of a natural fatty acid or a residue of a natural fatty acid ester.

[0022] In some embodiments, R1, R2, or R3is each independently selected from a residue of a C12-C30 natural fatty acid or a residue of a C12-C30 natural fatty acid ester.

[0023] In some embodiments, the natural fatty acid or natural fatty acid ester is selected from at least one of the compounds shown below:

[0024] wherein e, f, j, h, g, k are each independently selected from an integer from 1 to 30, i is selected from an integer from 1 to 5, L1is selected from -C=0-, -C(=0)0-, -OC(=0)0-, or -OC(=0)-, and R4is selected from a C1-C20 linear alkyl group or a C3-C30 branched alkyl group.

[0025] In some embodiments, the natural fatty acid or natural fatty acid ester is selected from at least one of the compounds shown below:

[0026] In some embodiments, R1, R2, or R3is each independently selected from a group, indicates the point of attachment of the group:

[0027] In some preferred embodiments, R1, R2, or R3is each independently selected from a residue of linoleic acid, oleic acid, stearic acid, or decanediolic acid monooctyl ester.

[0028] In some embodiments, m is 0, 1, 2, or 3.

[0029] In some embodiments, n is 1, 2, or 3.

[0030] In some embodiments, p is 1, 2, or 3.

[0031] In some embodiments, e is selected from an integer from 1 to 30, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any value therebetween. Preferably, e is selected from an integer from 11 to 30.

[0032] In some embodiments, f is selected from an integer from 1 to 30, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any value in between. Preferably, f is selected from an integer from 1 to 10.

[0033] In some embodiments, j is selected from an integer from 1 to 30, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any value in between. In some embodiments, j is selected from an integer from 5 to 30.

[0034] In some embodiments, h is selected from an integer from 1 to 30, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any value in between. Preferably, h is selected from an integer from 1 to 10.

[0035] In some embodiments, g is selected from an integer from 1 to 30, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any value in between. Preferably, g is selected from an integer from 5 to 30.

[0036] In some embodiments, k is selected from an integer from 1 to 30, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any value in between. Preferably, k is selected from an integer from 1 to 10.

[0037] In some embodiments, A1is hydrogen, A2is hydroxyl, and m, n, q are each independently 1, 2, or 3.

[0038] In some embodiments, A1is hydrogen, A2is and m, n, q are each independently 1, 2, or 3.

[0039] In some embodiments, A1is a residue of a natural amino acid, A2is hydroxyl, and m, n, q are each independently 1, 2, or 3.

[0040] In some embodiments, A1is a residue of a natural amino acid, A2is and m, n, q are each independently 1, 2 or 3.

[0041] In some embodiments, A1is A2is absent, and m is 0, n, q are each independently 1, 2 or 3.

[0042] In some embodiments, the compound has a structure represented by Formula (II):

[0043] wherein A1is defined as A1in Formula (I).

[0044] In some preferred embodiments, A1is selected from hydrogen, a histidine residue, an arginine residue or a lysine residue, a glycine residue, a leucine residue, an isoleucine residue or a serine residue.

[0045] In some embodiments, the compound is selected from:

[0046] The compound having a structure represented by Formula (I) provided herein has a natural amino acid head and a natural fatty acid or fatty acid ester tail connected by a degradable linker (connected by an in vivo readily degradable ester bond or amide bond), which is completely biodegradable.

[0047] In some embodiments, the sterol includes at least one of cholesterol, coprostanol, desmosterol, sitosterol, ergosterol, elaidosterol, stigmasterol, brassicasterol, campesterol, tomatidine, ursolic acid, alpha-tocopherol, a corticosteroid, and derivatives thereof. Preferably, the sterol is cholesterol.

[0048] In some embodiments, the polyethylene glycol lipid includes at least one of a polyethylene glycol-modified phosphatidylethanolamine, a polyethylene glycol-modified phosphatidic acid, a polyethylene glycol-modified ceramide, a polyethylene glycol-modified dialkylamine, a polyethylene glycol-modified diacylglycerol, and a polyethylene glycol-modified dialkylglycerol. Preferably, the polyethylene glycol lipid is DMG-PEG2000.

[0049] In some embodiments, the lipid nanoparticle does not include a helper phospholipid, including but not limited to at least one of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), palmitoyloleyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin (SM), ceramide, sterols and derivatives thereof, and the like.

[0050] In some embodiments, the mole fraction of the ionizable lipid is 25-70%, preferably 35-60%, based on the total moles of the lipid nanoparticle. In some embodiments, the mole fraction of the ionizable lipid is 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or any value therebetween, based on the total moles of the lipid nanoparticle.

