A ros-responsive ionizable lipid and its use for delivering nucleic acids

Abstract

The application discloses a ROS-responsive ionizable lipid and a use thereof for delivering nucleic acid. The ionizable lipid disclosed by the application has a tertiary amine group as a head group, a thio-ketone and a peroxo-oxalic acid bond as a ROS-responsive connecting chain, and an alkane structure as a hydrophobic tail chain. The structure is easy to synthesize, suitable for large-scale production, has good biocompatibility, can respond to a ROS environment, enhances the release of nucleic acid in a high ROS environment in cells, is suitable for the delivery of various nucleic acids, and the combination with a functional adamantane tail chain lipid significantly improves the cell transfection efficiency. Lipid nanoparticles prepared from the ionizable lipid of the application have good stability, and provide a safe and effective non-viral carrier platform for the delivery of various nucleic acids.

Description

Technical Field

[0001] This invention relates to the field of new formulation materials, specifically to ROS-responsive ionizable lipids and their application in nucleic acid delivery. Background Technology

[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 or abnormal genes. Currently, gene therapy has broad application prospects. However, nucleic acid drugs are easily degraded by nucleases, leading to a loss of activity in vivo. Simultaneously, 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 in entering cells to improve transfection efficiency is currently a research focus. Common delivery methods for nucleic acid drugs mainly include viral vectors and non-viral vectors.

[0003] In addition to nucleic acid components, LNPs also include four lipid components: ionizable lipids, neutral phospholipids, sterol lipids, and polyethylene glycol lipids. These four components collectively influence nucleic acid encapsulation, LNP particle size, charge properties, and stability. Among them, ionizable lipids are the most important component of LNPs. The properties of ionizable lipids give LNPs a strong positive charge in acidic buffer solutions, which is beneficial for loading nucleic acids; under physiological conditions (pH 7.4), they are nearly electroneutrally neutral, exhibiting good safety; after being endocytosed into acidic endosomes (pH 5.0–6.0), ionizable lipids can be rapidly protonated and acquire a strong positive charge, effectively promoting endosome escape.

[0004] Currently, the endosomal escape efficiency of LNPs is relatively low (below 15%), one possible reason being that different cells have different phenotypes and functions, resulting in different endosomal environments. For example, tumor cells have higher ROS levels compared to normal cells. Therefore, designing ionizable lipid structures to target different endosomal environments is crucial for improving the transfection efficiency of LNPs for specific cell types. Simultaneously, improving the biodegradability of ionizable lipids is also closely related to their safety in vivo application. Summary of the Invention

[0005] To address the aforementioned shortcomings in this field, the present invention provides a class of ROS-responsive ionizable lipids that enhance nucleic acid release by responding to intracellular ROS, while simultaneously improving the biodegradability of ionizable lipids.

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

[0007]

[0008] Where m = 1 or 2;

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

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

[0011]

[0012] Where X = CH or N, r represents an integer from 2 to 4, s represents an integer from 1 to 3, R4 represents methyl, ethyl, hydroxyethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and R5 represents methyl, ethyl, isopropyl or hydrogen atom.

[0013] Preferably, in the ROS-responsive lipids, m = 2; R1 is selected from... n = 1 or 2; R2 is selected from q = integers between 6 and 8; R3 is selected from... r = 3 or 4, s = 1 or 2, R4 is selected from methyl or ethyl, and R5 is selected from methyl or ethyl.

[0014] The ROS-responsive ionizable lipids provided by this invention use tertiary amine groups as head groups, enabling them to be protonated in endosomes; and employ thioacetate and peroxyoxalate bonds as linking chains, enabling them to release nucleic acids in response to intracellular ROS environments, enhancing nucleic acid release in high ROS environments, and simultaneously improving the biodegradability of the lipid material. Through these two designs, the release of nucleic acids in high ROS environments is further enhanced.

[0015] The second objective of this 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 method for synthesizing ROS-responsive ionizable lipids, as shown in Formula I, is to... 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℃), and reflux for 1-5 h. After stopping heating, cool the reaction system to room temperature, then add fatty alcohol R2OH, and continue heating to 120-160℃ and reflux overnight. After the reaction is complete, remove toluene by rotary evaporation at 40-60℃, redissolve the crude product in dichloromethane, and wash successively with appropriate amounts of water, saturated sodium bicarbonate aqueous solution, and saturated saline aqueous solution. Dry the organic phase with anhydrous sodium sulfate, filter and concentrate to obtain a dark red oily crude product. Purify by column chromatography with petroleum ether / ethyl acetate to obtain the dicarboxylic acid ester (I-2).

[0018] I-2 synthesis reaction formula:

[0019]

[0020] b. Dissolve 2,2'-[propane-2,2-diyldithio]diacetic acid in dichloromethane, then add HOBt and EDCI sequentially under ice-water bath conditions (0℃), stirring for 5-10 min. The reaction solution is then transferred to room temperature and stirred for 1-3 h to obtain reaction solution A. Dissolve I-1 in dichloromethane, add triethylamine at room temperature, and stir for 1-3 h to obtain reaction solution B. Slowly add reaction solution B dropwise to reaction solution A, stirring overnight at room temperature. After the reaction is complete, wash the reaction solution sequentially with water, citric acid aqueous solution, and saturated brine. After drying with anhydrous sodium sulfate, filter and concentrate 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 sequentially under ice-water bath conditions (0℃), stir for 5-10 min, then transfer the reaction solution to room temperature and continue stirring for 1-3 h to obtain reaction solution A; dissolve R3 in dichloromethane, add pyridine at room temperature, stir for 1-2 h to obtain reaction solution B; slowly add reaction solution B dropwise to reaction solution A, stir overnight at room temperature. After the reaction is complete, purify by dichloromethane / methanol column chromatography to obtain I.

[0024] I. Synthesis reaction formula:

[0025]

[0026] A third objective of this invention is to provide the application of such ROS-responsive ionizable lipids in the construction of lipid nanoparticles.

