Bile acid-based ionizable lipid, preparation method thereof and lipid nanoparticles

Abstract

The invention discloses an ionizable lipid based on bile acid, a preparation method of the ionizable lipid and lipid nanoparticles, and belongs to the technical field of biological medicines. The bile acid-based ionizable lipid provided by the invention has a structure as shown in a formula (I), x1 is a hydrogen atom or a hydroxyl group; x2 is a hydrogen atom or a hydroxyl group; x3 is a hydrogen atom or a hydroxyl group; y is selected from-O-or-NH-; n1 is an integer from 1 to 10; n2 is an integer from 1 to 8; r1 and R2 are each independently selected from a hydrocarbon group CxH2x + y; x is an integer from 5 to 30, and y is 1,-1,-3,-5,-7,-9 or-11. The transfection agent can be rapidly hydrolyzed by enzyme in vivo, has good biocompatibility, biodegradability and low toxicity, and is high in transfection efficiency. The lipid nano-particles prepared from the lipid nano-particles are good in stability and low in cytotoxicity, and effective delivery of intracellular nucleic acid drugs can be achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an ionizable lipid based on bile acid, a preparation method thereof, and lipid nanoparticles. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Nucleic acid drugs, as key vectors for gene therapy, offer significant advantages in modern biomedicine, including simple synthesis, easily modifiable structures, and remarkable target specificity. However, compared to traditional small-molecule drugs, nucleic acid drugs face significant challenges in tissue delivery and target cell uptake in vivo due to their large molecular weight, strong hydrophilicity, and negative charge. Their effective accumulation in target tissues is typically less than 1% of the total dose, severely limiting their therapeutic effectiveness.

[0004] To address this delivery challenge, lipid nanoparticle (LNP) technology has gradually developed into a mainstream strategy for nucleic acid drug delivery. Ionizable lipids serve as core functional components, and their molecular structure design plays a decisive role in the efficiency and biosafety of the delivery system. However, current ionizable lipid materials synthesized through epoxy ring opening or Michael addition reactions generally suffer from technical drawbacks such as a single metabolic pathway and the accumulation of degradation products in the body. Therefore, it is necessary to develop an ionizable lipid molecule that combines efficient delivery capabilities with good biodegradability. Summary of the Invention

[0005] In view of this, the present invention provides an ionizable lipid based on bile acid, a preparation method thereof, and lipid nanoparticles. The ionizable lipid based on bile acid provided by the present invention has an ester bond or an amide bond and good biocompatibility. At the same time, the lipid nanoparticles prepared therefrom can efficiently encapsulate nucleic acid drugs and have a good transfection effect.

[0006] In a first aspect, the present invention provides an ionizable lipid based on bile acid, which has a structure shown in formula (I):

[0007] wherein X1 is a hydrogen atom or a hydroxyl group; X2 is a hydrogen atom or a hydroxyl group; X3 is a hydrogen atom or a hydroxyl group; Y is selected from -O- or -NH-; n1 is an integer from 1 to 10; n2 is an integer from 1 to 8; R1 and R2 are each independently selected from a hydrocarbon group C x H 2x+y ; x is an integer from 5 to 30, and y is 1, -1, -3, -5, -7, -9 or -11.

[0008] In a second aspect, the present invention provides a method for preparing the above-mentioned ionizable lipid based on bile acid, comprising: The compound represented by formula (II) and the compound represented by formula (III) are obtained by esterification or amidation reaction; ; Wherein, Y' is an amino group or a hydroxyl group.

[0009] In a third aspect, the present invention provides a lipid nanoparticle comprising the bile acid-based ionizable lipid, a helper lipid, and a PEG-lipid.

[0010] In a fourth aspect, the present invention provides the use of the above-mentioned lipid nanoparticles as a gene drug delivery vector.

[0011] In a fifth aspect, the present invention provides a drug-loaded liposome complex, comprising the above-mentioned liposome nanoparticles and a drug loaded on the liposome nanoparticles.

[0012] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) The present invention provides an ionizable lipid synthesized based on bile acid, which is connected by ester bonds and amide bonds and can be rapidly hydrolyzed by enzymes in the body; secondly, the bile acid, the raw material for its synthesis, is an endogenous substance produced by the body, which gives the lipid good biocompatibility, biodegradability and low toxicity, and high transfection efficiency.

[0013] (2) The lipid nanoparticles prepared using the ionizable lipids provided by the present invention are electrically neutral at pH 7.0, which increases the in vivo stability of the nanoparticles and reduces the cytotoxicity caused by excessive positive charge. Furthermore, they can also load high-molecular-weight, highly hydrophilic, and negatively charged nucleic acid drugs, achieving effective intracellular delivery. At the cellular level, they are superior to the currently marketed product consisting of DLin-MC3-DMA and have promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The drawings, which constitute part of the present invention, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute undue limitations thereon. It is obvious that one of ordinary skill in the art could derive other drawings based on these drawings without inventive effort.

[0015] Figure 1 is a transmission electron micrograph of CA-NO12 lipid nanoparticles prepared in Example 15 of the present invention; Figure 21 is a graph showing the results of Raw 264.7 cell transfection with different mRNA-loaded lipid nanoparticles in Example 16 of the present invention; Figure 3 This is a diagram showing the results of 293T cell transfection using different mRNA-loaded lipid nanoparticles in Example 16 of the present invention; Figure 4 This is a graph showing the results of in vitro cytotoxicity studies of different mRNA-loaded lipid nanoparticles in Example 17 of the present invention. DETAILED DESCRIPTION

[0016] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0017] The present invention provides an ionizable lipid based on bile acid, which has a structure shown in formula (I):

[0018] wherein X1 is a hydrogen atom or a hydroxyl group; X2 is a hydrogen atom or a hydroxyl group; X3 is a hydrogen atom or a hydroxyl group; Y is selected from -O- or -NH-; n1 is an integer from 1 to 10; n2 is an integer from 1 to 8; R1 and R2 are each independently selected from a hydrocarbon group C x H 2x+y ; x is an integer from 5 to 30, and y is 1, -1, -3, -5, -7, -9 or -11.

