A lipo-compound based on a lipoic acid derivative, compositions thereof, and methods of making and using the same

Abstract

The application provides a lipids compound based on lipoic acid derivative, a composition, and a preparation method and application thereof. When the lipoic acid derivative lipids provided by the application is used as a delivery carrier of nucleic acid drugs, due to the cleavage of the disulfide bond in the compound under the action of intracellular glutathione, the compound is degraded, which not only triggers the timely release of the nucleic acid, but also well reduces the cytotoxicity caused by the accumulation of the compound in the cell, and has good biocompatibility. When the lipids nanoparticle composition of the application is used as a carrier to deliver nucleic acid drugs, the nucleic acid drugs can be preferentially delivered to the spleen, and the lipids nanoparticle composition has excellent delivery efficiency and excellent spleen targeting.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a lipid compound, composition, preparation method and application based on thioctic acid derivative. Background Technology

[0002] In recent years, gene therapy has shown great potential in biomedical applications. For nucleic acid drugs to function in vivo, safe, efficient, and stable delivery systems are needed to protect nucleic acids from degradation and allow for cellular uptake and release. To date, various materials have been developed for nucleic acid delivery, such as lipids, polymers, peptides, and inorganic nanoparticles. Among these carriers, lipid nanoparticles (LNPs) have shown great promise as nucleic acid carriers. In particular, in 2020, two mRNA vaccines developed by Pfizer BioNTech and Moderna both used lipid nanoparticles to deliver antigen mRNA.

[0003] LNPs typically consist of ionizable lipids, polyethylene glycol-modified lipids, cholesterol, and cofactor phospholipids. Ionizable lipids have been shown to be a key component of LNPs, significantly impacting nucleic acid delivery efficiency. They provide a positive charge, enabling the encapsulation of negatively charged nucleic acids. However, endosome escape remains a fundamental obstacle to LNP-mediated nucleic acid delivery systems. Therefore, there is an urgent need to develop novel ionizable lipids with highly efficient endosome escape capabilities to improve nucleic acid delivery both in vitro and in vivo.

[0004] By rationally designing the chemical structure of ionizable lipids to increase cellular uptake and endosome escape, the delivery efficiency of the LNP system can be improved. Lipoic acid is an essential factor in human cellular energy metabolism; it is a disulfide compound and is completely biodegradable in the intracellular reducing environment. Furthermore, studies have shown that disulfide bonds significantly contribute to thiol-mediated uptake, effectively transporting cargo into the cytosol. Therefore, this invention, based on the chemical modification of lipoic acid, rationally designs 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. To achieve therapeutic effects, nucleic acid molecules must reach specific target cells and produce sufficient target proteins. However, targeted delivery and endosome escape remain challenging for nucleic acid delivery systems. Therefore, there is an urgent need to develop safer and more efficient LNP nucleic acid delivery materials, and the key to achieving this goal lies in developing novel ionizable lipid chemical structures.

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

[0007] The present invention aims to provide a lipid compound, composition, preparation method, and application based on a lipoic acid derivative. The lipid compound and composition based on the lipoic acid derivative possess highly efficient nucleic acid delivery capabilities.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0009] In a first aspect, the present invention provides a lipid compound based on a lipoic acid derivative, said lipid compound having the structure shown in Formula I:

[0010]

[0011] Wherein, R1 is selected from any one of substituted or unsubstituted C8-C18 alkyl or substituted or unsubstituted C8-C18 alkenyl groups; R2 is selected from hydroxy-substituted C1-C12 alkyl, amino-substituted C1-C12 alkyl, alkylamino-substituted C1-C12 alkyl, hydroxyalkylamino-substituted C1-C12 alkyl, aryl-substituted C1-C12 alkyl, nitrogen-substituted C1-C12 alkyl, and nitrogen-oxygen-substituted C1-C12 alkyl; n is an integer from 0 to 5.

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

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

[0014]

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

[0016]

[0017] Among them, R 21 Selected from C2-C6 straight-chain 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 groups, or C1-C4 straight-chain or branched alkoxy groups.