[0027] The application of the ROS-responsive ionizable lipids or the ROS-responsive ionizable lipids combined with functional adamantane-tailed lipid AD8 in the preparation of nucleic acid-loaded lipid nanoparticle delivery systems, preferably the application of the ROS-responsive ionizable lipids combined with functional adamantane-tailed lipid AD8 in the preparation of nucleic acid-loaded lipid nanoparticle delivery systems, wherein the functional adamantane-tailed lipid AD8 has the following structural formula:

[0028]

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

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

[0031] The PEGylated lipids of the lipid nanoparticles are selected from: 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol (PEG-DMG), 1,2-distearate-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), polyethylene glycol-cholesterol (PEG-Chol), and bis(tetradecyl(4-methoxypolyethylene glycol-2000-4-oxobutyryl)glutamate (PEG). 2000 -Suc-TA2).

[0032] The neutral phospholipids mentioned are selected from: soybean lecithin (SPC), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphocholine (SOPC), 1-palmitoyl-2-oleoyl lecithin (POPC), dimyristoyl phosphatidylcholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-distearyl-sn-glycerol-3-phosphoethanolamine (DSPE), disorhoyl lecithin (DEPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), and 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC).

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

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

[0035] The lipid nanoparticles are prepared by methods including ethanol injection, microfluidic methods, or membrane extrusion. The preferred microfluidic method is as follows: Appropriate amounts of ROS-responsive ionizable lipids, neutral phospholipids, cholesterol, and polyethylene glycol-modified lipids are weighed according to the nitrogen-to-phosphorus ratio, and then dissolved in anhydrous ethanol to form the ethanol phase. Nucleic acid is dissolved in citrate buffer solution (pH = 4.0) to form the aqueous phase. Using a microfluidic device, the aqueous and ethanol phases are mixed in a microfluidic chip at a flow rate of 1–30 mL / min and a ratio of 1:1–4:1. The mixture is then dialyzed against ultrapure water or PBS at room temperature for 2–4 h. The collected solution is the lipid nanoparticle (LNP) solution.

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

[0037] A fourth objective of this invention is to provide the application of such ROS-responsive ionizable lipids in cell transfection.

[0038] The lipid nanoparticles are used for transfection of immune cells or tumor cells, including immune cells such as macrophages, dendritic cells, and T cells, and tumor cells such as 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.

[0039] Beneficial effects:

[0040] The ionizable lipids disclosed in this invention use a tertiary amine group as the head group, a thioacetate and a peroxyoxalate bond as the ROS-responsive linking chain, and an alkane structure as the hydrophobic tail chain. This structure is easy to synthesize, suitable for large-scale production, has good biocompatibility, can respond to ROS environments, enhances the release of nucleic acids in intracellular high ROS environments, and is suitable for the delivery of various nucleic acids. Its combination with functional adamantane-tailed lipids significantly improves cell transfection efficiency. The lipid nanoparticles prepared from the ionizable lipids of this invention exhibit good stability and provide a safe and effective non-viral vector platform for the delivery of various nucleic acids. Attached Figure Description

[0041] Figure 1 The response and release of TH series ionizable lipids in 5mM H2O2 in Example 25;

[0042] Figure 2 The HR-MS results for the tertiary amine head groups obtained after the OX series ionizable lipids in Example 26 were released in response to 25 mM H2O2;

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

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

[0045] Figure 5 To observe the expression of EGFP in human cervical cancer HeLa cells after transfection with lipid nanoparticles carrying EGFP-mRNA in Example 31 using an inverted fluorescence microscope (scale bar: 100 μm);

[0046] Figure 6 To detect the expression of EGFP in human cervical cancer HeLa cells after transfection with lipid nanoparticles loaded with EGFP-mRNA prepared from TH series ionizable lipids in Example 31 using flow cytometry.

[0047] Figure 7 To detect the expression of EGFP in human cervical cancer HeLa cells after transfection with lipid nanoparticles loaded with EGFP-mRNA prepared from OX series ionizable lipids in Example 31 using flow cytometry;

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

[0049] Figure 9 To observe the expression of EGFP in HeLa cells after transfection with lipid nanoparticles loaded with EGFP-mRNA prepared from the preferred ionizable lipid AD8 with adamantane tail chain in Example 32 using an inverted fluorescence microscope (scale bar: 100 μm).

[0050] Figure 10 To detect the expression of EGFP in HeLa cells after transfection with lipid nanoparticles loaded with EGFP-mRNA prepared from the preferred ionizable lipid AD8 with adamantane tail chain in Example 32 using flow cytometry.

[0051] Figure 11 To observe the expression of EGFP in HeLa cells after transfecting them with siRNA-loaded lipid nanoparticles prepared from the preferred ionizable lipid AD8 with adamantane tail chain in Example 33 using an inverted fluorescence microscope (scale bar: 100 μm).

[0052] Figure 12To detect the gene silencing efficiency of lipid nanoparticles prepared from the preferred ionizable lipid AD8 with introduced adamantane tail chain in Example 33 after transfection with EGFP-HeLa cells loaded with siRNA using flow cytometry.

[0053] Figure 13 To observe the lipid nanoparticles prepared by introducing the preferred ionizable lipid AD8 with adamantane tail chain in Example 34 using an inverted fluorescence microscope, which were then loaded with Pmax-GFP plasmid and transfected with human primary CD3 cells, + T cell post-crystal CD3 + EGFP expression in T cells (scale bar: 100 μm);

[0054] Figure 14 To detect the effects of flow cytometry on lipid nanoparticles prepared from the preferred ionizable lipid AD8 with an adamantane tail chain in Example 33, which were then transfected with human primary CD3 cells, the Pmax-GFP plasmid was loaded with the nanoparticles. + T cell post-crystal CD3 + EGFP expression in T cells;

[0055] Figure 15 The expression of luciferase in HeLa cells after transfection with lipid nanoparticles carrying Luci-mRNA of different neutral phospholipids in Example 36.

[0056] Figure 16 The expression of luciferase in HeLa cells after transfection with Luci-mRNA loaded with lipid nanoparticles of different polyethylene glycol lipid molar ratios in Example 36.

