Lipoic acid derivative-based lipid compound, composition and preparation method and application thereof

Abstract

The invention provides a lipid compound based on a lipoic acid derivative, a composition as well as a preparation method and application of the lipid compound. When the lipoic acid-derived lipid provided by the invention is used as a delivery carrier of a nucleic acid drug, because disulfide bonds in the compound are split under the action of glutathione in cells, the compound is degraded, so that the timely release of nucleic acid is triggered, the cytotoxicity caused by accumulation of the compound in the cells is well reduced, and the bioavailability of the nucleic acid drug is improved. The biocompatibility is good. When the lipid nanoparticle composition is used as a carrier to deliver nucleic acid drugs, the lipid nanoparticle composition has excellent delivery efficiency and excellent spleen targeting performance, and the nucleic acid drugs can be preferentially delivered to the spleen.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a lipid compound based on lipoic acid derivatives, a composition, and a preparation method and application thereof. Background Art

[0002] In recent years, gene therapy has shown great potential in biomedical applications. In order to work in vivo, nucleic acid drugs require a safe, effective and stable delivery system to protect nucleic acids from degradation and allow cellular uptake and nucleic acid release. To date, a variety of materials for nucleic acid delivery have been developed, such as lipids, polymers, peptides, and inorganic nanoparticles. But among these carriers, lipid nanoparticles (LNPs) as nucleic acid carriers show great promise. In particular, in 2020, two mRNA vaccines developed by Pfizer BioNTech and Moderna both used lipid nanoparticles to deliver antigen mRNA.

[0003] LNPs are usually composed of ionizable lipids, PEGylated lipids, cholesterol, and auxiliary phospholipids. Among them, ionizable lipids have been shown to be the key components of LNPs and have an important impact on the efficiency of nucleic acid delivery. Ionizable lipids provide positive charges and can encapsulate negatively charged nucleic acids. However, to date, endosomal escape remains a fundamental obstacle to LNP-mediated nucleic acid delivery systems. Therefore, there is still an urgent need to develop new ionizable lipids with efficient endosomal escape capabilities to improve nucleic acid delivery in vitro and in vivo.

[0004] By rationally designing the chemical structure of ionizable lipids, increasing cellular uptake and endosomal escape, the delivery efficiency of the LNP system can be improved. Lipoic acid is an essential factor for human cell energy metabolism and is a disulfide compound that is completely biodegradable in the intracellular reducing environment. Studies have shown that disulfide bonds contribute greatly to thiol-mediated uptake and can effectively deliver cargo to the cytosol. Therefore, the present invention is based on chemical modification of lipoic acid to rationally design a series of lipoic acid derivative lipid compounds.

[0005] Currently, gene therapy has shown great potential in a range of applications, including protein replacement therapy, cancer immunotherapy, cell reprogramming and genome editing. In order to achieve therapeutic effects, nucleic acid molecules must reach specific target cells and produce sufficient target proteins. However, targeted delivery and endosomal escape remain challenging for nucleic acid delivery systems. Therefore, we urgently need to develop safer and more efficient LNP nucleic acid delivery materials, and the key to achieving this goal lies in the development of new ionizable lipid chemical structures.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a lipid compound based on lipoic acid derivatives, a composition, a preparation method and an application thereof. The lipid compound and the composition based on lipoic acid derivatives have high-efficiency nucleic acid delivery capability.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] In a first aspect, the present invention provides a lipid compound based on lipoic acid derivatives, wherein the lipid compound based on lipoic acid derivatives has a structure as shown in the following formula I:

[0010]

[0011] Among them, R1 is selected from any one of substituted or unsubstituted C8~C18 alkyl or substituted or unsubstituted C8~C18 alkenyl; R2 is selected from C1~C12 alkyl substituted with hydroxyl, C1~C12 alkyl substituted with amino, C1~C12 alkyl substituted with alkylamino, C1~C12 alkyl substituted with hydroxyalkylamino, C1~C12 alkyl substituted with aryl, C1~C12 alkyl substituted with nitrogen heterocycle, C1~C12 alkyl substituted with nitrogen and oxygen heterocycle; n is an integer from 0 to 5.

[0012] Preferably, the R1 is selected from a C8-C18 straight chain or branched alkyl group, or a C8-C18 straight chain or branched alkenyl group.

[0013] Preferably, the R1 is selected from any one of the following groups; wherein, Representative group connection position:

[0014]

[0015] Preferably, the R2 is selected from any one or a combination of at least two of the following groups; wherein, Representative group connection position:

[0016]

[0017] Among them, R 21 is selected from C2 to C6 straight or branched alkyl groups, R 22 or R 23 Each is independently selected from any one of hydrogen, C1-C4 linear or branched alkyl, and C1-C4 linear or branched alkoxy.

[0018] Preferably, the R2 is selected from any one or a combination of at least two of the following groups; wherein, Representative group connection position:

[0019]

[0020] Preferably, the lipid compound based on lipoic acid derivatives comprises any one of the following compounds:

[0021]

[0022]

[0023] In a second aspect, the present invention provides a method for preparing a lipid compound based on lipoic acid derivatives as described in the first aspect, the preparation method comprising the following steps:

[0024] Alkyl carboxylic acid compounds and 6-bromohexanol undergo esterification reaction to obtain compound A; compound A and primary amine compounds undergo substitution reaction to obtain compound B; the reaction formula is as follows:

[0025]

[0026] Lipoic acid and bromoalkyl alcohol compounds are subjected to esterification reaction to obtain compound C; the reaction formula is shown below:

[0027]

[0028] Compound B reacts with compound C to obtain a lipid compound based on a lipoic acid derivative as shown in Formula I; the reaction formula is as follows:

[0029]

[0030] The selection of R1, R2 and n is consistent with that in the first aspect.

[0031] Preferably, the molar ratio of the alkyl carboxylic acid compound to 6-bromohexanol is 1:(0.8-1.2).

[0032] Preferably, in the process of preparing the compound A, the temperature of the esterification reaction is 10 to 40° C., and the time of the esterification reaction is 12 to 36 hours.

[0033] Preferably, in the process of preparing the compound A, the esterification reaction is carried out in the presence of a condensing agent, and the molar ratio of the alkyl carboxylic acid compound to the condensing agent is 1:(1-4).

[0034] Preferably, the condensing agent is EDC.

[0035] Preferably, in the process of preparing the compound A, the esterification reaction is carried out in the presence of a base, and the molar ratio of the alkyl carboxylic acid compound to the base is 1:(0.1-5).

[0036] Preferably, the base is DMAP.

[0037] Preferably, the molar ratio of the compound A to the primary amine compound is 1:(0.8-1.2).

[0038] Preferably, in the process of preparing the compound B, the temperature of the substitution reaction is 50 to 70° C., and the time of the substitution reaction is 12 to 24 hours.

[0039] Preferably, the molar ratio of lipoic acid to bromoalkyl alcohol compound is 1:(0.8-1.2).

[0040] Preferably, in the process of preparing the compound C, the temperature of the esterification reaction is 10 to 40° C., and the time of the esterification reaction is 12 to 36 hours.