[0019] The above-mentioned ionizable lipids with tertiary amine groups provided by the present invention can undergo structural reconstruction in response to the acidic microenvironment of the lysosome (pH 4.5-5.5) after entering the cell, become protonated and positively charged, and electrostatically bind to the negatively charged phospholipids on the lysosomal membrane to form non-double-layer ion pair complexes, destroying the integrity of the lysosomal membrane and forming transient pores, thereby promoting the efficient escape of the lipid nanoparticles prepared therefrom and the drugs encapsulated therein from the lysosomal cavity into the cytoplasm, thereby achieving effective intracellular delivery.

[0020] In the present invention, R1 and R2 are each independently selected from a C5 to C30 alkyl group. The saturated fatty alcohols forming the above R1 and R2 are independently selected from the group consisting of hexanol (C6 alcohol), grape flower alcohol (C7 alcohol), octanol (C8 alcohol), nonanol (C9 alcohol), decanol (C10 alcohol), undecanol (C11 alcohol), lauryl alcohol (C12 alcohol), tridecanol (C13 alcohol), myristyl alcohol (C14 alcohol), pentadecanol (C15 alcohol), palmityl alcohol (C16 alcohol), pearlescent alcohol (C17 alcohol), stearyl alcohol (C18 alcohol), nonadecanol (C19 alcohol), arachidyl alcohol (C20 alcohol), heneicosanol (C21 alcohol), behenyl alcohol (C22 alcohol), tricosanol (C23 alcohol), wood wax alcohol (wood tar alcohol, C24 alcohol), pentacosanol (C25 alcohol), wax alcohol (C26 alcohol), heptacosanol (C27 alcohol), montanyl alcohol (C28 alcohol), nonacosanol (C29 alcohol), and myricol (C30 alcohol).

[0021] In one or more embodiments of the present invention, the bile acid-based ionizable lipid is selected from any one of the structures shown in Formula I-1 to Formula I-12: (I-1); (I-2); (I-3); (I-4); (I-5); (I-6); (I-7); (I-8); (I-9); (I-10); (I-11); (I-12).

[0022] The present invention also provides a method for preparing the above-mentioned ionizable lipid based on bile acid, comprising: The compound represented by formula (II) and the compound represented by formula (III) are obtained by esterification or amidation reaction; ; Wherein, Y' is an amino group or a hydroxyl group.

[0023] Specifically, 1-hydroxybenzotriazole (HOBT) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) are used to catalyze the amidation reaction; or p-toluenesulfonic acid is used to catalyze the esterification reaction. Furthermore, the esterification reaction temperature is 40-50°C.

[0024] In some embodiments, the compound of formula (II) comprises cholic acid, deoxycholic acid, lithocholic acid, hyodeoxycholic acid, ursodeoxycholic acid, or chenodeoxycholic acid.

[0025] The present invention also provides a lipid nanoparticle, which comprises the bile acid-based ionizable lipid, an auxiliary lipid and a PEG-lipid.

[0026] The lipid nanoparticles of the present invention are positively charged under acidic conditions and uncharged under neutral conditions, exhibiting excellent in vivo stability. The lipid nanoparticles have a particle size of 10 to 900 nm, preferably 100 to 200 nm.

[0027] In the present invention, the helper lipid is selected from dioleoylphosphatidylethanolamine (DOPE), trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), trimethyl-2,3-dioleyloxypropylammonium chloride (DOTMA), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylcholine (DOPC), phosphatidylcholine (POPC), palmitoyloleylphosphatidylethanolamine (POPE), dilauroylphosphatidylcholine (DLPC), diethyl pyrocarbonate (DEPC), dimyristoylphosphatidylcholine (DMPC) and egg yolk lecithin (EPC), hydrogenated soybean phosphatidylcholine (HSPC) or sphingomyelin (SM) One or more, preferably DOPE.

[0028] In the present invention, the PEG-lipid is selected from one or more of dimethacrylate-polyethylene glycol (DMA-PEG), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), or dimyristylglycerol-polyethylene glycol (DMG-PEG), preferably DMG-PEG.

[0029] In the present invention, the lipid nanoparticles further comprise a hydrophobic lipid. Specifically, the hydrophobic lipid is a sterol. The sterol is preferably cholesterol.

[0030] In one or more embodiments of the present invention, the molar ratio of the bile acid-based ionizable lipid, the helper lipid, the hydrophobic lipid and the PEG-lipid is (3~55):(5~60):(0~50):(0.4~25).

[0031] The present invention does not impose any particular limitation on the preparation method of lipid nanoparticles. The preparation method of lipid nanoparticles commonly used in the art can be adopted, for example, nanoprecipitation method, thin film hydration method, impact jet mixing method, liposome extrusion method, microfluidics, etc., preferably nanoprecipitation method.

[0032] The present invention also provides the use of the lipid nanoparticles as gene drug delivery vectors.

[0033] The present invention also provides a drug-loaded liposome complex, comprising the above-mentioned liposome nanoparticles and a drug loaded on the liposome nanoparticles.

[0034] Preferably, the drug is selected from one or more of a small molecule drug, a protein drug, or a nucleic acid drug. When the drug is a nucleic acid, the nucleic acid may be siRNA, mRNA, microRNA, long non-coding RNA, cDNA, plasmid DNA, or ASO, etc., preferably mRNA.