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

[0019]

[0020] Preferably, the lipid compound based on lipoic acid derivatives includes 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 a lipoic acid derivative as described in the first aspect, the method comprising the following steps:

[0024] An alkyl carboxylic acid compound and 6-bromohexanol undergo esterification to give compound A; compound A and a primary amine compound undergo substitution to give compound B; the reaction formulas are shown below:

[0025]

[0026] The esterification of lipoic acid and bromoalkyl alcohols yields compound C; the reaction formula is shown below:

[0027]

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

[0029]

[0030] The choices of R1, R2, and n are consistent with those shown in the first aspect.

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

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

[0033] Preferably, in the preparation of 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 preparation of 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 compound A to the primary amine compound is 1:(0.8 to 1.2).

[0038] Preferably, in the preparation of compound B, the temperature of the substitution reaction is 50–70°C, and the time of the substitution reaction is 12–24 h.

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

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

[0041] Preferably, in the preparation of 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).

[0042] Preferably, the condensing agent is EDC.

[0043] Preferably, in the preparation of compound C, the esterification reaction is carried out in the presence of a base, and the molar ratio of lipoic acid to 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 to 1.2).

[0046] Preferably, the reaction temperature of compound B and compound C is 80-100°C, and the reaction time 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, wherein the amount of the iodine salt catalyst added is 0.01 to 10 mol% of the molar amount of compound B.

[0048] Preferably, the reaction between 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] Thirdly, the present invention provides an application of thioctic acid derivatives as described in the first aspect in the use of nucleic acid delivery media.

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

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

[0052] The thioctic acid derivative lipids include any one or a combination of at least two of the lipid compounds based on thioctic acid derivatives as described in the first aspect.

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

[0054] The composition includes 10%–70% lipoic acid derivative lipids, 1%–25% polyethylene glycol lipids, 10%–50% steroids, and 5%–30% auxiliary lipids.

[0055] 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-MPEG2000.

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

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

[0058] Fifthly, the present invention provides a nanoparticle composition encapsulating a nucleic acid drug, the nanoparticle composition encapsulating a nucleic acid drug comprising a nucleic acid drug and a lipoic acid derivative lipid;

[0059] The lipoic acid derivative lipids include any one or a combination of at least two of the lipid compounds based on lipoic acid derivatives as described in the first aspect.

[0060] Alternatively, the nanoparticle composition encapsulating a nucleic acid drug may include a nucleic acid drug and the lipid nanoparticle composition described in the fourth aspect, wherein the lipid nanoparticle composition includes a nucleic acid drug, lipoic acid derivative lipids, polyethylene glycol lipids, steroids, and auxiliary lipids.

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

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

[0063] Sixthly, the present invention provides a method for preparing a nanoparticle composition encapsulating a nucleic acid drug as described in the fourth aspect, the method comprising:

[0064] Lipoic acid derivative lipids, polyethylene glycol lipids, steroids and auxiliary lipids were dissolved in a solvent and mixed to obtain an organic phase liposome solution.

[0065] The nucleic acid drug was dissolved 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 were mixed and purified to obtain the nanoparticle composition encapsulating the nucleic acid drug.

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

[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 to 9.

[0071] Preferably, in the aqueous nucleic acid drug solution, the concentration of the nucleic acid drug is 0.05–2 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 includes ultrafiltration and / or dialysis.

[0074] [Terminology Explanation]

[0075] The various aspects and features of the present invention will be further described below.

[0076] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Nevertheless, this invention still aims to provide a more detailed explanation and interpretation of these terms and phrases. In the event of any inconsistency between the mentioned terms and their known meanings and those of the present invention, the meaning expressed in this invention shall prevail. Below are definitions of various terms used in this invention. These definitions apply to all terms used throughout this specification, unless otherwise specified in the specific context. The following provides definitions of various groups in the compounds of this invention, which, unless otherwise defined, are used consistently throughout the specification.

[0077] As mentioned in this 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, when "C1 to C12 alkyl" is mentioned, it refers to a straight-chain alkyl group or a branched alkyl group having 1 to 20 carbon atoms. Specific groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, etc., and similar groups.

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

[0079] As mentioned in this invention, the term "hydroxyl group" refers to -OH.

[0080] As mentioned in this invention, the term "primary amino" refers to -NH2.

[0081] As mentioned in this invention, the term "alkylamine" refers to an alkyl-substituted amino group, for example... and / or R 22 or R 23 This refers to the alkyl group mentioned above.

[0082] As mentioned in this invention, the term "hydroxyalkylamine" refers to a hydroxyalkyl-substituted amino group, for example... and / or R 22 or R 23 This refers to the alkyl group mentioned above.