[0041] Preferably, in the process of preparing the compound C, the esterification reaction is carried out in the presence of a condensing agent, and the molar ratio of lipoic acid to the condensing agent is 1:(1-4).

[0042] Preferably, the condensing agent is EDC.

[0043] Preferably, in the process of preparing the compound C, the esterification reaction is carried out in the presence of a base, and the molar ratio of lipoic acid to the base is 1:(0.1-5).

[0044] Preferably, the base is DMAP.

[0045] Preferably, the molar ratio of compound B to compound C is 1:(0.8-1.2).

[0046] Preferably, the temperature for the reaction of compound B and compound C is 80-100° C., and the time for the reaction of compound B and compound C is 42-54 h.

[0047] Preferably, the reaction of compound B and compound C is carried out in the presence of an iodine salt catalyst, and the added amount of the iodine salt catalyst is 0.01 to 10 mol% of the molar amount of compound B.

[0048] Preferably, the reaction of compound B and compound C is carried out in the presence of a base, and the molar ratio of compound B to the base is 1:(1-5).

[0049] In a third aspect, the present invention provides a use of the lipoic acid derivative as described in the first aspect as a nucleic acid delivery medium.

[0050] Wherein, the nucleic acid includes any one of DNA, mRNA, siRNA, microRNA, antisense nucleic acid or circular RNA.

[0051] In a fourth aspect, the present invention provides a lipid nanoparticle composition, the lipid nanoparticle composition comprising lipoic acid derivative lipids, polyethylene glycol lipids, steroids and auxiliary lipids;

[0052] Wherein, the lipoic acid derivative lipid includes any one or a combination of at least two of the lipoic acid derivative-based lipid compounds described in the first aspect.

[0053] Preferably, the nanoparticle composition comprises the following components by mass percentage:

[0054] Lipoic acid derivative lipids 10% to 70%, polyethylene glycol lipids 1% to 25%, steroids 10% to 50%, and helper lipids 5% to 30%.

[0055] Preferably, the polyethylene glycol lipid is selected from any one of DMG-PEG 2000, DSPE-MPEG 2000 or DPPE-MPEG2000, or a combination of at least two thereof.

[0056] Preferably, the steroid is selected from any one or a combination of at least two of cholesterol, hydroxycholesterol, fucosterol, campesterol, ergosterol, glycocholic acid, taurocholic acid, stigmasterol or stigmasterol.

[0057] Preferably, the helper lipid is selected from any one or a combination of at least two of DOPC, DOPE, DSPC, DPPG, POPE, DSPE or DGTS.

[0058] In a fifth aspect, the present invention provides a nanoparticle composition encapsulating a nucleic acid drug, wherein the nanoparticle composition encapsulating a nucleic acid drug comprises a nucleic acid drug and a lipoic acid derivative lipid;

[0059] Wherein, the lipoic acid derivative lipid comprises any one or a combination of at least two of the lipoic acid derivative-based lipid compounds described in the first aspect;

[0060] Alternatively, the nanoparticle composition encapsulating the nucleic acid drug comprises the nucleic acid drug and the lipid nanoparticle composition described in the fourth aspect, that is, the lipid nanoparticle composition comprises the nucleic acid drug, lipoic acid derivative lipid, polyethylene glycol lipid, steroid and auxiliary lipid.

[0061] Preferably, the nucleic acid drug accounts for 5% to 20% of the total mass of the nanoparticle composition encapsulating the nucleic acid drug.

[0062] Preferably, the nucleic acid drug comprises any one of Luc-mRNA, OVA-mRNA, GFP-mRNA, PD-1-mRNA, siRNA or DNA, or a combination of at least two thereof.

[0063] In a sixth aspect, the present invention provides a method for preparing the nanoparticle composition containing nucleic acid drugs as described in the fourth aspect, the preparation method comprising:

[0064] Dissolving lipoic acid derivative lipid, polyethylene glycol lipid, steroid and auxiliary lipid in a solvent and mixing them to obtain an organic phase liposome solution;

[0065] Dissolving the nucleic acid drug in a pH buffer solution to obtain an aqueous nucleic acid drug solution;

[0066] The organic phase liposome solution and the aqueous phase nucleic acid drug solution are mixed and purified to obtain the nanoparticle composition containing the nucleic acid drug.

[0067] Preferably, the solvent includes any one of methanol, ethanol, tetrahydrofuran or dimethyl sulfoxide, or a combination of at least two thereof.

[0068] Preferably, in the organic phase liposome solution, the concentration of the lipoic acid derivative lipid is 5-50 mg / mL.

[0069] Preferably, the pH buffer solution comprises a citric acid / sodium citrate solution.

[0070] Preferably, the pH of the pH buffer solution is 3-9.

[0071] Preferably, in the aqueous nucleic acid drug solution, the concentration of the nucleic acid drug is 0.05 to 2 mg / mL.

[0072] Preferably, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid drug is 1:(1-20).

[0073] Preferably, the purification comprises ultrafiltration and / or dialysis.

[0074] [Term explanation]

[0075] Various aspects and features of the present invention are further described below.

[0076] The various terms and phrases used in the present invention have the general meanings known to those skilled in the art. Even so, the present invention still hopes to provide a more detailed description and explanation of these terms and phrases. If the terms and phrases mentioned are inconsistent with the known meanings, the meanings expressed in the present invention shall prevail. Below are the definitions of various terms used in the present invention, which apply to the terms used throughout the specification of this application, unless otherwise specified in specific circumstances. The following provides definitions of various groups of the compounds of the present invention, which are used uniformly in the specification unless otherwise defined.

[0077] As mentioned in the present invention, the term "alkyl" refers to an alkyl group having a specified number of carbon atoms, which can be a straight-chain alkyl group or a branched alkyl group. For example, the "C1-C12 alkyl group" refers to a straight-chain alkyl group or a branched alkyl group having 1 to 20 carbon atoms, and specific groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, etc., and similar groups.

[0078] As mentioned in the present invention, the term "alkenyl" refers to an alkenyl group (a hydrocarbon group having one or more C=C double bonds) with a specified number of carbon atoms, which can be a straight-chain alkyl group or a branched alkenyl group. For example, the "C8-C18 alkenyl" refers to a straight-chain alkyl group or a branched alkenyl group with 8-18 carbon atoms, and specific groups include 1-octenyl, 2-octenyl, 2,4-octadienyl, 4-methyl-1-heptenyl, 2-nonenyl, 2-decenyl, etc., and similar groups.

[0079] As referred to in the present invention, the term "hydroxyl" refers to -OH.

[0080] As referred to in the present invention, the term "primary amino group" refers to -NH2.

[0081] As mentioned in the present invention, the term "alkylamino" refers to an alkyl substituted amino group, such as and / or R 22 or R 23 represents the above-mentioned alkyl group.

[0082] As mentioned in the present invention, the term "hydroxyalkylamino" refers to a hydroxyalkyl substituted amino group, such as and / or R 22 or R 23 represents the above-mentioned alkyl group.

[0083] As used herein, the term "aryl" refers to monocyclic or polycyclic (eg, having 2, 3, or 4 fused rings) aromatic hydrocarbons, such as phenyl, naphthyl, anthracenyl, phenanthrenyl, indenyl, and the like.

