An ionizable lipid based on an antioxidant hydrophobic scaffold, lipid nanoparticles and their use in nucleic acid delivery

Abstract

The application discloses an ionizable lipid based on an antioxidant hydrophobic skeleton, a lipid nanoparticle and application of the ionizable lipid in nucleic acid delivery, and the molecular structure of the ionizable lipid is composed of three parts, namely, a symmetrical antioxidant hydrophobic tail, an environment-sensitive connecting arm and a multifunctional ionizable amino head. The ionizable lipid provided by the application can remove ROS in situ in cells, reshape the oxidative microenvironment in cells, relieve cell oxidative stress and significantly improve the translation efficiency of nucleic acids. The nucleic acid delivery carrier constructed from the ionizable lipid has high nucleic acid encapsulation rate, excellent target cell uptake capacity and outstanding in vivo protein expression level.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and nucleic acid delivery vector technology, specifically to an ionizable lipid based on an antioxidant hydrophobic framework, lipid nanoparticles, and their application in nucleic acid delivery. Background Technology

[0002] Messenger RNA (mRNA)-mediated protein replacement therapy and gene regulation technology have become important development directions in the biomedical field in recent years. By delivering in vitro transcribed functional nucleic acids to the cytoplasm of host cells, the expression of target proteins or the regulation of endogenous gene expression can be induced. This has shown broad application prospects in multiple fields such as infectious disease prevention, tumor immunotherapy, protein supplementation for hereditary diseases, and in vivo gene editing. With the gradual maturation of nucleic acid sequence modification technology and synthesis processes, delivery systems have become a core bottleneck restricting the in vivo drug-likeness of nucleic acid drugs. Naked nucleic acid molecules are easily degraded by nucleases in vivo, and their negative charge and large molecular size make it difficult to penetrate cell membranes for intracellular delivery. Therefore, safe and efficient delivery carriers are a key foundation for the clinical translation of nucleic acid drugs.

[0003] Lipid nanoparticles (LNPs) are currently a relatively mature nucleic acid delivery carrier system for clinical translation. They are typically formed by the self-assembly of four components: ionizable lipids, helper phospholipids, cholesterol, and polyethylene glycol (PEG)-modified lipids. They can encapsulate nucleic acid molecules through electrostatic interactions, protecting them from enzymatic degradation in vivo, and mediate nucleic acid entry into cells via endocytosis. As nucleic acid therapy expands from preventative vaccines to the treatment of solid tumors, inflammatory diseases, and degenerative diseases, the delivery performance of lipid nanoparticles in complex pathological microenvironments faces new technical demands. Pathological tissues often exhibit abnormal microenvironmental characteristics, including high concentrations of reactive oxygen species, a slightly acidic environment, and immunosuppression, forming a biological barrier for the delivery carrier and affecting its tissue penetration and cellular uptake. Simultaneously, during the endosome escape process after the carrier enters the cell, the released lipid components and exogenous nucleic acids may induce intracellular oxidative stress, causing not only cytotoxicity but also affecting ribosome translation function, inhibiting the protein expression efficiency of exogenous nucleic acids, and ultimately limiting the therapeutic effect. Therefore, how to regulate the intracellular oxidative microenvironment while delivering nucleic acids and relieve the inhibition of translation by oxidative stress is an important research and development direction for improving the therapeutic effect of nucleic acid drugs.

[0004] To address the high oxidative stress in the pathological microenvironment, two main approaches have emerged in this field. One approach involves introducing reactive oxygen species (ROS)-sensitive chemical bonds into the lipid or polymer backbone to construct a microenvironment-responsive delivery system. This system uses high concentrations of ROS at the pathological site to trigger backbone breakage, enabling the responsive release of nucleic acids or drugs. However, this approach cannot continuously provide antioxidant activity, making it difficult to effectively improve intracellular oxidative stress, and ribosome translation function remains limited. Furthermore, the backbone is prone to premature breakage during tissue penetration, leading to a decrease in the effective nucleic acid dose reaching the target cell cytoplasm. The other approach involves introducing small-molecule antioxidants into lipid bilayers (LNPs) through physical doping, non-covalently encapsulating them in the lipid matrix to quench free radicals and regulate the local microenvironment. However, exogenously doped small molecules can interfere with the ordered arrangement of the lipid bilayer, disrupting the stability of the four-component self-assembly, resulting in decreased nucleic acid encapsulation efficiency, particle aggregation, and drug burst release. Simultaneously, physically encapsulated antioxidants are prone to premature leakage, failing to release synchronously with nucleic acids and thus hindering their synergistic protective effect during the critical translation window.

[0005] In summary, there is currently a lack of delivery materials in this field that can integrate antioxidant functions with an ionizable lipid backbone. Existing technologies struggle to simultaneously achieve the self-assembly stability of the carrier, efficient nucleic acid delivery capabilities, and precise regulation of the intracellular oxidative microenvironment. Developing novel functionally integrated ionizable lipids that can achieve in-situ regulation of the intracellular oxidative microenvironment without affecting carrier assembly performance and delivery efficiency has significant technological value for improving the in vivo expression efficiency and therapeutic efficacy of nucleic acid drugs. Summary of the Invention

[0006] The purpose of this invention is to solve the technical problem that nucleic acid delivery carriers in the prior art are prone to oxidative stress in sensitive cells, leading to translational inhibition. This invention provides an ionizable lipid based on an antioxidant hydrophobic framework, lipid nanoparticles, and their application in nucleic acid delivery.

[0007] The above-mentioned objective of the present invention is achieved through the following technical solution: The first aspect of this invention provides an ionizable lipid, the structural formula of which is as follows: ; Wherein, X is dimethylaminoethyl, dimethylaminopropyl, dimethylaminoethanol, dimethylaminopropanol, 4-dimethylaminobutyl, diethylaminopropanol, pyrrolylethyl, pyrrolylpropanol, or piperidinylpropanol; R1 is an antioxidant hydrophobic tail group, wherein the antioxidant hydrophobic tail group is a residue formed after dehydrogenation or functional group derivatization of an antioxidant hydrophobic compound; the antioxidant hydrophobic compound is selected from α-tocopherol, eicosapentaenoic acid, retinol, cholecalciferol, coenzyme Q10 derivatives, and propyl gallate.

[0008] The ionizable lipid molecule structure provided by this invention consists of three parts: a symmetrical antioxidant hydrophobic tail, an environmentally sensitive linker arm, and a multifunctional ionizable amino head. Addressing the shortcomings of traditional straight-chain alkane-delivered lipids, which are prone to intracellular degradation, generating toxicity and inducing severe cellular oxidative stress, this invention balances low cytotoxicity with the need for intracellular microenvironment regulation by introducing a symmetrical antioxidant backbone with reactive oxygen species scavenging function as the hydrophobic tail. The hydrophobic tail chain of this symmetrical antioxidant backbone can be selected from one of the following: benzodihydropyran derivatives (such as α-tocopherol), polyunsaturated fatty acids (such as eicosapentaenoic acid), fat-soluble vitamins (such as retinol and cholecalciferol), coenzymes, and responsive fat-soluble modified polyphenols (such as polyphenol acetal fragments). Furthermore, the phosphate ester bonds within the ionizable lipid molecule can be catalyzed and hydrolyzed by intracellular phosphatases, releasing antioxidant active substances in situ, effectively alleviating the inhibition of translation by oxidative stress in sensitive cells, and reshaping the intracellular microenvironment suitable for efficient nucleic acid expression.

[0009] Specifically, X is a protonable amino head group selected from groups containing tertiary amine structures: dimethylaminoethyl, dimethylaminopropyl, dimethylaminoethanol, dimethylaminopropanol, 4-dimethylaminobutyl, diethylaminopropanol, pyrrolylethyl, pyrrolylpropanol, piperidinylpropanol, with the following structural formula: The wavy line indicates the connection point.

[0010] The aforementioned groups are connected to the phosphorous acid linker through their terminal -NH-, -OH, or -CH2- sites, and the intramolecular tertiary amine skeleton is fully preserved, serving as ionizable protonation sites. For example, when the amino head is dimethylaminoethyl, X is -NH-CH2CH2-N(CH3)2; when the amino head is dimethylaminopropanol, X is -O-CH2CH2CH2-N(CH3)2.

[0011] Furthermore, the residues are linked to the molecular skeleton via hydroxyl, phenolic hydroxyl, carboxyl, or other derivatizable sites, and are introduced into the molecular skeleton via oxygen atoms, carbonyl groups, ester bonds, ether bonds, phosphate ester bonds, phosphite ester bonds, or other covalent linkages.

