An ionizable lipid and its applications

Abstract

This invention discloses an ionizable lipid and its applications; specifically, this invention provides a compound as shown in Formula I or a pharmaceutically acceptable salt thereof. The liposome nanoparticles prepared from the ionizable lipid provided by this invention are (1) easily degraded and metabolized rapidly; (2) have good transfection effects; (3) significantly target the spleen, which helps to enhance the immune effect; and (4) have low side effects.

Description

Technical Field

[0001] This invention relates to an ionizable lipid and its applications. Background Technology

[0002] mRNA vaccine technology has surpassed inactivated and protein vaccines in terms of antigen-antibody titer, efficacy, duration of protection, and broad-spectrum protection, becoming the mainstream direction for subsequent vaccine development. mRNA vaccines mainly consist of two parts: nucleic acid sequences and lipid nanoparticles.

[0003] Lipid nanoparticles generally consist of four components: cationic / ionizable lipids, phosphate lipids, cholesterol, and PEG lipids. Cationic / ionizable lipids are the main components of LNPs and play a crucial role in the assembly, delivery, and expression of nucleic acid vaccines. Exogenous nucleic acids, once inside the body, are easily degraded by nucleases or cleared by the immune system, rendering them ineffective. Nucleic acid-lipid nanoparticle complexes, by loading nucleic acid drugs within the particles during preparation, can protect nucleic acids from degradation and clearance by the in vivo environment. The complex enters the body fluid system, circulates, and binds to target tissues or cells, releasing the corresponding nucleic acids to exert therapeutic or immune effects.

[0004] Among the currently approved lipid nanoparticle products, the ionizable lipids used mainly include the following three types: MC3, ALC-0315, and SM-102. Analysis of existing lipid components reveals that ionizable lipids generally contain three important structures: a hydrophilic ionizable head with an amino group, a long-chain hydrophobic tail, and a linker connecting the hydrophilic head and the hydrophobic tail. The ionizable head plays a crucial role in binding nucleic acids to form a nanocore, interacting with the cell membrane, and releasing nucleic acids after ionization by disrupting the endosome membrane. The hydrophobic tail contributes to the structural stability of the nanoparticles. The linker determines the stability of the ionizable lipid molecule itself. With further research, in newly developed ionizable lipids, the linker is generally designed as a degradable group to facilitate rapid clearance of the ionizable lipids, reducing their biotoxicity and metabolic cycle. Among the other three lipid components, phospholipids and cholesterol generally assist in the encapsulation of nucleic acid drugs by lipid nanoparticles and also help stabilize the lipid nanoparticle membrane structure. PEG lipids are typically distributed on the outer layer of lipid nanoparticles, forming a hydration layer through hydrophilic PEG chains, thus preventing the lipid nanoparticles from agglomerating in solution. Lipid nanoparticle technology effectively overcomes the instability and degradation problems of nucleic acid drugs; however, this technology also brings new challenges. For example, the transfection efficiency of its core component, ionizable lipids, is not yet ideal; the positive charge carried by lipids may lead to cytotoxicity; and some components may also pose a risk of triggering an immune response. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the problems of general transfection efficiency, local cytotoxicity or systemic injection side effects caused by ionizable lipids in the prior art, and to provide a lipid molecule with low toxicity and side effects, easy degradation or high delivery efficiency.

[0006] The present invention solves the above-mentioned technical problems through the following technical solution:

[0007] This invention provides a compound as shown in Formula I or a pharmaceutically acceptable salt thereof:

[0008] ;

[0009] Among them, R 1 and R 2 Independently for C 8-24 Alkyl, C 8-20 alkenyl or C 8-20 alkynyl group, the C 8-20 The number of alkene bonds in the alkenyl group is 1, 2, or 3; the C 8-20 The number of alkyne bonds in an alkyne group can be 1, 2, or 3;

[0010] R 3 and R 4 Independently or ;

[0011] Or, R 3 and R 4 Connected, forming or .

[0012] R a R b R c R d R e and R f Independently for H and C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl, C 3-10 cycloalkyl, or a C with one CH2 group replaced by an O group 1-6 alkyl;

[0013] Or, R a R b The nitrogen atom attached to it forms 3-10-membered heterocyclic alkyl groups, 5-6-membered heteroaryl groups, and 5-6-membered heterocyclic alkenyl groups, which are then bonded by R. N1 Substituted 3-10 membered heterocyclic alkyl groups, R N2 Substituted 5-6 heteroaryl groups or R N3 Substituted 5-6 membered heterocyclic alkenyl groups;

[0014] Or R c R d The nitrogen atom attached to it forms 3-10-membered heterocyclic alkyl groups, 5-6-membered heteroaryl groups, and 5-6-membered heterocyclic alkenyl groups, which are then bonded by R. N1 Substituted 3-10 membered heterocyclic alkyl groups, R N2 Substituted 5-6 heteroaryl groups or R N3 Substituted 5-6 membered heterocyclic alkenyl groups;

[0015] R N1 R N2 and R N3 Independently for C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl, with one C2 replaced by O 1-6 Alkyl groups or C groups with one CH2 group replaced by an O group 1-6 Hydroxyalkyl;

[0016] n is independently 0-8;

[0017] n1 and n2 are independently 0-10;

[0018] The heteroatoms in the heterocyclic alkyl, heteroaryl, and heterocyclic alkenyl groups are one or more of N, O, and S, and the number of heteroatoms is 1 to 4.

[0019] In some implementation schemes, R 1 and R 2 In, the C 8-24 Alkyl groups are independently , or i1 is 1-11, i2, j2, k2, i3, j3, k3 and l3 are independently 0-10, i2+j2+k2≤20, i3+j3+k3+l3≤17.

[0020] In some implementation schemes, R 1 and R 2 In, the C 8-20 The alkenyl group is a straight-chain or branched C. 8-20 Alkenyl group; preferably straight-chain or branched C 12-18 Alkenyl; more preferably, a straight-chain C with two alkene bonds. 18 Alkenyl group.

[0021] In some implementation schemes, R 1 and R 2 In, the C 8-20 The alkynyl group is a straight-chain or branched C. 8-20 Alkyne group; preferably straight-chain or branched C 12-18 Alkyne group.

[0022] In some implementations, n1 and n2 are independently 0, 1, or 3.

[0023] In some implementations, n1 and n2 are the same.

[0024] In some implementation schemes, R 1 and R 2 same.

[0025] In some implementation schemes, R a R b R c R d R e and R f Independently for H and C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Hydroxyalkyl, with one C2 replaced by O 1-4 Alkyl or C 3-4 Cycloalkyl.

[0026] In some implementation schemes, R a R b And the nitrogen atom connected to it forms , , , 、Being R N1 Replacement 、Being R N1 Replacement 、Being R N2 Replacement Or be R N3 Replacement N* represents the relationship between R and R. a and R b Connected N.

[0027] In some implementation schemes, R c R d And the nitrogen atom connected to it forms , , , 、Being R N1 Replacement 、Being R N1 Replacement 、Being R N2 Replacement Or be R N3 Replacement N* represents the relationship between R and R. c and R d Connected N.

