Benzamide ionizable lipids or salts thereof, nanodelivery systems, and uses thereof

Abstract

This invention discloses a benzamide-based ionizable lipid or its salt, a nanodelivery system, and its applications, the structural formula of which is shown in Formula I: wherein R0 is selected from L-type or D-type amino acid side chain groups (R groups); R1-R6 are each independently selected from substituted or unsubstituted C6-C groups. 24 Alkyl; or, substituted or unsubstituted C6-C 24 Alkenyl; or, substituted or unsubstituted C6-C 24 Alkyne group; R7 is selected from H, substituted or unsubstituted C1-C. 10 Alkyl group; m is selected from integers from 1 to 8; X is selected from O or NH. It can efficiently encapsulate nucleic acid molecules, forming LNPs with uniform particle size and stable structure. Furthermore, the prepared LNPs can effectively promote lysosomal escape of nucleic acid molecules, resulting in high transfection efficiency and good biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a benzamide-based ionizable lipid or its salt, a nanodelivery system, and its applications. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Nucleic acid drugs (including DNA and RNA drugs) are drugs with specific base sequences. DNA drugs include plasmids and antisense oligonucleotides, while RNA drugs include messenger RNA (mRNA) and small interfering RNA (siRNA). Because nucleic acid molecules are readily and rapidly cleared from the bloodstream, the safe and efficient delivery of specific genes into cells is crucial for the therapeutic effect of these drugs.

[0004] Currently, lipid nanoparticle (LNP)-mediated nucleic acid drug delivery is one of the most widely used delivery systems. The LNP system consists of four main classes of lipids: ionizable lipids (or cationic lipids), accessory lipids, cholesterol, and polyethylene glycol-modified lipids (PEG-lipids). Ionizable lipids are a key component of the formulation; they are neutral at physiological pH but positively charged at the acidic pH of the endosomal environment to minimize potential toxicity. However, to date, only 1%–2% of the nucleic acid payload has managed to escape from late endosomal regions into the cytoplasm via LNPs. Therefore, there remains a pressing need to develop ionizable lipids with highly efficient endosomal escape capabilities to improve nucleic acid drug delivery both in vitro and in vivo.

[0005] Ionizable lipids, as multi-component molecules, require precise design of each part to safely and effectively package and deliver nucleic acids. Typically, ionizable lipids consist of three parts: an amino head, a linker, and a hydrophobic tail. The amino head primarily participates in encapsulating nucleic acids, stabilizing LNPs, interacting with cell membranes, and promoting endosome escape. The linker connects the head and tail, influencing the stability and biodegradability of the LNP. The hydrophobic tail affects lipid fluidity and incorporation, thus impacting LNP formation and potency. Modification and optimization of these three parts of ionizable lipids determine the ionization behavior and surface charge of LNPs, further influencing their stability and toxicity. Therefore, while ensuring good endosome escape ability for LNPs prepared from ionizable lipids, it is necessary to develop ionizable lipids with higher biocompatibility and stronger in vivo safety. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a benzamide-based ionizable lipid or its salt, a nanodelivery system, and its applications. The novel benzamide-based ionizable lipid provided by this invention can efficiently encapsulate nucleic acid molecules, forming uniformly sized and structurally stable LNPs. Furthermore, the prepared LNPs effectively promote lysosomal escape of nucleic acid molecules, exhibiting high transfection efficiency and good biocompatibility.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] In a first aspect, the present invention provides a benzamide-based ionizable lipid or a salt thereof, the structural formula of which is shown in Formula I:

[0009]

[0010] R0 is selected from L-type or D-type amino acid side chain groups (R groups);

[0011] R1-R6 are each independently selected from substituted or unsubstituted C6-C. 24 Alkyl; or, substituted or unsubstituted C6-C 24 Alkenyl; or, substituted or unsubstituted C6-C 24 alkynyl group;

[0012] R7 is selected from H, substituted or unsubstituted C1-C. 10 alkyl;

[0013] m is selected from integers from 1 to 8;

[0014] X is selected from O or NH.