[0012] Furthermore, the structural formula of the antioxidant hydrophobic tail group is as follows: ; in, Indicates the connection location.

[0013] A second aspect of this invention provides a method for preparing lipid nanoparticles, comprising the following steps: (1) Dissolve the ionizable lipids, auxiliary phospholipids, cholesterol and polyethylene glycol-modified lipids in an organic solvent to prepare a lipid organic phase solution; (2) The aqueous solution is mixed with the lipid organic phase solution to obtain a lipid nanoparticle dispersion by self-assembly; (3) The lipid nanoparticle dispersion was replaced with a neutral buffer solution to obtain the lipid nanoparticles.

[0014] Further, in step (1), the molar ratio of the ionizable lipid, cofactor phospholipid, cholesterol and polyethylene glycol-modified lipid is (10-40):(20-50):(20-50):(0.25-1).

[0015] Further, in step (1), the auxiliary phospholipid is distearylphosphatidylcholine (DSPC) and / or dioleoylphosphatidylethanolamine (DOPE).

[0016] Further, in step (1), the polyethylene glycol-modified lipid is 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000).

[0017] Further, in step (1), the organic solvent is ethanol.

[0018] Further, in step (2), the aqueous solution is a sodium citrate buffer solution with a pH of 3.0-5.0, preferably pH=4.0.

[0019] Furthermore, in step (2), mixing is carried out by rapid pipetting, vortexing, or a combination of both.

[0020] Furthermore, in step (3), a neutral buffer solution of phosphate (PBS) with a pH of 6.5-8.0 is used.

[0021] Further, in step (3), the buffer replacement is performed by at least one of dialysis, ultrafiltration, centrifugal ultrafiltration or tangential flow filtration.

[0022] The third aspect of the present invention provides lipid nanoparticles obtained by the preparation method described in the second aspect.

[0023] The lipid nanoparticles provided by this invention can be used as nucleic acid delivery carriers.

[0024] A fourth aspect of this invention provides a method for preparing lipid nanoparticles loaded with nucleic acids, comprising the following steps: (1) Dissolve the ionizable lipids, auxiliary phospholipids, cholesterol and polyethylene glycol-modified lipids in an organic solvent to prepare a lipid organic phase solution; (2) The aqueous solution containing nucleic acid is mixed with the lipid organic phase solution to obtain a lipid nanoparticle dispersion loaded with nucleic acid by self-assembly; (3) The lipid nanoparticle dispersion containing nucleic acid was replaced with a neutral buffer solution to remove the solvent and obtain the lipid nanoparticles containing nucleic acid.

[0025] The ionizable lipids provided by this invention can scavenge ROS in situ within cells, reshape the intracellular oxidative microenvironment, alleviate cellular oxidative stress, and significantly improve nucleic acid translation efficiency. The nucleic acid delivery vectors constructed from these lipids possess high nucleic acid encapsulation efficiency, excellent target cell uptake capacity, and outstanding in vivo protein expression levels.

[0026] Further, in step (1), the molar ratio of the ionizable lipids, cofactor phospholipids, cholesterol, and polyethylene glycol-modified lipids is (10-40):(20-50):(20-50):(0.25-1). Within this ratio range, the system can achieve excellent nucleic acid encapsulation efficiency and endosomal escape efficiency, while ensuring good storage stability of the nucleic acid delivery system.

[0027] Further, in step (1), the auxiliary phospholipid is DSPC and / or DOPE.

[0028] Further, in step (1), the polyethylene glycol-modified lipid is 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000).

[0029] Further, in step (1), the organic solvent is ethanol.

[0030] Further, in step (2), the nucleic acid is selected from one or more of mRNA, siRNA, DNA and circular RNA, specifically messenger RNA (mRNA), circular RNA (circRNA), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotide (ASO), plasmid DNA (pDNA) or a combination thereof.

[0031] Further, in step (2), the aqueous solution is a sodium citrate buffer solution with a pH of 3.0-5.0, preferably pH=4.0.

[0032] Further, in step (2), the volume ratio of the aqueous phase solution containing nucleic acid to the lipid organic phase solution is (1-5):1, preferably 3:1.

[0033] Furthermore, in step (2), mixing is carried out by rapid pipetting, vortexing, or a combination of both.

[0034] Specifically, in step (2), after the lipid organic phase solution is added to the aqueous phase solution containing nucleic acid, it is immediately subjected to rapid pipetting, vortexing, or a combination of both to form a lipid nanoparticle dispersion loaded with nucleic acid.

[0035] Furthermore, in step (3), a neutral buffer solution, PBS buffer, with a pH of 6.5-8.0, is used.

[0036] Further, in step (3), the buffer replacement is performed by at least one of dialysis, ultrafiltration, centrifugal ultrafiltration or tangential flow filtration.

[0037] The fifth aspect of the present invention provides a lipid nanoparticle loaded with nucleic acid obtained by the preparation method described in the fourth aspect.

[0038] The sixth aspect of this invention provides the application of the ionizable lipids described in the first aspect, the lipid nanoparticles described in the third aspect, or the nucleic acid-encapsulated lipid nanoparticles described in the fifth aspect in the preparation of nucleic acid delivery formulations.

[0039] The above-described technical solution of the present invention has the following beneficial effects: 1. This invention integrates antioxidant active groups into ionizable lipid molecules via covalent bonds to construct a symmetrical molecular framework. This symmetrical structure avoids the local steric hindrance caused by asymmetric lipids, promoting the orderly arrangement of lipid molecules within lipid nanoparticles (LNPs), thus endowing LNPs with excellent self-assembly stability and thermodynamic stability. Simultaneously, this molecular configuration enhances macromolecular stacking parameters, promoting spermatogonial cell membrane structural rearrangement, exhibiting superior intracellular delivery performance compared to traditional LNPs, demonstrating that antioxidant hydrophobic framework modification can improve spermatogonial cell transfection efficiency. This invention designs various protonable tertiary amine structures at the amino head of ionizable lipids, building a diverse library of ionizable lipids, enriching the chemical diversity of lipid molecules, and providing ample material for screening ionizable lipids with high transfection activity and low cytotoxicity. This invention achieves an integrated design of antioxidant functional units and carrier framework, significantly improving the overall stability of LNPs.

[0040] 2. The phosphate ester bonds contained in the ionizable lipids provided by this invention can be catalyzed and hydrolyzed by phosphatases in vivo, releasing antioxidant active components and efficiently clearing excess ROS in the lesion microenvironment. Relying on the dual synergistic effect of intracellular microenvironment remodeling and antioxidation, the expression level of nucleic acids is greatly improved: on the one hand, it effectively protects the molecular structure of exogenous nucleic acids released into the cytoplasm from oxidative degradation and significantly prolongs their half-life of functioning in the cell; on the other hand, it effectively protects the structural and functional integrity of ribosomes and completely relieves the strong inhibition of ribosome translation process by oxidative stress. Attached Figure Description

[0041] Figure 1 Particle size and PDI data for ToCAP2-LNP, SM102-DSPC, and MC3-DSPC are shown in the figure.

[0042] Figure 2 The diagram shows the zeta potential data for ToCAP2-LNP, SM102-DSPC, and MC3-DSPC.

[0043] Figure 3 This is a heatmap showing the delivery efficiency of each ToCAP-LNP group in Test Example 2.

[0044] Figure 4 A comparison of the mRNA delivery efficiency of ToCAP2-LNP, SM102-LNP, MC3-LNP and Lipo3000 in GC-1 mouse spermatogonia.

[0045] Figure 5 A comparison of the mRNA delivery efficiency of ToCAP2-LNP, SM102-LNP, MC3-LNP and Lipo3000 in Hep3B human liver cancer cells.

[0046] Figure 6 The left figure shows the time-series quantitative curve of the average radiance of the region of interest in the mouse testis after injection of lipid nanoparticles in each group in test case 4, and the right figure shows the comparison of the area under the curve (AUC).

[0047] Figure 7 The images show in vivo whole-body fluorescence images of mice 6 h, 12 h, 24 h, 48 h, and 72 h after injection of lipid nanoparticles in test case 4.

[0048] Figure 8 The images (left) and the bar chart (right) showing the in vitro luminescence of mouse testes and major organs after injection of lipid nanoparticles in each group for 12 h and 72 h in Test Example 4 are shown.

[0049] Figure 9 Photographs of the testes of mice isolated 72 h after injection of lipid nanoparticles in test case 4 (left side is the testis of the drug-treated side, right side is the testis of the untreated side).

