Lipids and uses thereof

Abstract

The present disclosure provides an ionizable amine-containing lipid. In particular, the ionizable amine-containing lipids are capable of delivering nucleic acids.

Description

Technical Field

[0001] The present disclosure relates to lipids and the use of these lipids for the delivery of active agents. Background Art

[0002] Lipid nanoparticles (LNPs) composed of ionizable amine-containing lipids are expected to be used for the delivery of bioactive agents, especially for the delivery of nucleotides such as antisense oligonucleotides (ASOs), siRNAs, miRNAs, mRNAs, and guide RNAs (gRNAs) into cells. These LNPs with ionizable amine-containing lipids play an important role in promoting the efficient transport of oligonucleotide agents across cell membranes. In addition, these LNPs also provide an important method for introducing gene editing components and compositions into living cells, thereby promoting the development of the field of gene manipulation.

[0003] The successful cellular uptake of bioactive agents, such as proteins, nucleic acid-based drugs and their derivatives, remains a huge challenge and is even more difficult for drugs containing larger oligonucleotides such as mRNA. Summary of the invention

[0004] The present disclosure provides an ionizable amine-containing lipid / composition that can enhance the delivery of gene editing technologies into cells, such as the delivery of components of the CRISPR / Cas9 system. One example of an ionizable amine-containing lipid / composition involves the use of an mRNA encoding a nuclease and its corresponding guide RNA as a means of achieving effective delivery.

[0005] The present disclosure demonstrates the use of ionizable amine-containing lipids and salts thereof (including pharmaceutically acceptable salts) for the effective delivery of biologically active agents. These ionizable amine-containing lipids and salts are valuable for delivering these active agents into cells, thereby promoting the production of engineered cells. In addition, the disclosed ionizable amine-containing lipids have demonstrated their applicability as lipids with ionizable properties in the development of lipid nanoparticle-based compositions.

[0006] In one aspect, the present disclosure provides a lipid of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer or isotopically enriched derivative thereof,

[0007]

[0008] in:

[0009] n is 1 or 2;

[0010] R 1 is amino, monoalkylamino, dialkylamino, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidinyl or pyrinyl;

[0011] R 2 and R 3 Each is independently an alkyl, alkenyl or alkynyl group having 5 to 30 carbon atoms; in some embodiments, R 2 and R 3 the same or different; in another embodiment, the alkyl, alkenyl or alkynyl group may be linear or branched;

[0012] L 1 is an alkyl group having 2 to 6 carbon atoms;

[0013] L 2 and L 3 are each independently an alkyl group having 2 to 9 carbon atoms; and

[0014] X and Y are each independently -C(O)-O- or -OC(O)-.

[0015] In some embodiments,

[0016] n is 1;

[0017] R 1 is hydroxyl group;

[0018] R 2 and R 3 Each is independently -CH((CH2) p CH3)((CH2) q CH3) or -

[0019] (CH2) m CH((CH2) p CH3)((CH2) q CH3), wherein m is an integer from 1 to 3, and p and q are each independently an integer from 1 to 12;

[0020] L 1 is an alkyl group having 2 to 6 carbon atoms;

[0021] L 2 and L 3 are each independently an alkyl group having 2 to 6 carbon atoms; and

[0022] X and Y are each independently -C(O)-O- or -OC(O)-.

[0023] In some embodiments,

[0024] n is 1;

[0025] R 1 is hydroxyl group;

[0026] R 2 and R 3 Each is independently -CH((CH2) p CH3)((CH2) q CH3) or -CH2CH((CH2) p CH3)((CH2) q CH3), wherein p and q are each independently an integer from 3 to 9;

[0027] L 1 is an alkyl group having 2 to 6 carbon atoms;

[0028] L 2 and L 3 are each independently an alkyl group having 2 to 6 carbon atoms; and

[0029] X and Y are each independently -C(O)-O- or -OC(O)-.

[0030] Examples of lipids include, but are not limited to, diheptadecan-9-yl 3,3'-(((3R,4S)-1-(2-hydroxyethyl)pyrrolidine-3,4-diyl)bis(oxy))dipropionate, diheptadecan-9-yl 3,3'-((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))dipropionate, diheptadecan-9-yl 5,5'-(((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))divalerate, diheptadecan-9-yl 5,5'-(((3S,4R)-1-(2-hydroxyethyl)pyrrolidine-3,4-diyl)bis(oxy))divalerate, diheptadecan-9-yl bis(2 -Hexyldecanoic acid)(((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl) ester, bis(2-hexyldecanoic acid)(((3S,4R)-1-(2-hydroxyethyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl) ester, 3,3'-(((3R,4S)-1-(4-hydroxybutyl)pyrrolidine-3,4-diyl)bis(oxy))dipropionate(heptadecan-9-yl) ester, bis(2-hexyldecanoic acid)(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(butane-4,1-diyl) ester, bis(2-hexyldecanoic acid)(( (3S,4R)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl) ester, bis(2-hexyldecanoic acid)(((3S,4R)-1-(6-hydroxyhexyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl) ester, 6,6'-(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoic acid diheptadecan-9-yl) ester, 6,6'-(((3R,4S)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoic acid diheptadecan-9-yl) ester, 6,6'-(((3R,4S)-1-(4-hydroxy bis(((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl) bis(hexyldecanoate), bis(((3S,4R)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl) bis(2-hexyldecanoate), bis(((3S,4R)-1-(4-hydroxybutyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl) bis(2-hexyldecanoate), bis(((3S,4R)-1-(4-hydroxybutyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl) bis(2-hexyldecanoate), bis(((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl) bis(2-hexyldecanoate), bis(((3S,4R)-1-(4-hydroxybutyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl) bis(5,5'-(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,bis(2-octyldodecyl)bis(((3R,4S)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))bis(2-octyldodecyl)bis(pentyl)bis(((3R,4S)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentyl)bis(2-octyldodecyl)bis(pentyl)bis(((3R,4S)-1-(6-hydroxyhex ...5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentyl)bis(2-octyldodecyl)bis(pentyl)bis(((3R,4S)-1-(6-hydroxyhexyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentyl)bis(2-octyldodecyl)bis(pentyl)bis(((3R,4S )-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))divalerate bis(2-butyloctyl) ester, 5,5'-(((3R,4S)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))divalerate bis(2-butyloctyl) ester and 5,5'-(((3R,4S)-1-(6-hydroxyhexyl)pyrrolidine-3,4-diyl)bis(oxy))divalerate bis(2-butyloctyl) ester.

[0031] In some embodiments, the pK of the protonated form of the lipid is a From about 5.0 to about 9.0.

[0032] In another aspect, the present disclosure provides an active agent delivery vehicle comprising a lipid as disclosed herein or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer or isotopically enriched derivative thereof.

[0033] Examples of active agent delivery vehicles include, but are not limited to, liposomes, single emulsions, micelles, nanoemulsions, lipid nanoparticles (LNP), nanostructured lipid carriers (NLC), and self-emulsifying drug delivery system (SEDDS).

[0034] In some embodiments of the present disclosure, the active agent delivery vehicle further comprises one or more lipids. Examples of lipids include, but are not limited to, neutral lipids, steroid lipids, and PEGylated lipids.

[0035] Examples of neutral lipids include, but are not limited to, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DP ...DOPC), 1,2-diste -glycerol-3-phosphatidylcholine (1,2-distearoyl-sn-glycero-3-phosphatidylcholine, DSPC), 1,2-diundecanoyl-sn-glycero-3-phosphatidylcholine (1,2-diundecanoyl-sn-glycero-3-phosphatidylcholine, DUPC), 1-plamitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphatidylcholine (1,2-di-O-octadecenyl-sn-glycero-3-phosphatidylcholine, 18:0Diether PC), 1-oleoyl-2-cholesterylhemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-O-hexadecyl-sn-glycero-3-phosphatidylcholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphatidylcholine, 1,2-diamidinoyl-sn-glycero-3-phosphatidylcholine (1,2-dilinolenoyl-sn-glycero-3-phosphatidylcholine), 1,2-diamidinoyl-sn-glycero-3-phosphatidylcholine (1,2-diarachidonoyl-sn-glycero-3-phosphatidylcholines), 1,2-didecosahexaenoyl-sn-glycero-3-phosphocholines, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamines (DOPE), 1,2-diphytanyl-sn-glycero-3-phosphoethanolamines (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamines, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamines, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamines, 1,2-diarachidonic acid 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamines, 1,2-didecosahexaenoyl-sn-glycero-3-phosphoethanolamines, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol)sodium salts,DOPG), dioleoylphosphatidylserines (DOPS), dipalmitoylphosphatidylglycerols (DPPG), palmitoyloleoylphosphatidylethanolamines (POPE), distearoylphosphatidylethanolamines (DPPE),DSPE), dipalmitoylphosphatidylethanolamines (DPPE), dimyristoleoylphosphoethanolamines (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamines (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelins, phosphatidylcholine, phosphatidylethnolamines, phosphatidylserines, phosphatidylinositols, phosphatidic acid, palmitoyloleoylphosphatidylcholine phosphatidylcholines), lysophosphatidylcholines and lysophosphatidylethanolamines (LPE), and combinations thereof.