[0028] In some implementation schemes, R N1 R N2 and R N3 Independently for C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Hydroxyalkyl, with one C2 replaced by O 1-6 Alkyl groups or C groups with one CH2 group replaced by an O group 1-6 Hydroxyalkyl.

[0029] In some implementation schemes, R 3 and R 4 Independently

[0030] , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or ; where each y is independently 0-4.

[0031] In some implementation schemes, R 1 and R 2 Independently , Or C 8-20 Alkenyl group, preferably, i1 is 9, 10 or 11; i2 is 0, j2 and k2 are independently 6-9, for example 8; the C 8-20 The alkenyl group is a C group with two alkene bonds. 16-18 alkenyl, for example .

[0032] In some implementation schemes, R 1 and R 2 Independently , , , or .

[0033] In some implementation schemes, R a R b R c R d R e and R f Independently for C 1-4 Alkyl or C 1-6 Hydroxyalkyl, such as methyl or .

[0034] In some implementation schemes, R 3 and R 4 Independently , , , or ;

[0035] Or, R 3 and R 4 Connected, forming .

[0036] In some implementation schemes, R 3 and R 4 same.

[0037] In some embodiments, the compound as shown in Formula I is , , , , , , , , , , , , , , , , or .

[0038] The present invention also provides the use of a compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of a nucleic acid delivery vector; preferably, the nucleic acid is mRNA.

[0039] The present invention also provides lipid nanoparticles comprising the aforementioned compounds as shown in Formula I or pharmaceutically acceptable salts thereof, phosphate lipids, PEG lipids, and cholesterol.

[0040] In some embodiments, the phospholipid is selected from one or more of DSPC and DOPE, such as DOPE.

[0041] In some embodiments, the PEG lipid is DMG-PEG2000.

[0042] In some embodiments, the lipid nanoparticles have an average particle size of 40-400 nm, preferably 80-150 nm; for example, 110-117 nm.

[0043] In some embodiments, the PDI of the lipid nanoparticles is <0.2, for example, 0.10 to 0.16.

[0044] In some embodiments, the surface potential of the lipid nanoparticles is -20 to 20 mV, preferably -5 to 5 mV; for example, -2.78 to -0.5 mV.

[0045] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is >80%, for example >90%.

[0046] In some embodiments, the lipid nanoparticles contain a molar percentage of 10% to 30% (e.g., 20%) of the compound of Formula I or a pharmaceutically acceptable salt thereof, a molar percentage of 15% to 35% (e.g., 25%) of the phospholipid, a molar percentage of 30% to 65% (e.g., 51%) of the cholesterol, and a molar percentage of 0.5% to 3% (e.g., 2.5%) of the PEG lipid.

[0047] In some embodiments, the nitrogen-to-phosphorus ratio of the lipid nanoparticles is 4 to 10; preferably 6 to 8.

[0048] In some embodiments, the lipid nanoparticles are prepared by a method comprising the following steps: mixing the compound of Formula I or a pharmaceutically acceptable salt thereof, the phosphate lipid, the cholesterol, the PEG lipid and ethanol into a solution, assembling it with a nucleic acid solution to obtain a stock solution, and then concentrating and replacing the solution by ultrafiltration.

[0049] In some embodiments, the nucleic acid solution is an mRNA buffer, such as a citrate-sodium citrate buffer for mRNA; preferably, the pH of the mRNA buffer is 4, and the mRNA concentration of the mRNA buffer is 70-90 μg / ml, for example, 80 μg / ml.

[0050] The lipid nanoparticles are prepared using existing mature technologies. The prepared ethanol mixture and nucleic acid assembly are generally prepared using mature microfluidic control processes, or they can be prepared using other processes such as manual mixing, ultrasonication, reverse phase evaporation, injection, freeze drying, high pressure homogenization, rotary evaporation, emulsification, etc.

[0051] In some embodiments, the volume ratio of the ethanol mixture to the nucleic acid solution is 1:(2.5~3.5), for example 1:3.

[0052] In some embodiments, the lipid nanoparticles are prepared by a microfluidic method in which the lipid concentration of the ethanol mixture is 6-9 mg / ml (e.g., 7.5 mg / ml) and the flow rate is 2-4 mL / min (e.g., 3 mL / min); and the flow rate of the nucleic acid solution is 8-10 mL / min (e.g., 9 mL / min).

[0053] In some embodiments, the ultrafiltration concentration and fluid exchange includes the following steps: diluting the stock solution with 15 to 25 (e.g., 20) times the volume of PBS buffer solution to obtain a diluted solution, which is then concentrated using a 50 kDa ultrafiltration tube; the PBS buffer solution may be a 1X PBS buffer solution.

[0054] Unless otherwise specified, the terms used in this invention have the following meanings:

[0055] The term "alkyl" refers to a straight-chain or branched alkyl group having a specified number of carbon atoms (e.g., C1 to C6). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, etc.

[0056] The term "hydroxyalkyl" refers to a group formed by replacing the aforementioned "alkyl" with one or more hydroxyl groups (e.g., 1, 2 or 3 hydroxyl groups).

[0057] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group that contains one or more carbon-carbon double bonds (e.g., 1, 2, or 3 carbon-carbon double bonds).

[0058] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group that contains one or more carbon-carbon triple bonds (e.g., 1, 2, or 3 carbon-carbon triple bonds).

[0059] The term "heterocyclic alkyl" refers to a cyclic group having a specified number of ring atoms (e.g., 5 to 10), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatom (one or more of N, O, and S), which can be monocyclic, bridged, or spirocyclic, and each ring is saturated. A bridged ring is a polycyclic ring that shares two or more atoms between monocyclic rings. A spirocyclic ring is a polycyclic ring that shares one atom between monocyclic rings. Heterocyclic alkyl groups include, but are not limited to, nitrogen-containing heterocyclic butyl, tetrahydropyrrolyl, tetrahydrofuranyl, morpholinyl, and piperidinyl.

[0060] The term "heteroaryl" refers to a cyclic group having a specified number of ring atoms (e.g., 5 to 10), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatom (one or more of N, O, and S). It can be monocyclic or polycyclic, and at least one ring is aromatic (conforming to Hückel's rule). Heteroaryl groups are linked to other segments of a molecule via aromatic or non-aromatic rings. Heteroaryl groups include, but are not limited to, furanyl, pyrroleyl, thiopheneyl, pyrazolyl, imidazoyl, oxazolyl, thiazoyl, pyridinyl, pyrimidinyl, and indoleyl.

[0061] The term “treatment” refers to any of the following: (1) alleviating one or more biological manifestations of a disease; (2) interfering with one or more points in the biological cascade that triggers the disease; or (3) slowing the development of one or more biological manifestations of a disease.

[0062] The term "prevention" refers to reducing the risk of developing a disease.

[0063] The positive and progressive effects of the present invention are as follows: the liposome nanoparticles prepared by ionizable lipids provided by the present invention are (1) easy to degrade and metabolized quickly; (2) have good transfection effect; (3) have significant spleen targeting, which helps to enhance the immune effect; and (4) have low side effects. Attached Figure Description

[0064] Figure 1 Fluorescence imaging images of lipid nanoparticles transfected with eGFP-mRNA prepared in Examples 1-7 and positive control lipo / eGFP-mRNA.

