Ionizable lipid molecules as well as composition and application thereof

Abstract

The invention belongs to the technical field of drug carriers, and relates to an ionizable lipid molecule as well as a composition and application thereof. The lipid molecule comprises an amino acid structure part and a tocopherol lipid structure part which are indirectly connected through a hydrolyzable ester bond, and an ionizable group is a basic group of the amino acid structure part. In a preferred embodiment, the indirect linking of the amino acid moiety to the tocopherol lipid moiety is an alkylene ether; and the amino acid structural part is lysine, histidine, arginine or residues of derivatives thereof. The invention also discloses a composition prepared from one or more ionizable lipid molecules and a therapeutic agent or a prophylactic agent, and an application of the lipid molecules or the composition in preparation of nucleic acid drugs, small molecule drugs, polypeptide or protein drugs, and the prepared RNA-loaded lipid nanoparticles can express or silence proteins in vivo, and can be used for preparing nucleic acid drugs, small molecule drugs, polypeptide drugs or protein drugs. The invention has the advantages of good stability, high transfection efficiency, low toxicity, safety, effectiveness and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug carriers, and relates to a class of ionizable lipid molecule carriers, and specifically to a class of ionizable lipid molecules of amino acid tocopherol oxyalkyl esters and a preparation method thereof, or a pharmaceutically acceptable salt thereof, and a composition and application thereof. Background Art

[0002] Nucleic acid drugs are not very stable both in vivo and in vitro, and are easily degraded by nucleases in body fluids. Therefore, carriers are often needed to complete the delivery of drugs in the body. At present, common carriers include two categories: viral vectors and non-viral vectors. Due to safety issues such as cytotoxicity, immunogenicity and carcinogenicity of viral vectors, as well as limited loading capacity and high production costs, non-viral vectors have gradually replaced viral vectors. Among them, ionizable lipids can form nanoparticles with negatively charged nucleic acid drugs through electrostatic interactions, protecting nucleic acid drugs from degradation by nucleases and achieving stable delivery of nucleic acid drugs in the body.

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

[0004]

[0005] However, these compounds containing tertiary amine groups and fatty chain structures still have the following shortcomings:

[0006] (1) The ionization adjustability of tertiary amine groups is poor, which may lead to certain biosafety risks and biotoxicity issues.

[0007] (2) These ionizable lipid molecules are lipid molecules containing a single ionizable group. However, different nucleic acid molecules have different chemical structures and compositions, and their interactions with these lipid molecules containing a single ionizable group are somewhat different. This leads to differences in the effects and effects of these lipid molecules containing a single ionizable group on loading and delivering different nucleic acids, and the loading amount and delivery effect are not stable enough. On the other hand, it also leads to deviations in the loading and delivery effects of certain nucleic acids and is difficult to improve. Summary of the invention

[0008] In order to solve the above problems, the first object of the present invention is to provide a class of ionizable lipid molecules and a preparation method thereof, wherein the ionizable lipid is prepared by indirectly connecting an amino acid structure part and a tocopherol lipid structure part through a hydrolyzable ester bond. In a preferred embodiment of the present invention, the amino acid part is a residue of lysine, arginine, histidine, or a derivative thereof, whereby the ionizable lipid contains multiple ionizable sites, has good adjustability, a wide range of applications, and has good biosafety.

[0009] The 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 agent or a preventive agent, and the use of the ionizable lipid molecules, or pharmaceutically acceptable salts thereof, or the composition in the preparation of nucleic acid drugs, small molecule drugs, polypeptides or protein drugs.

[0010] The technical solution of the present invention is as follows:

[0011] The present invention discloses an ionizable lipid molecule or a pharmaceutically acceptable salt thereof, wherein the ionizable lipid molecule comprises an amino acid structure portion and a tocopherol lipid structure portion, wherein the amino acid structure portion and the tocopherol lipid structure portion are indirectly connected via a hydrolyzable ester bond, wherein the ionizable group is a basic group of the amino acid structure portion.

[0012] In one embodiment of the present invention, the indirect connection between the amino acid structure and the tocopherol lipid structure is an alkylene ether, preferably Wherein, n is 2-10; q and l are 1-5, and more preferably, n is 2-3, q and l are 1; and the amino acid structure part is the residue of lysine, histidine, arginine, or its derivatives.

[0013] The present invention provides an ionizable lipid molecule or a pharmaceutically acceptable salt thereof, the general formula of which is shown as (I):

[0014]

[0015] Among them, B is R is n is 2-10; q and l are 1-5;

[0016] T is

[0017] The present invention also discloses a method for preparing the above-mentioned ionizable lipid, comprising the following steps:

[0018] (1) adding a halogenated alkanol to a solution containing α-tocopherol and a base to obtain a tocopherol oxyalkanol through a substitution reaction;

[0019] A preferred solution is to dropwise add halogenated alkyl alcohol into a DMF solution mixed with α-tocopherol and a base at 80-100° C., and stir the reaction for 90-110 hours to obtain tocopherol oxyalkyl alcohol;

[0020] (2) esterifying tocopherol oxyalkyl alcohol and base-protected amino acid to obtain base-protected amino acid tocopherol oxyalkyl ester;

[0021] A preferred solution is to mix CH 2 Cl 2 The solution is placed at room temperature to react to obtain base-protected amino acid tocopherol oxyalkyl esters;

[0022] (3) removing the protecting group on the base of the tocopherol oxyalkyl amino acid ester protected by the base under the action of a deprotecting agent to obtain an amino acid tocopherol oxyalkyl ester;

[0023] A preferred solution is to add a CH 2 Cl 2 An organic acid is added dropwise to the solution, and the reaction is stirred for 3-5 hours. After the base is deprotected, the organic acid is neutralized with a saturated sodium bicarbonate solution to obtain amino acid tocopherol oxyalkyl esters.

[0024] In one embodiment of the present invention, preferably, the halogenated alkanol in step (1) is a primary halogenated alkanol or a secondary halogenated alkanol; and the base is an alkali metal hydroxide, such as sodium hydroxide, potassium hydroxide or lithium hydroxide.

[0025] 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); the molar ratio of dihalogenated secondary alkanol, α-tocopherol and base is (0.1-10):1:(0.1-10).

[0026] In one embodiment of the present invention, preferably, the amino acid described in step (2) is a residue of lysine, arginine, histidine, or a derivative thereof, and its base is protected by a base protecting group on a common amino acid molecule, which is specifically screened by a person skilled in the art according to experimental conditions. The present invention preferably uses: tert-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl or 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl to protect the base. At the same time, the base protecting group can be removed under the action of an acid to obtain an amino acid tocopherol ester.