[0015] In some embodiments, the amino acids of the side chain groups of R0 are selected from glycine, alanine, proline, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, asparagine, glutamine, lysine, histidine, arginine, aspartic acid, or glutamic acid.

[0016] Preferably, the amino acid of the side chain group of R0 is selected from glycine, alanine, proline, valine, leucine, isoleucine, methionine, lysine, histidine, or arginine.

[0017] More preferably, the amino acid of the side chain group of R0 is leucine or methionine.

[0018] In some embodiments, R1-R6 are each independently selected from substituted or unsubstituted C6-C. 24 alkyl.

[0019] Preferably, R1-R6 are each independently selected from C8-C 14Straight-chain alkyl groups.

[0020] In some embodiments, R7 is H; X is NH; and m is 4.

[0021] In some embodiments, the benzamide-based ionizable lipids are selected from one of the following compounds:

[0022]

[0023]

[0024] In a second aspect, the present invention provides a nanodelivery system comprising the aforementioned ionizable benzamide lipid or its salt.

[0025] In some embodiments, the nanodelivery system further includes one or more of auxiliary lipids, cholesterol and its derivatives, or PEG lipids.

[0026] Preferably, the auxiliary lipid is a phospholipid or a derivative thereof.

[0027] More preferably, the auxiliary lipid is selected from one or more of distearylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), diethyl pyrocarbonate (DEPC), dilauroylphosphatidylcholine (DLPC), phosphatidylcholine (POPC), egg yolk lecithin (EPC), hydrogenated soybean phosphatidylcholine (HSPC), sphingomyelin (SM), or myristoylphosphatidylcholine (DMPC).

[0028] Preferably, the cholesterol derivative is selected from one or more of sterols and their derivatives, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, α-tocopherol, or corticosteroids.

[0029] Preferably, the PEG lipid is selected from one or more of DSPE-PEG, DMG-PEG, DPPE-PEG, or DMA-PEG.

[0030] In some embodiments, the nanodelivery system includes the benzamide-based ionizable lipid or its salt, auxiliary lipid, cholesterol or its derivative or PEG lipid, wherein the molar ratio of the benzamide-based ionizable lipid or its salt, auxiliary lipid, cholesterol or its derivative and PEG lipid is 20-50:20-60:10-40:0.5-10.

[0031] Thirdly, the present invention provides the application of the nanodelivery system in the preparation of nucleic acid drugs.

[0032] In some embodiments, the nucleic acid molecules in the nucleic acid drug are selected from one or more of small interfering RNA (siRNA), messenger RNA (mRNA), microRNA (miRNA), circular mRNA, long non-coding RNA (lncRNA), plasmid DNA, mini circle DNA (mcDNA), antisense oligonucleotides (ASOs), small activating RNA (saRNA), or nucleic acid aptamers.

[0033] Preferably, the nucleic acid molecule is a small interfering RNA (siRNA), a messenger RNA (mRNA), or a circular mRNA.

[0034] In some embodiments, in the nucleic acid drug, the mass ratio of benzamide-type ionizable lipids or their salts to nucleic acid molecules is 1-100:1.

[0035] Preferably, the particle size of the nucleic acid drug is 1-1000 nm.

[0036] Preferably, the nucleic acid drug includes a targeting molecule, which is located in or on the surface of the nucleic acid drug.

[0037] More preferably, the targeting molecule is selected from proteins, peptides, glycoproteins, lipids, small molecules, or nucleic acids. Examples include antibodies, antibody fragments, low-density lipoprotein (LDL), transferrin, asialoglycoprotein, receptor ligands, sialic acid, aptamers, etc.

[0038] Nucleic acid drugs can be prepared using any method known in the art. These methods include (but are not limited to) liposome extrusion, thin-film differentiation, nanoprecipitation, microfluidics, impingement jet mixing, and other methods well known to those skilled in the art.