[0050] Figure 10 The FITC-ROS fluorescence intensity histograms were used to detect the ROS levels in GC-1 spg cells of each group by flow cytometry. The left figure shows the comparison of ROS fluorescence distribution between the normal control group and the H2O2 model group, and the right figure shows the comparison of ROS fluorescence distribution in cells after intervention with ToCAP2-LNP, MC3-LNP, and SM102-LNP under the H2O2 damage model. Detailed Implementation

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0052] This invention provides an ionizable lipid, the structural formula of which is as follows: ; Wherein, X is dimethylaminoethyl, dimethylaminopropyl, dimethylaminoethanol, dimethylaminopropanol, 4-dimethylaminobutyl, diethylaminopropanol, pyrrolylethyl, pyrrolylpropanol, or piperidinylpropanol; R1 is an antioxidant hydrophobic tail group, wherein the antioxidant hydrophobic tail group is a residue formed after dehydrogenation or functional group derivatization of an antioxidant hydrophobic compound; the antioxidant hydrophobic compound is selected from α-tocopherol, eicosapentaenoic acid, retinol, cholecalciferol, coenzyme Q10 derivatives, and propyl gallate.

[0053] In one specific embodiment of the present invention, the method for preparing ionizable lipids includes the following steps: an antioxidant hydrophobic compound and diphenyl phosphite undergo transesterification, followed by an Atherton-Todd reaction with various amino-substituted substrates under oxidative reaction conditions to obtain the ionizable lipids.

[0054] In one specific embodiment of the present invention, the antioxidant hydrophobic compound is α-tocopherol, with its skeleton as the antioxidant hydrophobic tail and phosphate ester bonds as molecular linking arms, respectively bonded to various amino heads. The preparation method includes the following steps: (1) α-Tocopherol reacts with diphenyl phosphite via transesterification to synthesize bis(α-tocopherol) phosphite (structural formula: ); (2) Under oxidative reaction conditions, bis(α-tocopherol) phosphite is reacted with various amino-substituted substrates in an Atherton-Todd reaction to obtain an ionizable lipid, denoted as ToCAP.

[0055] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0057] In the following examples, α-tocopherol, pyridine, ethyl acetate, dichloromethane, methanol, petroleum ether, carbon tetrachloride, and triethylamine were purchased from Shanghai Titan Technology Co., Ltd.; diphenyl phosphite, 4-dimethylaminopyridine, N,N-dimethylethylenediamine, 3-dimethylaminopropylamine, 3-diethylamino-1-propanol, 1-(3-hydroxypropyl)pyrrolidine, N-methyl-2-(2-hydroxyethyl)pyrrolidine, and 1-piperidinpropanol were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; and 3-dimethylamino-1-propanol was purchased from Beijing Yinuo. Kai Technology Co., Ltd.; 8-[(2-hydroxyethyl)(6-oxo-6-undecyloxyhexyl)amino]octanoic acid heptadecan-9-yl ester (SM102) and 4-(N,N-dimethylamino)butyrate dilinoleyl alcohol ester (MC3) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; DSPC was purchased from MedChemexpress Biotechnology Co., Ltd., USA; DOPE was purchased from Beijing Solarbio Technology Co., Ltd.; DMG-PEG2000 was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0058] The preparation method of bis(α-tocopherol) phosphite in the following examples includes the following steps: Diphenyl phosphite (20 mmol) was dissolved in 10.0 mL of anhydrous pyridine to form a homogeneous solution. Then, α-tocopherol (40 mmol) was added to the solution, and the mixture was stirred at room temperature until fully combined. The resulting reaction system was heated to 75 °C and stirred overnight (16 h). After the reaction was complete, the solvent pyridine was removed under reduced pressure to obtain the reaction residue. 100 mL of dichloromethane was added to the residue for dissolution, and the resulting organic phase was washed successively with 10 mL of 1 mol / L sodium hydroxide aqueous solution and 10 mL of deionized water to remove residual acidic impurities and pyridine salts. The organic layer was separated and collected, and the organic solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using a petroleum ether mixture containing 5% ethyl acetate as the eluent. After collecting the target product fraction and removing the solvent, 6.0 g of bis(α-tocopherol) phosphite was obtained. The product was a colorless to pale yellow liquid with a yield of 32%. The product bis(α-tocopherol) phosphite (C...) 58 H 99 The NMR data for O5P are as follows: 1H NMR (400 MHz, CDCl3) δ 2.59 (t, J = 6.9 Hz, 4H), 2.21 (s, 6H), 2.18 (s, 6H), 2.09 (s, 6H), 1.88 – 1.70 (m, 4H), 1.58 – 1.46(m, 6H), 1.45 – 1.30 (m, 10H), 1.30 – 1.23 (m, 12H), 1.23 (s, 6H), 1.18 –1.00 (m, 14H), 0.87 (s, 6H), 0.86 – 0.84 (m, 15H), 0.83 (s, 3H). 31 P NMR (162MHz, CDCl3) δ 1.71; The signal of one active hydrogen in the molecule is extremely weak due to the sensitivity of the NMR instrument and the proton exchange effect of the deuterated solvent, and cannot be effectively collected.

[0059] Example 1 An ionizable lipid (ToCAP1), with the following structural formula: .

[0060] The preparation method of ToCAP1 includes the following steps: To a flame-dried flask, bis(α-tocopherol) phosphite (0.55 mmol) and 4.0 mL of carbon tetrachloride were added. Under vigorous stirring at 25 °C, a mixed solution of triethylamine (3.6 mmol), 4-dimethylaminopyridine (0.05 mmol), and N,N-dimethylethylenediamine (2.7 mmol) dissolved in 3.0 mL of anhydrous dichloromethane was added dropwise. After stirring the reaction mixture for 12 h, it was diluted with 50 mL of dichloromethane and washed three times with 50 mL of saturated brine, followed by vacuum distillation to remove the solvent. The residue was purified by silica gel column chromatography (dichloromethane / methanol, 95:5, v / v) to give 35 mg of the liquid compound ToCAP1 in a yield of 6%. ToCAP1 (C 62 H 109 The NMR data for N2O5P are as follows: 1H NMR (400 MHz, CDCl3) δ 3.58(s, 1H), 3.10 (p, J=5.9, 2H), 2.56 (t, J=6.9, 4H), 2.27 (t, J=5.8, 2H), 2.19(s, 6H), 2.15 (d, J=3.3, 12H), 2.07 (s, 6H), 1.83 – 1.72 (m, 4H), 1.56 – 1.46(m, 6H), 1.43 – 1.30 (m, 10H), 1.29 – 1.23 (m, 10H), 1.22 (s, 10H), 1.16 –1.14 (m, 2H), 1.13 – 1.00 (m, 10H), 0.87 (s, 6H), 0.85 (d, J=3.0, 12H), 0.84– 0.82 (m, 6H).

[0061] Example 2 An ionizable lipid (ToCAP2), with the following structural formula: .

[0062] The preparation method of ToCAP2 includes the following steps: To a flame-dried flask, bis(α-tocopherol) phosphite (0.55 mmol) and 4.0 mL of carbon tetrachloride were added. Under vigorous stirring at 25 °C, a mixed solution of triethylamine (3.6 mmol), 4-dimethylaminopyridine (0.05 mmol), and 3-dimethylaminopropylamine (2.7 mmol) dissolved in 3.0 mL of anhydrous dichloromethane was added dropwise. The reaction mixture was stirred for 12 h, diluted with 50 mL of dichloromethane, and washed three times with 50 mL of saturated brine. The solvent was then removed by vacuum distillation. The residue was purified by silica gel column chromatography (dichloromethane / methanol, 98:2, v / v) to give 40 mg of the liquid compound ToCAP2 in a yield of 7.2%. ToCAP2 (C 63 H 111 The NMR data for N2O5P are as follows: 1 H NMR (400 MHz, CDCl3) δ 3.87 (s,1H), 3.24 – 3.11 (m, 2H), 2.55 (t, J =7.0, 4H), 2.46 (d, J=7.0, 2H), 2.35 (s,6H), 2.19 (s, 6H), 2.14 (s, 6H), 2.05 (s, 6H), 1.80 – 1.68 (m, 6H), 1.50 (dp, J =13.4, 6.9, 6H), 1.42 – 1.29 (m, 10H), 1.27 – 1.22 (m, 12H), 1.20 (d, J =2.7,10H), 1.12 – 1.01 (m, 10H), 0.86 (s, 6H), 0.84 (s, 6H), 0.83 (s, 6H), 0.82(d, J =3.4, 6H). 31 P NMR (162 MHz, CDCl3) δ -7.39.