[0036] Examples of steroid lipids include, but are not limited to, cholesterol, coprostanols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterols, tomatidines, ursolic acids, and α-tocopherol, and combinations thereof.

[0037] Examples of PEGylated lipids include, but are not limited to, 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-cholesterol, PEG-diacylglycamide (PEG-DAG), and PEG-dialkyloxypropyl (PEG-DAA), and combinations thereof.

[0038] In another aspect, the present disclosure provides a composition comprising an active agent delivery vehicle as disclosed herein and an active agent. In some embodiments of the present disclosure, the composition is a pharmaceutical composition.

[0039] In some embodiments of the present disclosure, the active agent is encapsulated in an active agent delivery vehicle.

[0040] In some embodiments of the present disclosure, the active agent comprises a nucleic acid. Examples of nucleic acids include, but are not limited to, antisense oligonucleotides, plasmids, interfering nucleic acids, aptamers, microRNAs (miRNAs), Dicer-substrate RNAs (dsRNAs), small hairpin RNAs (shRNAs), messenger RNAs (mRNAs), guide RNAs (gRNAs), miRNA inhibitors (antagomirs), and ribonucleases, and combinations thereof.

[0041] In some embodiments of the present disclosure, the mRNA encodes an enzyme, an antigen, an antibody, or an antigen-binding fragment.

[0042] In some embodiments of the present disclosure, the composition further comprises one or more pharmaceutically acceptable excipients or adjuvants.

[0043] In another aspect, the present disclosure provides a method for treating or preventing a disease or disorder, comprising administering to a subject in need thereof a composition as disclosed herein.

[0044] In another aspect, the present disclosure provides a method for introducing a nucleic acid into a target cell or transfecting the target cell with the nucleic acid, comprising contacting the composition as disclosed herein with the target cell.

[0045] In another aspect, the present disclosure provides a method for regulating the expression of a target gene in a cell, comprising providing the cell with a composition as disclosed herein.

[0046] The present invention is described in detail in the following sections. Other features, objects and advantages of the present invention can be found in the detailed description and claims. DETAILED DESCRIPTION

[0047] The disclosure may be more easily understood with reference to the following detailed description of various embodiments, examples, and chemical formulae and tables with their associated descriptions of the disclosure. It should be understood that, as is well known to those of ordinary skill in the art, unless otherwise specifically indicated by the claims, the disclosure is not limited to a specific method of preparation, carrier, or formulation, or a specific mode of formulating the extracts of the disclosure into a product or composition intended for topical, oral, or parenteral administration, although such factors may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and not for limitation.

[0048] As utilized in accordance with the present disclosure, unless otherwise specified, the following terms shall be understood to have the following meanings:

[0049] Typically, ranges are expressed herein as from "about" one particular value and / or to "about" another particular value. When such ranges are expressed, an embodiment includes a range from one particular value and / or to another particular value. Similarly, when values ​​are expressed as approximations by using the word "about," it is understood that the particular value forms another embodiment. It should be further understood that it is important that the endpoints of the ranges be related to each other or independent of each other.

[0050] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur. For example, the phrase "optionally comprising an anagent" means that the anagent may or may not be present.

[0051] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular.

[0052] As used herein, the term "subject" refers to any animal, preferably a mammal, and more preferably a human. Examples of subjects include humans, non-human primates, rodents, guinea pigs, rabbits, sheep, pigs, goats, cows, horses, dogs, and cats.

[0053] As used herein, the terms "treating" or "treatment" means reversing, alleviating, inhibiting the development of, or ameliorating the disorder, disease or condition to which such terms apply, or one or more symptoms of such disorder, disease or condition.

[0054] The terms "preventing" or "prevention" are recognized in the art and when used in conjunction with a condition, include the administration of an agent prior to the onset of the condition that reduces the frequency or severity of symptoms of the medical condition or delays the onset of the condition in a subject relative to a subject not receiving the agent.

[0055] The term "pharmaceutical composition" refers to a drug and / or vaccine for treating and / or preventing one or more diseases or conditions in an individual in need. The pharmaceutical composition can act systemically or locally. For this purpose, the pharmaceutical composition can be administered by appropriate routes, such as injection (e.g., intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection, including instillation) and transdermal delivery, and can also be administered by oral, buccal, nasal, transmucosal or topical routes, or in the form of ophthalmic preparations, or by inhalation. For these routes of administration, the pharmaceutical composition of the present invention can be administered in a suitable dosage form. These dosage forms include, but are not limited to: tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs and syrups. The term "vaccine" refers to a preventive or therapeutic substance that provides at least one antigen or antigenic function. Antigens or antigenic functions can stimulate the body's adaptive immune system to provide an adaptive immune response.

[0056] As used herein, the term "excipient" refers to any substance that is added to a formulation to improve its handling or storage characteristics or to allow or facilitate the formation of dosage units of a composition into discrete articles, such as capsules or tablets suitable for oral administration, which is not itself a therapeutic agent. Suitable excipients are well known to those of ordinary skill in the art of manufacturing pharmaceutical formulations or foods. By way of illustration and not limitation, excipients may include buffers, diluents, disintegrants, binders, adhesives, wetting agents, polymers, lubricants, glidants, substances added to mask or counteract unpleasant tastes or odors, flavoring agents, dyes, fragrances, and substances added to improve the appearance of the composition. Acceptable excipients include citrate buffer, phosphate buffer, acetate buffer, bicarbonate buffer, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric acid and sulfate, magnesium carbonate, talc, gelatin, gum arabic, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starch, gelatin, cellulosic materials (such as cellulose esters of alkanoic acid and cellulose alkyl esters), low melting wax cocoa butter, amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), ethylenediaminetetraacetic acid (EDTA), dimethyl sulfoxide (DMSO), sodium chloride or other salts, lipid particles, mannitol, sorbitol, glycerol or powders, polymers (such as polyvinyl pyrrolidone, polyvinyl alcohol and polyethylene glycol) and other pharmaceutically acceptable materials.

[0057] The term "adjuvant" or "adjuvant component" is generally a (e.g., pharmacological or immunological) agent or composition that can change (e.g., enhance) the efficacy of other agents (e.g., drugs or vaccines). As is known, the term in the context of the present invention refers to a compound or composition that acts as an auxiliary substance for an immunogen and / or other pharmaceutically active compound. It should be interpreted in a broad sense and refers to a wide range of substances that can increase the immunogenicity of an antigen incorporated into or co-administered with the adjuvant in question. In the context of the present invention, the adjuvant will preferably enhance the specific immunogenic effect of the active agent of the present invention. Typically, "adjuvant" or "adjuvant component" have the same meaning and can be used interchangeably. The adjuvant can be dispersed in, for example, an immunopotentiator, an antigen delivery system, or even a combination thereof.

[0058] The term "delivery" refers to the delivery of an entity to a target, such as the delivery of an active agent, drug, therapeutic agent and / or prophylactic agent to a subject, which is a tissue and / or cell of a human and / or other animal.

[0059] The term "delivery vehicle" refers to a substance that can be used to administer or deliver one or more agents to a cell, tissue, or individual (specifically, a human individual) with or without the agent to be delivered. A delivery vehicle can preferentially deliver an agent to a specific subset or type of cell. Selective or preferential delivery achieved by a delivery vehicle can be achieved by the properties of the vehicle or by a moiety that is bound to, associated with, or contained in the delivery vehicle, which specifically or preferentially binds to a specific subset of cells. A delivery vehicle can also increase the in vivo half-life of the agent to be delivered, the efficiency of delivering the agent, and / or the bioavailability of the agent to be delivered, compared to delivery without a delivery vehicle.

[0060] As used herein, the term "nucleic acid" is intended to include any oligonucleotide or polynucleotide. Fragments containing up to 20 nucleotides are generally referred to as oligonucleotides, and longer fragments are referred to as polynucleotides. In a particular embodiment, the oligonucleotides of the invention are 20 to 50 nucleotides in length.

[0061] In the context of the present disclosure, the terms "polynucleotide" and "oligonucleotide" refer to polymers or oligomers of nucleotide or nucleoside monomers composed of naturally occurring bases, sugars, and intersugar (backbone) linkages. The terms "polynucleotide" and "oligonucleotide" also include functionally similar polymers or oligomers comprising non-naturally occurring monomers or portions thereof. Such modified or substituted oligonucleotides are often superior to the native form due to properties such as enhanced cellular uptake and increased stability in the presence of nucleases.

[0062] Oligonucleotides can be classified as deoxyribonucleotides or ribonucleotides. Deoxyribonucleotides consist of a five-carbon sugar called deoxyribose covalently attached to a phosphate at the 5' and 3' carbons to form an alternating unbranched polymer. Ribonucleotides consist of a similar repeating structure in which the five-carbon sugar is ribose.