[0039] Preferably, the method for preparing the nucleic acid drug includes: dissolving one or more of the lipid compound, the auxiliary lipid, the cholesterol, and the PEG lipid in ethanol to obtain a lipid-mixed ethanol phase; fully dispersing nucleic acid molecules in a citrate buffer solution at pH=4 to obtain a drug aqueous phase; rapidly mixing the lipid-mixed ethanol phase and the drug aqueous phase using microfluidics to prepare a solution containing lipid nanoparticles; and then obtaining lipid nanoparticles, i.e., the nucleic acid drug, through steps such as dialysis and ultrafiltration.

[0040] The nucleic acid drug can be used for the prevention and treatment of various diseases in humans and / or animals via oral, rectal, intravenous, intramuscular, vaginal, intranasal, subcutaneous, intraperitoneal, buccal, or by oral, injection, or inhalation.

[0041] Preferably, the nucleic acid drug is used for the prevention and / or treatment of cancer, inflammation, fibrotic diseases, autoimmune diseases, infections, mental illnesses, blood diseases, chromosomal diseases, genetic diseases, connective tissue diseases, digestive diseases, ear, nose and throat diseases, endocrine diseases, eye diseases, reproductive diseases, heart diseases, kidney diseases, lung diseases, metabolic diseases, oral diseases, musculoskeletal diseases, newborn screening, nutritional diseases, parasitic diseases, or skin diseases.

[0042] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows:

[0043] 1. The benzamide-based ionizable lipids provided by this invention, using amino acids (such as methionine and leucine) as linking groups and amide bonds as connecting bonds, exhibit good biodegradability, and the resulting lipid compounds and their salts show lower cytotoxicity. Furthermore, the phenyl groups in these lipid compounds can undergo π-π stacking interactions with bases in nucleic acid molecules, enhancing the interaction between lipid and nucleic acid molecules and facilitating efficient compression of nucleic acid molecules.

[0044] 2. The benzamide-based ionizable lipids provided in this invention are uncharged under physiological conditions (pH = 7.4), but positively charged under acidic conditions. The lipid nanoparticles undergo protonation with changes in endosome pH, resulting in a positively charged lipid compound. This compound, combined with its hydrophobic tail, forms an unstable inverted hexagonal phase with the negatively charged lipid, promoting the fusion of LNPs with the endosome membrane and facilitating the endosome escape of nucleic acid molecules.

[0045] 3. This invention can increase the structural diversity of lipid compounds in existing delivery systems, enrich existing nucleic acid drug delivery systems, and is beneficial to the development and application of nucleic acid drugs. Attached Figure Description

[0046] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0047] Figure 1 The particle size and PDI of the lipid nanoparticles prepared in Example 3 of this invention are shown.

[0048] Figure 2 This is the Zeta potential characterization of the lipid nanoparticles prepared in Example 3 of the present invention.

[0049] Figure 3 It is the encapsulation efficiency of the lipid nanoparticles prepared in Example 3 of this invention.

[0050] Figure 4 This is a transmission electron microscope image of the lipid nanoparticles prepared in Example 3 of this invention.

[0051] Figure 5 This is the result of the transfection efficiency evaluation of the lipid nanoparticles prepared in Example 3 of this invention. Detailed Implementation

[0052] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0053] Terminology Explanation:

[0054] The term "alkyl" refers to a saturated straight-chain or branched hydrocarbon group. In some embodiments, the alkyl group has 6-24 carbons and is also referred to as C6-C24 alkyl. The alkyl group can be straight-chain or branched, and can be substituted by one or more groups selected from halogen, hydroxyl, amino, oxo, alkoxycarbonyl, amide, alkylamide, dialkylamide, nitro, amino, alkylamide, dialkylamide, carboxyl, thioalkyl, and thioalkyl groups.

