Class of ionizable lipid molecules, composition thereof, and use thereof

Abstract

A class of ionizable lipid molecules, a composition, and use. The lipid molecule comprises an amino acid structural moiety and a tocopherol lipid structural moiety indirectly linked by means of a hydrolyzable ester bond, wherein an ionizable group is a basic group of the amino acid structural moiety. In a preferred embodiment, the indirect linkage between the amino acid structural moiety and the tocopherol lipid structural moiety is an alkylene ether; the amino acid structural moiety is a residue of lysine, histidine, arginine, or a derivative thereof. A composition prepared from one or more ionizable lipid molecules and a therapeutic agent or a prophylactic agent, and use of the lipid molecule or the composition in the preparation of a nucleic acid drug, a small molecule drug, a polypeptide drug, or a protein drug, wherein the prepared RNA-loaded lipid nanoparticle enables protein expression or silencing in vivo, and has the advantages of good stability, high transfection efficiency, low toxicity, safety, effectiveness, etc.

Description

A class of ionizable lipid molecules, their compositions and applications Technical Field

[0001] This invention belongs to the field of drug carrier technology, and relates to a class of ionizable lipid molecular carriers, specifically to a class of ionizable lipid molecules of amino acid tocopheryl oxyalkyl esters and their preparation methods, or pharmaceutically acceptable salts thereof, as well as compositions containing the same and their applications. Background Technology

[0002] Nucleic acid drugs are unstable both in vivo and in vitro, and are easily degraded by nucleases in body fluids. Therefore, carriers are often needed to deliver these drugs into the body. Currently, common carriers are broadly classified into viral carriers and non-viral carriers. Due to safety concerns such as cytotoxicity, immunogenicity, and carcinogenicity, as well as limitations in loading capacity and high production costs, non-viral carriers have gradually replaced viral carriers. Among these, ionizable lipids can form nanoparticles with negatively charged nucleic acid drugs through electrostatic interactions, protecting the nucleic acid drugs from degradation by nucleases and achieving stable delivery of nucleic acid drugs in vivo.

[0003] For example, MC3 is used in Onpattro, the world's first RNA (siRNA) lipid nanoparticle drug approved by the U.S. Food and Drug Administration (FDA) in October 2018; ALC-0315 is used in Comirnay, the world's first mRNA lipid nanoparticle COVID-19 vaccine jointly developed by Pfizer and BioNTech, approved by the FDA in August 2021; and SM-102 is Spikevax, an mRNA lipid nanoparticle COVID-19 vaccine developed by Moderna, approved by the FDA in January 2022. The ionizable groups of these ionizable lipid molecules are all tertiary amine groups, and the lipid portion has a fatty chain structure. Most other reported ionizable lipids are structural analogs or modifications of ALC-0315, SM-102, or MC3. These compounds containing tertiary amine groups and fatty chain structures can ionize in acidic environments, becoming positively charged, and thus recombine with negatively charged RNA through electrostatic interactions to load RNA.

[0004] However, these compounds containing tertiary amine groups and aliphatic chain structures still have the following shortcomings:

[0005] (1) The ionization of tertiary amine groups is poorly adjustable, posing certain risks to biosafety and biotoxicity.

[0006] (2) These ionizable lipid molecules are lipid molecules containing a single ionizable group. However, different nucleic acid molecules have different chemical structures and compositions, resulting in certain differences in their interactions with these lipid molecules containing a single ionizable group. This leads to variations in the loading and delivery effects of these lipid molecules containing a single ionizable group on different nucleic acids, resulting in unstable loading and delivery effects. Furthermore, it also causes deviations in the loading and delivery effects of certain nucleic acids that are difficult to improve. Summary of the Invention

[0007] To address the aforementioned problems, the primary objective of this invention is to provide a class of ionizable lipid molecules and a method for their preparation. This ionizable lipid is obtained by indirectly linking an amino acid structural moiety and a tocopherol lipid structural moiety via a hydrolyzable ester bond. In a preferred embodiment of this invention, the amino acid moiety is a residue of lysine, arginine, histidine, or their derivatives. Accordingly, this ionizable lipid contains multiple ionizable sites, exhibits good tunability, a wide range of applications, and good biocompatibility.

[0008] A second object of the present invention is to provide a composition comprising one or more of the ionizable lipid molecules or pharmaceutically acceptable salts thereof, and a therapeutic or preventative agent. It also includes the use of the ionizable lipid molecule, or a pharmaceutically acceptable salt thereof, or the composition in the preparation of nucleic acid drugs, small molecule drugs, peptide or protein drugs.

[0009] The technical solution of the present invention is as follows:

[0010] The present invention discloses an ionizable lipid molecule or a pharmaceutically acceptable salt thereof, the ionizable lipid molecule comprising an amino acid structural moiety and a tocopherol lipid structural moiety, wherein the amino acid structural moiety and the tocopherol lipid structural moiety are indirectly linked by a hydrolyzable ester bond, wherein the ionizable group is a basic group of the amino acid structural moiety.

[0011] In one embodiment of the present invention, the indirect link between the amino acid structural portion and the tocopherol lipid structural portion is preferably an alkylene ether. Wherein, n is 2-10; q and l are 1-5, and more preferably, n is 2-3 and q and l are 1; the amino acid structural part is lysine, histidine, arginine, or residues of their derivatives.

[0012] This invention provides an ionizable lipid molecule or a pharmaceutically acceptable salt thereof, with the general formula shown in (I):

[0013] Where B is R is n is 2-10; q and l are 1-5;

[0014] T is

[0015] This invention also discloses a method for preparing the above-mentioned ionizable lipids, comprising the following steps:

[0016] (1) Add haloalkanol to a solution containing α-tocopherol and base, and prepare tocopherol oxyalkanol by substitution reaction;

[0017] A preferred embodiment is to add haloalkanol dropwise to a DMF solution containing α-tocopherol and alkali at 80-100℃, and stir the reaction for 90-110 hours to obtain tocopherol oxyalkanol.

[0018] (2) To prepare base-protected amino acid tocopheroxane by esterification reaction of tocopheroxane alcohol and base-protected amino acid.

[0019] A preferred embodiment is to react a CH2Cl2 solution containing EDCI, DMAP, tocopheryl oxyalkanol and base-protected amino acids at room temperature to obtain base-protected amino acid tocopheryl oxyalkanol ester.

[0020] (3) Base-protected tocopheryl oxyalkyl amino acid esters are deprotected by a deprotecting agent to obtain amino acid tocopheryl oxyalkyl esters.

[0021] A preferred embodiment is to add an organic acid dropwise to a CH2Cl2 solution containing a base-protected amino acid tocopheroxane ester at room temperature, stir the reaction for 3-5 hours, deprotect the base, and then neutralize the organic acid with a saturated sodium bicarbonate solution to obtain the amino acid tocopheroxane ester.

[0022] In one embodiment of the present invention, preferably, the haloalkanol mentioned in step (1) is a primary haloalkanol or a secondary haloalkanol; the base is an alkali metal hydroxide, such as sodium hydroxide, potassium hydroxide or lithium hydroxide.

[0023] In one embodiment of the present invention, preferably, the molar ratio of monohalogenated primary alkanol, α-tocopherol and base is (0.1-5):1:(0.1-5); and the molar ratio of dihalogenated secondary alkanol, α-tocopherol and base is (0.1-10):1:(0.1-10).

[0024] In one embodiment of the present invention, preferably, the amino acid described in step (2) is a residue of lysine, arginine, histidine, or their derivatives, and its base is protected by a commonly used base protecting group on amino acid molecules. The specific protection group is selected by those skilled in the art based on experimental conditions. The present invention preferably uses tert-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl, or 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl to protect the base. Simultaneously, the base protecting group can be removed under acidic conditions to obtain an amino acid tocopherol oxyalkylene ester.