[0051] In some embodiments, the mole fraction of the steroidal compound is 25-70%, preferably 35-63%, based on the total moles of the lipid nanoparticle. In some embodiments, the mole fraction of the steroidal compound is 25%, 30%, 35%, 37.9%, 38.2%, 38.5%, 40%, 41.5%, 42.5%, 43.2%, 47.5%, 48.2%, 50%, 52.5%, 53.2%, 55%, 58%, 60%, 63%, 65%, 70%, or any value therebetween, based on the total moles of the lipid nanoparticle.

[0052] In some embodiments, the mole fraction of the polyethylene glycol lipid is 1-5%, preferably 1.5-5%, based on the total moles of the lipid nanoparticle. In some embodiments, the mole fraction of the polyethylene glycol lipid is 1%, 1.2%, 1.5%, 1.8%, 2%, 2.1%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or any value therebetween, based on the total moles of the lipid nanoparticle.

[0053] In some embodiments, the molar ratio between the ionizable lipid, the steroid compound and the PEG lipid in the lipid nanoparticle is 50:48.5:1.5, 35:63:2, 40:58:2, 45:53.2:1.8, 45:52.5:2.5, 50:47.5:2.5, 50:48.2:1.8, 55:43.2:1.8, 55:42.5:2.5, 55:41.5:3.5, 60:35:5, 60:38.5:1.5, 60:38.2:1.8, 60:37.9:2.1.

[0054] In some embodiments, the average particle size of the lipid nanoparticle is 50-200 nm, preferably 50-160 nm. In some embodiments, the average particle size of the lipid nanoparticle is 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, or any value therebetween.

[0055] In some embodiments, the polydispersity index (PDI) of the lipid nanoparticle is 0.05-0.5, preferably 0.05-0.3. In some embodiments, the polydispersity index (PDI) of the ionizable lipid nanoparticle is 0.05, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.35, 0.4, 0.45, 0.5, or any value therebetween.

[0056] According to another aspect of the present disclosure, a method for preparing the lipid nanoparticle is also provided, comprising the following steps:

[0057] (1) Dissolve the ionizable lipid, steroid compound and PEG lipid in an alcohol organic solvent such as anhydrous ethanol in a proportion to prepare an oil phase solution, with a total lipid concentration of 10-20 mM;

[0058] (2) Dissolve the active pharmaceutical ingredient such as nucleic acid (mRNA, DNA, siRNA, etc.) in a citric acid-sodium citrate buffer (e.g., 40-60 mM) with pH = 3-6 to form an aqueous phase solution;

[0059] (3) Use a microfluidic system or an impact mixer to mix the oil phase and the aqueous phase at a volume ratio of 1:2-1:7 to form a nucleic acid-encapsulated lipid nanoparticle; preferably, the mixing speed is 10-20 mL / min;

[0060] (4) dialysis in Tris-HCl buffer or PBS buffer (e.g. 4-16 hours) by 3 kDa-50 kDa dialysis bag to obtain the lipid nanoparticle.

[0061] According to yet another aspect of the present disclosure, there is also provided use of the lipid nanoparticle of the present disclosure in delivery of a pharmaceutically active ingredient.

[0062] In some embodiments, the pharmaceutically active ingredient is selected from the group consisting of nucleic acid, small molecule compound, polypeptide and / or protein.

[0063] In some embodiments, the nucleic acid comprises DNA and / or RNA. Preferably, the nucleic acid comprises one or more of mRNA, siRNA, rRNA, tRNA, snRNA, miRNA, cicleRNA and plasmid.

[0064] In some embodiments, the pharmaceutically active ingredient is encapsulated within or electrostatically bound to the lipid nanoparticle.

[0065] According to yet another aspect of the present disclosure, there is also provided a composition comprising the lipid nanoparticle, and a pharmaceutically active ingredient.

[0066] In some embodiments, the pharmaceutically active ingredient is selected from the group consisting of nucleic acid, small molecule compound, polypeptide and / or protein.

[0067] In some embodiments, the nucleic acid comprises DNA and / or RNA. Preferably, the nucleic acid comprises one or more of mRNA, siRNA, rRNA, tRNA, snRNA, miRNA, cicleRNA and plasmid.

[0068] In some embodiments, the pharmaceutically active ingredient is encapsulated within or electrostatically bound to the lipid nanoparticle.

[0069] In some embodiments, the pharmaceutical composition further comprises an adjuvant.

[0070] In some embodiments, the adjuvant comprises one or more of a pharmaceutically acceptable carrier, excipient or diluent.

[0071] According to yet another aspect of the present disclosure, there is also provided use of the lipid nanoparticle of the present disclosure or the pharmaceutical composition of the present disclosure in preparation of a vaccine or a gene vector.

[0072] In some embodiments, the lipid nanoparticle or composition is used in preparation of a gene vector, which has better cellular immune response and humoral immune response.

