ROS (reactive oxygen species) response type ionizable lipid and application of ROS response type ionizable lipid to delivery of nucleic acid

Abstract

The invention discloses ROS (reactive oxygen species) response type ionizable lipid and application of the ROS response type ionizable lipid to delivery of nucleic acid. According to the ionizable lipid disclosed by the invention, a tertiary amine group is used as a head group, thioketal and a peroxyoxalic acid bond are used as an ROS response connecting chain, and an alkane structure is a hydrophobic tail chain. The structure is easy to synthesize, suitable for large-scale production, good in biocompatibility, capable of responding to the ROS environment and enhancing release of nucleic acid in the intracellular high ROS environment, and suitable for delivery of various nucleic acids, and the cell transfection efficiency is obviously improved by combining the structure with functional adamantane tail chain lipid. The lipid nanoparticles prepared from the ionizable lipid have good stability, and a safe and effective non-viral vector platform can be provided for delivery of various nucleic acids.

Description

Technical Field

[0001] The present invention relates to the field of new preparation materials, and in particular to ROS-responsive ionizable lipids and applications thereof in nucleic acid delivery. Background Art

[0002] Gene therapy refers to the introduction of exogenous normal genes (DNA or RNA, etc.) into target cells to correct or compensate for diseases caused by defective and abnormal genes. At present, the application prospects of gene therapy are broad. However, nucleic acid drugs are susceptible to degradation by nucleases, resulting in the loss of their activity in vivo. At the same time, the negative charge of nucleic acid molecules makes it difficult for nucleic acid drugs to cross the negatively charged cell membrane and enter the cytoplasm / nucleus to exert their effects. Therefore, protecting and assisting nucleic acid drugs to enter cells to improve transfection efficiency is the current research focus. Common delivery methods of nucleic acid drugs mainly include viral vectors and non-viral vectors.

[0003] In addition to nucleic acid components, LNP also includes four lipid components: ionizable lipids, neutral phospholipids, sterol lipids and polyethylene glycol lipids. These four components jointly affect the encapsulation of nucleic acids, LNP particle size, charge properties and stability. Among them, ionizable lipids are the most important components of LNP. The properties of ionizable lipids make LNPs have a strong positive charge in acidic buffers, which is conducive to loading nucleic acids; under physiological conditions (pH 7.4), they are nearly electrically neutral and have good safety; after being internalized into acidic endosomes (pH 5.0-6.0), ionizable lipids can be quickly protonated and carry a strong positive charge, effectively promoting endosomal escape.

[0004] At present, the endosomal escape efficiency of LNP is low (less than 15%). One of the reasons may be that different cells have different phenotypes and functions, and the endosomal environment is also different. For example, compared with normal cells, the ROS content in tumor cells is higher. Therefore, it is crucial to improve the transfection efficiency of LNP for specific types of cells by designing the structure of ionizable lipids to target different endosomal environments. At the same time, improving the biodegradability of ionizable lipids is also closely related to the safety of their in vivo application. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the art, the present invention provides a class of ROS-responsive ionizable lipids, which enhance the release of nucleic acids by responding to intracellular ROS and improve the biodegradability of the ionizable lipids.

[0006] The first object of the present invention is to provide a ROS-responsive ionizable lipid having a structure of general formula (I).

[0007]

[0008] Wherein, m = 1 or 2;

[0009] Wherein, n represents an integer from 1 to 3;

[0010] Wherein p represents an integer from 1 to 17, and q represents an integer from 1 to 8;

[0011]

[0012] Wherein X=CH or N, r represents an integer of 2-4, s represents an integer of 1-3, R 4 represents methyl, ethyl, hydroxyethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, R 5 represents a methyl, ethyl, isopropyl or hydrogen atom;

[0013] Preferably, in the ROS-responsive lipid, m=2; R 1 Selected from n=1 or 2; R 2 Selected from q = integer of 6-8; R 3 Selected from r=3 or 4,s=1 or 2,R 4 is selected from methyl or ethyl, R 5 Selected from methyl or ethyl.

[0014] The ROS-responsive ionizable lipid provided by the present invention has a tertiary amine group as the head group, which enables it to have the ability to be protonated in the endosome; with thioketone and peroxyoxalic acid bonds as the connecting chain, it can respond to the intracellular ROS environment to release nucleic acids, enhance the release of nucleic acids in a high ROS environment, and at the same time improve the biodegradability of lipid materials. Through the above two designs, the release of nucleic acids in a high ROS environment is further enhanced.

[0015] The second object of the present invention is to provide a synthetic route for ROS-responsive ionizable lipids, which are easy to synthesize and suitable for large-scale production.

[0016] The synthesis method of the ROS-responsive ionizable lipid shown in the general formula I is as follows: For example, the synthesis steps are as follows:

[0017] a. Dissolve the dicarboxylic acid (I-1) in anhydrous toluene, add p-toluenesulfonic acid monohydrate under stirring in an oil bath (120-160°C), and reflux for 1-5 hours. After stopping heating, cool the reaction system to room temperature, and then add fatty alcohol R 2OH, continue to heat to 120-160℃ and reflux to react overnight. After the reaction is completed, remove toluene by rotary evaporation at 40-60℃, redissolve the crude product in dichloromethane, wash with appropriate amount of water, saturated sodium bicarbonate aqueous solution and saturated salt water solution in sequence, dry the organic phase with anhydrous sodium sulfate, filter and concentrate to obtain a reddish red oily crude product. Purify by petroleum ether / ethyl acetate column chromatography to obtain dicarboxylic acid ester (I-2).

[0018] I-2 synthesis reaction formula:

[0019]

[0020] b. Dissolve 2,2'-[propane-2,2-diyldisulfide]diacetic acid in dichloromethane, then add HOBt and EDCI in an ice-water bath (0°C), stir for 5-10 minutes, then move the reaction solution to room temperature and stir for 1-3 hours to obtain reaction solution A; dissolve I-1 in dichloromethane, add triethylamine at room temperature, stir for 1-3 hours to obtain reaction solution B; slowly add reaction solution B dropwise into reaction solution A, and stir at room temperature overnight. After the reaction is completed, the reaction solution is washed with water, citric acid aqueous solution and saturated brine in turn, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a pale yellow oily crude product, which is purified by petroleum ether / ethyl acetate column chromatography to obtain I-3.

[0021] I-3 synthesis reaction formula:

[0022]

[0023] c. Dissolve I-3 in dichloromethane, add HOBt and EDCI in an ice-water bath (0°C) in turn, stir for 5-10 min, then move the reaction solution to room temperature and continue stirring for 1-3 h to obtain reaction solution A; R 3 Dissolve in dichloromethane, add pyridine at room temperature, stir and react for 1-2 hours to obtain reaction solution B; slowly add reaction solution B dropwise into reaction solution A, stir at room temperature overnight. After the reaction is completed, purify by dichloromethane / methanol column chromatography to obtain I.

[0024] I Synthesis reaction formula:

[0025]

[0026] The third object of the present invention is to provide the use of such ROS-responsive ionizable lipids in the construction of lipid nanoparticles.

[0027] The use of the ROS-responsive ionizable lipid of the present invention or the ROS-responsive ionizable lipid combined with the functional adamantane tail chain lipid AD8 in the preparation of a lipid nanoparticle delivery system for nucleic acid loading, preferably the use of the ROS-responsive ionizable lipid combined with the functional adamantane tail chain lipid AD8 in the preparation of a lipid nanoparticle delivery system for nucleic acid loading, wherein the functional adamantane tail chain lipid AD8 has the following structural formula:

[0028]

[0029] The lipid nanoparticles all contain ROS-responsive ionizable lipids, neutral phospholipids, cholesterol and pegylated lipids. The molar ratio of ROS-responsive ionizable lipids: neutral phospholipids: cholesterol: pegylated lipids is 10-70: 5-40: 10-60: 0.5-10; preferably, the molar ratio of ROS-responsive ionizable lipids: neutral phospholipids: cholesterol: pegylated lipids is 30-50: 10-25: 40-60: 0.5-3.

[0030] The lipid nanoparticles can be used in combination with the functional adamantane tail lipid AD8. The molar ratio of ROS-responsive ionizable lipid: AD8: neutral phospholipid: cholesterol: PEGylated lipid is 10-50: 10-50: 5-40: 10-60: 0.5-10; preferably, the molar ratio of ROS-responsive ionizable lipid: adamantane tail lipid: neutral phospholipid: cholesterol: PEGylated lipid is 10-30: 10-30: 10-25: 30-60: 0.5-3.

[0031] The PEGylated lipid of the lipid nanoparticle is selected from: 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), polyethylene glycol-cholesterol (PEG-Chol), ditetradecyl (4-methoxypolyethylene glycol-2000-4-oxobutyryl) glutamate (PEG 2000 -Suc-TA 2 ).

[0032] The neutral phospholipid is selected from: soybean lecithin (SPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), dimyristoyl phosphatidylcholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), dierucoyl phosphatidylcholine (DEPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0033] The nucleic acid of the lipid nanoparticle is DNA or RNA, and the RNA is selected from siRNA, saRNA, shRNA, microRNA or mRNA, etc.

