Ionizable lipids and lipid nanoparticles comprising the same

Abstract

Ionizable lipids of formula (I) or pharmaceutically acceptable salts thereof, or stereoisomers of any of them; lipid nanoparticles comprising said ionizable lipids, in particular as encapsulating agents, optionally comprising a pharmaceutically active agent; and pharmaceutical compositions comprising said lipid nanoparticles. Lipid nanoparticles for use in medicine or pharmaceutical compositions comprising the same, and use of said lipid nanoparticles as encapsulating agents. Formula (I)

Description

Technical Field

[0001] This disclosure relates to ionizable lipids comprising at least one thioester moiety and at least one amide moiety, and lipid nanoparticles (LNPs) comprising said ionizable lipids. These LNPs can be used as nonviral carriers for delivering active ingredients, including polynucleotides, to cells. Background Technology

[0002] Today, various diseases (such as infectious diseases) are undergoing rapid transformation in their treatment methods, especially as novel polynucleotide delivery systems are continuously designed and optimized to improve their efficiency, becoming useful alternatives to traditional therapies. Nucleic acid therapies are an excellent model for rapid vaccine design. More importantly, they avoid pathogen culture.

[0003] Polynucleotides (such as RNA) for systemic delivery can be successfully encapsulated in a gene delivery system containing the polynucleotide. The delivery system must meet several requirements, such as being safe and non-toxic, having a nanoscale design, providing protection against polynucleotide degradation, remaining in the system long enough to reach its target, and being readily degradable once the load is released.

[0004] Generally, using RNA in gene therapy and vaccination is considered safer than using DNA. On one hand, RNA does not involve the risk of being stably integrated into the genome of transfected cells. On the other hand, RNA is more easily degraded in the body, thus reducing the risk of generating unwanted anti-RNA antibodies, which can reduce efficacy and potentially cause very serious side effects. RNA-based therapies have two main limitations: low transfection rates and limited protein production efficiency. For these reasons, higher doses are needed to achieve the desired therapeutic effect, but these higher doses also mean higher costs, and more importantly, the potential for unwanted side effects.

[0005] LNPs have emerged as one of the more efficient intracellular delivery technologies for encapsulating and delivering active ingredients, such as genetic material like mRNA in vaccines. Currently, LNPs used in commercial mRNA vaccines typically contain four types of lipids in their composition: ionizable or cationic lipids, structural lipids (which are sterols such as cholesterol), PEG-modified lipids, and non-cationic lipids such as phospholipids. Notably, ionizable lipids are crucial in LNPs.

[0006] The properties of ionizable lipids have a significant impact on protecting the genetic material encapsulated within LNPs, as they maintain the structure and physicochemical properties of the genetic material until the LNPs reach the target of the genetic material to be released (e.g., tissues, cells).

[0007] Ionizable lipids typically exhibit the following common structure: Lipophilic moiety ---- Linking group ---- Bridging ---- Hydrophilic moiety Numerous examples exist where the hydrophilic moiety contains an ionizable tertiary amine, a functional group whose formal charge is affected by pH changes. Furthermore, the presence of readily enzymatically hydrolyzable ester groups has been reported to promote the degradation of ionizable lipids once genetic material has been released into target tissues / cells, thus improving their biocompatibility and biodegradability. The proximity of the ester group to the hydrophilic moiety has also been described as having a significant impact on lipid potency.

[0008] As exemplary examples, a lipid commercially known as SM-102 and a lipid known as ALC-0315 (shown below) are contained in the commercial SARS-CoV-2 vaccine formulations Spikevax (Moderna) and Comirnaty (Pfizer), respectively. In both cases, their structures contain an ionizable tertiary amine and two ester groups.

[0009]

[0010]

[0011] Molla MR et al. (see Molla MR et al., “One-Pot Parallel Synthesis of LipidLibrary via Thiolactone Ring Opening and Screening for Gene Delivery”; Bioconjug. Chem. 2018, Vol. 29, No. 4, pp. 992-999, doi: 10.1021 / acs.bioconjchem.8b00007) disclosed a lipid combinatorial library with a hydrophobic tail containing a reducible disulfide group, which yields stable liposomes.

[0012] Despite continuous improvements in such LNP delivery systems, efficient and specific delivery of the targeting agent remains a challenge. Biodegradability and toxicity are also two interrelated aspects that need to be considered in order to provide alternative ionizable lipids and / or LNPs to overcome some of the aforementioned drawbacks in the prior art, particularly exhibiting good stability, high transfection efficiency, and safety. Summary of the Invention

[0013] The inventors have developed a novel ionizable lipid that allows for the production of lipid nanoparticles (LNPs) that can be effectively used as non-viral carriers for the delivery of active ingredients, including polynucleotides, to cells. In particular, LNPs containing the ionizable lipid of this disclosure exhibit enhanced / improved transfection rates and biodegradability without compromising their stability.

[0014] The inventors have discovered ionizable lipids of formula (I) as defined below: Formula (I); It comprises a polar head, at least one thioester moiety, at least one stereocenter, at least one amide moiety, and moiety X selected from amides, esters, or thioesters, wherein Q, X, and R 1、 R2, m, p, and t, as defined in this disclosure, are particularly useful for preparing LNPs capable of encapsulating active agents. Furthermore, the inventors have found that the in vivo transfection rate of LNPs prepared using the ionizable lipids of this disclosure and containing active agents is unexpectedly high compared to other commercial or standard compositions known in the art.

[0015] Therefore, it is evident from the data provided in the embodiments that the ionizable lipids of this disclosure offer a new tool to overcome some of the limitations of known LNPs.

[0016] The higher transfection efficiency of the LNPs disclosed herein allows for a reduction in the therapeutic dose of polynucleotides (such as RNA) required in gene therapy and vaccination. The high degradability of the thioester moiety may be detrimental to the stability of the ionizable lipids of this disclosure. However, the inventors have found that not only is the biodegradability and thus cytotoxicity superior to other known alternatives, but the stability of the ionizable lipids of formula (I) is unexpectedly good, providing easier handling and storage conditions.

[0017] Therefore, the first aspect of this disclosure relates to ionizable lipids of formula (I): Formula (I); Or a pharmaceutically acceptable salt thereof, or a stereoisomer of either thereof, wherein R' is selected from H, methyl, and ethyl; X is selected from -NH-C(O)-, -C(O)-NH-, -C(O)-O-, -OC(O)-, -SC(O)-; -C(O)-S-; m is selected from 0, 1, 2, 3, 4, 5, and 6; p is selected from 0, 1, 2, 3, 4, 5, and 6; t is selected from 1, 2, and 3; Q is a heterocycle containing at least one N atom, or alternatively, Q is:

[0018] Where R a and R b It is independently a straight-chain or branched C1-C6 alkyl group, which may optionally be substituted with a hydroxyl group; Furthermore, R1 is selected from straight-chain C11-C30 alkyl or branched C6-C30 alkyl, C6-C30 alkenyl and C6-C30 alkynyl, and R2 is selected from straight-chain or branched C6-C30 alkyl, C6-C30 alkenyl or C6-C30 alkynyl, wherein R1 and R2 are optionally substituted by one or more substituents selected from -OH, -COOR4 and -C(=O)SR4, wherein R4 is C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl.

[0019] Another aspect of the invention relates to LNPs comprising ionizable lipids as defined herein (I).

[0020] On the other hand, the LNP disclosed herein may also contain a pharmaceutically active agent and thus be formulated into a drug with excipients and a carrier.

[0021] Therefore, another aspect of the present invention relates to a pharmaceutical composition comprising the LNP (which comprises a pharmaceutically active agent as defined herein) and a pharmaceutically acceptable excipient or carrier.

[0022] LNPs containing pharmaceutically active agents or pharmaceutical compositions disclosed herein may be used in medical applications.

[0023] Therefore, another aspect of the present invention relates to LNPs or pharmaceutical compositions comprising a pharmaceutically active agent, used in medicine, particularly in methods for treating a disease or condition in a subject with such need; or in methods for inducing an immune response in a subject, in methods for therapeutic immunization of a subject, as a vaccine, or in gene therapy. The methods include administering a therapeutically effective amount of the nanoparticle composition or the pharmaceutical composition to the subject.

[0024] Another aspect of the invention relates to the use of LNP as an encapsulating agent for active ingredients, as defined herein.

[0025] Brief description of the attached figures Figure 1 (Referring to Example 4) shows the protein expression in mice administered LNPs prepared with the following lipids of the present invention: VC-LC-1272, VC-LC-1285 and VC-LC-1289, which contain mRNA encoding luciferase as an active ingredient.

[0026] Figure 2(Related to Example 5) Shows a comparison of the hydrolytic cleavage of thioester bonds and ester bonds in ionizable lipids VC-LC-1282, SM-102 and VC-LC-0729. Detailed Implementation

[0027] Unless otherwise stated, all terms used in this application shall be understood in their ordinary meaning as known in the art. Further more specific definitions of certain terms used in this application are set forth below and are intended to be uniformly applied throughout the specification and claims, unless otherwise expressly given, which provides for a broader interpretation.

[0028] As used herein, the indefinite article “a” and “an” are synonymous with “at least one” or “one or more”. Unless otherwise indicated, the definite article used herein, such as “as stated”, also includes the plural form of the noun.

[0029] The term "and / or" means that any one of the options involved is possible, or that at least two options occur simultaneously.

[0030] The term "part" refers to a specific segment or functional group of a molecule or compound.

[0031] As used in this article, the term “subject” refers to any mammal, including both human and non-human mammals.

[0032] As used herein, the term "C1-C# alkyl" refers to a saturated straight-chain or branched hydrocarbon containing 1 to # carbon atoms, and optionally substituted. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, neopentyl, n-hexyl, decyl, isodecyl, undecyl, dodecyl, tetradecyl, and hexadecyl.

[0033] The term "C2-C# alkenyl" refers to an unsaturated straight-chain or branched hydrocarbon chain containing 2 to # carbon atoms and having at least one or more double bonds, and optionally substituted. Examples of alkenyl groups include, but are not limited to, vinyl (i.e., vinyl), allyl, propenyl, butenyl, pentenyl and hexenyl, dodecenyl, tetradecenyl and hexadecenyl.

[0034] The term "C2-C# ynyl" refers to an unsaturated straight-chain or branched hydrocarbon chain containing 2 to # carbon atoms and having at least one or more triple bonds, and optionally substituted. Examples of ynyl groups include, but are not limited to, ethynyl, prop-1-ynyl, prop-2-ynyl, 1-methylprop-2-ynyl, but-1-ynyl, but-2-ynyl, and but-3-ynyl.

[0035] The term "heterocycle containing at least one N atom" refers to a ring system with optional mono- or poly-substituted rings, comprising one or more rings, wherein at least one ring contains at least one nitrogen atom.

[0036] The term "optional substitution" means that the number of substituents may be equal to or not equal to zero. Unless otherwise stated, an optionally substituted group may be replaced by as many optional substituents as possible, as long as any available carbon or nitrogen atom is replaced with a non-hydrogen substituent.

[0037] The terms "polynucleotide" and "nucleic acid" are used interchangeably, referring to polymers of nucleotides, whether ribonucleotides or deoxyribonucleotides. Polynucleotides formed from ribonucleotides can be called "RNA polynucleotides," "ribonucleic acid," or simply "RNA"; polynucleotides formed from deoxyribonucleotides can be called "DNA polynucleotides," "deoxyribonucleic acid," or simply "DNA." These polynucleotides can be single-stranded or double-stranded, optionally incorporating synthetic, non-natural, or modified nucleotides capable of incorporating into DNA or RNA. "Artificial polynucleotides" refer to polynucleotides whose sequences are not naturally occurring or have been artificially altered.

[0038] As used herein, the term "messenger RNA" (abbreviated as "mRNA") refers to any RNA polynucleotide that encodes a target polypeptide and can be translated in vitro, in vivo, or ex vivo to produce the encoded target polypeptide. Typically, mRNA is single-stranded and contains an ORF in its structure.

[0039] As used in this article, "open reading frame" or "ORF" refers to a sequence of three nucleotide triplets that encode a polypeptide, that is, a sequence that can be translated into a polypeptide sequence.

[0040] As used herein, “DNA construct” refers to an artificial polynucleotide containing a target sequence operatively linked to an expression promoter that controls the expression of the target sequence.

[0041] As used herein, "expression vector" refers to a vector used to introduce a specific nucleic acid (typically a DNA construct) into a target cell for expression of that nucleic acid. Examples of suitable expression promoters and expression vectors include those routinely used in molecular biology and known to those skilled in the art.

[0042] The term "polypeptide" refers to any peptide or protein that consists of two or more amino acids linked together by peptide bonds or modified peptide bonds (i.e., peptide isosteres). "Polypeptide" can refer to both short chains (often called peptides, oligopeptides, or oligomers) and longer chains (often called proteins).