[0073] The present disclosure provides a kind of lipid nanoparticle (LNP) containing only three kinds of lipids, using the ionizable lipid of specific structure replaces traditional ionizable lipid such as SM102, and removes the auxiliary phospholipid in classic LNP prescription.Compared with the traditional LNP four-component lipid prescription, the LNP prescription provided by the present application contains only three kinds of lipid components, which reduces the complexity of LNP preparation process and the risk of quality control.At the same time, compared with the LNP using SM102 as ionizable lipid, the three-component LNP provided by the present application also improves the encapsulation efficiency of mRNA. BRIEF DESCRIPTION OF DRAWINGS

[0074] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of compound SV-101 prepared in Example 1 of the present application.

[0075] Figure 2 is the mass spectrum of compound SV-101 prepared in Example 1 of the present application.

[0076] Figure 3 is the nuclear magnetic resonance hydrogen spectrum of compound SV-201 prepared in Example 1 of the present application.

[0077] Figure 4 is the mass spectrum of compound SV-201 prepared in Example 1 of the present application.

[0078] Figure 5 is the nuclear magnetic resonance hydrogen spectrum of compound SV-202 prepared in Example 1 of the present application.

[0079] Figure 6 is the mass spectrum of compound SV-202 prepared in Example 1 of the present application.

[0080] Figure 7 is the nuclear magnetic resonance hydrogen spectrum of compound SV-203 prepared in Example 1 of the present application.

[0081] Figure 8 is the mass spectrum of compound SV-203 prepared in Example 1 of the present application.

[0082] Figure 9 shows the comparison results of in vivo transfection efficiency of three-component LNP and four-component LNP in Example 7 of the present application.

[0083] Figure 10 shows the comparison results of binding antibody titers of herpes zoster vaccine using three-component LNP and four-component LNP as carriers in Example 8 of the present application. DETAILED DESCRIPTION

[0084] In order to make the purpose, technical scheme and advantages of the present application more clear and explicit, the present application will be further described in detail below in combination with the drawings and examples. The specific examples described herein are only used to explain the present application and do not constitute any limitation on the present application. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present disclosure. Such structures and technologies are also described in many publications.

[0085] TERMS

[0086] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0087] Herein, the term "alkyl" refers to aliphatic hydrocarbon groups, which can be branched or straight chain alkyl groups. In the present disclosure, for example, "C1-C20 straight chain alkyl" includes, but is not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, n-icosyl, and the like. "C3-C30 branched alkyl" includes, but is not limited to, isopropyl, neopentyl, isopentyl, and the like.

[0088] Lipid nanoparticles (LNPs) have a structure comprising a single or double layer of lipids that encapsulate a compound in a solid phase. Unlike liposomes, lipid nanoparticles do not have an aqueous or other liquid phase inside them, but rather the lipids from the double or single layer shell directly complex with the interior compound, thereby encapsulating it in a solid core. Lipid nanoparticles are typically spherical vesicles with a relatively uniform shape and size distribution. It is generally believed that the diameter of a lipid nanoparticle can range from 10 nm to 1000 nm.

[0089] For a lipid nanoparticle nucleic acid delivery system, the lipid shell can be formulated to comprise an ionizable ionizable lipid that can complex and associate with the negatively charged backbone of the nucleic acid core. At physiological pH, the lipid nanoparticle can adopt a relatively neutral exterior, thereby significantly increasing the circulating half-life of the particle after intravenous administration. In the context of nucleic acid delivery, lipid nanoparticles have many advantages over other lipid-based nucleic acid delivery systems, including high nucleic acid encapsulation efficiency, potent transfection, improved penetration into tissues to deliver therapeutic agents, and low levels of cytotoxicity and immunogenicity.

[0090] The pharmaceutical composition can be formulated for administration by a particular route of administration. For example, the pharmaceutical composition can be formulated for intravenous, intratumoral, intraperitoneal, intradermal, subcutaneous, intranasal, or other routes of administration.

[0091] The dosage form of the pharmaceutical composition herein includes, but is not limited to, injection, aerosol, drop pill, external use liniment, controlled release or sustained release or nano preparation.

[0092] As used herein, the term“delivery” encompasses both local delivery and systemic delivery. For example, delivery of mRNA encompasses situations in which the mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as“local distribution” or“local delivery”), as well as situations in which the mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into the patient’s circulatory system (e.g., serum), and is systemically distributed and taken up by other tissues (also referred to as“systemic delivery”).“Local delivery” refers to tissue-specific delivery or distribution. Typically, local delivery requires that the protein or peptide encoded by the mRNA is translated and expressed within the cell or with limited secretion, which avoids entry into the patient’s circulatory system. The term“target tissue” refers to any tissue affected by the disease to be treated. In some embodiments, the target tissue includes those tissues that exhibit disease-related pathology, symptoms, or characteristics.

[0093] As used herein, “pharmaceutically acceptable carrier, excipient, or diluent” refers to any ingredient other than the compound described herein (e.g., a vehicle which is capable of suspending, compounding, or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient. Excipients can include, for example, antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and water of hydration. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, crosscarmellose, crosslinked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methyl cellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C, xylitol, and other substances disclosed herein.