[0065] Figure 2 To detect the positive rate of LNP by flow cytometry

[0066] Figure 3 Images of in vivo animal and organ imaging in animal experiments.

[0067] Figure 4 The results show the fluorescence quantitative distribution of mRNA in various organs. Detailed Implementation

[0068] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Unless otherwise specified, the methods and techniques involved in the following examples are conventional methods.

[0069] Unless otherwise specified, all experimental reagents, instruments, materials, and consumables involved in the following examples are commercially available, standard products.

[0070] The following table is a list of English abbreviations and their Chinese translations:

[0071] .

[0072] The following table lists the cell lines used:

[0073] .

[0074] Example 1

[0075] .

[0076] Synthesis of compound (2):

[0077] A magnetic stir bar was added to a dry, single-necked reaction flask, followed by the sequential addition of 9-heptadecyl alcohol (2 eq, 20 mmol), bis-Boc-cysteine ​​(1 eq, 10 mmol), DCC (2.5 eq, 25 mmol), DMAP (0.2 eq, 4 mmol), and 20 mL of acetonitrile. The mixture was stirred at room temperature for four hours after the addition was complete. The reaction solution was filtered, extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated. The product was separated by silica gel column chromatography; it was a white to pale yellow solid with a yield of approximately 80%. Mass spectrum: m / z = 916.66.

[0078] Synthesis of compound (3):

[0079] A magnetic stir bar was added to the reaction flask, and compound (2) (1 eq, 10 mmol) was added. The mixture was dissolved at room temperature by stirring with 10 mL of DCM. The reaction was placed in an ice bath, and 2 mL of a prepared trifluoroacetic acid / DCM solution was added dropwise. After the addition was complete, the mixture was allowed to return to room temperature and stirred continuously for 4 hours. The reaction solution was diluted with dichloromethane, and the pH was adjusted to alkaline with excess saturated sodium bicarbonate solution. The solution was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The product was separated from the concentrate using a silica gel column chromatography. The product was a white to pale yellow solid with a yield of approximately 80%. Spectrum: Mass spectrum m / z = 716.56.

[0080] Synthesis of compound (4):

[0081] A magnetic stir bar was added to a dry reaction flask, followed by the sequential addition of compound (3) (1 eq, 10 mmol), (2-bromoethyl)dimethylamine (2.2 eq, 22 mmol), potassium carbonate (6.6 eq, 66 mmol), cesium carbonate (3.3 eq, 33 mmol), and potassium iodide (0.2 eq, 2 mmol), and DMF. The mixture was stirred at room temperature for 8 h. Water was added to dissolve the salts, and the mixture was extracted with dichloromethane. The extract was washed with saturated brine and dried over anhydrous sodium sulfate. After concentration, the product was separated by silica gel column chromatography; it was a pale yellow solid with a yield of approximately 30%. Spectrum: Mass spectrum m / z = 858.70. Nuclear magnetic resonance spectra: 1H: 0.98 (t, 12H), 1.28 (m, 48H), 1.56 (m, 8H), 2.30 (s, 12H), 2.48–2.91 (m, 8H), 2.91–3.30 (m, 4H), 3.59 (dt, 2H), 4.93 (m, 2H); 13C: 14.16, 22.71, 25.25, 29.30, 29.54, 31.90, 33.86, 34.04, 45.29, 45.63, 58.09, 58.96, 75.65, 173.16.

[0082] Weigh out compound (4) (12.8 mg), DOPE (14.9 mg), cholesterol (14.9 mg), and DMG-PEG2000 (3.76 mg), and dissolve them in ethanol (6 mL) for later use. Take 1 mg / mL of eGFP-mRNA product and dilute it with citrate-sodium citrate buffer (100 mM, pH = 4) to prepare an 80 μg / mL mRNA buffer. Measure the organic phase and aqueous phase at a 1:3 volume ratio, and prepare lipid nanoparticles by controlling the nitrogen-phosphorus ratio to 6-8 using a microfluidic method (lipid fraction concentration 7.5 mg / mL, flow rate 3 mL / min; nucleic acid fraction concentration 80 μg / mL, flow rate 9 mL / min). Dilute the prepared stock solution 20 times with 1X PBS buffer solution. Concentrate the diluted solution using a 50 kDa ultrafiltration tube. The concentrate can be diluted in 2-8 mL of water. o Store under C conditions for one week.

[0083] The prepared lipid nanoparticles were diluted 10-fold by volume, and the encapsulation efficiency and actual encapsulated nucleic acid concentration within the lipid nanoparticles were determined using the Thermo Fisher Quant-iT RiboGreen RNA Quantitative Reduction Kit. The particle size, particle size distribution, and surface potential were measured using a Malvern Nano-ZSZEN 3600 particle size analyzer. The measured average particle size was 112 nm, the PDI was 0.10, the surface potential was approximately -0.5 mV, and the encapsulation efficiency was approximately 95%.

[0084] Example 2

[0085] .

[0086] Synthesis of compound (5):

[0087] A magnetic stir bar was added to a dry, single-necked reaction flask, followed by the sequential addition of 9-heptadecyl alcohol (2 eq, 20 mmol), bis-Boc-cysteine ​​(1 eq, 10 mmol), DCC (2.5 eq, 25 mmol), DMAP (0.2 eq, 4 mmol), and 20 mL of acetonitrile. The mixture was stirred at room temperature for four hours after the addition was complete. The reaction solution was filtered, extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated. The product was separated by silica gel column chromatography; it was a white to pale yellow solid with a yield of approximately 80%. Mass spectrum: m / z = 916.66.

[0088] Synthesis of compound (6):

[0089] A magnetic stir bar was added to the reaction flask, and compound (2) (1 eq, 10 mmol) was added. The mixture was dissolved at room temperature by stirring with 10 mL of DCM. The reaction was placed in an ice bath, and a prepared trifluoroacetic acid / DCM solution (2 mol / 2 mL) was added dropwise. After the addition was complete, the mixture was allowed to return to room temperature and stirred continuously for 4 hours. The reaction solution was diluted with dichloromethane, and the pH was adjusted to alkaline with excess saturated sodium bicarbonate solution. The solution was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The product was separated from the concentrate using a silica gel column chromatography. The product was a white to pale yellow solid with a yield of approximately 80%. Spectrum: Mass spectrum m / z = 716.56.

[0090] Synthesis of compound (7):

[0091] A magnetic stir bar was added to a dry reaction flask, followed by the sequential addition of compound (6) (1 eq, 10 mmol), dimethylaminopropionic acid (2.2 eq, 22 mmol), DCC (2.5 eq, 25 mmol), DMAP (0.2 eq, 4 mmol), and 20 mL LDM. The mixture was stirred at room temperature for 6 hours after addition. The reaction solution was filtered, extracted with dichloromethane, and then washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated by silica gel column chromatography as a white to pale yellow solid, with a yield of approximately 50%. Mass spectrum: m / z = 914.69. Nuclear magnetic resonance (NMR) spectra: 1H: 0.91 (t, 12H), 1.29 (m, 48H), 1.57 (dt, 8H), 2.35 (s, 12H), 2.46 (m, 4H), 2.64 (m, 4H), 3.23 (t, 4H), 4.88 (m, 2H), 4.93 (m, 2H); 13C: 14.18, 22.73, 25.24, 25.31, 29.33, 29.55, 29.59, 31.92, 32.71, 33.87, 41.63, 44.57, 51.90, 54.92, 76.33, 170.23, 172.32.