[0025] In one embodiment of the present invention, preferably, the molar ratio of the base-protected amino acid and tocopherol, EDCI, and DMAP in step (2) is 1:(0.1-10):(0.1-10):(0.1-5).

[0026] The present invention also discloses a composition comprising a therapeutic agent or a preventive agent and a carrier for delivering the therapeutic agent or preventive agent, wherein the therapeutic agent or preventive agent is one or more of nucleic acid molecules such as siRNA, shRNA, miRNA and mRNA, polypeptides or proteins; and the carrier comprises one or more of the above-mentioned ionizable lipid molecules or pharmaceutically acceptable salts thereof.

[0027] In one embodiment of the present invention, preferably, the mass ratio of the carrier to the therapeutic agent or preventive agent is 5-30:1, more preferably (10-15):1.

[0028] In one embodiment of the present invention, preferably, the composition is lipid nanoparticles, the average particle size of the lipid nanoparticles is 60nm to 600nm, preferably 100 to 400nm; the polydispersity index of the lipid nanoparticles is less than 0.5, preferably less than 0.3.

[0029] In one embodiment of the present invention, preferably, the carrier further includes auxiliary lipids, cholesterol, and PEGylated lipids.

[0030] In one embodiment of the present invention, preferably, the molar ratio of the ionizable lipid molecule, the auxiliary lipid, cholesterol and the PEGylated lipid is 50:(5-20):(10-50):(1-5).

[0031] In one embodiment of the present invention, preferably, the auxiliary lipid is distearyl phosphatidylcholine (DSPC), distearyl phosphatidylethanolamine, dipalmitoyl phosphatidylcholine, or dipalmitoyl phosphatidylethanolamine; and the PEGylated lipid is polyethylene glycol 2000 vitamin E succinate (TPGS2000), polyethylene glycol 1000 vitamin E succinate (TPGS1000), or dimyristicoglycerol-polyethylene glycol 2000 (DMG2000).

[0032] The present invention also discloses the use of the ionizable lipid molecule represented by the above general formula (I), or a pharmaceutically usable salt thereof, or the above composition in the preparation of nucleic acid drugs, small molecule drugs, polypeptide or protein drugs.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] This invention discloses a class of ionizable lipid molecules using essential and highly safe tocopherols and amino acids as raw materials. These are synthesized via ester bonds to obtain amino acid tocopheroxane esters, such as amino acid tocopheroxane propyl ester, amino acid tocopheroxane ethyl ester, and amino acid 1,3-ditocopheroxane-2-propyl ester. These amino acid tocopheroxane esters can serve as ionizable lipids, acting as lipid nanoparticle carriers for delivering siRNA, shRNA, miRNA, mRNA, and other nucleic acid molecules, peptides, or proteins. The average particle size of the prepared lipid nanoparticles is 60 nm to 600 nm; the polydispersity index of the lipid nanoparticles is less than 0.5. Effective transfection was achieved in mice by injection of Fluc-mRNA lipid nanoparticles, and PCSK9 silencing was achieved by injection of PCSK9 siRNA lipid nanoparticles. Meanwhile, when the amino acid structure is selected from residues of lysine, histidine, arginine, or their derivatives, the ionizable lipid of the present invention has multiple ionizable sites. One or more suitable amino acid tocopherol esters can be selected according to the structural characteristics, content composition, and delivery requirements of the therapeutic or preventive agent. It has good tunability, wide applicability, and good biosafety.

[0035] Specifically: First, the amino acid ester containing a tocopherol lipid structure provided by the present invention can be hydrolyzed to generate the corresponding amino acid and tocopherol lipid structure moiety, and has good biosafety.

[0036] Secondly, when the amino acid ester containing a tocopherol lipid structure provided by this invention is a lysine ester, histidine ester, or arginine ester containing a tocopherol lipid structure, in addition to the amino group, weakly basic groups such as imidazole or guanidinium groups in the amino acid structure can also combine with hydrogen ions and undergo ionization. The degree of ionization of these groups varies under physiological conditions; and the degree of electrostatic interaction between lipid molecules containing groups with different degrees of ionization and negatively charged nucleic acids is different, resulting in different capacities for loading and releasing nucleic acids. This allows technicians to specifically select a suitable amino acid ester containing a tocopherol lipid structure, or to select a combination of multiple amino acid esters and adjust the appropriate ratio between the various amino acid esters, based on the nucleic acid structure and composition and its delivery requirements, thereby achieving stable and efficient delivery of different types of nucleic acids. Attached Figure Description

[0037] Figure 1 shows the tocopheryloxypropanol (TPOH) of Example 1 of the present invention.1 H NMR spectrum;

[0038] Figure 2 shows 1,3-ditocopherol-2-propanol (dTPOH) from Example 2 of the present invention. 1 H NMR spectrum;

[0039] Figure 3 shows the histidine tocopheryl oxypropyl ester (HTP) of Example 3 of the present invention. 1 H NMR spectrum;

[0040] Figure 4 is the MS spectrum of histidine tocopheryl oxypropyl ester (HTP) in Example 3 of the present invention;

[0041] Figure 5 shows the lysine tocopheryl oxypropyl ester (LTP) of Example 4 of the present invention. 1 H NMR spectrum;

[0042] Figure 6 is the MS spectrum of lysine tocopheryl oxypropyl ester (LTP) in Example 4 of the present invention;

[0043] Figure 7 shows the arginine tocopheryl oxypropyl ester (ATP) of Example 5 of the present invention. 1 H NMR spectrum;

[0044] Figure 8 shows the lysine 1,3-ditocopheroloxy-2-propyl ester (L2T) of Example 6 of the present invention. 1 H NMR spectrum;

[0045] Figure 9 is the MS spectrum of lysine 1,3-ditocopheroloxy-2-propyl ester (L2T) in Example 6 of the present invention.

[0046] Figure 10 shows histidine 1,3-ditocopheroloxy-2-propyl ester (H2T) from Example 7 of the present invention. 1 H NMR spectrum;

[0047] Figure 11 is the MS spectrum of histidine 1,3-ditocopheroloxy-2-propyl ester (H2T) in Example 7 of the present invention.

[0048] Figure 12 shows the lysine tocopheryl oxyethyl ester (LTE) of Example 8 of the present invention. 1 H NMR spectrum;

[0049] Figure 13 shows the histidine tocopherol oxyethyl ester (HTE) of Example 9 of the present invention. 1 H NMR spectrum;

[0050] Figure 14 is the MS spectrum of histidine tocopherol oxyethyl ester (HTE) in Example 9 of the present invention;

[0051] Figure 15 is a particle size distribution curve of the mRNA lipid nanoparticles of Example 10 of the present invention;

[0052] Figure 16 is a particle size distribution curve of the mRNA lipid nanoparticles of Example 11 of the present invention;

[0053] Figure 17 is a particle size distribution curve of the mRNA lipid nanoparticles of Example 12 of the present invention;

[0054] Figure 18 is a particle size distribution curve of the mRNA lipid nanoparticles of Example 13 of the present invention;

[0055] Figure 19 is an in vivo imaging image of mice transfected with Fluc-mRNA LNP-HTE in Example 14 of the present invention, where a is a dose of 3.5 μg and b is a dose of 7.0 μg.

[0056] Figure 20 is an in vivo imaging image of mice transfected with Fluc-mRNA LNP-HTP in Example 14 of the present invention, where a is a dose of 3.5 μg and b is a dose of 7.0 μg. Detailed Implementation

[0057] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0058] Numerous 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 other ways different from 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.