[0063] Example 3 An ionizable lipid (ToCAP3), with the following structural formula: .

[0064] The preparation method of ToCAP3 includes the following steps: Bis(α-tocopherol) phosphite (0.55 mmol) and 4.0 mL of carbon tetrachloride were added to a flame-dried flask. Under vigorous stirring at 25 °C, a mixed solution of triethylamine (3.6 mmol), 4-dimethylaminopyridine (0.05 mmol), and 3-dimethylamino-1-propanol (2.7 mmol) dissolved in 3.0 mL of anhydrous dichloromethane was added dropwise. The reaction mixture was stirred for 12 h, diluted with 50 mL of dichloromethane, and washed three times with 50 mL of saturated brine. The solvent was then removed by vacuum distillation. The residue was purified by silica gel column chromatography (dichloromethane / methanol, 98:2, v / v) to give 200 mg of the liquid compound ToCAP3, in a yield of 36.1%. ToCAP3 (C 63 H 110 The NMR data for NO6P are as follows: 1 H NMR (400 MHz, CDCl3) δ5.28 (s, 2H), 4.09 (dt, J =7.7, 6.2, 2H), 2.55 (t, J=6.9, 4H), 2.18 (s, 6H), 2.14 (s, 6H), 2.13 (s, 6H), 2.06 (s, 6H), 1.81 – 1.65 (m, 6H), 1.57 – 1.45(m, 6H), 1.36 (tdd, J =16.7, 8.0, 4.6, 10H), 1.28 – 1.22 (m, 12H), 1.21 (s,6H), 1.16 – 1.01 (m, 14H), 0.86 (s, 6H), 0.84 (s, 6H), 0.84 – 0.81 (m, 12H). 31 P NMR (162 MHz, CDCl3) δ -8.67.

[0065] Example 4 An ionizable lipid (ToCAP4), with the following structural formula: .

[0066] The preparation method of ToCAP4 includes the following steps: To a flame-dried flask, bis(α-tocopherol) phosphite (0.55 mmol) and 4.0 mL of carbon tetrachloride were added. Under vigorous stirring at 25 °C, a mixed solution of triethylamine (3.6 mmol), 4-dimethylaminopyridine (0.05 mmol), and 3-diethylamino-1-propanol (2.7 mmol) dissolved in 3.0 mL of anhydrous dichloromethane was added dropwise. The reaction mixture was stirred for 12 h, diluted with 50 mL of dichloromethane, and washed three times with 50 mL of saturated brine. The solvent was then removed by vacuum distillation. The residue was purified by silica gel column chromatography (dichloromethane / methanol, 98:2, v / v) to give 30 mg of the liquid compound ToCAP4 in a yield of 5.3%. ToCAP4 (C 65 H 114 The NMR data for NO6P are as follows: 1 H NMR (400 MHz, CDCl3) δ 4.12(dt, J =7.9, 6.1, 2H), 2.54 (q, J =6.8, 7H), 2.42 (t, J =7.7, 2H), 2.18 (s, 6H), 2.14 (s, 6H), 2.06 (s, 6H), 1.81 – 1.71 (m, 6H), 1.51 (dq, J =13.3, 6.7, 6H), 1.35 (td,J =16.9, 15.0, 9.0, 10H), 1.29 – 1.22 (m, 12H), 1.20 (s, 10H), 1.15 –1.04 (m, 10H), 1.01 (t, J =7.2, 7H), 0.86 (s, 6H), 0.84 (d, J =3.3, 12H), 0.82(d, J =4.0, 6H).

[0067] Example 5 An ionizable lipid (ToCAP5) with the following structural formula: .

[0068] The preparation method of ToCAP5 includes the following steps: To a flame-dried flask, bis(α-tocopherol) phosphite (0.55 mmol) and 4.0 mL of carbon tetrachloride were added. Under vigorous stirring at 25 °C, a mixed solution of triethylamine (3.6 mmol), 4-dimethylaminopyridine (0.05 mmol), and 1-(3-hydroxypropyl)pyrrolidine (2.7 mmol) dissolved in 3.0 mL of anhydrous dichloromethane was added dropwise. The reaction mixture was stirred for 12 h, diluted with 50 mL of dichloromethane, and washed three times with 50 mL of saturated brine. The solvent was then removed by vacuum distillation. The residue was purified by silica gel column chromatography (dichloromethane / methanol, 98:2, v / v) to give 200 mg of the liquid compound ToCAP5, in a yield of 35.1%. ToCAP5 (C 65 H 112 The NMR data for NO6P are as follows: 1 H NMR (400 MHz, CDCl3) δ4.03 – 4.00 (m, 2H), 2.71 – 2.35 (m, 4H), 2.27 – 2.09 (m, 12H), 2.03 (t, J =6.1 Hz, 8H), 1.89 – 1.66 (m, 9H), 1.60 – 1.45 (m, 8H), 1.37 (t, J = 9.0 Hz,10H), 1.30 – 1.21 (m, 14H), 1.18 (t, J = 6.4 Hz, 7H), 1.08 (dt, J = 28.6, 8.5 Hz, 14H), 0.83 (q, J= 9.5, 7.5 Hz, 24H).

[0069] Example 6 An ionizable lipid (ToCAP6), with the following structural formula: .

[0070] The preparation method of ToCAP6 includes the following steps: To a flame-dried flask, bis(α-tocopherol) phosphite (0.55 mmol) and 4.0 mL of carbon tetrachloride were added. Under vigorous stirring at 25 °C, a mixed solution of triethylamine (3.6 mmol), 4-dimethylaminopyridine (0.05 mmol), and N-methyl-2-(2-hydroxyethyl)pyrrolidine (2.7 mmol) dissolved in 3.0 mL of anhydrous dichloromethane was added dropwise. The reaction mixture was stirred for 12 h, diluted with 50 mL of dichloromethane, and washed three times with 50 mL of saturated brine. The solvent was then removed by vacuum distillation. The residue was purified by silica gel column chromatography (dichloromethane / methanol, 98:2, v / v) to give 200 mg of the liquid compound ToCAP6, in a yield of 35.15%. ToCAP6 (C 65 H 112 The NMR data for NO6P are as follows: 1 H NMR (400 MHz, CDCl3)δ 4.28 – 4.06 (m, 2H), 2.56 (t, J =6.9, 4H), 2.35 (s, 3H), 2.18 (d, J =4.5, 6H),2.17 – 2.14 (m, 7H), 2.06 (s, 6H), 1.89 (dd, J =10.5, 4.3, 2H), 1.80 – 1.67 (m,6H), 1.50 (tdd, J =13.1, 8.7, 5.3, 8H), 1.37 (qd, J =12.3, 9.9, 4.0, 10H), 1.29 –1.24 (m, 10H), 1.21 (s, 12H), 1.12 – 1.00 (m, 12H), 0.86 (s, 6H), 0.84 (d, J =3.1, 12H), 0.82 (d, J =4.2, 6H).

[0071] Example 7 An ionizable lipid (ToCAP7), with the following structural formula: .

[0072] The preparation method of ToCAP7 includes the following steps: To a flame-dried flask, bis(α-tocopherol) phosphite (0.55 mmol) and 4.0 mL of carbon tetrachloride were added. Under vigorous stirring at 25 °C, a mixed solution of triethylamine (3.6 mmol), 4-dimethylaminopyridine (0.05 mmol), and 1-piperidinpropanol (2.7 mmol) dissolved in 3.0 mL of anhydrous dichloromethane was added dropwise. The reaction mixture was stirred overnight, diluted with 50 mL of dichloromethane, and washed three times with 50 mL of saturated brine. The solvent was then removed by vacuum distillation. The residue was purified by silica gel column chromatography (dichloromethane / methanol, 98:2, v / v) to give 200 mg of the liquid compound ToCAP7, in a yield of 34.7%. ToCAP7 (C 66 H 114 The NMR data for NO6P are as follows: 1 H NMR (400 MHz, CDCl3) δ 4.10 (q, J =6.5,2H), 2.56 (t, J =6.8, 4H), 2.42 – 2.26 (m, 4H), 2.23 (dd, J =9.7, 5.4, 2H), 2.18(s, 6H), 2.14 (s, 6H), 2.06 (s, 6H), 1.77 (dq, J =13.0, 6.9, 6H), 1.55 (ddt, J =33.3, 13.3, 6.2, 12H), 1.37 (q, J =6.4, 5.9, 8H), 1.28 – 1.18 (m, 22H), 1.13 –1.00 (m, 12H), 0.86 (s, 6H), 0.85 – 0.81 (m, 18H).