[0057] Figure 17 The expression of luciferase in HeLa cells after transfection with lipid nanoparticles loaded with Luci-mRNA at different DOPE molar ratios in Example 36.

[0058] Figure 18 The expression of luciferase in HeLa cells after transfection with lipid nanoparticles loaded with Luci-mRNA at different TH10 and AD8 molar ratios in Example 36. Detailed Implementation

[0059] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

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

[0061]

[0062] TH1 was prepared from compounds OA2-TH-COOH (1 g, 1.17 mmol) and 3-dimethylaminopropylamine (143.52 mg, 1.40 mmol). OA2-TH-COOH (1 g, 1.17 mmol) was dissolved in dichloromethane, and HOBt (189.80 mg, 1.40 mmol) and EDCI (260.26 mg, 1.40 mmol) were added sequentially under ice-water bath conditions (0 °C) and stirred for 5 min. The reaction solution was then transferred to room temperature and stirred for 3 h to obtain reaction solution A. 3-Dimethylaminopropylamine (143.52 mg, 1.40 mmol) was dissolved in dichloromethane, and pyridine (277.76 mg, 3.51 mmol) was added at room temperature and stirred for 1 h to obtain reaction solution B. Reaction solution B was slowly added dropwise to reaction solution A, and the mixture was stirred overnight at room temperature. After the reaction was completed, the product was purified by column chromatography (dichloromethane:methanol = 30:1) to give 640 mg of a pale yellow oily product, with a yield of 58.26%. 1 HNMR (300MHz, CDCl3): 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),2.7 9(t,J=7.1Hz,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= 6.8Hz,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, CDCl3): 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 .03,37.82,34.76,32.57,31.86,30.26,29.72,29.48,29.39,29.28,29 .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] Using OA2-TH-COOH (1 g, 1.17 mmol) and N-(3-aminopropyl)diethanolamine (227.87 mg, 1.40 mmol) as raw materials, TH2 was prepared according to the method for TH1, yielding 540 mg of a pale yellow oily product with a yield of 46.20%. 1 HNMR (500MHz, CDCl3): δ (ppm) 7.18 (d, J = 8.6 Hz, 1H), 6.68 (t, J = 4.9 Hz, 1H), 5.37 (m, 4H), 4.31 (dt, J = 8.6, 6.8 Hz,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=6.0,4.9 Hz,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, CDCl3): δ(ppm)172.67,171.67,170.59,169.65,156.37,142.72,130 .37,129.90,129.68,127.89,125.28,124.59,118.09,110.97,65.72,64.88,57.57 ,56.24,55.78,52.11,36.83,34.76,34.64,32.55,31.85,30.26,29.71,29.63,29.47,29.40,29.27,29.20,28.54,28.46,27.16,25.86,25.81,23.99,22.63,14.08.

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

[0067]

[0068] Using OA2-TH-COOH (1g, 1.17mmol) and 3-diethylaminopropylamine (182.93mg, 1.40mmol) as raw materials, TH3 was prepared according to the method for TH1, yielding 620mg of a pale yellow oily product with a yield of 54.80%. 1 HNMR (300MHz, CDCl3): δ (ppm) 7.86 (s, 1H), 7.46 (d, J = 7.9Hz, 1H), 5.35 (td, J = 6.3, 3.4Hz, 4 H),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) ,1.62(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, CDCl3): 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 was prepared from OA2-TH-COOH (1 g, 1.17 mmol) and 1-methyl-3-aminopyrrolidine (140.69 mg, 1.40 mmol) using the same method as TH1, yielding 610 mg of a pale yellow oily product with a yield of 55.65%. 1HNMR (300MHz, CDCl3): δ (ppm) 8.38 (s, 1H), 7.80 (d, J = 7.8Hz, 1H), 5.45-5.24 (m, 4H), 4 .86(s,1H),4.56(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) ,2.00(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, CDCl3): 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, 5 4.89,52.06,34.82,32.58,31.87,30.88,30.14,29.73,29.49,29.41,29.29,29.21,28.56,28.47,27.18,25.80,22.65,14.10.

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

[0073]

[0074] Using OA2-TH-COOH (1 g, 1.17 mmol) and N-methyl-2-(2-aminoethyl)-pyrrolidine (140.69 mg, 1.40 mmol) as raw materials, TH5 was prepared according to the method for TH1, yielding 580 mg of a pale yellow oily product with a yield of 51.73%. 1HNMR (300MHz, CDCl3): 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),4.59(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, CDCl3): 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, 34.69,32.54,31.86,30.74,30.32,29.72,29.64,29.48,29.39,29.27,29.1 9,28.56,28.48,27.31,27.17,25.86,25.78,22.63,22.11,14.06.HRMS,ESI + ,m / z:CalcdforC 55 H 101 N3O6S2[M+H] + ,964.7192; found,964.7196.

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

[0076]

[0077] Using OA2-TH-COOH (1g, 1.17mmol) and (1-methyl-4-piperidin-)methylamine (180.09mg, 1.40mmol) as raw materials, TH6 was prepared according to the method for TH1, yielding 630mg of a pale yellow oily product with a yield of 55.80%. 1HNMR (300MHz, CDCl3): 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 ,6.4Hz,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, CDCl3): δ(ppm)172.67,171.63,169.94,169.39,156.19,156.13,1 30.35,129.89,129.66,127.92,125.41,124.43,118.23,110.73,65.66,64.84, 57.61,52.13,43.54,34.75,32.51,31.82,30.34,30.27,29.67,29.60,29.43,2 9.35,29.22,29.16,28.51,28.44,27.13,25.83,25.77,22.59,14.04.HRMS,ESI + ,m / z:CalcdforC 55 H 101 N3O6S2[M+H] + ,964.7193; found,964.7190.

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

[0079]

[0080] Using OA2-TH-COOH (1 g, 1.17 mmol) and 4-methyl-1-piperazinepropylamine (220.89 mg, 1.40 mmol) as raw materials, TH7 was prepared according to the method for TH1, yielding 650 mg of a pale yellow oily product with a yield of 55.89%. 1HNMR (300MHz, CDCl3): 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=2.2 Hz,2H),3.35(d,J=6.8Hz,4H),2.48(dd,J=14.2,7.8Hz,11H),2.31(s,3H),2.0 1(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, CDCl3): δ(ppm)172.67,171.59,169.05,157.26,156.15,129.92,12 9.69,123.66,77.32,65.77,64.91,57.77,56.41,54.35,52.36,52.05,45.36,39. 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 N4O6S2[M+H] + ,993.7473; found,993.7476.