[0083] As used in this invention, the term "aryl" refers to a monocyclic or polycyclic aromatic hydrocarbon (e.g., having 2, 3, or 4 fused rings), such as phenyl, naphthyl, anthracene, phenanthryl, indene, and similar groups.

[0084] As used in this invention, 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: pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furanyl, thiopheneyl, imidazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, 1,2,4-thiadiazolyl, pyrroleyl, pyrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, benzofuranyl, benzothiopheneyl, benzothiazolyl, indoleyl, and similar groups.

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

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

[0087] (2) The lipoic acid lipid of the present invention is composed of ester bonds and disulfide bonds. This unique structure endows it with excellent endosome 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 and cytotoxicity, which significantly improve the effectiveness and safety of nucleic acid delivery. Attached Figure Description

[0089] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

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

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

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

[0093] Figure 4 This is the 1H NMR spectrum of the lipoic acid lipid in Example 1 of this invention.

[0094] Figure 5 This 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 lipid nanoparticle composition based on thioctic acid after intravenous administration in Application Example 1 of the present invention.

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

[0098] Figure 9 These are confocal microscope images taken at 2, 4, and 6 hours after mOVA treatment of DC2.4 cells in Example 2 of this invention. Detailed Implementation

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

[0100] It should be noted that specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many ways other than those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the 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 with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0102] In a first aspect, the present invention provides a lipid compound based on a lipoic acid derivative, said lipid compound having the structure shown in Formula I:

[0103]

[0104] Wherein, R1 is selected from any one of substituted or unsubstituted C8-C18 (e.g., C8, C10, C12, C14, C16, C18, etc.) alkyl or substituted or unsubstituted C8-C18 (e.g., C8, C10, C12, C14, C16, C18, etc.) alkenyl groups; R2 is selected from hydroxylated C1-C12 (e.g., C1, C2, C4, C6, C8, C10, C12, etc.) alkyl, amino-substituted C1-C12 (e.g., C1, C2, C4, C6, C8, C10, C12, etc.) alkyl, alkylamino-substituted C1-C12 (e.g., C1, C2, C4, C6, C8, C10, C12, etc.) alkyl, alkylamino-substituted C1-C12 (e.g., C8, C2, C4, C6, C8, C10, C12, etc.) It is an alkyl group substituted with alkyl, hydroxyalkylamine group, C1-C12 alkyl group (e.g., C1, C2, C4, C6, C8, C10, C12, etc.), an aryl group substituted with alkyl, nitrogen heterocyclic group substituted with alkyl, nitrogen oxycyclic group substituted with alkyl; n is an integer from 0 to 5 (e.g., 0, 1, 2, 3, 4, 5, preferably 1 or 2).

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

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

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

[0108]

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

[0110]

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

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

[0113]

[0114] In a preferred embodiment, R2 is a 4-hydroxyalkyl group. or 2-hydroxyalkyl

[0115]

[0116] As an optional implementation, 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 a lipoic acid derivative as described in the first aspect, the method comprising the following steps:

[0120] An alkyl carboxylic acid compound and 6-bromohexanol undergo esterification to give compound A; compound A and a primary amine compound undergo substitution to give compound B; the reaction formulas are shown below:

[0121]

[0122] The esterification of lipoic acid and bromoalkyl alcohols yields compound C; the reaction formula is shown below:

[0123]

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

[0125]

[0126] The choices of R1, R2, and n are consistent with those specified in the first aspect.

[0127] As an optional implementation, the molar ratio of the alkyl carboxylic acid compound to 6-bromohexanol is 1:(0.8 to 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 implementation, in the process of preparing compound A, the temperature of the esterification reaction is 10-40°C, for example, 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, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, etc.

[0129] As an optional implementation, in the preparation of 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), for example, 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 implementation, in the preparation of compound A, the condensing agent includes any one or a combination of at least two of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbothiourea), DCC (1,3-dicyclohexylcarbodiimide), or DIC (N,N'-diisopropylcarbodiimide), preferably EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide).

[0131] As an optional implementation, in the preparation of 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 preparation of compound A, 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).