[0084] As used herein, the term "nitrogen heterocycle" refers to an aromatic heterocycle or aliphatic ring having at least one heteroatom ring member such as O, N or S. Examples of preferred "heteroaryl" groups include, but are not limited to, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furanyl, thienyl, imidazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, 1,2,4-thiadiazolyl, pyrrolyl, pyrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, benzofuranyl, benzothienyl, benzothiazolyl, indolyl, and the like.

[0085] Compared with the prior art, the present invention has the following beneficial effects:

[0086] (1) The compound provided by the scheme of the present invention is a novel lipid compound, the core of which is based on chemical modification of lipoic acid to make it have good biocompatibility;

[0087] (2) The lipoic acid lipid of the present invention is composed of ester bonds and disulfide bonds. This unique structure gives it excellent endosomal escape and rapid release of nucleic acid drugs.

[0088] (3) The lipoic acid lipids of the present invention have excellent in vitro and in vivo cell transfection efficiency, low cytotoxicity and other characteristics, which significantly improve the effectiveness and safety of nucleic acid delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0090] Figure 1 It is the H NMR spectrum of the intermediate product A in Example 1 of the present invention.

[0091] Figure 2 It is the H NMR spectrum of the intermediate product B in Example 1 of the present invention.

[0092] Figure 3 It is the H NMR spectrum of the intermediate product C in Example 1 of the present invention.

[0093] Figure 4 It is the nuclear magnetic hydrogen spectrum of lipoic acid lipid in Example 1 of the present invention.

[0094] Figure 5 It is the mass spectrum of lipoic acid lipid in Example 1 of the present invention.

[0095] Figure 6 This is a graph showing the in vivo mRNA delivery efficiency of the lipoic acid-based lipid nanoparticle composition and ALC-0315 lipid nanoparticles after intramuscular administration in Application Example 1 of the present invention.

[0096] Figure 7 This is a graph showing the in vivo mRNA delivery efficiency of the lipoic acid-based lipid nanoparticle composition after intravenous administration in Application Example 1 of the present invention.

[0097] Figure 8 This is a graph of the bioluminescence intensity of ALC-0315 injected into the tail vein of the present invention.

[0098] Fig. 9 These are confocal microscope images of DC2.4 cells at 2, 4, and 6 hours after 2mOVA treatment in Example 2 of the present invention. DETAILED DESCRIPTION

[0099] Unless otherwise defined herein, scientific and technological terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0100] It should be noted that specific details are described in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in a variety of other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0101] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0102] In a first aspect, the present invention provides a lipid compound based on lipoic acid derivatives, wherein the lipid compound based on lipoic acid derivatives has a structure as shown in the following formula I:

[0103]

[0104] Wherein, R1 is selected from any one of substituted or unsubstituted C8-C18 (for example, C8, C10, C12, C14, C16, C18, etc.) alkyl or substituted or unsubstituted C8-C18 (for example, C8, C10, C12, C14, C16, C18, etc.) alkenyl; R2 is selected from any one of hydroxy-substituted C1-C12 (for example, C1, C2, C4, C6, C8, C10, C12, etc.) alkyl, amino-substituted C1-C12 (for example, C1, C2, C4, C6, C8, C10, C12, etc.) alkyl, alkylamino-substituted C1-C12 (for example, C1, C2, C4, C6, C8, C10, C12, etc.) alkyl It is C1, C2, C4, C6, C8, C10, C12, etc.) alkyl, C1~C12 (for example, it can be C1, C2, C4, C6, C8, C10, C12, etc.) alkyl substituted with hydroxyalkylamine, C1~C12 (for example, it can be C1, C2, C4, C6, C8, C10, C12, etc.) alkyl substituted with aryl, C1~C12 alkyl substituted with nitrogen heterocycle, C1~C12 (for example, it can be C1, C2, C4, C6, C8, C10, C12, etc.) alkyl substituted with nitrogen and oxygen heterocycle; n is an integer from 0 to 5 (for example, it can be 0, 1, 2, 3, 4, 5, preferably 1 or 2).

[0105] In the present invention, lipoic acid is chemically modified to have good biocompatibility, and the lipoic acid lipid is composed of ester bonds and disulfide bonds. This unique structure gives it excellent endosomal escape and rapid release of nucleic acid drugs. The lipoic acid lipid of the structure shown in Formula I has excellent in vitro and in vivo cell transfection efficiency, low cytotoxicity and other characteristics, which significantly improves the effectiveness and safety of nucleic acid delivery.

[0106] As an optional embodiment, the R1 is selected from C8-C18 straight chain or branched alkyl, or C8-C18 straight chain or branched alkenyl.

[0107] As an optional embodiment, the R1 is selected from any one of the following groups; wherein, Representative group connection position:

[0108]

[0109] As an optional embodiment, the R2 is selected from any one or a combination of at least two of the following groups; wherein, Representative group connection position:

[0110]

[0111] Among them, R 21 is selected from C2 to C6 straight or branched alkyl groups, R 22 or R 23Each is independently selected from any one of hydrogen, C1-C4 linear or branched alkyl, and C1-C4 linear or branched alkoxy.

[0112] As an optional embodiment, the R2 is selected from any one or a combination of at least two of the following groups; wherein, Representative group connection position:

[0113]

[0114] As a preferred embodiment, R2 is 4-hydroxyalkyl or 2-hydroxyalkyl

[0115]

[0116] As an optional embodiment, the lipid compound based on lipoic acid derivatives includes any one of the following compounds:

[0117]

[0118]

[0119] In a second aspect, the present invention provides a method for preparing a lipid compound based on lipoic acid derivatives as described in the first aspect, the preparation method comprising the following steps:

[0120] Alkyl carboxylic acid compounds and 6-bromohexanol undergo esterification reaction to obtain compound A; compound A and primary amine compounds undergo substitution reaction to obtain compound B; the reaction formula is as follows:

[0121]

[0122] Lipoic acid and bromoalkyl alcohol compounds are subjected to esterification reaction to obtain compound C; the reaction formula is shown below:

[0123]

[0124] Compound B reacts with compound C to obtain a lipid compound based on a lipoic acid derivative as shown in Formula I; the reaction formula is as follows:

[0125]

[0126] Wherein, the selection of R1, R2 and n is consistent with that defined in the first aspect.

[0127] As an optional embodiment, the molar ratio of the alkyl carboxylic acid compound to 6-bromohexanol is 1:(0.8-1.2), for example, it can be 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, etc.

[0128] As an optional embodiment, in the process of preparing the compound A, the temperature of the esterification reaction is 10-40°C, for example, it can be 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, etc., and the time of the esterification reaction is 12-36h, for example, it can be 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, etc.

[0129] As an optional embodiment, in the process of preparing the compound A, the esterification reaction is carried out in the presence of a condensation agent, and the molar ratio of the alkyl carboxylic acid compound to the condensation agent is 1:(1-4), for example, it can be 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.6, 1:3.8, 1:4, etc.

[0130] As an optional embodiment, in the process of preparing the compound A, the condensing agent includes any one of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbothiourea), DCC (1,3-dicyclohexylcarbodiimide) or DIC (N,N'-diisopropylcarbodiimide) or a combination of at least two of them, preferably EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide).