[0092] Weigh out compound (7) (13.7 mg), DOPE (14.89 mg), cholesterol (14.9 mg), and DMG-PEG2000 (3.76 mg), and dissolve in ethanol (6 mL). Take 1 mg / mL of eGFP-mRNA product and dilute with citrate-sodium citrate buffer (100 mM, pH = 4) to prepare an 80 μg / mL mRNA buffer. Measure the organic phase and aqueous phase at a 1:3 volume ratio and prepare lipid nanoparticles using a microfluidic method (lipid fraction concentration 7.5 mg / mL, flow rate 3 mL / min; nucleic acid fraction concentration 80 μg / mL, flow rate 9 mL / min), controlling the nitrogen-phosphorus ratio at 6-8. Dilute the prepared stock solution 20 times with 1X PBS buffer solution. Concentrate the diluted solution using a 50 kDa ultrafiltration tube. The concentrate can be diluted in 2-8 mL of water. o Store under C conditions for one week.

[0093] The prepared lipid nanoparticles were diluted 10-fold by volume, and the encapsulation efficiency and actual encapsulated nucleic acid concentration within the lipid nanoparticles were determined using the Thermo Fisher Quant-iT RiboGreen RNA Quantitative Reduction Kit. The particle size, particle size distribution, and surface potential were measured using a Malvern Nano-ZSZEN 3600 particle size analyzer. The measured average particle size was 117 nm, the PDI was 0.15, the surface potential was approximately -2.78 mV, and the encapsulation efficiency was approximately 92%.

[0094] Example 3

[0095] .

[0096] Synthesis of compound (3-1):

[0097] A magnetic stir bar was added to a dry, single-necked reaction flask, followed by the sequential addition of hexadecyl alcohol (2 eq, 20 mmol), bis-Boc-cysteine ​​(1 eq, 10 mmol), DCC (2.5 eq, 25 mmol), DMAP (0.2 eq, 4 mmol), and 20 mL of acetonitrile. The mixture was stirred at room temperature for four hours after the addition was complete. The reaction solution was filtered, extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated. The product was separated by silica gel column chromatography; it was a white to pale yellow solid with a yield of approximately 80%. Mass spectrum: m / z = 889.39.

[0098] Synthesis of compound (3-2):

[0099] A magnetic stir bar was added to the reaction flask, and compound (3-1) (1 eq, 10 mmol) was added. The mixture was dissolved at room temperature with 10 mL of DCM. The reaction was placed in an ice bath, and a prepared trifluoroacetic acid / DCM solution (2 mol / 2 mL) was added dropwise. After the addition was complete, the mixture was allowed to return to room temperature and stirred continuously for 4 hours. The reaction solution was diluted with dichloromethane, and the pH was adjusted to alkaline with excess saturated sodium bicarbonate solution. The solution was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The product was separated from the concentrate using a silica gel column chromatography. The product was a white to pale yellow solid with a yield of approximately 80%. Spectrum: Mass spectrum m / z = 689.16.

[0100] Synthesis of compound (3-3):

[0101] A magnetic stir bar was added to a dry reaction flask, followed by the sequential addition of compound (3-2) (1 eq, 10 mmol), dimethylaminopropionic acid (2.2 eq, 22 mmol), DCC (2.5 eq, 25 mmol), DMAP (0.2 eq, 4 mmol), and 20 mL LDM. The mixture was stirred at room temperature for 6 hours after the addition was complete. The reaction solution was filtered, extracted with dichloromethane, and then washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated by silica gel column chromatography; it was a white to pale yellow solid with a yield of approximately 50%. Mass spectrum: m / z = 887.42. Nuclear magnetic resonance (NMR) spectra: 1H: 0.90(t, 6H), 1.28(m, 52H), 1.57(dt, 4H), 2.33(s, 12H), 2.34(t, 4H), 2.51(dd, 4H), 3.20(t, 4H), 4.15(t, 4H), 4.89(m, 2H); 13C: 14.17, 22.74, 25.92, 28.55, 29.30, 29.41, 29.59, 29.66, 29.71, 29.75, 31.97, 32.74, 33.87, 41.08, 44.58, 51.48, 54.96, 66.90, 170.70, 172.54.

[0102] Weigh out compound (3-3) (13.3 mg), DOPE (14.9 mg), cholesterol (14.9 mg), and DMG-PEG2000 (3.76 mg), and dissolve in ethanol (6 mL). Take 1 mg / mL of eGFP-mRNA product and dilute with citrate-sodium citrate buffer (100 mM, pH = 4) to prepare an 80 μg / mL mRNA buffer. Measure the organic phase and aqueous phase at a 1:3 volume ratio and prepare lipid nanoparticles using a microfluidic method (lipid fraction concentration 7.5 mg / mL, flow rate 3 mL / min; nucleic acid fraction concentration 80 μg / mL, flow rate 9 mL / min), controlling the nitrogen-phosphorus ratio at 6-8. Dilute the prepared stock solution 20 times with 1X PBS buffer. Concentrate the diluted solution using a 50 kDa ultrafiltration tube. The concentrate can be diluted within 2-8 seconds. o Store under C conditions for one week.

[0103] The prepared lipid nanoparticles were diluted 10-fold by volume, and the encapsulation efficiency and actual encapsulated nucleic acid concentration within the lipid nanoparticles were determined using the Thermo Fisher Quant-iT RiboGreen RNA Quantitative Reagent Kit. The particle size, particle size distribution, and surface potential were measured using a Malvern Nano-ZSZEN 3600 particle size analyzer. The measured average particle size was 110 nm, the PDI was 0.15, the surface potential was approximately -2.4 mV, and the encapsulation efficiency was approximately 95%.

[0104] Example 4

[0105] .

[0106] Synthesis of compound (4-1):

[0107] A magnetic stir bar was added to a dry, single-necked reaction flask, followed by the sequential addition of octadecyl alcohol (2 eq, 20 mmol), bis-Boc-cysteine ​​(1 eq, 10 mmol), DCC (2.5 eq, 25 mmol), DMAP (0.2 eq, 4 mmol), and 20 mL of acetonitrile. The mixture was stirred at room temperature for four hours after the addition was complete. The reaction solution was filtered, extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated. The product was separated by silica gel column chromatography; it was a white to pale yellow solid with a yield of approximately 80%. Mass spectrum: m / z = 945.5.