[0059] All raw materials used in this invention are commercially available. Among them, tocopherol is the commonly used, highly active D-α-tocopherol, and lysine, histidine, and arginine are commonly used L-type amino acids. Under the concept of this invention, other configurations of tocopherol and amino acids can also be selected as raw materials. Those skilled in the art can still prepare the amino acid tocopheryl oxyalkyl esters of this invention by selecting other configurations of tocopherol and amino acid raw materials, such as amino acid tocopheryl oxypropyl ester, amino acid tocopheryl oxyethyl ester, amino acid 1,3-ditocopheryl oxy-2-propyl ester, etc., and can prepare lipid nanoparticles, which can be used as carriers for delivering therapeutic or preventative agents, such as lipid nanoparticle carriers for delivering nucleic acid molecules, peptides, or proteins such as siRNA, shRNA, miRNA, and mRNA.

[0060] This invention discloses a class of ionizable lipid molecules, their preparation methods, compositions, and applications. The composition includes one or more of the ionizable lipid molecules or pharmaceutically acceptable salts thereof, and therapeutic or preventative agents. It also includes the application of the ionizable lipid molecules, pharmaceutically acceptable salts thereof, or the composition in the preparation of nucleic acid drugs, small molecule drugs, peptide drugs, or protein drugs.

[0061] In a preferred embodiment, the present invention relates to amino acid esters with monotocopherol lipid structures and ditocopherol lipid structures, specifically, for example, amino acid tocopheroloxypropyl ester, amino acid 1,3-ditocopheroloxy-2-propyl ester, etc.

[0062] This invention utilizes one or more ionizable lipid molecules, along with other lipid molecules such as cofactor lipid molecules, cholesterol, and PEGylated lipids, to prepare lipid nanoparticle compositions loaded with nucleic acid drugs, small molecule drugs, peptides, or protein drugs. The drug-loaded compositions can express or silence proteins in vivo, exhibiting characteristics such as good stability, high transfection efficiency, low toxicity, and safety and efficacy.

[0063] In a preferred embodiment, the aforementioned amino acid ester molecule is a lysine ester containing two amino groups, a histidine ester containing one amino group and one imidazole group, or an arginine ester containing one amino group and one guanidine group. In such amino acid esters, in addition to the amino group, weakly basic groups such as the imidazole or guanidine groups can also combine with hydrogen ions and undergo ionization, and the degree of ionization of these groups varies under physiological conditions. Lipid molecules containing groups with different degrees of ionization exhibit different electrostatic interactions with negatively charged RNA, resulting in different abilities to load and release RNA. For example, the pKa of the imidazole group is around 6, which can increase the buffering capacity of the nanocarrier in endosomes and lysosomes through the proton sponge effect, enhancing its escape ability; the guanidine group has strong cell penetration ability, which can enhance its affinity for the cell membrane and thus improve cellular uptake efficiency.

[0064] D-α-Tocopherol is the most widely distributed, abundant, and highly active form of vitamin E in nature. Vitamin E is a fat-soluble antioxidant and, as an essential nutrient for the human body, is mainly obtained through exogenous foods.

[0065] Among the essential amino acids for the human body, there are three basic amino acids: arginine contains a guanidine group and an amino group, histidine contains an imidazole group and an amino group, and lysine contains two amino groups.

[0066] This invention utilizes one or more ionizable lipid molecules, along with other lipid molecules such as cofactor lipid molecules, cholesterol, and PEGylated lipids, to prepare lipid nanoparticle compositions loaded with nucleic acid drugs, small molecule drugs, peptides, or protein drugs. Furthermore, based on the fact that lysine esters, arginine esters, and histidine esters exhibit different degrees of ionization under physiological conditions, the lipid nanoparticles provided by this invention have the advantage of allowing for the targeted selection of suitable amino acid tocopherol esters, or compositions of multiple amino acid tocopherol esters designed in specific ratios, according to the structure and composition of the loaded nucleic acid and its delivery requirements.

[0067] Furthermore, the amino acid tocopherol oxyalkyl esters provided by this invention can be hydrolyzed in vivo to generate the corresponding amino acids and tocopherol lipid structural moieties, exhibiting good biocompatibility.

[0068] This invention also provides a method for preparing the above-mentioned ionizable lipid molecule, namely, a method for synthesizing an amino acid ester by linking the tocopherol lipid structure portion and the amino acid structure portion through a hydrolyzable ester bond via a linking group, comprising the following steps:

[0069] (i) Tocopherol oxyalkanol is prepared by a substitution reaction of tocopherol with haloalkanol or dihaloalkanol, wherein the general formula for the chemical reaction of tocopherol with haloalkanol is as follows:

[0070] X: Halogen atom, such as bromine, chlorine or iodine; n = 2 to 10;

[0071] The general formula for the chemical reaction between tocopherol and dihaloalkanols is as follows:

[0072] X: Halogen atom, such as bromine, chlorine or iodine; q, l = 1-5;

[0073] (ii) Tocopheryl oxyalkyl alcohols are esterified with base-protected amino acids (such as lysine, histidine, or arginine) to prepare base-protected tocopheryl oxyalkyl amino acid esters. The general chemical reaction formula is as follows:

[0074] RCOOH: Base-protected amino acids (such as lysine, histidine, or arginine); n = 2–10;

[0075] or

[0076] RCOOH: Base-protected amino acids (such as lysine, histidine, or arginine); q, l = 1-5;

[0077] (iii) Preparation of tocopherol oxyalkyl amino acid esters by deprotection reaction, the general chemical reaction formula is as follows:

[0078] n = 2 to 10;

[0079] or,

[0080] q, l = 1-5;

[0081] B is a residue of an amino acid or an amino acid derivative, preferably at least one of the following three:

[0082] In addition to the three amino acids mentioned above, B can also be a residue of other amino acids or derivatives of the aforementioned amino acids.

[0083] The present invention also provides a method for preparing a lipid nanoparticle composition using the above-mentioned ionizable lipid molecules.

[0084] In one embodiment, the ionizable lipid molecule (also referred to as an ionizable lipid carrier) together with an auxiliary lipid molecule, cholesterol, and a PEGylated lipid molecule forms a carrier for delivering a therapeutic or preventative agent. The ionizable lipid molecule may be one, two, or more; the auxiliary lipid molecule may be distearylphosphatidylcholine, distearylphosphatidylethanolamine, or dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylethanolamine, etc.; the PEGylated lipid molecule may be polyethylene glycol 2000 vitamin E succinate, 1000 vitamin E succinate, or dimyristoylglycerol-polyethylene glycol 2000, etc.

[0085] In typical embodiments, the lipid nanoparticle composition is prepared using microfluidic, microfluidic, or high-speed homogenization methods. Preferably, when preparing the composition, the mass ratio of the carrier to the therapeutic or preventative agent is 5-30:1, more preferably (10-15):1; the molar ratio of ionizable lipids, cofactor lipids, cholesterol, and PEGylated lipids is 50:(5-20):(10-50):(1-5). The nanoparticle solution prepared by the above methods can be purified by removing the organic solvent or by concentration through dialysis, ultrafiltration, or tangential flow.

[0086] The prepared nanoparticle solution loaded with nucleic acid drugs, small molecule drugs, peptides or protein drugs can be used for intramuscular injection, intravenous injection or local administration to exert therapeutic or preventive effects.

