Ionizable lipid carrier as well as composition and application thereof

Abstract

The invention belongs to the technical field of drug carriers, and relates to an ionizable lipid carrier as well as a composition and application thereof. The lipid carrier is one or more amino acid tocopheryl esters or pharmaceutically acceptable salts thereof and is used for delivering a therapeutic agent or a prophylactic agent, and the therapeutic agent or the prophylactic agent is one or more of siRNA, shRNA, miRNA and mRNA nucleic acid molecules, polypeptides or proteins. In a preferred embodiment, the amino acid moiety is a residue of lysine, histidine, arginine, or a derivative thereof. The invention also discloses a composition prepared from one or more ionizable lipid carriers and a therapeutic agent or a prophylactic agent, and an application of the lipid carrier 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 carriers, and specifically to an ionizable lipid carrier of an amino acid tocopherol ester or a pharmaceutically acceptable salt thereof, as well as a preparation method of the amino acid tocopherol ester, 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 an application of an amino acid tocopherol ester or a pharmaceutically acceptable salt thereof as an ionizable lipid carrier, wherein one or more amino acid tocopherol esters or a pharmaceutically acceptable salt thereof are used as an ionizable lipid carrier for delivering a therapeutic agent or a preventive agent, and the ionizable lipid carrier is prepared by directly 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 carrier 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 carriers or pharmaceutically acceptable salts thereof, and a therapeutic agent or a preventive agent, and the use of the ionizable lipid carriers, 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 provides an application of an amino acid tocopherol ester or a pharmaceutically acceptable salt thereof as an ionizable lipid carrier, wherein one or more of the amino acid tocopherol esters or pharmaceutically acceptable salts thereof are used as an ionizable lipid carrier for delivering a therapeutic agent or a 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 ionizable lipid carrier 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 directly connected via a hydrolyzable ester bond, and wherein the ionizable group is a basic group of the amino acid structure part.

[0012] In one embodiment of the present invention, the amino acid moiety is a residue of lysine, histidine, arginine, or a derivative thereof.

[0013] In one embodiment of the present invention, the amino acid tocopherol ester or a pharmaceutically acceptable salt thereof of the present invention has the general formula as shown in (I):

[0014]

[0015] Among them, B is T is

[0016] The present invention also provides a method for preparing the above-mentioned amino acid tocopherol ester, comprising the following steps:

[0017] (1) subjecting tocopherol and a base-protected amino acid to an esterification reaction to obtain a base-protected amino acid tocopherol ester;

[0018] A preferred solution is to react a CH2Cl2 solution containing EDCI, DMAP, tocopherol and base-protected amino acids at room temperature for 24-48 hours to obtain base-protected amino acid tocopherol esters;

[0019] (3) removing the protecting group on the base of the amino acid tocopherol ester protected by the base under the action of a deprotecting agent to obtain the amino acid tocopherol ester;

[0020] A preferred solution is to add an organic acid dropwise to a CH2Cl2 solution containing base-protected amino acid tocopherol ester at room temperature, stir the reaction for 1-5 hours, and then neutralize the organic acid with a saturated sodium bicarbonate solution after base deprotection to obtain amino acid tocopherol ester.

[0021] The present invention also provides a composition, comprising 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 amino acid tocopherol esters or pharmaceutically acceptable salts thereof.

[0022] 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.

[0023] 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.

[0024] In one embodiment of the present invention, the carrier further comprises auxiliary lipids, cholesterol and PEGylated lipids.

[0025] In one embodiment of the present invention, preferably, the molar ratio of the ionizable lipid carrier, the auxiliary lipid, the cholesterol and the PEGylated lipid is 50:(5-20):(10-50):(1-5).

[0026] In one embodiment of the present invention, 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).

[0027] The present invention also provides a use of the above-mentioned amino acid tocopherol ester or a pharmaceutically acceptable salt thereof, or the above-mentioned composition in the preparation of nucleic acid drugs, small molecule drugs, polypeptides or protein drugs.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention discloses an ionizable lipid carrier, which uses tocopherol and amino acids, which are essential to the human body and highly safe, as raw materials, and obtains amino acid tocopherol esters, such as lysine tocopherol ester, arginine tocopherol ester, and histidine tocopherol ester, through ester bond construction. The amino acid tocopherol ester can be used as an ionizable lipid carrier, as a lipid nanoparticle carrier for delivering nucleic acid molecules such as siRNA, shRNA, miRNA and mRNA, polypeptides or proteins, and 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 selects lysine, histidine, arginine, or residues of their derivatives, the ionizable lipid of the present invention has multiple ionizable sites, and one or more suitable amino acid tocopherol esters can be selected according to the structural characteristics, content composition, and delivery requirements of the therapeutic agent or preventive agent, with good adjustability, wide application range, and good biosafety.