[0034] The nitrogen-phosphorus ratio of the ionizable lipid to the nucleic acid in the lipid nanoparticles is 1:1 to 30:1, preferably 3:1 to 10:1.

[0035] The preparation method of the lipid nanoparticles includes an ethanol injection method, a microfluidics method or a transmembrane extrusion method. The microfluidics method is preferably prepared as follows: an appropriate amount of ROS-responsive ionizable lipids, neutral phospholipids, cholesterol and pegylated lipids are weighed according to the nitrogen-phosphorus ratio, and then dissolved in anhydrous ethanol as the ethanol phase. The nucleic acid is dissolved in a citrate buffer solution (pH=4.0) as the aqueous phase. Through a microfluidic device, the aqueous phase and the ethanol phase are mixed in a microfluidic chip at a flow rate of 1 to 30 mL / min and a ratio of 1:1 to 4:1, and then dialyzed with ultrapure water or PBS for 2 to 4 hours at room temperature, and the dialyzed solution is collected as a lipid nanoparticle (LNP) solution.

[0036] The lipid nanoparticles have an average particle size of 50 to 500 nm and a Zeta potential of -20 mV to +20 mV.

[0037] A fourth object of the present invention is to provide use of such ROS-responsive ionizable lipids in cell transfection.

[0038] The lipid nanoparticles are used for transfection of immune cells or tumor cells, wherein the immune cells include macrophages, dendritic cells, T cells, etc., and the tumor cells include human cervical cancer Hela cells, melanoma B16-F10 cells, breast cancer MCF-7 cells, pancreatic cancer Panc-01 cells, lung cancer A549 cells, liver cancer HepG2 cells, etc.

[0039] Beneficial effects:

[0040] The ionizable lipid disclosed in the present invention has a tertiary amine group as the head group, a thioketal and peroxyoxalic acid bond as the ROS response connecting chain, and an alkane structure as the hydrophobic tail chain. The structure is easy to synthesize, suitable for large-scale production, has good biocompatibility, can respond to the ROS environment, enhances the release of nucleic acids in the intracellular high ROS environment, is suitable for the delivery of a variety of nucleic acids, and is used in combination with a functional adamantane tail chain lipid to significantly improve the cell transfection efficiency. The lipid nanoparticles prepared with the ionizable lipid of the present invention have good stability and can provide a safe and effective non-viral vector platform for the delivery of a variety of nucleic acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The TH series of ionizable lipids in Example 25 were dissolved in 5 mM H 2 O 2 Response release situation in;

[0042] Figure 2 The OX series of ionizable lipids in Example 26 were ionized in 25 mM H 2 O 2 HR-MS results of the tertiary amine head group obtained after the response release;

[0043] Figure 3 This is the agarose gel electrophoresis diagram of lipid nanoparticles loaded with mRNA prepared from the TH series ionizable lipids in Example 29;

[0044] Figure 4 This is the agarose gel electrophoresis diagram of lipid nanoparticles loaded with mRNA prepared from OX series ionizable lipids in Example 30;

[0045] Figure 5 The expression of EGFP in Hela cells after the lipid nanoparticles loaded with EGFP-mRNA in Example 31 were transfected into human cervical cancer Hela cells was observed using an inverted fluorescence microscope (scale bar: 100 μm);

[0046] Figure 6 The expression of EGFP in Hela cells was detected by flow cytometry after the lipid nanoparticles prepared by TH series ionizable lipids in Example 31 were loaded with EGFP-mRNA and transfected into human cervical cancer Hela cells;

[0047] Figure 7 The expression of EGFP in human cervical cancer Hela cells was detected by flow cytometry after lipid nanoparticles prepared by OX series ionizable lipids in Example 31 were loaded with EGFP-mRNA and transfected into the cells;

[0048] Figure 8This is an agarose gel electrophoresis diagram of lipid nanoparticles loaded with EGFP-mRNA prepared by introducing the preferred ionizable lipid of adamantane tail lipid AD8 in Example 32;

[0049] Fig. 9 The expression of EGFP in Hela cells after the lipid nanoparticles prepared by introducing the preferred ionizable lipid of the adamantane tail chain lipid AD8 in Example 32 and loaded with EGFP-mRNA were transfected into Hela cells using an inverted fluorescence microscope (scale bar: 100 μm);

[0050] Fig.10 The expression of EGFP in Hela cells was detected by flow cytometry after the lipid nanoparticles prepared by introducing the preferred ionizable lipid of adamantane tail chain lipid AD8 in Example 32 were loaded with EGFP-mRNA and transfected into Hela cells;

[0051] Fig.11 The expression of EGFP in Hela cells after the lipid nanoparticles prepared by introducing the preferred ionizable lipid of the adamantane tail chain lipid AD8 in Example 33 were loaded with siRNA and transfected into EGFP-Hela cells was observed using an inverted fluorescence microscope (scale bar: 100 μm);

[0052] Fig.12 The gene silencing efficiency of lipid nanoparticles prepared by introducing the preferred ionizable lipid of adamantane tail chain lipid AD8 in Example 33 and then loaded with siRNA and transfected into EGFP-Hela cells was detected by flow cytometry;

[0053] Fig.13 In order to observe the lipid nanoparticles prepared by the preferred ionizable lipids introduced with the adamantane tail lipid AD8 in Example 34 using an inverted fluorescence microscope, the lipid nanoparticles loaded with the Pmax-GFP plasmid were transfected with human primary CD3 + T cell CD3 + Expression of EGFP in T cells (scale bar: 100 μm);

[0054] Fig.14 In order to detect the lipid nanoparticles prepared by the preferred ionizable lipids introduced with the adamantane tail chain lipid AD8 in Example 33 by flow cytometry, the Pmax-GFP plasmid was loaded and then transfected into human primary CD3 + T cell CD3 + Expression of EGFP in T cells;

[0055] Fig.15 The expression of luciferase in Hela cells after the lipid nanoparticles of different neutral phospholipids loaded with Luci-mRNA were transfected into Hela cells in Example 36;

[0056] Fig.16 The expression of luciferase in Hela cells after the lipid nanoparticles with different PEGylated lipid molar ratios loaded with Luci-mRNA were transfected into Hela cells in Example 36;

[0057] Fig.17 The expression of luciferase in Hela cells after the lipid nanoparticles with different DOPE molar ratios loaded with Luci-mRNA were transfected into Hela cells in Example 36;

[0058] Fig.18 The expression of luciferase in Hela cells after Hela cells were transfected with Luci-mRNA loaded with lipid nanoparticles with different molar ratios of TH10 and AD8 in Example 36. DETAILED DESCRIPTION

[0059] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0060] Example 1 Preparation of ROS-responsive ionizable lipid TH1

[0061]

[0062] TH1 with compound OA 2 -TH-COOH (1 g, 1.17 mmol) and 3-dimethylaminopropylamine (143.52 mg, 1.40 mmol) were used as raw materials. 2 -TH-COOH (1g, 1.17mmol) was dissolved in dichloromethane, and HOBt (189.80mg, 1.40mmol) and EDCI (260.26mg, 1.40mmol) were added in turn under ice-water bath conditions (0℃) and stirred for 5min, then the reaction solution was moved to room temperature and continued to stir for 3h to obtain reaction solution A; 3-dimethylaminopropylamine (143.52mg, 1.40mmol) was dissolved in dichloromethane, pyridine (277.76mg, 3.51mmol) was added at room temperature, and stirred for 1h to obtain reaction solution B; reaction solution B was slowly added dropwise to reaction solution A and stirred at room temperature overnight. After the reaction was completed, it was purified by column chromatography (dichloromethane: methanol = 30: 1) to obtain 640mg of a light yellow oily product with a yield of 58.26%. 1 HNMR (300MHz, CDCl 3): δ (ppm) 7.67 (s, 1H), 7.53 (d, J = 7.9Hz, 1H), 5.36 (q, J = 5.8Hz, 4H), 4.57 (td, J = 8.0, 5.1Hz, 1H) ,4.11(t,J=6.8Hz,2H),4.05(t,J=6.8Hz,2H),3.42(s,2H),3.38(d,J=7.5Hz,4H),2.79(t,J=7.1 Hz,2H),2.56(s,6H),2.38(dd,J=8.1,6.3Hz,2H),2.21(dq,J=13.8,7.4Hz,2H),2.01(q,J=6.8H z,9H),1.88(q,J=6.6Hz,2H),1.60(d,J=4.1Hz,10H),1.40-1.20(m,50H),0.88(t,J=6.5Hz,6H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)172.68,171.67,169.56,169.17,129.93,129.71,124.64,118.78,110.71,65.71,64.88,57.69,56.59,52.05,44.03,37.8 2,34.76,32.57,31.86,30.26,29.72,29.48,29.39,29.28,29.20,28.55,28.47,27.17,25.86,25.79,25.21,22.64,14.09.HRMS,ESI + ,m / z:CalcdforC53H99N3O6S2[M+H] + ,938.7040;found,938.7044.