[0087] Example 1: Preparation of Tocopheryloxypropanol (TPOH)

[0088] 10.00 g of D-α-tocopherol and 1.39 g of sodium hydroxide were added to a flask containing 30 mL of DMF (N,N-dimethylformamide). Under magnetic stirring in a 90 °C oil bath, 10 mL of DMF solution containing 4.03 g of 3-bromopropanol was slowly added dropwise. After reacting for 90 h, the reaction mixture was added to 200 mL of pure water and extracted three times with 50 mL of methyl tert-butyl ether. The organic phases were combined, and methyl tert-butyl ether was removed by rotary evaporation. A small amount of dichloromethane was added to dissolve the crude product, and a dichloromethane solution was prepared for column chromatography.

[0089] The dichloromethane solution containing the crude product was purified by separation using a silica gel column (300g 200-300 mesh silica gel, 70mm diameter). The column was eluted sequentially with a mixed solvent of petroleum ether / ethyl acetate at a volume ratio of 8:1 and 5:1, and the eluates were collected. The eluates containing only TPOH were collected and combined, and the solvent was removed by rotary evaporation to obtain a deep yellow viscous liquid, i.e., TPOH, with a yield of 75.0%.

[0090] 1 ¹H NMR [δ / ppm] (400MHz, DMSO-D6): 0.72-0.90 (CH₃ at 12H, 20, 25, 29, 30), 1.17 (CH₃ at 3H, 31), 1.86 (CH₂ at 2H, 33), 1.93-2.11 (CH₃ at 9H, 7, 9, 10), 3.56-3.67 (CH₂ at 4H, 32, 34). 1 The H NMR spectrum is shown in Figure 1, and the structural formula is shown in Equation (1).

[0091] Example 2: Preparation of 1,3-ditocopherol-2-propanol (dTPOH)

[0092] 10.00 g of D-α-tocopherol and 1.11 g of sodium hydroxide were added to a flask containing 50 mL of DMF. Under magnetic stirring in a 90 °C oil bath, 10 mL of DMF solution containing 2.02 g of 1,3-dibromo-2-propanol was slowly added dropwise. After reacting for 60 h, the reaction solution was rotary evaporated at 55 °C using an oil pump until the volume no longer decreased. 60 mL of diethyl ether was added, and the mixture was transferred to a separatory funnel and extracted with an equal volume of pure water. The ether layer and aqueous layer were separated. The aqueous layer was washed twice with an equal volume of diethyl ether, and the ether layers were combined. Anhydrous sodium sulfate was added to the ether layer until no clumping occurred. The mixture was allowed to stand overnight, filtered, and the filtrate was rotary evaporated at room temperature until the volume no longer decreased to obtain the crude product. A small amount of dichloromethane was added to dissolve the crude product, and a dichloromethane solution was prepared for column chromatography.

[0093] The dichloromethane solution containing the crude product was purified by separation using a silica gel column (300g 200-300 mesh silica gel, 70mm diameter). Elution was performed sequentially with different ratios of petroleum ether / dichloromethane mixed solvents (volume ratios of 2:1, 1:1, 1:2, and 1:3), and the eluates were collected. The eluates containing only dTPOH were collected and combined, and the solvent was removed by rotary evaporation to obtain a yellow, transparent, oily liquid with a yield of 83.8%.

[0094] 1¹H NMR (400MHz, Chloroform-d) δ 4.43 (CH at 1H, 63), 3.77–3.95 (CH₂ at 4H, 65, 66), 2.56 (CH₂ at 4H, 14, 42), 1.99–2.27 (CH₃ at 18H, 7, 9, 10, 38, 39, 40), 1.77 (CH₂ at 4H, 13, 43), 0.78–0.93 (CH₃ at 24H, 20, 25, 29, 30, 59, 60, 61, 62), its 1 The H NMR spectrum is shown in Figure 2, and the structural formula is shown in Equation (2).

[0095] Example 3: Preparation of histidine tocopheryl oxypropyl ester (HTP)

[0096] 2.00 g TPOH, 1.74 g N,N'-di-tert-butoxycarbonyl-L-histidine, 1.18 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and 0.20 g 4-dimethylaminopyridine (DMAP) were added to a 50 mL dry flask containing 10 mL of anhydrous dichloromethane. The mixture was magnetically stirred at room temperature under a sealed, light-protected environment. After 90 h of reaction, the solution was rotary evaporated to approximately 3 mL. The 3 mL dichloromethane solution containing the crude product was purified by column chromatography using a silica gel column (90 g 200-300 mesh silica gel, 40 mm diameter). The solution was eluted with a mixture of dichloromethane, dichloromethane, and methanol (v / v) at a ratio of 1:20:1, and the eluent was collected. The eluent containing only the product (N,N'-di-tert-butoxycarbonyl-L-histidine tocopheroloxypropyl ester, BHTP) was collected and combined. The solvent was removed by rotary evaporation, and the product was dried under vacuum to constant weight to obtain a dark yellow semi-solid, which is BHTP, with a yield of 86.9%.

[0097] 2.94 g of BHTP was added to a flask containing 30 mL of dry dichloromethane. 10 mL of trifluoroacetic acid was slowly added dropwise. The flask was sealed and protected from light. The mixture was magnetically stirred and reacted at room temperature for 3 h. The reaction mixture was then removed by rotary evaporation at 40 °C to remove the solvent and trifluoroacetic acid, yielding the crude product. The crude product was dissolved in 5 mL of dichloromethane to prepare a dichloromethane solution for column chromatography purification. The dichloromethane solution containing the crude product was purified using a silica gel column (90 g 200-300 mesh silica gel, 40 mm diameter). Elution was performed sequentially with dichloromethane / methanol mixed solvents at volume ratios of 40:1, 20:2, and 20:3, and the eluates were collected. The eluates containing only HTP trifluoroacetate were collected and combined. The solvent was removed by rotary evaporation, and the product was dried under vacuum to constant weight to obtain a deep yellow semi-solid, HTP trifluoroacetate, with a yield of 81.9%.

[0098] 1.16 g of HTP trifluoroacetate was added to 12 mL of dichloromethane and extracted with 6 mL of saturated sodium bicarbonate solution. The organic phase was dried with anhydrous sodium sulfate, centrifuged, filtered, and the organic solvent was removed by rotary evaporation. The filtrate was then dried under vacuum to obtain a pale yellow semi-solid, namely HTP, with a yield of 83.7%. Its structural formula is shown in formula (3). 1 The H NMR spectrum is shown in Figure 3, and the MS spectrum is shown in Figure 4.

[0099] 1 ¹H NMR (400MHz, DMSO-d6): 0.63–0.91 (CH₃ on 12H, 20, 25, 29, 30), 1.17 (CH₃ on 3H, 31), 1.94–2.10 (CH₃ on 9H, 7, 9, 10), 2.64–2.89 (CH₂ on 2H, 38), 4.23 (CH₂ on 2H, 34), 6.77 (CH on 1H, 45), 7.49 (CH on 1H, 43).

[0100] MS (ESI+): The theoretical m / z value of HTP(C38H63N3O4H)[M+H]+ is 626.4897, and the measured value is 627.4897; the theoretical m / z value of HTP(C38H63N3O4Na)[M+Na]+ is 648.4716, and the measured value is 648.4715. The errors are both within 5 ppm, and these are the major components.

[0101] Example 4: Preparation of Lysine Tocopheryl Acetate (LTP)

[0102] 1.00 g TPOH, 0.85 g (S)-2,6-di-tert-butoxycarbonylaminohexanoic acid, 0.59 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and 0.10 g 4-dimethylaminopyridine (DMAP) were added to a 50 mL dry flask containing 5 mL of anhydrous dichloromethane. The flask was sealed and protected from light, and the reaction was carried out at room temperature with magnetic stirring. After 72 h of reaction, the solution was rotary evaporated to approximately 3 mL. The 3 mL dichloromethane solution containing the crude product was purified by column chromatography using a silica gel column (45 g 200-300 mesh silica gel, 40 mm diameter). The solution was eluted sequentially with dichloromethane / methanol mixed solvents at a volume ratio of 1:0 and 20:1, and the eluates were collected. The eluates containing only the product ((S)-2,6-di-tert-butoxycarbonylaminohexanoic acid tocopheryloxypropyl ester, BLTP) were collected and combined. The solvent was removed by rotary evaporation, and the product was dried under vacuum to constant weight to obtain a dark yellow semi-solid, which is BLTP, with a yield of 90.4%.