[0027] In one embodiment of the present invention, preferably, the molar ratio of the base-protected amino acid to tocopherol oxide, EDCI, and DMAP in step (2) is preferably 1:(0.1-10):(0.1-10):(0.1-5).

[0028] The present invention also discloses a composition, which comprises a therapeutic agent or a preventive agent and a carrier for delivering the therapeutic agent or the preventive agent, wherein the therapeutic agent or the preventive agent is one or more of nucleic acid molecules, polypeptides or proteins such as siRNA, shRNA, miRNA and mRNA; and the carrier comprises one or more of the above-mentioned ionizable lipid molecules or pharmaceutically acceptable salts thereof.

[0029] 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, preferably (10-15):1.

[0030] 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.

[0031] In one embodiment of the present invention, preferably, the carrier further comprises auxiliary lipids, cholesterol and PEGylated lipids.

[0032] In one embodiment of the present invention, preferably, the molar ratio of the ionizable lipid molecule, the auxiliary lipid, the cholesterol and the PEGylated lipid is 50:(5-20):(10-50):(1-5).

[0033] In one embodiment of the present invention, preferably, the auxiliary lipid is distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylethanolamine; the PEGylated lipid is polyethylene glycol 2000 vitamin E succinate (TPGS2000), polyethylene glycol 1000 vitamin E succinate (TPGS1000) or dimyristoylglycerol-polyethylene glycol 2000 (DMG2000).

[0034] The present invention also discloses the use of the ionizable lipid molecule represented by the general formula (I) above, or its pharmaceutically acceptable salt, or the above composition in the preparation of nucleic acid drugs, small molecule drugs, polypeptides or protein drugs.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention discloses a type of ionizable lipid molecules, which use tocopherol and amino acids, which are essential to the human body and highly safe, as raw materials, and obtain amino acid tocopherol oxyalkyl esters, such as amino acid tocopherol oxypropyl ester, amino acid tocopherol oxyethyl ester, amino acid 1,3-ditocopherol oxy-2-propyl ester, etc., through ester bond construction. The amino acid tocopherol oxyalkyl ester can be used as an ionizable lipid, as a lipid nanoparticle carrier for delivering siRNA, shRNA, miRNA and mRNA, and other nucleic acid molecules, polypeptides or proteins. The average particle size of the prepared lipid nanoparticles is 60nm to 600nm; the polydispersity index of the lipid nanoparticles is less than 0.5. Injection of Fluc-mRNA lipid nanoparticles in mice achieves effective transfection, and injection of PCSK9 siRNA lipid nanoparticles achieves silencing of PCSK9. At the same time, 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. According to the structural characteristics, content composition, and delivery requirements of the therapeutic agent or preventive agent, one or more suitable amino acid tocopherol esters can be selected. It has good adjustability, a wide range of applications, and good biosafety.

[0037] Specifically: First, the amino acid ester containing tocopherol lipid structure provided by the present invention can be hydrolyzed to generate corresponding amino acids and tocopherol lipid structure parts, and has good biological safety;

[0038] Second, when the amino acid ester containing tocopherol lipid structure provided by the present invention is lysine ester, histidine ester or arginine ester containing tocopherol lipid structure, in the amino acid structure, in addition to amino group, weakly basic groups such as imidazole or guanidine can also be combined with hydrogen ions to be ionized, and the degree of ionization of these groups under physiological conditions is different; and the electrostatic interaction degree of lipid molecules containing groups of different ionization degrees with negatively charged nucleic acids is different, and the ability to load and release nucleic acids is also different. This allows technicians to select a suitable amino acid ester containing tocopherol lipid structure according to the structure and composition of nucleic acids and their delivery requirements, or select a composition of multiple amino acid esters and adjust the appropriate ratio between various amino acid esters, so as to achieve stable and efficient delivery of different types of nucleic acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1The tocopherol oxypropanol (TPOH) of Example 1 of the present invention is 1 H NMR spectrum;

[0040] Figure 2 1,3-ditocopherol-2-propanol (dTPOH) of Example 2 of the present invention 1 H NMR spectrum;

[0041] Figure 3 The histidine tocopherol oxypropyl ester (HTP) of Example 3 of the present invention 1 H NMR spectrum;

[0042] Figure 4 This is the MS spectrum of histidine tocopherol oxypropyl ester (HTP) of Example 3 of the present invention;

[0043] Figure 5 The lysine tocopherol oxypropyl ester (LTP) of Example 4 of the present invention 1 H NMR spectrum;

[0044] Figure 6 is the MS spectrum of lysine tocopherol oxypropyl ester (LTP) of Example 4 of the present invention;

[0045] Figure 7 is the arginine tocopherol oxypropyl ester (ATP) of Example 5 of the present invention 1 H NMR spectrum;

[0046] Figure 8 The lysine 1,3-ditocopheryloxy-2-propyl ester (L2T) of Example 6 of the present invention is 1 H NMR spectrum;

[0047] Fig. 9 The MS spectrum of lysine 1,3-ditocopheryloxy-2-propyl ester (L2T) of Example 6 of the present invention;

[0048] Fig.10 The histidine 1,3-ditocopheryl oxygen-2-propyl ester (H2T) of Example 7 of the present invention 1 H NMR spectrum;

[0049] Fig.11 The MS spectrum of histidine 1,3-ditocopheryloxy-2-propyl ester (H2T) of Example 7 of the present invention;

[0050] Fig.12 is the lysine tocopherol oxyethyl ester (LTE) of Example 8 of the present invention 1 H NMR spectrum;

[0051] Fig.13The histidine tocopherol oxyethyl ester (HTE) of Example 9 of the present invention 1 H NMR spectrum;

[0052] Fig.14 is the MS spectrum of histidine tocopherol oxyethyl ester (HTE) of Example 9 of the present invention;

[0053] Fig.15 is a particle size distribution curve of the mRNA lipid nanoparticles of Example 10 of the present invention;

[0054] Fig.16 is a particle size distribution curve of the mRNA lipid nanoparticles of Example 11 of the present invention;

[0055] Fig.17 is a particle size distribution curve of the mRNA lipid nanoparticles of Example 12 of the present invention;

[0056] Fig.18 is a particle size distribution curve of the mRNA lipid nanoparticles of Example 13 of the present invention;

[0057] Fig.19 This is a live imaging diagram of transfection of Fluc-mRNA LNP-HTE mice injected in Example 14 of the present invention, wherein a is a dose of 3.5 μg and b is a dose of 7.0 μg;

[0058] Fig. 20 This is a live imaging diagram of mice transfected with Fluc-mRNA LNP-HTP injected in Example 14 of the present invention, wherein a is a dose of 3.5 μg and b is a dose of 7.0 μg. DETAILED DESCRIPTION

[0059] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0060] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited to the specific implementation disclosed below.