[0043] As used herein, the term "therapeuticly effective amount" means that the amount of a compound, when administered, is sufficient to prevent or, to a certain extent, alleviate one or more symptoms of the disease it targets. The specific dosage of the compound administered according to the invention is, of course, determined by the specific circumstances of the case, including the compound administered, the route of administration, the specific disease being treated, the specific circumstances of the individual subject to be treated, and similar considerations. The term "medicine" also encompasses the concept of "veterinary composition." Therefore, these refer to compositions that are therapeutically effective when administered to any animal (including humans) via any desired or applicable route.

[0044] According to the present invention, the term "antigen" is a compound that can be recognized by the immunoglobulin receptor of B cells, or by the receptor of T cells when complexed with MHC. Preferably, the "antigen" is a polypeptide.

[0045] As used herein, the term "nanoparticle" refers to a particle with at least two dimensions at the nanoscale, particularly all three dimensions at the nanoscale, wherein the nanoscale ranges from about 1 nm to about 500 nm. Specifically, when the nanoparticle is substantially rod-shaped and has a substantially circular cross-section (e.g., nanowire or nanotube), "nanoparticle" refers to a particle with at least two dimensions at the nanoscale, which are the cross-section of the nanoparticle. As used herein, in the context of a nanoparticle composition, "size" or "average size" refers to the average diameter of the nanoparticle composition. As used herein, the term "lipid nanoparticle" refers to a nanoparticle whose outer coating is entirely or partially composed of lipids.

[0046] As used in this paper, the polydispersity index (PDI) is a ratio that describes the uniformity of the particle size distribution of a system. Smaller values ​​(e.g., less than 0.3) indicate a narrow particle size distribution.

[0047] As used herein, the term "Z-potential" refers to the electrokinetic potential of lipids (e.g., in particulate compositions). Z-potential is also defined as the potential difference between the dispersion medium and the fluid quiescent layer attached to the dispersed particles. It is generally considered a quantification of charge magnitude and is often the only available way to characterize bilayer properties.

[0048] As used herein, “apparent pKa” refers to an experimentally determined value derived from the average ratio of all ionized groups to deionized groups in a nanoparticle. Apparent pKa differs from the intrinsic pKa of any single molecule and is an important parameter for the performance of nanoparticles encapsulating RNA. The apparent pKa of nanoparticles can be measured using various techniques known in the art. For example, acid-base titration and 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS) fluorescence are widely used in the field. Nanoparticles with optimal pKa carry a negligible charge at physiological pH, which avoids nonspecific binding and toxicity in vivo. The optimal pKa of nanoparticles plays an important role in endosome escape mechanisms and the release of RNA from the cytosol to exert therapeutic effects.

[0049] Ionizable lipids As described above, the first aspect of the present invention relates to ionizable lipids of formula (I): Formula (I); Or a pharmaceutically acceptable salt thereof, or a stereoisomer of either thereof, as defined above.

[0050] As used herein, the term "pharmaceutically acceptable salt" encompasses any salt formed from a pharmaceutically acceptable non-toxic acid (including inorganic or organic acids). There are no limitations on the salts mentioned, except that they must be pharmaceutically acceptable if used for therapeutic purposes. The preparation of pharmaceutically acceptable salts of the ionizable lipids of this disclosure can be carried out by methods known in the art. For example, they can be prepared from a parent compound containing a basic or acidic moiety by conventional chemical methods. Typically, such salts are prepared, for example, by reacting the free acidic or free base form of the ionizable lipids of this disclosure with a stoichiometric amount of a suitable pharmaceutically acceptable base or acid in water, in an organic solvent, or in a mixture thereof.

[0051] Examples of pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, or phosphoric acid; and acid addition salts formed with organic acids such as succinic acid, maleic acid, acetic acid, fumaric acid, citric acid, tartaric acid, benzoic acid, malic acid, lactic acid, formic acid, propionic acid, glycolic acid, camphor sulfuric acid, mandelic acid, benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid, methanesulfonic acid, or naphthalenesulfonic acid; and base addition salts formed with alkali metals and alkaline earth metals and organic bases such as N,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, lysine, and procaine. The ionizable lipids and their salts disclosed herein may differ in some physical properties, but they are equivalent for the purposes of this invention.

[0052] The ionizable lipids disclosed herein have an asymmetric center, thus allowing for a variety of stereoisomers. As used herein, the term "stereoisomer" refers to all isomers of a single ionizable lipid, differing only in the spatial orientation of their atoms. The term stereoisomer includes enantiomers, racemates, racemic mixtures, geometric isomers (cis / trans or cis / trans or E / Z), and diastereomers. This invention relates to each of these stereoisomers and mixtures thereof.

[0053] The preparation methods described herein can be modified to obtain enantiomeric pure compounds and mixtures of stereoisomers. Specific stereoisomers or mixtures can be prepared by various methods, including the use of stereospecific reagents or the introduction of chiral centers into the compound during preparation. Furthermore, once the compound has been prepared, the stereoisomers can be separated using standard resolution techniques known to those skilled in the art.

[0054] In all embodiments of the present invention relating to the ionizable lipids of this disclosure, the ionizable lipid or a pharmaceutically acceptable salt or stereoisomer thereof is always taken into consideration, even if not explicitly mentioned.

[0055] The ionizable lipids disclosed herein also include isotopes of the described structures. "Isotope" refers to atoms with the same atomic number but different mass numbers due to differences in the number of neutrons in their nuclei. For example, isotopes include, but are not limited to, tritium, deuterium, and... 13 C or 14 C or 15 N. Furthermore, the compounds or salts disclosed herein can be combined with solvents or water molecules to form solvates and hydrates by conventional methods.

[0056] The ionizable lipids of this disclosure are characterized by their retention time, mass spectrometry, particle size distribution, polydispersity index, and Z-potential. These parameters can be measured using methods well known in the art. Some of these are described in more detail in the examples below.

[0057] In one implementation, R1 is selected from linear C. 11 -C 30 Alkyl or branched C6-C 30 Alkyl, C6-C3 alkenyl and C6-C 30 The alkynyl group, and R2 is selected from straight-chain or branched C6-C. 30 Alkyl, C6-C 30 alkenyl or C6-C 30 The alkynyl group, wherein R1 and R2 are optionally substituted by one or more substituents selected from -OH, -COOR4 and -C(=O)SR4, wherein R4 is a C1-C6 alkyl, C2-C6 alkenyl or -C2-C6 alkynyl.

[0058] According to one embodiment of formula (I), optionally combined with one or more features of the above embodiments, R' is selected from H and methyl, and m is selected from 0, 1, 2, 3 and 4.

[0059] In one embodiment, the ionizable lipid of formula (I) is a compound of formula (IA): Formula (IA) R', X, A, R1, R2 and m are defined as above.

[0060] According to another embodiment, X may optionally be combined with one or more features of the above embodiments, wherein X is -NH-C(O)- or -C(O)-NH-; or X is -C(O)-O- or -OC(O)-; or X is -C(O)-S- or -SC(O)-.

[0061] According to another implementation scheme, Q is optionally combined with one or more features of the above-described implementation schemes, and Q is: ; Ra and Rb are independently straight-chain or branched C1-C6 alkyl groups, optionally substituted with hydroxyl groups.

[0062] According to another embodiment, optionally in combination with one or more features of the various embodiments described above, Q is a 5-membered or 6-membered ring containing an N atom and optionally a second heteroatom, the second heteroatom preferably selected from N and O.

[0063] In another implementation, optionally in combination with one or more features of the various implementations described above, Q is selected from the following structures:

[0064]

[0065]

[0066] In a preferred embodiment of formula (I), Q is N(Me)2; m=t=2; p=0; R' is H; X is selected from -NH-C(O)- and -C(O)-NH-; R1 is selected from C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 branched alkyl groups; and R2 is selected from C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 branched alkyl groups, and C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 alkenyl or dienyl groups. In a more preferred embodiment of formula (I), Q is N(Me)2; m=t=2; p=0; R' is H; X is selected from -NH-C(O)- and -C(O)-NH-; R1 is selected from C11, C15 and C19 branched alkyl groups; and R2 is selected from C11, C15 and C17 branched alkyl groups, and C17 alkenyl or dienyl groups.

[0067] In another preferred embodiment of formula (I), Q is N(Me)2; m=t=2; p=0; R' is H; X is selected from -C(O)-O- and -OC(O)-; R1 is selected from C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 branched alkyl groups; and R 2 is selected from C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 branched alkyl groups, and C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 straight-chain alkenyl or dienyl groups.

[0068] In a more preferred embodiment of formula (I), Q is N(Me)2; m=t=2; p=0; R' is H; X is selected from -C(O)-O- and -OC(O)-; R1 is selected from C12, C16 and C24 branched alkyl groups; and R2 is selected from C15 branched alkyl groups and C17 straight-chain alkenyl groups.

[0069] In another preferred embodiment of formula (I), Q is N(Me)2; m=t=2; p=0; R' is H; X is selected from -C(O)-S- and -SC(O)-; R1 is selected from C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 branched alkyl; and R2 is C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 branched alkyl.

[0070] In a more preferred embodiment of formula (I), Q is N(Me)2; m=t=2; p=0; R' is H; X is selected from -C(O)-S- and -SC(O)-; R1 is a C8 branched alkyl group; and R2 is a C15 branched alkyl group.

[0071] In another preferred embodiment of formula (I), Q is N(Et)2; m=t=2; p=0; R' is H; X is selected from -C(O)-O- and -OC(O)-; R1 is selected from C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 branched alkyl groups; and R 2 is selected from C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 branched alkyl groups, and C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 straight-chain alkenyl or dienyl groups.

[0072] In a more preferred embodiment of formula (I), Q is N(Et)2; m=t=2; p=0; R' is H; X is selected from -C(O)-O- and -OC(O)-; R1 is a C15 branched alkyl group; and R2 is a C17 alkenyl group.

[0073] In another preferred embodiment of formula (I), Q is a heterocycle containing at least one N atom, m=t=2; p=0; R' is H; X is selected from -NH-C(O)- and -C(O)-NH-; R1 is selected from C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 branches. Alkyl; and R2 is selected from C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 or C24 branched alkyl, and C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 and C24 straight-chain alkenyl or dienyl.

[0074] In a more preferred embodiment of formula (I), Q is a five-membered or six-membered heterocycle containing one or two N atoms, m=t=2; p=0; R' is H; X is selected from -NH-C(O)- and -C(O)-NH-; R1 is a C15 branched alkyl group; and R2 is a C17 straight-chain alkenyl group. Examples of ionizable lipids of formula (I) include the compounds shown in Table 1 below.

[0075] Table 1

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083] In one embodiment, the ionizable lipid of this disclosure is a compound selected from the following: (VC-LC-1272), (VC-LC-1284), (VC-LC-1285), (VC-LC-1286), (VC-LC-1287), (VC-LC-1288), (VC-LC-1289), (VC-LC-1293), (VC-LC-1297), (VC-LC-1311), (VC-LC-1369), (VC-LC-1371), (VC-LC-1373), (VC-LC-1374), (VC-LC-1376), (VC-LC-1377), (VC-LC-1378), (VC-LC-1539), (VC-LC-1540), (VC-LC-1541), (VC-LC-1543), and (VC-LC-1545).

[0084] In another embodiment, the ionizable lipid of formula (IA) is a compound selected from the following compounds described herein: (VC-LC-1272), (VC-LC-1284), (VC-LC-1285), (VC-LC-1286), (VC-LC-1287), (VC-LC-1288), (VC-LC-1289), (VC-LC-1293), (VC-LC-1297), (VC-LC-1311), (VC-L C-1369), (VC-LC-1371), (VC-LC-1373), (VC-LC-1374), (VC-LC-1376), (VC-LC-1377), (VC-LC-1378), (VC-LC-1539), (VC-LC-1540), (VC-LC-1541), (VC-LC-1543), (VC-LC-1545); or pharmaceutically acceptable salts thereof, or stereoisomers of any of them.

[0085] Preparation method The method for preparing ionizable lipids of formula (I) is also part of this invention.

[0086] All reactants and solvents required to prepare the compounds disclosed herein are commercially available.

[0087] As an example, the ionizable lipid of formula (I) can be prepared according to any of the following synthetic schemes. Those skilled in the art will recognize which reactants are required to obtain any particular ionizable lipid according to this disclosure, following the synthetic methods described below or similar methods.

[0088] The ionizable lipid of formula (I), where X is the amide moiety, can be prepared by the synthetic method described in Scheme 1 below or a similar method.

[0089]

[0090] Option 1 The ionizable lipid of formula (I), where X is the ester moiety, can be prepared by the synthetic method described in Scheme 2 below or a similar method.