[0030] Specifically: First, the ionizable lipid carrier of the present invention is an amino acid tocopherol ester, which can be hydrolyzed to generate corresponding amino acids and tocopherol lipid structural parts and has good biological safety;

[0031] Second, when the amino acid tocopherol ester provided by the present invention is a lysine ester, histidine ester or arginine ester containing a tocopherol lipid structure, in the amino acid structure, in addition to the amino group, weakly basic groups such as imidazole 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; and the electrostatic interaction degree of lipid molecules containing groups of different ionization degrees and negatively charged RNA is different, and the ability to load and release RNA is also different. This allows technicians to select a suitable amino acid tocopherol ester according to RNA structure and composition, and its delivery requirements, or select a composition of multiple amino acid tocopherol esters and adjust the appropriate ratio between each amino acid tocopherol ester, so as to achieve stable and efficient delivery of different types of RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the MS spectrum of lysine tocopherol ester of Example 1 of the present invention;

[0033] Figure 2 The lysine tocopherol ester hydrochloride of Example 1 of the present invention 1 H NMR spectrum;

[0034] Figure 3 is the MS spectrum of histidine tocopherol ester of Example 2 of the present invention;

[0035] Figure 4 The histidine tocopherol ester hydrochloride of Example 2 of the present invention 1 H NMR spectrum;

[0036] Figure 5 is the MS spectrum of arginine tocopherol ester of Example 3 of the present invention;

[0037] Figure 6 The arginine tocopherol ester hydrochloride of Example 3 of the present invention 1 H NMR spectrum;

[0038] Figure 7 is a particle size distribution curve diagram of the mRNA lipid nanoparticles of Example 4 of the present invention;

[0039] Figure 8 is a particle size distribution curve of the mRNA lipid nanoparticles of Example 5 of the present invention;

[0040] Fig. 9 is a particle size distribution curve diagram of the mRNA lipid nanoparticles of Example 6 of the present invention;

[0041] Fig.10 is a particle size distribution curve of the mRNA lipid nanoparticles of Example 7 of the present invention;

[0042] Fig.11 These are the in vivo imaging images of mouse transfection according to Example 9 of the present invention, wherein a / c represents the saline group, a represents the imaging image 4 hours after injection, c represents the imaging image 8 hours after injection, and b / d represents the lipid nanoparticle group, b represents the imaging image 4 hours after injection, and d represents the imaging image 8 hours after injection. DETAILED DESCRIPTION

[0043] 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.

[0044] 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.

[0045] The raw materials used in the present invention can be obtained commercially, 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 esters of the present invention, such as lysine tocopherol ester, arginine tocopherol ester, and histidine tocopherol ester by selecting tocopherol and amino acid raw materials of other configurations, and can prepare lipid molecule nanoparticles, which are used as carriers for delivering therapeutic agents or preventive agents, and lipid nanoparticle carriers for delivering siRNA, shRNA, miRNA and mRNA nucleic acid molecules, polypeptides or proteins.

[0046] The present invention discloses a class of ionizable lipid carriers, and a preparation method, composition, and application thereof. The composition comprises one or more ionizable lipid carriers or pharmaceutically acceptable salts thereof, and a therapeutic agent or a preventive agent. And the lipid carrier, or a pharmaceutically acceptable salt thereof, or the application of the composition in the preparation of nucleic acid drugs, small molecule drugs, polypeptides, or protein drugs.

[0047] In a preferred embodiment, the present invention relates to an ionizable lipid carrier of an amino acid tocopherol ester, specifically, lysine tocopherol ester, arginine tocopherol ester, histidine tocopherol ester.

[0048] The present invention can select one or more ionizable lipid carriers, 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.

[0049] 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.

[0050] 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.

[0051] Among the essential amino acids for the human body, there are three basic amino acids, 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.

[0052] The present invention can select one or more ionizable lipid carriers, 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 can be selected according to the structure and composition of the RNA loaded and its delivery requirements, or a composition of multiple amino acid tocopherol esters can be designed through specific proportions.

[0053] Furthermore, the amino acid tocopherol 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.