[0094] The lipid nanoparticles of the present application are further described below in connection with examples. However, the present application is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are the conventional conditions in the industry. The technical features involved in each embodiment of the present application can be combined with each other as long as there is no conflict between them. It is particularly pointed out that the person skilled in the art can make corresponding modifications and optimizations to the present application, and these equivalent forms are also included in the protection scope of the claims attached to the present disclosure.

[0095] In the specific embodiments of the present application, the raw materials used can be obtained by market purchase.

[0096] Examples

[0097] Example 1, synthesis of ionizable lipids

[0098] 1): synthesis route of compound SV-101

[0099] (1) Tris(hydroxymethyl)aminomethane (1 mmol), di-tert-butyl dicarbonate (1.2 mmol) were dissolved in a mixed solvent of MeOH and H2O, and reacted at room temperature for 12 h. After concentration under reduced pressure, extraction and column chromatography separation, SV-101-1 was obtained with a yield of 89%.

[0100] (2) SV-101-1 (1 mmol), linoleic acid (2 mmol), EDCI (1.1 mmol), DMAP (1.2 mmol) were dissolved in DCM, and reacted at room temperature. After filtration, extraction and column chromatography separation, SV-101-2 was obtained with a yield of 78%.

[0101] (3) SV-101-2 (1 mmol) was dissolved in DCM, and TFA (1 mL) was added dropwise under ice bath. The mixture was stirred at room temperature for 5 h, extracted, and column chromatography separated (dichloromethane:methanol = 10:1) to obtain SV-101 with a yield of 92%.

[0102] It was tested that the hydrogen nuclear magnetic resonance spectrum of SV-101 is shown in Figure 1, and the mass spectrum is shown in Figure 2.

[0103] 2): synthesis route of compound SV-201

[0104] (1) SV-101-1 (1 mmol), linoleic acid (4 mmol), EDCI (1.1 mmol), DMAP (1.2 mmol) were dissolved in DCM, and reacted at room temperature. After filtration, extraction and column chromatography separation, SV-201-1 was obtained with a yield of 75%.

[0105] (2) SV-201-1 (1 mmol) was dissolved in DCM, and TFA (1 mL) was added dropwise thereto under ice-bath, and the mixture was stirred at room temperature for 5 hours, extracted, and column chromatography was performed (dichloromethane:methanol=20:1) to obtain SV-201 at a yield of 90%.

[0106] SV-201 was tested, and the nuclear magnetic resonance hydrogen spectrum thereof is shown in FIG. 3, and the mass spectrum thereof is shown in FIG. 4.

[0107] 3) Synthesis route of compound SV-202

[0108] (1) A reaction flask of tris(hydroxymethyl)aminomethane (1 mmol), imidazole (2 mmol), and DMF (40 mL) was added with TBDMS-Cl (1.1 mmol), and stirred at room temperature overnight, poured into water, extracted, and column chromatography was performed to obtain SV-202-1 at a yield of 75%.

[0109] (2) SV-202-1, boc-protected arginine (1.5 mmol), HATU (1.5 mmol), and DIPEA (4 mmol) were dissolved in DCM, and reacted at room temperature for 5 hours. Diluted with ice-cold water, extracted, and column chromatography was performed to obtain SV-202-2 at a yield of 62%.

[0110] (3) Tetrabutylammonium fluoride (TBAF, 1M THF solution) (2 mmol) was added to a solution of SV-202-2 (1 mmol) in THF, and the mixture was stirred at room temperature for 18 hours, and column chromatography was performed after the reaction solution was concentrated under reduced pressure to obtain SV-202-3 at a yield of 85%.

[0111] (4) SV-202-3 (1 mmol), linoleic acid (4 mmol), EDCI (1.1 mmol), and DMAP (1.2 mmol) were dissolved in DCM, and reacted at room temperature, filtered, extracted, and column chromatography was performed to obtain SV-202-4 at a yield of 81%.

[0112] (5) SV-202-4 (1 mmol) was dissolved in DCM, and TFA (1 mL) was added dropwise thereto under ice-bath, and the mixture was stirred at room temperature for 5 hours, extracted, and column chromatography was performed (dichloromethane:methanol=20:1) to obtain SV-202 at a yield of 93%.

[0113] SV-202 was tested, and the nuclear magnetic resonance hydrogen spectrum thereof is shown in FIG. 5, and the mass spectrum thereof is shown in FIG. 6.

[0114] 4) Synthesis route of compound SV-203

[0115] Synthesis steps of SV-203:

[0116] (1) SV-201 (1 mmol), boc protected histidine (1.5 mmol), HATU (1.5 mmol), DIPEA (4 mmol) were dissolved in DCM, and reacted at room temperature for 4 hours. Diluted with ice-cold water, extracted, and separated by column chromatography to obtain SV-203-1 with a yield of 62%.