[0091]

[0092] Option 2 The ionizable lipid of formula (I), where X is the thioester moiety, can be prepared by the synthetic method described in Scheme 3 below or a similar method.

[0093]

[0094] Option 3 lipid nanoparticles As described above, the ionizable lipids of this disclosure can form LNPs in solution.

[0095] Therefore, the present invention also relates to LNPs comprising ionizable lipids as defined herein (I).

[0096] All implementation schemes indicated for ionizable lipids of formula (I) are also applicable to LNP.

[0097] Typically, LNPs have a core-shell structure, comprising a core and an outer shell. LNP formulations containing multiple lipid components of different properties (e.g., ionizable lipids, sterols, PEGylated lipids, and non-cationic lipids (also known as "accessory lipids") such as phospholipids) comprise multiple phases separated into a hydrophobic core region formed by ionizable lipids and cholesterol, and a surrounding outer shell formed by accessory lipids, cholesterol, and PEGylated lipids covering the surface. More specifically, the ionizable lipids of formula (I) constitute part of the LNP inner shell, and optionally, the pharmaceutical active agent is encapsulated or loaded within the core.

[0098] In one embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP further comprises at least one lipid selected from: non-cationic lipids; sterols, steroid precursors or steroid derivatives; and PEG-modified lipids.

[0099] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP comprises the ionizable lipids and noncationic lipids of this disclosure. Examples of non-cationic lipids include, but are not limited to, 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate choline (SOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-eicosanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), and 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 Diether PC), 1-oleoyl-2-cholesterol hemisuccinoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicyl-sn-glycerol-3-phosphate choline, 1,2-docosahexaenoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0) The non-cationic lipids include 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, sodium 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol)phosphate (DOPG), sphingomyelin, and mixtures thereof. In one embodiment, the non-cationic lipid is DSPC. In a particular embodiment, the non-cationic lipid is DOPE. In another particular embodiment, the lipid-containing particles comprise both DSPC and DOPE.

[0100] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP further comprises a sterol or a sterol precursor.

[0101] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP comprises ionizable lipids and sterols, steroid precursors or steroid derivatives of this disclosure.

[0102] Examples of sterols include, but are not limited to, cholesterol, coccidosterol, sitosterol, ergosterol, campesterol, stigmasterol, phytosterol, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above, the sterol is cholesterol. Examples of sterol precursors include, but are not limited to, triterpenes, triterpenoids, or such steroid precursors. Non-limiting examples of triterpenes, triterpenoids, and other steroid precursors include squalene, achilleol, polypodatetrane, lanosterane, cucurbitacin, hopane, oleanane, chamoecydin, lupine, and mixtures thereof. The term "steroid derivative" refers to the simplest steroid derivative containing a cyclopentane polyhydrophenanthrene core (also known as a sterane) containing seventeen carbon atoms arranged in four fused rings. Steroid derivatives may include various modifications, such as different functional groups attached to the four-ring core or the oxidation state of the rings. Examples of steroidal derivatives include, but are not limited to, cholic acid, lanosterol, and β-sitosterol.

[0103] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP comprises ionizable lipids of this disclosure, non-cationic lipids as defined above, and sterols, steroid precursors, or steroid derivatives as defined above.

[0104] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP further comprises a PEG-modified lipid. The term "PEG-modified lipid" refers to a lipid comprising a polyethylene moiety. Examples of PEG-modified lipids include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified phosphatidylcholine, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also known as PEG-DOMG), PEG-DSG, PEG-DPG, or combinations thereof.

[0105] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP further comprises conjugated lipids.

[0106] Examples of conjugated lipids include, but are not limited to, polysarcosine (pSar) lipids and their derivatives, such as N-tetradecyl-pSar25, N-hexadecyl-pSar25, N-octadecyl-pSar25, N-dodecyl-pSar25, DMG-pSar25, 18:1 PE (DOPE) pSar25, N-TETAMINE-pSar25, N-TETAMINE-pSar35, N-TETAMINE-pSar45, and N-TETAMINE-pSar45-maleimide.

[0107] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP comprises ionizable lipids of this disclosure, non-cationic lipids as defined above, and PEG-modified lipids or conjugated lipids as defined above.

[0108] In one embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP comprises a lipid component, which comprises or consists of ionizable lipids, noncationic lipids, sterols, and PEG-modified lipids as disclosed herein.

[0109] In one particular embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP comprises a lipid component comprising an ionizable lipid as disclosed herein, and at least one of distearate phosphatidylcholine (DSPC), cholesterol, and DMG-PEG2000. In a more particular embodiment, the lipid component comprises or consists of an ionizable lipid as disclosed herein, DSPC, cholesterol, and DMG-PEG2000.

[0110] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP comprises 25 to 60 mol% ionizable lipids; 0.1 to 10 mol% PEG-modified lipids or alternatively conjugated lipids, particularly PEG-modified lipids; 10 to 45 mol% non-cationic lipids; and 10 to 40 mol% sterols. Specifically, the LNP comprises 32 to 50 mol% ionizable lipids, 1 to 8 mol% PEG-modified lipids or conjugated lipids, particularly PEG-modified lipids; 12.5 to 42 mol% non-cationic lipids; and 15 to 38.5 mol% sterols. More specifically, the LNP comprises 35 to 47 mol% ionizable lipids, 1 to 4 mol% PEG-modified lipids, 30 to 38 mol% non-cationic lipids, and 15 to 25 mol% sterols.

[0111] In another embodiment of the LNP, optionally in combination with one or more features of the above embodiments, the amount of the ionizable lipid is 25 to 64 mol%, the amount of the PEG-modified lipid is 0.1 to 1.5 mol%, and the amount of the sterol is 35 to 74 mol%.

[0112] As used in this article, “mol%” refers to the molar percentage of a component relative to the total number of moles of all lipid components in the LNP (i.e., the total number of moles of ionized lipids, PEG-modified lipids, non-cationic lipids, and sterols).

[0113] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the molar ratio of ionizable lipids to non-cationic lipids ranges from 6:1 to 1:2, or from 2:1 to 1:1.

[0114] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the molar ratio of ionizable lipids to sterols ranges from 5:1 to 1:2, or from 2:1 to 1:1.

[0115] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the molar ratio of ionizable lipids to PEG-modified lipids ranges from 120:1 to 2:1, or from 100:1 to 10:1.

[0116] Surfactant As described above, the LNP of the present invention may contain one or more pharmaceutically active agents.

[0117] As used herein, the term "pharmaceutical active agent" refers to a drug that has pharmacological activity and is intended to cure, alleviate, treat or prevent a disease in a subject (particularly a human).

[0118] For the purposes of this invention, pharmaceutical active agents include low molecular weight drugs, polynucleotides, peptides, antibodies, proteins, and combinations thereof.

[0119] As used herein, the term "polynucleotide" refers to natural or artificial deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The polynucleotide may contain at least one chemical modification selected from: pseudouridine, N1-methylpseudouridine (also known as 1-methylpseudouridine or m1Ψ), N6-methyladenosine (also known as m6A), 2-thiouridine (also known as s2U), 4'-thiouridine, and 5-methylcytosine (also known as 5mC). ), 2-thio-1-methyl-1-deaza-pseuuridine, 2-thio-1-methyl-pseuuridine, 2-thio-5-aza-uridine, 2-thio-dihydropseuuridine, 2-thio-dihydrouridine, 2-thio-pseuuridine, 4-methoxy-2-thio-pseuuridine, 4-methoxy-pseuuridine, 4-thio-1-methyl-pseuuridine, 4-thio-pseuuridine, 5-aza-uridine, dihydropseuuridine, 5-methyluridine (also known as m5U), 5-methoxyuridine, 2'-O-methyluridine, and combinations thereof. Specifically, the chemical modification is N1-methylpseuuridine, 5-methoxyuridine, or combinations thereof; particularly, the chemical modification is N1-methylpseuuridine.

[0120] The polynucleotide is partially modified with at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of N1-methylpseuuridine, or is completely modified with N1-methylpseuuridine, 5-methoxyuridine, or a combination thereof.

[0121] In one particular embodiment, optionally in combination with any of the above embodiments, the active agent is selected from polynucleotides, DNA constructs comprising a promoter operatively linked to a sequence encoding a polynucleotide, and expression vectors comprising a DNA construct containing a promoter operatively linked to a sequence encoding a polynucleotide.

[0122] In one particular embodiment, optionally in combination with any of the embodiments described above, the polynucleotide is ribonucleic acid (RNA).

[0123] Specifically, the RNA is selected from short interfering RNA (siRNA), self-replicating RNA (srRNA), circular RNA (circRNA), self-amplifying RNA (saRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), small interfering RNA (siRNA), small RNA (sRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

[0124] In one particular implementation, optionally in combination with any of the above implementations, the RNA is mRNA.

[0125] Those skilled in the art know how to produce the polynucleotide, DNA construct, or expression vector using conventional methods well known in the art (e.g., through chemical synthesis or molecular biology techniques) without any inventive effort.

[0126] In one particular embodiment, optionally in combination with any of the above embodiments, the ratio of ionizable lipids to RNA in the LNP (N / P; where N represents the number of moles of amines present in the ionizable lipids and P represents the number of moles of phosphate esters present in the polynucleotide backbone) ranges from 20:1 to 2:1, particularly from 10:1 to 3:1.

[0127] In one particular embodiment, optionally in combination with any of the embodiments described above, the polynucleotide is an isolated artificial polynucleotide.

[0128] In one embodiment, optionally in combination with one or more features of the various embodiments described above, the LNP comprises ionizable lipids, noncationic lipids, sterols, PEG-modified lipids, and polynucleotides as defined herein.

[0129] LNPs containing one or more polynucleotides can be prepared by standard methods, such as microfluidic mixing as disclosed in Hassett, KJ et al. (“Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines”, 2019, Mol. Ther. Nucleic Acid, Vol. 15, pp. 1-11), or manual / batch mixing as disclosed in Wang X., Liu S., Sun Y. et al. (“Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery”, 2022, Nat. Protoc., doi:10.1038 / s41596-022-00755-x). Both methods are known in the art, and those skilled in the art will understand how to operate in each specific situation.

[0130] Typically, methods for preparing LNPs include: i) preparing a first alcoholic mixture in a suitable alcohol (e.g., ethanol), the first alcoholic mixture comprising the ionizable lipids of this disclosure and optionally at least one lipid selected from non-cationic lipids, sterols, and PEG-modified lipids; ii) preparing a second aqueous composition comprising a polynucleotide and an acidification buffer; and iii) mixing i) and ii) in a microfluidic mixer. The microfluidic mixer allows for thorough and rapid mixing of the lipid and polynucleotide phases in a microscale device. Depending on process parameters, particularly the total flow rate, those skilled in the art will be able to adjust the size of the LNP.

[0131] In one particular embodiment, optionally in combination with any of the embodiments described above, the polynucleotide encodes a polypeptide, particularly, wherein the polypeptide is an antigen. More particularly, the antigen is selected from viral proteins, bacterial proteins, and tumor-associated antigens.

[0132] In one particular embodiment, optionally in combination with any of the embodiments described above, the polypeptide is an antibody or a fragment thereof. In a more specific embodiment, the antibody or a fragment thereof is a therapeutic antibody or a fragment thereof.

[0133] In another embodiment, optionally in combination with any of the above embodiments, the antigen is a SARS-CoV-2 antigen, particularly the SARS-CoV-2 spike antigen.

[0134] The methods for preparing the above-described lipid, LNP, and pharmaceutical compositions are described in this document and / or are known in the art. Those skilled in the art will know which LNP to use to encapsulate each pharmaceutical active agent based on the intended use of the composition. In particular, methods for synthesizing compositions formed from LNPs encapsulating RNA are well known to those skilled in the art and have been appropriately established in molecular biology protocols. Specific conditions are given in the examples.

[0135] As described above, another aspect of the present invention relates to a pharmaceutical composition comprising an LNP as defined herein and a pharmaceutically acceptable excipient or carrier.

[0136] The phrase "pharmaceuticalally acceptable excipient or carrier" refers to a pharmaceutically acceptable material, composition, or carrier. Each component must be pharmaceutically acceptable in the sense of compatibility with the other components of the pharmaceutical composition. It must also be suitable for contact with human and non-human animal tissues or organs without excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0137] Examples of suitable pharmaceutically acceptable excipients include solvents, dispersion media, diluents or other liquid carriers, dispersants or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc. Unless any conventional excipient medium is incompatible with the substance or its derivatives, for example by producing any undesirable biological effects or interacting in a harmful manner with any other component of the pharmaceutical composition, its use is considered within the scope of this invention.