[0054] The present invention also provides a method for preparing the above-mentioned amino acid tocopherol ester, comprising the following steps:

[0055] (i) Tocopherol and a base-protected amino acid (such as lysine, histidine or arginine) are reacted by esterification to prepare a base-protected amino acid tocopherol ester. The general chemical reaction formula is as follows:

[0056]

[0057] RCOOH: base-protected amino acids (such as lysine, histidine or arginine);

[0058] (ii) Deprotection reaction to prepare amino acid tocopherol esters, the chemical reaction formula is as follows:

[0059]

[0060] B is the residue of an amino acid or an amino acid derivative, preferably at least one of the following three:

[0061]

[0062] The present invention also provides a method for preparing a lipid nanoparticle composition using the ionizable lipid carrier.

[0063] In one embodiment, the ionizable lipid carrier is composed of 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 carrier is one, two or more; the auxiliary lipid molecule can be distearoylphosphatidylcholine, distearoylphosphatidylethanolamine or dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylethanolamine, etc.; the PEGylated lipid molecule can be polyethylene glycol 2000 vitamin E succinate, 1000 vitamin E succinate or dimyristoylglycerol-polyethylene glycol 2000, etc.

[0064] 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.

[0065] 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.

[0066] Example 1 Preparation of lysine tocopherol ester (LE)

[0067] N,N'-di-tert-butyloxycarbonyl-L-lysine (1.38g) and D-α-tocopherol (2.06g), EDCI (1.16g), DMAP (0.10g) were added to 10ml of dichloromethane. After reacting for 24 hours at room temperature under anhydrous, oxygen-free and light-proof conditions, the reaction solution was concentrated by rotary evaporation and N,N'-di-tert-butyloxycarbonyl-L-lysine D-α-tocopherol ester was separated and purified by silica gel column chromatography. The mixture was eluted with 100ml, 120ml and 480ml of petroleum ether / ethyl acetate (20:2, 20:3 and 20:4, volume ratio) mixed solvents in turn. The separated N,N'-di-tert-butyloxycarbonyl-L-lysine D-α-tocopherol ester solution was detected by thin layer chromatography and the solvent was removed by rotary evaporation to obtain N,N'-di-tert-butyloxycarbonyl-L-lysine D-α-tocopherol ester (BLE) with a yield of 61.2%.

[0068] A certain amount of N,N'-di-tert-butyloxycarbonyl-L-lysine D-α-tocopherol ester (BLE) was dissolved in dichloromethane, trifluoroacetic acid (BLE: trifluoroacetic acid is 1:30, molar ratio) was added, and the deprotection reaction was carried out under magnetic stirring at room temperature under light-proof conditions for about 1 hour. The deprotection reaction solution was neutralized with a saturated sodium bicarbonate aqueous solution, and then repeatedly extracted with dichloromethane, separated and dried with anhydrous sodium sulfate, and dichloromethane was removed by rotary evaporation. Lysine tocopherol ester (LE) was obtained in a yield of 81.5% by vacuum drying at room temperature to constant weight.

[0069] An appropriate amount of lysine tocopherol ester (LE) is added to a 0.05M hydrochloric acid aqueous solution to dissolve it, and the obtained aqueous solution is freeze-dried to obtain lysine tocopherol ester hydrochloride, the structural formula of which is shown below.

[0070]

[0071] The mass spectrum of tocopheryl lysine (LE) is as follows Figure 1 As shown, the molecular formula of lysine tocopherol ester is C35 H63 N2O3, the ion mode is [M+H]+, and the measured m / z value is 559.4833, which is consistent with the calculated value of 559.4839 based on the LE molecular formula.

[0072] Lysine Tocopheryl Ester Hydrochloride 1 H NMR spectrum (400 MHz, DMSO-d6) Figure 2 As shown, the methyl hydrogen at positions 20, 25, 29, and 30 in lysine tocopherol ester was used for calibration (peak area calibration was 12.0), and the proton peaks in the 1H NMR spectrum of lysine tocopherol ester hydrochloride were analyzed and assigned, as shown in Table 1. The results of the nuclear magnetic resonance hydrogen spectrum analysis of the prepared LE hydrochloride showed that the prepared LE hydrochloride had the expected structure.

[0073] Table 1

[0074]

[0075] Example 2 Preparation of Histidine Tocopherol Ester (HE)

[0076] N,N'-di-tert-butyloxycarbonyl-L-histidine (1.42g) and D-α-tocopherol (2.07g), EDCI (1.15g), DMAP (0.10g) were added to 10ml of dichloromethane, and reacted under magnetic stirring at room temperature for 24 hours under anhydrous, oxygen-free and light-proof conditions. The reaction solution was concentrated by rotary evaporation, and N,N'-di-tert-butyloxycarbonyl-L-histidine D-α-tocopherol ester was separated and purified by silica gel column chromatography, and eluted with 100ml, 250ml and 500ml of petroleum ether / ethyl acetate (20:2, 20:3 and 20:4, volume ratio) mixed solvents in sequence. The N,N'-di-tert-butyloxycarbonyl-L-histidine D-α-tocopherol ester solution obtained by thin layer chromatography was detected and the solvent was removed by rotary evaporation to obtain N,N'-di-tert-butyloxycarbonyl-L-histidine D-α-tocopherol ester with a yield of 51.2%.