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

[0082]

[0083] Using OA2-TH-COOH (1 g, 1.17 mmol) and 4-methyl-1-piperazineethylamine (201.18 mg, 1.40 mmol) as raw materials, TH8 was prepared according to the method for TH1, yielding 680 mg of a pale yellow oily product with a yield of 59.31%. 1HNMR (300MHz, CDCl3): 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, CDCl3): δ (ppm) 172.68, 171.56, 169.16, 169.03, 156.10, 12 9.96,129.70,125.74,118.50,110.58,65.84,64.98,57.68,56.07,53.35 ,52.13,50.10,43.74,36.06,34.76,31.86,30.32,29.71,29.64,29.48, 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 N4O6S2[M+Na] + ,1001.7101;found,1001.7104.

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

[0085]

[0086] Using OA2-TH-COOH (1 g, 1.17 mmol) and 4-dimethylaminopiperidine (180.09 mg, 1.40 mmol) as raw materials, TH9 was prepared according to the method for TH1, yielding 720 mg of a pale yellow oily product with a yield of 63.77%. 1HNMR (300MHz, CDCl3): 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, CDCl3): 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 N3O6S2[M+H] + ,964.7193; found,964.7196.

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

[0088]

[0089] TH10 was prepared from compound OA2-TH-COOH (1 g, 1.17 mmol) and 3-(2-methylpiperidin-1-yl)propyl-1-amine (219.50 mg, 1.4 mmol) according to the method for preparing TH1, yielding 710 mg of a pale yellow oily product, with a yield of 61.11%. 1HNMR (300MHz, CDCl3): 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), 3.0 3-2.81(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, CDCl3): δ (ppm) 172.72, 171.72, 170.24, 169.38, 129.93, 129.71, 65.60, 64.82, 31.85, 3 0.46,30.29,29.71,29.47,29.39,29.26,29.20,28.56,28.48,27.17,25.86,22.63,14.06.HRMS,ESI + ,m / z:CalcdforC 57 H 105 N3O6S2[M+H] + ,992.7499;found,992.7508.

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

[0091]

[0092] TH11 was prepared using compounds OA2-TH-COOH (1 g, 1.17 mmol) and 4-piperidinylpiperidine (236.37 mg, 1.4 mmol) as raw materials, and yielded 690 mg of a pale yellow oily product according to the method for preparing TH1, with a yield of 58.68%. 1HNMR (300MHz, CDCl3): 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,1H),2.86(s,4H),2.58(t,J=12.8Hz,1H),2.40(q,J=7.0Hz,2H),2.34-2.1 4(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, CDCl3): 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 N3O6S2[M+H] + ,1004.7495; found,1004.7498.

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

[0094]

[0095] TH12 was prepared from compounds OA2-TH-COOH (1 g, 1.17 mmol) and N-(3-aminopropyl)pyrrole (180.09 mg, 1.4 mmol) according to the method for preparing TH1, yielding 750 mg of a pale yellow oily product with a yield of 66.43%. 1HNMR (300MHz, CDCl3): 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, CDCl3): 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 N3O6S2[M+H] + ,964.7190; found,964.7192.

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

[0097]

[0098] OX1 was prepared from compounds OA2-TH2 (1 g, 1.54 mmol) and 3-dimethylaminopropylamine (189.20 mg, 1.85 mmol). 3-Dimethylaminopropylamine (189.20 mg, 1.85 mmol) was dissolved in dichloromethane, and pyridine (244.12 mg, 3.09 mmol) was added and stirred at room temperature to obtain reaction solution A. Oxaloyl chloride (979.22 mg, 7.72 mmol) was slowly added dropwise to dichloromethane under stirring at room temperature. Subsequently, under ice-water bath conditions, a dichloromethane solution of OA2-NH2 (1 g, 1.54 mmol) was slowly added dropwise, and the reaction was carried out at low temperature for 5 min. The reaction system was then transferred to room temperature and stirred for 3 h. After the reaction was complete, the solvent was evaporated under vacuum at 45°C. During this process, dichloromethane was added five times in small amounts to azeotropically remove excess oxalyl chloride until no oxalyl chloride remained. No further treatment was required, yielding a yellow oily product. This yellow oily substance was dissolved in dichloromethane to obtain reaction solution B. Under ice-water bath stirring, reaction solution B was slowly added dropwise to reaction solution A, and the reaction was carried out at low temperature for 5 minutes. Subsequently, the reaction system was transferred to room temperature and stirred overnight. After the reaction was completed, the product was purified by column chromatography (dichloromethane:methanol = 35:1) to obtain 560 mg of a pale yellow oily product, with a yield of 45.12%. 1 HNMR (300MHz, CDCl3): 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=6.1Hz,2H),2.88(t,J=7.5Hz,2H),2.66(s,6H),2.39(q,J=6.9Hz,2H),2.02(dt ,J=12.8,6.4Hz,10H),1.72-1.54(m,4H),1.43-1.07(m,47H),0.88(t,J=6.5Hz,6H). 13 CNMR (75MHz, CDCl3): 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:CalcdforC48 H 89 N3O6[M+H] + ,804.6807;found,804.6810.

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

[0100]

[0101] OX2 was prepared by using compound OX-OA2 (1.1 g, 1.49 mmol) and N-(3-aminopropyl)diethanolamine (300.40 mg, 1.85 mmol) as raw materials, and following the method for preparing OX1, yielding 350 mg of a pale yellow oily substance 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, CDCl3): 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 N3O8[M+H] + ,865.7098; found,865.7159.