[0063] Example 2 Preparation of ROS-responsive ionizable lipid TH2

[0064]

[0065] TH2 with compound OA 2 -TH-COOH (1 g, 1.17 mmol) and N-(3-aminopropyl)diethanolamine (227.87 mg, 1.40 mmol) were used as raw materials, and according to the method for preparing TH1, 540 mg of a light yellow oily product was obtained with a yield of 46.20%. 1 HNMR (500MHz, CDCl 3): δ(ppm)7.18(d,J=8.6Hz,1H),6.68(t,J=4.9Hz,1H),5.37(m,4H),4.31(dt,J=8.6,6.8Hz,1H),4.18-4 .06(m,2H),4.02-3.92(m,4H),3.64(q,J=5.9Hz,4H),3.50-3.39(m,4H),3.20(td,J=6.0,4.9Hz,2H),2. 75(t,J=5.7Hz,4H),2.59(t,J=6.0Hz,2H),2.50-2.32(m,2H),2.03(ddt,J=11.0,7.3,3.7Hz,9H),1.72- 1.61(m,4H),1.48-1.37(m,2H),1.39-1.33(m,3H),1.36-1.29(m,3H),1.32-1.24(m,46H),0.86(m,6H). 13 CNMR (75MHz, CDCl 3 ):δ(ppm)172.67,171.67,170.59,169.65,156.37,142.72,130.37,129.90,1 29.68,127.89,125.28,124.59,118.09,110.97,65.72,64.88,57.57,56.24,5 5.78,52.11,36.83,34.76,34.64,32.55,31.85,30.26,29.71,29.63,29.47,29.40,29.27,29.20,28.54,28.46,27.16,25.86,25.81,23.99,22.63,14.08.

[0066] Example 3 Preparation of ROS-responsive ionizable lipid TH3

[0067]

[0068] TH3 with compound OA 2 -TH-COOH (1 g, 1.17 mmol) and 3-diethylaminopropylamine (182.93 mg, 1.40 mmol) were used as raw materials, and according to the method for preparing TH1, 620 mg of a light yellow oily product was obtained with a yield of 54.80%. 1 HNMR (300MHz, CDCl 3): δ(ppm)7.86(s,1H),7.46(d,J=7.9Hz,1H),5.35(td,J=6.3,3.4Hz,4H),4.58(td,J= 8.0,5.1Hz,1H),4.20-3.99(m,4H),3.46-3.26(m,6H),2.77-2.56(m,6H),2.40(q,J=7 .6Hz,2H),2.26-2.13(m,2H),2.03(dq,J=12.5,7.5Hz,9H),1.75(q,J=6.3Hz,2H),1.6 2(d,J=10.7Hz,10H),1.42-1.18(m,49H),1.11(t,J=7.2Hz,6H),0.88(t,J=6.4Hz,6H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)172.68,171.62,169.03,157.57,156.44,129.94,129.72,65.73,64.87,57.81,52.02,51.46,46.62,34.85,34 .70,31.87,30.29,29.72,29.49,29.40,29.29,29.21,28.48,27.18,25.87,25.79,25.26,22.65,14.09,10.91.HRMS,ESI + ,m / z:CalcdforC55H103N3O6S2[M+H] + ,966.7343;found,966.7347.

[0069] Example 4 Preparation of ROS-responsive ionizable lipid TH4

[0070]

[0071] TH4 with compound OA 2 -TH-COOH (1 g, 1.17 mmol) and 1-methyl-3-aminopyrrolidine (140.69 mg, 1.40 mmol) were used as raw materials, and according to the method for preparing TH1, 610 mg of a light yellow oily product was obtained, with a yield of 55.65%. 1 HNMR (300MHz, CDCl 3): δ (ppm) 8.38 (s, 1H), 7.80 (d, J = 7.8Hz, 1H), 5.45-5.24 (m, 4H), 4.86 (s, 1H), 4.5 6(s,1H),4.07(dt,J=16.5,6.8Hz,4H),3.43(q,J=4.9Hz,2H),3.35(d,J=3.6Hz,2H ),2.90(d,J=3.9Hz,3H),2.40(d,J=7.9Hz,2H),2.23(dd,J=16.6,8.4Hz,2H),2.0 0(p,J=6.9Hz,8H),1.72-1.44(m,10H),1.44-1.00(m,43H),0.87(d,J=7.0Hz,7H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)172.77,171.77,156.45,129.95,129.72,125.76,124.74,118.30,110.72,65.72,64.91,57.61,54.89,52. 06,34.82,32.58,31.87,30.88,30.14,29.73,29.49,29.41,29.29,29.21,28.56,28.47,27.18,25.80,22.65,14.10.

[0072] Example 5 Preparation of ROS-responsive ionizable lipid TH5

[0073]

[0074] TH5 with compound OA 2 -TH-COOH (1 g, 1.17 mmol) and N-methyl-2-(2-aminoethyl)-pyrrolidine (140.69 mg, 1.40 mmol) were used as raw materials, and according to the method for preparing TH1, 580 mg of a light yellow oily product was obtained, with a yield of 51.73%. 1 HNMR (300MHz, CDCl 3): δ(ppm)7.61(d,J=7.8Hz,1H),7.37(d,J=7.9Hz,1H),5.35(td,J=6.4,3.4Hz,3H),4.5 9(td,J=7.9,5.0Hz,1H),4.22-3.98(m,4H),3.55-3.42(m,1H),3.37(d,J=14.9Hz,4H), 3.28(d,J=9.2Hz,2H),2.47(s,3H),2.38(t,J=7.7Hz,2H),2.26-2.16(m,1H),2.03(dq, J=12.3,7.1Hz,10H),1.61(t,J=5.9Hz,10H),1.49-1.13(m,45H),0.88(t,J=6.4Hz,6H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)172.66,171.59,168.98,156.18,130.41,129.94,129.71,65 .77,65.09,64.90,57.75,56.74,52.06,40.20,37.04,34.83,34.69,32 .54,31.86,30.74,30.32,29.72,29.64,29.48,29.39,29.27,29.19,28 .56,28.48,27.31,27.17,25.86,25.78,22.63,22.11,14.06.HRMS,ESI + ,m / z:CalcdforC 55 H 101 N 3 O 6 S 2 [M+H] + ,964.7192;found,964.7196.

[0075] Example 6 Preparation of ROS-responsive ionizable lipid TH6

[0076]

[0077] TH6 with compound OA 2 -TH-COOH (1 g, 1.17 mmol) and (1-methyl-4-piperidinyl-)methylamine (180.09 mg, 1.40 mmol) were used as raw materials, and according to the method for preparing TH1, 630 mg of a light yellow oily product was obtained, with a yield of 55.80%. 1 HNMR (300MHz, CDCl 3): δ (ppm) 7.32 (t, J = 7.2Hz, 2H), 7.20 (s, 1H), 5.47-5.24 (m, 4H), 4.56 (q, J = 7.6Hz, 1H),4.12(t,J=6.8Hz,2H),4.05(t,J=6.8Hz,2H),3.51(s,2H),3.39(d,J=1.7Hz,4H ),3.20(s,2H),2.75(d,J=5.5Hz,3H),2.40(q,J=7.2Hz,2H),2.20(dd,J=13.8,6.4 Hz,2H),2.10-1.93(m,8H),1.88(s,4H),1.43-1.16(m,41H),0.88(t,J=6.4Hz,6H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)172.67,171.63,169.94,169.39,156.19,156.13,130.35,129.89 ,129.66,127.92,125.41,124.43,118.23,110.73,65.66,64.84,57.61,52 .13,43.54,34.75,32.51,31.82,30.34,30.27,29.67,29.60,29.43,29.35 ,29.22,29.16,28.51,28.44,27.13,25.83,25.77,22.59,14.04.HRMS,ESI + ,m / z:CalcdforC 55 H 101 N 3 O 6 S 2 [M+H] + ,964.7193;found,964.7190.

[0078] Example 7 Preparation of ROS-responsive ionizable lipid TH7

[0079]

[0080] TH7 with compound OA 2 -TH-COOH (1 g, 1.17 mmol) and 4-methyl-1-piperazinepropylamine (220.89 mg, 1.40 mmol) were used as raw materials, and according to the method for preparing TH1, 650 mg of a light yellow oily product was obtained with a yield of 55.89%. 1 HNMR (300MHz, CDCl 3): δ(ppm)7.57(d,J=5.3Hz,1H),7.36(d,J=7.9Hz,1H),5.42-5.26(m,4H) ,4.59(td,J=7.9,5.0Hz,1H),4.18-4.01(m,4H),3.39(t,J=2.2Hz,2H),3. 35(d,J=6.8Hz,4H),2.48(dd,J=14.2,7.8Hz,11H),2.31(s,3H),2.01(q, J=6.3Hz,9H),1.75-1.54(m,12H),1.44-1.13(m,50H),0.95-0.79(m,6H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)172.67,171.59,169.05,157.26,156.15,129.92,129.69,123.66, 77.32,65.77,64.91,57.77,56.41,54.35,52.36,52.05,45.36,39.00,34.82 ,34.66,32.53,31.84,30.32,30.27,29.70,29.62,29.46,29.37,29.25,29. 17,28.54,28.46,27.25,27.15,25.84,25.77,25.22,22.61,14.05.HRMS,ESI + ,m / z:CalcdforC 56 H 104 N 4 O 6 S 2 [M+H] + ,993.7473;found,993.7476.