[0103] 1.51 g of BLTP was added to a flask containing 15 mL of dry dichloromethane, and 5 mL of trifluoroacetic acid was slowly added dropwise. The flask was sealed and protected from light, and the mixture was magnetically stirred. After reacting at room temperature for 3 h, the reaction mixture was removed by rotary evaporation at 40 °C and dried under vacuum to constant weight to obtain a dark yellow semi-solid, namely LTP trifluoroacetate, with a yield of 95.0%.

[0104] 1.69 g of LTP trifluoroacetate was added to 16 mL of dichloromethane and extracted with 8 mL of saturated sodium bicarbonate solution. The organic phase was dried with anhydrous sodium sulfate, centrifuged, filtered, and the organic solvent was removed by rotary evaporation. The filtrate was then dried under vacuum to obtain a pale yellow semi-solid, namely LTP, with a yield of 84.5%. Its structural formula is shown in formula (4). 1 The H NMR spectrum is shown in Figure 5, and the MS spectrum is shown in Figure 6.

[0105] 1 H NMR (400MHz, Chloroform-d): 0.85 (12H, CH3 on 22, 25, 29, 30), δ 3.96 (t, J = 5.6 Hz, 1H), 3.83 (t, J = 5.8 Hz, 1H),3.21(s,1H),2.57(t,J=6.6Hz,2H),2.20–2.11(m,4H),2.11(d,J=8.2Hz,1H),2.08(s,2H),2.09–1.9 8(m,2H),1.78(qt,J=13.3,6.7Hz,2H),1.52(dt,J=13.6,6.4Hz,2H),1.40(ddt,J=15.3,11.6,6.2Hz,2H ), 1.36 (s, 1H), 1.33–1.20 (m, 9H), 1.18–1.10 (m, 1H), 1.08 (dd, J = 9.1, 4.6Hz, 1H), 1.04 (t, J = 6.2Hz, 1H).

[0106] MS (ESI+): The theoretical m / z value of LTP(C38H69N2O4)[M+H]+ is 617.5257, and the measured value is 618.5254; the theoretical m / z value of LTP(C38H68N2O4Na)[M+Na]+ is 639.5077, and the measured value is 639.5078. The errors are all within 5 ppm, and these are the major components.

[0107] Example 5: Preparation of arginine tocopheryl oxypropyl ester (ATP)

[0108] 1.00 g TPOH, 1.29 g Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine, 0.59 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and 0.10 g 4-dimethylaminopyridine (DMAP) were added to a 50 mL dry flask containing 5 mL of anhydrous dichloromethane. The flask was sealed and protected from light, and the reaction was carried out at room temperature with magnetic stirring. After 110 h of reaction, the solution was rotary evaporated to approximately 3 mL. The 3 mL dichloromethane solution containing the crude product was purified by silica gel column chromatography (45 g 200-300 mesh silica gel, 40 mm diameter), eluting successively with petroleum ether / ethyl acetate mixed solvents at a volume ratio of 1:1 and 5:7, and the eluents were collected. The eluent containing only the product (Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine tocopheroxypropyl ester, BATP) was collected and combined. The solvent was removed by rotary evaporation at room temperature, and the residue was dried under vacuum at room temperature to constant weight to obtain a colorless semi-solid, which was BATP, with a yield of 65.2%.

[0109] 1.33 g of BATP was added to a flask containing 13 mL of dry dichloromethane. 4.3 mL of trifluoroacetic acid was slowly added dropwise. The flask was sealed and protected from light. The mixture was magnetically stirred and reacted at room temperature for 24 h. The reaction mixture was then removed by rotary evaporation at 40 °C to remove the solvent and trifluoroacetic acid, yielding the crude product. The crude product was dissolved in 5 mL of dichloromethane to prepare a dichloromethane solution for column chromatography purification. The dichloromethane solution containing the crude product was purified using a silica gel column (45 g 200-300 mesh silica gel, 40 mm diameter), eluted sequentially with methanol / dichloromethane mixed solvents at a volume ratio of 2:20 and 3:20, and the eluates were collected. The eluates containing only ATP trifluoroacetate were collected and combined. The solvent was removed by rotary evaporation at room temperature. The residue was dried under vacuum at room temperature to constant weight, yielding a pale yellow semi-solid, ATP trifluoroacetate, with a yield of 37.9%.

[0110] 0.18 g of ATP trifluoroacetate was added to 2 mL of dichloromethane and extracted with 1 mL of saturated sodium bicarbonate solution. The organic phase was dried with anhydrous sodium sulfate, centrifuged, filtered, and the organic solvent was removed by rotary evaporation. The filtrate was then dried under vacuum to obtain a pale yellow semi-solid, namely ATP, with a yield of 47.7%. Its structural formula is shown in formula (5). 1 The H NMR spectrum is shown in Figure 7.

[0111] 1¹H NMR (400MHz, DMSO-d6): 0.90 (CH₃ at 12H, 20, 25, 29, 30), 1.24 (CH₃ at 3H, 31), 3.18 (CH₂ at 2H, 42), 3.68 (CH₂ at 2H, 32), 4.40 (CH₂ at 2H, 34), 4.43 (CH₃ at 1H, 37).

[0112] Example 6: Preparation of lysine 1,3-ditocopheroloxy-2-propyl ester (L2T)

[0113] 415.7 mg N,N'-di-tert-butoxycarbonyl-L-lysine, 287.6 mg EDCI, 49.08 mg DMAP, and 917.5 mg 1,3-ditocopherol-2-propanol (dTPOH) were added to a dry flask containing 10 mL of dry dichloromethane. The mixture was magnetically stirred at room temperature and reacted for 60 h. The reaction solution was then concentrated to approximately 1.0 mL by rotary evaporation. 1 mL of the dichloromethane solution containing the crude product was purified by silica gel column chromatography (60 g 200-300 mesh silica gel, 35 mm diameter). The solution was eluted sequentially with a petroleum ether / ethyl acetate mixture at a volume ratio of 10:1 and 5:1, and the eluates were collected. The eluates containing only the product (N,N'-di-tert-butoxycarbonyl-L-lysine 1,3-ditocopherol-2-propanol, BL2T) were collected and combined. The eluent was rotary evaporated at 55°C until its volume no longer decreased, and then vacuum dried overnight at room temperature to obtain a yellow, transparent, oily liquid, namely BL2T, with a yield of 38.0%.

[0114] 473.3 mg of BL2T was dissolved in 2.5 mL of dichloromethane. 0.846 mL of trifluoroacetic acid was added dropwise under magnetic stirring. The mixture was sealed at room temperature and reacted under magnetic stirring for 3 hours. The reaction solution was concentrated by rotary evaporation and then added dropwise to 10 mL of cold diethyl ether, producing a large amount of white flocculent precipitate. The precipitate was centrifuged at 5000 rpm for 5 minutes, and the supernatant was discarded. The precipitate was washed three times with cold diethyl ether and dried under vacuum to obtain 351.5 mg of a white solid, which is the trifluoroacetic acid salt of lysine 1,3-ditocopheroloxy-2-propyl ester.