[0108] Synthesis of compound (4-2):

[0109] A magnetic stir bar was added to the reaction flask, and compound (4-1) (1 eq, 10 mmol) was added and dissolved with DCM (10 mL) at room temperature. The reaction was placed in an ice bath, and a prepared trifluoroacetic acid / DCM solution (2 mol / 2 mL) was added dropwise. After the addition was complete, the mixture was brought back to room temperature and stirred continuously for 4 hours. The reaction solution was diluted with dichloromethane, and the pH was adjusted to alkaline with excess saturated sodium bicarbonate solution. The solution was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The product was separated from the concentrate using a silica gel column chromatography, and was a white to pale yellow solid with a yield of approximately 80%. Spectrum: Mass spectrum m / z = 745.26.

[0110] Synthesis of compound (4-3):

[0111] A magnetic stir bar was added to a dry reaction flask, followed by the sequential addition of compound (4-2) (1 eq, 10 mmol), dimethylaminopropionic acid (2.2 eq, 22 mmol), DCC (2.5 eq, 25 mmol), DMAP (0.2 eq, 4 mmol), and 20 mL LDM. The mixture was stirred at room temperature for 6 hours after the addition was complete. The reaction solution was filtered, extracted with dichloromethane, and then washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated by silica gel column chromatography; it was a white to pale yellow solid with a yield of approximately 50%. Mass spectrum: m / z = 943.53. Nuclear magnetic resonance (NMR) spectra: ¹H: 0.90(t, 6H), 1.28(m, 56H), 1.57(dt, 4H), 2.33(s, 12H), 2.34(t, 4H), 2.51(dd, 4H), 3.20(t, 4H), 4.15(t, 4H), 4.89(m, 2H); ¹³C: 14.18, 22.74, 25.92, 28.55, 29.30, 29.41, 29.59, 29.66, 29.72, 29.76, 31.97, 32.72, 41.08, 44.57, 51.48, 54.94, 64.94, 65.91, 170.69, 172.50.

[0112] Weigh out compound (4-3) (14.14 mg), DOPE (14.9 mg), cholesterol (14.9 mg), and DMG-PEG2000 (3.76 mg), and dissolve in ethanol (6 mL). Take 1 mg / mL of eGFP-mRNA product and dilute with citrate-sodium citrate buffer (100 mM, pH = 4) to prepare an 80 μg / mL mRNA buffer. Measure the organic phase and aqueous phase at a 1:3 volume ratio and prepare lipid nanoparticles using a microfluidic method (lipid fraction concentration 7.5 mg / mL, flow rate 3 mL / min; nucleic acid fraction concentration 80 μg / mL, flow rate 9 mL / min), controlling the nitrogen-phosphorus ratio at 6-8. Dilute the prepared stock solution 20 times with 1X PBS buffer. Concentrate the diluted solution using a 50 kDa ultrafiltration tube. The concentrate can be obtained in 2-8 seconds. o Store under C conditions for one week.

[0113] The prepared lipid nanoparticles were diluted 10-fold by volume, and the encapsulation efficiency and actual encapsulated nucleic acid concentration within the lipid nanoparticles were determined using the Thermo Fisher Quant-iT RiboGreen RNA Quantitative Reduction Kit. The particle size, particle size distribution, and surface potential were measured using a Malvern Nano-ZSZEN 3600 particle size analyzer. The measured average particle size was 115 nm, the PDI was 0.16, the surface potential was approximately -2.0 mV, and the encapsulation efficiency was approximately 90%.

[0114] Example 5

[0115] .

[0116] Synthesis of compound (5-1):

[0117] A magnetic stir bar was added to a dry, single-necked reaction flask, followed by the sequential addition of octadecyl alcohol (2 eq, 20 mmol), bis-Boc-cysteine ​​(1 eq, 10 mmol), DCC (2.5 eq, 25 mmol), DMAP (0.2 eq, 4 mmol), and 20 mL of acetonitrile. The mixture was stirred at room temperature for four hours after the addition was complete. The reaction solution was filtered, extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated. The product was separated by silica gel column chromatography; it was a white to pale yellow solid with a yield of approximately 80%. Mass spectrum: m / z = 945.5.

[0118] Synthesis of compound (5-2):

[0119] A magnetic stir bar was added to the reaction flask, and compound (4-1) (1 eq, 10 mmol) was added and dissolved with DCM (10 mL) at room temperature. The reaction was placed in an ice bath, and a prepared trifluoroacetic acid / DCM solution (2 mol / 2 mL) was added dropwise. After the addition was complete, the mixture was brought back to room temperature and stirred continuously for 4 hours. The reaction solution was diluted with dichloromethane, and the pH was adjusted to alkaline with excess saturated sodium bicarbonate solution. The solution was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The product was separated from the concentrate using a silica gel column chromatography, and was a white to pale yellow solid with a yield of approximately 80%. Spectrum: Mass spectrum m / z = 745.26.

[0120] Synthesis of compound (5-3):

[0121] A magnetic stir bar was added to a dry reaction flask, followed by the sequential addition of compound (4-2) (1 eq, 10 mmol), dimethylaminovaleric acid (2.2 eq, 22 mmol), DCC (2.5 eq, 25 mmol), DMAP (0.2 eq, 4 mmol), and 20 mL LDM. The mixture was stirred at room temperature for 6 hours after the addition was complete. The reaction solution was filtered, extracted with dichloromethane, and then washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated by silica gel column chromatography; it was a white to pale yellow solid with a yield of approximately 50%. Mass spectrum: m / z = 999.64. Nuclear magnetic resonance (NMR) spectra: ¹H: 0.90(t, 6H), 1.28(m, 60H), 1.57(dt, 4H), 1.70(m, 8H), 2.27(s, 12H), 2.34(m, 8H), 3.22(t, 4H), 4.17(t, 4H), 4.87(m, 2H); ¹³C: 14.20, 22.76, 23.39, 25.92, 27.07, 28.51, 29.30, 29.43, 29.60, 29.68, 29.73, 29.78, 31.99, 36.11, 40.90, 45.47, 51.80, 59.26 66.21, 170.62, 172.90.

[0122] Weigh out compound (5-3) (15.25 mg), DOPE (14.9 mg), cholesterol (14.9 mg), and DMG-PEG2000 (3.76 mg), and dissolve in ethanol (6 mL). Take 1 mg / mL of eGFP-mRNA product and dilute with citrate-sodium citrate buffer (100 mM, pH = 4) to prepare an 80 μg / mL mRNA buffer. Measure the organic phase and aqueous phase at a 1:3 volume ratio and prepare lipid nanoparticles using a microfluidic method (lipid fraction concentration 7.5 mg / mL, flow rate 3 mL / min; nucleic acid fraction concentration 80 μg / mL, flow rate 9 mL / min), controlling the nitrogen-phosphorus ratio at 6-8. Dilute the prepared stock solution 20 times with 1X PBS buffer. Concentrate the diluted solution using a 50 kDa ultrafiltration tube. The concentrate can be obtained in 2-8 seconds. o Store under C conditions for one week.