[0081] Example 8 Preparation of ROS-responsive ionizable lipid TH8

[0082]

[0083] TH8 with compound OA 2 -TH-COOH (1 g, 1.17 mmol) and 4-methyl-1-piperazineethylamine (201.18 mg, 1.40 mmol) were used as raw materials, and according to the method for preparing TH1, 680 mg of a light yellow oily product was obtained with a yield of 59.31%. 1 HNMR (300MHz, CDCl 3): δ(ppm)7.84(d,J=8.2Hz,1H),7.65(d,J=8.0Hz,1H),7.45-7.28(m,2H),5.36(d ,J=5.8Hz,4H),4.57(q,J=7.5Hz,1H),4.17-4.00(m,4H),3.49(s,2H),3.38(d,J= 11.9Hz,4H),3.12(s,4H),2.93(s,3H),2.70(s,5H),2.40(q,J=7.4Hz,2H),2.01( d,J=6.4Hz,7H),1.61(d,J=4.8Hz,8H),1.41-1.18(m,46H),0.88(t,J=6.4Hz,6H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)172.68,171.56,169.16,169.03,156.10,129.96,129.70,125.74,118.50,110.58,65.84,64.98,57.68,56.07,53.35,52.13,5 0.10,43.74,36.06,34.76,31.86,30.32,29.71,29.64,29.48,29.27,29.19,28.56,28.48,27.18,25.86,25.79,22.63,14.07.HRMS,ESI + ,m / z:CalcdforC 55 H 102 N 4 O 6 S 2 [M+Na] + ,1001.7101;found,1001.7104.

[0084] Example 9 Preparation of ROS-responsive ionizable lipid TH9

[0085]

[0086] TH9 with compound OA 2 -TH-COOH (1 g, 1.17 mmol) and 4-dimethylaminopiperidine (180.09 mg, 1.40 mmol) were used as raw materials, and according to the method for preparing TH1, 720 mg of a light yellow oily product was obtained with a yield of 63.77%. 1 HNMR (300MHz, CDCl 3): δ(ppm)7.46(d,J=7.8Hz,1H),5.46-5.22(m,3H),4.76(d,J=13.6Hz,1H),4.57(d,J= 7.0Hz,1H),4.17-3.98(m,4H),3.51(d,J=3.9Hz,2H),3.39(d,J=2.2Hz,2H),3.14(t,J =13.2Hz,2H),2.66(s,6H),2.42(t,J=7.7Hz,2H),2.35-2.16(m,2H),2.04(dq,J=12.9 ,7.3Hz,8H),1.62(dd,J=11.9,5.8Hz,10H),1.47-0.98(m,43H),0.88(t,J=6.4Hz,6H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)171.63,168.87,167.57,157.36,156.19,129.92,129.69,57.66,52.07,45.01,39.94 ,34.81,32.86,31.84,30.38,29.69,29.45,29.24,28.54,27.15,25.84,22.61,14.06.HRMS,ESI + ,m / z:CalcdforC 55 H 101 N 3 O 6 S 2 [M+H] + ,964.7193;found,964.7196.

[0087] Example 10 Preparation of ROS-responsive ionizable lipid TH10

[0088]

[0089] TH10 with compound OA 2 -TH-COOH (1 g, 1.17 mmol) and 3-(2-methylpiperidin-1-yl)propan-1-amine (219.50 mg, 1.4 mmol) were used as raw materials, and according to the method for preparing TH1, 710 mg of a light yellow oily product was obtained with a yield of 61.11%. 1 HNMR (300MHz, CDCl 3): δ(ppm)7.96-7.55(m,2H),5.37-5.28(m,4H),4.54(s,1H),4.08(dt,J=16.9,6.8Hz,4H),3.62-3.10(m,8H),3.03-2.81(m, 2H), 2.61 (s, 1H), 2.43 (d, J = 7.4Hz, 2H), 2.31-1.73 (m, 18H), 1.70-1.53 ​​(m, 12H), 1.44-1.10 (m, 45H), 0.88 (t, J = 6.5Hz, 6H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)172.72,171.72,170.24,169.38,129.93,129.71,65.60,64.82,31.85,30.46,30. 29,29.71,29.47,29.39,29.26,29.20,28.56,28.48,27.17,25.86,22.63,14.06.HRMS,ESI + ,m / z:CalcdforC 57 H 105 N 3 O 6 S 2 [M+H] + ,992.7499;found,992.7508.

[0090] Example 11 Preparation of ROS-responsive ionizable lipid TH11

[0091]

[0092] TH11 with compound OA 2 -TH-COOH (1 g, 1.17 mmol) and 4-piperidinylpiperidine (236.37 mg, 1.4 mmol) were used as raw materials, and according to the method for preparing TH1, 690 mg of a light yellow oily product was obtained with a yield of 58.68%. 1 HNMR (300MHz, CDCl 3): δ (ppm) 7.50 (d, J = 7.9Hz, 1H), 5.44-5.23 (m, 4H), 4.64-4.51 (m, 1H), 4.17-4 .00(m,4H),3.50(d,J=3.7Hz,2H),3.39(d,J=1.4Hz,2H),3.12(t,J=12.9Hz,1 H),2.86(s,4H),2.58(t,J=12.8Hz,1H),2.40(q,J=7.0Hz,2H),2.34-2.14(m, 2H),2.09-1.91(m,10H),1.66(s,12H),1.44-1.15(m,44H),0.95-0.78(m,6H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)172.68,171.63,168.83,167.51,157.24,129.93,129.70,65.69,64.86,63.30,57.72,52.09,49.90,34 .84,31.85,30.25,29.71,29.63,29.47,29.38,29.26,29.19,28.49,27.17,25.79,23.66,22.62,14.06.HRMS,ESI + ,m / z:CalcdforC 58 H 105 N 3 O 6 S 2 [M+H] + ,1004.7495;found,1004.7498.

[0093] Example 12 Preparation of ROS-responsive ionizable lipid TH12

[0094]

[0095] TH12 with compound OA 2 -TH-COOH (1 g, 1.17 mmol) and N-(3-aminopropyl)pyrrole (180.09 mg, 1.4 mmol) were used as raw materials, and according to the method for preparing TH1, 750 mg of a light yellow oily product was obtained with a yield of 66.43%. 1 HNMR (300MHz, CDCl 3): δ (ppm) 7.80 (q, J = 8.4Hz, 1H), 7.68 (d, J = 7.8Hz, 1H), 5.35 (td, J = 6.2, 3.6Hz, 4H), 4.56 (td, J =8.0,5.0Hz,1H),4.08(dt,J=17.9,6.9Hz,4H),3.54-3.39(m,4H),3.40-3.35(m,2H),3.11(t, J=7.1Hz,2H),2.43(td,J=7.5,3.8Hz,2H),2.14(dq,J=9.0,5.4Hz,4H),2.05(d,J=4.1Hz,2H), 2.01(d,J=6.3Hz,4H),1.63(s,6H),1.61-1.43(m,4H),1.43-1.23(m,50H),0.93-0.83(m,6H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)172.68,171.71,170.04,169.32,157.23,156.08,129.91,129. 69,65.60,64.81,57.57,53.64,53.41,52.82,52.11,36.63,34.87,34.78 ,32.53,31.84,30.43,30.29,29.70,29.62,29.46,29.37,29.25,29.18, 28.55,28.48,27.15,25.85,25.79,25.62,23.25,22.61,14.04.HRMS,ESI + ,m / z:CalcdforC 55 H 101 N 3 O 6 S 2 [M+H] + ,964.7190;found,964.7192.

[0096] Example 13 Preparation of ROS-responsive ionizable lipid OX1

[0097]

[0098] OX1 is a compound OA 2 -TH 2(1g, 1.54mmol) and 3-dimethylaminopropylamine (189.20mg, 1.85mmol) were used as raw materials. 3-Dimethylaminopropylamine (189.20mg, 1.85mmol) was dissolved in dichloromethane, pyridine (244.12mg, 3.09mmol) was added and stirred at room temperature to obtain reaction solution A; oxalyl chloride (979.22mg, 7.72mmol) was slowly dripped into dichloromethane under stirring at room temperature, and then OA was slowly dripped into the solution under ice-water bath conditions. 2 -NH 2 (1g, 1.54mmol) of dichloromethane solution, react at low temperature for 5min, then move the reaction system to room temperature and stir for 3h. After the reaction is completed, the reaction system is vacuum evaporated at 45℃, and dichloromethane is added 5 times in small amounts to azeotropically rotate the excess oxalyl chloride until there is no oxalyl chloride residue. No other treatment is required to obtain a yellow oily product, which is dissolved in dichloromethane to obtain reaction solution B; under ice-water bath stirring conditions, reaction solution B is slowly added dropwise to reaction solution A, react at low temperature for 5min, and then the reaction system is moved to room temperature and stirred for overnight reaction. After the reaction is completed, it is purified by column chromatography (dichloromethane: methanol = 35: 1) to obtain 560mg of a light yellow oily product with a yield of 45.12%. 1 HNMR (300MHz, CDCl 3 ): δ (ppm) 8.15-8.01 (m, 1H), 7.95 (d, J = 8.5Hz, 1H), 5.45-5.25 (m, 4H), 4.58 (td ,J=8.2,4.7Hz,1H),4.15(t,J=6.8Hz,2H),4.06(t,J=6.8Hz,2H),3.48(q,J=6.1 Hz,2H),2.88(t,J=7.5Hz,2H),2.66(s,6H),2.39(q,J=6.9Hz,2H),2.02(dt,J=1 2.8,6.4Hz,10H),1.72-1.54(m,4H),1.43-1.07(m,47H),0.88(t,J=6.5Hz,6H). 13 CNMR (75MHz, CDCl 3): δ(ppm)172.37,170.65,159.65,159.61,157.93,130.36,129.89,129.66,65.91,64.89,56.03,52.00,43.52,37.42,32.53 ,31.84,30.14,29.69,29.61,29.46,29.36,29.25,29.17,28.52,28.42,27.14,25.84,25.72,24.75,22.61,14.04.HRMS,ESI + ,m / z:CalcdforC 48 H 89 N 3 O 6 [M+H] + ,804.6807;found,804.6810.