[0117] (2) SV-203-1 (1 mmol) was dissolved in dry DCM, and 6 mol / L hydrogen chloride dioxane solution (6 mmol) was added, and reacted at room temperature for 5 hours. The reaction system was concentrated, and separated by column chromatography (dichloromethane:methanol = 20:1) to obtain SV-203 with a yield of 93%.

[0118] It was tested that the nuclear magnetic resonance hydrogen spectrum of SV-203 is shown in FIG. 7, and the mass spectrum is shown in FIG. 8.

[0119] The synthesis processes of other compounds are basically the same as the synthesis process of the above-mentioned compounds, except that part of the process conditions are adjusted and part of the substrates are replaced.

[0120] Example 2, effect of lipid structure on formation of three-component LNP

[0121] The prescription of SM102 four-component LNP is: SM102 / DSPC / cholesterol / DMG-PEG2000 (50:10:38.5:1.5, molar ratio);

[0122] The prescription of SM102 three-component LNP is: SM102 / cholesterol / DMG-PEG2000 (50:48.5:1.5, molar ratio);

[0123] The prescription of SV-203 four-component LNP is: SV-203 / DSPC / cholesterol / DMG-PEG2000 (50:20:28.5:1.5, molar ratio);

[0124] The prescription of SV-203 three-component LNP is: SV-203 / cholesterol / DMG-PEG2000 (50:48.5:1.5, molar ratio);

[0125] Among them, SM-102 is purchased from Xiamen Senobangge Biotechnology Co., Ltd., DSPC, cholesterol and DMG-PEG 2000 are purchased from Avantor (Shanghai) Pharmaceutical Technology Co., Ltd.

[0126] The lipids (N / P = 6) of each prescription amount were precisely weighed and dissolved with anhydrous ethanol; the mRNA (Genbank: KJ561464) active ingredient was dissolved in 50 mM citric acid-sodium citrate buffer (pH = 4.0, mRNA concentration was 133 μg / mL); the organic phase dissolved with the lipid mixture and the aqueous phase dissolved with the mRNA ingredient were mixed at a volume ratio of 1:3 using a microfluidic system (Maynana (Shanghai) Instrument Technology Co., Ltd.) at a mixing speed of 12 mL / min; the nanoparticle solution was dialyzed in a Tris-HCl buffer using a dialysis box with a molecular weight cut-off of 10 kDa, and nucleic acid-lipid nanoparticles were obtained.

[0127] The particle size and polydispersity index (PDI) of the lipid nanoparticles were determined by dynamic light scattering using a Malvern Zetasizer Nano ZS, and the encapsulation efficiency and content of the lipid nanoparticles were detected by RiboGreen RNA quantitative detection kit (Thermo Fisher), and the results are shown in Table 1.

[0128] The results show that when SV203 is selected as the ionizable lipid, both the three-component lipid prescription and the four-component lipid prescription can form LNP with uniform particle size and high encapsulation efficiency; while selecting SM102 as the ionizable lipid, only the four-component lipid prescription can form LNP, and its encapsulation efficiency is lower than that of the LNP with SV203 as the ionizable lipid.

[0129] Table 1. Effect of lipid structure on formation of three-component LNP

[0130] Example 3 Preparation and characterization of three-component LNP

[0131] According to the prescription and method of SV-203 three-component LNP in Example 2, three-component LNP of different ionizable lipids was prepared, wherein only SV-203 was replaced with the ionizable lipids in Table 2 below.

[0132] The particle size, PDI and encapsulation efficiency of the prepared three-component LNP were detected by the same method as in Example 2, and the results are shown in Table 2.

[0133] Table 2: Particle size, PDI and encapsulation efficiency of different lipid samples

[0134] Example 4 Effect of the proportion of SV-203 in the prescription on the formation of LNP

[0135] Three-component lipid nanoparticles were prepared according to the gradient molar ratio of SV-203, with the prescription being SV-203 / cholesterol / DMG-PEG2000, and the molar ratios being 35:63:2, 40:58:2, 45:53.2:1.8, 50:48.2:1.8, and 55:43.2:1.8, respectively.

[0136] The lipid materials (N / P = 6) of each prescription were precisely weighed and dissolved in anhydrous ethanol; the mRNA (Genbank: KJ561464) active ingredient was dissolved in 50 mM citric acid-sodium citrate buffer (pH = 4.0, mRNA concentration of 133 μg / mL); the organic phase with dissolved lipid mixture and the aqueous phase with dissolved mRNA ingredient were mixed at a volume ratio of 1:3 using a microfluidic system, and the mixing speed was 12 mL / min; the nanoparticle solution was dialyzed in a Tris-HCl buffer using a dialysis cassette with a molecular weight cutoff of 10 kDa, to obtain nucleic acid-lipid nanoparticles.