[0077] A certain amount of N,N'-di-tert-butyloxycarbonyl-L-histidine D-α-tocopherol ester was dissolved in dichloromethane, and trifluoroacetic acid (N,N'-di-tert-butyloxycarbonyl-L-histidine D-α-tocopherol ester: trifluoroacetic acid was added in a molar ratio of 1:30). Under anhydrous, oxygen-free, and light-proof conditions, the deprotection reaction was carried out under magnetic stirring at room temperature for about 1 hour. The deprotection reaction solution was neutralized with a saturated sodium bicarbonate aqueous solution, and then repeatedly extracted with dichloromethane. After separation, it was dried with anhydrous sodium sulfate, and dichloromethane was removed by rotary evaporation. Histidine tocopherol ester (HE) was obtained by vacuum drying at room temperature to constant weight, and the yield was 93.1%.

[0078] An appropriate amount of histidine tocopherol (HE) is added to a 0.05M hydrochloric acid aqueous solution to dissolve it, and the obtained aqueous solution is freeze-dried to obtain histidine tocopherol hydrochloride, the structural formula of which is shown below.

[0079]

[0080] The mass spectrum of histidine tocopherol (HE) is as follows Figure 3 As shown, the molecular formula of histidine tocopherol ester is C35 H58 N3O3, the ion mode is [M+H]+, and the measured m / z value is 568.4484, which is consistent with the calculated value of 568.4478 based on the molecular formula of histidine tocopherol ester.

[0081] Histidine tocopherol hydrochloride 1 H NMR spectrum (400 MHz, DMSO-d6) Figure 4As shown, the 25, 26, 30, and 31 methyl hydrogens in the histidine tocopherol ester hydrochloride molecule were calibrated (peak area calibration was 12.0), and the proton peaks in the 1HNMR spectrum of HE hydrochloride were analyzed and attributed, as shown in Table 2. The results of the nuclear magnetic resonance hydrogen spectrum analysis of the prepared histidine tocopherol ester hydrochloride showed that the prepared HE hydrochloride had the expected structure.

[0082] Table 2

[0083]

[0084] Example 3 Preparation of Arginine Tocopherol Ester (AE)

[0085] Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine (2.11 g) and D-α-tocopherol (3.44 g), EDCI (1.16 g) and DMAP (0.18 g) were added to 14 ml of dichloromethane and 6 ml of dimethylformamide. The mixture was reacted under magnetic stirring at room temperature for 24 hours under anhydrous, oxygen-free and light-proof conditions. The reaction solution was concentrated by rotary evaporation and Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine D-α-tocopherol ester was separated and purified by silica gel column chromatography and eluted with 500 ml, 800 ml and 100 ml of a mixed solvent of petroleum ether / ethyl acetate (5:3, 5:5 and 5:7, volume ratio) in sequence. The separated Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine D-α-tocopherol ester solution was detected by thin layer chromatography and rotary evaporated to remove the solvent to obtain Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine D-α-tocopherol ester with a yield of 63.2%.

[0086] A certain amount of Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine D-α-tocopherol ester was dissolved in dichloromethane, and trifluoroacetic acid (Nα-Boc-Nω-(2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl)-L-arginine D-α-tocopherol ester: trifluoroacetic acid was 1:40, molar ratio) was added. Under anhydrous, oxygen-free and light-proof conditions, the deprotection reaction was carried out under magnetic stirring at room temperature for about 5 hours. The deprotection reaction solution was neutralized with a saturated sodium bicarbonate aqueous solution, repeatedly extracted with dichloromethane, dried with anhydrous sodium sulfate after separation, and dichloromethane was removed by rotary evaporation. Arginine tocopherol ester (AE) was obtained in a yield of 60.2% by vacuum drying at room temperature to constant weight.

[0087] An appropriate amount of arginine tocopherol (AE) is added to a 0.05M hydrochloric acid aqueous solution to dissolve it, and the obtained aqueous solution is freeze-dried to obtain arginine tocopherol hydrochloride, the structural formula of which is shown below.

[0088]

[0089] The mass spectrum of arginine tocopherol ester (AE) is as follows Figure 5 As shown, the molecular formula of arginine tocopherol ester is C35 H63 N4O3, the ion mode is [M+H]+, and the measured value of m / z is 587.4908, which is consistent with the calculated value of 587.4900 based on the molecular formula of arginine tocopherol ester.