[0073] Example 8 A method for preparing lipid nanoparticles loaded with nucleic acids includes the following steps: (1) ToCAP1-7, phospholipids (DSPC or DOPE), cholesterol and DMG-PEG2000 prepared in Examples 1-7 were dissolved in anhydrous ethanol in a specific ratio to prepare a lipid organic phase solution; In this example, multiple LNP formulations with different lipid molar ratios were set up, which were denoted as AP respectively. The lipid components and their specific molar ratios in each formulation are shown in Table 1: Table 1 Formulation Table

[0074] (2) Firefly luciferase mRNA was dissolved in 10 mM sodium citrate buffer (pH 3.0) to obtain an aqueous solution containing mRNA with a final mRNA concentration of 1.5 mg / mL. The lipid organic phase solution and the aqueous solution containing mRNA were fed into the micromixer through different injection channels using a microfluidic mixing process. The volume flow ratio of the aqueous solution containing mRNA to the lipid organic phase solution was 3:1 and the total flow rate was 12 mL / min. After mixing, a lipid nanoparticle dispersion encapsulating mRNA was formed.

[0075] (3) Add 1×PBS buffer to the lipid nanoparticle dispersion containing mRNA for buffer replacement to adjust the pH of the system to 7.2; transfer the resulting lipid nanoparticle suspension into a dialysis bag with a molecular weight cutoff of 10 kDa, and dialyze for 1 h with 1×PBS as the external dialysis solution to obtain the LNP preparation for injection, namely the lipid nanoparticles containing nucleic acid, denoted as ToCAP-LNP.

[0076] Example 9 An ionizable lipid (ECAP1), with the following structural formula: .

[0077] The preparation method of ECAP1 includes the following steps: (1) Diphenyl phosphite (20 mmol) was dissolved in 10.0 mL of anhydrous pyridine to form a homogeneous solution. Then, eicosapentaenoic acid (40 mmol) was added to the solution, and the mixture was stirred at room temperature until fully combined. The resulting reaction system was heated to 75 °C and stirred overnight (16 h) under this temperature condition. After the reaction was complete, the solvent pyridine was removed under reduced pressure to obtain the reaction residue. 100 mL of dichloromethane was added to the residue for dissolution, and the resulting organic phase was washed successively with 10 mL of 1 mol / L sodium hydroxide aqueous solution and 10 mL of deionized water to remove residual acidic impurities and pyridine salts. The organic layer was separated and collected, and the organic solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using a mixture of petroleum ether and 5% ethyl acetate as the eluent. After collecting the target product fraction and removing the solvent, 7.2 g of bis(eicosapentaenoic acid)phosphonate was obtained. The product was a colorless to pale yellow liquid with a yield of 55.3%, and its structural formula was [insert structural formula here]. bis(eicosapenoyl)phosphonate (C 40 H 59 The NMR data for O5P are as follows:1 H NMR (400 MHz, CDCl3)δ 5.64 (d, J = 14.3Hz, 1H), 5.39 (ddtt, J = 8.8, 6.4, 4.4, 2.6 Hz, 20H), 2.70 (q, J = 3.7 Hz,16H), 2.55 (t, J = 8.2 Hz, 4H), 2.24 – 1.99 (m, 8H), 1.72 (s, 4H), 1.06 –0.85 (m, 6H).

[0078] (2) To a flame-dried flask, add bis(eicosapenoyloxy)phosphonate (0.55 mmol) and 4.0 mL of carbon tetrachloride. While stirring vigorously at 25 °C, add dropwise a mixed solution of triethylamine (3.6 mmol), 4-dimethylaminopyridine (0.05 mmol), and N,N-dimethylethylenediamine (2.7 mmol) dissolved in 3.0 mL of anhydrous dichloromethane. After stirring the reaction mixture for 12 h, dilute with 50 mL of dichloromethane and wash three times with 50 mL of saturated brine. Then, remove the solvent by vacuum distillation. The residue was purified by silica gel column chromatography (dichloromethane / methanol, 95:5, v / v) to give 28 mg of a liquid compound, designated ECAP1, with a yield of 6.9%. ECAP1 (C 44 H 69 The NMR data for N2O5P are as follows: 1 H NMR (400 MHz, CDCl3)δ5.58–5.14 (m, 20H), 4.65 (t, J = 6.0 Hz, 1H), 3.11–2.93 (m, 2H), 2.85 (t, J =5.3 Hz, 2H), 2.76–2.66 (m, 16H), 2.46 (t, J = 8.3 Hz, 4H), 2.34 (s, 6H), 2.28–1.92 (m, 8H), 1.69 (pd, J = 8.0, 0.8 Hz, 4H), 1.16–0.77 (m, 6H).

[0079] Example 10 An ionizable lipid (ChoCAP1), with the following structural formula: .

[0080] The preparation method of ChoCAP1 includes the following steps: (1) Diphenyl phosphite (20 mmol) was dissolved in 10.0 mL of anhydrous pyridine to form a homogeneous solution. Cholecalciferol (40 mmol) was then added to the solution, and the mixture was stirred at room temperature until fully combined. The resulting reaction system was heated to 75 °C and stirred overnight (16 h) under these conditions. After the reaction was complete, the solvent pyridine was removed under reduced pressure to obtain the reaction residue. 100 mL of dichloromethane was added to the residue for dissolution. The resulting organic phase was washed successively with 10 mL of 1 mol / L saturated sodium bicarbonate (NaHCO3) aqueous solution and 10 mL of deionized water to remove residual acidic impurities and pyridine salts. The organic layer was separated and collected, and the organic solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using a mixture of petroleum ether and 5% ethyl acetate as the eluent. After collecting the target product component and removing the solvent, 6.3 g of bis(cholecalciferol-3-oxy)phosphonate was obtained. The product was a colorless to pale yellow liquid with a yield of 38.6%, and its structural formula was [structural formula would be inserted here]. bis(cholecalciferol-3-oxy)phosphonate (C 54 H 87 The NMR data for O3P are as follows: 1 H NMR (400 MHz, CDCl3)δ6.36 (dq, J = 8.2, 1.0 Hz, 2H), 6.01 (ddq,J = 8.3, 1.9, 0.9 Hz, 2H), 4.92 (dq, J = 14.9, 1.1 Hz, 4H), 4.45 (ddt, J =11.4, 5.4, 2.7 Hz, 2H), 2.99 (d, J = 14.3 Hz, 1H), 2.79–2.53 (m, 4H), 2.53–2.37 (m, 4H), 2.34–2.17 (m, 4H), 2.11–1.71 (m, 9H), 1.71–1.02 (m, 31H), 0.93 (dd, J = 6.4, 1.8 Hz, 6H), 0.84 (dd, J = 6.7, 1.2 Hz, 12H), 0.57 (s, 6H).

[0081] (2) To a flame-dried flask, add 0.55 mmol of bis(cholecalciferol-3-oxy)phosphonate and 4.0 mL of carbon tetrachloride. While stirring vigorously at 25 °C, add dropwise a mixed solution of triethylamine (3.6 mmol), 4-dimethylaminopyridine (0.05 mmol), and N,N-dimethylethylenediamine (2.7 mmol) dissolved in 3.0 mL of anhydrous dichloromethane. After stirring the reaction mixture for 12 h, dilute with 50 mL of dichloromethane and wash three times with 50 mL of saturated saline solution. Then, remove the solvent by vacuum distillation. The residue was purified by silica gel column chromatography (dichloromethane / methanol, 95:5, v / v) to give 16 mg of a liquid compound, designated ChoCAP1, with a yield of 3.2%. ChoCAP1(C 58 H 97 The NMR data for N2O3P are as follows: 1 H NMR (400 MHz, CDCl3) δ6.36 (dp, J = 8.2, 0.9 Hz, 2H), 6.02 (ddq, J = 8.3, 2.1, 1.0 Hz, 2H), 4.92 (dq, J = 14.9, 1.1 Hz, 4H), 4.46 (dtt, J = 8.2, 5.5, 2.8 Hz, 2H), 3.64(t, J = 5.5 Hz, 1H), 2.97 (q, J = 5.7 Hz, 2H), 2.85–2.69 (m, 4H), 2.62 (dddt,J = 13.5, 7.7, 4.9, 1.0 Hz, 2H), 2.51 (ddt, J = 15.0, 5.6, 1.0 Hz, 2H), 2.41(dddt, J = 12.3, 8.4, 5.8, 1.0 Hz, 2H), 2.33 (s, 10H), 2.07–1.02 (m, 40H), 0.93 (dd, J = 6.4, 1.7 Hz, 6H), 0.84 (dd, J = 6.7, 1.2 Hz, 12H), 0.57 (s,6H).