[0035] In the present invention, the mass ratio of the drug to the ionizable lipid in the liposome nanoparticles is 1:(5-15).

[0036] The present invention does not impose any particular limitation on the method for preparing the drug-loaded liposome complex. In one or more embodiments of the present invention, during the preparation of the drug-loaded liposome complex, the drug is dissolved in a buffer solution, and the ionizable lipid, helper lipid, and PEG-lipid are dissolved in an organic solvent, and the two are mixed to prepare the drug-loaded liposome complex.

[0037] In the present invention, the buffer solution is selected from one or more of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, potassium dihydrogen phosphate-sodium hydroxide buffer solution, citric acid buffer solution, citric acid-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, tris-hydrochloric acid buffer solution, glycine-hydrochloric acid buffer solution, boric acid-borax buffer solution, phthalic acid-hydrochloric acid buffer solution, or potassium hydrogen phthalate buffer solution. The buffer reagent is preferably a citric acid buffer solution. Specifically, when forming the drug-loaded liposome complex, the volume fraction of the buffer solution is 40-90% (v / v), preferably 70-80% (v / v).

[0038] In one or more embodiments of the present invention, the organic reagent is selected from one or more of methanol, ethanol, isopropanol, pentane, hexane, octane, benzene, toluene, xylene, chlorobenzene, dichlorobenzene, toluene cyclohexanone, cyclohexanone, cyclohexane, ether, dichloromethane, propylene oxide, acetone, methyl butyl ketone, methyl isobutyl ketone, acetonitrile, pyridine, phenol, styrene, perchloroethylene, trichloroethylene, triethanolamine or ethylene glycol ether, preferably ethanol.

[0039] The technical solution of the present invention is further described below with reference to specific examples. The present invention has no particular limitation on the sources of the reagents used in the following examples, and commercially available products known to those skilled in the art can be used.

[0040] Example 1 This embodiment provides a method for synthesizing didodecyl 3,3'-((2-((tert-butoxycarbonyl)amino)ethyl)azodiyl)dipropionate. The specific reaction formula is as follows: .

[0041] To a 250 mL round-bottom flask equipped with a magnetic rod was added dodecyl acrylate (8 g, 33.3 mmol), triethylamine (3.37 g, 33.3 mmol), N-tert-butoxycarbonyl-1,2-ethylenediamine (1.78 g, 11.1 mmol), and isopropanol (30 mL). Triethylamine was slowly added dropwise to the N-tert-butoxycarbonyl-1,2-ethylenediamine dissolved in isopropanol. After 1 h of reaction, dodecyl acrylate was added. A condenser was installed on the reaction flask, and the mixture was heated under reflux at 90°C for 5 h. The solvent was removed by rotary evaporation under reduced pressure, and the product was isolated and purified by thin-layer chromatography (eluent: methanol: dichloromethane, volume ratio 1:20) to obtain the intermediate product, didodecyl 3,3'-((2-((tert-butoxycarbonyl)amino)ethyl)azadiyl)dipropionate, in an 80% yield.

[0042] Example 2 This embodiment provides a method for synthesizing didodecyl 3,3'-((2-aminoethyl) azodiyl) dipropionate. The specific reaction formula is as follows: .

[0043] To a 100 mg round-bottom flask equipped with a magnetic ion, 1.0 g, 1.56 mmol of didodecyl 3,3'-((2-((tert-butoxycarbonyl)amino)ethyl)azepine diyl)dipropionate prepared in Example 1 was added. After dissolving in dichloromethane (16 mL), trifluoroacetic acid (4.1 g, 35.88 mmol) was slowly added dropwise. The mixture was cooled to 10°C and allowed to react for 5-6 h. The reaction solution was evaporated to dryness under reduced pressure, and an appropriate amount of saturated sodium bicarbonate solution was added. The mixture was extracted with dichloromethane (3 × 30 mL). The organic phase was dried over anhydrous sodium sulfate and filtered. The solvent was removed using a rotary evaporator to obtain the crude didodecyl 3,3'-((2-aminoethyl)azepine diyl)dipropionate, which was used in the next reaction without purification.

[0044] Example 3 This example provides a method for synthesizing didodecyl 3,3'-((((R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamido)methyl)nitrogendiyl)dipropionate (CA-NO12). The reaction formula is as follows: .

[0045] To a 100 ml three-necked flask under inert gas, cholic acid (563.8 mg, 1.38 mmol), EDCI (291.4 mg, 1.52 mmol), and HOBT (9.5 mg, 0.07 mmol) were added and dissolved in N,N-dimethylacetamide (15 ml). N-methylmorpholine (349 mg, 3.46 mmol) was then added, followed by N-(2-aminoethyl)-N-[3-(dodecyloxy)-3-oxopropyl]-β-alanine dodecyl ester (900 mg, 1.66 mmol). The reaction mixture was stirred at room temperature for 60 h. The mixture was then diluted with 2-methyltetrahydrofuran (50 mL) and cooled to 10°C. The mixture was quenched by the addition of 3 M HCl (30 mL). The contents were then transferred to a separatory funnel and the layers separated. The aqueous phase was removed, and the organic layer was washed with H2O, saturated sodium bicarbonate solution, and dilute brine. After evaporation to dryness under reduced pressure, the residual mixture was separated by silica gel column chromatography (eluent: methanol and dichloromethane in a volume ratio of 1:20) to obtain the target product as a white solid with a yield of 50%, recorded as CA-NO12. 1 H NMR (400 MHz, CDCl3) δ 5.30 (s, 1H), 4.06 (t, J = 6.9 Hz, 4H), 3.99 (s, 1H), 3.87 – 3.83 (m, 1H), 3.35 – 3.29 (m, 2H), 2.76 (t, J = 6.7 Hz, 4H), 2.54 (t, J = 5.5 Hz,2H), 2.42 (t, J = 6.6 Hz, 4H), 1.26 (s, 33H), 1.00 (d, J = 6.3 Hz, 3H), 0.91– 0.86 (m, 9H), 0.69 (s, 3H).