[0138] The relative amounts of the active pharmaceutical ingredient, pharmaceutically acceptable excipients, and / or any additional ingredients in the pharmaceutical compositions of the present invention will vary depending on the identity, weight, and / or condition of the subject being treated, and further depending on the route of administration of the composition.

[0139] Pharmaceutically acceptable excipients used in the preparation of pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Excipients such as colorants, coating agents, sweeteners and flavoring agents may be present in the composition at the formulator's discretion.

[0140] In one embodiment, optionally in combination with any of the above embodiments, the pharmaceutical compositions disclosed herein are administered orally, intranasally, intravenously, intraperitoneally, intramuscularly, intradermally, subcutaneously, topically, or intra-articularly.

[0141] The pharmaceutical compositions disclosed herein can be prepared by methods well known in the pharmaceutical industry. For example, pharmaceutical compositions intended for administration by injection can be prepared by combining the lipid nanoparticles of the present invention with sterile distilled water or other carriers to form a solution. Excipients or carriers may be added to promote the formation of a homogeneous solution or suspension. As described above, LNPs containing pharmaceutically active agents or pharmaceutical compositions of the present disclosure can be used for therapeutic applications. In particular, they can be used as universal non-viral vectors for biomedical applications such as vaccines or gene therapy, and are effective for transfecting genetic material into eukaryotic cells.

[0142] Therefore, one aspect of the present invention relates to a method of treating a disease or condition of a subject as defined herein with an LNP or pharmaceutical composition, the method comprising administering to the subject a therapeutically effective amount of the nanoparticle composition or pharmaceutical composition as defined herein.

[0143] Another aspect of the invention relates to LNPs or pharmaceutical compositions as defined herein, used in methods for inducing an immune response in a subject, in methods for therapeutic immunization of a subject, as a vaccine, or for use in gene therapy. Specifically, the subject is a human being.

[0144] In one embodiment, optionally in combination with any of the above embodiments, the disease or condition is selected from infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0145] When the active pharmaceutical ingredient is a polynucleotide, the LNP or pharmaceutical composition described herein may be used for vaccine therapy, to enhance the efficacy of conventional vaccines and / or as a novel vaccine form for protection against infectious pathogens (such as viruses, bacteria, fungi, protozoa, prions and worms); or for the treatment of diseases such as cancer and proliferative diseases.

[0146] In one embodiment, optionally in combination with any of the embodiments described above, the pharmaceutical composition is a vaccine. In a more specific embodiment, the pharmaceutical composition is a vaccine and also contains an adjuvant. Those skilled in the art will know, based on their general knowledge, which excipients, carriers, and adjuvants are included in a vaccine according to its intended use.

[0147] If one of the structures shown in this document does not match its chemical name, the chemical structure shall prevail in all cases.

[0148] Throughout the specification and claims, the word "comprising" and its variations are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprising" covers situations where something "consists of...".

[0149] The following embodiments and accompanying drawings are provided by way of illustration and are not intended to limit the invention. Furthermore, the invention covers all possible combinations of the specific and preferred embodiments described herein.

[0150] Example Reagents were purchased from Sigma-Aldrich, TCI Chemicals, Fluorochem, or VWR. All possible sensible combinations shown above were prepared according to a three-step reaction protocol.

[0151] Example 1 - Synthesis of lipid VC-LC-1272

[0152] Step 1. Synthesis of intermediate VC-LC-9018

[0153] Scheme 4: Synthetic pathway of step 1 in the preparation of lipid VC-LC-1272 DL-homocysteine ​​thiolactone hydrochloride (270.4 mg, 1.76 mmol) was dissolved in 5 mL of anhydrous dichloromethane at room temperature. Triethylamine (252 µL, 1.8 mmol) was then added, followed by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (249.2 mg, 1.3 mmol), 4-(dimethylamino)pyridine (24.4 mg, 0.2 mmol), and 2-hexyldecanoic acid (256.42 mg, 1 mmol). The reaction mixture was stirred overnight at room temperature under an argon atmosphere. The crude product was then washed twice with distilled water (2 × 10 mL) and finally with saturated brine (10 mL). The organic layer was dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The resulting residue was purified by rapid chromatography (hexane / ethyl acetate gradient: 100 / 0 to 0 / 100) to give VC-LC-9018, a pale yellow oil (yield 89%).

[0154] The product was characterized by HPLC-ELSD-MS. Retention time: 6.02 min, MS (ES): experimental value m / z [M+H]+ 356.44 {theoretical value m / z [M+H]+ 356.26}.

[0155] Step 2. Synthesis of intermediate VC-LC-9121

[0156] Scheme 5: Synthetic pathway of step 2 in the preparation of lipid VC-LC-1272 The product of step 1 (VC-LC-9018, 249 mg, 0.7 mmol) was dissolved in anhydrous tetrahydrofuran (1.5 mL) and added under an argon atmosphere to a solution of N,N-dimethylethylenediamine (780 µL, 7.03 mmol) in anhydrous tetrahydrofuran (1.0 mL). The solution was then stirred at room temperature for 30 minutes, followed by evaporation of the solvent under reduced pressure. The dried mixture was redissolved in ethyl acetate (20 mL) and washed with 0.1 M HCl (aqueous solution) (20 mL, three consecutive washes), then with distilled water (20 mL, three consecutive washes), and finally with saturated brine (20 mL, three consecutive washes; NaCl:water was prepared by dissolving 35 g NaCl in 100 mL of water). The combined organic fractions were dried over anhydrous MgSO4 and then evaporated under reduced pressure to give VC-LC-9121 as a pale yellow oil (quantitative yield).

[0157] The product was characterized by HPLC-ELSD-MS. Retention time: 3.58 min, MS (ES): experimental value m / z [M+H] + 444.55 {Theoretical value m / z [M+H]} + 444.36}.

[0158] Step 3: The final step in the synthesis of lipid VC-LC-1272.

[0159] Scheme 6. The final step in the synthesis of lipid VC-LC-1272.

[0160] The product from the previous step (VC-LC-9121, 177.5 mg, 0.4 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (101.7 mg, 0.52 mmol), 4-(dimethylamino)pyridine (9.8 mg, 0.08 mmol), and oleic acid (153.8 µL, 0.48 mmol) were dissolved in 2.5 mL of anhydrous dichloromethane at room temperature. The reaction mixture was stirred overnight at room temperature under an argon atmosphere. The crude product was then redissolved in 40 mL of dichloromethane, washed twice with distilled water (2 × 10 mL), and finally washed with saturated brine (10 mL). The organic layer was dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The resulting residue was purified by rapid chromatography (dichloromethane / eluent A gradient: 100 / 0 to 0 / 100). After removing the solvent under reduced pressure, VC-LC-1272 was obtained as an oil (yield 31%).

[0161] *Eluent A: Dichloromethane / Methanol / Ammonium hydroxide (80:20:1) The product was characterized by HPLC-ELSD-MS. Retention time: 8.33 min, MS (ES): experimental value m / z [M+H] + 708.80 {Theoretical value m / z [M+H]} + 708.61}. VC-LC-1272 was characterized by HPLC-ELSD.

[0162] The general procedure for characterizing the obtained product by HPLC-ELSD-MS (HPLC Waters Alliance ELSD2424, column X Bridge BEH C8, 4.6 mm, 50 mm, 2.5 µm. Injection volume 2 µL, column temperature 65℃): gradient:

[0163] Sample preparation for reaction monitoring: Dissolve 5 µL of the crude reaction product in 100 µL of HPLC-MS grade acetonitrile in an HPLC sample tube. Vortex the tube manually for 5 seconds and then place it in the HPLC sample chamber.

[0164] Final product sample preparation: Dissolve 1 µL of dry lipid in HPLC sample tube in 100 µL of HPLC-MS grade acetonitrile.

[0165] Successful detection of the analytes was achieved using an evaporative light scattering detector (Waters ELSD 2424) and a single quadrupole mass spectrometer (positive ion mode, equipped with a Waters Acquity QDa spectrophotometer).

[0166] Retention time: 8.33 minutes, MS (ES): experimental value m / z [M+H] + 708.7955 {Theoretical value m / z [M+H]} + 710.6071}.

[0167] Example 2 – Synthesis of lipid VC-LC-1285

[0168] Step 1. Synthesis of intermediate VC-LC-9099

[0169] Scheme 7. Synthetic route of step 1 in the preparation of lipid VC-LC-9099 2-Oxotetrahydrothiophene-3-carboxylic acid (730 mg, 5 mmol) was dissolved in 13 mL of anhydrous dichloromethane at room temperature. Then, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (870 mg, 4.51 mmol) was added, followed by dimethylaminopyridine (104 mg, 0.85 mmol) and 2-hexyl-1-decanol (1.03 g, 4.25 mmol). The reaction was stirred overnight at room temperature, and the reaction was checked for completion by TLC. The resulting product was purified by rapid chromatography using a hexane / ethyl acetate gradient (100 / 0 to 0 / 100). The fractions containing the product were combined and evaporated under reduced pressure to give VC-LC-9099, a pale yellow oil (1.33 g, 84%).

[0170] The product was characterized by HPLC-ELSD (retention time: 8.21 min) and thin-layer chromatography (hexane: ethyl acetate).

[0171] Steps 2 and 3. The final steps in the synthesis of lipid VC-LC-1285.

[0172]

[0173] Scheme 8. The combined synthetic pathway of steps 2 and 3 in the preparation of lipid VC-LC-1285.

[0174] To obtain VC-LC-1285, steps 2 and 3 were performed, which were similar to the corresponding steps followed in the synthesis of lipid VC-LC-1272 described in Example 1. This resulted in a 21% combinatorial yield (quantitative yield and 21% yield for each of steps 2 and 3).

[0175] VC-LC-1285 was characterized by HPLC-ELSD-MS. Retention time: 9.37 min, MS (ES): experimental value m / z [M+H] + 723.85 {Theoretical value m / z [M+H]} + 723.61}.

[0176] VC-LC-1285 was characterized by HPLC-ELSD-MS following the detailed general procedure described above. Successful detection of the analytes was performed using an evaporative light scattering detector and a single quadrupole mass spectrometer (positive ion mode, equipped with a WatersAcquity QDa spectrophotometer).

[0177] Retention time: 9.37 minutes, MS (ES): experimental value m / z [M+H] + 723.8485 {Theoretical value m / z [M+H]} + 723.6068}.

[0178] Example 3 – Synthesis of lipid VC-LC-1311

[0179] The complete synthesis steps are shown in Scheme 9 below:

[0180] Scheme 9. Complete synthetic route for the preparation of VC-LC-1311 Step 1. Synthesis of intermediate VC-LC-9240

[0181] Scheme 10. Synthetic scheme for the preparation of intermediate VC-LC-9240 2-Hexyldecanoic acid (534 µL, 1.7 mmol) was dissolved in excess thionyl chloride (2 mL, 27.5 mmol), and stirred at 60ºC for 2 hours under an argon atmosphere. After the reaction mixture was checked by TLC to confirm the completion of the reaction, 2 mL of toluene was added to the crude product, and then evaporated under reduced pressure. After drying, the product was redissolved in 2 mL of dichloromethane and evaporated under reduced pressure until dry to give VC-LC-9240 in quantitative yield. VC-LC-9240 was not characterized.

[0182] Step 2. Synthesis of intermediate VC-LC-9248

[0183] Scheme 11. Synthetic scheme for the preparation of intermediate VC-LC-9248 2-Oxotetrahydrothiophene-3-carboxylic acid (640 mg, 4.66 mmol) was dissolved in 1.4 mL of anhydrous tetrahydrofuran under an argon atmosphere. Then, N,N-dimethylethylenediamine (776 µL, 7 mmol) was added and the mixture was stirred for 3 hours. The reaction was then checked for completeness by HPLC-ELSD, and the solvent was evaporated under reduced pressure. The product was obtained in quantitative yield and used directly in step 3 without further purification.

[0184] VC-LC-9248 was characterized by HPLC-ELSD-MS. Retention time: 0.58 min, MS (ES): experimental value m / z [M+H] + 235.23 {Theoretical value m / z [M+H]} + 235.11}.

[0185] Step 3. Synthesis of intermediate VC-LC-9261

[0186] Scheme 12. Synthetic scheme for the preparation of intermediate VC-LC-9261 Trifluoroacetic acid (1 mL, 1.3 mmol) was added to the product VC-LC-9248 (492 mg, 2.1 mmol), and the mixture was stirred at 0ºC for 30 min under an argon atmosphere. Subsequently, VC-LC-9240 (577 mg, 2.1 mmol) was added to the reaction mixture, and the mixture was stirred overnight at room temperature. The trifluoroacetic acid was then removed by distillation under reduced pressure, and the resulting product was purified by rapid chromatography using a dichloromethane / eluent B gradient (100 / 0 to 0 / 100). The fractions containing the product were combined and evaporated under reduced pressure to give pure VC-LC-9261 (21% yield).