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

[0134] As an optional implementation, the molar ratio of compound A to the primary amine compound is 1:(0.8 to 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 implementation, in the process of preparing compound B, the temperature of the substitution reaction is 50-70°C, for example, 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, 12h, 14h, 16h, 18h, 20h, 22h, 24h, etc.

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

[0137] As an optional implementation, the molar ratio of the thioctic acid and the bromoalkyl alcohol compound is 1:(0.8 to 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 implementation, in the process of preparing compound C, the temperature of the esterification reaction is 10-40°C, for example, 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, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, etc.

[0139] As an optional implementation, in the preparation of 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, 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 implementation, in the preparation of compound C, the condensing agent includes any one or a combination of at least two of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbothiourea), DCC (1,3-dicyclohexylcarbodiimide), or DIC (N,N'-diisopropylcarbodiimide), preferably EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide).

[0141] As an optional implementation, in the preparation of compound C, the esterification reaction is carried out in the presence of a base, and the molar ratio of lipoic acid to 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 preparation of 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 implementation, in the preparation of compound C, the esterification reaction is carried out in a solvent, which includes any one or a combination of at least two of dichloromethane, chloroform, toluene, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, N,N-dimethylformamide, and N,N-dimethylacetamide, preferably dichloromethane.

[0144] As an optional implementation, the molar ratio of compound B to compound C is 1:(0.8 to 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 implementation, the reaction temperature of compound B and compound C is 80-100°C, for example, 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 42-54 hours, for example, 42 hours, 44 hours, 46 hours, 48 ​​hours, 50 hours, 52 hours, 54 hours, etc.

[0146] As an optional implementation, the reaction of compound B and compound C is carried out in the presence of an iodine salt catalyst, wherein the amount of the iodine salt catalyst added 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%, etc.

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

[0148] As an optional implementation, the reaction between 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 to 5), for example, 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 optional implementation, the alkali includes potassium carbonate.

[0150] Thirdly, the present invention provides an application of thioctic acid derivatives as described in the first aspect in the use of nucleic acid delivery media.

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

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

[0153] The thioctic acid derivative lipids include any one or a combination of at least two of the lipid compounds based on thioctic acid derivatives as described in the first aspect.

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

[0155] The composition includes 10%–70% lipoic acid derivative lipids, 1%–25% polyethylene glycol lipids, 10%–50% steroids, and 5%–30% auxiliary lipids.

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

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

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

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

[0160] As an optional implementation, 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.

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

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

[0163] Fifthly, the present invention provides a nanoparticle composition encapsulating a nucleic acid drug, the nanoparticle composition encapsulating a nucleic acid drug comprising a nucleic acid drug and a lipoic acid derivative lipid;

[0164] The lipoic acid derivative lipids include 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 a nucleic acid drug may include a nucleic acid drug and the lipid nanoparticle composition described in the fourth aspect.

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

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

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

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

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

[0171] Sixthly, the present invention provides a method for preparing a nanoparticle composition loaded with a nucleic acid drug according to the fifth aspect, the method comprising the following steps:

[0172] Lipoic acid derivative lipids, polyethylene glycol lipids, steroids and auxiliary lipids were dissolved in a solvent and mixed to obtain an organic phase liposome solution.

[0173] The nucleic acid drug was dissolved 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 were mixed and purified to obtain the nanoparticle composition encapsulating the nucleic acid drug.

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

[0176] As an optional embodiment, the concentration of the lipoic acid derivative lipid in the organic phase liposome solution is 5 to 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, etc.

[0177] As an optional implementation, the pH buffer solution comprises a citric acid / sodium citrate solution.

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

[0179] As an optional implementation, the concentration of the nucleic acid drug in the aqueous phase nucleic acid drug solution 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 implementation, the volume ratio of the organic phase liposome solution to 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 implementation, the purification includes ultrafiltration and / or dialysis.

[0182] The present invention will be further illustrated below through examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market. All raw materials used in the following examples of the present invention are commercially available products and were purchased from Aladdin Reagents.

[0183] Example 1

[0184] This embodiment provides a lipid compound based on a lipoic acid derivative, the structural formula of which is shown in Formula I-1 below:

[0185]

[0186] The synthetic route for lipid compounds based on lipoic acid derivatives, as 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 includes the following steps:

[0189] Preparation of S1 and compound A1:

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

[0191] Preparation of S2 and 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 separated between ethyl acetate and water. The organic layer was separated, washed with water and brine, dried over Na2SO4, and 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 B1 (NMR spectrum as shown in Figure 1). Figure 2 (As shown).