[0046] Example 4 This example provides the synthesis of didodecyl 3,3'-((((R)-4-((3R,5S,7R,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthracene-17-yl)pentanamido)methyl)nitrogendiyl)dipropionate (CDCA-NO12). The specific reaction formula is as follows: .

[0047] To a 100 ml three-necked flask under inert gas, chenodeoxycholic acid (541.7 mg, 1.38 mmol), EDCI (291.4 mg, 1.52 mmol), and HOBT (9.5 mg, 0.07 mmol) were added and dissolved in N,N-dimethylacetamide (15 ml). N-methylmorpholine (349 mg, 3.46 mmol) was then added, followed by N-(2-aminoethyl)-N-[3-(dodecyloxy)-3-oxopropyl]-β-alanine dodecyl ester (900 mg, 1.66 mmol). The reaction mixture was stirred at room temperature for 60 h. The mixture was then diluted with 2-methyltetrahydrofuran (50 mL) and cooled to 10°C. The mixture was quenched by the addition of 3 M HCl (30 mL). The contents were then transferred to a separatory funnel and the layers separated. The aqueous phase was removed, and the organic layer was washed with H2O, saturated sodium bicarbonate solution, and dilute brine. After evaporation to dryness under reduced pressure, the residual mixture was separated by silica gel column chromatography (eluent: methanol and dichloromethane in a volume ratio of 1:10) to obtain the target product as a white solid in a yield of 35%. 1 H NMR (400 MHz, CDCl3) δ 6.81 (t, J = 4.5 Hz, 1H), 4.07 (t, J = 6.1 Hz, 4H), 3.94 (d, J = 5.5Hz, 1H), 3.29 (td, J = 5.4, 4.5 Hz, 2H), 2.75 (t, J = 6.7 Hz, 4H), 2.53 (t, J = 5.5 Hz, 2H), 2.46 (t, J = 6.6 Hz, 4H), 1.29 – 1.23 (m, 33H), 0.93 (dd, J =6.7, 1.5 Hz, 3H), 0.91 – 0.85 (m, 9H), 0.69 (s, 3H).

[0048] Example 5 This example provides the synthesis of didodecyl 3,3'-((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthracene-17-yl)pentanamido)methylene)diazodiyl)dipropionate (DCA-NO12). The specific reaction formula is as follows: .

[0049] To a 100 ml three-necked flask under inert gas, deoxycholic acid (541.7 mg, 1.38 mmol), EDCI (291.4 mg, 1.52 mmol), and HOBT (9.5 mg, 0.07 mmol) were added and dissolved in N,N-dimethylacetamide (15 ml). N-methylmorpholine (349 mg, 3.46 mmol) was then added, followed by N-(2-aminoethyl)-N-[3-(dodecyloxy)-3-oxopropyl]-β-alanine dodecyl ester (900 mg, 1.66 mmol). The reaction mixture was stirred at room temperature for 60 h. The mixture was then diluted with 2-methyltetrahydrofuran (50 mL) and cooled to 10°C. The mixture was quenched by the addition of 3 M HCl (30 mL). The contents were then transferred to a separatory funnel and the layers separated. The aqueous phase was removed, and the organic layer was washed with H2O, saturated sodium bicarbonate solution, and dilute brine. After evaporation to dryness under reduced pressure, the residual mixture was separated by silica gel column chromatography (eluent: methanol and dichloromethane, volume ratio of 1:20) to obtain the target product as a white solid in a yield of 57%. 1H NMR (400 MHz, CDCl3)δ 6.54 (t, J = 5.3 Hz, 1H), 4.06 (t, J = 6.8 Hz, 4H), 3.99 (d, J = 3.2 Hz,1H), 3.32 (q, J = 5.4 Hz, 2H), 2.75 (t, J = 6.7 Hz, 4H), 2.53 (t, J = 5.5 Hz, 2H), 2.42 (t, J = 6.6 Hz, 4H), 1.26 (s, 33H), 0.99 (d, J = 6.3 Hz, 3H), 0.89(dd, J = 12.0, 5.8 Hz, 9H), 0.68 (s, 3H).

[0050] Example 6 This example provides the synthesis of didodecyl 3,3'-((R)-4-((3R,5R,8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthro[9,10-d]cyclopenten-17-yl)pentanamido)methylene)diazodiyl)dipropionate (LCA-NO12). The specific reaction formula is as follows: .