[0090] Arginine tocopheryl ester hydrochloride 1 H NMR spectrum (400 MHz, DMSO-d6) Figure 6 As shown, the methyl hydrogen at positions 20, 25, 29, and 30 in arginine tocopherol ester was used for calibration (peak area calibration was 12.0), and the proton peaks in the 1H NMR spectrum of arginine tocopherol ester hydrochloride were analyzed and attributed, as shown in Table 3. The results of the nuclear magnetic resonance hydrogen spectrum analysis of the prepared arginine tocopherol ester hydrochloride showed that the prepared arginine tocopherol ester hydrochloride had the expected structure.

[0091] Table 3

[0092]

[0093] Example 4 Preparation of mRNA-loaded lipid nanoparticles using arginine tocopherol as ionizable lipid

[0094] Prepare 18 mL acetate buffer (pH = 3.6) containing 300 μg Fluc-mRNA, 6 mL ethanol solution containing 4.5 mg arginine tocopherol and corresponding amounts of DSPC, cholesterol and DMG2000 (molar ratio of arginine tocopherol: DSPC: cholesterol: DMG2000 = 50: 6.7: 25.6: 1.0). Use a microfluidic mixer (ring chip) to mix the above-prepared 6 mL ethanol solution with 18 mL Fluc-mRNA acetate buffer at a total flow rate of 28 mL / min, and the flow rate ratio of acetate buffer to ethanol solution is 21:7 to prepare and collect 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 7 mL Fluc-mRNA / mL nanoparticle concentrated solution. The particle size was measured by dynamic laser light scattering, and the particle size of the nanoparticles was 97.54±1.13nm, and the polydispersity index (PDI) was 0.170±0.018. The particle size distribution curve is shown in Figure 7 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 97%.

[0095] Example 5 Preparation of mRNA-loaded lipid nanoparticles using lysine tocopherol, histidine tocopherol and arginine tocopherol (4:1:1, molar ratio) as ionizable lipids

[0096] 3 mL of citrate buffer (50 mM, pH = 4.0) containing 70 μg of Fluc-mRNA, 1 mL of ethanol solution containing 700 μg of lysine tocopherol, histidine tocopherol and arginine tocopherol (molar ratio of 4:1:1) and corresponding amounts of DSPC, cholesterol and TPGS1000 (molar ratio of (lysine tocopherol + histidine tocopherol + arginine tocopherol): DSPC: cholesterol: TPGS1000 = 50:10:38.5:1.5) were prepared. The above-prepared 1 mL of ethanol solution was mixed with 3 mL of citrate buffer using a microfluidic mixer (fishbone chip) at a total flow rate of 12 mL / min and a flow rate ratio of citrate buffer to ethanol solution of 3:1 to prepare and collect the nanoparticle solution. The prepared nanoparticle solution was dialyzed in a phosphate buffer solution (PBS, pH = 7.4) using a dialysis bag (molecular weight cutoff of 2500) for 8 times, with the solution replaced every 0.5 hours. The dialyzed nanosolution was transferred to an ultrafiltration tube (molecular weight cutoff of 3k) and ultrafiltered at low temperature with a speed of 3500 rpm for about 3 hours. A nanoparticle solution containing 44.28 μg Fluc-mRNA / mL was prepared. The particle size of the nanoparticles was measured using a dynamic laser light scattering instrument, and the particle size was measured to be 101.2±0.79 nm, and the polydispersity index (PDI) was 0.115±0.004. The particle size distribution curve is shown in Figure 8 The zeta potential was 8.51±1.11 mV. 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 98%.

[0097] Example 6 Preparation of mRNA-loaded lipid nanoparticles using lysine tocopherol, histidine tocopherol and arginine tocopherol (molar ratio of 1:1:4) as ionizable lipids