[0187] Eluent B: Methanol / Ammonium hydroxide (99:1) VC-LC-9261 was characterized by HPLC-ELSD-MS. Retention time: 4.17 min, MS (ES): experimental value m / z [M+H] + 473.65 {Theoretical value m / z [M+H]} + 473.34}.

[0188] Step 4. The final step in the synthesis of lipid VC-LC-1311.

[0189] Scheme 13. The final synthetic step in the preparation of lipid VC-LC-1311. The product from the previous step (VC-LC-9261, 137 mg, 0.29 mmol), EDC hydrochloride (74 mg, 0.38 mmol), 4-(dimethylamino)pyridine (7.1 mg, 0.06 mmol), and 2-ethylhexyl mercaptan (103 µL, 0.58 mmol) were dissolved in 2 mL of anhydrous dichloromethane at room temperature. The reaction mixture was stirred overnight at room temperature under an argon atmosphere. The crude product was then redissolved in 40 mL of dichloromethane, washed twice with distilled water (2 × 10 mL), and finally washed with saturated brine (10 mL). The organic layer was dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The resulting residue was purified by rapid chromatography (dichloromethane / eluent A gradient: 100 / 0 to 0 / 100). After solvent removal under reduced pressure, VC-LC-1311 was given as an oil (10% yield).

[0190] Eluent A: Dichloromethane / Methanol / Ammonium hydroxide (80:20:1) VC-LC-1311 was characterized by HPLC-ELSD-MS. Retention time: 6.98 min, MS (ES): experimental value m / z [M+H] + 601.56 {Theoretical value m / z [M+H]} + 601.44}.

[0191] Eluent A: Dichloromethane / Methanol / Ammonium hydroxide (80:20:1) VC-LC-9241 was characterized by HPLC-ELSD-MS following the detailed general procedures described above. Retention time: 6.98 min, MS (ES): experimental value m / z [M+H] + 601.56 {Theoretical value m / z [M+H]} + 601.44}.

[0192] RNA encapsulated in LNP Encapsulation of mRNA containing the sequence encoding luciferase from 5' to 3' direction into LNPs is performed in some cases via microfluidics, or alternatively by manual means.

[0193] The microfluidic mixer was used, following the same procedure as described in Hassett, KJ et al., “Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines”, 2019, Mol. Ther. Nucleic Acid, Vol. 15, pp. 1-11. In short, the purified mRNA was first diluted in sodium citrate buffer at pH 4 to a final concentration of 266 µg / ml. Additionally, the ionizable lipids of this invention:DSPC (Merk850365P):cholesterol (Sigma C3045):DMG-PEG2000 (Cayman 33945-1) were dissolved in ethanol at a molar percentage of 50:10:38.5:1.5, with a lipid nitrogen to phosphate ratio (N:P ratio) of 5.5:1.

[0194] Alternatively, when LNPs are obtained manually, such as by manual / batch mixing as disclosed in Wang X., Liu S., Sun Y., et al. (“Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery”, 2022, Nat. Protoc., doi:10.1038 / s41596-022-00755-x), an aqueous solution of mRNA is carefully added to an ethanol solution, and the resulting solution is homogenized by aspiration 4-5 times. The resulting LNPs are immediately diluted 1:1 with Tris buffer and dialyzed overnight against Tris buffer containing 15% sucrose. The resulting LNP solution is then collected, and the encapsulated mRNA is evaluated using a Quant-IT® Ribogreen (Invitrogen R11490) according to the manufacturer’s instructions.

[0195] The LNP solution was then adjusted to a final mRNA concentration of 100 µg / ml. Particle size distribution, polydispersity, and Z-potential were measured by dynamic light scattering (DLS) using Malvern Zetasizer Advance Lab Blue Label. RNA encapsulation efficiency was evaluated using Quant-IT® Ribogreen according to the manufacturer's instructions.

[0196] Finally, the LNP solution was filtered through a 0.22 µm filter and the LNP was stored at -80 °C until needed.

[0197] Table 6 provides results for several parameters of the LNPs for the specific ionizable lipids shown therein, using the following standard formulations: Ionizable lipids: noncationic lipids: sterols: PEG-modified lipids → 50:10:38.5:1.5.

[0198] Table 6

[0199] As shown in Table 6, each of the tested compounds has an acceptable particle size for the intended purpose. Encapsulation efficiency (measured as the percentage of RNA embedded in the LNP) ranges from approximately 100% to approximately 15%. However, this only indicates the ease with which the nanoparticle components interact to form an LNP capable of encapsulating RNA. It does not indicate intracellular transfection efficiency. Intracellular transfection efficiency of mRNA encoding luciferase expression is measured by fluorescence intensity and expressed as total flux (p·s⁻¹). -1 Total flux is a bioluminescence measurement in terms of photons per second, or the average radiance of each pixel integrated over the target area.

[0200] For clarity, encapsulation efficiency refers to the approximate percentage of RNA encapsulated upon contact with LNP components. The observed total throughput values ​​indicate that LNPs were not immediately degraded in vivo after injection, and that cell transfection was indeed very successful, thus the contents encapsulated within the LNPs effectively reached the cytosol after endosome escape.

[0201] Example 4 – Protein expression in mouse muscle following administration of LNP prepared using the lipids of the present invention and containing mRNA as the active ingredient. mRNA (LNP) was administered to mice. Female BALB / c mice (Charles River Laboratories), 8-10 weeks old, weighing 18-23 g, acclimatized to their new environment for 3-7 days after arriving at the experimental facility. Housing conditions included a room temperature of 20-24 ºC, humidity of 50-70%, light intensity of 60 lux, and a 12-hour light-dark cycle.

[0202] To measure the activity of firefly luciferase in mice, LNPs prepared as described above (ionizable lipids: helper lipids: sterols: PEG-modified lipids → 50:10:38.5:1.5) containing 1 µg of the mRNA in a final volume of 30 µl were injected intramuscularly.

[0203] Four to 72 hours after RNA-LNP inoculation, mice were anesthetized by inhalation of 4% isoflurane using an vaporizer. Anesthesia was maintained using 1.5% isoflurane. Then, D-luciferin (Quimigen, 12507) was administered intraperitoneally at 150 mg / kg, typically approximately 200 µL of a 15 mg / mL stock solution in PBS for 20 g mice. Luteinase images were acquired 10 minutes after inoculation using the IVIS Lumina XRMS imaging system, following the manufacturer's instructions.

[0204] Table 7 below shows selected examples of LNPs containing ionizable lipids of formula (I) (with different combinations of R1 and R2) to demonstrate that the general structure of formula (I) produces the desired effect regardless of the selected substituents. Figure 1 The protein expression of the compounds shown in Table 7 in mice is depicted.

[0205] Table 7

[0206] Table 7 demonstrates that LNPs containing ionizable lipids of formula (I) can effectively encapsulate polynucleotides and generate high levels of cell transfection in vivo after administration.

[0207] Figure 1 Images of the total flux measured in mice given LNPs formulated with ionizable lipids VC-LC-1272, VC-LC-1285, and VC-LC-1289 are shown.

[0208] Example 5 – Comparative Study of Hydrolysis and Cracks of Selected Functional Groups To monitor the hydrolytic cleavage of thioester and ester bonds, three ionizable lipids of the same concentration were incubated for more than 4 hours under acidic conditions (pH=5.2) similar to those in the endosome. Therefore, the acidic cleavage of the ionizable lipids VC-LC-1282, SM-102, and VC-LC-0729 was carried out according to the proposed hydrolysis scheme, generating fragments A, B, and C, respectively, as shown in Scheme 14 below:

[0209]

[0210]

[0211] Option 14. Recommended hydrolysis schemes for VC-LC-1284, SM-102 and VC-LC-0729.

[0212] To measure the occurrence of the hydrolysis reaction, the appearance of fragments A, B, and C was monitored using HPLC-MS single-ion recording, with theoretical m / z values ​​as follows. Therefore, 50 mM stock solutions of lipids VC-LC-1284, SM-102, and VC-LC-0729 were prepared using DMSO (TCI, >99% purity). Then, 20 µL of the stock solution was added to a vial containing 60 µL of 2.4 M aqueous buffer (sodium acetate, Sigma-Aldrich, >99%) at pH 5.2 and 20 µL of DMSO (TCI, >99% purity). Measurements were started immediately after mixing, and the vial was kept under stirring throughout the experiment, with HPLC injections performed every 42 minutes.

[0213] Experimental method for the synthesis of VC-LC-0729 Step 1. Synthesis of intermediate VC-LC-9018

[0214] Scheme 15. Synthetic route of step 1 in the preparation of lipid VC-LC-0729.

[0215] DL-homocysteine ​​thiolactone hydrochloride (270.4 mg, 1.76 mmol) was dissolved in 5 mL of anhydrous dichloromethane at room temperature. Triethylamine (1620 µL, 1.8 mmol) was then added, followed by EDC hydrochloride (249.2 mg, 1.3 mmol), 4-(dimethylamino)pyridine (24.4 mg, 0.2 mmol), and 2-hexyldecanoic acid (256.42 mg, 1 mmol). The reaction mixture was stirred overnight at room temperature under an argon atmosphere. The crude product was then washed twice with distilled water (2 × 10 mL) and finally with saturated brine (10 mL). The organic layer was dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by rapid chromatography (hexane / ethyl acetate gradient: 100 / 0 to 0 / 100) to give VC-LC-9018 as a pale yellow oil (89% yield).

[0216] The product was characterized by HPLC-ELSD-MS. Retention time: 6.02 min, MS (ES): experimental value m / z [M+H] + 356.44 {Theoretical value m / z [M+H]} +356.26}.

[0217] Step 2. Synthesis of intermediate VC-LC-9121

[0218] Scheme 16. Synthetic pathway of step 2 in the preparation of lipid VC-LC-0729.

[0219] The product of step 1 (VC-LC-9018, 249 mg, 0.7 mmol) was dissolved in anhydrous tetrahydrofuran (1.5 mL) and added under argon atmosphere to a solution of N,N-dimethylethylenediamine (756 µL, 7.03 mmol) in anhydrous tetrahydrofuran (1.0 mL). The solution was then stirred at room temperature for 30 minutes, followed by evaporation of the solvent under reduced pressure. The dried mixture was redissolved in ethyl acetate (20 mL) and washed with 0.1 M HCl (aqueous solution) (20 mL, three consecutive washes), then with distilled water (20 mL, three consecutive washes), and finally with saturated brine (20 mL, three consecutive washes; NaCl:water was prepared by dissolving 35 g NaCl in 100 mL of water). The combined organic fractions were dried over anhydrous MgSO4 and then evaporated under reduced pressure to give VC-LC-9121 as a pale yellow oil (quantitative yield).

[0220] The product was characterized by HPLC-ELSD-MS. Retention time: 3.58 min, MS (ES): experimental value m / z [M+H] + 444.55 {Theoretical value m / z [M+H]} + 444.36}.

[0221] Step 3. Synthesis of intermediate VC-LC-9076

[0222] Scheme 17. Synthetic route of step 3 in the preparation of lipid VC-LC-0729.

[0223] Acryloyl chloride (0.335 mL, 4 mmol) was added dropwise to 2-hexyl-1-decyl alcohol (1.315 mL, 4.4 mmol) and triethylamine (0.843 mL, 6 mmol) in a cooled (0 ºC) solution in anhydrous dichloromethane (15 mL). The resulting solution was protected from light and stirred overnight under an argon atmosphere, allowing it to slowly rise to room temperature. The reaction was monitored by TLC (hexane:ethyl acetate, 6:4) until completion. The reaction mixture was then washed first with water (2 × 6 mL) and finally with brine (6 mL). The organic phase was dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. It was used directly for the next step without further purification.

[0224] Step 4. The final step in the synthesis of lipid VC-LC-0729.

[0225] Scheme 18. The synthetic route of step 4 in the preparation of lipid VC-LC-0729.

[0226] The product from step 2 (VC-LC-9121, 133.12 mg, 0.3 mmol) was added to a solution of tetrahydrofuran (1 mL) with triethylamine (42 µL, 0.3 mmol), followed by VC-LC-9076 (88.94 mg, 0.3 mmol). The reaction mixture was stirred overnight at room temperature, and the reaction was checked for completion by TLC. The resulting product was purified by column chromatography using a dichloromethane / dichloromethane:methanol:ammonium hydroxide (80:20:1) gradient (100 / 0 to 0 / 100). The fractions containing the product were combined and evaporated under reduced pressure to give VC-LC-0729 as an oil (77% yield).