[0063] The nucleic acid present in the lipid-nucleic acid particles according to the present disclosure includes any known form of nucleic acid. The nucleic acid used herein may be single-stranded DNA or RNA, or double-stranded DNA or RNA, or a DNA-RNA mixture. Examples of double-stranded DNA include structural genes, genes including control regions and termination regions, and self-replicating systems such as viral or plasmid DNA. Examples of double-stranded RNA include siRNA and other RNA interference agents. Single-stranded nucleic acids include, for example, antisense oligonucleotides, ribozymes, microRNAs, and triplex-forming oligonucleotides.

[0064] The term "RNA" refers to a ribonucleic acid that can be naturally or non-naturally occurring. For example, RNA can include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, and linkers. RNA can include a cap structure, a chain terminating nucleoside, a stem loop, a polyadenylic acid sequence, and / or a polyadenylation signal. RNA can have a nucleotide sequence encoding a polypeptide of interest. For example, RNA can be a messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide, such as in vivo translation of the mRNA inside a mammalian cell, can produce the encoded polypeptide. RNA can be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), micro RNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA, including cas9 mRNA), guide RNA (gRNA, also known as single-guide RNA (sgRNA)), miRNA inhibitors (Anta Gou Miao), ribonucleases and mixtures thereof. Antisense nucleic acids and siRNA can inhibit the expression of target genes and target proteins in vitro or in vivo.

[0065] The term "modulating the expression of a target gene" refers to the ability of nucleic acid to silence, weaken or inhibit the expression of a target gene. In order to examine the degree of gene silencing, a test sample (e.g., a cell sample in a culture expressing the target gene) is contacted with a nucleic acid that inhibits the expression of the target gene. The expression of the target gene in the test sample or test animal is compared with the expression of the target gene in a control sample (e.g., a cell sample in a culture expressing the target gene) that is not contacted with nucleic acid or administered with these nucleic acids. The expression of the target gene in the control sample can be assigned a value of 100%. In a specific embodiment, when the target gene expression amount in the test sample or test mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0% relative to the target gene expression amount in the control sample or control mammal, the regulation of the expression of the target gene is achieved.

[0066] The term "transfecting" or "transfection" refers to the introduction of species (eg, RNA) into a cell. Transfection can occur, for example, in vitro, ex vivo, or in vivo.

[0067] The term "PEGylation" is the process of covalently and non-covalently attaching or fusing polyethylene glycol (PEG, known in pharmacy as macrogol) polymer chains to molecules and macromolecular structures (such as drugs, therapeutic proteins or vesicles (macrogols), which are then described as "PEGylated".

[0068] The present disclosure presents amine-containing lipids that are extremely valuable for formulating lipid nanoparticle (LNP) compositions. These LNP compositions provide advantageous features for the efficient delivery of nucleic acid cargoes (such as CRISPR / Cas gene editing components) into cells.

[0069] The present disclosure provides a lipid of formula (I),

[0070]

[0071] or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer or isotopically enriched derivative thereof,

[0072] in:

[0073] n is 1 or 2;

[0074] R 1 is amino, monoalkylamino, dialkylamino, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidinyl or pyrinyl;

[0075] R 2 and R 3 are each independently an alkyl, alkenyl or alkynyl group having 5 to 30 carbon atoms;

[0076] L 1 is an alkyl group having 2 to 6 carbon atoms;

[0077] L 2 and L 3 are each independently an alkyl group having 2 to 9 carbon atoms; and

[0078] X and Y are each independently -C(O)-O- or -OC(O)-.

[0079] In some embodiments, R 2 and R 3They may be the same as or different from each other. All alkyl, alkenyl and alkynyl groups may be linear or branched and may have 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms.

[0080] In some embodiments, L 1 It may be an alkyl group having 2, 3, 4, 5 or 6 carbon atoms.

[0081] In some embodiments, L 2 and L 3 They may be the same as or different from each other. The alkyl group may be linear or branched, and may have 2, 3, 4, 5, 6, 7, 8 or 9 carbon atoms.

[0082] In some embodiments, X and Y may be the same as or different from each other.

[0083] In some embodiments, n is 1; R 1 is hydroxyl group; R 2 and R 3 Each is independently -CH((CH2) p CH3)((CH2) q CH3) or -(CH2) m CH((CH2) p CH3)((CH2) q CH3), wherein m is 1, 2 or 3, p and q are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; L 1 is an alkyl group having 2 to 4 carbon atoms; L 2 and L 3 are each independently an alkyl group having 3 to 6 carbon atoms; and X and Y are each independently -C(O)-O- or -OC(O)-.

[0084] In some embodiments, n is 1; R 1 is hydroxyl group; R 2 and R 3 Each is independently -CH((CH2) p CH3)((CH2) q CH3) or -CH2CH((CH2) p CH3)((CH2) q CH3), wherein p and q are each independently 3, 4, 5, 6, 7, 8 or 9; L 1 is an alkyl group having 2 to 6 carbon atoms; L 2 and L 3are each independently an alkyl group having 2 to 6 carbon atoms; and X and Y are each independently -C(O)-O- or -OC(O)-.

[0085] In some embodiments, the lipid disclosed herein or its pharmaceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer or isotopically enriched derivative can be used as a constituent lipid of a lipid membrane structure for encapsulating an active agent. For example, the lipid disclosed herein can be used in liposomes or lipid nanoparticles (LNP) for encapsulation and delivery of nucleic acids.

[0086] In certain embodiments, the lipid disclosed herein may be ionizable. In certain embodiments, the lipid disclosed herein is an ionizable cationic lipid, which is positively charged (therefore in conjunction with nucleic acid, such as RNA) at low pH and becomes neutral at physiological pH, which helps to reduce the toxicity of LNP-RNA complexes in vivo. Lipids may provide two key functions, including promoting nucleic acid encapsulation in LNP and mediating endosomal membrane destruction so that nucleic acid can be released into the cytosol. In certain embodiments, lipids may directly achieve endosomal uptake (endosomal uptake) by interaction between their positive charge and negatively charged cell membranes or by binding to plasma proteins that support cell uptake.

[0087] In some embodiments, the lipid can be deprotonated under neutral conditions and can be ionized at a temperature below its acid dissociation constant (pK a ) has a positive charge under pH conditions. pK a The pK value should be high enough to allow the lipid to be positively charged at low pH and to bind to negatively charged RNA molecules and form LNPs. Therefore, at low endosomal pH, the positive charge of the ionizable amine-containing lipid allows it to interact with endogenous anionic lipids, thereby causing the disruption of the endosomal structure and the release of the LNP cargo into the cytoplasm. At the same time, the pK value of the ionizable amine-containing lipid is a The value should be low enough so that at physiological pH, the surface charge of the LNP will remain relatively neutral, which can modulate the toxicity and immunogenicity of the resulting LNP and increase its circulation time. In some embodiments, the pK of the protonated form of the lipid is a From about 5.0 to about 9.0, such as about 5.5, about 6.0, about 6.5, about 7.0 or more, or about 8.5, about 8.0, about 7.5, about 7.0 or less. The pKa value of the lipid can be critical for efficient LNP delivery and transfection efficacy.

[0088] The present disclosure further provides an active agent delivery vehicle comprising a lipid or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer or isotopically enriched derivative thereof.

[0089] In some embodiments, the active agent delivery vehicle can be a drug delivery system, such as liposomes, single emulsions, micelles, nanoemulsions, lipid nanoparticles (LNPs), nanostructured lipid carriers (NLCs), and self-emulsifying drug delivery systems (SEDDS). In some embodiments, the active agent delivery vehicle can be prepared at an acidic pH (about 4 or 5) lower than the pKa value of the lipid, followed by a buffer / pH exchange step.

[0090] In some embodiments, the active agent delivery vehicle further comprises one or more neutral lipids, steroid lipids and / or pegylated lipids. In some embodiments, the active agent delivery vehicle comprises at least one neutral lipid, at least one steroid lipid and at least one pegylated lipid. These ingredients can contribute to the formation of monodisperse nanoparticles, improve nanoparticle stability, achieve efficient nucleic acid encapsulation, assist cell absorption and promote endosomal escape of nucleic acid cargo.

[0091] In some embodiments, neutral lipids are also referred to as auxiliary lipids or structural lipids. Neutral lipids can be any lipid species that is uncharged or exists in a neutral zwitterionic form at a selected pH. In some embodiments, neutral lipids can be phospholipids such as 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-diundecanoyl-sn-glycero-3-phosphatidylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphatidylcholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-O-hexadecyl-sn-glycero-3-phosphatidylcholine (C16 LysoPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, 1,2-diacetoyl-sn-glycero-3-phosphatidylcholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylglycerol (DPPG) The present invention relates to a kind of phospholipids of LNP. The phospholipids of LNP are phosphatidylcholine, phosphatidylethanolamine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine (LPE), and combinations thereof. The neutral lipids may be synthetic or natural. In certain embodiments, the preferred phospholipids of LNP are DSPC and DOPE.

[0092] Steroid lipids can improve the cellular entry of active agents encapsulated in the delivery vehicle. Examples of steroid lipids include, but are not limited to, cholesterol, coprosterol, stigmasterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, and α-tocopherol, and combinations thereof.