[0193] Preparation of S3 and compound C1:

[0194] Lipoic acid (2 g) and 6-bromohexanol (2.6 g) were dissolved in dichloromethane (50 mL), and EDC (1.8 g) and DMAP (0.2 g) were added. The mixture was stirred at room temperature for 24 h. After the reaction was complete, the solution was diluted with dichloromethane, washed with saturated sodium bicarbonate and sodium chloride, dried, and the excess solvent was removed by rotary evaporation. The residue was purified by silica gel chromatography (100% DCM) to obtain product C1 (NMR spectrum as shown in Figure 1). Figure 3 (As shown).

[0195] Preparation of lipid compounds based on lipoic acid derivatives as shown in S4 and I-1:

[0196] The acetonitrile (5 mL) solution of compound B1 (100 mg) and compound C1 (90 mg) was reacted with NaI (36 mg) and K2CO3 (100 mg) in an oil bath at 90 °C for 48 h with stirring. After the reaction was complete, the mixture was cooled to room temperature, the white precipitate was filtered, washed with dichloromethane, washed with saturated sodium bicarbonate aqueous solution and dichloromethane, 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 give the lipid compound based on lipoic acid derivative shown in I-1 (NMR spectrum as shown in Figure 1). Figure 4 As shown, the mass spectrum is as follows Figure 4 (As shown).

[0197] Example 2

[0198] This embodiment provides a lipid compound based on a lipoic acid derivative, the structural formula of which is shown in Formula I-2 below:

[0199]

[0200] The synthetic route for lipid compounds based on lipoic acid derivatives, as shown in Formula I-2, is as follows:

[0201]

[0202] Preparation of S1 and compound A2:

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

[0204] Preparation of S2 and 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 separated between ethyl acetate and water. The organic layer was separated, washed with water and brine, dried over Na2SO4, and 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 as shown in S3 and I-3:

[0207] The acetonitrile (5 mL) solution of compound B2 (100 mg) and compound C1 (90 mg) was reacted with NaI (36 mg) and K2CO3 (100 mg) in an oil bath at 90 °C for 48 h with stirring. After the reaction was complete, the mixture was cooled to room temperature, the white precipitate was filtered, washed with dichloromethane, washed with saturated sodium bicarbonate aqueous solution and dichloromethane, 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 give the lipid compound based on lipoic acid derivative shown in I-2.

[0208] Example 3

[0209] This embodiment provides a lipid compound based on a lipoic acid derivative, the structural formula of which is shown in Formula I-3 below:

[0210]

[0211] The synthetic route for lipid compounds based on lipoic acid derivatives, as shown in Formula I-3, is as follows:

[0212]

[0213] Preparation of S1 and compound A3:

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

[0215] Preparation of S2 and 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 separated between ethyl acetate and water. The organic layer was separated, washed with water and brine, dried over Na2SO4, and 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 as shown in S3 and I-3:

[0218] The acetonitrile (5 mL) solution of compound B3 (100 mg) and compound C1 (90 mg) was reacted with NaI (36 mg) and K2CO3 (100 mg) in an oil bath at 90 °C for 48 h with stirring. After the reaction was complete, the mixture was cooled to room temperature, the white precipitate was filtered, washed with dichloromethane, washed with saturated sodium bicarbonate aqueous solution and dichloromethane, 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 give the lipid compounds based on lipoic acid derivatives shown in I-3.

[0219] Example 4

[0220] This embodiment provides a lipid compound based on a lipoic acid derivative, the structural formula of which is shown in Formula I-4 below:

[0221]

[0222] The synthetic route for lipid compounds based on lipoic acid derivatives, as shown in Formula I-4, is as follows:

[0223]

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

[0225] Preparation of S2 and compound C2:

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

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

[0228] The acetonitrile (5 mL) solution of compound B2 (100 mg) and compound C2 (90 mg) was reacted with NaI (36 mg) and K2CO3 (100 mg) in an oil bath at 90 °C for 48 h with stirring. After the reaction was complete, the mixture was cooled to room temperature, the white precipitate was filtered, washed with dichloromethane, washed with saturated sodium bicarbonate aqueous solution and dichloromethane, 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 give the lipid compounds based on lipoic acid derivatives shown in I-4.