[0082] Example 11 An ionizable lipid (ReCAP1), with the following structural formula: .

[0083] The preparation method of ReCAP1 includes the following steps: (1) Diphenyl phosphite (20 mmol) was dissolved in 10.0 mL of anhydrous pyridine to form a homogeneous solution. Retinol (40 mmol) was then added to the solution, and the mixture was stirred at room temperature until fully combined. The resulting reaction system was heated to 75 °C and stirred overnight (16 h). After the reaction was complete, the solvent pyridine was removed under reduced pressure to obtain the reaction residue. 100 mL of dichloromethane was added to the residue for dissolution. The resulting organic phase was washed successively with 10 mL of 1 mol / L sodium hydroxide aqueous solution and 10 mL of deionized water to remove residual acidic impurities and pyridine salts. The organic layer was separated and collected, and the organic solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using a mixture of petroleum ether and 5% ethyl acetate as the eluent. After collecting the target product fraction and removing the solvent, 5.5 g of bis(retinoxy)phosphonate was obtained. The product was a colorless to pale yellow liquid with a yield of 44.4%, and its structural formula was [insert structural formula here]. Bis(retinoxy)phosphonates (C 40 H 59 The NMR data for O3P are as follows: 1 H NMR (400 MHz, CDCl3) δ6.73 (ddp, J = 14.5, 11.5, 0.9 Hz, 2H), 6.45–6.02 (m, 8H), 5.58 (tp, J = 4.1, 1.3 Hz, 2H), 4.56 (ddp, J = 7.5, 4.2,1.0 Hz, 4H), 2.39 (d, J = 14.3 Hz, 1H), 2.03–1.92 (m, 10H), 1.86 (p, J = 1.0Hz, 6H), 1.72 (s, 6H), 1.67–1.51 (m, 4H), 1.51–1.39 (m, 4H), 1.06 (s, 12H).

[0084] (2) To a flame-dried flask, add 0.55 mmol of bis(retinoxy)phosphonate and 4.0 mL of carbon tetrachloride. While stirring vigorously at 25 °C, add dropwise a mixed solution of triethylamine (3.6 mmol), 4-dimethylaminopyridine (0.05 mmol), and N,N-dimethylethylenediamine (2.7 mmol) dissolved in 3.0 mL of anhydrous dichloromethane. After stirring the reaction mixture for 12 h, dilute with 50 mL of dichloromethane and wash three times with 50 mL of saturated brine. Then, remove the solvent by vacuum distillation. The residue was purified by silica gel column chromatography (dichloromethane / methanol, 95:5, v / v) to give 10 mg of a liquid compound, designated ReCAP1, with a yield of 2.6%. ReCAP1 (C44 H 69 The NMR data for N2O3P are as follows: 1 H NMR (400 MHz, CDCl3)δ6.73 (ddp, J = 14.5, 11.5, 0.9 Hz, 2H), 6.48–6.01 (m, 8H), 5.48 (tp, J =4.1, 1.4 Hz, 2H), 4.63 (ddq, J = 8.5, 4.2, 1.0 Hz, 4H), 4.35 (t, J = 6.0 Hz,1H), 3.05–2.91 (m, 2H), 2.82 (t, J = 5.3 Hz, 2H), 2.33 (s, 6H), 2.11–1.92 (m,10H), 1.86 (p, J = 1.0 Hz, 6H), 1.72 (s, 6H), 1.65–1.52 (m, 4H), 1.52–1.39 (m, 4H), 1.06 (s, 12H).

[0085] Example 12 An ionizable lipid (GaCAP1) with the following structural formula: .

[0086] The preparation method of GaCAP1 includes the following steps: (1) Diphenyl phosphite (20 mmol) was dissolved in 10.0 mL of anhydrous pyridine to form a homogeneous solution. Then, 40 mmol of propanol hemiacetal of gallaldehyde was added to the solution, and the mixture was stirred at room temperature until fully mixed. The resulting reaction system was heated to 75 °C and stirred overnight (16 h) under these conditions. After the reaction was complete, the solvent pyridine was removed under reduced pressure to obtain the reaction residue. 100 mL of dichloromethane was added to the residue for dissolution. The resulting organic phase was washed successively with 10 mL of 1 mol / L saturated sodium bicarbonate (NaHCO3) aqueous solution and 10 mL of deionized water to remove residual acidic impurities and pyridine salts. The organic layer was separated and collected, and the organic solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using a mixture of petroleum ether and 5% ethyl acetate as the eluent. After collecting the target product components and removing the solvent, 7.1 g of bis(gallic acid propanol-oxy)phosphonate was obtained. The product was a colorless to pale yellow liquid with a yield of 74.8%, and its structural formula was [insert structural formula here]. bis(gallic acid propanol-oxy)phosphonate (C 20 H 27 O 11 The NMR data for P are: 1H NMR (400 MHz, CDCl3)δ6.79 (d, J = 0.7 Hz, 4H), 6.21–6.06 (m,6H), 5.36 (s, 2H), 4.49 (d, J = 14.3 Hz, 1H), 3.63 (dt, J = 11.2, 5.1 Hz,2H), 3.49 (dt, J = 11.2, 5.2 Hz, 2H), 1.75–1.42 (m, 4H), 0.91 (t, J = 7.8 Hz, 6H).

[0087] (2) To a flame-dried flask, add 0.55 mmol of bis(galactalpropanol-oxy)phosphonate and 4.0 mL of carbon tetrachloride. While stirring vigorously at 25 °C, add dropwise a mixed solution of triethylamine (3.6 mmol), 4-dimethylaminopyridine (0.05 mmol), and N,N-dimethylethylenediamine (2.7 mmol) dissolved in 3.0 mL of anhydrous dichloromethane. After stirring the reaction mixture for 12 h, dilute with 50 mL of dichloromethane and wash three times with 50 mL of saturated brine. Then, remove the solvent by vacuum distillation. The residue was purified by silica gel column chromatography (dichloromethane / methanol, 95:5, v / v) to give 14 mg of a liquid compound, designated GaCAP1, with a yield of 4.5%. GaCAP1 (C 24 H 37 N2O 11 The NMR data for P are: 1 H NMR (400 MHz, CDCl3) δ6.81 (s, 4H), 6.19 (dd, J = 8.1, 0.8 Hz, 2H), 6.13 (s, 4H), 5.36 (s,2H), 4.24 (t, J = 5.5 Hz, 1H), 3.61 (dt, J = 11.2, 5.2 Hz, 2H), 3.48 (dt, J =11.2, 5.2 Hz, 2H), 2.95 (q, J = 5.6 Hz, 2H), 2.82 (t, J = 5.4 Hz, 2H), 2.33(s, 6H), 1.73–1.50 (m, 4H), 0.91 (t, J = 7.8 Hz, 6H).

[0088] Example 13 An ionizable lipid (Q10CAP1), with the following structural formula: .

[0089] The preparation method of Q10CAP1 includes the following steps: (1) Diphenyl phosphite (20 mmol) was dissolved in 10.0 mL of anhydrous pyridine to form a homogeneous solution. Then, a derivative of reduced coenzyme Q10 (2,3-dimethyl-5-decapopyrene-1,4-hydroquinone) (40 mmol) was added to the solution, and the mixture was stirred at room temperature until fully combined. The resulting reaction system was heated to 75 °C and stirred overnight (16 h). After the reaction was complete, the solvent pyridine was removed under reduced pressure to obtain the reaction residue. 100 mL of dichloromethane was added to the residue for dissolution. The resulting organic phase was washed successively with 10 mL of 1 mol / L saturated sodium bicarbonate (NaHCO3) aqueous solution and 10 mL of deionized water to remove residual acidic impurities and pyridine salts. The organic layer was separated and collected, and the organic solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using a mixture of petroleum ether and 5% ethyl acetate as the eluent. After collecting the target product components and removing the solvent, 6.7 g of bis(panthenol derivative oxy)phosphonate was obtained. The product was a colorless to pale yellow liquid with a yield of 74.8%, and its structural formula was [structural formula would be inserted here]. Bis(panthenol derivative oxy)phosphonate (C 116 H 183 The NMR data for O7P are as follows: 1 H NMR (400 MHz, CDCl3) δ8.50 (d, J = 14.3 Hz, 1H), 5.27 (tq, J = 6.9, 1.2 Hz, 2H), 5.19–4.98 (m, 18H), 4.90 (dq, J = 3.6, 1.0 Hz, 2H), 4.64 (ddq, J = 5.4, 2.9, 1.5 Hz, 2H), 4.35(d, J = 5.5 Hz, 2H), 3.39 (d, J = 3.6 Hz, 2H), 3.01–2.60 (m, 4H), 2.27–1.89(m, 72H), 1.84 (dt, J = 11.1, 1.2 Hz, 12H), 1.71 (q, J = 1.0 Hz, 6H), 1.60 (d, J = 1.4 Hz, 60H).