[0051] To a 100 ml three-necked flask under inert gas, lithocholic acid (519.7 mg, 1.38 mmol), EDCI (291.4 mg, 1.52 mmol), and HOBT (9.5 mg, 0.07 mmol) were added and dissolved in N,N-dimethylacetamide (15 ml). N-methylmorpholine (349 mg, 3.46 mmol) was then added, followed by N-(2-aminoethyl)-N-[3-(dodecyloxy)-3-oxopropyl]-β-alanine dodecyl ester (900 mg, 1.66 mmol). The reaction mixture was stirred at room temperature for 60 h. The mixture was then diluted with 2-methyltetrahydrofuran (50 mL) and cooled to 10°C. The mixture was quenched by the addition of 3 M HCl (30 mL). The contents were then transferred to a separatory funnel and the layers separated. The aqueous phase was removed, and the organic layer was washed with H2O, saturated sodium bicarbonate, and dilute brine. After evaporation to dryness under reduced pressure, the residual mixture was separated by silica gel column chromatography (eluent: methanol and dichloromethane in a volume ratio of 1:20) to obtain the target product as a white solid in a yield of 45%. 1 H NMR (400 MHz, CDCl3) δ6.81 (t, J = 4.5 Hz, 1H), 4.07 (t, J = 6.1 Hz, 4H), 3.29 (td, J = 5.4, 4.5Hz, 2H), 2.75 (t, J = 6.7 Hz, 4H), 2.55 (t, J = 5.5 Hz, 2H), 2.42 (t, J = 6.6Hz, 4H), 1.26 (s, 33H), 0.93 (dd, J = 6.7, 1.5 Hz, 3H), 0.91 – 0.86 (m, 9H), 0.67 (s, 3H). Example 7 This example provides the synthesis of 3,3'-((R)-4-((3R,5R,6S,8S,9S,10R,13R,14S,17R)-3,6-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthro[9,10-d]cyclopenten-17-yl)pentanamido)methylenediazadipropionic acid didodecyl ester (HDCA-NO12). The specific reaction formula is as follows: .

[0052] To a 100 ml three-necked flask under inert gas, hyodeoxycholic acid (541.7 mg, 1.38 mmol), EDCI (291.4 mg, 1.52 mmol), and HOBT (9.5 mg, 0.07 mmol) were added and dissolved in N,N-dimethylacetamide (15 ml). N-methylmorpholine (349 mg, 3.46 mmol) was then added, followed by N-(2-aminoethyl)-N-[3-(dodecyloxy)-3-oxopropyl]-β-alanine dodecyl ester (900 mg, 1.66 mmol). The reaction mixture was stirred at room temperature for 60 h. The mixture was then diluted with 2-methyltetrahydrofuran (50 mL) and cooled to 10°C. The mixture was quenched by the addition of 3 M HCl (30 mL). The contents were then transferred to a separatory funnel and the layers separated. The aqueous phase was removed, and the organic layer was washed with H2O, saturated sodium bicarbonate, and dilute brine. After evaporation to dryness under reduced pressure, the residual mixture was separated by silica gel column chromatography (eluent: methanol and dichloromethane, volume ratio of 1:20) to obtain the target product as a white solid in a yield of 65%. 1 H NMR (400 MHz, CDCl3) δ 6.82 (d, J = 4.4 Hz, 1H), 4.07 (t, J = 6.1 Hz, 4H), 4.02 (dddq, J =6.4, 3.8, 2.7, 1.3 Hz, 1H), 3.29 (td, J = 5.4, 4.5 Hz, 2H), 2.76 (t, J = 6.7Hz, 4H), 2.53 (t, J = 5.5 Hz, 2H), 2.44 (t, J = 6.6 Hz, 4H), 1.29 – 1.24 (m,33H), 0.93 (dd, J = 6.7, 1.5 Hz, 3H), 0.90 – 0.84 (m, 9H), 0.67 (s, 3H).

[0053] Example 8 This example provides the synthesis of didodecyl 3,3'-((((R)-4-(((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentylamino)methyl)azodiazo)dipropionate (UDCA-NO12). The specific reaction formula is as follows: .

[0054] To a 100 ml three-necked flask under inert gas, ursodeoxycholic acid (541.4 mg, 1.38 mmol), EDCI (291.4 mg, 1.52 mmol), and HOBT (9.5 mg, 0.07 mmol) were added and dissolved in N,N-dimethylacetamide (15 ml). N-methylmorpholine (349 mg, 3.46 mmol) was then added, followed by N-(2-aminoethyl)-N-[3-(dodecyloxy)-3-oxopropyl]-β-alanine dodecyl ester (900 mg, 1.66 mmol). The reaction mixture was stirred at room temperature for 60 h. The mixture was then diluted with 2-methyltetrahydrofuran (50 mL) and cooled to 10°C. The mixture was quenched by the addition of 3 M HCl (30 mL). The contents were then transferred to a separatory funnel and the layers separated. The aqueous phase was removed, and the organic layer was washed with H2O, saturated sodium bicarbonate, and dilute brine. After evaporation to dryness under reduced pressure, the residual mixture was separated by silica gel column chromatography (eluent: methanol and dichloromethane in a volume ratio of 1:10) to obtain the target product as a white solid in a yield of 46.5%. 1 H NMR (400 MHz, CDCl3)δ 6.81 (t, J = 4.5 Hz, 1H), 4.07 (t, J = 6.1 Hz, 4H), 3.94 – 3.88 (m, 1H), 3.29 (td, J = 5.4, 4.5 Hz, 2H), 2.80 (t, J = 6.7 Hz, 4H), 2.54 (t, J = 5.5Hz, 2H), 2.50 (t, J = 6.6 Hz, 4H), 1.29 – 1.23 (m, 33H), 0.93 (dd, J = 6.7,1.5 Hz, 3H), 0.91 – 0.85 (m, 9H), 0.69 (s, 3H).

[0055] Example 9 This example provides the synthesis of didodecyl 3,3'-(((((R)-4-(((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoyl)oxy)methyl)nitrogendiyl)dipropionate (CA-OO12). The specific reaction formula is as follows: .