[0115] 25 mg of trifluoroacetate of lysine 1,3-ditocopheroloxy-2-propyl ester was dissolved in 1 mL of dichloromethane by ultrasonication in a water bath. Then, 0.5 mL of saturated sodium bicarbonate solution (pH approximately 8) was added, and the mixture was shaken and allowed to stand to separate into layers. The organic layer was collected. The aqueous layer was back-extracted three times with twice the volume of dichloromethane. The organic layers were combined, dried with anhydrous sodium sulfate, filtered, and the filtrate was rotary evaporated at 35°C until the volume no longer decreased. The filtrate was then vacuum dried to obtain a yellow, transparent, soft solid, which is lysine 1,3-ditocopheroloxy-2-propyl ester (L2T). Its structural formula is shown in formula (6). 1 The H NMR spectrum is shown in Figure 8, and the MS spectrum is shown in Figure 9.

[0116] 1 1H NMR (400MHz, DMSO-d6): 0.82 (CH3 at 24H, 20, 25, 29, 30, 59, 60, 61, 62), 1.17 (CH3 at 6H, 31, 47), 2.74 (CH2 at 2H, 74), 3.79-4.03 (CH2 at 4H, 64, 65), 4.14 (CH at 1H, 63), 5.60 (CH at 1H, 68), 7.82 (NH2 at 2H, 75), 8.56 (NH2 at 2H, 71).

[0117] MS(ESI+): The theoretical m / z value of L2T(C67H117N2O6)[M+H]+ is 1045.8912, and the measured value is 1045.8948. The errors are both within 5 ppm, and it is the main component.

[0118] Example 7: Preparation of histidine 1,3-ditocopheroloxy-2-propyl ester (H2T)

[0119] 426.4 mg N,N'-di-tert-butoxycarbonyl-L-histidine, 287.6 mg EDCI, 49.08 mg DMAP, and 917.5 mg 1,3-ditocopherol-2-propanol (dTOH) were added to a dry flask containing 10 mL of dry dichloromethane. The mixture was magnetically stirred at room temperature and reacted for 24 h. The reaction solution was then concentrated to approximately 1.0 mL by rotary evaporation. 1 mL of the dichloromethane solution containing the crude product was purified by silica gel column chromatography (60 g 200-300 mesh silica gel, 35 mm diameter). The eluents were successively eluted with petroleum ether / ethyl acetate mixed solvents at a volume ratio of 10:1 and 5:1, respectively, and the eluents were collected. The eluent containing only the product (N,N'-di-tert-butoxycarbonyl-L-histidine 1,3-ditocopherol-2-propanol, BH2T) was collected and combined. The eluent was rotary evaporated at 55°C until its volume no longer decreased, and then vacuum dried overnight at room temperature to obtain a yellow, transparent, oily liquid, namely BH2T, with a yield of 64.4%.

[0120] 807.7 mg of BH2T was dissolved in 4.3 mL of dichloromethane. While stirring magnetically, 1.43 mL of trifluoroacetic acid was added dropwise. The mixture was sealed at room temperature and reacted with magnetic stirring for 3 hours. The solvent was removed by rotary evaporation of the reaction solution, yielding 550.1 mg of a yellow transparent solid, which is the trifluoroacetic acid salt of histidine 1,3-ditocopheroloxy-2-propyl ester.

[0121] 25 mg of trifluoroacetate of histidine 1,3-ditocopheroloxy-2-propyl ester was dissolved in 1 mL of dichloromethane by ultrasonication in a water bath. Then, 0.5 mL of saturated sodium bicarbonate solution (pH approximately 8) was added, and the mixture was shaken and allowed to stand to separate into layers. The organic layer was collected. The aqueous layer was back-extracted three times with twice the volume of dichloromethane. The organic layers were combined, dried with anhydrous sodium sulfate, filtered, and the filtrate was rotary evaporated at 35°C until the volume no longer decreased. Vacuum drying yielded a yellow, transparent, soft solid, which is histidine 1,3-ditocopheroloxy-2-propyl ester (H2T). Its structural formula is shown in formula (7). 1 The H NMR spectrum is shown in Figure 10, and the MS spectrum is shown in Figure 11.

[0122] 1 ¹H NMR (400MHz, DMSO-d6): 0.81 (CH₃ at 24H, 20, 25, 29, 30, 59, 60, 61, 62), 3.61-4.01 (CH₂ at 4H, 64, 65), 4.30 (CH at 1H, 63), 5.50 (CH at 1H, 66), 6.95 (CH at 1H, 76), 7.67 (CH at 1H, 74).

[0123] MS(ESI+): The theoretical m / z value of H2T(C67 H112 N3 O6)[M+H]+ is 1054.8551, and the measured value is 1054.8535. The error is within 5 ppm, and it is the main component.

[0124] Example 8: Preparation of lysine tocopheryl oxyethyl ester (LTE)

[0125] The preparation method of tocopherol oxyethanol (TEOH) is described in Example 1.

[0126] 2.72 g of tocopheryl oxyethanol (TEOH), 2.38 g of (S)-2,6-di-tert-butoxycarbonylaminohexanoic acid, 1.65 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and 0.28 g of 4-dimethylaminopyridine (DMAP) were added to a 50 mL dry flask containing 13.6 mL of anhydrous dichloromethane. The flask was sealed and protected from light, and the reaction was carried out at room temperature with magnetic stirring. After 66 h of reaction, the solution was rotary evaporated to approximately 3 mL. The 3 mL dichloromethane solution containing the crude product was purified by silica gel column chromatography (40 g 200-300 mesh silica gel, 40 mm diameter) using a 50:1 dichloromethane / ethyl acetate mixture. The eluent was collected. The eluent containing only the product ((S)-2,6-di-tert-butoxycarbonylaminohexanoic acid tocopheryl oxyethyl ester, BLTE) was collected and combined. The solvent was removed by rotary evaporation at room temperature, and the residue was dried under vacuum at room temperature to constant weight to obtain a dark yellow semi-solid, which is BLTE, with a yield of 47.4%.

[0127] 2.02 g of BLTE was added to a flask containing 20 mL of dry dichloromethane, and 6.8 mL of trifluoroacetic acid was slowly added dropwise. The flask was sealed, protected from light, and magnetically stirred. After reacting at room temperature for 3 h, the solvent was removed by rotary evaporation at 40 °C. The residue was dried under vacuum at room temperature to constant weight to obtain a deep yellow semi-solid, namely LTE trifluoroacetate, with a yield of 92.0%.

[0128] 0.69 g of LTE trifluoroacetate was added to 6 mL of dichloromethane and extracted with 3 mL of saturated sodium bicarbonate solution. The organic phase was dried with anhydrous sodium sulfate, centrifuged, filtered, and the organic solvent was removed by rotary evaporation. The filtrate was then dried under vacuum to obtain a deep yellow semi-solid, namely LTE, with a yield of 85.5%. Its structural formula is shown in formula (8). 1 The H NMR spectrum is shown in Figure 12.

[0129] 1 1H NMR (400MHz, DMSO-d6): 0.83 (12H, CH3 at 20, 25, 29, 30), 1.98-2.08 (CH3 at 7, 9, 10), 3.05 (2H, CH2 at 42), 3.59 (2H, CH2 at 32), 3.68 (2H, CH2 at 33).