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

[0103]

[0104] OX3 was prepared from compound OX-OA2 (1.1 g, 1.49 mmol) and 3-diethylaminopropylamine (241.15 mg, 1.85 mmol) using the same method as OX1, yielding 580 mg of a pale yellow oily substance with a yield of 45.16%. 1 HNMR (300MHz, CDCl3): 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,CH2(oleoyl)),1.08(t,J=7.1Hz,7H),0.88(t,J=6.6Hz,7H). 13CNMR(75MHz, CDCl3)δ(ppm)172.19,170.48,159.77,159.12,130.36,129.89,129.66,65.87,64.86,56.01,54.67,52.12,52.07,45.32,36.34,3 2.47,31.81,30.17,29.67,29.58,29.42,29.32,29.22,29.13,29.07,28 .54,28.43,27.25,27.13,25.82,25.70,22.56,13.96.13CNMR(75MHz,CD Cl3): δ(ppm)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, 30.16,29.73,29.66,29.49,29.39,29.35,29.28,29.19,29.14,28.54,2 8.44,27.38,27.17,25.86,25.74,25.50,22.64,14.06,11.54.HRMS,ESI + m / z:Calcdfor C 50 H 93 N3O6[M+H] + ,832.7130; found,832.7133.

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

[0106]

[0107] OX4 was prepared from compound OX-OA2 (1.1 g, 1.49 mmol) and 1-methyl-3-aminopyrrolidine (185.47 mg, 1.85 mmol) using the same method as OX1, yielding 620 mg of a pale yellow oily substance with a yield of 50.09%. 1HNMR (300MHz, CDCl3): δ (ppm) 7.92 (d, J = 9.0Hz, 1H), 7.72 (d, J = 8.5Hz, 1H), 5.48-5.2 2(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 -1.91(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, CDCl3): δ(ppm)172.31,170.55,159.74,158.41,157.86,130.3 9,129.92,129.72,65.92,64.91,62.26,54.76,51.99,49.52,41.68,32.72, 32.69,32.57,31.88,30.14,29.73,29.65,29.49,29.39,29.36,29.29,29.2 0,29.14,28.55,28.44,27.31,27.18,25.86,25.74,22.65,14.07.HRMS,ESI + ,m / z:CalcdforC 48 H 87 N3O6[M+H] + ,804.6807;found,804.6810.

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

[0109]

[0110] OX5 was prepared using compound OX-OA2 (1.1 g, 1.49 mmol) and N-methyl-2-(2-aminoethyl)-pyrrolidine (237.42 mg, 1.85 mmol) as raw materials, following the method for preparing OX1, to obtain 590 mg of a pale yellow oil, with a yield of 46.05%. 1HNMR (300MHz, CDCl3): 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,1H),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=6.4 Hz,10H),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, CDCl3): 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, 31.8 7,31.03,30.17,29.72,29.54,29.48,29.38,29.28,29.19,29.13,28.55,28.45,27.35,27.17,25.86,25.74,22.63,22.30,14.05.HRMS,ESI + ,m / z:CalcdforC 50 H 91 N3O6[M+H] + ,830.6907;found,830.6976.

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

[0112]

[0113] Using compound OX-OA2 (1.1 g, 1.49 mmol) and (1-methyl-4-piperidin-)methylamine (237.42 mg, 1.85 mmol) as raw materials, OX6 was prepared according to the method for preparing OX1, yielding 640 mg of a pale yellow oil, with a yield of 49.95%. 1HNMR (300MHz, CDCl3): 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, CDCl3): 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 .41,32.53,31.84,30.14,29.70,29.63,29.59,29.46,29.37,29.33,29.25 ,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 N3O6[M+H] + ,829.6907;found,830.6975.

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

[0115]

[0116] OX7 was prepared using compound OX-OA2 (1.1 g, 1.49 mmol) and 4-methyl-1-piperazinepropylamine (291.19 mg, 1.85 mmol) as raw materials, following the method for preparing OX1, yielding 610 mg of a pale yellow oily substance, with a yield of 46.00%. 1HNMR (300MHz, CDCl3): δ (ppm) 8.59 (t, J = 5.4Hz, 1H), 7.93 (d, J = 8.5Hz, 1H), 5.46-5.25 (m,4H),4.59(td,J=8.3,5.0Hz,1H),4.15(t,J=6.8Hz,2H),4.06(t,J=6.8Hz,2H),3.41 (q,J=6.1Hz,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, CDCl3): 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 N4O6[M+H] + ,859.7220; found,859.7228.

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

[0118]

[0119] Using OX-OA2 (1.1 g, 1.49 mmol) and 4-methyl-1-piperazineethylamine (265.22 mg, 1.85 mmol) as raw materials, OX8 was prepared according to the method for OX1, yielding 605 mg of a pale yellow oily substance 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, CDCl3): 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 N4O6[M+H] + ,845.7099;found,845.7110.

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

[0121]

[0122] Using compound OX-OA2 (1.1 g, 1.49 mmol) and 4-dimethylaminopiperidine (237.42 mg, 1.85 mmol) as raw materials, OX9 was prepared according to the method for preparing OX1, yielding 630 mg of a pale yellow oily substance with a yield of 49.17%. 1HNMR (300MHz, CDCl3): 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,2 H),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, CDCl3): δ (ppm) 172.45, 170.85, 161.58, 161.49, 160.46, 130.38, 1 29.91,129.69,65.86,64.92,62.06,61.75,51.78,50.42,45.07,42.14,41.13, 40.89,32.53,31.85,30.23,29.70,29.64,29.60,29.46,29.36,29.26,29.18,2 9.13,28.53,28.45,27.35,27.16,27.05,25.84,25.73,22.62,14.04.HRMS,ESI + ,m / z:CalcdforC 50 H 91 N3O6[M+H] + ,830.6907;found,830.6975.

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

[0124]

[0125] OX10 was prepared using compound OX-OA2 (1.1 g, 1.49 mmol) and 3-(2-methylpiperidin-1-yl)propyl-1-amine (347.24 mg, 1.85 mmol) as raw materials, and yielded 720 mg of a pale yellow oily substance according to the method for preparing OX1, with a yield of 45.30%. 1HNMR (300MHz, CDCl3): δ (ppm) 8.14 (s, 1H), 7.93 (d, J = 8.6Hz, 1H), 5.35 (td, J = 11.5, 6.3Hz,4H),4.59(td,J=8.2,4.7Hz,1H),4.15(t,J=6.8Hz,2H),4.06(t,J=6.8Hz,2H) ,3.55-3.31(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, CDCl3): 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. 45,27.40,27.18,25.86,25.74,24.88,24.59,22.88,22.64,17.72,14.06.