[0227] The product was characterized by HPLC-ELSD-MS. Retention time: 8.19 min, MS (ES): experimental value m / z [M+H] + 740.85 {Theoretical value m / z [M+H]} + 740.63}.

[0228] HPLC Waters Alliance ELSD 2424 Column: X Bridge – Waters - BEH C8, 4.6 mm, 50 mm, 2.5 µm Gradient: (A: water containing 0.01% TFA; B: acetonitrile containing 0.01% TFA), 15 minutes, constant flow rate = 1 mL / min)

[0229] SM-102 is commercially available and was purchased from BOCSI (95% purity, catalog number B2699-358154).

[0230] Sample preparation for reaction monitoring: In an HPLC sample tube, dissolve 5 µL of the crude reaction product in 100 µL of HPLC-MS grade tetrahydrofuran. Then, manually vortex the sample tube for 5 seconds and place it in the HPLC sample chamber.

[0231] Final product sample preparation: In an HPLC sample tube, 1 µL of dried lipid was dissolved in 100 µL of HPLC-MS grade tetrahydrofuran. Then, the sample tube was manually vortexed for 5 seconds and placed in the HPLC sample chamber.

[0232] Injection volume: 2 µL Column temperature: 65 ℃.

[0233] Detection: Evaporative light scattering detector plus single quadrupole mass spectrometer (positive ion mode) (WatersAcquity QDa).

[0234] ELSD: Evaporative Light Scattering Detector (Waters ELSD 2424).

[0235] Characterization (MS): Single quadrupole mass spectrometer detector (Waters Acquity QDa).

[0236] The general procedure for characterizing the obtained fragments by HPLC-ELSD-MS (HPLC Waters Alliance ELSD2424, column X Bridge BEH C8, 4.6 mm, 50 mm, 2.5 µm. Injection volume 2 µL, column temperature 65 °C): Gradient: (A: water containing 0.01% TFA; B: acetonitrile containing 0.01% TFA), 15 minutes, constant flow rate = 1 mL / min.

[0237]

[0238] Table 8

[0239]

[0240] Table 9

[0241] Tables 8 and 9 summarize the data obtained using selected ion monitoring (SIR) HPLC-MS for the appearance of expected fragments generated during the hydrolysis of ionizable lipids.

[0242] The peak area of ​​each segment obtained in this experiment is as follows: Figure 2 As shown. Therefore, it is evident that the fragments corresponding to the hydrolysis of VC-LC-1284 appear at a much higher concentration compared to SM-102 and VC-LC-0729, indicating that VC-LC-1284 hydrolyzes more rapidly. This demonstrates that the ionizable lipids of this disclosure, comprising at least one amide moiety and one thioester moiety, exhibit superior degradability compared to SM-102 and VC-LC-0729, which are free of both amide and thioester (SM-102), or contain either an amide moiety but not a thioester moiety (VC-LC-0729). Importantly, VC-LC-1284 demonstrates that the ionizable lipids of this disclosure do not degrade before reaching their target and releasing their cargo into the system, but when they do degrade, their degradation occurs more rapidly, which is advantageous in avoiding several problems, such as a strong immune response.

[0243] Example 6 – A selected example of LNP prepared from ionizable lipids containing a shorter R1 (excluding formula (I)) in a study of protein expression after administration to mouse muscle.

[0244] Two ionizable lipids comprising R1 (straight-chain butyl and straight-chain nonyl) with shorter R1 alkyl substituents than those defined in Formula (I) or Formula (IA) were synthesized according to the methods described in this disclosure and administered to mice according to the same steps described in Example 4 above.

[0245] The structures and characteristic retention times of ionizable lipids VC-LC-1544 and VC-LC-1546 are summarized in Table 10 below.

[0246] Table 10

[0247] The mRNA containing the sequence encoding luciferase from 5' to 3' direction was encapsulated into an LNP and carried out via microfluidics in the same manner as described in Example 3 above.

[0248] Table 11 provides results for several parameters of the LNPs containing the specific ionizable lipids shown therein, using the following standard formulations: Ionizable lipids: noncationic lipids: sterols: PEG-modified lipids → 50:10:38.5:1.5.

[0249] Table 11

[0250] As shown in Table 11, the tested compounds exhibit acceptable particle size, PDI, Z potential, apparent pKa, and encapsulation efficiency exceeding 90% for both VC-LC-1544 and VC-LC-1546.

[0251] To measure the activity of firefly luciferase in mice, LNPs prepared as described above (ionizable lipids: helper lipids: sterols: PEG-modified lipids → 50:10:38.5:1.5) containing 1 µg of the mRNA (final volume 30 µl) were administered intramuscularly in the same manner as described in Example 4.

[0252] Table 12

[0253] Table 12 shows that LNPs containing ionizable lipids VC-LC-1544 and VC-LC-1546 (containing linear butyl or linear nonyl as R1 substituents) effectively encapsulate polynucleotides, but produce very low levels of cell transfection in vivo after administration. The observed total throughput indicates that cell transfection is three orders of magnitude lower compared to that observed with LNPs containing ionizable lipids of formulas (I) and (IA).

[0254] Example 7 – In vivo biodegradability test of LNP containing ionizable lipids of formula (I) encapsulating mRNA.

[0255] To evaluate the biodegradability of LNPs containing the ionizable lipids of this disclosure compared to commercially available ionizable lipids (as a comparison), LNPs containing either ionizable lipid VC-LC-1272 or SM-102 and 25 µg of the aforementioned mRNA (firefly luciferase), formulated with a standard composition (ionizable lipids: cofactor lipids: sterols: PEG-modified lipids → 50:10:38.5:1.5), were intravenously injected into 8-week-old BALB / c mice (n=3, female). The LNPs were diluted in Tris buffer containing 15% sucrose and administered via tail vein injection at a final volume of 250 µL using a 27 G syringe. Mice were euthanized by CO2 inhalation, and liver samples were collected at different time points post-injection, particularly at t=0, 1, 4, 24, and 120 hours post-injection.

[0256] 0.4 mg of liver tissue was diluted in 300 µL of isopropanol / ethanol, with an internal standard added. The liver tissue was dissociated using a GentleMACS dissociator (Miltenyi). The sample was sonicated for 15 min and centrifuged at 10000 g for 15 min. The supernatant was collected, concentrated using a SpeedVac system, diluted with a 1:1 mixture of isopropanol and ethanol, and analyzed against calibration standards. Chromatographic separation and quantification were performed using a liquid chromatography-MS system. The sample was injected and separated on a Bridgewaters-BEH C8 column equilibrated with 95% solvent A (acetonitrile:CAN:TFA 99:1) containing 0.01% trifluoroacetic acid. Signal detection was performed using a single quadrupole MS system (Waters Acquity QDa) in positive ion mode under SIR conditions.

[0257] The quantitative results of VC-LC-1272, metabolite M1 (which can be detected after hydrolysis of VC-LC-1272, as shown below), and SM-102 are summarized in Table 13 below.

[0258] Metabolite M1 Table 13

[0259] The results indicate that, on the one hand, the ionizable lipid VC-LC-1272 was only detectable at the very beginning of the experiment (t=0); it became undetectable only 1 hour after injection. On the other hand, the metabolite M1, generated from the partial hydrolysis of thioesters in VC-LC-1272, was detectable immediately after injection (t=0) and remained detectable until 4 hours after injection, demonstrating that the physiological conditions had an immediate effect and that LNPs degraded very rapidly in vivo.

[0260] In contrast, LNPs formulated with the commercially available ionizable lipid SM-102 were also monitored. Measurements showed that SM-102 was detectable from t=0, with concentrations increasing over the first 4 hours. A significant amount of SM-102 was still observed in mice after 24 hours. Finally, at t=120 hours post-injection, SM-102 was either undetectable or detected in negligible amounts.

[0261] Given the results shown in Table 13, it is clear that the biodegradability of LNPs formulated with ionizable lipids of formula (I) occurs much faster than that of LNPs formulated with commercially available ionizable lipids that do not contain a thioester moiety in their structure.

[0262] Example 8 – Stability of LNP over time LNPs for firefly luciferase mRNA encapsulated with an ionizable lipid comprising formula (I) of this disclosure (ionizable lipid VC-LC-1272:helper lipid:sterol:PEG-modified lipid → 50:10:38.5:1.5) were prepared as previously described. The resulting LNP solutions were collected and either lyophilized or kept in solution. In both cases, the samples were stored at T=4°C. The encapsulated mRNA was evaluated in mice via Quant-IT® Ribogreen (Invitrogen R11490) or intramuscular injection, following the manufacturer's instructions, in the same manner as described in Example 4.

[0263] LNPs were characterized at t=0 and t=30 days, and activity / transfection was assessed; the observed results are summarized in Table 14 below.

[0264] Table 14

[0265] As shown in Table 14, the LNP retained its properties with only a slight increase in particle size, and transfection and protein expression were maintained after 30 days at T=4°C, regardless of storage conditions (in solution or lyophilized). This experiment demonstrates that although the LNP of this disclosure contains an ionizable lipid of formula (I) with a thioester moiety (a more readily hydrolyzed functional group), it exhibits a long shelf life when stored at T=4°C. This is a surprisingly long lifespan compared to the rapid biodegradability test shown in Example 7.

[0266] Example 9 – Toxicity of LNPs Containing Ionizable Lipids of Formula (I) The concentrations of liver enzymes alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in mice (as indicators of abnormal liver function), as well as albumin and urea (as markers of liver synthetic function and kidney function), were measured to determine the toxicity of the ionizable lipids of formula (I) of this disclosure.

[0267] The encapsulation of an ionizable lipid (ionizable lipid VC-LC-1272:helper lipid:sterol:PEG-modified lipid → 50:10:38.5:1.5) comprising formula (I) of this disclosure for use as an LNP for firefly luciferase mRNA was prepared as described above.

[0268] Eight-week-old BALB / c mice (n=3, female) were intravenously injected with 50 µg FLuc mRNA encapsulated in LNP, or as a control, naked buffer was injected. LNP was diluted in Tris buffer containing 15% sucrose and administered via tail vein to a final volume of 250 µL using a 27 G syringe.

[0269] Blood samples were collected from the submandibular vein 48 hours after vaccination. The blood was allowed to clot and then centrifuged at 6500g and 4°C for 10 minutes to collect serum. Serum biochemical analysis was performed using a Cobas c-311 automated analyzer (Roche Diagnostics).

[0270] The results are summarized in Table 15 below.

[0271] Table 15

[0272] The results showed that only slight increases in the measured values ​​were observed in some cases. Fluctuations in these results are expected due to the variability in mice. However, none of the LNP compositions produced significant values ​​suggesting abnormal liver activity or damage, and all obtained values ​​were within the established reference limits for that mouse strain. Therefore, these tests confirm that LNP is safe for systemic administration in vivo.

[0273] This invention includes the following provisions: 1. Ionizable lipids of formula (I): Formula (I); Or its pharmaceutically acceptable salt, or a stereoisomer of any of them, wherein R' is selected from H, methyl, and ethyl; X is selected from -NH-C(O)-, -C(O)-NH-, -C(O)-O-, -OC(O)-, -SC(O)-, -C(O)-S-; m and p are independently selected from 0, 1, 2, 3, 4, 5 and 6; t is selected from 1, 2, and 3; Q is a heterocycle containing at least one N atom, or alternatively, Q is:

[0274] Ra and Rb are independently straight-chain or branched C1-C6 alkyl groups, which may optionally be substituted with hydroxyl groups; Furthermore, R1 and R2 are independently straight-chain or branched C1-C30 alkyl, C2-C30 alkenyl or C2-C30 alkynyl, wherein R1 and R2 are optionally substituted by one or more substituents selected from -OH, -COOR4 and -C(=O)SR4, wherein R4 is C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl; The condition is to exclude compounds: ; ; S-oleoyl-N-acetyl-L-cysteine-3-(dimethylaminopropylamine)-amide; and S-oleoyl-N-acetyl-L-cysteine-3-(dimethylaminopropylamine)-amide hydrochloride.

[0275] 2. The ionizable lipid described in Clause 1 or a pharmaceutically acceptable salt thereof, or a stereoisomer of any of them, wherein R1 is selected from straight-chain C11-C30 alkyl or branched C6-C30 alkyl, C6-C30 alkenyl, and C6-C30 alkynyl, and R2 is selected from straight-chain or branched C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl, wherein R1 and R2 are optionally substituted with one or more substituents selected from -OH, -COOR4, and -C(=O)SR4, wherein R4 is C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl.

[0276] 3. An ionizable lipid or a pharmaceutically acceptable salt thereof, or a stereoisomer of any of them, as described in Clause 1 or 2, wherein R' is selected from H and methyl; and m is selected from 1, 2, 3, and 4.