[0061] The raw materials used in the present invention can be obtained from the market, wherein tocopherol is commonly used D-α-tocopherol with high activity, and lysine, histidine, and arginine are all commonly used L-type amino acids. Under the conception of the present invention, tocopherol and amino acids of other configurations can also be selected as raw materials. Those skilled in the art can still prepare the amino acid tocopherol oxyalkyl esters of the present invention by selecting tocopherol and amino acid raw materials of other configurations, such as amino acid tocopherol oxypropyl ester, amino acid tocopherol oxyethyl ester, amino acid 1,3-ditocopherol oxy-2-propyl ester, etc., and lipid molecule nanoparticles can be prepared, which are used as carriers for delivering therapeutic agents or preventive agents, and lipid nanoparticle carriers for delivering nucleic acid molecules, polypeptides or proteins such as siRNA, shRNA, miRNA and mRNA.

[0062] The present invention discloses a class of ionizable lipid molecules, and a preparation method, composition, and application thereof. The composition comprises one or more ionizable lipid molecules or pharmaceutically acceptable salts thereof, and a therapeutic agent or a preventive agent. The ionizable lipid molecules, or pharmaceutically acceptable salts thereof, or application of the composition in the preparation of nucleic acid drugs, small molecule drugs, polypeptides, or protein drugs are also disclosed.

[0063] In a preferred embodiment, the present invention relates to amino acid esters of mono-tocopherol lipid structures and di-tocopherol lipid structures, such as amino acid tocopherol oxypropyl ester, amino acid 1,3-di-tocopherol oxy-2-propyl ester, and the like.

[0064] The present invention can select one or more ionizable lipid molecules, together with other lipid molecules, such as auxiliary lipid molecules, cholesterol and PEGylated lipids, to prepare lipid nanoparticle compositions loaded with nucleic acid drugs, small molecule drugs, polypeptides or protein drugs. The drug-loaded composition can express or silence proteins in vivo, and has the characteristics of good stability, high transfection efficiency, low toxicity, safety and effectiveness.

[0065] In a preferred embodiment, the above-mentioned amino acid ester molecule is a lysine ester containing 2 amino groups, a histidine ester containing 1 amino group and 1 imidazole group, or an arginine ester containing 1 amino group and a guanidine group. In such amino acid esters, in addition to the amino group, weakly basic groups such as imidazole groups or guanidine groups can also be combined with hydrogen ions to be ionized, and the degree of ionization of these groups under physiological conditions is different. The lipid molecules containing groups with different ionization degrees have different degrees of electrostatic interaction with negatively charged RNA, and the ability to load and release RNA is also different. For example, the pKa of imidazole groups is about 6, which can increase the buffering capacity of nanocarriers in endosomes and lysosomes through the proton sponge effect and enhance their escape ability; the cell penetration ability of guanidine groups is strong, which can enhance the affinity with cell membranes and thus improve the efficiency of cell uptake.

[0066] D-α-tocopherol is the most widely distributed, most abundant and most active form of vitamin E in nature. Vitamin E is a fat-soluble antioxidant and an essential nutrient for the human body, which is mainly obtained through exogenous food.

[0067] There are three basic amino acids among the essential amino acids for the human body, such as arginine contains a guanidine group and an amino group, histidine contains an imidazole group and an amino group, and lysine contains two amino groups.

[0068] The present invention can select one or more ionizable lipid molecules, together with other lipid molecules, such as auxiliary lipid molecules, cholesterol and PEGylated lipids, to prepare a lipid nanoparticle composition loaded with nucleic acid drugs, small molecule drugs, polypeptides or protein drugs. And based on the different degrees of ionization of lysine esters, arginine esters and histidine esters under physiological conditions, the advantage of the lipid nanoparticles provided by the present invention is that suitable amino acid tocopherol esters or a composition of multiple amino acid tocopherol esters designed with specific ratios can be selected according to the structure and composition of the loaded nucleic acid and its delivery requirements.

[0069] Furthermore, the amino acid tocopherol oxyalkyl ester provided by the present invention can be hydrolyzed in vivo to generate corresponding amino acids and tocopherol lipid structural parts, and has good biological safety.

[0070] The present invention also provides a method for preparing the above-mentioned ionizable lipid molecule, that is, a method for synthesizing an amino acid ester in which the above-mentioned tocopherol lipid structure part and the amino acid structure part are connected via a hydrolyzable ester bond via a linking group, comprising the following steps:

[0071] (i) Tocopherol and halogenated alkanol or dihalogenated alkanol are reacted to prepare tocopherol oxyalkanol through substitution reaction, wherein the general formula of chemical reaction between tocopherol and halogenated alkanol is as follows:

[0072]

[0073] X: halogen atom, such as bromine, chlorine or iodine; n = 2 to 10;

[0074] The general formula for the chemical reaction between tocopherol and dihaloalkanol is as follows:

[0075]

[0076] X: halogen atom, such as bromine, chlorine or iodine; q, l = 1-5;

[0077] (ii) Tocopherol oxide alkanol and base-protected amino acids (such as lysine, histidine or arginine) are reacted by esterification to prepare base-protected tocopherol oxide alkyl amino acid esters. The general chemical reaction formula is as follows:

[0078]

[0079] RCOOH: base-protected amino acid (such as lysine, histidine or arginine); n = 2 to 10;

[0080] or

[0081]

[0082] RCOOH: base-protected amino acid (such as lysine, histidine or arginine); q, l = 1-5;

[0083] (iii) Deprotection reaction to prepare tocopherol oxyalkyl amino acid ester, the chemical reaction formula is as follows:

[0084]

[0085] n = 2 to 10;

[0086] or,

[0087]

[0088] q, l = 1-5;

[0089] B is the residue of an amino acid or an amino acid derivative, preferably at least one of the following three:

[0090]

[0091] In addition to the above three amino acids, B may also be the residue of other amino acids or derivatives of the above amino acids.

[0092] The present invention also provides a method for preparing a lipid nanoparticle composition using the ionizable lipid molecules.