[0093] In some embodiments, PEGylated lipids are molecules containing both lipid and PEG moieties, also known as polyethylene glycol (PEG)-lipid conjugates. PEGylated lipids can improve the stability and circulation time of delivery vehicles by preventing serum protein binding. In some embodiments, the PEGylated lipids can be 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-cholesterol, PEG-diacylglyceramide (PEG-DAG), PEG-dialkoxypropyl (PEG-DAA), and combinations thereof. Specifically, the PEGylated lipid may be one or more of the following: PEG500-dipalmitoylphosphatidylcholine, PEG2000-dipalmitoylphosphatidylcholine, PEG500-stearylphosphatidylethanolamine, PEG2000-distearylphosphatidylethanolamine, PEG500-1 ,2-dioleoylphosphatidylethanolamine (PEG500-1,2-dioleoylphosphatidylethanolamine), PEG2000-1,2-dioleoylphosphatidylethanolamine (PEG2000-1,2-dioleoylphosphatidylethanolamine), PEG2000-2,3-distearoylglycerol (PEG2000-DMG) and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, DMG-PEG2000).

[0094] The present disclosure also provides a composition comprising an active agent delivery vehicle and an active agent encapsulated by the active agent delivery vehicle.

[0095] In some embodiments, the composition is a pharmaceutical composition useful as a drug for treating any disease such as cancer and malignancies, anti-infective agents, anti-viral agents, anti-fungal agents, and vaccines.

[0096] In some embodiments, the active agent is encapsulated in an active agent delivery vehicle. In some embodiments, the active agent includes one or more nucleic acids. Examples of nucleic acids include DNA, RNA, antisense oligonucleotides, plasmids, interfering nucleic acids, aptamers, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-subject RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), guide RNA (gRNA), miRNA inhibitors (Anta Gou Miao), ribonucleases and combinations thereof. In some embodiments, mRNA encodes enzymes, antigens, antibodies or antigen-binding fragments.

[0097] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients or adjuvants. In some embodiments, the pharmaceutically acceptable excipient may comprise deionized water, ultrapure water, phosphate buffer or physiological saline, preferably phosphate buffer or physiological saline.

[0098] The present disclosure further provides a method for treating or preventing a disease or disorder comprising administering a pharmaceutical composition to a subject in need thereof.

[0099] In some embodiments, the pharmaceutical composition according to the present disclosure is administered locally or systemically by any method known in the art, including but not limited to intramuscular, intradermal, intravenous, subcutaneous, intraperitoneal, intranasal, oral, transmucosal or external routes. Appropriate routes, formulations and administration schedules can be determined by those skilled in the art. In the present disclosure, the pharmaceutical composition can be formulated in various ways according to the corresponding administration route, such as liquid solutions, suspensions, emulsions, syrups, tablets, pills, capsules, sustained release formulations, powders, granules, ampoules, injections, infusions, sets, ointments, lotions, liniments, creams or combinations thereof. If necessary, it can be sterilized or mixed with any pharmaceutically acceptable excipient, many of which are generally known to those skilled in the art.

[0100] As used herein, external routes are also referred to as topical administration, which include, but are not limited to, administration by insufflation and inhalation. Examples of various types of formulations for topical administration include ointments, lotions, creams, gels, foams, formulations for delivery via a transdermal patch, powders, sprays, aerosols, capsules or cartridges for use in an inhaler or insufflator or drops (e.g., eye drops or nose drops), solutions / suspensions for nebulization, suppositories, pessaries, retention enemas, and chewable or inhalable lozenges or pellets or liposomes or microencapsulated formulations.

[0101] The present disclosure further provides a method for introducing a nucleic acid into a target cell or transfecting the target cell with the nucleic acid, comprising contacting a composition with the target cell. In some embodiments, an active agent delivery vehicle encapsulating the nucleic acid can be administered to an individual so that the vehicle for introducing the nucleic acid can contact the target cell of the individual.

[0102] In another aspect, the present disclosure provides a method for regulating the expression of a target gene in a cell, comprising providing the cell with a composition as disclosed herein.

[0103] The following examples are given for the purpose of illustration and are not intended to limit the scope of the present disclosure.

[0104] Synthesis of Examples

[0105] General procedure for the synthesis of ionizable amine-containing lipids

[0106]

[0107] Process 1

[0108] Synthesis of (3S,4R)-3,4-dihydroxypyrrolidine-1-carboxylic acid tert-butyl ester (A-1)

[0109]

[0110] A solution of tributyl 2,5-dihydro-1H-pyrrole-1-carboxylate (2875 mg; 16.98 mmol) in acetone (30 mL) was added dropwise to a mixture of NMO (2188 mg; 18.68 mmol, 1.1 equiv.), osmium tetroxide (4% in water) (25 mg, 0.004 mmol) and water (30 mL) while keeping the temperature below 25 ° C. The reaction mixture was then stirred at ambient temperature for 16 hours. The acetone was evaporated and the organic phase was extracted with ethyl acetate. The combined organic phases were washed with water and brine in sequence and dried over magnesium sulfate. The solvent was evaporated under vacuum, and the residue was a crude product (A-1) (2865 mg, yield: 83%) as a brown oil, which was used in the next reaction without further purification. 1H NMR (600MHz, CDCl3) δ4.30-4.21 (m, 2H), 3.60 (ddt, J = 15.8, 11.4, 5.3Hz, 2H), 3.3 6(ddd,J=27.5,11.6,4.0Hz,2H),2.77(b,1H),1.67(b,1H),1.47(d,J=2.1Hz,9H).

[0111] Synthesis of 3,3'-(((3S,4R)-1-(tert-butyloxycarbonyl)pyrrolidine-3,4-diyl)bis(oxy))(2E,2'E)-diethyl diacrylate (A-2)

[0112]

[0113] The crude product (A-1) (2070 mg, 10.08 mmol) and DABCO (340 mg, 3.02 mmol, 0.3 eq) were dissolved in 20 mL of anhydrous DCM. This solution was then slowly added dropwise to a mixture of ethyl propiolate (5936 mg, 60.51 mmol, 6.0 eq) and 25 mL of anhydrous DCM in an ice / water bath under a nitrogen atmosphere. After the reaction was complete, NH4Cl was added. (aq) The mixture was stirred for 2 hours at 4 ℃ for 10 minutes.Then 1H-2O-butylene glycol (A-2) was added to 4% ethyl acetate (EtOAc) and 1% ethyl acetate (EtOAc). ... 1 H NMR (600MHz, CDCl3) δ7.51(d,J=12.5Hz,2H),5.33(d,J=12.6Hz,2H),4.61(h,J=3.3,2.8Hz,2H),4.22-4.17(m,4H),3.76(dq,J=11 .6,6.8,5.9Hz,2H),3.58(dd,J=11.8,4.3Hz,1H),3.53-3.48(m,1H),1.48(dd,J=3.3,1.4Hz,9H),1.31-1.28(m,6H); LCMS(ESI): C 19 H 29 NO8+H] + The calculated m / z value is 399.44, the experimental value is 399.9.

[0114] Synthesis of 3,3'-(((3S,4R)-1-(tert-butyloxycarbonyl)pyrrolidine-3,4-diyl)bis(oxy))dipropionic acid (A-3)

[0115]

[0116] Under hydrogen conditions, a solution of (A-2) (1700 mg, 4.25 mmol) and 10% Pd-C (170 mg) in a mixed solvent of ethyl acetate and methanol at a volume ratio of 5:1 was added to a round-bottomed flask and reacted at room temperature for 4 hours. The product was filtered through celite, and the organic layer was taken out and concentrated to dryness to obtain a colorless oily product.

[0117] A solution of the above crude product in MeOH / H2O (1 / 1, 40 mL) and 2M NaOH (6 mL) was added to a round-bottomed flask and reacted at room temperature for 16 hours. After the reaction was completed, a portion of the solvent was discharged, and the residual solution was cooled in an ice bath and then adjusted to pH 4 with 2N HCl. The resulting solution was extracted twice with EtOAc and the combined organic layers were dried (MgSO4). The dried product was filtered and concentrated in vacuo to obtain a crude substance (A-3) (1123 mg, yield: 76%) as a viscous colorless oily product. 1 H NMR (600MHz, CDCl3) δ4.20-4.11(m,1H),3.99(dd,J=22.2,4.3Hz,2H),3.82(ddd,J=13.9,7 .7,3.2Hz,4H),3.57-3.46(m,2H),3.38-3.30(m,1H),2.75-2.55(m,4H),1.49-1.46(m,9H). LCMS(ESI):C 19 H 33 NO8+H] + The calculated value of m / z is 403.47; the experimental value is 404.0 [M+H] + .