[0055] The term "alkenyl" refers to a straight-chain, cyclic, or branched hydrocarbon group containing at least one carbon-carbon double bond. In some embodiments, the alkenyl group has 6-24 carbons and is also referred to as a C6-C24 alkenyl group. The alkenyl group may be substituted by one or more substituents selected from those defined above for substituted alkyl groups.

[0056] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group containing at least one carbon-carbon triple bond. In some embodiments, the alkynyl group has 6-24 carbons and is also referred to as a C6-C24 alkynyl group. The alkynyl group may be substituted by one or more substituents selected from those defined above for substituted alkyl groups.

[0057] "Substitution" refers to the independent replacement of one or more hydrogen atoms in a group by a corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions, and those skilled in the art can determine (through experiment or theory) possible substitutions without much effort.

[0058] In the context of this invention, the alkyl, olefin, and alkyne portions as defined herein may further include one or more heteroatoms, for example, carbon in the alkyl, olefin, or alkyne chain may be replaced by heteroatoms such as those selected from nitrogen, oxygen, or sulfur.

[0059] The terms “nucleic acid” and “nucleic acid molecule” are used interchangeably herein. The term refers to polymers of deoxyribonucleotides (DNA), ribonucleotides (RNA), and modified forms thereof, in the form of individual fragments or as components of larger constructs, in the form of straight or branched strands, single-stranded, double-stranded, triple-stranded, or hybrids thereof.

[0060] The structural formulas of compounds L1, L2, L3, L4, M1, M2, M3 and M4 are shown in Table 1.

[0061] Table 1

[0062]

[0063]

[0064] The present invention will be further described below with reference to the embodiments.

[0065] Example 1

[0066]

[0067] Synthesis of compound L2

[0068] Synthesis of Compound 1: N-tert-butoxycarbonyl-1,2-butanediamine (10 mmol), 1-bromodecane (22 mmol), and potassium carbonate (20 mmol) were dissolved in acetonitrile (60 mL), heated to 80 °C, and refluxed with stirring for 72 h. After TLC detection, the solvent was removed by rotary evaporation under reduced pressure. 50 mL of ethyl acetate was added, followed by washing three times with an equal volume of saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate for 30 min, and the solvent was removed by rotary evaporation under reduced pressure. The mixture was then purified by column chromatography (eluent: methanol:dichloromethane, volume ratio = 1:20) to obtain Compound 1 in 74% yield.

[0069] Synthesis of Compound 2: Compound 1 (5 mmol) was dissolved in dichloromethane (30 mL), and trifluoroacetic acid (10 mmol) was added. The mixture was stirred at room temperature for 2 h. After TLC monitoring, the solvent was removed by rotary evaporation under reduced pressure. Saturated potassium carbonate solution was added to adjust the pH to 7. 50 mL of ethyl acetate was added, followed by washing three times with an equal volume of saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate for 30 min, and the solvent was removed by rotary evaporation under reduced pressure to obtain Compound 2 in 95% yield. 1 H NMR(400MHz,Chloroform-d)δ5.21(d,J=6.4Hz,1H),3.11(d,J=5.7Hz,2H),2.43(q,J= 7.9Hz, 4H), 1.49 (s, 4H), 1.44 (s, 12H), 1.31 (d, J = 7.1Hz, 28H), 0.88 (t, J = 6.7Hz, 6H).

[0070] Synthesis of Compound 3: BOC-L-leucine (10 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 20 mmol), and 1-hydroxybenzotriazole (HOBT, 10 mmol) were dissolved in dichloromethane (60 ml), and the compound (8 mmol) was added.

[0071] The reaction was stirred at room temperature for 12 h. After TLC analysis, the solvent was removed by rotary evaporation under reduced pressure. 50 mL of ethyl acetate was added, followed by washing three times with an equal volume of saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate for 30 min, and the solvent was removed by rotary evaporation under reduced pressure. The mixture was then purified by column chromatography (eluent: methanol: dichloromethane, volume ratio = 1:50) to obtain compound 3, with a yield of 67%.