[0099] Example 14 Preparation of ROS-responsive ionizable lipid OX2

[0100]

[0101] OX2 is a compound OX-OA 2 (1.1 g, 1.49 mmol) and N-(3-aminopropyl)diethanolamine (300.40 mg, 1.85 mmol) were used as raw materials, and according to the method for preparing OX1, 350 mg of a light yellow oil was obtained, with a yield of 26.24%. 1 HNMR (300MHz, DMSO): δ (ppm) 7.40 (d, J = 9.5Hz, 1H), 7.00 (t, J = 5.4Hz, 1H), 5.38-5.30 (m, 4H), 4.38 (dt, J = 9 .3,6.8Hz,1H),4.18-4.06(m,2H),4.02-3.92(m,4H),3.64(q,J=5.9Hz,4H),3.22(td,J=6.1,5.3Hz,2H),2. 75(t,J=5.7Hz,4H),2.59(t,J=6.0Hz,2H),2.52-2.32(m,2H),2.12-1.98(m,9H),1.74(p,J=6.0Hz,2H),1. 70-1.61(m,1H),1.48-1.36(m,3H),1.39-1.33(m,3H),1.36-1.29(m,2H),1.43-1.06(m,46H),0.85(m,6H). 13 CNMR (75MHz, CDCl 3): δ(ppm)172.35,170.43,160.63,158.91,155.98,129.93,129.74,66.02,64.98,59.90,57.18,54.60 ,52.11,39.51,31.88,30.13,29.74,29.50,29.29,28.54,27.19,25.87,25.75,22.65,14.08.HRMS,ESI + ,m / z:CalcdforC 50 H 93 N 3 O 8 [M+H] + ,865.7098;found,865.7159.

[0102] Example 15 Preparation of ROS-responsive ionizable lipid OX3

[0103]

[0104] OX3 is a compound OX-OA 2 (1.1 g, 1.49 mmol) and 3-diethylaminopropylamine (241.15 mg, 1.85 mmol) were used as raw materials, and according to the method for preparing OX1, 580 mg of a light yellow oil was obtained, with a yield of 45.16%. 1 HNMR (300MHz, CDCl 3 ): δ (ppm) 8.75 (s, 1H), 7.93 (d, J = 8.6Hz, 1H), 5.44-5.26 (m, 4H), 4.59 (td, J=8.3,4.8Hz,1H),4.14(t,J=6.8Hz,2H),4.06(t,J=6.8Hz,2H),3.41(q,J =6.0Hz,2H),2.65-2.52(m,6H),2.46-2.19(m,3H),2.01(q,J=6.7Hz,8H), 1.74(p,J=6.4Hz,2H),1.61(dd,J=11.3,6.4Hz,4H),1.36-1.22(m,50H,CH 2 (oleoyl)),1.08(t,J=7.1Hz,7H),0.88(t,J=6.6Hz,7H). 13 CNMR (75MHz, CDCl 3)δ(ppm)172.19,170.48,159.77,159.12,130.36,129.89,129.66,65.87,64.86,56.01,54.67,52.12,52.07,45.32,36.34,32.47,31.81,3 0.17,29.67,29.58,29.42,29.32,29.22,29.13,29.07,28.54,28.43,27.25,27.13,25.82,25.70,22.56,13.96.13CNMR(75MHz,CDCl3):δ(p pm)172.30,170.63,159.88,159.05,156.78,130.37,129.90,129.70 ,65.82,64.84,52.13,51.87,46.79,39.86,32.56,31.87,31.39,30.1 6,29.73,29.66,29.49,29.39,29.35,29.28,29.19,29.14,28.54,28.44,27.38,27.17,25.86,25.74,25.50,22.64,14.06,11.54.HRMS,ESI + ,m / z:Calcdfor C 50 H 93 N 3 O 6 [M+H] + ,832.7130;found,832.7133.

[0105] Example 16 Preparation of ROS-responsive ionizable lipid OX4

[0106]

[0107] OX4 is a compound OX-OA 2 (1.1 g, 1.49 mmol) and 1-methyl-3-aminopyrrolidine (185.47 mg, 1.85 mmol) were used as raw materials, and according to the method for preparing OX1, 620 mg of a light yellow oil was obtained, with a yield of 50.09%. 1 HNMR (300MHz, CDCl 3): δ(ppm)7.92(d,J=9.0Hz,1H),7.72(d,J=8.5Hz,1H),5.48-5.22(m,3H),4.58( qd,J=6.7,3.6Hz,1H),4.46(s,1H),4.19-4.10(m,2H),4.06(td,J=6.8,2.1Hz,2H ),2.93(s,1H),2.71(d,J=6.2Hz,2H),2.40(s,3H),2.38-2.21(m,4H),2.11-1.9 1(m,8H),1.62(p,J=6.5Hz,4H),1.46-1.05(m,44H),0.88(td,J=6.7,2.2Hz,6H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)172.31,170.55,159.74,158.41,157.86,130.39,129.92,129 .72,65.92,64.91,62.26,54.76,51.99,49.52,41.68,32.72,32.69,32 .57,31.88,30.14,29.73,29.65,29.49,29.39,29.36,29.29,29.20,29 .14,28.55,28.44,27.31,27.18,25.86,25.74,22.65,14.07.HRMS,ESI + ,m / z:CalcdforC 48 H 87 N 3 O 6 [M+H] + ,804.6807;found,804.6810.

[0108] Example 17 Preparation of ROS-responsive ionizable lipid OX5

[0109]

[0110] OX5 is a compound OX-OA 2 (1.1 g, 1.49 mmol) and N-methyl-2-(2-aminoethyl)-pyrrolidine (237.42 mg, 1.85 mmol) were used as raw materials, and according to the method for preparing OX1, 590 mg of a light yellow oil was obtained, with a yield of 46.05%. 1 HNMR (300MHz, CDCl 3): δ(ppm)8.10(s,1H),7.94(d,J=8.5Hz,1H),5.43-5.27(m,3H),4.58(td,J=8.1,4.6Hz,1 H),4.15(t,J=6.8Hz,2H),4.06(t,J=6.8Hz,2H),3.49(dt,J=12.2,6.4Hz,1H),3.43-3.18( m,2H),2.44(d,J=2.8Hz,3H),2.37(t,J=6.6Hz,2H),2.33-2.19(m,2H),2.01(q,J=6.4Hz,1 0H),1.85(s,2H),1.63(dt,J=12.0,6.5Hz,4H),1.48-1.03(m,41H),0.88(t,J=6.4Hz,6H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)172.29,170.57,159.87,159.01,129.93,129.71,65.90,64.90,64.32,57.00,51.99,40.38,36.89,32.55,31.87,31.03, 30.17,29.72,29.54,29.48,29.38,29.28,29.19,29.13,28.55,28.45,27.35,27.17,25.86,25.74,22.63,22.30,14.05.HRMS,ESI + ,m / z:CalcdforC 50 H 91 N 3 O 6 [M+H] + ,830.6907;found,830.6976.

[0111] Example 18 Preparation of ROS-responsive ionizable lipid OX6

[0112]

[0113] OX6 is a compound OX-OA 2 (1.1 g, 1.49 mmol) and (1-methyl-4-piperidinyl-)methylamine (237.42 mg, 1.85 mmol) were used as raw materials, and according to the method for preparing OX1, 640 mg of a light yellow oil was obtained, with a yield of 49.95%. 1 HNMR (300MHz, CDCl 3): δ(ppm)7.96(d,J=8.4Hz,1H),7.52(t,J=6.4Hz,1H),5.49-5.18(m,4H),4.59(dd,J=8.5,5.2Hz,1H),4.22-4.00(m,4H),3.38-3.10( m,4H),2.54(s,3H),2.48-2.19(m,5H),2.02(p,J=7.8Hz,8H),1.64(dd,J=12.5,6.7Hz,6H),1.45-1.00(m,43H),0.88(t,J=6.3Hz,7H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)172.31,170.54,159.71,159.40,156.82,155.57,130.38,129.91,129.67,65.95,64.92,54.66,52.06,45.08,44.61,34.41,32.5 3,31.84,30.14,29.70,29.63,29.59,29.46,29.37,29.33,29.25,29.17,29.11,28.53,28.42,27.15,25.84,25.72,22.61,14.03.HRMS,ESI + ,m / z:CalcdforC 50 H 91 N 3 O 6 [M+H] + ,829.6907;found,830.6975.