[0131] As an optional embodiment, in the process of preparing the compound A, the esterification reaction is carried out in the presence of a base, and the molar ratio of the alkyl carboxylic acid compound to the base is 1:(1-5), for example, it can be 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.6, 1:3.8, 1:4, 1:4.2, 1:4.4, 1:4.6, 1:4.8, 1:5, etc.

[0132] As an optional embodiment, in the process of preparing the compound A, the base includes any one of DMAP (4-dimethylaminopyridine), TEA (triethylamine), DIPEA (N,N-diisopropylethylamine), DIMPA (N,N-dimethylisopropylamine), 4-PPY (4-pyrrolidinylpyridine) or DABCO (1,4-diazabicyclo[2.2.2]octane) or a combination of at least two, preferably DMAP (4-dimethylaminopyridine).

[0133] As an optional embodiment, in the process of preparing the compound A, the esterification reaction is carried out in a solvent, and the solvent includes any one of dichloromethane, chloroform, toluene, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide or a combination of at least two thereof, preferably dichloromethane.

[0134] As an optional embodiment, the molar ratio of the compound A to the primary amine compound is 1:(0.8-1.2), for example, it can be 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, etc.

[0135] As an optional embodiment, in the process of preparing the compound B, the temperature of the substitution reaction is 50-70°C, for example, it can be 50°C, 52°C, 54°C, 56°C, 58°C, 60°C, 62°C, 64°C, 66°C, 68°C, 70°C, etc., and the time of the substitution reaction is 12-24h, for example, it can be 12h, 14h, 16h, 18h, 20h, 22h, 24h, etc.

[0136] As an optional embodiment, in the process of preparing the compound B, the substitution reaction is carried out in a solvent, and the solvent includes any one of methanol, ethanol, tetrahydrofuran, 2-methyltetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide or a combination of at least two thereof, preferably ethanol.

[0137] As an optional embodiment, the molar ratio of lipoic acid to bromoalkyl alcohol compound is 1:(0.8-1.2), for example, it can be 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, etc.

[0138] As an optional embodiment, in the process of preparing the compound C, the temperature of the esterification reaction is 10-40°C, for example, it can be 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, etc., and the time of the esterification reaction is 12-36h, for example, it can be 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, etc.

[0139] As an optional embodiment, in the process of preparing the compound C, the esterification reaction is carried out in the presence of a condensing agent, and the molar ratio of thioctic acid to the condensing agent is 1:(1-4), for example, it can be 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.6, 1:3.8, 1:4, etc.

[0140] As an optional embodiment, in the process of preparing the compound C, the condensing agent includes any one of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbothiourea), DCC (1,3-dicyclohexylcarbodiimide) or DIC (N,N'-diisopropylcarbodiimide) or a combination of at least two of them, preferably EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide).

[0141] As an optional embodiment, in the process of preparing the compound C, the esterification reaction is carried out in the presence of a base, and the molar ratio of thioctic acid to the base is 1:(1-5), for example, it can be 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.6, 1:3.8, 1:4, 1:4.2, 1:4.4, 1:4.6, 1:4.8, 1:5, etc.

[0142] As an optional embodiment, in the process of preparing the compound C, the base includes any one or a combination of at least two of DMAP (4-dimethylaminopyridine), TEA (triethylamine), DIPEA (N,N-diisopropylethylamine), DIMPA (N,N-dimethylisopropylamine), 4-PPY (4-pyrrolidinylpyridine) or DABCO (1,4-diazabicyclo[2.2.2]octane), preferably DMAP (4-dimethylaminopyridine).

[0143] As an optional embodiment, in the process of preparing the compound C, the esterification reaction is carried out in a solvent, and the solvent includes any one of dichloromethane, chloroform, toluene, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide or a combination of at least two thereof, preferably dichloromethane.

[0144] As an optional embodiment, the molar ratio of compound B to compound C is 1:(0.8-1.2), for example, it can be 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, etc.

[0145] As an optional embodiment, the reaction temperature of compound B and compound C is 80-100°C, for example, it can be 80°C, 82°C, 84°C, 86°C, 88°C, 90°C, 92°C, 94°C, 96°C, 98°C, 100°C, etc., and the reaction time of compound B and compound C is 42-54h, for example, it can be 42h, 44h, 46h, 48h, 50h, 52h, 54h, etc.

[0146] As an optional embodiment, the reaction of compound B and compound C is carried out in the presence of an iodine salt catalyst, and the added amount of the iodine salt catalyst is 0.01 to 10 mol% of the molar amount of compound B, for example, it can be 0.01 mol%, 0.1 mol%, 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol% and the like.

[0147] As an optional embodiment, the iodine salt catalyst includes NaI.

[0148] As an optional embodiment, the reaction of compound B and compound C is carried out in the presence of a base, and the molar ratio of compound B to the base is 1:(1-5), for example, it can be 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.6, 1:3.8, 1:4, 1:4.2, 1:4.4, 1:4.6, 1:4.8, 1:5, etc.

[0149] As an alternative embodiment, the base comprises potassium carbonate.

[0150] In a third aspect, the present invention provides a use of the lipoic acid derivative as described in the first aspect as a nucleic acid delivery medium.

[0151] As an optional embodiment, the nucleic acid includes any one of DNA, mRNA, siRNA, microRNA, antisense nucleic acid or circular RNA.

[0152] In a fourth aspect, the present invention provides a lipid nanoparticle composition, the lipid nanoparticle composition comprising lipoic acid derivative lipids, polyethylene glycol lipids, steroids and auxiliary lipids;

[0153] Wherein, the lipoic acid derivative lipid includes any one or a combination of at least two of the lipoic acid derivative-based lipid compounds described in the first aspect.

[0154] As an optional embodiment, the nanoparticle composition comprises the following components by mass percentage:

[0155] Lipoic acid derivative lipids 10% to 70%, polyethylene glycol lipids 1% to 25%, steroids 10% to 50%, and helper lipids 5% to 30%.

[0156] Taking the total mass of the nanoparticle composition as 100%, the content of the lipoic acid derivative lipid is 10% to 70%, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, etc.

[0157] Taking the total mass of the nanoparticle composition as 100%, the content of the polyethylene glycol lipid is 1% to 25%, for example, 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 25%, etc.

[0158] Taking the total mass of the nanoparticle composition as 100%, the content of the steroid is 10% to 50%, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, etc.

[0159] Taking the total mass of the nanoparticle composition as 100%, the auxiliary lipid is 5% to 30%, for example, 5%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, etc.

[0160] As an optional embodiment, the polyethylene glycol lipid is selected from any one of DMG-PEG 2000, DSPE-MPEG 2000 or DPPE-MPEG 2000, or a combination of at least two of them.

[0161] As an optional embodiment, the steroid is selected from any one or a combination of at least two of cholesterol, hydroxycholesterol, fucosterol, campesterol, ergosterol, glycocholic acid, taurocholic acid, stigmasterol or stigmasteranol;

[0162] As an optional embodiment, the auxiliary lipid is selected from any one or a combination of at least two of DOPC, DOPE, DSPC, DPPG, POPE, DSPE or DGTS.