[0056] To a 100 mL round-bottom flask equipped with a magnetic rod, cholic acid (1.0 g, 2.45 mmol), 2-(dodecylamino)-1-ethanol (3.05 g, 7.66 mmol), and p-toluenesulfonic acid (0.19 g, 1.10 mmol) were added and dissolved in N,N-dimethylacetamide (15 mL). A condenser was placed over the reaction flask and the mixture was heated at reflux at 45°C for 15 h. The contents were transferred to a separatory funnel and the layers separated. The aqueous phase was removed, and the organic layer was washed with dilute brine. After evaporation to dryness under reduced pressure, the residual mixture was separated by silica gel column chromatography (eluent: methanol: dichloromethane, volume ratio 1:20) to obtain the desired product as a white solid in a 45% yield. 1 H NMR(400 MHz, CDCl3) δ 4.28 (t, J = 6.1 Hz, 2H), 4.07 (t, J = 6.1 Hz, 4H), 4.04(d, J = 5.5 Hz, 1H), 3.91 – 3.83 (m, 1H), 3.48 (qq, J = 2.1, 1.2 Hz, 2H), 2.81 (t, J = 6.8 Hz, 4H), 2.61 (t, J = 5.0 Hz, 2H), 2.46 (t, J = 6.8 Hz, 4H), 1.30 – 1.23 (m, 33H), 0.92 (s, 3H), 0.91 – 0.85 (m, 9H), 0.74 (s, 3H).

[0057] Example 10 This example provides the synthesis of didodecyl 3,3'-(((((R)-4-(((3R,5S,7R,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)oxy)methyl)azodiazo)dipropionate (CDCA-OO12). The specific reaction formula is as follows: .

[0058] To a 100 mL round-bottom flask equipped with a magnetic rod, chenodeoxycholic acid (1.0 g, 2.55 mmol), 2-(dodecylamino)-1-ethanol (3.17 g, 7.97 mmol), and p-toluenesulfonic acid (0.20 g, 1.15 mmol) were added and dissolved in N,N-dimethylacetamide (15 mL). A condenser was placed over the reaction flask and the mixture was heated at reflux at 45°C for 15 h. The contents were transferred to a separatory funnel and the layers separated. The aqueous phase was removed, and the organic layer was washed with dilute brine. After evaporation to dryness under reduced pressure, the residual mixture was separated by silica gel column chromatography (eluent: methanol: dichloromethane, volume ratio 1:20) to obtain the desired product as a white solid in a 38% yield. 1 HNMR (400 MHz, CDCl3) δ 4.28 (t, J = 6.1 Hz, 2H), 4.07 (t, J = 6.1 Hz, 4H), 3.94 (d, J = 5.5 Hz, 1H), 3.49 – 3.47 (m, 2H), 2.82 (t, J = 6.8 Hz, 4H), 2.61(t, J = 5.0 Hz, 2H), 2.47 (t, J = 6.8 Hz, 4H), 1.29 – 1.23 (m, 33H), 0.93(dd, J = 6.3, 1.9 Hz, 3H), 0.91 – 0.86 (m, 9H), 0.69 (s, 3H).

[0059] Example 11 This example provides the synthesis of didodecyl 3,3'-(((((R)-4-(((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)oxy)methyl)azodiazo)dipropionate (DCA-OO12). The specific reaction formula is as follows: .

[0060] Deoxycholic acid (1.0 g, 2.55 mmol), 2-(dodecylamino)-1-ethanol (3.17 g, 7.97 mmol), and p-toluenesulfonic acid (0.20 g, 1.15 mmol) were added to a 100 mL round-bottom flask equipped with a magnetic rod. The mixture was dissolved in N,N-dimethylacetamide (15 mL). A condenser was placed over the reaction flask and the mixture was heated under reflux at 45°C for 15 h. The contents were transferred to a separatory funnel and the layers were separated. The aqueous phase was removed, and the organic layer was washed with dilute brine. After evaporation to dryness under reduced pressure, the residual mixture was separated by silica gel column chromatography (eluent: methanol: dichloromethane, volume ratio 1:20) to obtain the desired product as a white solid in a 44% yield. 1 H NMR(400 MHz, CDCl3) δ 4.28 (t, J = 6.1 Hz, 2H), 4.07 (t, J = 6.1 Hz, 4H), 3.75(dddd, J = 8.8, 5.9, 3.3, 1.9 Hz, 1H), 3.72 – 3.68 (m, 2H), 2.82 (t, J = 6.8Hz, 4H), 2.62 (t, J = 5.0 Hz, 2H), 2.47 (t, J = 6.8 Hz, 4H), 1.28 – 1.24 (m,33H), 0.91 – 0.89 (m, 3H), 0.89 – 0.86 (m, 9H), 0.71 (s, 3H).

[0061] Example 12 This example provides the synthesis of didodecyl 3,3'-(((((R)-4-(((3R,5R,8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)oxy)methyl)azodiazo)dipropionate (LCA-OO12). The specific reaction formula is as follows: .

[0062] To a 100 mL round-bottom flask equipped with a magnetic rod, lithocholic acid (1.0 g, 2.66 mmol), 2-(dodecylamino)-1-ethanol (3.31 g, 8.31 mmol), and p-toluenesulfonic acid (0.21 g, 1.20 mmol) were added and dissolved in N,N-dimethylacetamide (15 mL). A condenser was placed over the reaction flask and the mixture was heated under reflux at 45°C for 15 h. The contents were transferred to a separatory funnel and the layers separated. The aqueous phase was removed, and the organic layer was washed with dilute brine. After evaporation to dryness under reduced pressure, the residual mixture was separated by silica gel column chromatography (eluent: methanol: dichloromethane, volume ratio 1:20) to obtain the desired product as a white solid in a 48% yield. 1 H NMR(400 MHz, CDCl3) δ 4.28 (t, J = 6.1 Hz, 2H), 4.07 (t, J = 6.1 Hz, 4H), 2.82(t, J = 6.8 Hz, 4H), 2.61 (t, J = 5.0 Hz, 2H), 2.45 (t, J = 6.8 Hz, 4H), 1.26(s, 33H), 0.93 (dd, J = 6.6, 1.5 Hz, 3H), 0.91 – 0.87 (m, 9H), 0.67 (s, 3H).