[0093] In one embodiment, the ionizable lipid molecule (also referred to as an ionizable lipid carrier) is combined with an auxiliary lipid molecule, cholesterol and a PEGylated lipid molecule to form a carrier for delivering a therapeutic agent or a preventive agent, wherein the ionizable lipid molecule is one, two or more than two; the auxiliary lipid molecule may be distearoylphosphatidylcholine, distearoylphosphatidylethanolamine 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.

[0094] In a common embodiment, the lipid nanoparticle composition is prepared by a microfluidics, microjetting or high-speed homogenization method. Preferably, when preparing the composition, the mass ratio of the carrier to the therapeutic agent or preventive agent is 5-30:1, preferably (10-15):1; the molar ratio of the ionizable lipid, auxiliary lipid, cholesterol and PEGylated lipid is: 50:(5-20):(10-50):(1-5). The nanoparticle solution prepared by the above method can be removed from the organic solvent or concentrated by dialysis, ultrafiltration or tangential flow.

[0095] The prepared nanoparticle solution loaded with nucleic acid drugs, small molecule drugs, polypeptides or protein drugs can be used for intramuscular injection, intravenous injection or local administration to exert therapeutic or preventive effects.

[0096] Example 1: Preparation of Tocopherol Oxypropanol (TPOH)

[0097] D-α-tocopherol (10.00 g) and 1.39 g of sodium hydroxide were added to a flask containing 30 mL of DMF (N,N-dimethylformamide). In a 90°C oil bath, 10 mL of DMF solution containing 4.03 g of 3-bromopropanol was slowly added dropwise under magnetic stirring. 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 the methyl tert-butyl ether was removed by rotary evaporation. A small amount of dichloromethane was added to dissolve the crude product to prepare a dichloromethane solution for column chromatography.

[0098] The dichloromethane solution containing the crude product was separated and purified by a silica gel column (300 g 200-300 mesh silica gel, 70 mm in diameter), and eluted with a mixed solvent of petroleum ether / ethyl acetate in a volume ratio of 8:1 and 5:1, respectively, and the eluent was collected. The eluent containing only TPOH was collected and combined, and the solvent was removed by rotary evaporation to obtain a dark yellow viscous liquid, namely TPOH, with a yield of 75.0%.

[0099] 1 H NMR [δ / ppm] (400 MHz, DMSO-D6): 0.72-0.90 (CH3 on 12H, 20, 25, 29, 30), 1.17 (CH3 on 3H, 31), 1.86 (CH2 on 2H, 33), 1.93-2.11 (CH3 on 9H, 7, 9, 10), 3.56-3.67 (CH2 on 4H, 32, 34), 1 H NMR spectrum Figure 1 As shown, the structural formula is shown in formula (1).

[0100]

[0101] Example 2: Preparation of 1,3-ditocopherol-2-propanol (dTPOH)

[0102] D-α-tocopherol (10.00 g) and 1.11 g of sodium hydroxide were added to a flask containing 50 mL of DMF. 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 under magnetic stirring. After 60 hours of reaction, the reaction solution was evaporated with an oil pump at 55°C until the volume no longer decreased. 60 mL of ether was added, transferred to a separatory funnel, and extracted with an equal volume of pure water. The ether layer and the water layer were separated, and the water layer was washed twice with an equal volume of ether, and the ether layer was combined. Anhydrous sodium sulfate was added to the ether layer until no lumps were formed, and the mixture was allowed to stand overnight, filtered, and the filtrate was evaporated at room temperature until the volume no longer decreased to obtain a crude product. A small amount of dichloromethane was added to dissolve the crude product, and a dichloromethane solution was prepared for column chromatography.

[0103] The dichloromethane solution containing the crude product was separated and purified using a silica gel column (300 g 200-300 mesh silica gel, 70 mm in diameter), and eluted with different proportions of petroleum ether / dichloromethane mixed solvents (volume ratio of 2:1, 1:1, 1:2, 1:3) in sequence, and the eluents were collected. The eluents 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%.

[0104] 1 H NMR (400 MHz, Chloroform-d) δ 4.43 (CH on 1H, 63), 3.77-3.95 (CH2 on 4H, 65, 66), 2.56 (CH2 on 4H, 14, 42), 1.99-2.27 (CH3 on 18H, 7, 9, 10, 38, 39, 40), 1.77 (CH2 on 4H, 13, 43), 0.78-0.93 (CH3 on 24H, 20, 25, 29, 30, 59, 60, 61, 62), which 1 HNMR spectrum Figure 2 As shown, the structural formula is shown in formula (2).

[0105]

[0106] Example 3: Preparation of Histidine Tocopherol Oxypropyl Ester (HTP)

[0107] 2.00 g TPOH, 1.74 g N,N'-di-tert-butyloxycarbonyl-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 anhydrous dichloromethane, and the mixture was sealed and protected from light and reacted with magnetic stirring at room temperature. After 90 h of reaction, the mixture was rotary evaporated to about 3 mL. 3 mL of dichloromethane solution containing the crude product was separated and purified using a silica gel column (90 g 200-300 mesh silica gel, 40 mm in diameter), and eluted with a mixed solvent of dichloromethane, dichloromethane and methanol in a volume ratio of 1:20:1, and the eluate was collected. The eluate containing only the product (N,N'-di-tert-butyloxycarbonyl-L-histidine tocopheryl oxypropyl ester, BHTP) was collected and combined. The solvent was removed by rotary evaporation and vacuum dried to constant weight to obtain a dark yellow semi-solid substance, namely BHTP, with a yield of 86.9%.

[0108] 2.94 g BHTP was added to a flask containing 30 mL dry dichloromethane, 10 mL trifluoroacetic acid was slowly added dropwise, the flask was sealed to avoid light, magnetic stirring was applied, and the reaction mixture was reacted at room temperature for 3 h, and then the solvent and trifluoroacetic acid were removed by rotary evaporation at 40 ° C to obtain a crude product. The crude product was dissolved in 5 mL dichloromethane to prepare a dichloromethane solution for column chromatography separation and purification. The dichloromethane solution containing the crude product was separated and purified using a silica gel column (90 g 200-300 mesh silica gel, 40 mm in diameter), and eluted with a dichloromethane / methanol mixed solvent with a volume ratio of 40:1, 20:2 and 20:3, and the eluent was collected. The eluent containing only HTP trifluoroacetate was collected and combined. The solvent was removed by rotary evaporation, and vacuum dried to constant weight to obtain a dark yellow semisolid, i.e., HTP trifluoroacetate, with a yield of 81.9%.