[0137] The particle size and polydispersity index (PDI) of the lipid nanoparticles were determined by dynamic light scattering, and the encapsulation efficiency and content of the lipid nanoparticles were detected by RiboGreen, and the results are shown in Table 2.

[0138] The results show that the SV-203 three-component can form lipid nanoparticles with uniform size distribution and stable encapsulation efficiency, and as the molar ratio of SV-203 increases, the particle size of the lipid nanoparticles also increases, which indicates that the particle size of the lipid nanoparticles can be controlled by adjusting the molar ratio of SV-203 in the lipid nanoparticles.

[0139] Table 2. Effect of the proportion of SV-203 in the prescription on the formation of LNP

[0140] Example 5, Effect of the proportion of cholesterol in the prescription on the formation of LNP

[0141] Three-component lipid nanoparticles were prepared according to the gradient molar ratio of cholesterol, with the prescription being SV-203 / cholesterol / DMG-PEG2000, and the molar ratios being 45:52.5:2.5, 50:47.5:2.5, 55:42.5:2.5, 55:41.5:3.5, and 60:35:5, respectively.

[0142] The lipids (N / P = 6) of each prescription amount were precisely weighed and dissolved with anhydrous ethanol; the mRNA (Genbank: KJ561464) active ingredient was dissolved in 50 mM citric acid-sodium citrate buffer (pH = 4.0, mRNA concentration of 133 μg / mL); the organic phase with the dissolved lipid mixture and the aqueous phase with the dissolved mRNA ingredient were mixed at a volume ratio of 1:3 using a microfluidic system, and the mixing speed was 12 mL / min; the nanoparticle solution was dialyzed in a Tris-HCl buffer using a dialysis box with a molecular weight cut-off of 10 kDa, and nucleic acid-lipid nanoparticles were obtained.

[0143] The particle size and polydispersity index (PDI) of the lipid nanoparticles were determined by dynamic light scattering, and the encapsulation efficiency and content of the lipid nanoparticles were detected by RiboGreen, and the results are shown in Table 3.

[0144] The results show that the molar proportion of cholesterol in the three components between 35% and 55% can form lipid nanoparticles with uniform particle size, distribution, and high encapsulation efficiency.

[0145] Table 3, the effect of the proportion of cholesterol in the prescription on the formation of LNP

[0146] Example 6, the effect of the proportion of DMG-PEG2000 in the prescription on the formation of LNP

[0147] Three-component lipid nanoparticles were prepared according to the gradient molar ratio of DMG-PEG2000, and the prescription was SV-203 / cholesterol / DMG-PEG2000, and the molar ratio was 60:38.5:1.5, 60:38.2:1.8, and 60:37.9:2.1, respectively.

[0148] The lipids (N / P = 6) of each prescription amount were precisely weighed and dissolved with anhydrous ethanol; the mRNA (Genbank: KJ561464) active ingredient was dissolved in 50 mM citric acid-sodium citrate buffer (pH = 4.0, mRNA concentration of 133 μg / mL); the organic phase with the dissolved lipid mixture and the aqueous phase with the dissolved mRNA ingredient were mixed at a volume ratio of 1:3 using a microfluidic system, and the mixing speed was 12 mL / min; the nanoparticle solution was dialyzed in a Tris-HCl buffer using a dialysis box with a molecular weight cut-off of 10 kDa, and nucleic acid-lipid nanoparticles were obtained.

[0149] The particle size and polydispersity index (PDI) of the lipid nanoparticles were determined by dynamic light scattering, and the encapsulation efficiency and content of the lipid nanoparticles were detected by RiboGreen, and the results are shown in Table 4.

[0150] The results show that the molar ratio of DMG-PEG2000 in the three components is between 1.5% and 2.5%, and the lipid nanoparticles with uniform particle size, distribution and high encapsulation efficiency can be formed.

[0151] Table 4, the effect of the ratio of DMG-PEG2000 in the prescription on LNP formation

[0152] Example 7, the effect of the structure of the ionizable lipid on the formation of LNP

[0153] Lipid nanoparticles of various ionizable lipids were prepared, with the prescription being SV-201 / cholesterol / DMG-PEG2000 (50:48.5:1.5, molar ratio), SV-202 / cholesterol / DMG-PEG2000 (50:48.5:1.5, molar ratio), and SV-203 / cholesterol / DMG-PEG2000 (50:48.5:1.5, molar ratio).

[0154] The lipid materials (N / P=6) of each prescription were precisely weighed and dissolved in anhydrous ethanol; the mRNA (Genbank: KJ561464) active ingredient was dissolved in a 50 mM citric acid-sodium citrate buffer (pH=4.0, mRNA concentration of 133 μg / mL); the organic phase with dissolved lipid mixture and the aqueous phase with dissolved mRNA components were mixed at a volume ratio of 1:3 using a microfluidic system, and the mixing speed was 12 mL / min; the nanoparticle solution was dialyzed in a Tris-HCl buffer using a dialysis cassette with a molecular weight cutoff of 10 kDa, and nucleic acid-lipid nanoparticles were obtained.