[0229] Application Example 1

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

[0231] (A) The lipoic acid derivative (the lipid compound based on the lipoic acid derivative shown in Formula I-1 prepared in Example 1), cholesterol, DSPE, and DMG-PEG2000 were dissolved in ethanol respectively to prepare 10 mg / mL ethanol solutions, and then mixed in a mass ratio of 59%:24%:13%:6% to obtain a mixture.

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

[0233] (C) Under vortex conditions, the organic phase was added dropwise to the aqueous phase at a volume ratio of 1:3, and the lipids and mRNA were mixed at a mass ratio of 10:1 to obtain a slightly white solution. Ethanol was then removed by dialyzing. This yielded a lipid nanoparticle composition based on lipoic acid compounds encapsulating mRNA.

[0234] Application Example 2

[0235] This application example provides a nanoparticle composition encapsulating a nucleic acid drug. The only difference from Application Example 1 is that the lipid compound based on thioctic acid derivative shown in Formula I-1 is replaced with an equal mass of the lipid compound based on thioctic acid derivative shown in Formula I-2. All other steps are completely consistent with Example 1.

[0236] Application Example 3

[0237] This application example provides a nanoparticle composition encapsulating a nucleic acid drug. The only difference from Application Example 1 is that the lipid compound based on thioctic acid derivative shown in Formula I-1 is replaced with an equal mass of the lipid compound based on thioctic acid derivative shown in Formula I-3. All other steps are completely consistent with Example 1.

[0238] Application Example 4

[0239] This application example provides a nanoparticle composition encapsulating a nucleic acid drug. The only difference from Application Example 1 is that the lipid compound based on thioctic acid derivative shown in Formula I-1 is replaced with an equal mass of the lipid compound based on thioctic acid derivative shown in Formula I-4. All other steps are completely consistent with Example 1.

[0240] Application Example 5

[0241] This application example provides a nanoparticle composition encapsulating a nucleic acid drug. The only difference from Application Example 1 is that cholesterol is no longer added, but is supplemented to 100% with a thioctic acid derivative. All other steps are exactly the same as in Example 1.

[0242] Application Example 6

[0243] This application example provides a nanoparticle composition encapsulating a nucleic acid drug. The only difference from Application Example 1 is that DSPE is no longer added, and the amount is made up to 100% with a thioctic acid derivative. 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. The only difference from application example 1 is that DMG-PEG2000 is no longer added, and the amount is made up to 100% with a thioctic acid derivative. The other steps are completely consistent with example 1.

[0246] Test Example 1

[0247] Evaluation of in vivo delivery performance of luciferase mRNA

[0248] Test samples: Nanoparticle compositions containing nucleic acid drugs provided in Examples 1-7;

[0249] Test method:

[0250] (1) Intramuscular injection: The lipid nanoparticle composition prepared by intramuscular injection was administered at a dosage of 5 μg mRNA per mouse. The molar ratio of lipoic acid lipids, DOPE, cholesterol and DMGPEG2000 was 57:13:24:6; the mass ratio of lipoic acid lipids to mRNA was 10:1 or 5:1. After 12 h, 100 μL of 30 mg / mL D-fluorescein potassium salt was injected intraperitoneally into the mice. After 5 min, the mice were placed under an in vivo imaging system to observe and photograph the bioluminescence intensity of the mouse's lower leg.

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

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

[0253] Table 1

[0254] sample Intramuscular injection bioluminescence intensity Tail vein injection bioluminescence intensity 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) shows the lipid compound based on lipoic acid derivatives as shown in Formula I-1, and the right shows the bioluminescence intensity comparison of ALC-0315 after intramuscular injection. Figure 7 The bioluminescence intensity of the lipid compound based on thioctic acid derivatives shown in I-1 after tail vein injection. Figure 8 ALC-0315 tail vein injection bioluminescence intensity.

[0256] As shown in Table 1 and Figures 6-8 As shown, the compound provided by the present invention is a novel lipid compound, the core of which is based on the chemical modification of lipoic acid to give it good biocompatibility; the lipoic acid lipid of the present invention is composed of ester bonds and disulfide bonds, and this unique structure endows it with 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 Example 1;

[0259] Test method:

[0260] The cell internalization process of the complex was observed using a confocal laser scanning microscope (Leica STELLARIS 5). Cells were co-incubated with the lipid / Cy5-mRNA complex, and observations were performed at 2 h, 4 h, and 6 h after treatment.