[0098] 3 mL of citrate buffer (50 mM, pH 4.0) containing 70 μg of Fluc-mRNA, 1 mL of ethanol solution containing 700 μg of lysine tocopherol, histidine tocopherol and arginine tocopherol (molar ratio of 4:1:1) and corresponding amounts of DSPC, cholesterol and TPGS1000 (molar ratio of (lysine tocopherol + histidine tocopherol + arginine tocopherol): DSPC: cholesterol: TPGS1000 = 50:10:38.5:1.5) were prepared. The 1 mL of ethanol solution prepared above was mixed with 3 mL of citrate buffer using a microfluidic mixer (fishbone chip) at a total flow rate of 12 mL / min and a flow rate ratio of citrate buffer to ethanol solution of 3:1 to prepare and collect the nanoparticle solution. The prepared nanoparticle solution was dialyzed in a phosphate buffer solution (PBS, pH 7.4) using a dialysis bag (molecular weight cutoff of 2500) for 8 times, with the solution replaced every 0.5 hours. The dialyzed nanosolution was transferred to an ultrafiltration tube (molecular weight cutoff of 3k) and centrifuged for ultrafiltration at 4°C and 3500rpm for about 3h. A nanoparticle solution containing 45.16μg Fluc-mRNA / mL was prepared. The particle size of the nanoparticles was measured using a dynamic laser light scattering instrument, and the particle size was measured to be 107.2±0.46nm, and the polydispersity index (PDI) was 0.233±0.103. The particle size distribution curve is shown in Fig. 9 The zeta potential was 7.41±0.48 mV. 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 97%.

[0099] Example 7 Preparation of mRNA-loaded lipid nanoparticles using lysine tocopherol ester and histidine tocopherol ester (molar ratio of 1:1) as ionizable lipids

[0100] 3 mL of acetate buffer (pH 4.0) containing 100 μg of Fluc-mRNA, 1 mL of ethanol solution containing 2000 μg of lysine tocopherol ester and histidine tocopherol ester (molar ratio of 1:1) and corresponding amounts of DSPC, cholesterol and DMG2000 (molar ratio of (lysine tocopherol ester + histidine tocopherol ester): DSPC: cholesterol: DMG2000 = 50:10:38.5:1.5) were prepared. The above-prepared 1 mL of ethanol solution was mixed with 3 mL of acetate buffer using a microfluidic mixer (fishbone chip) at a total flow rate of 12 mL / min and a flow rate ratio of acetate buffer to ethanol solution of 3:1 to prepare and collect the nanoparticle solution. The prepared nanoparticle solution was dialyzed in a phosphate buffer solution (PBS, pH 7.4) using a dialysis bag (molecular weight cutoff of 2500) for 4 times, with the solution replaced every 0.5 hours. The dialyzed nanoparticle solution was transferred to an ultrafiltration tube (molecular weight cutoff of 3k), centrifuged and ultrafiltered at 4°C and 4000 rpm, and concentrated to prepare a Fluc-mRNA nanoparticle solution. The particle size of the nanoparticles was measured using a dynamic laser light scattering instrument, and the particle size was measured to be 113.2±0.655, and the polydispersity index (PDI) was 0.104±0.014. The particle size distribution curve is shown in Fig.10 The zeta potential was -0.954±0.339mV. TM RNA kit was used, and the mRNA was quantitatively analyzed by an ELISA instrument to determine the encapsulation efficiency. The encapsulation efficiency of Fluc-mRNA was found to be 95.4%.

[0101] Example 8 Preparation of mRNA-loaded lipid nanoparticles using histidine tocopherol ester and in vivo transfection

[0102] 3 mL of citrate buffer (50 mM, pH 4.0) containing 70 μg of Fluc-mRNA, 1 mL of ethanol solution containing 700 μg of lysine tocopherol and corresponding amounts of DSPC, cholesterol and TPGS1000 (molar ratio of (lysine tocopherol + histidine tocopherol + arginine tocopherol): DSPC: cholesterol: TPGS1000 = 50:10:38.5:1.5). The above-prepared 1 mL of ethanol solution was mixed with 3 mL of citrate buffer using a microfluidic mixer (fishbone chip) at a total flow rate of 12 mL / min and a flow rate ratio of citrate buffer to ethanol solution of 3:1 to prepare and collect the nanoparticle solution. The prepared nanoparticle solution was dialyzed in phosphate buffer solution (PBS, pH 7.4) using a dialysis bag (molecular weight cutoff of 2500) for 8 times, with the solution changed every 0.5 hours. The dialyzed nano solution was transferred to an ultrafiltration tube (molecular weight cutoff 3k) and centrifuged at 4°C, 3500 rpm, for about 3 hours to prepare a Fluc-mRNA nanoparticle solution. The nanoparticle size was measured by dynamic laser light scattering, and the particle size was 96.48±0.64nm, the polydispersity index (PDI) was 0.103±0.091, and the zeta potential was 5.03±0.51mV. 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 96.2%.