[0155] The particle size and polydispersity index (PDI) of the lipid nanoparticles were determined by dynamic light scattering, and the encapsulation efficiency and content of the lipid nanoparticles were detected by RiboGreen, and the results are shown in Table 5.

[0156] The results show that the ionizable lipids of the present disclosure can all form LNP using a three-component lipid prescription.

[0157] Table 5, the effect of the structure of the ionizable lipid on the formation of LNP

[0158] Example 8, comparison of the in vivo transfection efficiency of three-component LNP and four-component LNP

[0159] Select 6-8 week old female BALB / c mice (purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.), randomly divided into 2 groups (SV203 three-component LNP group and SM102 four-component LNP group, 2 in each group), and raised in the experimental conditions of 22±2℃ and relative humidity of 45-75%, light / dark cycle of 12 hours. The SV203 three-component LNP and SM102 four-component LNP prepared in Example 3 were injected into the mice by muscle injection, the injection dose was 0.25mg / kg, 6 hours later, 100μL of D-fluorescein solution with a concentration of 40mg / mL was injected intraperitoneally, and the in vivo transfection efficiency of different LNP groups was detected by a small animal live imaging instrument, and the results are shown in Figure 9.

[0160] The results show that the three-component LNP of the application has similar transfection efficiency at the injection site compared with the four-component LNP with SM102 as the ionizable lipid, and can reduce unintended liver accumulation.

[0161] Example 9, Comparison of the immunogenicity of herpes zoster mRNA vaccine with three-component LNP and four-component LNP as carriers

[0162] Synthesize mRNA encoding herpes zoster virus gE protein, and prepare herpes zoster mRNA vaccine (Vaccine, 2020, 38(36):5793-5802) in the manner of Example 3. Prepare three-component LNP with SV203 as ionizable lipid into herpes zoster mRNA vaccine, and detect the immunogenicity difference with four-component LNP with SM102 as ionizable lipid.

[0163] Select 6-8 week old female C57BL / 6 mice (purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.) and randomly divide them into 2 groups (SV203 three-component LNP group and SM102 four-component LNP group), 10 in each group, and each group is immunized according to the immunization dose of 10μg / one at day 0 and day 21, and the mouse serum is collected at day 28 after immunization, and the binding antibody titer of different LNP administration groups is detected, and the results are shown in Figure 10.

[0164] The results show that there is no significant difference in the binding antibody titer produced by SV203 three-component LNP and SM102 four-component LNP.

[0165] Example 10, Comparison of the protection rate of rabies mRNA vaccine with three-component LNP and four-component LNP as carriers

[0166] Synthetic mRNA encoding rabies virus (GenBank: ACR39382.1), rabies mRNA vaccine was prepared in the manner as in Example 3. Three-component LNP with SV203 as ionizable lipid and four-component LNP with SM102 as ionizable lipid were prepared as rabies virus mRNA vaccine respectively.

[0167] KM mice (purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.) were selected and randomly divided into two groups (SV203 three-component LNP group and SM102 four-component LNP group), 10 mice in each group, 0.03ml CVS strain (50xLD50) was injected into the brain of mice on the 14th day after single immunization, and the mice were observed for 14 days after challenge, and the death of mice was recorded.

[0168] The results show that the challenge protection rates of SV203 three-component LNP and SM102 four-component LNP on mice are close.

[0169] The technical solutions of the present application are not limited to the above specific examples, and any technical variations made according to the technical solutions of the present application fall within the protection scope of the present application.

Claims

1. A lipid nanoparticle comprising an ionizable lipid selected from the group consisting of a compound having a structure according to Formula (I) and / or a pharmaceutically acceptable salt thereof, a steroidal compound, and a polyethylene glycol lipid, wherein, A1 is selected from hydrogen or a residue of a natural amino acid, wherein the -C=O- in the residue of the natural amino acid is bonded to the -NH in formula (I) to form an amide bond; A2is absent, or is selected from hydroxyl or R1, R2 or R3 are each independently selected from a residue of a natural fatty acid or a residue of a natural fatty acid ester; m is selected from an integer from 0 to 3, n, q are each independently selected from an integer from 1 to 3.

2. The lipid nanoparticle of claim 1, wherein, The lipid nanoparticle does not comprise a helper phospholipid; Preferably, the lipid component in the lipid nanoparticle consists of the ionizable lipid, the sterol compound and the polyethylene glycol lipid.