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

[0127]

[0128] OX11 was prepared using compound OX-OA2 (1.1 g, 1.49 mmol) and 4-piperidinylpiperidine (311.60 mg, 1.85 mmol) as raw materials, and 700 mg of a pale yellow oil was obtained according to the method for preparing OX1, with a yield of 52.12%. 1HNMR (300MHz, CDCl3): 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) ,2.01(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, CDCl3): 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 .54,31.86,30.20,29.72,29.64,29.47,29.38,29.35,29.27,29.19,29.1 4,28.55,28.46,27.53,27.16,25.97,25.85,25.74,24.44,22.63,14.05.

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

[0130]

[0131] OX12 was prepared by using compound OX-OA2 (1.1 g, 1.49 mmol) and N-(3-aminopropyl)pyrrole (261.16 mg, 1.85 mmol) as raw materials, and obtaining 640 mg of a pale yellow oily substance according to the method for preparing OX1, with a yield of 45.41%. 1HNMR (300MHz, CDCl3): 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, CDCl3): 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, 32.52,31.84,30.16,29.69,29.63,29.59,29.45,29.37,29.24,29.18,2 9.12,28.53,28.43,27.15,25.84,25.74,23.30,22.61,14.03.HRMS,ESI + ,m / z:CalcdforC 50 H 91 N3O6[M+H] + ,830.6949;found,830.6956.

[0132] Example 25: ROS responsiveness of TH series lipids

[0133] The responsiveness of TH-series lipids to hydrogen peroxide was investigated using 5,5'-dithiobis-(2-nitrobenzoic acid), DTNB, and quantitative detection was performed. DTNB, also known as Ellman's reagent, can be used to quantitatively detect free thiol groups in solution. DTNB reacts quantitatively with free thiol groups in solution at a 1:1 ratio under conditions of pH 7.5-8.6, generating disulfide and 2-nitro-5-thiobenzoic acid (TNB). TNB has a significant absorption peak at a wavelength of 412 nm. Since the amount of TNB generated is the same as the amount of thiol groups consumed, it can be used for the quantitative detection of thiol groups. TH-series lipids use thiophene as the ROS-responsive linker, which breaks in response to hydrogen peroxide, producing two molecules of free thiol groups and one molecule of acetone. Therefore, the DTNB method was used to investigate 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 rapidly break down within the first 2 hours after the reaction begins, exposing free thiol groups, indicating that TH series lipids have good ROS responsiveness.

[0134] Example 26: ROS responsiveness of OX series lipids

[0135] Since OX-series lipids do not generate free thiol groups after ROS-responsive cleavage, they cannot be quantitatively detected using the DTNB method. HRMS was used for qualitative detection of ROS-responsive cleavage. Due to the relatively complex structure of the tail chain and the presence of carbon-carbon double bonds susceptible to strong oxidants, molecular ion peaks of the tail chain are difficult to observe in HRMS results. Therefore, the less affected head group was selected as the target molecule. Results are as follows... Figure 2 As shown, all OX series lipids can undergo responsive cleavage in 25 mM H2O2 solution, and the molecular ion peak of the corresponding head group of each OX lipid was successfully detected, proving its ROS responsiveness.

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

[0137] According to the molar ratio of TH lipids / DOPE / Chol / PEG2000-Suc-TA2 = 36 / 15 / 46.5 / 2.5, the required ROS-responsive ionizable lipids TH1-TH12, neutral phospholipids, cholesterol, and PEGylated lipids were weighed and dissolved in anhydrous ethanol to prepare the ethanol phase. EGFP-mRNA was dissolved in 10mM citrate buffer (pH = 4.0) to prepare the aqueous phase. Under vigorous stirring, the ethanol phase was rapidly and uniformly injected into the aqueous phase at a volume ratio of 3:1. After injection, the sample was dialyzed against ultrapure water at room temperature for 4 hours to obtain the LNP solution. The particle size, polydispersity index (PDI), and zeta potential of the LNPs were determined using an Omni particle size potentiometer, and the results are shown in Table 1.

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

[0139]

[0140]

[0141] The above data show that the LNPs of the TH series of this invention have a particle size between 110 nm and 180 nm, a potential between +10 mV and +20 mV, and a PDI of less than 0.3. This indicates that the above-mentioned ionizable lipids can form stable LNPs.

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

[0143] The desired ROS-responsive ionizable lipids OX1-OX12, neutral phospholipids, cholesterol, and PEGylated lipids were weighed out according to the molar ratio of OX lipids / DOPE / Chol / PEG2000-Suc-TA2 = 36 / 15 / 46.5 / 2.5 and dissolved in anhydrous ethanol to form the ethanol phase. LNPs were prepared according to the method in Example 27. The particle size, PDI, and potential of the LNPs were determined using an Omni particle size potentiometric analyzer, and the results are shown in Table 2.

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

[0145]

[0146] The above data show that the LNPs of the OX series of this invention have a particle size between 80 nm and 130 nm, a potential between +10 mV and +20 mV, and a PDI of less than 0.3. This indicates that the above-mentioned ionizable lipids can form stable LNPs.

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

[0148] Lipid nanoparticles with different ROS-responsive ionizable lipids carrying EGFP-mRNA at N / P = 7 were prepared according to the ethanol injection method in Example 27. The ability of the lipid nanoparticles to load mRNA was examined by agarose gel electrophoresis. Figure 3 As shown in the figure. The results indicate that TH series lipid nanoparticles can stably load mRNA at N / P = 7, and can be further used for cell transfection experiments.