[0261] Test results:

[0262] like Figure 9 As shown, cellular uptake behavior was observed 2 hours after drug administration. The complex successfully escaped from endosomes within 6 hours. The dosage 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 lipids provided by this invention are used as delivery carriers for nucleic acid drugs, the disulfide bonds within the compound are cleaved by intracellular glutathione, leading to compound degradation. This not only triggers the timely release of nucleic acids but also effectively reduces the cytotoxicity caused by the accumulation of compounds within cells, demonstrating good biocompatibility. Furthermore, the lipid nanoparticle composition of this invention, when used as a carrier for delivering nucleic acid drugs, exhibits excellent delivery efficiency and superior spleen targeting, preferentially delivering 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, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 a lipoic acid derivative, characterized in that, The lipid compound based on lipoic acid derivative has the structure shown in Formula I: Formula I Where n is an integer from 0 to 5; Wherein, R1 is selected from any one of the following groups; wherein, Representative group connection positions: ； Wherein, R2 is selected from any one of the following groups; wherein, Representative group connection positions: 。 2. The lipid compound based on lipoic acid derivatives according to claim 1, characterized in that, The lipid compound based on lipoic acid derivatives is any one of the following compounds:

3. A method for preparing a lipid compound based on a lipoic acid derivative according to claim 1 or 2, characterized in that, The preparation method includes the following steps: An alkyl carboxylic acid compound and 6-bromohexanol undergo esterification to give compound A; compound A and a primary amine compound undergo substitution to give compound B; the reaction formulas are shown below: The esterification of lipoic acid and bromoalkyl alcohols yields compound C; the reaction formula is shown below: Compound B reacts with compound C to yield a lipid compound based on a lipoic acid derivative, as shown in Formula I; the reaction formula is as follows: ； The choices of R1, R2, and n are consistent with those defined in claim 1.

4. The method for preparing lipid compounds based on lipoic acid derivatives according to claim 3, characterized in that, The molar ratio of the alkyl carboxylic acid compound to 6-bromohexanol is 1:(0.8~1.2).

5. The method for preparing lipid compounds based on lipoic acid derivatives according to claim 3, characterized in that, In the preparation of compound A, the esterification reaction is carried out at a temperature of 10-40°C for 12-36 h.

6. The method for preparing lipid compounds based on lipoic acid derivatives according to claim 3, characterized in that, In the preparation of 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).

7. The method for preparing lipid compounds based on lipoic acid derivatives according to claim 3, characterized in that, In the preparation of 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).

8. The method for preparing lipid compounds based on lipoic acid derivatives according to claim 3, characterized in that, The molar ratio of compound A to the primary amine compound is 1:(0.8~1.2).

9. The method for preparing lipid compounds based on lipoic acid derivatives according to claim 3, characterized in that, In the preparation of compound B, the temperature of the substitution reaction is 50~70℃ and the time of the substitution reaction is 12~24 h.

10. The method for preparing lipid compounds based on lipoic acid derivatives according to claim 3, characterized in that, The molar ratio of the thioctic acid and the bromoalkyl alcohol compound is 1:(0.8~1.2).

11. The method for preparing lipid compounds based on lipoic acid derivatives according to claim 3, characterized in that, In the preparation of compound C, the esterification reaction is carried out at a temperature of 10-40°C for 12-36 h.

12. The method for preparing lipid compounds based on lipoic acid derivatives according to claim 3, characterized in that, In the preparation of 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).

13. The method for preparing lipid compounds based on lipoic acid derivatives according to claim 3, characterized in that, In the preparation of compound C, the esterification reaction is carried out in the presence of a base, and the molar ratio of lipoic acid to base is 1:(0.1~5).

14. The method for preparing lipid compounds based on lipoic acid derivatives according to claim 3, characterized in that, The molar ratio of compound B to compound C is 1:(0.8~1.2).

15. The method for preparing lipid compounds based on lipoic acid derivatives according to claim 3, characterized in that, The reaction temperature of compound B and compound C is 80~100℃, and the reaction time of compound B and compound C is 42~54 h.