[0063] Example 13 This example provides the synthesis of didodecyl 3,3'-(((((R)-4-(((3R,5R,6S,8S,9S,10R,13R,14S,17R)-3,6-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)oxy)methyl)azodiazo)dipropionate (HDCA-OO12). The specific reaction formula is as follows: .

[0064] To a 100 mL round-bottom flask equipped with a magnetic rod, hyodeoxycholic acid (1.0 g, 2.55 mmol), 2-(dodecylamino)-1-ethanol (3.17 g, 7.97 mmol), and p-toluenesulfonic acid (0.20 g, 1.15 mmol) were added and dissolved in N,N-dimethylacetamide (15 mL). A condenser was placed over the reaction flask and the mixture was heated at reflux at 45°C for 15 h. The contents were transferred to a separatory funnel and the layers separated. The aqueous phase was removed, and the organic layer was washed with dilute brine. After evaporation to dryness under reduced pressure, the residual mixture was separated by silica gel column chromatography (eluent: methanol: dichloromethane, volume ratio 1:20) to obtain the desired product as a white solid in a yield of 27.9%. 1HNMR (400 MHz, CDCl3) δ 4.28 (t, J = 6.1 Hz, 2H), 4.07 (t, J = 6.1 Hz, 4H), 4.03 – 3.99 (m, 1H), 2.80 (t, J = 6.8 Hz, 4H), 2.62 (t, J = 5.0 Hz, 2H), 2.47(t, J = 6.8 Hz, 4H), 1.28 – 1.23 (m, 33H), 0.93 (dd, J = 6.6, 1.3 Hz, 3H), 0.90 – 0.83 (m, 9H), 0.67 (s, 3H).

[0065] Example 14 This example provides the synthesis of didodecyl 3,3'-(((((R)-4-(((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)oxy)methyl)azodiazo)dipropionate (UDCA-OO12). The specific reaction formula is as follows: .

[0066] Ursodeoxycholic acid (1.0 g, 2.55 mmol), 2-(dodecylamino)-1-ethanol (3.17 g, 7.97 mmol), and p-toluenesulfonic acid (0.20 g, 1.15 mmol) were added to a 100 mL round-bottom flask equipped with a magnetic rod. The mixture was dissolved in N,N-dimethylacetamide (15 mL). A condenser was placed over the reaction flask and the mixture was heated at reflux at 45°C for 15 h. The contents were transferred to a separatory funnel and the layers were separated. The aqueous phase was removed, and the organic layer was washed with dilute brine. After evaporation to dryness under reduced pressure, the residual mixture was separated by silica gel column chromatography (eluent: methanol: dichloromethane, volume ratio 1:20) to obtain the desired product as a white solid in a yield of 42.7%. 1HNMR (400 MHz, CDCl3) δ 4.28 (t, J = 6.1 Hz, 2H), 4.07 (t, J = 6.1 Hz, 4H), 3.90 (d, J = 5.5 Hz, 1H), 2.82 (t, J = 6.8 Hz, 4H), 2.61 (t, J = 5.0 Hz, 2H), 2.47 (t, J = 6.8 Hz, 4H), 1.29 – 1.23 (m, 33H), 0.93 (dd, J = 6.2, 2.0 Hz, 3H), 0.91 – 0.86 (m, 9H), 0.69 (s, 3H).

[0067] Example 15 This example provides the preparation of lipid nanoparticles.

[0068] The lipid compounds synthesized in Examples 3-14 (CA-NO12, CDCA-NO12, DCA-NO12, LCA-NO12, HDCA-NO12, UDCA-NO12, CA-OO12, CDCA-OO12, DCA-OO12, LCA-OO12, HDCA-OO12, and UDCA-OO12) were selected as ionizable lipid components. The ionizable lipids, cholesterol, DOPE, and DMG-PEG were dissolved in ethanol at a molar ratio of 15:15:20:0.5 to prepare the organic phase. EGFP mRNA (Shanghai Jiying Biotechnology Co., Ltd., Cat. No. JYSJ-015-002; mRNA:ionizable lipid mass ratio = 1:10) was then dissolved in 50 mM citrate buffer at pH 4.0 to prepare the aqueous phase (the buffer volume was 3 times that of the organic phase). mRNA-loaded lipid nanoparticles were prepared by rapidly mixing the organic phase solution with the aqueous phase solution in an enzyme-free centrifuge tube, and finally purified by dialyzing against PBS buffer at 4°C overnight.

[0069] The synthesized mRNA-loaded lipid nanoparticles were further characterized. The nanoparticle size and polydispersity index (PDI) were characterized by a dynamic light scattering laser particle size analyzer (Malvern Zetasizer Nano ZS). The results are shown in Table 1. The 12 mRNA-loaded lipid nanoparticles have uniform particle sizes (PDI < 0.3) ranging from 100 to 200 nm. In addition, the CA-NO12 LNP of Example 3 was used as a representative mRNA-loaded lipid nanoparticle, and its morphology was characterized by transmission electron microscopy. The results showed that the mRNA-loaded lipid nanoparticles were spherical, as shown in Figure 1. Figure 1 shown.

[0070] Table 1 Nanosize and polydispersity coefficient of mRNA-loaded lipid nanoparticles with different formulations

[0071] Example 16 This example provides an investigation into the in vitro transfection efficiency of mRNA-loaded lipid nanoparticles prepared in Example 15.