[0072] Synthesis of Compound 4: Compound 3 (5 mmol) was dissolved in dichloromethane (30 mL), and trifluoroacetic acid (10 mmol) was added. The mixture was stirred at room temperature for 2 h. After TLC monitoring, the solvent was removed by rotary evaporation under reduced pressure. Saturated potassium carbonate solution was added to adjust the pH to 7. 50 mL of ethyl acetate was added, followed by washing three times with an equal volume of saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate for 30 min, and the solvent was removed by rotary evaporation under reduced pressure to obtain Compound 4, with a yield of 90%.

[0073] Synthesis of compound L2: Compound 4 (3.6 mmol) and triethylamine (3.6 mmol) were dissolved in dichloromethane (30 mL), and 1,3,5-benzenetricarboxyl chloride (1 mmol) was added. The mixture was stirred at room temperature for 12 h. After TLC detection, the solvent was removed by rotary evaporation under reduced pressure. 50 mL of ethyl acetate was added, followed by washing three times with an equal volume of saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate for 30 min, and the solvent was removed by rotary evaporation under reduced pressure. The mixture was then purified by column chromatography (eluent: methanol:dichloromethane, volume ratio = 1:30) to obtain compound L2 in 80% yield. 1 H NMR(400MHz,Chloroform-d)δ8.78(s,3H),8.72(s,3H),7.85(s,3H),4.60(s,3H),3.45–3.08(m,6H),3.03– 2.80(m,18H),1.93–1.64(m,21H),1.28(d,J=23.9Hz,96H),0.93(t,J=5.7Hz,18H),0.87(t,J=6.7Hz,18H).

[0074] Example 2

[0075] Synthesis of compound M2

[0076]

[0077] The synthesis of compound 5 was as described in Example 1 for the synthesis of compound 3, except that BOC-L-leucine was replaced with BOC-L-methionine; other reaction conditions and steps were consistent with Example 1. The single-step yield of compound 5 was 71%, that of compound 6 was 94%, and that of compound M2 was 82%. 1 H NMR(400MHz,Chloroform-d)δ8.80(s,3H),8.45(s,3H),7.92(s,3H),4.70(d,J=10.4Hz,3H),3.38(d,J=27.1Hz,6H),2.98–2.80(m,18H),2.6 8–2.60(m,6H),2.25(td,J=14.6,13.9,7.7Hz,6H),2.11(s,9H),1.81( s,6H),1.68–1.65(m,6H),1.42–1.13(m,96H),0.87(t,J=6.7Hz,18H).

[0078] Compounds L1, L3, L4, M1, M3, and M4 can also be prepared using the methods described in Examples 1 and 2, and will not be repeated here. The proton NMR spectra of some compounds are shown in Table 2.

[0079] Table 2

[0080]

[0081]

[0082] Example 3

[0083] Preparation and characterization of lipid nanoparticles

[0084] A lipid ethanol solution was prepared by dissolving lipid compound M2, DOPE, cholesterol, and DMG-PEG prepared in Example 2 in anhydrous ethanol at a molar ratio of 35:30:25:0.5, wherein the concentration of lipid compound M2 was 10 mg / ml. An aqueous mRNA solution was prepared by dissolving EGFP (or Luciferase) mRNA in citrate buffer (10-50 mM, pH=4). The lipid ethanol solution and the aqueous mRNA solution were rapidly mixed using a NanoAssemblr microfluidic device (Precision Nanosystems) to prepare a solution containing lipid nanoparticles. The weight ratio of lipid compound to mRNA was 10:1. Subsequently, lipid nanoparticles encapsulating mRNA were obtained through dialysis and ultrafiltration.

[0085] The morphology and physicochemical properties of the lipid nanoparticles were then further characterized. Transmission electron microscopy was used to characterize the morphology of the lipid nanoparticles, while a Malvern Zetasizer Nano ZS was used to characterize the nanosize, polydispersity index (PDI), and zeta potential. The encapsulation efficiency was determined using the Quant-iT RiboGreen RNAAssay Kit.