[0103] 100ul of nanoparticle solution containing 6μg Fluc-mRNA was drawn with an insulin needle and injected intramuscularly at a depth of 3mm on the inner side of the thigh muscle of two BALB / C mice. The expression of mRNA was detected using a small animal in vivo imager (IVISspectrum, PerkinElmer) at 4h, 8h, and 24h after injection. 10min 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 was about 5min). After the mouse was anesthetized, the mouse was placed with its abdomen facing up, its limbs spread out, and it lay flat on the imager panel, and the bioluminescence mode was selected. The average fluorescence intensity (unit: p / sec / cm2 / sr) of the mouse thigh was quantitatively analyzed using IVIS spectrum software, as shown in Table 4.

[0104] Table 4

[0105]

[0106] The in vivo transfection results showed that the fluorescence intensity of the prepared Fluc-mRNA nanoparticles at the injection site was stronger at 4h, 8h and 24h after injection than at 0h (before injection of nanoparticles, i.e., no injection of nanoparticle solution), indicating that there was more fluorescent protein expression at the injection site after the injection of nanoparticles.

[0107] Example 9 Preparation of mRNA-loaded lipid nanoparticles using lysine tocopherol, histidine tocopherol and arginine tocopherol (molar ratio of 1:1:1) and in vivo transfection thereof

[0108] 3 mL of citrate buffer (50 mM, pH 4.0) containing 70 μg of Fluc-mRNA, 1 mL of ethanol solution containing 700 μg of lysine tocopherol, histidine tocopherol and arginine tocopherol (molar ratio of 1:1:1) and corresponding amounts of DSPC, cholesterol and TPGS1000 (molar ratio of (lysine tocopherol + histidine tocopherol + arginine tocopherol): DSPC: cholesterol: TPGS1000 = 50:10:38.5:1.5) were prepared. The 1 mL of ethanol solution prepared above was mixed with 3 mL of citrate buffer using a microfluidic mixer (fishbone chip) at a total flow rate of 12 mL / min and a flow rate ratio of citrate buffer to ethanol solution of 3:1 to prepare and collect the nanoparticle solution. The prepared nanoparticle solution was dialyzed in a phosphate buffer solution (PBS, pH 7.4) using a dialysis bag (molecular weight cutoff of 2500) for 8 times, with the solution changed every 0.5 hours. The dialyzed nanoparticle solution was transferred to an ultrafiltration tube (molecular weight cutoff of 3k) and centrifuged for ultrafiltration at 4°C and 3500rpm for about 3 hours to prepare a nanoparticle solution containing 32.48μg Fluc-mRNA / mL. The particle size of the nanoparticles was measured using a dynamic laser light scattering instrument, and the particle size was measured to be 60.93±0.54nm, the polydispersity index (PDI) was 0.199±0.091, and the zeta potential was 0.516±0.507mV. 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 98.5%.

[0109] 100ul of nanoparticle solution containing 6μg Fluc-mRNA was drawn up with an insulin needle and injected intramuscularly at a depth of 3mm on the inner side of the thigh muscle of two BALB / C mice. The expression of mRNA was detected using a small animal in vivo imager (IVISspectrum, PerkinElmer) at 4h and 8h after injection. 10min 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 the anesthetic (anesthesia time was about 5min). After the mouse was anesthetized, the mouse was placed with its abdomen facing up, its limbs spread out, and it lay flat on the imager panel. The bioluminescence mode was selected to image and photograph the mouse, such as Fig.11 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 5.

[0110] The in vivo transfection results showed that the fluorescence intensity of the prepared Fluc-mRNA nanoparticles was stronger at the injection site at 4h and 8h after injection than that of the saline injection group, but not obvious in the liver, indicating that there was more fluorescent protein expression at the injection site and little expression in the liver.

[0111] Table 5

[0112]

[0113] Example 10 Preparation of PCSK9-loaded siRNA lipid nanoparticles using lysine tocopherol, histidine tocopherol and arginine tocopherol (molar ratio of 1:1:1) and their silencing effect on PCSK9

[0114] 3 mL of acetate buffer (pH 4.0) containing 100 μg of siRNA targeting PCSK9, 1000 μg of lysine tocopherol, histidine tocopherol and arginine tocopherol (molar ratio of 1:1:1) and corresponding amounts of DSPC, cholesterol and DMG2000 (molar ratio of (lysine tocopherol + histidine tocopherol + arginine tocopherol): DSPC: cholesterol: DMG2000 = 50:10:38.5:1.5 (molar ratio)) were prepared. MC3 was used as a comparison, with a molar ratio of MC3: DSPC: cholesterol: DMG2000 = 50:10:38.5:1.5, MC3: siRNA = 10:1 (w / w).