[0118] Synthesis of (3S,4R)-3,4-bis(4-cyanobutyloxy)pyrrolidine-1-carboxylic acid tert-butyl ester (A-4)

[0119]

[0120] The crude product of (A-1) (1900 mg, 9.35 mmol), 5-bromovaleronitrile (6057 mg, 37.39 mmol, 4.0 equiv) and TBAB (605 mg, 1.87 mmol, 0.2 equiv) were mixed in 40W% NaOH (6 mL) in a round bottom flask. The mixture was warmed to 65 ° C and the reaction was stirred for 24 hours. The mixture was cooled in an ice bath and diluted with water (20 mL), then adjusted to pH = 5 to 6 with 12N HCl. The resulting solution was extracted twice with EtOAc, and the combined organic layers were dried (MgSO4). The dried product was filtered and concentrated in vacuo to give the crude product. The crude product was purified by CombiFlash MPLC using a gradient of hexane / EtOAC (EtOAC = 28% to 31%), and then concentrated in vacuo to obtain a colorless oil product (A-4) (1776 mg, yield: 52%). 1 H NMR (600MHz, CDCl3) δ3.92 (q, J=4.8, 4.2Hz, 2H), 3.63-3.46 (m, 6H), 3.44 (dd, J=11.5, 3.8Hz, 1H), 3.32 (dd ,J=10.8,5.2Hz,1H),2.51-2.35(m,4H),1.78(tq,J=10.3,6.0Hz,8H),1.48(d,J=0.7Hz,9H); LCMS (ESI): C 19 H 31 N3O+H] + The calculated m / z value is 365.47, the experimental value is 366.3 [M+H] + .

[0121] Synthesis of 5,5'-(((3S,4R)-1-(tert-butyloxycarbonyl)pyrrolidine-3,4-diyl)bis(oxy))divaleric acid (A-5)

[0122]

[0123] The product (A-4) (1300 mg, 3.55 mmol) and KOH (1944 mg, 35.54 mmol, 10 equiv) were mixed in EtOH / H2O (1 / 1, 20 mL) in a round bottom flask. The mixture was warmed to 110°C and the reaction was stirred for 32 hours. The mixture was then cooled to room temperature and the EtOH was evaporated. The resulting mixture was cooled in an ice-water bath and diluted with water (10 mL), then adjusted to pH 4-5 with 36N HCl. The resulting solution was extracted twice with EtOAc and the combined organic layers were dried (MgSO4). The dried product was filtered and concentrated in vacuo to obtain the viscous colorless product (A-5) (1219 mg, yield: 85%). 1H NMR (600MHz, CDCl3) δ3.96-3.86 (m, 2H), 3.64-3.43 (m, 7H), 3.34 (dd, J = 11.0, 5.5Hz, 1H), 2.41 (dq,J=7.4,4.1,3.0Hz,4H),1.76(d,J=15.0Hz,4H),1.68(dq,J=12.5,6.3Hz,4H),1.47(s,9H). LCMS(ESI):C 19 H 33 NO8+H] + The calculated m / z value is 403.47, the experimental value is 404.1 [M+H] + .

[0124] Synthesis of (3S,4S)-3,4-bis((5-hydroxypentyl)oxy)pyrrolidine-1-carboxylic acid tert-butyl ester (A-6)

[0125]

[0126] Borane dimethyl sulfide (2M in THF) (2.9 mL, 4.0 equiv.) was added to a solution of (A-5) (600 mg, 1.49 mmol) in anhydrous THF (10 mL) cooled in an ice-water bath under nitrogen over a period of 5 minutes. The mixture was warmed to 65 ° C and then stirred for 3 hours. The mixture was then cooled in an ice-water bath under nitrogen, and MeOH (10 mL) was carefully and slowly added to the mixture over a period of 5 minutes. The mixture was again warmed to 65 ° C and then stirred for 15 minutes. The mixture was then cooled to room temperature and the solvent was evaporated. The residue was extracted twice with EtOAc and the combined organic layers were dried (MgSO4). The dried product was filtered and concentrated in vacuo to obtain a viscous colorless product (A-6) (497 mg, yield: 89%). 1 H NMR(600MHz, CDCl3) δ3.85(ddt,J=4.8,3.1,1.8Hz,2H),3.67(tt,J=6.7,2.6Hz,4H),3.56-3.42(m,7 H),3.38-3.31(m,1H),1.60(td,J=7.1,6.7,2.9Hz,8H),1.48(s,9H),1.46-1.40(m,4H); LCMS(ESI): C 19 H 37 NO6+H] + The calculated m / z value is 375.51, the experimental value is 376.0 [M+H] + .

[0127] Synthesis of 5,5'-(((3S,4R)-pyrrolidine-3,4-diyl)bis(oxy))divalerate di(heptadecan-9-yl)ester (A-7)

[0128]

[0129] To a suspension of (A-5) (730 mg, 1.81 mmol) in anhydrous DCM (10 mL) was added EDC-HCl (1038 mg, 5.43 mmol, 3.0 equiv), 9-heptadecanol (925 mg, 3.62 mmol, 2.0 equiv), DIPEA (1403 mg, 10.86 mmol, 6.0 equiv) and DMAP (88 mg, 0.72 mmol, 0.4 equiv) in sequence. The mixture was stirred at room temperature under nitrogen overnight, and then poured into water and extracted twice with DCM. The organic layer was separated, washed with brine, and dried over MgSO4. The dried product was filtered and concentrated in vacuo to obtain the crude product. The crude product was purified by CombiFlash MPLC using a gradient of hexane / EtOAc (EtOAc=8% to 13%), and then concentrated in vacuo to obtain a colorless oily product. To a solution of the crude product (1140 mg, 1.29 mmol) in anhydrous dichloromethane (15 mL) was added TFA (1.8 mL). The mixture was stirred at room temperature under nitrogen for 1.5 hours. The mixture was cooled in an ice-water bath and diluted with DCM (10 mL), followed by NaHCO 3(aq) The pH was adjusted to 8. The resulting solution was extracted with DCM and the combined organic layers were dried (MgSO 4 ). The dried product was filtered and concentrated in vacuo to obtain the product (A-7) (776 mg, yield: 55%) as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.88 (p, J=6.3Hz, 2H), 4.07 (q, J=3.9, 3.3Hz, 2H), 3.61 (d t,J=9.0,6.1Hz,2H),3.56-3.51(m,2H),3.44(dd,J=11.6,5.0Hz,2H),3.31(dd ,J=11.8,4.7Hz,2H),2.37-2.30(m,4H),1.75-1.69(m,4H),1.67-1.61(m,4H), 1.52 (d, J=6.3Hz, 8H), 1.33-1.24 (m, 48H), 0.90 (t, J=7.0Hz, 12H); LCMS (ESI): C 48 H 93 NO6+H] + The calculated m / z value is 780.27, the experimental value is 781.0 [M+H] + .

[0130] Synthesis of bis(2-hexyldecanoate)(((3S,4R)-pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)ester (A-8)

[0131]

[0132] The synthesis steps are the same as described in (A-7). 1 H NMR (600MHz, CDCl3) δ4.09 (t, J=6.8Hz, 4H), 4.01 (q, J=4.0Hz, 2H), 3.58 (dt, J=9.1 ,6.5Hz,2H),3.51(dt,J=9.0,6.5Hz,2H),3.33(dd,J=11.7,5.0Hz,2H),3.23(dd,J= 11.8,4.5Hz,2H),2.33(tt,J=8.9,5.3Hz,2H),1.63(ddd,J=15.9,10.6,7.2Hz,12H) ,1.49-1.41(m,8H),1.33-1.23(m,40H),0.90(td,J=7.0,1.6Hz,12H); LCMS(ESI):C 46 H 89 NO6+H] + The calculated m / z value is 752.22, the experimental value is 753.0 [M+H] + .

[0133] Example 1

[0134] di(heptadecan-9-yl)3,3'-(((3R,4S)-1-(2-hydroxyethyl)pyrrolidine-3,4-diyl)bis(oxy))dipropionate

[0135]

[0136] 1H NMR (600MHz, CDCl3) δ4.94-4.85(m,2H),3.97(ddt,J=7.8,5.5,2.8Hz,2H),3.87-3.74(m,4H),3.61(dd,J=6.2,4.4Hz,2H),3.07-3.00 (m,2H),2.72-2.67(m,2H),2.66-2.56(m,6H),1.57-1.48(m,8H),1.36-1.22(m,48H),0.90(t,J=7.0Hz,12H); LCMS(ESI): 768.6[M+H] + .

[0137] Example 2

[0138] di(heptadecan-9-yl)3,3'-((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))dipropionate

[0139]

[0140] 1 H NMR (600MHz, CDCl3) δ4.96-4.83(m,2H),3.93(ddt,J=8.0,5.5,2.8Hz,2H),3.86-3.70(m,6H),3.18(dd,J=10.0,5.4Hz,2H),2.77 (t,J=5.8Hz,2H),2.65-2.50(m,6H),1.75-1.62(m,2H),1.53(dd,J=10.4,5.1Hz,8H),1.36-1.21(m,48H),0.90(t,J=7.0Hz,12H). LCMS(ESI):783.4[M+H] + .