[0109] 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 filtrate was rotary evaporated to remove the organic solvent, and vacuum dried to obtain a light yellow semi-solid, i.e., HTP, with a yield of 83.7%. Its structural formula is shown in formula (3), 1 H NMR spectrum Figure 3 As shown in the MS spectrum Figure 4 shown.

[0110] 1H NMR (400 MHz, 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).

[0111] MS (ESI+): The theoretical value of m / z for HTP (C38 H63 N3 O4H) [M+H]+ is 626.4897, and the measured value is 627.4897; the theoretical value of m / z for HTP (C38 H63 N3 O4Na) [M+Na]+ is 648.4716, and the measured value is 648.4715. The errors are within 5ppm, and it is the main component.

[0112]

[0113] Example 4: Preparation of lysine tocopherol oxypropyl ester (LTP)

[0114] 1.00 g TPOH, 0.85 g (S)-2,6-di-tert-butyloxycarbonylaminocaproic 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 anhydrous dichloromethane, sealed and protected from light, and reacted under magnetic stirring at room temperature. After 72 h of reaction, the solution was rotary evaporated to about 3 mL. 3 mL of dichloromethane solution containing the crude product was separated and purified using a silica gel column (45 g 200-300 mesh silica gel, 40 mm in diameter), and eluted with a dichloromethane / methanol mixed solvent with a volume ratio of 1:0 and 20:1, respectively, and the eluent was collected. The eluent containing only the product ((S)-2,6-di-tert-butyloxycarbonylaminocaproic acid tocopheryloxypropyl ester, BLTP) was collected and combined. The solvent was removed by rotary evaporation and vacuum dried to constant weight to obtain a dark yellow semi-solid substance, namely BLTP, with a yield of 90.4%.

[0115] 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 to avoid light and magnetically stirred. After reacting at room temperature for 3 h, the reaction mixture was rotary evaporated at 40 °C to remove the solvent and vacuum dried to constant weight to obtain a dark yellow semisolid, i.e., LTP trifluoroacetate, with a yield of 95.0%.

[0116] 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 filtrate was rotary evaporated to remove the organic solvent, and vacuum dried to obtain a light yellow semi-solid, namely LTP, with a yield of 84.5%. Its structural formula is shown in formula (4), 1 H NMR spectrum Figure 5 As shown in the MS spectrum Figure 6 shown.

[0117] 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).

[0118] MS (ESI+): The theoretical m / z value of LTP (C38 H69 N2 O4) [M + H] + is 617.5257, and the measured value is 618.5254; the theoretical m / z value of LTP (C38 H68 N2 O4Na) [M + Na] + is 639.5077, and the measured value is 639.5078. The errors are within 5ppm, and it is the main component.

[0119]

[0120] Example 5: Preparation of arginine tocopherol oxypropyl ester (ATP)

[0121] 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 anhydrous dichloromethane, sealed and protected from light, and reacted under magnetic stirring at room temperature. After 110 h of reaction, rotary evaporation was performed to about 3 mL. 3 mL of dichloromethane solution containing the crude product was separated and purified using a silica gel column (45 g 200-300 mesh silica gel, 40 mm in diameter), and eluted with a petroleum ether / ethyl acetate mixed solvent with a volume ratio of 1:1 and 5:7, and the eluate was collected. The eluent containing only the product (Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine tocopheryl oxypropyl ester, BATP) was collected and combined. The solvent was removed by rotary evaporation at room temperature, and the residue was vacuum dried at room temperature to constant weight to obtain a colorless semi-solid substance, namely BATP, with a yield of 65.2%.

[0122] 1.33 g BATP was added to a flask containing 13 mL dry dichloromethane, 4.3 mL trifluoroacetic acid was slowly added dropwise, the flask was sealed to avoid light, magnetic stirring was applied, and the reaction mixture was reacted at room temperature for 24 h, and then the solvent and trifluoroacetic acid were removed by rotary evaporation at 40 °C to obtain a crude product. The crude product was dissolved in 5 mL dichloromethane to prepare a dichloromethane solution for column chromatography separation and purification. The dichloromethane solution containing the crude product was separated and purified using a silica gel column (45 g 200-300 mesh silica gel, 40 mm in diameter), and eluted with a methanol / dichloromethane mixed solvent with a volume ratio of 2:20 and 3:20, respectively, and the eluent was collected. The eluent containing only ATP trifluoroacetate was collected and combined. The solvent was removed by rotary evaporation at room temperature, and the residue was vacuum dried at room temperature to constant weight to obtain a light yellow semisolid, i.e., ATP trifluoroacetate, with a yield of 37.9%.

[0123] 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 filtrate was rotary evaporated to remove the organic solvent, and vacuum dried to obtain a light yellow semisolid, i.e., ATP, with a yield of 47.7%. Its structural formula is shown in formula (5), 1 H NMR spectrum Figure 7 shown.

[0124] 1H NMR (400 MHz, DMSO-d6): 0.90 (12H, 20, 25, 29, 30 of CH3), 1.24 (3H, 31), 3.18 (2H, 42 of CH2), 3.68 (2H, 32 of CH2), 4.40 (2H, 34 of CH2), 4.43 (1H, 37 of CH).

[0125]

[0126] Example 6: Preparation of lysine 1,3-ditocopheryloxy-2-propyl ester (L2T)

[0127] 415.7 mg N, N'-di-tert-butyloxycarbonyl-L-lysine, 287.6 mg EDCI, 49.08 mg DMAP and 917.5 mg 1,3-di-tocopherol-2-propanol (dTPOH) were added to a dry flask containing 10 mL of dry dichloromethane, and stirred magnetically at room temperature. After 60 hours of reaction, the reaction solution was concentrated by rotary evaporation to about 1.0 mL. 1 mL of dichloromethane solution containing the crude product was separated and purified using a silica gel column (60 g 200-300 mesh silica gel, 35 mm in diameter). The mixture was eluted with a mixed solvent of petroleum ether / ethyl acetate in a volume ratio of 10:1 and 5:1, and the eluent was collected. The eluent containing only the product (N, N'-di-tert-butyloxycarbonyl-L-lysine 1,3-di-tocopherol-2-propanol, BL2T) was collected and combined. The eluate was rotary evaporated at 55°C until the volume stopped decreasing, and then vacuum dried at room temperature overnight to obtain a yellow transparent oily liquid, namely BL2T, with a yield of 38.0%.