[0086] The results are as follows Figure 1-3 As shown, the lipid nanoparticles are spherical with a particle size within 200 nm, and their surface charge is close to electroneutrality. They exhibit good encapsulation efficiency (>80%), indicating that lipid nanoparticles composed of benzamide-based ionizable lipids, cholesterol, phospholipids, and PEG lipids can effectively encapsulate mRNA.

[0087] In vitro transfection experiment of lipid nanoparticles

[0088] The prepared lipid compounds L1-L4 and M1-M4 were used to prepare lipid nanoparticle solutions containing EGFP mRNA according to Example 3 (lipid compound:mRNA weight ratio of 10:1), and were named LLNP1, LLNP2, LLNP3, LLNP4, MLNP1, MLNP2, MLNP3, and MLNP4, respectively.

[0089] RAW264.7 cells in the logarithmic growth phase were seeded into 96-well plates containing DMEM medium at a density of 1 x 10⁶ cells per well. 5 Cells were collected and prepared for transfection after cell adhesion (37℃, approximately 12 hours). Lipid nanoparticles containing 0.2 μg mRNA were added to each well, with three replicates per group. 24 hours after transfection, the transfection efficiency of each group was assessed using flow cytometry. The transfection efficiencies for LLNP1-4 and MLNP1-4 were 53.64%, 78.99%, 62.64%, 51.51%, 59.99%, 85.38%, 68.04%, and 64.29%, respectively.

[0090] Transfection results as follows Figure 5 As shown, the transfection effects of LNPs prepared by benzamide-based ionizable lipids with different linking amino acids and tail chain lengths vary. Lipids containing methionine may interact with groups in RNA through hydrogen bonding via methionine groups, thus enabling better delivery of nucleic acid molecules.

[0091] Biocompatibility experiments of lipid nanoparticles

[0092] Cell viability was determined using the CCK-8 (cell counting kit-8) assay. Logarithmically growing RAW264.7 cells were seeded into 96-well plates containing DMEM medium at a density of 1 x 102 cells per well. 5 Cells were incubated in a cell culture incubator for 24 hours. Then, the original cell culture medium was replaced with 100 μL of LNP (containing 20 μg / mL mRNA) cell culture medium, and incubated for a total of 4 hours. Subsequently, the cell supernatant was removed, fresh cell culture medium was added, and incubation continued for 20 hours. Finally, the supernatant in the well plate was removed, 100 μL of fresh cell culture medium containing CCK-8 working solution (10 μL / mL) was added, and incubation continued for 2 hours. Cell viability of each group of LNPs was detected using a multifunctional microplate: LLNP1-4 and MLNP1-4 showed viability of 96%, 98%, 95%, 94%, 97%, 96%, 96%, and 95%, respectively.

[0093] Cell viability (%) = [A1 - A0] / [A2 - A0] × 100; A1 is the absorbance of the drug-treated group, A0 is the absorbance of the blank group, and A2 is the absorbance of the control group. Cells not treated with LNP were used as the control group, and their cell viability was set to 100%.

[0094] Cell viability results showed that, within the specified LNP concentration range, none of the LNPs prepared from benzamide-based ionizable lipids exhibited significant cytotoxicity and good biocompatibility.

[0095] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A benzamide-based ionizable lipid or its salt, characterized in that: Its structural formula is shown in Formula I: Ⅰ； R0 is selected from L-type or D-type amino acid side chain groups; R1-R6 are each independently selected from unsubstituted C6-C. 24 Alkyl; or, unsubstituted C6-C 24 alkenyl; or, unsubstituted C6-C 24 alkynyl group; R7 is selected from H, unsubstituted C1-C. 10 alkyl; m is selected from integers from 1 to 8; X is selected from O or NH.