[0123] The prepared lipid nanoparticles were diluted 10-fold by volume, and the encapsulation efficiency and actual encapsulated nucleic acid concentration within the lipid nanoparticles were determined using the Thermo Fisher Quant-iT RiboGreen RNA Quantitative Reduction Kit. The particle size, particle size distribution, and surface potential were measured using a Malvern Nano-ZSZEN 3600 particle size analyzer. The measured average particle size was 115 nm, the PDI was 0.10, the surface potential was approximately -1.2 mV, and the encapsulation efficiency was approximately 90%.

[0124] Example 6

[0125] .

[0126] Synthesis of compound (6-1):

[0127] A magnetic stir bar was added to a dry, single-necked reaction flask, followed by the sequential addition of 9-heptadecyl alcohol (2 eq, 20 mmol), bis-Boc-cysteine ​​(1 eq, 10 mmol), DCC (2.5 eq, 25 mmol), DMAP (0.2 eq, 4 mmol), and 20 mL of acetonitrile. The mixture was stirred at room temperature for four hours after the addition was complete. The reaction solution was filtered, extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated. The product was separated by silica gel column chromatography; it was a white to pale yellow solid with a yield of approximately 80%. Mass spectrum: m / z = 916.66.

[0128] Synthesis of compound (6-2):

[0129] A magnetic stir bar was added to the reaction flask, and compound (2) (1 eq, 10 mmol) was added. The mixture was dissolved at room temperature by stirring with 10 mL of DCM. The reaction was placed in an ice bath, and a prepared trifluoroacetic acid / DCM solution (2 mol / 2 mL) was added dropwise. After the addition was complete, the mixture was allowed to return to room temperature and stirred continuously for 4 hours. The reaction solution was diluted with dichloromethane, and the pH was adjusted to alkaline with excess saturated sodium bicarbonate solution. The solution was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The product was separated from the concentrate using a silica gel column chromatography. The product was a white to pale yellow solid with a yield of approximately 80%. Spectrum: Mass spectrum m / z = 716.56.

[0130] Synthesis of compound (6-3):

[0131] A magnetic stir bar was added to a dry reaction flask, followed by the sequential addition of compound (6) (1 eq, 10 mmol), dimethylaminopropionic acid (2.2 eq, 22 mmol), DCC (2.5 eq, 25 mmol), DMAP (0.2 eq, 4 mmol), and 20 mL of DMF. The mixture was stirred at room temperature for 6 hours after the addition was complete. The reaction solution was filtered, extracted with dichloromethane, and then washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated by silica gel column chromatography; it was a white to pale yellow solid with a yield of approximately 50%. Mass spectrum: m / z = 887.42. Nuclear magnetic resonance spectra: 1H: 0.88 (t, 12H), 1.26 (m, 48H), 1.55 (dt, 8H), 2.34 (s, 12H), 2.99 (s, 4H), 3.21 (dd, 4H), 4.90 (m, 4H); 13C: 14.15, 22.69, 22.71, 25.24, 25.31, 29.28, 29.31, 29.50, 29.53, 31.88, 33.85, 41.27, 46.07, 51.51, 62.97, 170.10, 170.63.

[0132] Weigh out compound (6-3) (13.29 mg), DOPE (14.89 mg), cholesterol (14.9 mg), and DMG-PEG2000 (3.76 mg), and dissolve in ethanol (6 mL). Take 1 mg / mL of eGFP-mRNA product and dilute with citrate-sodium citrate buffer (100 mM, pH = 4) to prepare an 80 μg / mL mRNA buffer. Measure the organic phase and aqueous phase at a 1:3 volume ratio and prepare lipid nanoparticles using a microfluidic method (lipid fraction concentration 7.5 mg / mL, flow rate 3 mL / min; nucleic acid fraction concentration 80 μg / mL, flow rate 9 mL / min), controlling the nitrogen-phosphorus ratio at 6-8. Dilute the prepared stock solution 20 times with 1X PBS buffer. Concentrate the diluted solution using a 50 kDa ultrafiltration tube. The concentrate can be obtained in 2-8 seconds. o Store under C conditions for one week.

[0133] The prepared lipid nanoparticles were diluted 10-fold by volume, and the encapsulation efficiency and actual encapsulated nucleic acid concentration within the lipid nanoparticles were determined using the Thermo Fisher Quant-iT RiboGreen RNA Quantitative Reduction Kit. The particle size, particle size distribution, and surface potential were measured using a Malvern Nano-ZSZEN 3600 particle size analyzer. The measured average particle size was 117 nm, the PDI was 0.15, the surface potential was approximately -2.78 mV, and the encapsulation efficiency was approximately 92%.

[0134] Example 7

[0135] .

[0136] Synthesis of compound (7-1):

[0137] A magnetic stir bar was added to a dry, single-necked reaction flask, followed by the sequential addition of octadecyl alcohol (1.2 eq, 12 mmol), bis-Boc-cysteine ​​(1 eq, 10 mmol), DCC (1.5 eq, 15 mmol), DMAP (0.1 eq, 1 mmol), and 10 mL of acetonitrile. The mixture was stirred at room temperature for four hours after the addition was complete. The reaction solution was filtered, extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated. The product was separated by silica gel column chromatography; it was a white to pale yellow solid with a yield of approximately 70%. Mass spectrum: m / z = 693.01.

[0138] Synthesis of compound (7-2):

[0139] A magnetic stir bar was added to a dry single-necked reaction flask, followed by the sequential addition of 9-17 alcohol (1.2 eq, 12 mmol), compound (7-1) (1 eq, 10 mmol), DCC (1.5 eq, 15 mmol), DMAP (0.1 eq, 1 mmol), and 10 mL of acetonitrile. The mixture was stirred at room temperature for four hours after the addition was complete. The reaction solution was filtered, extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated. The product was separated by silica gel column chromatography; it was a white to pale yellow solid with a yield of approximately 80%. Mass spectrum: m / z = 931.47.

[0140] Synthesis of compound (7-3):

[0141] A magnetic stir bar was added to the reaction flask, and compound (7-2) (1 eq, 10 mmol) was added. The mixture was dissolved at room temperature with 10 mL of DCM. The reaction was placed in an ice bath, and a prepared trifluoroacetic acid / DCM solution (2 mol / 2 mL) was added dropwise. After the addition was complete, the mixture was allowed to return to room temperature and stirred continuously for 4 hours. The reaction solution was diluted with dichloromethane, and the pH was adjusted to alkaline with excess saturated sodium bicarbonate solution. The solution was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The product was separated from the concentrate using a silica gel column chromatography. The product was a white to pale yellow solid with a yield of approximately 80%. Spectrum: Mass spectrum m / z = 731.24.

[0142] Synthesis of compound (7-4):

[0143] A magnetic stir bar was added to a dry reaction flask, followed by the sequential addition of compound (7-3) (1 eq, 10 mmol), dimethylaminopropionic acid (2.2 eq, 22 mmol), DCC (2.5 eq, 25 mmol), DMAP (0.2 eq, 4 mmol), and 20 mL LDM. The mixture was stirred at room temperature for 6 hours after the addition was complete. The reaction solution was filtered, extracted with dichloromethane, and then washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated by silica gel column chromatography; it was a white to pale yellow solid with a yield of approximately 50%. Mass spectrum: m / z = 929.50. Nuclear magnetic resonance (NMR) spectra: ¹H: 0.91 (t, 9H), 1.26 (m, 52H), 1.56 (m, 4H), 1.84 (tt, 2H), 2.28 (s, 12H), 2.39 (m, 8H), 2.99 (s, 4H), 3.21 (dd, 4H), 4.85 (m, 2H), 4.92 (m, 2H); ¹³C: 14.17, 22.74, 22.94, 25.27, 25.30, 29.36, 29.57, 29.62, 31.93, 31.96, 33.86, 34.63, 41.32, 45.24, 52.27, 58.82. 170.35, 172.91.