[0090] (2) To a flame-dried flask, add 0.55 mmol of bis(panthenol derivative oxy)phosphonate and 4.0 mL of carbon tetrachloride. While stirring vigorously at 25 °C, add dropwise a mixed solution of triethylamine (3.6 mmol), 4-dimethylaminopyridine (0.05 mmol), and N,N-dimethylethylenediamine (2.7 mmol) dissolved in 3.0 mL of anhydrous dichloromethane. After stirring the reaction mixture for 12 h, dilute with 50 mL of dichloromethane and wash three times with 50 mL of saturated brine. Then, remove the solvent by vacuum distillation. The residue was purified by silica gel column chromatography (dichloromethane / methanol, 95:5, v / v) to give 17 mg of a liquid compound, designated Q10CAP1, with a yield of 1.75%. Q10CAP1 (C 120 H 193 The NMR data for N2O7P are as follows: 1 H NMR (400 MHz, CDCl3)δ 5.27 (ddq, J = 8.2, 5.6, 1.2 Hz, 2H), 5.09 (tq, J = 7.0, 1.2 Hz, 18H), 4.90 (dh, J = 4.1, 1.0 Hz, 2H), 4.65 (dh, J = 5.3, 1.3 Hz, 2H), 4.50(t, J = 6.0 Hz, 1H), 4.28 (d, J = 5.5 Hz, 2H), 3.46 (d, J = 3.8 Hz, 2H), 3.00(q, J = 5.5 Hz, 2H), 2.96–2.71 (m, 6H), 2.34 (s, 6H), 2.19–1.98 (m, 68H), 1.94 (tq, J = 7.3, 1.1 Hz, 4H), 1.84 (dt, J = 16.9, 1.2 Hz, 12H), 1.71 (q, J= 1.0 Hz, 6H), 1.60 (d, J = 1.4 Hz, 60H).

[0091] Test Example 1 Dynamic light scattering (DLS) and electrophoretic light scattering were used to characterize the physicochemical properties of each group of ToCAP2-LNP (with DSPC as the auxiliary phospholipid), SM102-LNP and MC3-LNP. DLS was used to determine the hydrodynamic particle size and polydispersity index (PDI) of the particles, and electrophoretic light scattering was used to detect the zeta potential of the particle surface.

[0092] The corresponding test results are as follows Figure 1 and Figure 2As shown, the average particle size distribution of each ToCAP2-LNP group ranged from 80 to 200 nm, with a PDI of 0.15 to 0.25 and a Zeta potential of 5 to 30 mV. Compared with the two traditional control LNPs, SM102-DSPC and MC3-DSPC, the ToCAP series LNPs of this invention have controllable particle size, lower dispersion, and moderate surface positive potential, demonstrating that the quaternary lipid ratio system of this invention can produce nucleic acid delivery vectors with uniform particle size and excellent structural stability.

[0093] Test Example 2 An orthogonal screening system was designed using the four-component lipid molar ratio, ionizable lipids (ToCAP1-7), and helper phospholipids (DSPC / DOPE) as variables. In vitro high-throughput orthogonal screening experiments were conducted to screen for the optimal ionizable lipid molecular structure and quaternary lipid component formulation. The targeted transfection specificity of each ToCAP-LNP group at the in vitro cellular level was evaluated in a GC-1 mouse spermatogonial model. The testing methods are as follows: (1) GC-1 mouse spermatogonia were subjected to a treatment of 2×10⁻⁶ 5 Inoculate 10 cells / well into a 96-well plate and culture in DMEM medium containing 10% fetal bovine serum and 1% antibiotics for 12 h at 37 ℃ and 5% CO2.

[0094] (2) ToCAP-LNP was prepared in the experimental group by using ToCAP1-7 with helper phospholipids (DSPC / DOPE), cholesterol, and DMG-PEG2000 according to the formulation in Table 1. The control group was prepared by using commercially available ionizable lipids SM102 and MC3 according to the preparation method in Example 8 to prepare corresponding SM102-LNP and MC3-LNP, and commercially available transfection reagent Lipo3000 was set as a positive transfection control. An equal volume of lipid nanoparticle suspension or Lipo3000 / mRNA transfection complex was added to each experimental well so that the equivalent dose of mRNA loaded or complexed in each group was 0.1 μg / well.

[0095] (3) After transfection, the cells were cultured for another 18 h. Then, luciferase detection reagent was added, and the relative luminescence intensity (RLU) was measured in luminescence mode using a multifunctional microplate reader to quantitatively characterize the in vitro mRNA delivery efficiency of different lipid nanoparticles in GC-1 cells.

[0096] (4) Data normalization plotting: Based on the delivery efficiency of SM102-LNP, the RLU values ​​of all experimental groups were normalized and a delivery efficiency heatmap was plotted.

[0097] Test results are as follows Figure 3As shown, the type of ionizable lipid, the type of helper phospholipid, and the lipid ratio all significantly affect the mRNA delivery efficiency in GC-1 cells, and different combinations of formulations show significant differences in transfection activity. Among them, the formulation using ToCAP2 as an ionizable lipid, DSPC as a helper phospholipid, and a corresponding molar ratio of ToCAP2:DSPC:cholesterol:DMG-PEG2000=20:20:40:0.25 showed the highest RLU among all screened formulations, indicating the best transfection performance. Simultaneously, this formulation maintained a GC-1 cell viability of over 90% and exhibited extremely low cytotoxicity. Therefore, this optimal quaternary lipid ratio system was selected as the preferred nucleic acid delivery vector for subsequent cell-targeting specificity verification and in vivo animal experiments.

[0098] Test Example 3 Cell selectivity of ToCAP-LNP was tested. GC-1 mouse spermatogonia were selected as the target cell model of the reproductive system, and Hep3B human liver cancer cells were selected as the non-target control cell model. The testing method was the same as in test case 2. Three types of vectors (ToCAP2-LNP, SM102-LNP, and MC3-LNP, all with DSPC helper phospholipids) were used in the experiment. All three lipid nanoparticles carried an equal amount of firefly luciferase mRNA. After transfection and culture, RLU was measured in each group to quantitatively compare the mRNA delivery efficiency of the three types of LNPs in GC-1 and Hep3B cells.

[0099] Test results are as follows Figure 4 and Figure 5 As shown, in Hep3B cells, SM102-LNP and MC3-LNP exhibited high luciferase expression levels, while ToCAP2-LNP showed significantly lower expression levels, indicating weaker transfection ability in non-target cells. However, in GC-1 cells, ToCAP-LNP significantly increased luciferase mRNA expression levels, with its RLU value increasing by approximately 15-fold compared to the SM102-LNP group and by approximately 20-fold compared to the MC3-LNP group. These results demonstrate that, compared to commercially available SM102-LNP and MC3-LNP, the ToCAP-LNP of this invention possesses outstanding mRNA delivery capability in GC-1 mouse spermatogonia. After local administration to the testes, it can efficiently remain in the target tissue and achieve long-term expression, exhibiting good local delivery and expression selectivity. Simultaneously, its transfection activity in Hep3B non-target cells is significantly lower, confirming that this lipid nanoparticle possesses reproductive system target cell delivery selectivity.

[0100] Test Example 4 To evaluate the in vivo targeted delivery and long-term expression capabilities of the lipid nanoparticles of this invention in the complex microenvironment of living tissue, a healthy male C57BL / 6 mouse model aged 6-8 weeks was constructed for in vivo validation testing. The testing method was as follows: The experimental group consisted of ToCAP2-LNP (DSPC was the helper phospholipid, and the lipid molar ratio was ToCAP2:DSPC:cholesterol:DMG-PEG2000=20:20:40:0.25), while the control group consisted of commercially available lipid-constructed SM102-LNP and MC3-LNP. Using a precision microinjection pump and glass microcapillaries (outer diameter approximately 40-60 μm), under the assistance of a stereomicroscope, suspensions of lipid nanoparticles carrying firefly luciferase mRNA (Luc-mRNA) were precisely injected into the efferent ducts of mouse testes, gradually filling the seminiferous tubule network. The absolute mRNA dose injected into each testis was 3 μg, and the injection volume was controlled at 20 μL. After injection, the testes were sutured back in situ. Following drug administration, specific time points (6 h, 12 h, 24 h, 48 h, and 72 h) were established, and D-fluorescein substrate (dose: 150 mg / kg) was administered intraperitoneally. Ten minutes after injection, mice were placed in a small animal in vivo fluorescence imaging system for bioluminescence signal acquisition. The mean radiance of the region of interest (ROI) in the testis was quantitatively analyzed using the system's built-in software, and time-series quantitative curves and area under the curve (AUC) comparisons were plotted. At 12 h and 72 h, the testes and major organs were harvested for in vitro bioluminescence imaging to clarify the retention characteristics of LNP in the target organs.