[0141] Example 3

[0142] di(heptadecan-9-yl)5,5'-(((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate

[0143]

[0144] 1 H NMR(600MHz, CDCl3) δ4.88(p,J=6.2Hz,2H),3.88(t,J=4.3Hz,2H),3.83-3.78(m,2H),3.56 (dt,J=9.2,6.3Hz,2H),3.47(dt,J=9.2,6.3Hz,2H),3.20(s,2H),2.81(s,2H),2.61-2.51(m ,2H),2.33(t,J=7.4Hz,4H),1.72(ddd,J=9.0,6.7,4.5Hz,6H),1.64(dd,J=8.5,5.7Hz,4H) ,1.52(d,J=6.0Hz,8H),1.35-1.22(m,48H),0.90(t,J=7.0Hz,12H); LCMS(ESI):838.9[M+H] + .

[0145] Example 4

[0146] di(heptadecan-9-yl)5,5'-(((3S,4R)-1-(2-hydroxyethyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate

[0147]

[0148] 1 H NMR (600MHz, CDCl3) δ4.88 (p, J = 6.3Hz, 2H), 3.87-3.79 (m, 2H), 3.63 (t, J = 5.4Hz, 2H), 3.47 (ddt, J=26.6,9.1,6.3Hz,4H),2.93(dd,J=9.9,6.0Hz,2H),2.68(dt,J=12.3,5.5Hz,1H),2.60(ddd,J= 10.0,8.9,4.6Hz,3H),2.33(t,J=7.4Hz,4H),1.71(dtd,J=9.1,7.9,7.2,5.9Hz,4H),1.66-1.58( m,4H),1.52(d,J=5.9Hz,8H),1.36-1.22(m,48H),0.90(t,J=7.0Hz,12H); LCMS(ESI):825.1[M+H]+ .

[0149] Example 5

[0150] Bis(2-hexyldecanoate)(((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)bis(2-hexyldecanoate)

[0151]

[0152] 1 H NMR (600MHz, CDCl3) δ4.08(t,J=6.7Hz,4H),3.87(d,J=5.0Hz,2H),3.82-3.77(m,2H),3.54(q,J =7.6Hz,2H),3.46(q,J=7.5Hz,2H),3.26-3.13(m,2H),2.79(t,J=5.7Hz,2H),2.54(dd,J=10.4, 4.7Hz,2H),2.32(ddd,J=8.9,6.0,2.8Hz,2H),1.72(q,J=5.6Hz,2H),1.64(ddq,J=26.2,14.4,7 .6Hz,12H),1.44(d,J=8.8Hz,8H),1.27(s,40H),0.90(t,J=7.0Hz,12H); LCMS(ESI):810.9[M+H] + .

[0153] Example 6

[0154] Bis(pentane-5,1-diyl)bis(2-hexyldecanoate)(((3S,4R)-1-(2-hydroxyethyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)bis(2-hexyldecanoate)

[0155]

[0156] 1H NMR(600MHz, CDCl3) δ4.09(t,J=6.7Hz,4H),3.91(t,J=4.3Hz,2H),3.62(t,J=5.3Hz,2H),3 .55(dt,J=9.3,6.6Hz,2H),3.47(dt,J=9.2,6.7Hz,2H),3.08(dd,J=9.7,5.3Hz,2H),2.71( t,J=5.3Hz,2H),2.64(dd,J=9.7,4.6Hz,2H),2.32(td,J=9.1,4.6Hz,2H),1.68-1.63(m,12 H),1.49-1.39(m,8H),1.34-1.22(m,40H),0.90(t,J=6.9Hz,12H); LCMS(ESI):796.4[M+H] + .

[0157] Example 7

[0158] di(heptadecan-9-yl)3,3'-(((3R,4S)-1-(4-hydroxybutyl)pyrrolidine-3,4-diyl)bis(oxy))dipropionate

[0159]

[0160] 1 H NMR(600MHz, CDCl3)δ4.90(p,J=6.3Hz,2H),3.99(s,2H),3.86-3.81(m,2H),3.80-3.74(m,2H),3.59(s,2H),3 .20(s,2H),2.66-2.56(m,6H),1.67(s,4H),1.52(t,J=6.4Hz,8H),1.35-1.23(m,50H),0.90(t,J=6.9Hz,12H). LCMS(ESI):796.9[M+H] + .

[0161] Example 8

[0162] Bis(2-hexyldecanoate)(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(butane-4,1-diyl)bis(2-hexyldecanoate)

[0163]

[0164] 1 H NMR (600MHz, CDCl3) δ4.11 (t, J = 6.5 Hz, 4H), 3.92 (t, J = 4.6 Hz, 2H), 3.83-3. 78(m,2H),3.59(dt,J=9.1,6.2Hz,2H),3.50(dt,J=9.1,6.2Hz,2H),3.29(s ,2H),2.87(s,2H),2.63(s,2H),2.33(tt,J=8.9,5.4Hz,2H),1.79-1.64(m, 12H), 1.64-1.55 (m, 6H), 1.35-1.21 (m, 40H), 0.90 (td, J = 7.0, 1.8Hz, 12H). LCMS (ESI): 782.8 [M+H] + .

[0165] Example 9

[0166] Bis(pentane-5,1-diyl)bis(2-hexyldecanoate)(((3S,4R)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)bis(2-hexyldecanoate)

[0167]

[0168] 1H NM(600MHz, CDCl3)δ4.09(t,J=6.7Hz,4H),3.67(t,J=6.3Hz,2H),3.60(s,2H),3.49(q,J=7.2Hz,2H),2.33(tt,J=9. 0,5.3Hz,2H),1.69-1.58(m,24H),1.44(dt,J=17.7,6.7Hz,10H),1.35-1.20(m,40H),0.90(td,J=7.0,1.5Hz,12H). LCMS(ESI):839.0[M+H] + .

[0169] Example 10

[0170] Bis(pentane-5,1-diyl)bis(2-hexyldecanoate)(((3S,4R)-1-(6-hydroxyhexyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)bis(2-hexyldecanoate)

[0171]

[0172] 1 H NMR (600MHz, CDCl3) δ4.08(t,J=6.7Hz,4H),3.93(s,2H),3.66(t,J=6.5Hz,2H),3.61-3.52(m,2H),3.47(dt,J=9.3,6.7Hz, 2H), 2.33(tt,J=8.9,5.3Hz,2H),1.71-1.55(m,20H),1.48-1.36(m,12H),1.34-1.22(m,40H),0.90(td,J=7.1,1.6Hz,12H). LCMS(ESI):853.2[M+H] + .

[0173] Example 11

[0174] di(heptadecan-9-yl)6,6'-(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate

[0175]

[0176] 1 H NMR (600MHz, CDCl3) δ4.88(p,J=6.2Hz,2H),3.89(q,J=4.9,4.4Hz,2H),3.85-3.75(m,2H),3.54( qd,J=6.8,3.3Hz,2H),3.45(td,J=8.5,7.9,6.0Hz,2H),3.33-3.15(m,2H),2.83(s,2H),2.64-2. 48(m,2H),2.30(t,J=7.6Hz,4H),1.73(p,J=5.5Hz,2H),1.64(ddd,J=20.2,14.7,7.3Hz,8H),1.5 2(q,J=6.6,6.0Hz,8H),1.40(tt,J=9.9,6.3Hz,4H),1.33-1.24(m,48H),0.90(t,J=7.0Hz,12H). LCMS(ESI):867.1[M+H] + .

[0177] Example 12

[0178] di(heptadecan-9-yl)6,6'-(((3R,4S)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate

[0179]

[0180] 1 H NMR (600MHz, CDCl3) δ4.88(p,J=6.3Hz,2H),3.97(s,2H),3.67(t,J=6.3Hz,2H),3.57(d,J=8.1Hz,2H),3.47(dt,J=9.0,6.6Hz,2H) ,2.31(t,J=7.6Hz,4H),1.71-1.58(m,19H),1.54-1.51(dd,8H),1.44-1.36(m,4H),1.28(d,J=9.8Hz,48H),0.90(t,J=7.0Hz,12H). LCMS(ESI):895.1[M+H] + .

[0181] Example 13

[0182] di(heptadecan-9-yl)6,6'-(((3R,4S)-1-(4-hydroxybutyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate

[0183]

[0184] 1 H NMR (600MHz, CDCl3) δ4.93-4.82(m,2H),3.96-3.85(m,2H),3.58(t,J=4.7Hz,2H),3.5 4(dt,J=9.4,6.6Hz,2H),3.45(dt,J=9.2,6.7Hz,2H),3.22(d,J=7.0Hz,2H),2.66-2.5 8(m,2H),2.53(dd,J=10.1,4.9Hz,2H),2.30(t,J=7.6Hz,4H),1.72-1.59(m,12H),1.5 2(d,J=6.3Hz,8H),1.44-1.36(m,4H),1.28(d,J=9.6Hz,48H),0.90(t,J=7.0Hz,12H). LCMS(ESI):881.2[M+H] + .