[0072] In this example, RAW 264.7 cells were used as model cells to detect the EGFP mRNA transfection efficiency of CA-NO12 LNP, CDCA-NO12 LNP, DCA-NO12 LNP, LCA-NO12 LNP, HDCA-NO12 LNP, UDCA-NO12 LNP, CA-OO12 LNP, CDCA-OO12 LNP, DCA-OO12 LNP, LCA-OO12 LNP, HDCA-OO12 LNP, and UDCA-OO12 LNP prepared in Example 15. The specific operation method is as follows: RAW 264.7 cells in the logarithmic growth phase were inoculated in a 96-well plate containing DMEM complete medium and cultured overnight. LNPs containing 100 ng of mRNA were added to each well and incubated with RAW 264.7 cells, and 3 replicates were set for each group. After 24 h of transfection, the proportion of EGFP-positive cells was detected by flow cytometry. The commercially available ionizable lipid DLin-MC3-DMA LNP was used as a positive control. Figure 2 The results show that the mRNA-loaded lipid nanoparticles prepared with the ionizable lipids of the embodiments of the present invention can effectively transfect RAW 264.7 cells, among which the transfection efficiency of CA-NO12 LNP, DCA-NO12 LNP and UDCA-NO12LNP in RAW 264.7 cells is significantly better than that of DLin-MC3-DMA LNP, and all are more than twice the transfection efficiency of the latter.

[0073] In order to further investigate the transfection efficiency of the mRNA-loaded lipid nanoparticles prepared by the present invention in other cells, 293T cells were used as model cells to detect the EGFP mRNA transfection efficiency of three high-performance LNPs, CA-NO12 LNP, DCA-NO12 LNP and UDCA-NO12 LNP. The specific operation method is as follows: 293T cells in the logarithmic growth phase were inoculated into a 96-well plate and cultured overnight. LNPs containing 100 ng of mRNA were added to each well and incubated with 293T cells, and 3 replicates were set up for each group. 24 hours after transfection, the proportion of EGFP-positive cells was detected by flow cytometry. The commercially available ionizable lipid DLin-MC3-DMA LNP was also used as a positive control. The results are shown in Figure 2. Figure 3As shown in the data, CA-NO12 LNP, DCA-NO12 LNP and UDCA-NO12 LNP can effectively transfect 293T cells, and the transfection efficiency of the three is also significantly better than that of DLin-MC3-DMA LNP. The transfection efficiency of DCA-NO12 LNP with the best transfection performance is more than 3 times that of DLin-MC3-DMA LNP.

[0074] Example 17 Investigation of cytotoxicity of ionizable lipid nanoparticles in vitro: RAW 264.7 cells were used as model cells to investigate the cytotoxicity of the mRNA-loaded lipid nanoparticles prepared in Example 15. RAW 264.7 cells in the logarithmic growth phase were seeded in a 96-well plate filled with DMEM complete medium and cultured overnight. Subsequently, lipid nanoparticles (CA-NO12 LNP, CDCA-NO12 LNP, DCA-NO12 LNP, LCA-NO12 LNP, HDCA-NO12 LNP, UDCA-NO12 LNP, CA-OO12 LNP, CDCA-OO12 LNP, DCA-OO12 LNP, LCA-OO12 LNP, HDCA-OO12 LNP, UDCA-OO12 LNP) containing 100 ng of mRNA were incubated with RAW 264.7 cells for 24 h, and cell activity was detected by CCK-8 kit. The results are shown in Figure 2. Figure 4 As shown, it can be seen that the above 12 lipid nanoparticles did not produce obvious cytotoxicity, indicating that the mRNA-loaded lipid nanoparticles provided by the present invention have good biocompatibility.

[0075] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An ionizable lipid based on bile acid, characterized in that It has the structure shown in formula (I): wherein X1 is a hydrogen atom or a hydroxyl group; X2 is a hydrogen atom or a hydroxyl group; X3 is a hydrogen atom or a hydroxyl group; Y is selected from -O- or -NH-; n1 is an integer from 1 to 10; n2 is an integer from 1 to 8; R1 and R2 are each independently selected from a hydrocarbon group C x H 2x+y ; x is an integer from 5 to 30, and y is 1, -1, -3, -5, -7, -9 or -11.

2. The ionizable lipid based on bile acid according to claim 1, wherein R1 and R2 are each independently selected from a C5 to C30 alkyl group.

3. The ionizable lipid based on bile acid according to claim 2, wherein The bile acid-based ionizable lipid is selected from any one of the structures shown in Formula I-1 to Formula I-12: (I-1)； (I-2)； (I-3)； (I-4)； (I-5)； (I-6)； (I-7)； (I-8)； (I-9)； (I-10)； (I-11)； (I-12)。 4. The method for preparing ionizable lipids based on bile acid according to claim 1, wherein include: The compound represented by formula (II) and the compound represented by formula (III) are obtained by esterification or amidation reaction; ； Wherein, Y' is an amino group or a hydroxyl group.

5. A lipid nanoparticle, characterized in that The lipid nanoparticles include the bile acid-based ionizable lipid, a helper lipid, and a PEG-lipid.

6. The lipid nanoparticle according to claim 5, wherein The lipid nanoparticles further comprise a hydrophobic lipid.

7. The lipid nanoparticle according to claim 6, wherein The molar ratio of the bile acid-based ionizable lipid, the auxiliary lipid, the hydrophobic lipid and the PEG-lipid is (3-55): (5-60): (0-50): (0.4-25).

8. Use of the lipid nanoparticles according to any one of claims 5 to 7 as a gene drug delivery vector.

9. A drug-loaded liposome complex, characterized in that: The method comprises the liposome nanoparticles according to any one of claims 5 to 7 and a drug loaded on the liposome nanoparticles.

10. The drug-loaded liposome complex according to claim 9, wherein The drug is selected from one or more of small molecule drugs, proteins or nucleic acids.

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