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

[0150] Lipid nanoparticles with different ROS-responsive ionizable lipids carrying EGFP-mRNA at N / P = 7 were prepared according to the ethanol injection method in Example 27. The ability of the lipid nanoparticles to load mRNA was examined by agarose gel electrophoresis. Figure 4 As shown in the figure. The results indicate that OX series lipid nanoparticles can stably load mRNA at N / P = 7, and can be further used for 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 in Example 27. One day before transfection, 1×10⁻⁶... 6 Human cervical cancer HeLa cells were seeded into 24-well plates at a density of cells / mL. 500 μL of DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was added to each well. The plates were incubated at 37°C with 5% CO2 for 24 h. The medium was then discarded, and 500 μL of DMEM medium and 100 μL of lipid nanoparticles were added. The plates were cultured for another 6 h. The culture medium was then discarded, and 500 μL of DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was added again. After 48 h, 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 assessed using flow cytometry. Results are as follows: 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 analysis by flow cytometry. The mRNA-LNP prepared from the TH4, TH9, TH10, and TH11 ionizable lipids and the OX4, OX9, OX10, and OX11 ionizable lipids of this invention exhibits significant green fluorescence indicating EGFP-mRNA expression in HeLa cells.

[0153] Example 32: Transfection of cervical cancer HeLa cells with lipid nanoparticles containing adamantane-tailed lipid AD8 loaded with EGFP-mRNA.

[0154] Lipid nanoparticles TH4AD8LNP, TH9AD8LNP, TH10AD8LNP, TH11AD8LNP, OX4AD8LNP, OX9AD8LNP, OX10AD8LNP, and OX11AD8LNP, loaded with adamantane-tailed lipid AD8 (patent CN114436994B) and expressing eGFP mRNA, were prepared according to the method in Example 27. The formulation composition was TH lipid (OX lipid) / AD8 / DOPE / chol / PEG2000-Suc-TA2 = 18 / 18 / 15 / 46.5 / 2.5 (mol:mol). The particle size, PDI, and potential of the LNPs were determined using an Omni particle size and potential analyzer. The results are shown in Table 3. The introduction of AD8 significantly reduced the particle size of most LNPs. Agarose gel electrophoresis results are shown below. Figure 8 As shown, the introduction of AD8 does not affect the stability of EGFP-mRNA loading. Transfection was performed according to the method in Example 31, and the results are as follows. Figure 9 and Figure 10 As shown, LNPs containing AD8 have a stronger EGFP fluorescence intensity than LNPs without AD8, indicating that the introduction of AD8 can significantly increase the transfection efficiency of mRNA.

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

[0156]

[0157] Example 33: Transfection of EGFP-HeLa cells with lipid nanoparticles containing adamantane-tailed lipid AD8 loaded with siRNA.

[0158] Lipid nanoparticles TH10AD8LNP, TH11AD8LNP, and OX12AD8LNP containing adamantane-tailed lipid AD8 and loaded with siRNA were prepared according to the method in Example 27. The formulation composition was TH lipid (OX lipid) / AD8 / DOPE / chol / PEG2000-Suc-TA2 = 18 / 18 / 15 / 46.5 / 2.5 (mol:mol). Human cervical cancer EGFP-HeLa cells stably expressing enhanced green fluorescent protein (EGFP) were subjected to a 1×10⁻⁶ ppm in vitro. 5Cells / mL were seeded in 24-well plates, with 500 μL of RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin added to each well. After 24 h of incubation, the medium was discarded, and 440 μL of RPMI 1640 medium and 60 μL of lipid nanoparticles were added. The cells were cultured for another 6 h, after which the medium was discarded. Then, 500 μL of RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was added, and the cells were cultured for another 48 h. After 48 h of culture, the expression of green fluorescent protein (GFP) in Hela-eGFP cells was observed using an inverted fluorescence microscope, and the molecular weight index (MFI) of GFP in the cells was quantitatively assessed using flow cytometry. Results are as follows: Figure 11 and Figure 12 As shown, the siRNA-LNP prepared by combining the lipids of the present invention with AD8 exhibits a gene silencing efficiency of more than 60%, which is superior to the commercial transfection reagent Lipofectamine2000.

[0159] Example 34: Lipid nanoparticles containing adamantane-tailed lipid AD8 loaded with Pmax-GFP plasmid and used to target human primary CD3... + T cell transfection

[0160] Lipid nanoparticles TH10AD8LNP and TH11AD8LNP loaded with Pmax-GFP plasmid were prepared according to the method in Example 27. The formulation composition was TH lipid / AD8 / DOPE / chol / PEG2000-Suc-TA2 = 18 / 18 / 15 / 46.5 / 2.5 (mol:mol). Human CD3... + T cells at 1×10 6 Cells / mL seeding (ultra-low adsorption suspension cell culture plate, six-well plate), followed by 1×10⁶ cells / well. 6 Cells were seeded at a density of 100 cells / mL in 24-well plates. 500 μL of T-cell culture medium containing human IL-2 (100 IU / mL) and human CD3 / CD28 antibody was added to each well, along with 100 μL of LNP. After incubation for 48 h, GFP expression was observed using an inverted fluorescence microscope, and GFP expression was assessed by flow cytometry. + T cell positivity rate and MFI of GFP. Results are as follows: Figure 13 and Figure 14 As shown, the Pmax-LNP prepared by combining the lipids of the present invention with AD8 exhibits good T cell transfection efficiency.

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

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

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

[0164]

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

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

[0167] Table 5 Properties of lipid nanoparticles with different polyethylene glycol-modified lipid molar ratios (n=3)

[0168]

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

[0170] Following the method of Example 27, with a fixed N / P ratio of 7, the ionizable lipids and polyethylene glycol lipids in the formulation accounted for 36% and 1.5% of the total lipid molars, respectively. By adjusting the molar ratio of cholesterol and DOPE, DOPE accounted for 6%, 10%, 15%, 20%, and 25% of the total lipid molars, respectively, LNPs loaded with Fluc-mRNA were prepared. The particle size, PDI, and potential of the LNPs were measured using an Omni particle size and 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 LNPs exhibited particle sizes between 120 and 150 nm, potentials between +10 and +20 mV, and PDI values ​​all less than 0.3. This indicates that stable LNPs can be prepared from neutral lipids within a certain range.