[0163] In a fifth aspect, the present invention provides a nanoparticle composition encapsulating a nucleic acid drug, wherein the nanoparticle composition encapsulating a nucleic acid drug comprises a nucleic acid drug and a lipoic acid derivative lipid;

[0164] Wherein, the lipoic acid derivative lipid comprises any one or a combination of at least two of the lipid compounds based on lipoic acid derivatives as described above;

[0165] Alternatively, the nanoparticle composition encapsulating the nucleic acid drug comprises the nucleic acid drug and the lipid nanoparticle composition described in the fourth aspect.

[0166] As an optional embodiment, the nanoparticle composition encapsulating the nucleic acid drug includes the nucleic acid drug, thioctic acid derivative lipids, polyethylene glycol lipids, steroids and auxiliary lipids; wherein the thioctic acid derivative lipids include any one or a combination of at least two of the thioctic acid derivative-based lipid compounds shown in Formula I.

[0167] As an optional embodiment, the nucleic acid drug accounts for 5% to 20% of the total mass of the nanoparticle composition encapsulating the nucleic acid drug, for example, it can be 5%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, etc.

[0168] As an optional embodiment, the nanoparticle composition containing nucleic acid drugs comprises the following components by mass percentage:

[0169] Nucleic acid drugs 5% to 20%, lipoic acid derivative lipids 10% to 70%, polyethylene glycol lipids 1% to 25%, steroids 10% to 50%, and auxiliary lipids 5% to 30%.

[0170] As an optional embodiment, the nucleic acid drug includes any one of Luc-mRNA, OVA-mRNA, GFP-mRNA, PD-1-mRNA, siRNA or DNA, or a combination of at least two of them.

[0171] In a sixth aspect, the present invention provides a method for preparing a nanoparticle composition containing a nucleic acid drug according to the fifth aspect, wherein the method for preparing the nanoparticle composition containing a nucleic acid drug comprises the following steps:

[0172] Dissolving lipoic acid derivative lipid, polyethylene glycol lipid, steroid and auxiliary lipid in a solvent and mixing them to obtain an organic phase liposome solution;

[0173] Dissolving the nucleic acid drug in a pH buffer solution to obtain an aqueous nucleic acid drug solution;

[0174] The organic phase liposome solution and the aqueous phase nucleic acid drug solution are mixed and purified to obtain the nanoparticle composition containing the nucleic acid drug.

[0175] As an optional embodiment, the solvent includes any one of methanol, ethanol, tetrahydrofuran or dimethyl sulfoxide, or a combination of at least two thereof.

[0176] As an optional embodiment, in the organic phase liposome solution, the concentration of the thioctic acid derivative lipid is 5-50 mg / mL, for example, it can be 5 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL and the like.

[0177] As an optional embodiment, the pH buffer solution includes citric acid / sodium citrate solution.

[0178] As an optional embodiment, the pH of the pH buffer solution is 3 to 9, for example, 3, 4, 5, 6, 7, 8, 9, etc.

[0179] As an optional embodiment, in the aqueous nucleic acid drug solution, the concentration of the nucleic acid drug is 0.05 to 2 mg / mL, for example, it can be 0.05 mg / mL, 0.06 mg / mL, 0.08 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2 mg / mL, etc.

[0180] As an optional embodiment, the volume ratio of the organic phase liposome solution and the aqueous phase nucleic acid drug is 1:(1-20), for example, it can be 1:1, 1:2, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:16, 1:18, 1:20, etc.

[0181] As an optional embodiment, the purification comprises ultrafiltration and / or dialysis.

[0182] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market. The raw materials used in the following examples of the present invention are all commercially available products, all purchased from Aladdin Reagents.

[0183] Example 1

[0184] This embodiment provides a lipid compound based on lipoic acid derivatives, and the structural formula of the lipid compound based on lipoic acid derivatives is shown in the following formula I-1:

[0185]

[0186] The synthetic route of the lipid compound based on lipoic acid derivatives shown in Formula I-1 is as follows:

[0187]

[0188] The method for preparing the lipid compound based on lipoic acid derivatives shown in Formula I-1 comprises the following steps:

[0189] S1. Preparation of compound A1:

[0190] 2-Heptyldecanoic acid (2 g) and 6-bromohexanol (2.5 g) were dissolved in dichloromethane (50 mL), and EDC (1.6 g) and DMAP (0.25 g) were added, and stirred at room temperature for 24 h. After the reaction was complete, the mixture was diluted with dichloromethane, washed with saturated sodium bicarbonate and sodium chloride, and dried, and the excess solvent was removed by rotary evaporation. The residue was purified by silica gel chromatography (100% DCM) to obtain compound A (H NMR spectrum as shown in FIG. Figure 1 shown).

[0191] S2. Preparation of compound B1:

[0192] A solution of compound A1 (1.5 g) and 4-aminobutanol (5 mL) in ethanol (2 mL) was heated to 65°C and stirred for 18 h. The reaction mixture was concentrated under vacuum and the residue was partitioned between ethyl acetate and water. The organic layer was separated, washed with water and brine, dried over Na2SO4, and the excess solvent was removed by rotary evaporation. The residue was purified by silica gel chromatography (100% DCM to 100% 80:20:1 DCM / MeOH / ammonium hydroxide) to obtain compound B1 (H NMR spectrum as shown in Figure 2 shown).

[0193] S3. Preparation of compound C1:

[0194] Thioctic acid (2 g) and 6-bromohexanol (2.6 g) were dissolved in dichloromethane (50 mL), EDC (1.8 g) and DMAP (0.2 g) were added, and stirred at room temperature for 24 h. After the reaction was complete, the mixture was diluted with dichloromethane, washed with saturated sodium bicarbonate and sodium chloride, and dried, and the excess solvent was removed by rotary evaporation. The residue was purified by silica gel chromatography (100% DCM) to obtain the product C1 (H NMR spectrum as shown in FIG. Figure 3 shown).

[0195] Preparation of the lipid compound based on lipoic acid derivatives shown in S4.1-1:

[0196] NaI (36 mg) and K2CO3 (100 mg) were added to a solution of compound B1 (100 mg) and compound C1 (90 mg) in acetonitrile (5 mL), and the mixture was stirred in an oil bath at 90°C for 48 h. After the reaction was complete, the mixture was cooled to room temperature, the white precipitate was filtered, washed with dichloromethane, filtered, washed with saturated sodium bicarbonate aqueous solution and dichloromethane, and dried, and the excess solvent was removed by rotary evaporation. The residue was purified by silica gel chromatography (100% DCM to 100% 80:20:1 DCM / MeOH / ammonium hydroxide) to obtain a lipid compound based on a lipoic acid derivative shown in I-1 (H NMR spectrum as shown in FIG. Figure 4 As shown in the mass spectrum Figure 4 shown).