[0128] 473.3 mg of BL2T was added to 2.5 mL of dichloromethane to dissolve it. Under magnetic stirring, 0.846 mL of trifluoroacetic acid was added dropwise. The mixture was sealed at room temperature and reacted for 3 h under magnetic stirring. The reaction solution was concentrated by rotary evaporation and then dropped into 10 mL of cold ether to produce a large amount of white floccules. The mixture was centrifuged at 5000 rpm for 5 min and the supernatant was discarded. The precipitate was washed 3 times with cold ether and dried under vacuum to obtain 351.5 mg of a white solid, which was the trifluoroacetate of 1,3-ditocopheryloxy-2-propyl lysine.

[0129] 25mg of lysine 1,3-ditocopheryloxy-2-propyl trifluoroacetate was added with 1mL of dichloromethane, dissolved in a water bath by ultrasonication, and then 0.5mL of saturated sodium bicarbonate solution (pH=about 8) was added. After oscillation and mixing, the mixture was allowed to stand for stratification and the organic layer was collected. The aqueous layer was extracted three times with twice the volume of dichloromethane, and the organic layers were combined, dried with anhydrous sodium sulfate, filtered, and the filtrate was evaporated at 35°C until the volume no longer decreased, and vacuum dried to obtain a yellow transparent soft solid, which is lysine 1,3-ditocopheryloxy-2-propyl ester (L2T). Its structural formula is shown in formula (6). 1 H NMR spectrum Figure 8 As shown in the MS spectrum Fig. 9 shown.

[0130] 1 H NMR (400 MHz, DMSO-d6): 0.82 (CH3 on 24H, 20, 25, 29, 30, 59, 60, 61, 62), 1.17 (CH3 on 6H, 31, 47), 2.74 (CH2 on 2H, 74), 3.79-4.03 (CH2 on 4H, 64, 65), 4.14 (CH on 1H, 63), 5.60 (CH on 1H, 68), 7.82 (NH2 on 2H, 75), 8.56 (NH2 on 2H, 71).

[0131] MS (ESI+): The theoretical m / z value of L2T (C67 H117 N2 O6) [M+H] + is 1045.8912, and the measured value is 1045.8948. The error is within 5ppm, and it is the main component.

[0132]

[0133] Example 7: Preparation of Histidine 1,3-Di-Tocopheryl Oxy-2-Propyl Ester (H2T)

[0134] 426.4 mg N, N'-di-tert-butyloxycarbonyl-L-histidine, 287.6 mg EDCI, 49.08 mg DMAP and 917.5 mg 1,3-di-tocopherol-2-propanol (dTOH) were added to a dry flask containing 10 mL of dry dichloromethane, and stirred magnetically at room temperature. After 24 hours of reaction, the reaction solution was concentrated by rotary evaporation to about 1.0 mL. 1 mL of dichloromethane solution containing the crude product was separated and purified using a silica gel column (60 g 200-300 mesh silica gel, 35 mm in diameter). The mixture was eluted with a mixed solvent of petroleum ether / ethyl acetate in a volume ratio of 10:1 and 5:1, respectively, and the eluent was collected. The eluent containing only the product (N, N'-di-tert-butyloxycarbonyl-L-histidine 1,3-di-tocopherol-2-propanol, BH2T) was collected and combined. The eluate was rotary evaporated at 55°C until the volume stopped decreasing, and then dried in vacuo at room temperature overnight to obtain a yellow transparent oily liquid, namely BH2T, with a yield of 64.4%.

[0135] 807.7 mg of BH2T was added to 4.3 mL of dichloromethane to dissolve it, and 1.43 mL of trifluoroacetic acid was added dropwise under magnetic stirring. The mixture was sealed at room temperature and reacted for 3 h under magnetic stirring. The reaction solution was evaporated to remove the solvent to obtain 550.1 mg of a yellow transparent solid, which was the trifluoroacetate of histidine 1,3-ditocopheryloxy-2-propyl ester.

[0136] Add 1 mL of dichloromethane to 25 mg of trifluoroacetate of histidine 1,3-ditocopheryloxy-2-propyl ester, dissolve in water bath ultrasound, add 0.5 mL of saturated sodium bicarbonate solution (pH=about 8), shake and mix, let stand to separate, and collect the organic layer. The aqueous layer is extracted three times with twice the volume of dichloromethane, the organic layers are combined, dried with anhydrous sodium sulfate, filtered, and the filtrate is evaporated at 35°C until the volume no longer decreases. Vacuum drying gives a yellow transparent soft solid, which is histidine 1,3-ditocopheryloxy-2-propyl ester (H2T). Its structural formula is shown in formula (7), 1 H NMR spectrum Fig.10 As shown in the MS spectrum Fig.11 shown.

[0137] 1 H NMR (400 MHz, DMSO-d6): 0.81 (CH3 of 24H, 20, 25, 29, 30, 59, 60, 61, 62), 3.61-4.01 (CH2 of 4H, 64, 65), 4.30 (CH of 1H, 63), 5.50 (CH of 1H, 66), 6.95 (CH of 1H, 76), 7.67 (CH of 1H, 74).

[0138] 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 errors are within 5ppm, and it is the main component.

[0139]

[0140]

[0141] Example 8: Preparation of lysine tocopherol oxyethyl ester (LTE)

[0142] The preparation method of tocopherol oxyethanol (TEOH) is shown in Example 1.

[0143] 2.72 g of tocopherol oxyethanol (TEOH), 2.38 g of (S)-2,6-di-tert-butyloxycarbonylaminocaproic 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, sealed and protected from light, and reacted under magnetic stirring at room temperature. After 66 h of reaction, the solution was rotary evaporated to about 3 mL. 3 mL of dichloromethane solution containing the crude product was separated and purified using a silica gel column (40 g of 200-300 mesh silica gel, 40 mm in diameter), eluted with a dichloromethane / ethyl acetate mixed solvent with a volume ratio of 50:1, and the eluate was collected. The eluate containing only the product ((S)-2,6-di-tert-butyloxycarbonylaminocaproic acid tocopherol 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, namely BLTE, with a yield of 47.4%.

[0144] 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 stirred magnetically. After reacting at room temperature for 3 h, the reaction mixture was rotary evaporated at 40° C. to remove the solvent. The residue was vacuum dried at room temperature to constant weight to obtain a dark yellow semisolid, i.e., LTE trifluoroacetate, with a yield of 92.0%.

[0145] 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 filtrate was rotary evaporated to remove the organic solvent, and vacuum dried to obtain a dark yellow semi-solid, namely LTE, with a yield of 85.5%. Its structural formula is shown in formula (8), 1 H NMR spectrum Fig.12 shown.