[0130] Example 9: Preparation of histidine tocopheryl oxyethyl ester (HTE)

[0131] 2.13 g TEOH, 1.92 g N,N'-di-tert-butoxycarbonyl-L-histidine, 1.29 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and 0.22 g 4-dimethylaminopyridine (DMAP) were added to a 50 mL dry flask containing 10.5 mL anhydrous dichloromethane. The flask was sealed and protected from light, and the reaction was carried out at room temperature with magnetic stirring. After 70 h of reaction, the solution was rotary evaporated to approximately 3 mL. The 3 mL dichloromethane solution containing the crude product was purified by silica gel column chromatography (90 g 200-300 mesh silica gel, 40 mm diameter) using a dichloromethane / methanol mixed solvent at a volume ratio of 40:1. The eluent was collected. The eluent containing only the product (N,N'-di-tert-butoxycarbonyl-L-histidine tocopherol oxyethyl ester, BHTE) was collected and combined. The solvent was removed by rotary evaporation at room temperature, and the residue was dried under vacuum at room temperature to constant weight to obtain a pale yellow semi-solid, which is BHTE, with a yield of 86.5%.

[0132] 3.15 g of BHTE was added to a flask containing 31.5 mL of dry dichloromethane. 10.5 mL of trifluoroacetic acid was slowly added dropwise. The flask was sealed and protected from light. The mixture was magnetically stirred and reacted at room temperature for 3 h. The reaction mixture was then removed by rotary evaporation at 40 °C to remove the solvent and trifluoroacetic acid, yielding the crude product. The crude product was dissolved in 5 mL of dichloromethane to prepare a dichloromethane solution for column chromatography purification. The dichloromethane solution containing the crude product was purified using a silica gel column (90 g 200-300 mesh silica gel, 40 mm diameter). The column was eluted sequentially with dichloromethane / methanol mixed solvents at volume ratios of 40:1, 20:2, and 20:3, and the eluates were collected. The eluates containing only HTE trifluoroacetate were collected and combined. The solvent was removed by rotary evaporation at room temperature. The residue was dried under vacuum at room temperature to constant weight, yielding a pale yellow semi-solid, HTE trifluoroacetate, with a yield of 87.8%.

[0133] 1.20 g of HTE trifluoroacetate was added to 12 mL of dichloromethane and extracted with 6 mL of saturated sodium bicarbonate solution. The organic phase was dried with anhydrous sodium sulfate, centrifuged, filtered, and the organic solvent was removed by rotary evaporation. The filtrate was then dried under vacuum to obtain a pale yellow semi-solid, namely HTE, with a yield of 82.9%. Its structural formula is shown in formula (9). 1 The H NMR spectrum is shown in Figure 13, and the MS spectrum is shown in Figure 14.

[0134] 1 1H NMR (400MHz, DMSO-d6): 0.83 (CH3 at 12H, 20, 25, 29, 30), 1.22 (CH3 at 3H, 31), 1.83-2.14 (CH3 at 9H, 7, 9, 10), 2.83-3.06 (CH2 at 2H, 37), 3.80 (CH2 at 2H, 32), 4.36 (CH2 at 2H, 33), 6.89 (CH at 1H, 44), 7.66 (CH at 1H, 42).

[0135] MS(ESI+):HTE(C37 H63 N3 O4)[M+H] + The theoretical m / z value is 612.4740, and the measured value is 612.4747. The errors are both within 5 ppm, and it is the main component.

[0136] Example 10: Preparation of mRNA-loaded lipid nanoparticles using HTE as an ionizable lipid

[0137] Prepare 18 mL of acetate buffer (pH = 3.6) containing 600 μg luciferase mRNA (Fluc-mRNA) and 6 mL of ethanol solution containing 9000 μg HTE and corresponding amounts of DSPC, cholesterol, and DMG2000 (molar ratio of HTE:DSPC:cholesterol:DMG2000 = 50:7.7:29.6:1.2). Use a microfluidic mixer (ring chip) to mix the prepared 6 mL ethanol solution with the 18 mL Fluc-mRNA acetate buffer at a total flow rate of 28 mL / min, with an acetate buffer to ethanol flow rate ratio of 21:7. Prepare and collect the nanoparticle solution. Dilute the prepared nanoparticle solution with 200 mL of HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) (pH = 7.4). Transfer the nanoparticle solution to an ultrafiltration tube (molecular weight cutoff of 100 kJ), and perform low-temperature centrifugation at 4000 rpm for approximately 1 min to prepare a concentrated nanoparticle solution. The particle size was determined using a dynamic laser light scattering (DLS) instrument, and the measured particle size was 126.4 ± 1.3 nm, with a polydispersity index (PDI) of 0.104 ± 0.008. The particle size distribution curve is shown in Figure 15. Quant-iT... TM RNA kit, microplate reader for quantitative analysis of mRNA, and determination of encapsulation efficiency; the encapsulation efficiency of Fluc-mRNA was found to be 95%.

[0138] Example 11: Preparation of mRNA-loaded lipid nanoparticles using HTP as an ionizable lipid

[0139] Prepare a 30 mL acetate buffer (pH = 3.6) containing 1000 μg Fluc-mRNA and a 10 mL ethanol solution containing 10 mg HTP and corresponding amounts of DSPC, cholesterol, and DMG2000 (molar ratio of HTP:DSPC:cholesterol:DMG2000 = 50:6.5:25.3:1.0). Use a microfluidic mixer (ring chip) to mix the prepared 10 mL ethanol solution with the 30 mL Fluc-mRNA acetate buffer at a total flow rate of 28 mL / min, with an acetate buffer to ethanol flow rate ratio of 21:7. Prepare and collect the nanoparticle solution. Dilute the prepared nanoparticle solution with 370 mL HEPES (pH = 7.4). Transfer the nanoparticle solution to an ultrafiltration tube (molecular weight cutoff of 100 kJ), and centrifuge at low temperature at 4000 rpm for approximately 2.5 min to prepare a concentrated nanoparticle solution. The particle size was determined using a dynamic laser light scattering (DLS) instrument, and the measured particle size was 123.6 ± 1.9 nm, with a polydispersity index (PDI) of 0.128 ± 0.017. The particle size distribution curve is shown in Figure 16. Quant-iT... TM An RNA kit was used to quantitatively analyze mRNA using an ELISA reader, and the encapsulation efficiency was measured. The encapsulation efficiency of Fluc-mRNA was found to be 96%.

[0140] Example 12: Preparation of mRNA-loaded lipid nanoparticles using LTE as an ionizable lipid

[0141] Prepare 18 mL of acetate buffer (pH = 3.6) containing 300 μg Fluc-mRNA, and 6 mL of ethanol solution containing 3 mg LTE and corresponding amounts of DSPC, cholesterol, and DMG2000 (molar ratio of LTE:DSPC:cholesterol:DMG2000 = 50:10:38.5:1.5). Use a microfluidic mixer (ring chip) to mix the prepared 6 mL ethanol solution with the 18 mL Fluc-mRNA acetate buffer at a total flow rate of 28 mL / min, with an acetate buffer to ethanol flow rate ratio of 21:7. Prepare and collect the nanoparticle solution. Dilute the prepared nanoparticle solution with 100 mL of HEPES (pH = 7.4). Transfer the nanoparticle solution to an ultrafiltration tube (molecular weight cutoff of 100 kJ), and centrifuge at low temperature at 4000 rpm for approximately 1 min to prepare a concentrated nanoparticle solution. The particle size was determined using a dynamic laser light scattering (DLS) instrument, and the measured particle size was 72.53 ± 1.22 nm, with a polydispersity index (PDI) of 0.306 ± 0.014. The particle size distribution curve is shown in Figure 17. Quant-iT... TM An RNA kit was used to quantitatively analyze mRNA using an ELISA reader, and the encapsulation efficiency was measured. The encapsulation efficiency of Fluc-mRNA was found to be 96%.