[0197] Example 2

[0198] This embodiment provides a lipid compound based on lipoic acid derivatives, and the structural formula of the lipid compound based on lipoic acid derivatives is shown in the following formula I-2:

[0199]

[0200] The synthetic route of the lipid compound based on lipoic acid derivatives shown in formula I-2 is as follows

[0201]

[0202] S1. Preparation of compound A2:

[0203] After dissolving pentadecan-6-enoic acid (2 g) and 6-bromohexanol (2.5 g) in dichloromethane (50 mL), EDC (1.6 g) and DMAP (0.25 g) were added and stirred at room temperature for 24 h. After the reaction was complete, the mixture was diluted with dichloromethane, washed with saturated sodium bicarbonate and sodium chloride, and dried, and the excess solvent was removed by rotary evaporation. The residue was purified by silica gel chromatography (100% DCM) to obtain compound A2.

[0204] S2. Preparation of compound B2:

[0205] A solution of compound A2 (1.5 g) and 4-aminobutanol (5 mL) in ethanol (2 mL) was heated to 65° C. and stirred for 24 h. The reaction mixture was concentrated under vacuum and the residue was partitioned between ethyl acetate and water. The organic layer was separated, washed with water, brine, dried over Na2SO4, and the excess solvent was removed by rotary evaporation. The residue was purified by silica gel chromatography (100% DCM to 100% 80:20:1 DCM / MeOH / ammonium hydroxide) to give compound B2.

[0206] Preparation of lipid compounds based on lipoic acid derivatives shown in S3 and I-3:

[0207] Compound B2 (100 mg) and compound C1 (90 mg) in acetonitrile (5 mL) solution were added with NaI (36 mg) and K2CO3 (100 mg) and the reaction was stirred in an oil bath at 90°C for 48 h. After the reaction was complete, the mixture was cooled to room temperature, the white precipitate was filtered, washed with dichloromethane, filtered, washed with saturated sodium bicarbonate aqueous solution and dichloromethane, and dried, and the excess solvent was removed by rotary evaporation. The residue was purified by silica gel chromatography (100% DCM to 100% 80:20:1 DCM / MeOH / ammonium hydroxide) to obtain a lipid compound based on lipoic acid derivatives shown in I-2.

[0208] Example 3

[0209] This embodiment provides a lipid compound based on lipoic acid derivatives, and the structural formula of the lipid compound based on lipoic acid derivatives is shown in the following formula I-3:

[0210]

[0211] The synthetic route of the lipid compound based on lipoic acid derivatives shown in Formula I-3 is as follows:

[0212]

[0213] S1. Preparation of compound A3:

[0214] 2-Hexyldecanoic acid (2 g) and 6-bromohexanol (2.5 g) were dissolved in dichloromethane (50 mL), and EDC (1.5 g) and DMAP (0.23 g) were added, and stirred at room temperature for 24 h. After the reaction was complete, it was diluted with dichloromethane, washed with saturated sodium bicarbonate and sodium chloride, and dried, and the excess solvent was removed by rotary evaporation. The residue was purified by silica gel chromatography (100% DCM) to obtain compound A2.

[0215] S2. Preparation of compound B3:

[0216] A solution of compound A3 (1.5 g) and 1-(3-aminopropyl)pyrrolidine (4 g) in ethanol (2 mL) was heated to 70°C and stirred for 24 h. The reaction mixture was concentrated under vacuum and the residue was partitioned between ethyl acetate and water. The organic layer was separated, washed with water, brine, dried over Na2SO4, and the excess solvent was removed by rotary evaporation. The residue was purified by silica gel chromatography (100% DCM to 100% 80:20:1 DCM / MeOH / ammonium hydroxide) to give compound B2.

[0217] Preparation of lipid compounds based on lipoic acid derivatives shown in S3 and I-3:

[0218] Compound B3 (100 mg) and compound C1 (90 mg) in acetonitrile (5 mL) solution were added with NaI (36 mg) and K2CO3 (100 mg) and the reaction was stirred in a 90°C oil bath for 48 h. After the reaction was complete, the mixture was cooled to room temperature, the white precipitate was filtered, washed with dichloromethane, filtered, washed with saturated sodium bicarbonate aqueous solution and dichloromethane, and dried, and the excess solvent was removed by rotary evaporation. The residue was purified by silica gel chromatography (100% DCM to 100% 80:20:1 DCM / MeOH / ammonium hydroxide) to obtain the lipid compound based on lipoic acid derivatives shown in I-3.

[0219] Example 4

[0220] This embodiment provides a lipid compound based on lipoic acid derivatives, and the structural formula of the lipid compound based on lipoic acid derivatives is shown in the following formula I-4:

[0221]

[0222] The synthetic route of the lipid compound based on lipoic acid derivatives shown in Formula I-4 is as follows:

[0223]

[0224] The preparation methods of S1, compounds A1 and B1 are carried out as described above;

[0225] S2. Preparation of compound C2:

[0226] Thioctic acid (2 g) and 4-bromo-1-butanol (1.5 g) were dissolved in dichloromethane (50 mL), EDC (1.8 g) and DMAP (0.2 g) were added, and stirred at room temperature for 24 h. After the reaction was complete, the mixture was diluted with dichloromethane, washed with saturated sodium bicarbonate and sodium chloride, and dried, and the excess solvent was removed by rotary evaporation. The residue was purified by silica gel chromatography (100% DCM) to obtain product C2.

[0227] Preparation of lipid compounds based on lipoic acid derivatives shown in S3 and I-4:

[0228] Compound B2 (100 mg) and compound C2 (90 mg) were added to an acetonitrile (5 mL) solution of NaI (36 mg) and K2CO3 (100 mg) and the mixture was stirred in an oil bath at 90°C for 48 h. After the reaction was complete, the mixture was cooled to room temperature, the white precipitate was filtered, washed with dichloromethane, filtered, washed with saturated sodium bicarbonate aqueous solution and dichloromethane, and dried, and the excess solvent was removed by rotary evaporation. The residue was purified by silica gel chromatography (100% DCM to 100% 80:20:1 DCM / MeOH / ammonium hydroxide) to obtain a lipid compound based on a lipoic acid derivative shown in I-4.

[0229] Application Example 1

[0230] This application example provides a nanoparticle composition encapsulating a nucleic acid drug, and the nanoparticle composition encapsulating a nucleic acid drug is prepared by the following steps:

[0231] (A) dissolving a lipoic acid derivative (a lipid compound based on a lipoic acid derivative represented by formula I-1 prepared in Example 1), cholesterol, DSPE, and DMG-PEG2000 in ethanol to prepare a 10 mg / mL ethanol solution, and then mixing them in a mass ratio of 59%:24%:13%:6% to obtain a mixed solution;

[0232] (B) The mRNA is Luc-mRNA, which is dissolved and diluted with a sodium citrate (100 mM) buffer solution at pH 3 to obtain an aqueous solution with an mRNA concentration of 1 mM.

[0233] (C) Under vortex conditions, the organic phase is dripped into the aqueous phase, the volume ratio of the organic phase solution to the aqueous phase solution is 1:3, and the lipids and mRNA are mixed in a ratio of 10:1 to obtain a slightly white solution. The ethanol is then removed by dialysis to obtain a lipid nanoparticle composition based on a lipoic acid compound encapsulating mRNA.