[0114] Example 19 Preparation of ROS-responsive ionizable lipid OX7

[0115]

[0116] OX7 is a compound OX-OA 2 (1.1 g, 1.49 mmol) and 4-methyl-1-piperazinepropylamine (291.19 mg, 1.85 mmol) were used as raw materials, and according to the method for preparing OX1, 610 mg of a light yellow oil was obtained, with a yield of 46.00%. 1 HNMR (300MHz, CDCl 3): δ (ppm) 8.59 (t, J = 5.4Hz, 1H), 7.93 (d, J = 8.5Hz, 1H), 5.46-5.25 (m, 4H), 4.59 (t d,J=8.3,5.0Hz,1H),4.15(t,J=6.8Hz,2H),4.06(t,J=6.8Hz,2H),3.41(q,J=6.1 Hz,2H),2.81-2.43(m,12H),2.37(d,J=2.9Hz,3H),2.33-2.20(m,2H),2.03(dq,J =12.5,6.5Hz,10H),1.85-1.43(m,8H),1.36-1.25(m,50H),0.88(t,J=6.4Hz,6H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)172.31,170.62,159.81,159.13,155.55,129.96,129.73,65.92,64.93,57.08,54.73,52.96,51.94,45.73,39 .56,31.87,30.18,29.72,29.49,29.28,29.20,28.56,28.46,27.44,27.18,25.87,25.74,25.01,22.64,14.06.HRMS,ESI + ,m / z:Calcdfor C 51 H 94 N 4 O 6 [M+H] + ,859.7220;found,859.7228.

[0117] Example 20 Preparation of ROS-responsive ionizable lipid OX8

[0118]

[0119] OX8 is a compound OX-OA 2 (1.1 g, 1.49 mmol) and 4-methyl-1-piperazineethylamine (265.22 mg, 1.85 mmol) were used as raw materials, and according to the method for preparing OX1, 605 mg of a light yellow oil was obtained, with a yield of 46.38%. 1HNMR (300MHz, DMSO): δ (ppm) 9.02 (d, J = 7.7Hz, 1H), 8.57 (d, J = 6.2Hz, 1H), 5.32 (t, J = 5.0Hz, 3H), 4.29 (t, J = 6.8Hz, 1H), 4.09-3.90 ( m,4H),2.45-2.27(m,8H),2.23(s,3H),2.05-1.88(m,8H),1.53(s,4H),1.24(d,J=5.2Hz,43H),1.06(s,2H),0.84(d,J=6.5Hz,6H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)172.19,170.48,159.77,159.12,130.36,129.89,129.66,65.87,64.86,56.01,54.67,52.12,52.07,45.32,36.34,32.47 ,31.81,30.17,29.67,29.58,29.42,29.32,29.22,29.13,29.07,28.54,28.43,27.25,27.13,25.82,25.70,22.56,13.96.HRMS,ESI + ,m / z:CalcdforC 50 H 92 N 4 O 6 [M+H] + ,845.7099;found,845.7110.

[0120] Example 21 Preparation of ROS-responsive ionizable lipid OX9

[0121]

[0122] OX9 is a compound OX-OA 2 (1.1 g, 1.49 mmol) and 4-dimethylaminopiperidine (237.42 mg, 1.85 mmol) were used as raw materials, and according to the method for preparing OX1, 630 mg of a light yellow oil was obtained, with a yield of 49.17%. 1 HNMR (300MHz, CDCl 3): δ(ppm)7.73(dd,J=14.1,8.1Hz,1H),5.45-5.24(m,4H),4.97(d,J=13.7Hz,1H),4 .72-4.45(m,2H),4.15(tt,J=6.9,3.8Hz,2H),4.07(td,J=6.8,1.6Hz,2H),3.08(q,J =12.6Hz,1H),2.82-2.60(m,2H),2.45--2.39(m,6H),2.26(dq,J=13.2,6.2Hz,2H),2 .02(p,J=8.5Hz,11H),1.71-1.50(m,6H),1.38-1.21(m,50H),0.88(t,J=6.3Hz,6H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)172.45,170.85,161.58,161.49,160.46,130.38,129.91,129.69 ,65.86,64.92,62.06,61.75,51.78,50.42,45.07,42.14,41.13,40.89,32 .53,31.85,30.23,29.70,29.64,29.60,29.46,29.36,29.26,29.18,29.13 ,28.53,28.45,27.35,27.16,27.05,25.84,25.73,22.62,14.04.HRMS,ESI + ,m / z:CalcdforC 50 H 91 N 3 O 6 [M+H] + ,830.6907;found,830.6975.

[0123] Example 22 Preparation of ROS-responsive ionizable lipid OX10

[0124]

[0125] OX10 is a compound OX-OA 2 (1.1 g, 1.49 mmol) and 3-(2-methylpiperidin-1-yl)propan-1-amine (347.24 mg, 1.85 mmol) were used as raw materials, and according to the method for preparing OX1, 720 mg of a light yellow oil was obtained. The yield was 45.30%. 1 HNMR (300MHz, CDCl 3): δ(ppm)8.14(s,1H),7.93(d,J=8.6Hz,1H),5.35(td,J=11.5,6.3Hz,4H),4.5 9(td,J=8.2,4.7Hz,1H),4.15(t,J=6.8Hz,2H),4.06(t,J=6.8Hz,2H),3.55-3.3 1(m,2H),3.10(s,1H),2.72(s,1H),2.48-2.20(m,3H),2.03(tq,J=15.2,6.7Hz ,11H),1.63(dt,J=12.2,6.6Hz,4H),1.34-1.23(m,44H),0.88(t,J=6.4Hz,6H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)172.33,170.65,159.70,159.30,129.94,129.72,65.90,64. 91,57.02,52.20,51.91,51.59,39.21,33.25,32.56,31.87,30.17,29 .72,29.64,29.49,29.40,29.36,29.28,29.20,29.14,28.55,28.45,2 7.40,27.18,25.86,25.74,24.88,24.59,22.88,22.64,17.72,14.06.

[0126] Example 23 Preparation of ROS-responsive ionizable lipid OX11

[0127]

[0128] OX11 is a compound OX-OA 2 (1.1 g, 1.49 mmol) and 4-piperidinylpiperidine (311.60 mg, 1.85 mmol) were used as raw materials, and according to the method for preparing OX1, 700 mg of a light yellow oil was obtained, with a yield of 52.12%. 1 HNMR (300MHz, CDCl 3): δ(ppm)7.71(dd,J=15.5,8.1Hz,1H),5.36(q,J=7.4Hz,4H),5.04(s,1H), 4.56(td,J=8.0,4.9Hz,1H),4.29-3.94(m,4H),3.04(q,J=12.4Hz,2H),2.96 -2.54(m,5H),2.40(tt,J=7.1,4.1Hz,2H),2.27(dt,J=13.7,5.9Hz,2H),2.0 1(q,J=5.7Hz,9H),1.81(s,12H),1.46-1.05(m,42H),0.88(t,J=6.3Hz,6H). 13 CNMR (75MHz, CDCl 3 ): δ(ppm)172.44,170.85,161.49,160.31,160.27,130.39,129.91,129.70,65.84,64.90,62.39,51.77,50.14,45.65,42.85,32.54,31.8 6,30.20,29.72,29.64,29.47,29.38,29.35,29.27,29.19,29.14,28 .55,28.46,27.53,27.16,25.97,25.85,25.74,24.44,22.63,14.05.

[0129] Example 24 Preparation of ROS-responsive ionizable lipid OX12

[0130]

[0131] OX12 is a compound OX-OA 2 (1.1 g, 1.49 mmol) and N-(3-aminopropyl)pyrrole (261.16 mg, 1.85 mmol) were used as raw materials, and according to the method for preparing OX1, 640 mg of a light yellow oil was obtained, with a yield of 45.41%. 1 HNMR (300MHz, CDCl 3): δ (ppm) 7.94 (d, J = 8.5Hz, 2H), 5.56-5.16 (m, 3H), 4.59 (td, J = 8.1, 4.7Hz, 1H), 4.23-3.96 (m, 4H), 3.48 (d, J = 5.3Hz, 4H), 3.05 (t, J = 7.6Hz, 2H),2.39(q,J=7.1Hz,2H),2.35-2.17(m,2H),2.21-1.88(m,13H),1.64(dd,J=12.6,6.4Hz,6H),1.47-0.98(m,44H),0.88(t,J=6.5Hz,6H). 13 CNMR (75MHz, CDCl 3 ): δ (ppm)172.39,170.67,159.80,159.51,155.50,129.91,129.67,65.94,64.91,53.75,53.03,52.02,50.35,37.11,32.78,32.52,31 .84,30.16,29.69,29.63,29.59,29.45,29.37,29.24,29.18,29.12,28.53,28.43,27.15,25.84,25.74,23.30,22.61,14.03.HRMS,ESI + ,m / z:CalcdforC 50 H 91 N 3 O 6 [M+H] + ,830.6949;found,830.6956.