[0146] 1 H NMR (400 MHz, DMSO-d6): 0.83 (12H, 20, 25, 29, 30 of CH3), 1.98-2.08 (7, 9, 10 of CH3), 3.05 (2H, 42 of CH2), 3.59 (2H, 32 of CH2), 3.68 (2H, 33 of CH2).

[0147]

[0148]

[0149] Example 9: Preparation of Histidine Tocopherol Ethyl Ester (HTE)

[0150] 2.13gTEOH, 1.92gN,N'-di-tert-butyloxycarbonyl-L-histidine, 1.29g1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 0.22g4-dimethylaminopyridine (DMAP) were added to a 50mL dry flask containing 10.5mL anhydrous dichloromethane, sealed and protected from light, and reacted under magnetic stirring at room temperature. After 70h of reaction, rotary evaporation was performed to about 3mL. 3mL of dichloromethane solution containing crude product was separated and purified by silica gel column (90g 200-300 mesh silica gel, diameter 40mm), eluted with dichloromethane / methanol mixed solvent with a volume ratio of 40:1, and the eluate was collected. The eluate containing only the product (N,N'-di-tert-butyloxycarbonyl-L-histidine tocopheryl 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 light yellow semi-solid substance, namely BHTE, with a yield of 86.5%.

[0151] 3.15 g BHTE was added to a flask containing 31.5 mL dry dichloromethane, 10.5 mL trifluoroacetic acid was slowly added dropwise, the flask was sealed to avoid light, magnetic stirring was applied, and the reaction mixture was reacted at room temperature for 3 h, and then the solvent and trifluoroacetic acid were removed by rotary evaporation at 40 ° C to obtain a crude product. The crude product was dissolved in 5 mL dichloromethane to prepare a dichloromethane solution for column chromatography separation and purification. The dichloromethane solution containing the crude product was separated and purified by a silica gel column (90 g 200-300 mesh silica gel, 40 mm in diameter), and eluted with a dichloromethane / methanol mixed solvent with a volume ratio of 40:1, 20:2 and 20:3, respectively, and the eluent was collected. The eluent containing only HTE trifluoroacetate was collected and combined, the solvent was removed by rotary evaporation at room temperature, and the residue was vacuum dried at room temperature to constant weight to obtain a light yellow semi-solid, i.e., HTE trifluoroacetate, with a yield of 87.8%.

[0152] 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 filtrate was rotary evaporated to remove the organic solvent, and vacuum dried to obtain a light yellow semi-solid, namely HTE, with a yield of 82.9%. Its structural formula is shown in formula (9), 1 H NMR spectrum Fig.13 As shown in the MS spectrum Fig.14 shown.

[0153] 1H NMR (400 MHz, DMSO-d6): 0.83 (CH3 on 12H, 20, 25, 29, 30), 1.22 (CH3 on 3H, 31) 1.83-2.14 (CH3 on 9H, 7, 9, 10), 2.83-3.06 (CH2 on 2H, 37), 3.80 (CH2 on 2H, 32), 4.36 (CH2 on 2H, 33), 6.89 (CH on 1H, 44), 7.66 (CH on 1H, 42).

[0154] MS(ESI+):HTE(C37 H63 N3 O4)[M+H] + The theoretical value of m / z is 612.4740, and the measured value is 612.4747. The error is within 5ppm, and it is the main component.

[0155]

[0156] Example 10: Preparation of mRNA-loaded lipid nanoparticles using HTE as an ionizable lipid

[0157] Prepare 18 mL acetate buffer (pH = 3.6) containing 600 μg of luciferase mRNA (Fluc-mRNA), 6 mL ethanol solution containing 9000 μg of 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 above-prepared 6 mL ethanol solution with 18 mL of Fluc-mRNA acetate buffer, with a total flow rate of 28 mL / min and a flow rate ratio of acetate buffer to ethanol solution of 21:7 to prepare and collect nanoparticle solution. Dilute the prepared nanoparticle solution with 200 mL HEPES (4-hydroxyethylpiperazineethanesulfonic acid) (pH = 7.4). Transfer the nanoparticle solution to an ultrafiltration tube (molecular weight cutoff of 100k), centrifuge and ultrafilter at low temperature at a speed of 4000 rpm for about 1 min to prepare a nanoparticle concentrated solution. The particle size was measured by dynamic laser light scattering, and the particle size of the nanoparticles was 126.4±1.3nm, and the polydispersity index (PDI) was 0.104±0.008. The particle size distribution curve is shown in Fig.15 As shown. Using Quant-iT TM RNA kit, microplate reader quantitatively analyzed mRNA, and determined the encapsulation efficiency. The encapsulation efficiency of Fluc-mRNA was found to be 95%.

[0158] Example 11: Preparation of mRNA-loaded lipid nanoparticles using HTP as an ionizable lipid

[0159] Prepare 30 mL acetate buffer (pH = 3.6) containing 1000 μg Fluc-mRNA, 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 above-prepared 10 mL ethanol solution with 30 mL Fluc-mRNA acetate buffer, with a total flow rate of 28 mL / min and a flow rate ratio of acetate buffer to ethanol solution of 21:7 to 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 100k), centrifuge and ultrafilter at low temperature at a speed of 4000 rpm for about 2.5 min to prepare a concentrated nanoparticle solution. The particle size was measured by dynamic laser light scattering, and the particle size of the nanoparticles was 123.6±1.9nm, and the polydispersity index (PDI) was 0.128±0.017. The particle size distribution curve is shown in Fig.16 As shown. Using Quant-iT TM RNA kit was used, and mRNA was quantitatively analyzed by microplate reader. The encapsulation efficiency was determined, and the encapsulation efficiency of Fluc-mRNA was 96%.

[0160] Example 12: Preparation of mRNA-loaded lipid nanoparticles using LTE as an ionizable lipid

[0161] Prepare 18 mL acetate buffer (pH = 3.6) containing 300 μg Fluc-mRNA, 6 mL 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 above-prepared 6 mL ethanol solution with 18 mL Fluc-mRNA acetate buffer, with a total flow rate of 28 mL / min and a flow rate ratio of acetate buffer to ethanol solution of 21:7 to prepare and collect the nanoparticle solution. Dilute the prepared nanoparticle solution with 100 mL HEPES (pH = 7.4). Transfer the nanoparticle solution to an ultrafiltration tube (molecular weight cutoff of 100k), centrifuge and ultrafilter at low temperature at a speed of 4000 rpm for about 1 min to prepare a concentrated nanoparticle solution. The particle size was measured by dynamic laser light scattering, and the particle size of the nanoparticles was 72.53±1.22nm, and the polydispersity index (PDI) was 0.306±0.014. The particle size distribution curve is shown in Fig.17 As shown. Using Quant-iT TM RNA kit was used, and mRNA was quantitatively analyzed by microplate reader. The encapsulation efficiency was determined, and the encapsulation efficiency of Fluc-mRNA was 96%.