[0234] Application Example 2

[0235] This application example provides a nanoparticle composition encapsulating a nucleic acid drug, which differs from Application Example 1 only in that the lipid compound based on a thioctic acid derivative shown in Formula I-1 is replaced with an equal mass of a lipid compound based on a thioctic acid derivative shown in Formula I-2, and the other steps are exactly the same as Example 1.

[0236] Application Example 3

[0237] This application example provides a nanoparticle composition encapsulating a nucleic acid drug, which differs from Application Example 1 only in that the lipid compound based on a thioctic acid derivative shown in Formula I-1 is replaced with an equal mass of a lipid compound based on a thioctic acid derivative shown in Formula I-3, and the other steps are exactly the same as Example 1.

[0238] Application Example 4

[0239] This application example provides a nanoparticle composition encapsulating a nucleic acid drug, which differs from Application Example 1 only in that the lipid compound based on a thioctic acid derivative shown in Formula I-1 is replaced with an equal mass of a lipid compound based on a thioctic acid derivative shown in Formula I-4, and the other steps are exactly the same as Example 1.

[0240] Application Example 5

[0241] This application example provides a nanoparticle composition encapsulating a nucleic acid drug, which differs from application example 1 only in that cholesterol is no longer added, and lipoic acid derivatives are used to make up to 100%, and the other steps are completely consistent with example 1.

[0242] Application Example 6

[0243] This application example provides a nanoparticle composition encapsulating a nucleic acid drug, which differs from application example 1 only in that DSPE is no longer added, and lipoic acid derivatives are used to make up to 100%, and the other steps are completely consistent with example 1.

[0244] Application Example 7

[0245] This application example provides a nanoparticle composition encapsulating a nucleic acid drug, which is different from application example 1 only in that DMG-PEG2000 is no longer added, and lipoic acid derivatives are used to make up to 100%, and other steps are completely consistent with example 1.

[0246] Test Example 1

[0247] Evaluation of luciferase mRNA delivery performance in vivo

[0248] Test sample: the nanoparticle composition containing nucleic acid drugs provided in Application Examples 1 to 7;

[0249] Test method:

[0250] (1) Intramuscular injection: The lipid nanoparticle composition prepared for intramuscular injection was used at a dosage of 5 μg mRNA per mouse. The molar ratio of lipoic acid lipid, DOPE, cholesterol and DMGPEG2000 was 57:13:24:6; the mass ratio of lipoic acid lipid to mRNA was 10:1 or 5:1. After 12 hours, 100 μL of 30 mg / mL D-luciferin potassium salt was injected intraperitoneally into the mouse. After 5 minutes, the mouse was placed under a live imaging system to observe and take pictures to record the bioluminescence intensity of the mouse calf.

[0251] (2) Tail vein injection: The lipid nanoparticle composition prepared was injected into the tail vein at a dosage of 5 μg mRNA per mouse. The molar ratio of lipoic acid lipid, DOPE, cholesterol and DMG-PEG2000 was 57:13:24:6; the mass ratio of lipoic acid lipid to mRNA was 10:1 or 5:1. After 12 hours, 100 μL of 30 mg / mL D-luciferin potassium salt was injected intraperitoneally into each mouse. After 5 minutes, the mice were placed under the in vivo imaging system to observe and take pictures to record the bioluminescence intensity of the mouse body and isolated organs.

[0252] The test results are shown in Table 1 and Figure 6 , Figure 7 and Figure 8 As shown:

[0253] Table 1

[0254] sample Intramuscular injection bioluminescence intensity Bioluminescence intensity after tail vein injection Application Example 1 <![CDATA[4.895×10 8 ]]> <![CDATA[9.306×10 7 ]]> Application Example 2 <![CDATA[5.067×10 7 ]]> <![CDATA[2.135×10 7 ]]> Application Example 3 <![CDATA[1.393×10 8 ]]> <![CDATA[3.286×10 7 ]]> Application Example 4 <![CDATA[1.549×10 8 ]]> <![CDATA[7.484×10 7 ]]> Application Example 5 <![CDATA[3.589×10 7 ]]> <![CDATA[5.355×10 6 ]]> Application Example 6 <![CDATA[8.718×10 7 ]]> <![CDATA[3.874×10 7 ]]> Application Example 7 <![CDATA[9.635×10 6 ]]> <![CDATA[2.534×10 6 ]]> ALC-0315 <![CDATA[1.151×10 8 ]]> <![CDATA[9.128×10 7 ]]>

[0255] Note: Figure 6 The middle (left) is a lipid compound based on lipoic acid derivatives as shown in formula I-1, and the (right) is a comparison of the bioluminescence intensity of ALC-0315 intramuscular injection. Figure 7 Bioluminescence intensity of the lipid compounds based on lipoic acid derivatives shown in I-1 after tail vein injection. Figure 8 Bioluminescence intensity of ALC-0315 injected into tail vein.

[0256] As shown in Table 1 and Figure 6 to Figure 8 As shown, the compound provided by the scheme of the present invention is a new type of lipid compound, the core of which is based on the chemical modification of lipoic acid to make it have good biocompatibility; the lipoic acid lipid of the scheme of the present invention is composed of ester bonds and disulfide bonds, and this unique structure gives it excellent endosomal escape and rapid release of nucleic acid drugs.

[0257] Test Example 2

[0258] Test sample: the nanoparticle composition containing nucleic acid drugs provided in Application Example 1;

[0259] Test method:

[0260] The cellular internalization process of the complex was observed by confocal laser scanning microscopy (Leica STELLARIS 5). Cells were incubated with lipid / Cy5-mRNA complexes and observed at 2h, 4h, and 6h after treatment.

[0261] Test results:

[0262] like Fig. 9 As shown, the cellular uptake behavior was observed 2 h after administration. The complex successfully escaped from the endosome within 6 h. The dose of Cy5-mRNA was 0.5 μg / well. mOVA was labeled with Cy5 (red), stained with Hoechst 33342 (blue), and endosomes / lysosomes were stained with LysoGreen (green).

[0263] In summary, when the lipoic acid-derived lipid provided by the present invention is used as a delivery carrier for nucleic acid drugs, the disulfide bonds in the compound are cleaved under the action of intracellular glutathione, so that the compound is degraded, which not only triggers the timely release of nucleic acids, but also well reduces the cytotoxicity caused by the accumulation of the compound in cells, and has good biocompatibility. When the lipid nanoparticle composition of the present invention is used as a carrier to deliver nucleic acid drugs, it has excellent delivery efficiency and excellent spleen targeting, and can preferentially deliver nucleic acid drugs to the spleen.

[0264] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A lipid compound based on lipoic acid derivatives, characterized in that, The lipid compound based on lipoic acid derivatives has a structure as shown in the following formula I: Among them, R1 is selected from any one of substituted or unsubstituted C8~C18 alkyl or substituted or unsubstituted C8~C18 alkenyl; R2 is selected from C1~C12 alkyl substituted with hydroxyl, C1~C12 alkyl substituted with amino, C1~C12 alkyl substituted with alkylamino, C1~C12 alkyl substituted with hydroxyalkylamino, C1~C12 alkyl substituted with aryl, C1~C12 alkyl substituted with nitrogen heterocycle, C1~C12 alkyl substituted with nitrogen and oxygen heterocycle; n is an integer from 0 to 5.