[0132] Example 25 ROS responsiveness of TH series lipids

[0133] 5,5'-Dithio-bis-(2-nitrobenzoicacid) (DTNB) was used to investigate the responsiveness of TH series lipids to hydrogen peroxide and to conduct quantitative detection. DTNB, also known as Ellman's reagent, can be used to quantitatively detect free thiols in solution. DTNB can react quantitatively with free thiols in solution at a pH of 7.5-8.6 in a 1:1 ratio to generate disulfides and 2-nitro-5-thiobenzoic acid (TNB), where TNB has an obvious absorption peak at a wavelength of 412nm. Since the amount of TNB generated is the same as the amount of thiol consumed, it can be used to quantitatively detect thiols. TH series lipids use thioketal as the ROS-responsive linker chain, which can break in response to hydrogen peroxide to produce two molecules of free thiol and one molecule of acetone. Therefore, the DTNB method was used to explore the ROS-responsiveness of TH series ROS-responsive lipids. The results are as follows. Figure 1 As shown, all TH series ROS-responsive ionizable lipids can be rapidly broken within the first 2 h after the start of the reaction to expose free thiol groups, indicating that the TH series lipids have good ROS-responsive ability.

[0134] Example 26 ROS responsiveness of OX series lipids

[0135] Since the OX series lipids do not produce free thiol groups after response cleavage, the DTNB method cannot be used for quantitative detection. HRMS is used for qualitative detection of ROS response cleavage. Since the tail chain part has a relatively complex structure and contains carbon-carbon double bonds that are easily affected by strong oxidants, it is difficult for the molecular ion peak of the tail chain part to appear in the HRMS results. The head group part that is not easily affected will be selected as the target molecule for detection. The results are as follows Figure 2 As shown, all OX series lipids can be 2 O 2 Responsive fragmentation was performed in solution, and the molecular ion peaks of the corresponding head groups of each OX lipid were successfully detected, demonstrating its ROS responsiveness.

[0136] Example 27 Preparation and Characterization of TH Series Lipid Nanoparticles

[0137] According to TH lipid / DOPE / Chol / PEG2000-Suc-TA 2=36 / 15 / 46.5 / 2.5 molar ratio, the required ROS-responsive ionizable lipids TH1-TH12, neutral phospholipids, cholesterol and pegylated lipids were weighed and dissolved in anhydrous ethanol as the ethanol phase. EGFP-mRNA was dissolved in 10mM citric acid buffer (pH=4.0) as the aqueous phase. Under vigorous stirring, the ethanol phase was quickly and evenly injected into the aqueous phase, and the volume ratio of the aqueous phase to the ethanol phase was 3:1. After the injection was completed, the sample was dialyzed with ultrapure water for 4h at room temperature to obtain the LNP solution. The particle size, polydispersity index (PDI) and Zeta potential of LNP were measured using an Omni particle size potential analyzer, and the results are shown in Table 1.

[0138] Table 1 Properties of TH series lipid nanoparticles of the present invention (n=3)

[0139]

[0140]

[0141] The above data show that the particle size of the TH series LNP of the present invention is between 110nm and 180nm, the potential is between +10mV and +20mV, and the PDI is less than 0.3, indicating that the above ionizable lipids can form stable LNPs.

[0142] Example 28 Preparation and Characterization of OX Series Lipid Nanoparticles

[0143] According to OX lipid / DOPE / Chol / PEG2000-Suc-TA 2 =36 / 15 / 46.5 / 2.5 molar ratio The required ROS-responsive ionizable lipids OX1-OX12, neutral phospholipids, cholesterol and pegylated lipids were weighed and dissolved in anhydrous ethanol as the ethanol phase. LNPs were prepared according to the method of Example 27. The particle size, PDI and potential of LNPs were measured using an Omni particle size potential analyzer. The results are shown in Table 2.

[0144] Table 2 Properties of OX series lipid nanoparticles of the present invention (n=3)

[0145]

[0146] The above data show that the particle size of the OX series LNP of the present invention is between 80nm and 130nm, the potential is between +10mV and +20mV, and the PDI is less than 0.3, indicating that the above ionizable lipids can form stable LNPs.

[0147] Example 29: Loading capacity of TH series lipid nanoparticles for mRNA

[0148] The TH series of lipid nanoparticles with different ROS-responsive ionizable lipids loaded with EGFP-mRNA at N / P=7 were prepared by the ethanol injection method of Example 27. The mRNA loading capacity of lipid nanoparticles was investigated by agarose gel electrophoresis. Figure 3 The results showed that TH series lipid nanoparticles could stably load mRNA when N / P=7 and could be further used in cell transfection experiments.

[0149] Example 30: Loading capacity of OX lipid nanoparticles for mRNA

[0150] The OX series of lipid nanoparticles with different ROS-responsive ionizable lipids loaded with EGFP-mRNA at N / P=7 were prepared by the ethanol injection method of Example 27. The mRNA loading capacity of lipid nanoparticles was investigated by agarose gel electrophoresis. Figure 4 The results showed that the OX series lipid nanoparticles could stably load mRNA when N / P=7 and could be further used in cell transfection experiments.

[0151] Example 31 Transfection of cervical cancer Hela cells with lipid nanoparticles loaded with EGFP-mRNA

[0152] Lipid nanoparticles TH1LNP-TH12LNP and OX1LNP-OX12LNP loaded with EGFP-mRNA were prepared according to the method of Example 27. One day before transfection, 1×10 6 Human cervical cancer Hela cells were inoculated in a 24-well plate at a density of cells / mL, and 500 μL of DMEM culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was added to each well. After culturing in a 37°C cell incubator containing 5% carbon dioxide for 24 hours, the culture medium was discarded, and 500 μL of DMEM culture medium and 100 μL of lipid nanoparticles were added, and the culture was continued for 6 hours. The culture medium was discarded, and 500 μL of DMEM culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was added, and the culture was continued. After 48 hours, the expression of green fluorescent protein in Hela-eGFP cells was observed using an inverted fluorescence microscope, and the mean fluorescence intensity (MFI) in Hela-eGFP cells was quantitatively examined using a flow cytometer. The results are shown in Figure 5 , Figure 6 and Figure 7 As shown, the expression of EGFP in Hela cells observed under an inverted fluorescence microscope is basically consistent with the results of quantitative investigation by flow cytometry. The mRNA-LNP prepared by the ionizable lipids of TH4, TH9, TH10, TH11 and OX4, OX9, OX10, OX11 of the present invention has significant green fluorescence of EGFP-mRNA expression on Hela cells.

[0153] Example 32 Transfection of cervical cancer Hela cells by lipid nanoparticles containing adamantane tail lipid AD8 loaded with EGFP-mRNA

[0154] Lipid nanoparticles TH4AD8LNP, TH9AD8LNP, TH10AD8LNP, TH11AD8LNP, OX4AD8LNP, OX9AD8LNP, OX10AD8LNP and OX11AD8LNP containing adamantane tail lipid AD8 (patent CN114436994B) and loaded with eGFP mRNA were prepared according to the method of Example 27, and the prescription composition was TH lipid (OX lipid) / AD8 / DOPE / chol / PEG2000-Suc-TA 2 =18 / 18 / 15 / 46.5 / 2.5 (mol:mol). The particle size, PDI and potential of LNP were measured using an Omni particle size potential analyzer. The results are shown in Table 3. The introduction of AD8 can significantly reduce the particle size of most LNPs. The agarose electrophoresis results are shown in Table 3. Figure 8 As shown in Figure 3, the introduction of AD8 does not affect the stability of EGFP-mRNA loading. Transfection was performed according to the method of Example 31. The results are shown in Figure 3 Fig. 9 and Fig.10 As shown, LNPs containing AD8 have stronger EGFP fluorescence intensity than LNPs without AD8, indicating that the introduction of AD8 can significantly increase the transfection effect of mRNA.

[0155] Table 3 Properties of lipid nanoparticles after introduction of AD8 (n=3)

[0156]

[0157] Example 33 Transfection of EGFP-Hela cells with lipid nanoparticles containing adamantane tail lipid AD8 loaded with siRNA

[0158] According to the method of Example 27, siRNA-loaded lipid nanoparticles TH10AD8LNP, TH11AD8LNP and OX12AD8LNP containing adamantane tail lipid AD8 were prepared, and the formulation composition was TH lipid (OX lipid) / AD8 / DOPE / chol / PEG2000-Suc-TA 2 =18 / 18 / 15 / 46.5 / 2.5 (mol:mol). Human cervical cancer EGFP-Hela cells stably expressing enhanced green fluorescent protein (EGFP) were cultured at 1×10 5The cells were inoculated at a density of 10 cells / mL in a 24-well plate, and 500 μL of RPMI1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was added to each well. After culturing in the incubator for 24 hours, the medium was discarded, and 440 μL of RPMI 1640 medium and 60 μL of lipid nanoparticles were added. After continuing to culture for 6 hours, the culture medium was discarded. Then 500 μL of RPMI1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was added and the culture was continued. After culturing for 48 hours, the expression of green fluorescent protein in Hela-eGFP cells was observed using an inverted fluorescence microscope, and the MFI of EGFP in the cell species was quantitatively examined using a flow cytometer. The results are shown in Fig.11 and Fig.12 As shown, the siRNA-LNP prepared by combining the lipid of the present invention with AD8 showed a gene silencing efficiency greater than 60%, which is better than the commercial transfection reagent Lipofectamine2000.