[0185] Example 14

[0186] Bis(hexyldecanoate)(((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl)bis(hexyldecanoate)

[0187]

[0188] 1H NMR(600MHz, CDCl3) δ4.08(t,J=6.7Hz,4H),3.90(q,J=4.9,4.4Hz,2H),3.81(t,J=5.3Hz,2H),3 .54(dt,J=9.0,6.6Hz,2H),3.46(dt,J=9.1,6.6Hz,2H),3.28(s,2H),2.86(s,2H),2.65-2.55(m, 2H),2.33(tt,J=9.0,5.3Hz,2H),1.75(p,J=5.5Hz,2H),1.61(dddd,J=16.6,12.9,10.0,7.1Hz,1 2H),1.48-1.41(m,4H),1.39(p,J=3.7Hz,8H),1.33-1.23(m,40H),0.90(td,J=7.0,1.8Hz,12H). LCMS(ESI):839.0[M+H] + .

[0189] Example 15

[0190] Bis(2-hexyldecanoate)(((3S,4R)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl)bis(2-hexyldecanoate)

[0191]

[0192] 1 H NMR (600MHz, CDCl3) δ4.08(t,J=6.7Hz,4H),3.96(s,2H),3.67(t,J=6.4Hz,2H),3.57(q,J=7.2Hz,2H),3.47(dt,J=9.1,6.6Hz,2H),2.33 (tt,J=9.0,5.3Hz,2H),1.66-1.59(m,22H),1.48-1.41(m,6H),1.39(p,J=3.5Hz,8H),1.33-1.22(m,40H),0.90(td,J=7.0,1.8Hz,12H). LCMS(ESI):867.0[M+H] + .

[0193] Example 16

[0194] Bis(2-hexyldecanoate)(((3S,4R)-1-(4-hydroxybutyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl)bis(2-hexyldecanoate)

[0195]

[0196] 1 H NMR(600MHz, CDCl3) δ4.08(t,J=6.7Hz,4H),3.92(t,J=4.4Hz,2H),3.59(d,J=5.1Hz,2H) ,3.54(dt,J=9.2,6.6Hz,2H),3.46(dt,J=9.1,6.6Hz,2H),3.26(s,2H),2.63(s,2H),2.59 -2.48(m,2H),2.33(tt,J=9.0,5.3Hz,2H),1.72-1.55(m,16H),1.45(dddt,J=11.5,9.8, 6.0, 3.0Hz, 4H), 1.39 (q, J = 3.7Hz, 8H), 1.32-1.22 (m, 40H), 0.90 (td, J = 7.0, 1.8Hz, 12H). LCMS(ESI):853.2[M+H] + .

[0197] Example 17

[0198] bis(2-octyldodecyl)5,5'-(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate

[0199]

[0200] 1H NMR (600MHz, CDCl3) δ3.99 (d, J = 5.8Hz, 4H), 3.90 (t, J = 4.4Hz, 2H), 3.85-3.73 (m, 2H),3.57(dt,J=9.2,6.3Hz,2H),3.48(dt,J=9.2,6.3Hz,2H),3.27(s,2H),2.85( t,J=5.9Hz,2H),2.60(dd,J=9.8,4.9Hz,2H),2.36(t,J=7.4Hz,4H),1.78-1.68(m ,6H),1.65(dt,J=9.4,6.3Hz,6H),1.29(d,J=7.9Hz,64H),0.91(t,J=7.0Hz,12H). LCMS (ESI): 923.2 [M+H] + .

[0201] Example 18

[0202] bis(2-octyldodecyl)5,5'-(((3R,4S)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate

[0203]

[0204] 1 H NMR (600MHz, CDCl3) δ3.99(d,J=5.8Hz,4H),3.68(td,J=6.4,1.8Hz,2H),3.59(d,J=8.1Hz,2H),3.50(ddt,J=11.8,8.5,3.9Hz,2H),2.3 6(t,J=7.4Hz,4H),1.72(dq,J=11.9,7.3Hz,4H),1.67-1.58(m,20H),1.46(d,J=7.1Hz,2H),1.36-1.23(m,62H),0.91(t,J=6.9Hz,12H). LCMS(ESI):951.2[M+H] + .

[0205] Example 19

[0206] Bis(2-octyldodecyl)5,5'-(((3R,4S)-1-(6-hydroxyhexyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate

[0207]

[0208] 1 H NMR (600MHz, CDCl3) δ3.98(d,J=5.8Hz,4H),3.67(t,J=6.5Hz,2H),3.60(d,J=8.4Hz,2H),3.54-3.41(m,2H),2.35(t,J= 7.4Hz, 4H), 1.71 (p, J = 7.2Hz, 4H), 1.62 (d, J = 18.8Hz, 22H), 1.40 (s, 4H), 1.29 (d, J = 8.6Hz, 60H), 0.90 (t, J = 6.9Hz, 12H). LCMS(ESI):965.4[M+H] + .

[0209] Example 20

[0210] bis(2-butyloctyl)5,5'-(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate

[0211]

[0212] 1 H NMR (600MHz, CDCl3) δ3.98(d,J=5.8Hz,4H),3.67(t,J=6.5Hz,2H),3.60(d,J=8.4Hz,2H),3.54-3.41(m,2H),2.35(t,J=7.4Hz,4H), 1.71(p,J=7.2Hz,4H),1.62(d,J=18.8Hz,22H),1.40(s,4H),1.29(d,J=8.6Hz,60H),0.90(t,J=6.9Hz,12H); LCMS(ESI): 698.8[M+H] + .

[0213] Example 21

[0214] bis(2-butyloctyl)5,5'-(((3R,4S)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate

[0215]

[0216] 1 H NMR (600MHz, CDCl3) δ3.99(d,J=5.8Hz,6H),3.66(t,J=6.4Hz,2H),3.59(dt,J=9.0,6.4Hz,2H),3.49(dt,J=9.1,6.3Hz,2H),2.35(t,J=7.4Hz ,4H),1.76-1.68(m,6H),1.67-1.57(m,14H),1.44(p,J=8.0Hz,2H),1.35-1.25(m,32H),0.91(td,J=6.7,5.1Hz,12H); LCMS(ESI): 726.7[M+H] + .

[0217] Example 22

[0218] bis(2-butyloctyl)5,5'-(((3R,4S)-1-(6-hydroxyhexyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate

[0219]

[0220] 1H NMR (600MHz, CDCl3) δ3.99(d,J=5.8Hz,4H),3.66(t,J=6.5Hz,2H),3.59(d,J=7.9Hz,2H),3.49(dt,J=9.2,6.2Hz,2H),2.35(t,J=7.4Hz,4H),1.7 5-1.68(m,4H),1.61(tq,J=21.1,6.3Hz,20H),1.39(d,J=14.3Hz,4H),1.34-1.25(m,30H),0.91(td,J=6.7,5.1Hz,12H); LCMS(ESI): 740.9[M+H] + .

[0221] Lipid Nanoparticle (LNP) Preparation

[0222] use IGNITE TM Lipid nanoparticles (LNPs) loaded with firefly luciferase (FLuc) reporter gene mRNA were prepared. Briefly, a mixture of lipids, cholesterol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) and 1,2-stearoyl-sn-glycero-3-phosphocholine (DSPC) was prepared in an ethanol solution at a molar percentage of 50:38.5:10:1.5. Three units of FLuc mRNA were dissolved in 25mM sodium acetate pH 5.0 buffer, and one unit of the lipid mixture was quickly combined in a microfluidic mixing cartridge at a constant amine to phosphate (N / P) ratio of 6, and the instrument flow rate was set to 12mL / min. The mRNA-LNP was diluted 40 times with calcium- and magnesium-free phosphate-buffered saline (PBS) to achieve pH neutrality. Next, the mRNA-LNP was diluted 40 times with calcium- and magnesium-free phosphate-buffered saline (PBS) to achieve pH neutrality. Next, the mRNA-LNP was added to the microfluidic mixing cartridge using a 50:38.5:10:1.5 molar percentage of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) and 1,2-stearoyl-sn-glycero-3-phosphocholine (DSPC). Ultra-15 centrifugal filter units were used to concentrate the LNPs.

[0223] mRNA (SEQ ID NO: 1) can be purchased from TRILINK Limited Liability Company.

[0224] Feature analysis

[0225] By ZETASIZER TM PRO BLUE(MALVERN ) were characterized in terms of hydrodynamic size (z-mean diameter), zeta potential, and polydispersity index. TM RIBOGREEN Analysis Kit Determination of mRNA concentration and encapsulation efficiency. Briefly, mRNA-LNPs were diluted in an equal volume of Tris / EDTA (TE) buffer or TE buffer containing 2% Triton X-100. Thereafter, RIBOGREEN reagent was added to each sample and incubated at 37°C in the absence of light for 15 minutes. TM The fluorescence intensity (Ex / Em 480 / 520 nm) was detected on a microplate reader. The encapsulation efficiency (EE) was calculated by the following formula:

[0226] EE (%) = (total mRNA concentration - unencapsulated mRNA concentration) / (total mRNA concentration) × 100%

[0227] In addition, the acid dissociation constant (pKa) of mRNA-LNP is determined using 6-(p-toluidine)-2-naphthalenesulfonyl chloride (TNS) binding analysis. Several pH range buffers (20mM citrate buffer has a range of pH 3.0 to 5.5; 20mM sodium phosphate buffer has a range of pH 6.0 to 8.0; 20mM Tris buffer has a range of pH 8.5 to 10.0) are prepared. LNP (0.5mM total lipid) is mixed with the above buffer solution and TNS reagent to make the final concentration of TNS reagent be 6 μM in a total volume of 0.2mL, and then cultivated for ten minutes. Fluorescence intensity values ​​(Ex / Em 321 / 447nm) are recorded by Infinite M200 PRO.