2. The lipid compound based on lipoic acid derivatives according to claim 1, characterized in that The R1 is selected from a C8-C18 straight chain or branched alkyl group, or a C8-C18 straight chain or branched alkenyl group; Preferably, the R1 is selected from any one of the following groups; wherein, Representative group connection position:

3. The lipid compound based on lipoic acid derivatives according to claim 1, characterized in that The R2 is selected from any one or a combination of at least two of the following groups; wherein, Representative group connection position: Among them, R 21 is selected from C2 to C6 straight or branched alkyl groups, R 22 or R 23 Each is independently selected from any one of hydrogen, C1-C4 straight chain or branched alkyl, C1-C4 straight chain or branched alkoxy; Preferably, the R2 is selected from any one or a combination of at least two of the following groups; wherein, Representative group connection position:

4. The lipid compound based on lipoic acid derivatives according to claim 1, characterized in that The lipid compound based on lipoic acid derivatives includes any one of the following compounds:

5. A method for preparing a lipid compound based on lipoic acid derivatives according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: Alkyl carboxylic acid compounds and 6-bromohexanol undergo esterification reaction to obtain compound A; compound A and primary amine compounds undergo substitution reaction to obtain compound B; the reaction formula is as follows: Lipoic acid and bromoalkyl alcohol compounds are subjected to esterification reaction to obtain compound C; the reaction formula is shown below: Compound B reacts with compound C to obtain a lipid compound based on a lipoic acid derivative as shown in Formula I; the reaction formula is as follows: Wherein, the selection of R1, R2 and n is consistent with that defined in claim 1.

6. The method for preparing a lipid compound based on lipoic acid derivatives according to claim 5, characterized in that: The molar ratio of the alkyl carboxylic acid compound to 6-bromohexanol is 1:(0.8-1.2); Preferably, in the process of preparing the compound A, the temperature of the esterification reaction is 10 to 40° C., and the time of the esterification reaction is 12 to 36 hours; Preferably, in the process of preparing the compound A, the esterification reaction is carried out in the presence of a condensing agent, and the molar ratio of the alkyl carboxylic acid compound to the condensing agent is 1:(1-4); Preferably, in the process of preparing the compound A, the esterification reaction is carried out in the presence of a base, and the molar ratio of the alkyl carboxylic acid compound to the base is 1:(0.1-5); Preferably, the molar ratio of the compound A to the primary amine compound is 1:(0.8-1.2); Preferably, in the process of preparing the compound B, the temperature of the substitution reaction is 50 to 70° C., and the time of the substitution reaction is 12 to 24 hours; Preferably, the molar ratio of lipoic acid to bromoalkyl alcohol compound is 1:(0.8-1.2); Preferably, in the process of preparing the compound C, the temperature of the esterification reaction is 10 to 40° C., and the time of the esterification reaction is 12 to 36 hours; Preferably, in the process of preparing the compound C, the esterification reaction is carried out in the presence of a condensing agent, and the molar ratio of lipoic acid to the condensing agent is 1:(1-4); Preferably, in the process of preparing the compound C, the esterification reaction is carried out in the presence of a base, and the molar ratio of lipoic acid to the base is 1:(0.1-5); Preferably, the molar ratio of compound B to compound C is 1:(0.8-1.2); Preferably, the temperature for the reaction of compound B and compound C is 80-100° C., and the time for the reaction of compound B and compound C is 42-54 h; Preferably, the reaction of compound B and compound C is carried out in the presence of an iodine salt catalyst, and the amount of the iodine salt catalyst added is 0.01 to 10 mol% of the molar amount of compound B; Preferably, the reaction of compound B and compound C is carried out in the presence of a base, and the molar ratio of compound B to the base is 1:(1-5).

7. A use of a lipoic acid derivative according to any one of claims 1 to 4 as a nucleic acid delivery medium; And / or, the nucleic acid comprises any one of DNA, mRNA, siRNA, microRNA, antisense nucleic acid or circular RNA.

8. A lipid nanoparticle composition, characterized in that: The lipid nanoparticle composition includes lipoic acid derivative lipids, polyethylene glycol lipids, steroids and helper lipids; Wherein, the lipoic acid derivative lipid comprises any one or a combination of at least two of the lipoic acid derivative-based lipid compounds described in any one of claims 1 to 4; Preferably, the nanoparticle composition comprises the following components by mass percentage: Lipoic acid derivative lipids 10% to 70%, polyethylene glycol lipids 1% to 25%, steroids 10% to 50%, auxiliary lipids 5% to 30%; Preferably, the polyethylene glycol lipid is selected from any one or a combination of at least two of DMG-PEG 2000, DSPE-MPEG 2000 or DPPE-MPEG 2000; Preferably, the steroid is selected from any one or a combination of at least two of cholesterol, hydroxycholesterol, fucosterol, campesterol, ergosterol, glycocholic acid, taurocholic acid, stigmasterol or stigmasteranol; Preferably, the helper lipid is selected from any one or a combination of at least two of DOPC, DOPE, DSPC, DPPG, POPE, DSPE or DGTS.

9. A nanoparticle composition containing a nucleic acid drug, characterized in that: The nanoparticle composition containing nucleic acid drugs comprises nucleic acid drugs and lipoic acid derivative lipids; Wherein, the lipoic acid derivative lipid comprises any one or a combination of at least two of the lipoic acid derivative-based lipid compounds described in any one of claims 1 to 4; Or, the nanoparticle composition encapsulating the nucleic acid drug comprises the nucleic acid drug and the lipid nanoparticle composition according to claim 8; Preferably, the nucleic acid drug accounts for 5% to 20% of the total mass of the nanoparticle composition containing the nucleic acid drug; Preferably, the nucleic acid drug comprises any one of Luc-mRNA, OVA-mRNA, GFP-mRNA, PD-1-mRNA, siRNA or DNA, or a combination of at least two thereof.

10. A method for preparing a nanoparticle composition containing a nucleic acid drug according to claim 9, characterized in that: The preparation method comprises: Dissolving lipoic acid derivative lipid, polyethylene glycol lipid, steroid and auxiliary lipid in a solvent and mixing them to obtain an organic phase liposome solution; Dissolving the nucleic acid drug in a pH buffer solution to obtain an aqueous nucleic acid drug solution; The organic phase liposome solution and the aqueous phase nucleic acid drug solution are mixed and purified to obtain the nanoparticle composition containing the nucleic acid drug; Preferably, the solvent comprises any one of methanol, ethanol, tetrahydrofuran or dimethyl sulfoxide or a combination of at least two thereof; Preferably, in the organic phase liposome solution, the concentration of the lipoic acid derivative lipid is 5 to 50 mg / mL; Preferably, the pH buffer solution comprises a citric acid / sodium citrate solution; Preferably, the pH of the pH buffer solution is 3 to 9; Preferably, in the aqueous nucleic acid drug solution, the concentration of the nucleic acid drug is 0.05 to 2 mg / mL; Preferably, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid drug is 1:(1-20); Preferably, the purification comprises ultrafiltration and / or dialysis.

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