[0159] Example 34 Effect of lipid nanoparticles containing adamantane tail lipid AD8 loaded with Pmax-GFP plasmid on primary human CD3 + Transfection of T cells

[0160] Lipid nanoparticles TH10AD8LNP and TH11AD8LNP loaded with Pmax-GFP plasmid were prepared according to the method of Example 27, and the formulation composition was TH lipid / AD8 / DOPE / chol / PEG2000-Suc-TA 2 =18 / 18 / 15 / 46.5 / 2.5 (mol:mol). + T cells were 1×10 6 1×10 cells / mL were plated (six-well culture plate for ultra-low attachment suspension cells), and then 1×10 6 The cells were inoculated in a 24-well plate at a density of 100 / mL. 500 μL of T cell culture medium containing human IL-2 (100 IU / mL) and human CD3 / CD28 antibodies were added to each well, and 100 μL of LNPs were added. After culturing in an incubator for 48 h, the expression of GFP was observed using an inverted fluorescence microscope, and the expression of GFP was examined by flow cytometry. + T cell positive rate and GFP MFI. Fig.13 and Fig.14 As shown, the Pmax-LNP prepared by combining the lipid of the present invention with AD8 showed good T cell transfection efficiency.

[0161] Example 35 Preparation and characterization of lipid nanoparticles with different formulations

[0162] According to the method and formulation of Example 27, LNPs loaded with Fluc-mRNA were prepared using DSPC or DOPE as neutral phospholipids. The particle size, PDI and potential of LNPs were measured using an Omni particle size potential analyzer. The results are shown in Table 4.

[0163] Table 4 Properties of lipid nanoparticles of different neutral phospholipids (n=3)

[0164]

[0165] The particle size of the above LNPs is between 100 and 200 nm, the potential is about +15 mV, and the PDI is less than 0.3, indicating that LNPs can be prepared using DOPE or DSPC as neutral phospholipids.

[0166] According to the method of Example 27, N / P was fixed to 7, and the ionizable lipid and DOPE in the formulation accounted for 36% and 15% of the total lipid molar amount, respectively. By adjusting the molar ratio of cholesterol and PEGylated lipid, the polyethylene glycol accounted for 0%, 0.5%, 1%, 1.5%, 2%, 2.5% and 4% of the total lipid molar amount, respectively, to prepare LNP loaded with Fluc-mRNA. The particle size, PDI and potential of LNP were measured using an Omni particle size potential analyzer, and the results are shown in Table 5.

[0167] Table 5 Properties of lipid nanoparticles with different PEGylated lipid molar ratios (n=3)

[0168]

[0169] The particle size of the LNPs is between 100 and 150 nm, the potential is between +10 and +20 mV, and the PDI is less than 0.3, indicating that PEGylated lipids within a certain range can be made into stable LNPs.

[0170] According to the method of Example 27, N / P was fixed at 7, and the ionizable lipid and polyethylene glycol lipid in the formulation accounted for 36% and 1.5% of the total lipid molar amount, respectively. By adjusting the molar ratio of cholesterol and DOPE, DOPE accounted for 6%, 10%, 15%, 20% and 25% of the total lipid molar amount, respectively, to prepare LNP loaded with Fluc-mRNA. The particle size, PDI and potential of LNP were measured using an Omni particle size potential analyzer, and the results are shown in Table 6.

[0171] Table 6 Properties of lipid nanoparticles with different DOPE molar ratios (n=3)

[0172]

[0173] The particle size of the LNPs is between 120 and 150 nm, the potential is between +10 and +20 mV, and the PDI is less than 0.3, indicating that neutral lipids within a certain range can be made into stable LNPs.

[0174] According to the method of Example 27, N / P was fixed at 7, DOPE and polyethylene glycol lipids accounted for 15% and 1.5% of the total lipid molar amount, respectively, and the ratio of TH10 and AD8 was 1:1. By adjusting the molar ratio of cholesterol to TH10 and AD8 as a whole, TH10 and AD8 accounted for 18%, 24%, 30%, 36%, 42%, 48% and 60% of the total lipid molar amount, LNP loaded with Fluc-mRNA was prepared. The particle size, PDI and potential of LNP were measured using an Omni particle size potential analyzer, and the results are shown in Table 7.

[0175] Table 7 Properties of lipid nanoparticles with different TH10 and AD8 molar ratios (n=3)

[0176]

[0177] The particle size of the LNPs is between 110 and 170 nm, the potential is between +15 and +20 mV, and the PDI is less than 0.3, which meets the requirements for gene vector transfection, indicating that ionizable lipids within a certain range can be made into stable LNPs.

[0178] Example 36 Transfection of cervical cancer Hela cells with Luci-mRNA loaded with lipid nanoparticles after optimized formulation ratio

[0179] According to the formula ratio of Example 35, lipid nanoparticles loaded with Luci-mRNA were prepared, and transfection was performed according to the method of Example 31. After transfection, Hela cells were lysed using a luciferase detection kit, and the chemiluminescence of the cell lysate mixed with the luciferin substrate was detected using an ELISA instrument. The results are shown in Fig.15 , Fig.16 , Fig.17 and Fig.18 The results showed that LNPs with different formulations all had a certain mRNA transfection effect.

Claims

1. ROS-responsive ionizable lipid, characterized in that The chemical structure is shown in (I): Wherein, m = 1 or 2; Wherein, n represents an integer from 1 to 3; Wherein p represents an integer from 1 to 17, and q represents an integer from 1 to 8; Wherein X=CH or N, r represents an integer of 0-4, s represents an integer of 1-3, R4 represents methyl, ethyl, hydroxyethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and R5 represents methyl, ethyl, isopropyl or a hydrogen atom.

2. The method according to claim 1, characterized in that: in, m = 1 or 2; Wherein, n represents an integer from 1 to 3; Where q = 8; Wherein X=CH, r represents an integer of 0-4, s represents 1 or 2, R4 represents a methyl group, and R5 represents a methyl group.

3. A method for synthesizing the ROS-responsive ionizable lipid of general formula I in claim 1, wherein the synthetic route is as follows:

4. Use of the ROS-responsive ionizable lipid according to claim 1 or 2 or the ROS-responsive ionizable lipid according to claim 1 or 2 combined with a functional adamantane tail chain lipid AD8 in the preparation of a lipid nanoparticle delivery system for nucleic acid loading, preferably use of the ROS-responsive ionizable lipid according to claim 1 or 2 combined with a functional adamantane tail chain lipid AD8 in the preparation of a lipid nanoparticle delivery system for nucleic acid loading, wherein the functional adamantane tail chain lipid AD8 has the following structural formula:

5. A lipid nanoparticle delivery system for nucleic acid loading, characterized in that: The lipid nanoparticle delivery system contains the ROS-responsive ionizable lipid and neutral phospholipid, cholesterol and pegylated lipid according to claim 1 or 2; the molar ratio of ROS-responsive ionizable lipid: neutral phospholipid: cholesterol: pegylated lipid is 10-70:5-40:10-60:0.5-10; preferably, the molar ratio of ROS-responsive ionizable lipid: neutral phospholipid: cholesterol: pegylated lipid is 30-50:10-25:40-60:0.5-3.

6. The nucleic acid-loaded lipid nanoparticle delivery system according to claim 5, characterized in that: The nucleic acid-loaded lipid nanoparticle delivery system contains ROS-responsive ionizable lipids, functional adamantane tail chain lipids AD8, neutral phospholipids, cholesterol and pegylated lipids; wherein the molar ratio of ROS-responsive ionizable lipids: AD8: neutral phospholipids: cholesterol: pegylated lipids is 10-50:10-50:5-40:10-60:0.5-10; preferably, the molar ratio of ROS-responsive ionizable lipids: adamantane tail chain lipids: neutral phospholipids: cholesterol: pegylated lipids is 10-30:10-30:10-25:30-60:0.5-3.

7. The nucleic acid-loaded lipid nanoparticle delivery system according to claim 5 or 6, wherein: The nucleic acid is DNA or RNA, and the RNA is selected from siRNA, saRNA, shRNA, microRNA or mRNA.

8. The nucleic acid-loaded lipid nanoparticle delivery system according to claim 5 or 6, wherein: The PEGylated lipid is selected from the group consisting of 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)], polyethylene glycol-cholesterol, and ditetradecyl(4-methoxypolyethylene glycol-2000-4-oxobutyryl)glutamate; the neutral phospholipid is selected from the group consisting of soybean lecithin, 1-stearoyl-2-oleoyl-sn-glycero-3 -phosphocholine, 1-palmitoyl-2-oleoylphosphatidylcholine, dimyristoylphosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, dierucoylphosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and 1,2-distearoyl-sn-glycero-3-phosphocholine.

9. Use of the nucleic acid-loaded lipid nanoparticle delivery system of claim 5 or 6 in DNA transfection, mRNA transfection, siRNA gene silencing, and shRNA gene silencing.

10. The nucleic acid-loaded lipid nanoparticle delivery system of claim 5 or 6 is used for transfection of immune cells or tumor cells, wherein the immune cells are selected from macrophages, dendritic cells, and T cells, and the tumor cells are selected from human cervical cancer Hela cells, melanoma B16-F10 cells, breast cancer MCF-7 cells, pancreatic cancer Panc-01 cells, lung cancer A549 cells, and liver cancer HepG2 cells.

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