16. The method for preparing lipid compounds based on lipoic acid derivatives according to claim 3, characterized in that, The reaction of compound B and compound C is carried out in the presence of an iodine salt catalyst, wherein the amount of the iodine salt catalyst added is 0.01 to 10 mol of the molar amount of compound B.

17. The method for preparing lipid compounds based on lipoic acid derivatives according to claim 3, characterized in that, The reaction between 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).

18. A lipid nanoparticle composition, characterized in that, The lipid nanoparticle composition includes lipoic acid derivative lipids, polyethylene glycol lipids, steroids, and auxiliary lipids; The lipoic acid derivative lipids include any one or a combination of at least two of the lipid compounds based on lipoic acid derivatives as described in claim 1 or 2.

19. The lipid nanoparticle composition according to claim 18, characterized in that, The nanoparticle composition comprises the following components by mass percentage: Lipoic acid derivative lipids 10%~70%, polyethylene glycol lipids 1%~25%, steroids 10%~50%, and auxiliary lipids 5%~30%.

20. The lipid nanoparticle composition according to claim 18 or 19, characterized in that, 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.

21. The lipid nanoparticle composition according to claim 18 or 19, characterized in that, The steroid is selected from any one or a combination of at least two of cholesterol, hydroxycholesterol, fucosterol, brassosterol, ergosterol, glycocholic acid, taurine, stigmasterol, or stigmasterol.

22. The lipid nanoparticle composition according to claim 18 or 19, characterized in that, The assisting lipid is selected from any one or a combination of at least two of DOPC, DOPE, DSPC, DPPG, POPE, DSPE, or DGTS.

23. A nanoparticle composition encapsulating a nucleic acid drug, characterized in that, The nanoparticle composition encapsulating nucleic acid drugs includes nucleic acid drugs and lipoic acid derivative lipids; Wherein, the lipoic acid derivative lipid includes any one or a combination of at least two of the lipid compounds based on lipoic acid derivatives as described in claim 1 or 2; Alternatively, the nanoparticle composition encapsulating a nucleic acid drug may include a nucleic acid drug and a lipid nanoparticle composition as described in any one of claims 18 to 22.

24. The nanoparticle composition encapsulating a nucleic acid drug according to claim 23, characterized in that, The nucleic acid drug accounts for 5% to 20% of the total mass of the nanoparticle composition containing the nucleic acid drug.

25. The nanoparticle composition encapsulating a nucleic acid drug according to claim 23, characterized in that, The nucleic acid drug includes any one or a combination of at least two of Luc-mRNA, OVA-mRNA, GFP-mRNA, PD-1-mRNA, siRNA, or DNA.

26. A method for preparing a nanoparticle composition encapsulating a nucleic acid drug according to any one of claims 23-25, characterized in that, The preparation method includes: Lipoic acid derivative lipids, polyethylene glycol lipids, steroids and auxiliary lipids were dissolved in a solvent and mixed to obtain an organic phase liposome solution. The nucleic acid drug was dissolved 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 were mixed and purified to obtain the nanoparticle composition encapsulating the nucleic acid drug.

27. The method for preparing the nanoparticle composition encapsulating nucleic acid drugs according to claim 26, characterized in that, The solvent includes any one or a combination of at least two of methanol, ethanol, tetrahydrofuran, or dimethyl sulfoxide.

28. The method for preparing the nanoparticle composition encapsulating nucleic acid drugs according to claim 26, characterized in that, In the organic phase liposome solution, the concentration of the lipoic acid derivative lipid is 5-50 mg / mL.

29. The method for preparing the nanoparticle composition encapsulating nucleic acid drugs according to claim 26, characterized in that, The pH buffer solution includes a citric acid / sodium citrate solution.

30. The method for preparing the nanoparticle composition encapsulating nucleic acid drugs according to claim 26, characterized in that, The pH of the pH buffer solution is 3 to 9.

31. The method for preparing the nanoparticle composition encapsulating nucleic acid drugs according to claim 26, characterized in that, In the aqueous nucleic acid drug solution, the concentration of the nucleic acid drug is 0.05~2 mg / mL.

32. The method for preparing the nanoparticle composition encapsulating nucleic acid drugs according to claim 26, characterized in that, The volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid drug is 1:(1~20).

33. The method for preparing the nanoparticle composition encapsulating nucleic acid drugs according to claim 26, characterized in that, The purification includes ultrafiltration and / or dialysis.

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