[0277] 4. The ionizable lipid according to Clause 1, wherein the compound of formula (I) is a compound of formula (IA): Formula (IA); Q, R', X, R1, R2 and m are as defined in Clause 1.

[0278] 5. The ionizable lipid according to Clause 4, wherein X is -NH-C(O)- or -C(O)-NH-.

[0279] 6. The ionizable lipid according to Clause 4, wherein X is -C(O)-O- or -OC(O)-.

[0280] 7. The ionizable lipid according to Clause 4, wherein X is -SC(O)- or -C(O)-S-.

[0281] 8. The ionizable lipid according to any one of clauses 5 to 7, wherein Q is: ; Ra and Rb are as disclosed in Clause 1.

[0282] 9. An ionizable lipid according to any one of clauses 5 to 7, wherein Q is a 5-membered or 6-membered ring comprising an N atom and optionally, preferably, a second heteroatom selected from N and O; Or, alternatively Where Q is selected from the following structure:

[0283]

[0284] .

[0285] 10. An ionizable lipid according to any one of clauses 1 to 9, wherein Q is selected from the following structures: .

[0286] 11. An ionizable lipid according to any one of clauses 4 to 10, wherein Q is N(Me)2; m=t=2; p=0; R' is H; X is selected from -NH-C(O)- and -C(O)-NH-; R1 is selected from C8-C24 branched alkyl; and R2 is selected from C10-C24 branched alkyl, as well as C10-C24 alkenyl or dienyl.

[0287] 12. An ionizable lipid according to any one of clauses 4 to 10, wherein Q is N(Me)2; m=t=2; p=0; R' is H; X is selected from -NH-C(O)- and -C(O)-NH-; R1 is selected from C11, C15 and C19 branched alkyl; and R2 is selected from C11, C15 and C17 branched alkyl, as well as C17 alkenyl or dienyl.

[0288] 13. An ionizable lipid according to any one of clauses 4 to 10, wherein Q is N(Me)2; m=t=2; p=0; R' is H; X is selected from -C(O)-O- and -OC(O)-; R1 is selected from C8-C24 branched alkyl; and R2 is selected from C10-C24 branched alkyl and C10-C24 straight-chain alkenyl or dienyl.

[0289] 14. An ionizable lipid according to any one of clauses 4 to 10, wherein Q is N(Me)2; m=t=2; p=0; R' is H; X is selected from -C(O)-O- and -OC(O)-; R1 is selected from C12, C16 and C24 branched alkyl groups; and R2 is selected from C15 branched alkyl groups and C17 straight-chain alkenyl groups.

[0290] 15. An ionizable lipid according to any one of clauses 4 to 10, wherein Q is N(Me)2; m=t=2; p=0; R' is H; X is selected from -C(O)-S- and -SC(O)-; R1 is selected from C8-C24 branched alkyl; and R2 is C10-C24 branched alkyl.

[0291] 16. An ionizable lipid according to any one of clauses 4 to 10, wherein Q is N(Me)2; m=t=2; p=0; R' is H; X is selected from -C(O)-S- and -SC(O)-; R1 is a C8 branched alkyl group; and R2 is a C15 branched alkyl group.

[0292] 17. An ionizable lipid according to any one of clauses 4 to 10, wherein Q is N(Et)2; m=t=2; p=0; R' is H; X is selected from -C(O)-O- and -OC(O)-; R1 is selected from C8-C24 branched alkyl; and R2 is selected from C10-C24 branched alkyl and C10-C24 straight-chain alkenyl or dienyl.

[0293] 18. An ionizable lipid according to any one of clauses 4 to 10, wherein Q is N(Et)2; m=t=2; p=0; R' is H; X is selected from -C(O)-O- and -OC(O)-; R1 is a C15 branched alkyl group; and R2 is a C17 alkenyl group.

[0294] 19. An ionizable lipid according to any one of clauses 4 to 10, wherein Q is a heterocycle containing at least one N atom, m=t=2; p=0; R' is H; X is selected from -NH-C(O)- and -C(O)-NH-; R1 is selected from C8-C24 branched alkyl; and R2 is selected from C10-C24 branched alkyl and C10-C24 straight-chain alkenyl or dienyl.

[0295] 20. An ionizable lipid according to any one of clauses 4 to 10, wherein Q is a five-membered or six-membered heterocycle containing one or two N atoms, m=t=2; p=0; R' is H; X is selected from -NH-C(O)- and -C(O)-NH-; R1 is a C15 branched alkyl group; and R2 is a C17 straight-chain alkenyl group.

[0296] 21. The ionizable lipid according to Clause 1, wherein the lipid is selected from the group consisting of: S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-hexyldecanoyl)-4-oxobutyl) (Z)-octadecano-9-enylthioate (VC-LC-1272), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-hexyldecanoyl)-4-oxobutyl) 2-Hexyldecylthioester (VC-LC-1284), 2-Hexyldecyl 2-((2-(dimethylamino)ethyl)carbamoyl)-4-(oleoylthio)butyrate (VC-LC-1285), 2-Hexyldecyl 2-((2-(dimethylamino)ethyl)carbamoyl)-4-((2-hexyldecyl)thio)butyrate (VC-LC-1286), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-hexyldecamido)-4-oxobutyl) (9Z,12Z)-octadec-9,12-Dienylthioester (VC-LC-1287), 2-Butyloctyl 2-((2-(dimethylamino)ethyl)carbamoyl)-4-(oleoylthio)butyrate (VC-LC-1288), 2-Butyloctyl 2-((2-(dimethylamino)ethyl)carbamoyl)-4-((2-hexyldecanoyl)thio)butyrate (VC-LC-1289), 2-Decyltetradecyl 2-((2-( Dimethylamino)ethyl)carbamoyl)-4-((2-hexyldecanoyl)thio)butyrate (VC-LC-1293), 2-decyltetradecyl 2-((2-(dimethylamino)ethyl)carbamoyl)-4-(oleoylthio)butyrate (VC-LC-1297), S-(4-((2-(dimethylamino)ethyl)amino)-3-(((2-ethylhexyl)thio)carbonyl)-4-oxobutyl) 2-Hexyldecylthioester (VC-LC-1311), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-hexyldecamido)-4-oxobutyl) 2-Butyloctylthioester (VC-LC-1369), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-hexyldecamido)-4-oxobutyl) 2-Heptylundecylthioester (VC-LC-1371), S-(3-(2-butyloctamido)-4-((2-(dimethylamino)ethyl)amino)-4-oxobutyl) 2-Hexyldecylthioester (VC-LC-1373), S-(3-(2-butyloctamido)-4-((2-(dimethylamino)ethyl)amino)-4-oxobutyl)(Z)-octadec-9-enthioester (VC-LC-1374), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-octyldodecanoamido)-4-oxobutyl) (Z)-octadec-9-enthioester (VC-LC-1376), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-octyldodecanoamido)-4-oxobutyl) (9Z,12Z)-octadec-9,12-Dienylthioester (VC-LC-1377), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-octyldodecanoamide)-4-oxobutyl) 2-Hexyldecylthioester (VC-LC-1378), S-(3-(2-hexyldecanoamide)-4-oxo-4-((2-(pyrrolidin-1-yl)ethyl)amino)butyl) (Z)-Octadeca-9-enylthioester (VC-LC-1539), S-(3-(2-hexyldecanoamide)-4-oxo-4-((2-(piperidin-1-yl)ethyl)amino)butyl) (Z)-Octadeca-9-enthioester (VC-LC-1540), S-(4-((2-(1H-imidazol-1-yl)ethyl)amino)-3-(2-hexyldecanoyl)-4-oxobutyl) (Z)-Octadeca-9-enthioester (VC-LC-1541), S-(3-(2-hexyldecanoyl)-4-((1-methylpiperidin-4-yl)amino)-4-oxobutyl) (Z)-Octadeca-9-enthioester (VC-LC-1543), S-(4-((2-(diethylamino)ethyl)amino)-3-(2-hexyldecanoyl)-4-oxobutyl) (Z)-Octadeca-9-enthioester (VC-LC-1545).

[0297] 22. Lipid nanoparticles comprising ionizable lipids of formula (I): Formula (I), Or its pharmaceutically acceptable salt, or a stereoisomer of any of them, wherein R' is selected from H, methyl, and ethyl; X is selected from -NH-C(O)-, -C(O)-NH-, -C(O)-O-, -OC(O)-, -SC(O)-, -C(O)-S-; m and p are independently selected from 0, 1, 2, 3, 4, 5 and 6; t is selected from 1, 2, and 3; Q is a heterocycle containing at least one N atom, or alternatively, Q is...

[0298] Ra and Rb are independently straight-chain or branched C1-C6 alkyl groups, which may optionally be substituted with hydroxyl groups; Furthermore, R1 and R2 are independently straight-chain or branched C1-C30 alkyl, C2-C30 alkenyl or C2-C30 alkynyl, wherein R1 and R2 are optionally substituted by one or more substituents selected from -OH, -COOR4 and -C(=O)SR4, wherein R4 is C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl; Or any one of the ionizable lipids described in Clauses 2 to 21.

[0299] 23. The lipid nanoparticles of claim 22, wherein the compounds S-oleoyl-N-acetyl-L-cysteine-3-(dimethylaminopropylamine)-amide and S-oleoyl-N-acetyl-L-cysteine-3-(dimethylaminopropylamine)-amide hydrochloride are excluded from the ionizable lipids of formula (I).

[0300] 24. The lipid nanoparticles according to clause 22 or 23 further comprise: i) Non-cationic lipids; Optionally, the non-cationic lipid is selected from: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate choline (SOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearate-sn- Glyceryl-3-phosphate choline (DSPC), 1,2-eicosanoyl-sn-glyceryl-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glyceryl-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glyceryl-3-phosphate choline (18:0 diether PC), 1-oleoyl-2-cholesterol hemisuccinoyl-sn-glyceryl-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glyceryl-3-phosphate choline (C16 Lyso PC), 1,2-dilinoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidanoyl-sn-glycerol-3-phosphate choline, 1,2-docosahexaenoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0) PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof; Optionally, the non-cationic lipid is DOPE; and / or the non-cationic lipid is DSPC; ii) Sterols, steroid precursors, or steroid derivatives; Optionally, the sterol is selected from cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, squalene, tomatine, ursolic acid, α-tocopherol, and mixtures thereof; Further optionally, said sterol is cholesterol; and iii) Conjugated lipids or alternatively PEG-modified lipids; Optionally, the PEG-modified lipid is selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified phosphatidylcholine, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

[0301] 25. The lipid nanoparticles according to Clause 24, wherein the amount of ionizable lipid is 25 to 60 mol%, the amount of PEG-modified lipid or conjugated lipid is 0.1 to 10 mol%, the amount of non-cationic lipid is 10 to 45 mol%, and the amount of sterol is 10 to 40 mol%. Or, alternatively The amount of the ionizable lipid is 25 to 64 mol, the amount of the PEG-modified lipid or the conjugated lipid is 0.1 to 1.5 mol, and the amount of the sterol, steroid precursor or steroid derivative is 35 to 74 mol.

[0302] 26. The lipid nanoparticles according to any one of clauses 22 to 25 further comprise a pharmaceutically active agent.

[0303] 27. The lipid nanoparticles according to Clause 26, wherein the pharmaceutical active agent is selected from polynucleotides, DNA constructs comprising a promoter operatively linked to a sequence encoding a polynucleotide, and expression vectors comprising a DNA construct containing a promoter operatively linked to a sequence encoding a polynucleotide.

[0304] 28. The lipid nanoparticles according to Clause 27, wherein the polynucleotide is a natural or artificial deoxyribonucleic acid (DNA) or a natural or artificial ribonucleic acid (RNA). Further optionally, the polynucleotide comprises at least one chemical modification selected from pseudouridine, N1-methylpseudouridine (also known as 1-methylpseudouridine or m1Ψ), N6-methyladenosine (also known as m6A), 2-thiouridine (also known as s2U), 4'-thiouridine, 5-methylcytidine (also known as 5mC), 2-thio-1-methyl-1-deazopseudouridine, 2-thio-1-methylpseudouridine, 2-thio-5-azauridine, 2-thiodihydro... Pseudouridine, 2-thiodihydrouridine, 2-thiopseudouridine, 4-methoxy-2-thiopseudouridine, 4-methoxypseudouridine, 4-thio-1-methylpseudouridine, 4-thiopseudouridine, 5-azauridine, dihydropseudouridine, 5-methyluridine (also known as m5U), 5-methoxyuridine, 2'-O-methyluridine, and combinations thereof; specifically, the chemical modification is N1-methylpseudouridine, 5-methoxyuridine, or combinations thereof; specifically, the chemical modification is N1-methylpseudouridine. Further optionally, the polynucleotide is ribonucleic acid (RNA). Further optionally, the RNA is selected from short interfering RNA (siRNA), self-replicating RNA (srRNA), circular RNA (circRNA), self-amplifying RNA (saRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), small interfering RNA (siRNA), small RNA (sRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof; specifically, the RNA is mRNA.