[0162] Example 13: Preparation of mRNA-loaded lipid nanoparticles using HTE as an ionizable lipid

[0163] Prepare 30 mL acetate buffer (pH = 3.6) containing 1000 μg Fluc-mRNA, 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 above-prepared 10 mL ethanol solution with 30 mL of Fluc-mRNA acetate buffer at a total flow rate of 28 mL / min and a flow rate ratio of acetate buffer to ethanol solution of 21:7 to prepare and collect the nanoparticle solution. Dilute the prepared nanoparticle solution with 280 mL HEPES (pH = 7.4). Concentrate the nanoparticle solution by tangential flow (molecular weight cutoff of 100k) ultrafiltration to prepare a concentrated nanoparticle solution. The particle size was measured by dynamic laser light scattering, and the particle size of the nanoparticles was 137.9±0.5nm, and the polydispersity index (PDI) was 0.183±0.016. The particle size distribution curve is shown in Fig.18 As shown. Using Quant-iT TM RNA kit, microplate reader quantitatively analyzed mRNA, and determined the encapsulation efficiency. The encapsulation efficiency of Fluc-mRNA was found to be 95%.

[0164] Example 14 In vivo transfection of mRNA-loaded lipid nanoparticles prepared using HTE or HTP as ionizable lipids

[0165] Prepare 300 mL acetate buffer (pH 3.6) containing 10 mg Fluc-mRNA, 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 above-prepared 100 mL ethanol solution with 300 mL of Fluc-mRNA acetate buffer, with a total flow rate of 80 mL / min and a flow rate ratio of acetate buffer to ethanol solution of 60:20 to prepare and collect 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 100k) ultrafiltration to prepare a concentrated solution of nanoparticles loaded with Fluc-mRNA. Dynamic laser light scattering was used to measure the particle size. Quant-iT TM RNA kit, ELISA instrument quantitatively analyzed mRNA, and the encapsulation efficiency was determined. The test results are shown in Table 1. The nanoparticles prepared with HTE and HTP were labeled as Fluc-mRNA LNP-HTE and Fluc-mRNA LNP-HTP, respectively.

[0166]

[0167] Use an insulin needle to draw 100ul of the above-mentioned Fluc-mRNA LNP-HTE and Fluc-mRNA LNP-HTP nanoparticle solutions, respectively, and inject them intramuscularly at a depth of 3mm on the inner side of the posterior thigh muscle of BALB / C mice. 8 and 24 hours after the injection of the nanoparticle solution, the expression of mRNA was detected using a small animal in vivo imager (IVIS spectrum, PerkinElmer). 10 minutes before the test, 200ul of 15mg / mL fluorescein potassium salt PBS solution was injected into the abdominal cavity (right lower abdomen), and the mouse was anesthetized using a mouse gas anesthesia instrument with isoflurane as an anesthetic (anesthesia time is about 5 minutes). After the mouse is anesthetized, turn the mouse abdomen up, spread its limbs, lie flat on the imaging panel, select the bioluminescence mode, image and take pictures of the mouse, and the imaging picture of the injected Fluc-mRNA LNP-HTE nanoparticle solution is shown as follows Fig.19 As shown, the imaging of the nanoparticle solution injected with Fluc-mRNA LNP-HTP, as Fig. 20 The IVIS spectrum software was used to quantitatively analyze the average fluorescence intensity (unit: p / sec / cm2 / sr) of the left leg of the mouse, as shown in Table 2.

[0168] The in vivo transfection results showed that the fluorescence intensity of the Fluc mRNA-loaded nanoparticles prepared with HTE or HTP as ionizable lipids was stronger at the injection site at 8h and 24h after injection compared with the saline injection group, indicating that more fluorescent protein was expressed at the injection site.

[0169] Table 2

[0170]

[0171] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. An ionizable lipid molecule or a pharmaceutically acceptable salt thereof, characterized in that: The ionizable lipid molecule comprises an amino acid structure part and a tocopherol lipid structure part, wherein the amino acid structure part and the tocopherol lipid structure part are indirectly connected via a hydrolyzable ester bond, wherein the ionizable group is a basic group of the amino acid structure part.

2. The ionizable lipid molecule or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The amino acid structure part and the tocopherol lipid structure part are indirectly connected by alkylene ether; the amino acid structure part is the residue of lysine, histidine, arginine, or its derivatives.

3. The ionizable lipid molecule or a pharmaceutically acceptable salt thereof according to claim 1, wherein the general formula is as 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 an ionizable lipid molecule as claimed in claim 1, characterized in that: The following steps are involved: (1) adding a halogenated alkanol to a solution containing α-tocopherol and a base to obtain a tocopherol oxyalkanol through a substitution reaction; (2) esterifying tocopherol oxyalkyl alcohol and base-protected amino acid to obtain base-protected amino acid tocopherol oxyalkyl ester; (3) The amino acid tocopherol ester protected by the base is deprotected by a deprotecting agent to obtain an amino acid tocopherol ester.

5. The method for preparing ionizable lipid molecules according to claim 4, characterized in that: The halogenated alkanol described in step (1) is a monohalogenated primary alkanol or a dihalogenated secondary alkanol; and 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 alkyl alcohol, α-tocopherol and base is (0.1-5):1:(0.1-5); the molar ratio of dihalogenated secondary alkyl alcohol, 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-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl or 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, and 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 agent or a preventive agent, and a carrier for delivering the therapeutic agent or the preventive agent, wherein the therapeutic agent or the preventive 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 or pharmaceutically acceptable salts thereof as described in any one of claims 1 to 3.

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

5.

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

11. A composition according to claim 10, characterized in that The auxiliary lipid is distearoylphosphatidylcholine, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylethanolamine; the PEGylated lipid is polyethylene glycol 2000 vitamin E succinate, polyethylene glycol 1000 vitamin E succinate or dimyristoylglycerol-polyethylene glycol 2000.

12. Use of the ionizable lipid molecule according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, or the composition according to any one of claims 8 to 11 in the preparation of nucleic acid drugs, small molecule drugs, polypeptides or protein drugs.

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