[0115] Use a microfluidic mixer (fishbone chip) to mix 1 mL of the above-configured ethanol solution with 3 mL of acetate buffer at a total flow rate of 12 mL / min, and the flow rate ratio of acetate buffer to ethanol solution is 3:1 to prepare and collect nanoparticle solution. The prepared nanoparticle solution was dialyzed in a phosphate buffer solution (PBS, pH 7.4) using a dialysis bag (molecular weight cutoff of 2500) for 4 times, with the solution changed every 0.5 hours to obtain a nanoparticle dilute solution. The nanoparticle dilute solution was transferred to an ultrafiltration tube (molecular weight cutoff of 3k), centrifuged and ultrafiltered at 4°C and 4000 rpm to obtain a nanoparticle concentrate. The nanoparticles prepared with MC3 are designated as siRNA LNP-0, and the nanoparticles prepared with a combination of lysine tocopherol ester, histidine tocopherol ester and arginine tocopherol ester are designated as siRNA LNP-1. The particle size of the nanoparticles was measured using a dynamic laser scattering instrument. Quant-iT TM RNA kit, ELISA instrument quantitatively analyzes siRNA. The test results are shown in Table 6:

[0116] Table 6

[0117]

[0118] The prepared siRNA-loaded nanoparticle solution was co-incubated with HepG2 cells, and the decreased level of PCSK9 expression in the cells was detected by Western blot: HepG2 cells were cultured at 2.5×10 7 The density of cells / well was planted in a 6-well plate and cultured at 37°C and 5% CO2. When the density of adhesion reached about 70%, the culture medium was replaced with a low serum culture medium (1% serum) and cultured for 24 hours. The final concentration of siRNA was 60nM in each well. After 6 hours of transfection, the medium was changed to DMEM+10% FBS culture medium and culture was continued. After 72 hours of administration, the cells were lysed and the decreased level of PCSK9 expression in the cells was detected by Western blot. The cell culture medium in the six-well plate was removed, and the phosphate buffer solution was gently rinsed once. The PBS was discarded, and 176μL RIPA+PMSF lysis solution was added to each well, and the cell plate was gently shaken. The cell solution in each well was aspirated into the corresponding marked 2mL Ep tube with a pipette. Lyse on ice for 30 minutes (vortex once every 5 minutes), centrifuge at 12000rpm for 20-30 minutes, and take the supernatant. Western blot was used to detect the silencing efficiency of PCSK9. The efficiency of LNP-0 in silencing PCSK9 was 50.6±3.5%, and the efficiency of LNP-1 in silencing PCSK9 was 51.9±2.6%.

[0119] 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. Use of amino acid tocopherol ester or a pharmaceutically acceptable salt thereof as an ionizable lipid carrier, characterized in that: One or more of the amino acid tocopherol esters or pharmaceutically acceptable salts thereof are used as ionizable lipid carriers for delivering therapeutic agents or preventive agents, wherein the therapeutic agent or preventive agent is one or more of siRNA, shRNA, miRNA and mRNA nucleic acid molecules, polypeptides or proteins, and the ionizable lipid carrier 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 directly connected through a hydrolyzable ester bond, and wherein the ionizable group is a basic group of the amino acid structure part.

2. Use of the amino acid tocopherol ester or a pharmaceutically acceptable salt thereof according to claim 1 as an ionizable lipid carrier, characterized in that: The amino acid structure part is the residue of lysine, histidine, arginine, or their derivatives.

3. Use of the amino acid tocopherol ester or a pharmaceutically acceptable salt thereof according to claim 1 as an ionizable lipid carrier, characterized in that: Amino acid tocopherol ester or a pharmaceutically acceptable salt thereof, the general formula of which is shown as (I): Among them, B is T is 4. A method for preparing amino acid tocopherol ester according to claim 1, characterized in that: The following steps are involved: (1) subjecting tocopherol and a base-protected amino acid to an esterification reaction to obtain a base-protected amino acid tocopherol ester; (2) The amino acid tocopherol ester protected by the base is deprotected by a deprotecting agent to obtain the amino acid tocopherol ester.

5. 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 amino acid tocopherol esters or pharmaceutically acceptable salts thereof as described in any one of claims 1 to 3.

6. A composition according to claim 5, 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.

7. A composition according to claim 5, characterized in that The carrier also includes auxiliary lipids, cholesterol and PEGylated lipids.

8. A composition according to claim 7, 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.

9. A composition according to claim 8, characterized in that The molar ratio of the ionizable lipid carrier, auxiliary lipid, cholesterol and PEGylated lipid is 50:(5-20):(10-50):(1-5).

10. Use of the amino acid tocopherol ester or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 3, or the composition as claimed in any one of claims 5 to 9 in the preparation of nucleic acid drugs, small molecule drugs, polypeptides or protein drugs.

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