[0101] Test results are as follows Figures 6-9 As shown, Figure 6 Time-series quantitative data and Figure 7 In vivo whole-body imaging revealed highly significant differences in local mRNA expression levels among the three vector groups after orthotopic injection into the testes. In the SM102-LNP group, only a very weak bioluminescent signal was detected in the testes at all detection time points; the MC3-LNP group showed low fluorescence signal intensity and a short peak duration; the ToCAP2-LNP group of this invention produced a high-intensity bioluminescent signal in the testes, with an average radiance at each time point 5-10 times higher than SM102-LNP and 3-5 times higher than MC3-LNP. The protein expression signal remained stable until 72 hours after administration, and the area under the curve was significantly higher than that of the two control vectors. No obvious fluorescence signal was observed in non-target organ regions such as the liver and spleen in the in vivo whole-body scanning images, demonstrating that ToCAP2-LNP has no significant organ off-target effect. Figure 8 Imaging of ex vivo organs and Figure 9 The results of the appearance observation showed that the testicular tissue of the ToCAP2-LNP group still maintained a high level of luciferase expression 72 h after administration; the testes of mice treated with SM102-LNP and MC3-LNP showed obvious congestion and edema, while the testicular tissue of the ToCAP2-LNP group had normal morphology and no congestion or inflammation, indicating that the lipid nanoparticles of the present invention have lower in vivo immunogenicity and better tissue compatibility.

[0102] The combined results of in vivo imaging quantification and tissue morphology confirm that ToCAP2-LNP can achieve long-term high expression of mRNA in the seminiferous tubules of the testis, with outstanding targeted enrichment effect and low accumulation in non-target organs. It also has the advantages of low inflammation and low immune stimulation in vivo application.

[0103] Test Example 5 To evaluate the ability of the antioxidant ionizable lipid nanoparticles of this invention to alleviate oxidative stress damage, an oxidative stress model was established using GC-1 spg cells induced by hydrogen peroxide (H2O2), and intracellular reactive oxygen species (ROS) levels were detected using the DCFH-DA fluorescent probe combined with flow cytometry. The testing method was as follows: GC-1 spg cells were seeded in 6-well plates. Once cell confluence reached 70%-80%, an oxidative stress model was established using H2O2 treatment. The experiment included a normal control group, an H2O2 model group, an H2O2+SM102-LNP group, an H2O2+MC3-LNP group, and an H2O2+ToCAP2-LNP group (DSPC was the helper phospholipid, and the lipid molar ratio was ToCAP2:DSPC:cholesterol:DMG-PEG2000=20:20:40:0.25). All groups were treated with the same mRNA dose. After treatment, cells were incubated in DCFH-DA working solution (10 μM, dissolved in serum-free DMEM medium) at 37 ℃ in the dark for 30 min. After washing three times with PBS, cells were digested and collected. Flow cytometry was used to detect fluorescein isothiocyanate (FITC) fluorescence signals, and the proportion of ROS-positive cells was used as an indicator of intracellular ROS levels.

[0104] Test results are as follows Figure 10As shown, flow cytometry results indicated that the ROS level in the GC-1 spg cells of the normal control group was low, with only 0.23% of cells being ROS-positive. After induction with H2O2, the proportion of ROS-positive cells in the H2O2 model group increased to 90.6%, indicating a large accumulation of reactive oxygen species in the cells and the successful construction of the oxidative stress model. Compared with the H2O2 model group, the proportions of ROS-positive cells in the commercial H2O2+SM102-LNP group and the H2O2+MC3-LNP group were 99.8% and 100%, respectively, showing no significant decrease compared with the H2O2 model group. This indicates that traditional lipid nanoparticles have almost no alleviating effect on H2O2-induced oxidative stress, and the intracellular ROS level did not decrease but instead showed a further increasing trend. This suggests that traditional commercial lipid nanoparticles did not show the ability to alleviate ROS accumulation under oxidative stress and may even further aggravate the cellular oxidative stress state. In contrast, after treatment with ToCAP2-LNP of this invention, the proportion of ROS-positive cells decreased to 66.9%, the flow cytometry fluorescence peak shifted significantly to the left, and the intracellular ROS level decreased significantly, indicating that ToCAP2-LNP can effectively remove excessive reactive oxygen species generated under oxidative stress conditions.

[0105] The above results demonstrate that by introducing antioxidant natural products into the hydrophobic tail of ionizable lipids, the resulting lipid nanoparticles possess excellent antioxidant capabilities while maintaining superior nucleic acid delivery performance, effectively reducing intracellular ROS accumulation under oxidative stress. Compared to traditional commercial lipid nanoparticles, the ToCAP2-LNP of this invention not only avoids the continuous increase in ROS under oxidative stress but also significantly alleviates cellular oxidative damage, providing strong support for its application in oxidative stress-related diseases, especially in the treatment of male infertility.

[0106] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art should understand that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. An ionizable lipid, characterized in that, The structural formula of the ionizable lipid is as follows: ； Wherein, X is dimethylaminoethyl, dimethylaminopropyl, dimethylaminoethanol, dimethylaminopropanol, 4-dimethylaminobutyl, diethylaminopropanol, pyrrolylethyl, pyrrolylpropanol, or piperidinylpropanol; R1 is an antioxidant hydrophobic tail group, wherein the antioxidant hydrophobic tail group is a residue formed after dehydrogenation or functional group derivatization of an antioxidant hydrophobic compound; the antioxidant hydrophobic compound is selected from α-tocopherol, eicosapentaenoic acid, retinol, cholecalciferol, coenzyme Q10 derivatives, and propyl gallate.

2. The ionizable lipid according to claim 1, characterized in that, The structural formula of the antioxidant hydrophobic tail group is as follows: ； in Indicates the connection location.

3. A method for preparing lipid nanoparticles, characterized in that, Includes the following steps: (1) Dissolve the ionizable lipid, auxiliary phospholipid, cholesterol and polyethylene glycol-modified lipid as described in claim 1 or 2 in an organic solvent to prepare a lipid organic phase solution; (2) The aqueous solution is mixed with the lipid organic phase solution to obtain a lipid nanoparticle dispersion by self-assembly; (3) The lipid nanoparticle dispersion was replaced with a neutral buffer solution to obtain the lipid nanoparticles.

4. The preparation method according to claim 3, characterized in that, In step (1), the molar ratio of the ionizable lipid, cofactor phospholipid, cholesterol and polyethylene glycol-modified lipid is (10-40):(20-50):(20-50):(0.25-1).

5. The preparation method according to claim 3, characterized in that, In step (1), the auxiliary phospholipid is distearylphosphatidylcholine and / or dioleoylphosphatidylethanolamine; the PEGylated lipid is 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000.

6. Lipid nanoparticles obtained by the preparation method according to any one of claims 3-5.

7. A method for preparing lipid nanoparticles loaded with nucleic acids, characterized in that, Includes the following steps: (1) Dissolve the ionizable lipid, auxiliary phospholipid, cholesterol and polyethylene glycol-modified lipid as described in claim 1 or 2 in an organic solvent to prepare a lipid organic phase solution; (2) The aqueous solution containing nucleic acid is mixed with the lipid organic phase solution to obtain a lipid nanoparticle dispersion loaded with nucleic acid by self-assembly; (3) The lipid nanoparticle dispersion containing nucleic acid was replaced with a neutral buffer solution to obtain the lipid nanoparticles containing nucleic acid.

8. The preparation method according to claim 7, characterized in that, In step (2), the nucleic acid is selected from one or more of mRNA, siRNA, DNA and circular RNA; the volume ratio of the aqueous phase solution containing nucleic acid to the lipid organic phase solution is (1-5):

1.

9. A lipid nanoparticle loaded with nucleic acid obtained by the preparation method of claim 7 or 8.

10. The use of an ionizable lipid according to claim 1 or 2, a lipid nanoparticle according to claim 6, or a nucleic acid-loaded lipid nanoparticle according to claim 9 in the preparation of a nucleic acid delivery formulation.

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