[0144] Weigh out compound (7-4) (13.93 mg), DOPE (14.89 mg), cholesterol (14.9 mg), and DMG-PEG2000 (3.76 mg), and dissolve in ethanol (6 mL). Take 1 mg / mL of eGFP-mRNA product and dilute with citrate-sodium citrate buffer (100 mM, pH = 4) to prepare an 80 μg / mL mRNA buffer. Measure the organic phase and aqueous phase at a 1:3 volume ratio and prepare lipid nanoparticles using a microfluidic method (lipid fraction concentration 7.5 mg / mL, flow rate 3 mL / min; nucleic acid fraction concentration 80 μg / mL, flow rate 9 mL / min), controlling the nitrogen-phosphorus ratio at 6-8. Dilute the prepared stock solution 20 times with 1X PBS buffer. Concentrate the diluted solution using a 50 kDa ultrafiltration tube. The concentrate can be obtained in 2-8 seconds. o Store under C conditions for one week.

[0145] The prepared lipid nanoparticles were diluted 10-fold by volume, and the encapsulation efficiency and actual encapsulated nucleic acid concentration within the lipid nanoparticles were determined using the Thermo Fisher Quant-iT RiboGreen RNA Quantitative Reduction Kit. The particle size, particle size distribution, and surface potential were measured using a Malvern Nano-ZSZEN 3600 particle size analyzer. The measured average particle size was 117 nm, the PDI was 0.15, the surface potential was approximately -2.78 mV, and the encapsulation efficiency was approximately 92%.

[0146] Example 8: Evaluation of in vitro transfection of lipid nanoparticles using fluorescence microscopy

[0147] HeLa cells were seeded in 96-well plates (approximately 15,000 cells / well), with 100 μL of freshly prepared cell culture medium (DMEM in 10% FBS) added to each well. The plates were incubated at 37°C. o The cells were cultured at 5% carbon dioxide concentration for 24 hours until the cell density reached approximately 70%. The lipid nanoparticles prepared in Examples 1-7 were seeded into 96-well plates with 0.1 μg / well of actual encapsulated eGFP-mRNA. Simultaneously, 0.1 μg of eGFP-mRNA was transfected into each well using commercially available Lipofectamine 3000 as a positive control (the transfection procedure was performed according to the manufacturer's instructions). The transfection status of the lipid nanoparticles was observed using a fluorescence microscope after 24 hours. Figure 1 The images show fluorescence imaging of the lipid nanoparticles / eGFP-mRNA prepared in Examples 1-7, and the positive control lipo / eGFP-mRNA, respectively. Green indicates that the lipid nanoparticles successfully transfected eGFP-mRNA into cells and expressed eGFP green fluorescent protein. ImageJ software was used to quantify the specific transfection values ​​for each example; the specific results are shown in Table 1.

[0148] Table 1

[0149]

[0150] Example 9: Evaluation of the positive rate of in vitro transfection of lipid nanoparticles by flow cytometry

[0151] Cell transfection: Collect 1× 10⁻⁶ cells one day in advance. 5100,000 HeLa cells in the logarithmic growth phase were seeded into 24-well cell culture plates, and DMEM medium containing antibiotics + 10% FBS was added to 0.5 mL. The cells were mixed and incubated overnight in a cell culture incubator at 37 °C and 5% CO2. On the second day, the lipid nanoparticles prepared in Example 2 were seeded into 24-well plates with 1 μg / well of actual encapsulated eGFP-mRNA. The commercial cationic lipid SM-102 was used as a positive control, and the untransfected group was set as a negative control. The cells were mixed by cross-shaking. The cell culture plates were incubated in a cell culture incubator at 37 °C and 5% CO2.

[0152] Flow cytometry assay: After 24 h of transfection incubation, cells were washed with 0.5 mL PBS, and 80 μL of trypsin was added to each well to digest the cells. An equal volume of complete culture medium was added to stop the digestion, and the cells were collected into 1.5 mL centrifuge tubes. The centrifuge tubes were then washed with 500 μL PBS, centrifuged at 4 ℃ and 800 rpm for 3 min, and the supernatant was carefully discarded after centrifugation. The cells were resuspended in 300 μL PBS, passed through a 300-mesh sieve, and then analyzed by flow cytometry. Figure 2 The transfection positivity rates of LNP prepared in Example 2, the positive control SM-102, and the negative control group in HeLa cells were respectively shown. Flow cytometry results showed that the positivity rate of the negative control group was 0.175%, the positivity rate of the SM-102 group was 93.402%, and the positivity rate of Example 2 was 95.037%, indicating that most of the mRNA was transfected into the cells and expressed.

[0153] Example 10 Animal Experiment

[0154] Experimental materials: C57BL6 mice (6 weeks old)

[0155] Following the method in Example 2, eGFP-mRNA was replaced with Luc-mRNA to prepare LNPs encapsulated with Luc-mRNA. The LNPs were concentrated to a concentration of 200 μg / mL, and the administration volume was 50 μL. Each mouse was injected once via the tail vein. The overall fluorescence intensity of the mice was observed using an in vivo imaging system at 6h, 12h, 24h, and 48h (30 min prior to injection of Luc luminescent substrate fluorescein potassium solution). After 48h, the mice were sacrificed, and their major organs (heart, liver, spleen, lungs, and kidneys) were imaged and quantitatively analyzed using fluorescence. Animal experimental results showed that SM-102 was mainly expressed in the liver, with Example 2 demonstrating a clear spleen-targeting trend, and very low expression levels in other organs. Animal in vivo imaging and organ imaging are as follows: Figure 3 As shown, the quantitative fluorescence results of mRNA distribution in various organs are as follows: Figure 4 As shown in Table 2.

[0156] Table 2

[0157]

[0158] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but all such changes and modifications fall within the scope of protection of the present invention.