3. The lipid nanoparticle of claim 1 or 2, wherein, The natural amino acid is selected from a non-polar hydrophobic amino acid, a polar neutral amino acid, an acidic amino acid or a basic amino acid; preferably, the non-polar hydrophobic amino acid is selected from alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), methionine (Met), tryptophan (Trp) or phenylalanine (Phe), the polar neutral amino acid is selected from glutamine (Gln), serine (Ser), threonine (Thr), cysteine (Cys), asparagine (Asn), tyrosine (Tyr) or glycine (Gly), the acidic amino acid is selected from aspartic acid (Asp) or glutamic acid (Glu), and the basic amino acid is selected from lysine (Lys), arginine (Arg) or histidine (His); and / or R1, R2 or R3 are each independently selected from a residue of a natural fatty acid or a residue of a natural fatty acid ester; Preferably, R1, R2 or R3 are each independently selected from a residue of a C12-C30 natural fatty acid or a residue of a C12-C30 natural fatty acid ester; Preferably, the natural fatty acid or natural fatty acid ester is selected from at least one of the following compounds: wherein e, f, j, h, g, k are each independently selected from an integer from 1 to 30, and i is selected from an integer from 1 to 5, L1is selected from -C=0-, -C(=0)0-, -OC(=0)0-, or -OC(=0)-, R4is selected from a C1-C20 linear alkyl group or a C3-C30 branched alkyl group; preferably, the natural fatty acid or natural fatty acid ester is selected from at least one of the compounds shown below: Preferably, R1, R2 or R3 are each independently selected from a residue of linoleic acid, oleic acid, stearic acid or decanediolic acid monooctyl ester.

4. The compound of any one of claims 1-3, wherein, The compound has a structure shown in formula (II): A1 is as defined in claim 1 or 2, preferably A1 is selected from hydrogen, a histidine residue, an arginine residue or a lysine residue, a glycine residue, a leucine residue, an isoleucine residue or a serine residue.

5. The compound of any one of claims 1-4, wherein, The compound is selected from:

6. The lipid nanoparticle of any one of claims 1-5, wherein, The sterol compound comprises at least one of cholesterol, coprostanol, desmosterol, sitosterol, ergosterol, elaidosterol, stigmasterol, brassicasterol, tomatine, ursolic acid, alpha-tocopherol, a corticosteroid and derivatives thereof, preferably cholesterol; and / or The polyethylene glycol lipid comprises at least one of a polyethylene glycol-modified phosphatidylethanolamine, a polyethylene glycol-modified phosphatidic acid, a polyethylene glycol-modified ceramide, a polyethylene glycol-modified dialkylamine, a polyethylene glycol-modified diacylglycerol, a polyethylene glycol-modified dialkylglycerol, preferably DMG-PEG2000.

7. The lipid nanoparticle of any one of claims 1-6, wherein, The ionizable lipid has a molar percentage of 25% to 70%, preferably 35% to 60%, based on the total moles of the lipid nanoparticle; and / or The sterol compound has a molar percentage of 25% to 70%, preferably 35% to 63%, based on the total moles of the lipid nanoparticle; and / or The polyethylene glycol lipid has a molar percentage of 5% to 20%, preferably 10% to 15%, based on the total moles of the lipid nanoparticle. the polyethylene glycol lipid is in a molar ratio of 1-5%, preferably 1.5-5%, based on the total moles of the lipid nanoparticle; and / or the average particle size of the lipid nanoparticle is 50-200 nm, preferably 50-160 nm; and / or the polydispersity index (PDI) of the lipid nanoparticle is 0.05-0.5, preferably 0.05-0.

3.

8. The lipid nanoparticle according to any one of claims 1-7 for use in the delivery of a pharmaceutically active ingredient, preferably, the pharmaceutically active ingredient comprises one or more of nucleic acids, small molecule compounds, polypeptides and proteins; preferably, the nucleic acids comprise DNA and / or RNA, more preferably one or more of mRNA, siRNA, rRNA, tRNA, snRNA, miRNA, cicleRNA and plasmid; preferably, the pharmaceutically active ingredient is encapsulated within or electrostatically bound to the lipid nanoparticle.

9. A composition comprising the lipid nanoparticle according to any one of claims 1-7, and a pharmaceutically active ingredient; preferably, the pharmaceutical composition further comprises an adjuvant; more preferably, the adjuvant comprises one or more of a pharmaceutically acceptable carrier, excipient or diluent; more preferably, the pharmaceutically active ingredient comprises one or more of nucleic acids, small molecule compounds, polypeptides and proteins; further preferably, the nucleic acids comprise DNA and / or RNA, more preferably one or more of mRNA, siRNA, rRNA, tRNA, snRNA, miRNA, cicleRNA and plasmid; further preferably, the pharmaceutically active ingredient is encapsulated within or electrostatically bound to the lipid nanoparticle.

10. Use of the lipid nanoparticle according to any one of claims 1-7 or the pharmaceutical composition of claim 9 in the manufacture of a vaccine or a gene vector.

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