[0228] Table 1: Composition analysis

[0229]

[0230] Transfection efficiency and cytotoxicity in HEPG2 cell line

[0231] To investigate the potential and cytotoxicity of mRNA-LNPs made from novel lipids, FLuc mRNA Encapsulated in LNPs and transfected into cell lines. HEPG2 cells were maintained in DMEM medium with 10% fetal bovine serum at 37°C under 5% CO2. Cells were plated at 4×10 4 Cells / well were seeded and treated with Fluc mRNA-LNP for 18 hours, while the cell confluence reached 70%. To investigate the transfection efficiency, HEPG2 cells were treated with D-luciferin and incubated at 37°C for another 5 minutes with thorough mixing. The M200 microplate reader was used. Record the intensity of cold light. ) to assess cell viability and measure absorbance at 450 nm. The results are shown in Table 2.

[0232] Table 2: Transfection efficiency and cytotoxicity

[0233]

[0234] The toxicity of compounds 11 and 14 was less than 10%.

[0235] Although the present disclosure has been described and illustrated with reference to its specific embodiments, these descriptions and illustrations are not restrictive. Those skilled in the art will understand that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present disclosure as defined by the appended claims. The description may not be drawn to scale. Due to manufacturing processes and tolerances, there may be differences between the process reproduction in the present disclosure and the actual device. There may be other embodiments of the present disclosure that are not specifically described. This specification should be considered illustrative rather than restrictive. Modifications may be made to adapt specific circumstances, materials, material compositions, methods or procedures to the goals, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the claims attached hereto. Although the methods disclosed herein have been described with reference to specific operations performed in a specific order, it should be understood that these operations may be combined, subdivided, or reordered to form equivalent methods without departing from the teachings of the present disclosure. Therefore, unless specifically indicated herein, the order and grouping of operations are not limitations of the present disclosure.

Claims

1. A lipid of formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer or isotopically enriched derivative thereof, in: n is 1 or 2; R 1 is amino, monoalkylamino, dialkylamino, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidinyl or pyrinyl; R 2 and R 3 are each independently an alkyl, alkenyl or alkynyl group having 5 to 30 carbon atoms; L 1 is an alkyl group having 2 to 6 carbon atoms; L 2 and L 3 are each independently an alkyl group having 2 to 9 carbon atoms; and X and Y are each independently -C(O)-O- or -OC(O)-.

2. The lipid of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer or isotopically enriched derivative thereof, wherein: n is 1; R 1 is hydroxyl group; R 2 and R 3 Each is independently -CH((CH2) p CH3)((CH2) q CH3) or -(CH2) m CH((CH2) p CH3)((CH2) q CH3), wherein m is an integer from 1 to 3, and p and q are each independently an integer from 1 to 12; L 1 is an alkyl group having 2 to 6 carbon atoms; L 2 and L 3 are each independently an alkyl group having 2 to 6 carbon atoms; and X and Y are each independently -C(O)-O- or -OC(O)-.

3. The lipid of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer or isotopically enriched derivative thereof, wherein: n is 1; R 1 is hydroxyl group; R 2 and R 3 Each is independently -CH((CH2) p CH3)((CH2) q CH3) or -CH2CH((CH2) p CH3)((CH2) q CH3), wherein p and q are each independently an integer from 3 to 9; L 1 is an alkyl group having 2 to 6 carbon atoms; L 2 and L 3 are each independently an alkyl group having 2 to 6 carbon atoms; and X and Y are each independently -C(O)-O- or -OC(O)-.

4. The lipid of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer or isotopically enriched derivative thereof, wherein the lipid is selected from the group consisting of: 3,3'-(((3R,4S)-1-(2-hydroxyethyl)pyrrolidine-3,4-diyl)bis(oxy))dipropionate di(heptadecan-9-yl) ester, 3,3'-((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))dipropionic acid di(heptadecan-9-yl) ester, 5,5'-(((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoic acid di(heptadecan-9-yl) ester, 5,5'-(((3S,4R)-1-(2-hydroxyethyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoic acid di(heptadecan-9-yl) ester, Bis(2-hexyldecanoate)(((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)ester, Bis(2-hexyldecanoate)(((3S,4R)-1-(2-hydroxyethyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)ester, 3,3'-(((3R,4S)-1-(4-hydroxybutyl)pyrrolidine-3,4-diyl)bis(oxy))dipropionate diheptadecan-9-yl, Bis(2-hexyldecanoate)(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(butane-4,1-diyl)ester, Bis(2-hexyldecanoate)(((3S,4R)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)ester, Bis(2-hexyldecanoate)(((3S,4R)-1-(6-hydroxyhexyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)ester, 6,6'-(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoic acid di(heptadecan-9-yl) ester, 6,6'-(((3R,4S)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoic acid di(heptadecan-9-yl) ester, 6,6'-(((3R,4S)-1-(4-hydroxybutyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoic acid di(heptadecan-9-yl) ester, Bis(hexyldecanoate)(((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl)ester, Bis(2-hexyldecanoate)(((3S,4R)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl)ester, Bis(2-hexyldecanoate)(((3S,4R)-1-(4-hydroxybutyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl)ester, 5,5'-(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))divalerate bis(2-octyldodecyl) ester, 5,5'-(((3R,4S)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))divalerate bis(2-octyldodecyl) ester, 5,5'-(((3R,4S)-1-(6-hydroxyhexyl)pyrrolidine-3,4-diyl)bis(oxy))divalerate bis(2-octyldodecyl) ester, 5,5'-(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))divalerate bis(2-butyloctyl) ester, 5,5'-(((3R,4S)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))divalerate bis(2-butyloctyl) ester and Bis(2-butyloctyl)-5,5'-(((3R,4S)-1-(6-hydroxyhexyl)pyrrolidine-3,4-diyl)bis(oxy))divalerate.

5. The lipid of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer or isotopically enriched derivative thereof, wherein the pK of the protonated form of the lipid is a From about 5.0 to about 9.

0.

6. An active agent delivery vehicle comprising the lipid according to any one of claims 1 to 5 or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer or isotopically enriched derivative thereof.

7. The active agent delivery vehicle of claim 6, which is selected from the group consisting of liposomes, single emulsions, micelles, nanoemulsions, lipid nanoparticles, nanostructured lipid carriers, and self-emulsifying drug delivery systems.

8. The active agent delivery vehicle of claim 6, further comprising one or more lipids selected from the group consisting of neutral lipids, steroid lipids, and pegylated lipids.

9. The active agent delivery vehicle of claim 8, wherein the neutral lipids are selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphatidylcholine, 1,2-diondecanoyl-sn-glycero-3-phosphatidylcholine, 1,2-palmitoyl-2 -oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenyl-sn-glycero-3-phosphatidylcholine, 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine, 1-O-hexadecyl-sn-glycero-3-phosphatidylcholine, 1,2-dilinolenoyl-sn-glycero-3-phosphatidylcholine, 1,2-diacetoyl-sn-glycero-3-phosphatidylcholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn -glycero-3-phosphoethanolamine, 1,2-diphytyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1 -glycerol), sodium salt of dioleoylphosphatidylserine, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine, and combinations thereof.

10. The active agent delivery vehicle of claim 8, wherein the steroid lipids are selected from the group consisting of cholesterol, coproposterol, stigmasterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid and α-tocopherol, and combinations thereof.

11. The active agent delivery vehicle of claim 8, wherein the PEGylated lipids are selected from the group consisting of 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)], PEG-cholesterol, PEG-diacylglyceramide and PEG-dialkoxypropyl, and combinations thereof.

12. A composition comprising the active agent delivery vehicle according to claim 6 and an active agent.

13. The composition of claim 12, wherein the active agent is encapsulated in the active agent delivery vehicle.

14. The composition of claim 12, wherein the active agent comprises a nucleic acid.

15. The composition of claim 14, wherein the nucleic acid is selected from the group consisting of antisense oligonucleotides, plasmids, interfering nucleic acids, aptamers, microRNAs (miRNAs), Dicer-substrate RNAs, small hairpin RNAs, messenger RNAs (mRNAs), guide RNAs, miRNA inhibitors, and ribonucleases, and combinations thereof.

16. The composition of claim 15, wherein the mRNA encodes an enzyme, an antigen, an antibody, or an antigen-binding fragment.

17. The composition of claim 12, further comprising one or more pharmaceutically acceptable excipients or adjuvants.

18. Use of the composition of claim 12 for the manufacture of a medicament for treating or preventing a disease or condition.

19. A method for introducing a nucleic acid into a target cell or transfecting the target cell with the nucleic acid, comprising contacting the composition of claim 12 with the target cell.

20. A method for regulating the expression of a target gene in a cell, comprising providing the composition of claim 12 to the cell.

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