Claims

1. A compound as shown in Formula I or a pharmaceutically acceptable salt thereof: ； in, R 1 and R 2 Independently for C 8-24 Alkyl, C 8-20 alkenyl or C 8-20 alkynyl group, the C 8-20 The number of alkene bonds in the alkenyl group is 1, 2, or 3; the C 8-20 The number of alkyne bonds in an alkyne group can be 1, 2, or 3; R 3 and R 4 Independently or ; Or, R 3 and R 4 Connected, forming or ; R a R b R c R d R e and R f H and C independently 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl, C 3-10 cycloalkyl, or a C with one CH2 group replaced by an O group 1-6 alkyl; Or, R a R b The nitrogen atom attached to it forms 3-10-membered heterocyclic alkyl groups, 5-6-membered heteroaryl groups, and 5-6-membered heterocyclic alkenyl groups, which are then bonded by R. N1 Substituted 3-10 membered heterocyclic alkyl groups, R N2 Substituted 5-6 heteroaryl groups or R N3 Substituted 5-6 membered heterocyclic alkenyl groups; Or R c R d The nitrogen atom attached to it forms 3-10-membered heterocyclic alkyl groups, 5-6-membered heteroaryl groups, and 5-6-membered heterocyclic alkenyl groups, which are then bonded by R. N1 Substituted 3-10 membered heterocyclic alkyl groups, R N2 Substituted 5-6 heteroaryl groups or R N3 Substituted 5-6 membered heterocyclic alkenyl groups; R N1 R N2 and R N3 Independently for C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl, with one C2 replaced by O 1-6 Alkyl groups or C groups with one CH2 group replaced by an O group 1-6 Hydroxyalkyl; n is independently 0-8; n1 and n2 are independently 0-10; The heteroatoms in the heterocyclic alkyl, heteroaryl, and heterocyclic alkenyl groups are one or more of N, O, and S, and the number of heteroatoms is 1 to 4.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or more of the following conditions: (1) R 1 and R 2 In, the C 8-24 Alkyl groups are independently , or i1 is 1-11, i2, j2, k2, i3, j3, k3 and l3 are independently 0-10, i2+j2+k2≤20, i3+j3+k3+l3≤17; (2) R 1 and R 2 In, the C 8-20 The alkenyl group is a straight-chain or branched C. 8-20 Alkenyl group; preferably straight-chain or branched C 12-18 Alkenyl; more preferably, a straight-chain C with two alkene bonds. 18 alkenyl; (3) R 1 and R 2 In, the C 8-20 The alkynyl group is a straight-chain or branched C. 8-20 Alkyne group; preferably straight-chain or branched C 12-18 alkynyl group; (4) n1 and n2 are independently 0, 1 or 3; (5) n1 and n2 are the same; (6) R a R b R c R d R e and R f H and C independently 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Hydroxyalkyl, with one C2 replaced by O 1-4 Alkyl or C 3-4 cycloalkyl; Or, R a R b And the nitrogen atom connected to it forms , , , 、Being R N1 Replacement 、Being R N1 Replacement 、Being R N2 Replacement Or be R N3 Replacement N* represents the relationship between R and R. a and R b Connected N; Or R c R d And the nitrogen atom connected to it forms , , , 、Being R N1 Replacement 、Being R N1 Replacement 、Being R N2 Replacement Or be R N3 Replacement N* represents the relationship between R and R. c and R d Connected N; (7) R N1 R N2 and R N3 Independently for C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Hydroxyalkyl, with one C2 replaced by O 1-6 Alkyl groups or C groups with one CH2 group replaced by an O group 1-6 Hydroxyalkyl; Better location; R 3 and R 4 Independently , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or ; where each y is independently 0-4.

3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or two of the following conditions: (1) R 1 and R 2 Independently , Or C 8-20 Alkenyl group, preferably, i1 is 9, 10 or 11; i2 is 0, j2 and k2 are independently 6-9, for example 8; the C 8-20 The alkenyl group is a C group with two alkene bonds. 16-18 alkenyl, for example ; (2) R a R b R c R d R e and R f Independently for C 1-4 Alkyl or C 1-6 Hydroxyalkyl, such as methyl or .

4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or two of the following conditions: (1) R 1 and R 2 Independently , , , or ; (2) R 3 and R 4 Independently , , , or ; Or, R 3 and R 4 Connected, forming .

5. The compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The compound shown in Formula I is , , , , , , , , , , , , , , , , or .

6. The use of a compound of Formula I as described in any one of claims 1-5, or a pharmaceutically acceptable salt thereof, in the preparation of a nucleic acid delivery vector; preferably, the nucleic acid is mRNA.

7. A lipid nanoparticle, characterized in that, It includes compounds of Formula I as described in any one of claims 1-5, or pharmaceutically acceptable salts of the compounds thereof, phosphate lipids, PEG lipids, and cholesterol.

8. The lipid nanoparticles as described in claim 7, characterized in that, It meets one or more of the following conditions: (1) The phospholipid is selected from one or more of DSPC and DOPE, such as DOPE; (2) The PEG lipid is DMG-PEG2000; (3) The lipid nanoparticles have an average particle size of 40~400nm, preferably 80~150nm; for example, 110~117nm; (4) The PDI of the lipid nanoparticles is <0.2, for example, 0.10~0.16; (5) The surface potential of the lipid nanoparticles is -20~20mV, preferably -5~5mV; for example -2.78~-0.5mV; (6) The encapsulation efficiency of the lipid nanoparticles is >80%, for example >90%; (7) The nitrogen-to-phosphorus ratio of the lipid nanoparticles is 4-10; preferably 6-8; (8) In the lipid nanoparticles, the molar percentage of the compound of Formula I or its pharmaceutically acceptable salt is in the range of 10% to 30% (e.g., 20%), the molar percentage of the phospholipid is in the range of 15% to 35% (e.g., 25%), the molar percentage of the cholesterol is in the range of 30% to 65% (e.g., 51%), and the molar percentage of the PEG lipid is in the range of 0.5% to 3% (e.g., 2.5%). (9) The lipid nanoparticles are prepared by the following method, which includes the following steps: mixing the compound shown in Formula I or its pharmaceutically acceptable salt, the phosphate lipid, the cholesterol, the PEG lipid and ethanol to form an ethanol mixture, and then assembling it with a nucleic acid solution to obtain the stock solution, followed by ultrafiltration concentration and liquid replacement.

9. The lipid nanoparticles as described in claim 8, characterized in that, The preparation method satisfies one or more of the following conditions: (1) The nucleic acid solution is an mRNA buffer, such as citrate-sodium citrate buffer for mRNA; preferably, the pH of the mRNA buffer is 4, and the mRNA concentration of the mRNA buffer is 70~90 μg / ml, such as 80 μg / ml; (2) The assembly is carried out by microfluidic preparation process, manual mixing method, ultrasonic method, reverse phase evaporation method, injection method, freeze drying method and high pressure homogenization, rotary evaporation method or emulsification method; for example, microfluidic preparation process; (3) The ultrafiltration concentration and replacement includes the following steps: diluting the original solution with 15 to 25 (e.g., 20) times the volume of PBS buffer solution to obtain a diluted solution, and concentrating the diluted solution with a 50 kDa ultrafiltration tube; the PBS buffer solution can be a 1X PBS buffer solution; (4) The volume ratio of the ethanol mixture to the nucleic acid solution is 1:(2.5~3.5), for example 1:

3.

10. The lipid nanoparticles as described in claim 9, characterized in that, In the microfluidic preparation process, the lipid concentration of the ethanol mixture is 6-9 mg / ml (e.g., 7.5 mg / ml), and the flow rate is 2-4 mL / min (e.g., 3 mL / min); the flow rate of the nucleic acid solution is 8-10 mL / min (e.g., 9 mL / min).

Images