[0305] 29. A pharmaceutical composition comprising lipid nanoparticles as defined in any one of Clauses 22 to 28 and a pharmaceutically acceptable excipient or carrier.

[0306] 30. A method of treating a disease or condition in a subject as defined in any one of Clauses 22 to 28 or a pharmaceutical composition as defined in Clause 17, wherein the method comprises administering to the subject a therapeutically effective amount of the nanoparticle composition or the pharmaceutical composition; Optionally, the disease or condition is selected from infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases; Further, optionally, the subject is a human being; Alternatively, in methods for inducing an immune response in subjects, in therapeutic immunization methods for subjects, as a vaccine, or in gene therapy; Alternatively, it can be used to prevent or treat COVID-19.

[0307] 31. Use of lipid nanoparticles as encapsulating agents as defined in any of Clauses 22 to 25.

[0308] Reference List 1. Molla MR, et al. (cf. Molla MR, et al. "One-Pot Parallel Synthesis of Lipid Library via Thiolactone Ring Opening and Screening for GeneDelivery". Bioconjug Chem. 2018, vol. 29(4), pp. 992-999. doi: 10.1021 / acs.bioconjchem.8b00007. 2. Churusova, S. et al. (2021). Palladium(II) Pincer Complexes ofFunctionalized Amides with S-Modified Cysteine ​​and Homocysteine ​​Residues: Cytotoxic Activity and Different Aspects of Their Biological Effect on LivingCells. Inorganic Chemistry. 2021, vol. 60, pp. 9880−9898. 3. US4929736. 4. Garbiras, BJ; Marburg, S. "Preparation of Carboxythiolactonesand Their Active Derivatives". Synthesis, 1999, vol. 2, pp. 270–274; doi:10.1055 / s-1999-3377. 5. Hassett, K. J. et al., "Optimization of Lipid Nanoparticles forIntramuscular Administration of mRNA Vaccines", 2019, Mol. Ther. NucleicAcid, vol. 15, pp. 1–11, DOI:10.1016 / j.omtn.2019.01.013. 6. Wang X., Liu S., Sun Y., et al. “Preparation of selective organ-targeting (SORT) lipid nanaoparticles (LNPs) using multiple technical methodsfor tissue-specific mRNA delivery”, Nat. Protoc., 2022, doi:10.1038 / s41596-022-00755-x.

Claims

1. Ionizable lipids of formula (I): Equation (I); Or its pharmaceutically acceptable salt, or a stereoisomer of any of them, wherein R' is selected from H, methyl, and ethyl; X is selected from -NH-C(O)-, -C(O)-NH-, -C(O)-O-, -OC(O)-, -SC(O)-, -C(O)-S-; m and p are independently selected from 0, 1, 2, 3, 4, 5 and 6; t is selected from 1, 2, and 3; Q is a heterocycle containing at least one N atom, or alternatively, Q is: ； Ra and Rb are independently straight-chain or branched C1-C6 alkyl groups, which may optionally be substituted with hydroxyl groups; Furthermore, R1 is selected from straight-chain C11-C30 alkyl or branched C6-C30 alkyl, C6-C30 alkenyl and C6-C30 alkynyl, and R2 is selected from straight-chain or branched C6-C30 alkyl, C6-C30 alkenyl or C6-C30 alkynyl, wherein R1 and R2 are optionally substituted by one or more substituents selected from -OH, -COOR4 and -C(=O)SR4, wherein R4 is C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl.

2. The ionizable lipid according to claim 1 or a pharmaceutically acceptable salt thereof, or a stereoisomer of any of them, wherein R' is selected from H and methyl; and m is selected from 1, 2, 3 and 4.

3. The ionizable lipid according to claim 1, wherein the compound of formula (I) is a compound of formula (IA): Expression (IA); Wherein Q, R', X, R1, R2 and m are as defined in claim 1.

4. The ionizable lipid according to claim 3, wherein X is -NH-C(O)- or -C(O)-NH-; or wherein X is -C(O)-O- or -OC(O)-; or wherein X is -C(O)-S- or -SC(O)-.

5. The ionizable lipid according to claim 4, wherein Q is: ； Ra and Rb are independently straight-chain or branched C1-C6 alkyl groups, which may optionally be substituted with hydroxyl groups.

6. The ionizable lipid according to claim 4 or 5, wherein Q is a 5-membered or 6-membered ring comprising an N atom and optionally, preferably, a second heteroatom selected from N and O; Or, alternatively Where Q is selected from the following structure: ； 。 7. The ionizable lipid of claim 1, wherein the lipid is selected from the group consisting of: S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-hexyldecanoyl)-4-oxobutyl) (Z)-octadecano-9-enylthioate (VC-LC-1272), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-hexyldecanoyl)-4-oxobutyl) 2-Hexyldecylthioester (VC-LC-1284), 2-Hexyldecyl 2-((2-(dimethylamino)ethyl)carbamoyl)-4-(oleoylthio)butyrate (VC-LC-1285), 2-Hexyldecyl 2-((2-(dimethylamino)ethyl)carbamoyl)-4-((2-hexyldecyl)thio)butyrate (VC-LC-1286), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-hexyldecamido)-4-oxobutyl) (9Z,12Z)-octadec-9,12-Dienylthioester (VC-LC-1287), 2-Butyloctyl 2-((2-(dimethylamino)ethyl)carbamoyl)-4-(oleoylthio)butyrate (VC-LC-1288), 2-Butyloctyl 2-((2-(dimethylamino)ethyl)carbamoyl)-4-((2-hexyldecanoyl)thio)butyrate (VC-LC-1289), 2-Decyltetradecyl 2-((2-( Dimethylamino)ethyl)carbamoyl)-4-((2-hexyldecanoyl)thio)butyrate (VC-LC-1293), 2-decyltetradecyl 2-((2-(dimethylamino)ethyl)carbamoyl)-4-(oleoylthio)butyrate (VC-LC-1297), S-(4-((2-(dimethylamino)ethyl)amino)-3-(((2-ethylhexyl)thio)carbonyl)-4-oxobutyl) 2-Hexyldecylthioester (VC-LC-1311), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-hexyldecamido)-4-oxobutyl) 2-Butyloctylthioester (VC-LC-1369), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-hexyldecamido)-4-oxobutyl) 2-Heptylundecylthioester (VC-LC-1371), S-(3-(2-butyloctamido)-4-((2-(dimethylamino)ethyl)amino)-4-oxobutyl) 2-Hexyldecylthioester (VC-LC-1373), S-(3-(2-butyloctamido)-4-((2-(dimethylamino)ethyl)amino)-4-oxobutyl)(Z)-octadec-9-enthioester (VC-LC-1374), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-octyldodecanoamido)-4-oxobutyl) (Z)-octadec-9-enthioester (VC-LC-1376), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-octyldodecanoamido)-4-oxobutyl) (9Z,12Z)-octadec-9,12-Dienylthioester (VC-LC-1377), S-(4-((2-(dimethylamino)ethyl)amino)-3-(2-octyldodecanoamide)-4-oxobutyl) 2-Hexyldecylthioester (VC-LC-1378), S-(3-(2-hexyldecanoamide)-4-oxo-4-((2-(pyrrolidin-1-yl)ethyl)amino)butyl) (Z)-Octadeca-9-enylthioester (VC-LC-1539), S-(3-(2-hexyldecanoamide)-4-oxo-4-((2-(piperidin-1-yl)ethyl)amino)butyl) (Z)-Octadeca-9-enthioester (VC-LC-1540), S-(4-((2-(1H-imidazol-1-yl)ethyl)amino)-3-(2-hexyldecanoyl)-4-oxobutyl) (Z)-Octadeca-9-enthioester (VC-LC-1541), S-(3-(2-hexyldecanoyl)-4-((1-methylpiperidin-4-yl)amino)-4-oxobutyl) (Z)-Octadeca-9-enthioester (VC-LC-1543), S-(4-((2-(diethylamino)ethyl)amino)-3-(2-hexyldecanoyl)-4-oxobutyl) (Z)-Octadeca-9-enthioester (VC-LC-1545).

8. Lipid nanoparticles comprising ionizable lipids as defined in any one of claims 1 to 7.

9. The lipid nanoparticles according to claim 8, further comprising: i) Non-cationic lipids; Optionally, the non-cationic lipid is selected from: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate choline (SOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearate-sn- Glyceryl-3-phosphate choline (DSPC), 1,2-eicosanoyl-sn-glyceryl-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glyceryl-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glyceryl-3-phosphate choline (18:0 diether PC), 1-oleoyl-2-cholesterol hemisuccinoyl-sn-glyceryl-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glyceryl-3-phosphate choline (C16 Lyso PC), 1,2-dilinoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidanoyl-sn-glycerol-3-phosphate choline, 1,2-docosahexaenoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0) PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof; Optionally, the non-cationic lipid is DOPE; and / or the non-cationic lipid is DSPC; ii) Sterols, steroid precursors, or steroid derivatives; Optionally, the sterol is selected from cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, squalene, tomatine, ursolic acid, α-tocopherol, and mixtures thereof; Further optionally, said sterol is cholesterol; and iii) Conjugated lipids or alternatively PEG-modified lipids; Optionally, the PEG-modified lipid is selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified phosphatidylcholine, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

10. The lipid nanoparticles according to claim 9, wherein the amount of ionizable lipid is 25 to 60 mol%, the amount of PEG-modified lipid or conjugated lipid is 0.1 to 10 mol%, the amount of non-cationic lipid is 10 to 45 mol%, and the amount of sterol, steroid precursor or steroid derivative is 10 to 40 mol%. Or, alternatively The amount of the ionizable lipid is 25 to 64 mol, the amount of the PEG-modified lipid or the conjugated lipid is 0.1 to 1.5 mol, and the amount of the sterol, steroid precursor or steroid derivative is 35 to 74 mol.

11. The lipid nanoparticles according to any one of claims 8 to 10, further comprising a pharmaceutically active agent.

12. The lipid nanoparticles of claim 11, wherein the pharmaceutical active agent is selected from polynucleotides, DNA constructs comprising a promoter operably linked to a sequence encoding a polynucleotide, and expression vectors comprising a DNA construct containing a promoter operably linked to a sequence encoding a polynucleotide. Optionally, the polynucleotide is a natural or artificial deoxyribonucleic acid (DNA) or a natural or artificial ribonucleic acid (RNA). Further optionally, the polynucleotide comprises at least one chemical modification selected from pseudouridine, N1-methylpseudouridine (also known as 1-methylpseudouridine or m1Ψ), N6-methyladenosine (also known as m6A), 2-thiouridine (also known as s2U), 4'-thiouridine, 5-methylcytidine (also known as 5mC), 2-thio-1-methyl-1-deazopseudouridine, 2-thio-1-methylpseudouridine, 2-thio-5-azauridine, 2-thiodihydro... Pseudouridine, 2-thiodihydrouridine, 2-thiopseudouridine, 4-methoxy-2-thiopseudouridine, 4-methoxypseudouridine, 4-thio-1-methylpseudouridine, 4-thiopseudouridine, 5-azauridine, dihydropseudouridine, 5-methyluridine (also known as m5U), 5-methoxyuridine, 2'-O-methyluridine, and combinations thereof; specifically, the chemical modification is N1-methylpseudouridine, 5-methoxyuridine, or combinations thereof; specifically, the chemical modification is N1-methylpseudouridine. Further optionally, the polynucleotide is ribonucleic acid (RNA). Further optionally, the RNA is selected from short interfering RNA (siRNA), self-replicating RNA (srRNA), circular RNA (circRNA), self-amplifying RNA (saRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), small interfering RNA (siRNA), small RNA (sRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof; specifically, the RNA is mRNA.

13. A pharmaceutical composition comprising lipid nanoparticles as defined in any one of claims 11 to 12 and a pharmaceutically acceptable excipient or carrier.

14. A method of treating a disease or condition of a subject in need of a lipid nanoparticle as defined in any one of claims 11 to 12 or a pharmaceutical composition as defined in claim 13, the method comprising administering to the subject a therapeutically effective amount of the nanoparticle composition or the pharmaceutical composition; Optionally, the disease or condition is selected from infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases; Further, optionally, the subject is a human being; Alternatively, in methods for inducing an immune response in subjects, in therapeutic immunization methods for subjects, as a vaccine, or in gene therapy; Alternatively, it can be used to prevent or treat COVID-19.

15. Use of lipid nanoparticles as an encapsulating agent as defined in any one of claims 8 to 14.

Images