[0142] Example 13: Preparation of mRNA-loaded lipid nanoparticles using HTE as an ionizable lipid

[0143] Prepare a 30 mL acetate buffer (pH = 3.6) containing 1000 μg Fluc-mRNA, and a 10 mL ethanol solution containing 15 mg HTE and corresponding amounts of DSPC, cholesterol, and DMG2000 (molar ratio of HTE:DSPC:cholesterol:DMG2000 = 50:7.5:29.4:1.1). Use a microfluidic mixer (ring chip) to mix the prepared 10 mL ethanol solution with the 30 mL Fluc-mRNA acetate buffer at a total flow rate of 28 mL / min, with an acetate buffer to ethanol flow rate ratio of 21:7. Prepare and collect the nanoparticle solution. Dilute the prepared nanoparticle solution with 280 mL HEPES (pH = 7.4). Concentrate the nanoparticle solution using tangential flow (molecular weight cutoff of 100 kJ) ultrafiltration to obtain a concentrated nanoparticle solution. The particle size was determined using a dynamic laser light scattering (DLS) instrument, and the measured particle size was 137.9 ± 0.5 nm, with a polydispersity index (PDI) of 0.183 ± 0.016. The particle size distribution curve is shown in Figure 18. Quant-iT... TM RNA kit, microplate reader for quantitative analysis of mRNA, and determination of encapsulation efficiency; the encapsulation efficiency of Fluc-mRNA was found to be 95%.

[0144] Example 14: In vivo transfection of mRNA-loaded lipid nanoparticles using HTE or HTP as ionizable lipids

[0145] Prepare a 300 mL acetate buffer (pH 3.6) containing 10 mg Fluc-mRNA and a 100 mL ethanol solution containing 15 mg HTE or HTP and corresponding amounts of DSPC, cholesterol, and DMG2000 (molar ratio of HTE or HTP:DSPC:cholesterol:DMG2000 = 50:7.5:29.4:1.1). Use a microfluidic mixer (large-channel ring chip) to mix the 100 mL ethanol solution with the 300 mL Fluc-mRNA acetate buffer at a total flow rate of 80 mL / min and a flow rate ratio of acetate buffer to ethanol of 60:20. Prepare and collect the nanoparticle solution. Dilute the prepared nanoparticle solution with 2000 mL HEPES (pH 7.4). Concentrate the diluted nanoparticle solution by tangential flow (molecular weight cutoff of 100 kJ) ultrafiltration to prepare a concentrated Fluc-mRNA-loaded nanoparticle solution. Particle size was determined using a dynamic laser light scattering (DLS) spectrometer. Quant-iT... TM An RNA kit was used to quantitatively analyze mRNA using a microplate reader, and the encapsulation efficiency was determined. The test results are shown in Table 1. Nanoparticles prepared using HTE and HTP were labeled as Fluc-mRNA LNP-HTE and Fluc-mRNA LNP-HTP, respectively.

[0146] 100 μL of the Fluc-mRNA LNP-HTE and Fluc-mRNA LNP-HTP nanoparticle solutions were drawn into an insulin needle and injected intramuscularly into the medial thigh muscle of BALB / c mice at a depth of 3 mm. Eight and 24 hours after injection, mRNA expression was detected using an in vivo imaging system (IVIS spectrum, PerkinElmer). Ten minutes before detection, 200 μL of 15 mg / mL fluorescein potassium salt in PBS solution was injected intraperitoneally (right lower abdomen). Mice were anesthetized using isoflurane as an anesthetic (approximately 5 minutes) with a gas anesthesia device. After anesthesia, the mice were placed abdomen-up, limbs extended, and supine on the imaging system panel. The bioluminescence mode was selected, and images were taken. The image of the injected Fluc-mRNA LNP-HTE nanoparticle solution is shown in Figure 19, and the image of the injected Fluc-mRNA LNP-HTP nanoparticle solution is shown in Figure 20. The mean fluorescence intensity (unit: p / sec / cm2 / sr) of the left leg of the mouse was quantitatively analyzed using IVIS spectrum software, as shown in Table 2.

[0147] In vivo transfection results showed that, compared with the saline injection group, the fluorescence intensity of the Fluc mRNA-loaded nanoparticles prepared by HTE or HTP as ionizable lipids was stronger at the injection site at 8h and 24h after injection, indicating that there was more fluorescent protein expression at the injection site.

[0148] Table 2

[0149] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A class of ionizable lipid molecules or pharmaceutically acceptable salts thereof, characterized in that, The ionizable lipid molecule comprises an amino acid structural portion and a tocopherol lipid structural portion, wherein the amino acid structural portion and the tocopherol lipid structural portion are indirectly connected by a hydrolyzable ester bond, and wherein the ionizable group is a basic group of the amino acid structural portion.

2. The ionizable lipid molecule or its pharmaceutically acceptable salt according to claim 1, characterized in that, The amino acid structural portion indirectly connecting the tocopherol lipid structural portion is an alkylene ether; the amino acid structural portion is a residue of lysine, histidine, arginine, or their derivatives.

3. The ionizable lipid molecule according to claim 1, or a pharmaceutically acceptable salt thereof, has the general formula shown in (I): in, B is R is n is 2-10; q and l are 1-5; T is 4. A method for preparing ionizable lipid molecules as described in claim 1, characterized in that, Includes the following steps: (1) Add haloalkanol to a solution containing α-tocopherol and base, and prepare tocopherol oxyalkanol by substitution reaction; (2) To prepare base-protected amino acid tocopheroxane by esterification reaction of tocopheroxane alcohol and base-protected amino acid. (3) Base-protected amino acid tocopheroxane esters are deprotected by a deprotecting agent to obtain amino acid tocopheroxane esters.

5. The method for preparing ionizable lipid molecules according to claim 4, characterized in that, The haloalcohol mentioned in step (1) is a monohaloprimalcohol or a dihalosecondalcohol; the base is an alkali metal hydroxide.

6. The method for preparing ionizable lipid molecules according to claim 5, characterized in that, The molar ratio of monohalogenated primary alkanol, α-tocopherol, and base is (0.1–5):1:(0.1–5); the molar ratio of dihalogenated secondary alkanol, α-tocopherol, and base is (0.1–10):1:(0.1–10).

7. The method for preparing ionizable lipid molecules according to claim 4, characterized in that, The base protecting group in step (2) is tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl or 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl. The base protecting group is removed under the action of acid to obtain amino acid tocopherol oxyalkyl ester.

8. A composition, characterized in that, The composition comprises a therapeutic or preventative agent and a carrier for delivering the therapeutic or preventative agent, wherein the therapeutic or preventative agent is one or more of siRNA, shRNA, miRNA, and mRNA nucleic acid molecules, polypeptides, or proteins; and the carrier comprises one or more ionizable lipid molecules as described in any one of claims 1 to 3 or pharmaceutically acceptable salts thereof.

9. The composition according to claim 8, characterized in that, The composition is lipid nanoparticles, the average particle size of which is 60 nm to 600 nm; the polydispersity index of which is less than 0.

5.

10. The composition according to claim 8, characterized in that, The carrier also includes auxiliary lipids, cholesterol, and PEGylated lipids.

11. The composition according to claim 10, characterized in that, The auxiliary lipid is distearyl phosphatidylcholine, distearyl phosphatidylethanolamine, dipalmitoyl phosphatidylcholine, or dipalmitoyl phosphatidylethanolamine; the PEGylated lipid is polyethylene glycol 2000 vitamin E succinate, polyethylene glycol 1000 vitamin E succinate, or dimyristoyl glycerol-polyethylene glycol 2000.

12. The use of an ionizable lipid molecule as described in any one of claims 1 to 3, or a pharmaceutically usable salt thereof, or a composition as described in any one of claims 8 to 11, in the preparation of a nucleic acid drug, a small molecule drug, a polypeptide or a protein drug.

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