[0174] Following the method of Example 27, with a fixed N / P ratio of 7, DOPE and polyethylene glycol lipids accounted for 15% and 1.5% of the total lipid molars, respectively, and the ratio of TH10 to AD8 was 1:1. By adjusting the overall molar ratio of cholesterol and TH10, AD8, TH10 and AD8 accounted for 18%, 24%, 30%, 36%, 42%, 48%, and 60% of the total lipid molars, LNPs loaded with Fluc-mRNA were prepared. The particle size, PDI, and potential of the LNPs were measured using an Omni particle size and 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 LNPs exhibited particle sizes ranging from 110 to 170 nm, potentials from +15 to +20 mV, and PDI values ​​all less than 0.3, meeting the requirements for use as gene vectors for transfection. This indicates that ionizable lipids within a certain range can be used to prepare stable LNPs.

[0178] Example 36: Transfection of cervical cancer HeLa cells with lipid nanoparticles loaded with Luci-mRNA after optimization of formulation ratio.

[0179] Lipid nanoparticles loaded with Luci-mRNA were prepared according to the formulation ratio of Example 35 and transfected according to the method of Example 31. After transfection, HeLa cells were lysed using a luciferase assay kit, and the chemiluminescence of the mixture of cell lysate and luciferase substrate was detected using a microplate reader. The results are as follows: Figure 15 , Figure 16 , Figure 17 and Figure 18 As shown in the figure. The results indicate that LNPs with different formulations all have a certain mRNA transfection effect.

Claims

1. A ROS-responsive ionizable lipid, characterized in that, The chemical structure is shown in (I): ， Where m = 1 or 2; R1= , where n represents an integer from 1 to 3; R2= , or , where p represents an integer from 1 to 17, and q represents an integer from 1 to 8; R3= , , , , or Where X = CH or N, r represents an integer from 0 to 4, s represents an integer from 1 to 3, R4 represents methyl, ethyl, hydroxyethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and R5 represents methyl, ethyl, isopropyl or hydrogen atom.

2. The ROS-responsive ionizable lipid according to claim 1, characterized in that, in, m = 1 or 2; R1= , where n represents an integer from 1 to 3; R2= , where q=8; R3= , , or Where X = CH, r represents an integer from 0 to 4, s represents 1 or 2, R4 represents methyl, and R5 represents methyl.

3. The method for synthesizing the ROS-responsive ionizable lipid shown in general formula I of claim 1, the synthetic route of which is as follows: 。 4. The application of the ROS-responsive ionizable lipid of claim 1 or 2, or the ROS-responsive ionizable lipid of claim 1 or 2 combined with functional adamantane-tailed lipid AD8, in the preparation of a lipid nanoparticle delivery system loaded with nucleic acid, wherein the functional adamantane-tailed lipid AD8 has the following structural formula: 。 5. A lipid nanoparticle delivery system for loading nucleic acids, characterized in that, The lipid nanoparticle delivery system comprises the ROS-responsive ionizable lipids and neutral phospholipids, cholesterol, and polyethylene glycol-modified lipids as described in claim 1 or 2, wherein the molar ratio of ROS-responsive ionizable lipids: neutral phospholipids: cholesterol: polyethylene glycol-modified lipids is 10~70 : 5~40 : 10~60 : 0.5~10; or the nucleic acid-loaded lipid nanoparticle delivery system comprises the ROS-responsive ionizable lipids, functional adamantane-tailed lipid AD8, neutral phospholipids, cholesterol, and polyethylene glycol-modified lipids as described in claim 1 or 2; wherein the molar ratio of ROS-responsive ionizable lipids: functional adamantane-tailed lipid AD8 : neutral phospholipids: cholesterol: polyethylene glycol-modified lipids is 10~50 : 10~50 : 5~40 : 10~60 : 0.5~10; The structural formula of the functional adamantane tail-chain lipid AD8 is as follows: 。 6. The nucleic acid-loaded lipid nanoparticle delivery system according to claim 5, characterized in that, The molar ratio of ROS-responsive ionizable lipids: neutral phospholipids: cholesterol: PEGylated lipids is 30~50 : 10~25 : 40~60 : 0.5~3.

7. The nucleic acid-loaded lipid nanoparticle delivery system as described in claim 5, characterized in that, The molar ratio of ROS-responsive ionizable lipids: functional adamantane tail chain lipid AD8: neutral phospholipids: cholesterol: PEGylated lipids is 10~30 : 10~30 : 10~25 : 30~60 : 0.5~3.

8. The lipid nanoparticle delivery system for nucleic acid loading as described in any one of claims 5-7, characterized in that, The nucleic acid is DNA or RNA, and the RNA is selected from siRNA, saRNA, shRNA, microRNA, or mRNA.

9. The lipid nanoparticle delivery system for nucleic acid loading as described in any one of claims 5-7, characterized in that, The PEGylated lipids are selected from: 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol, 1,2-distearate-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)], polyethylene glycol-cholesterol, and bis(tetradecyl)glutamic acid (4-methoxy polyethylene glycol-2000-4-oxobutyryl)glutamic acid; the neutral phospholipids are selected from: soybean lecithin, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)], PEG-cholesterol, and bis(tetradecyl)glutamic acid (4-methoxy polyethylene glycol-2000-4-oxobutyryl)glutamic acid. - Phosphocholine, 1-palmitoyl-2-oleoyl lecithin, myristoyl phosphatidylcholine, 1,2-dioleoyl-sn-glycerol-3-phosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, 1,2-distearyl-sn-glycerol-3-phosphoethanolamine, disorhoyl lecithin, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine and 1,2-distearyl-sn-glycerol-3-phosphocholine.

10. The use of the nucleic acid-loaded lipid nanoparticle delivery system according to any one of claims 5-7 in the preparation of DNA transfection, mRNA transfection, siRNA gene silencing, and shRNA gene silencing reagents.

11. The use of the nucleic acid-loaded lipid nanoparticle delivery system according to any one of claims 5-7 in the preparation of transfection reagents for 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.

Images