2. The benzamide-based ionizable lipid or its salt according to claim 1, characterized in that: The amino acid of the side chain group of R0 is selected from glycine, alanine, proline, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, asparagine, glutamine, lysine, histidine, arginine, aspartic acid, or glutamic acid.

3. The benzamide-based ionizable lipid or its salt according to claim 2, characterized in that: The amino acid of the side chain group of R0 is selected from glycine, alanine, proline, valine, leucine, isoleucine, methionine, lysine, histidine, or arginine.

4. The benzamide-based ionizable lipid or its salt according to claim 3, characterized in that: The amino acid in the side chain group of R0 is leucine or methionine.

5. The benzamide-based ionizable lipid or its salt according to claim 1, characterized in that: R1-R6 are each independently selected from unsubstituted C6-C. 24 alkyl.

6. The benzamide-based ionizable lipid or its salt according to claim 5, characterized in that: R1-R6 are each independently selected from C8-C 14 Straight-chain alkyl groups.

7. The benzamide-based ionizable lipid or its salt according to claim 1, characterized in that: R7 is H; X is NH; m is 4.

8. The benzamide-based ionizable lipid or its salt according to claim 1, characterized in that: The benzamide-type ionizable lipids are selected from one of the following compounds: （L1）； (L2）； （L3）； （L4）； （M1）； （M2）； （M3）； （M4）。 9. A nanodelivery system, characterized in that: Includes the benzamide-type ionizable lipids or their salts as described in any one of claims 1-8.

10. The nanodelivery system according to claim 9, characterized in that: It also includes one or more of the following: auxiliary lipids, cholesterol and its derivatives, or PEG lipids.

11. The nanodelivery system according to claim 10, characterized in that: The auxiliary lipid is a phospholipid or its derivative.

12. The nanodelivery system according to claim 11, characterized in that: The auxiliary lipid is selected from one or more of distearyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, dipalmitoyl phosphatidylcholine, diethyl pyrocarbonate, dilauroyl phosphatidylcholine, phosphatidylcholine, egg yolk lecithin, hydrogenated soybean phosphatidylcholine, sphingomyelin, or dimyristoyl phosphatidylcholine.

13. The nanodelivery system according to claim 10, characterized in that: Cholesterol derivatives are selected from one or more of the following: sterols and their derivatives, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, α-tocopherol, or corticosteroids.

14. The nanodelivery system according to claim 10, characterized in that: The PEG lipid is selected from one or more of DSPE-PEG, DMG-PEG, DPPE-PEG or DMA-PEG.

15. The nanodelivery system according to claim 9, characterized in that: The product includes the ionizable lipids of the benzamide class or their salts, auxiliary lipids, cholesterol or their derivatives or PEG lipids, wherein the molar ratio of the ionizable lipids of the benzamide class or their salts, auxiliary lipids, cholesterol or their derivatives and PEG lipids is 20-50:20-60:10-40:0.5-10.

16. The use of the nanodelivery system according to any one of claims 9-15 in the preparation of nucleic acid drugs.

17. The application according to claim 16, characterized in that: The nucleic acid molecules in the nucleic acid drug are selected from one or more of small interfering RNA, messenger RNA, microRNA, circular mRNA, long non-coding RNA, plasmid DNA, mini circle DNA, antisense oligonucleotides, small activating RNA, or nucleic acid aptamers.

18. The application according to claim 17, characterized in that: The nucleic acid molecule is a small interfering RNA, messenger RNA, or circular mRNA.

19. The application according to claim 16, characterized in that: In the nucleic acid drug, the mass ratio of benzamide-type ionizable lipids or their salts to nucleic acid molecules is 1-100:

1.

20. The application according to claim 19, characterized in that: The particle size of the nucleic acid drug is 1-1000 nm.

21. The application according to claim 19, characterized in that: The nucleic acid drug includes a target molecule, which is located in or on the surface of the nucleic acid drug.

22. The application according to claim 21, characterized in that: The target molecule is selected from proteins, peptides, glycoproteins, lipids, small molecules, or nucleic acids.

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