Degradable ionizable lipid compound 2N12B as well as application and preparation method thereof

Abstract

The invention relates to a degradable ionizable lipid compound 2N12B as well as application and a preparation method thereof, nano-particles prepared from the compound have pKa of 6.50, are neutral in a physiological environment, have good in-vivo stability and biocompatibility, are protonated in a slightly acidic environment, are beneficial to targeted accumulation in a low-pH environment and lysosome escape in cells, and have good in-vivo stability and biocompatibility. According to the present invention, the ester bond and the disulfide bond respond to the microenvironment fracture in the cytoplasm, and the cleavage of the disulfide bond accelerates the ester bond fracture so as to improve the cytoplasm removal rate of the compound and reduce the chronic toxicity, and the prepared nano-drug has characteristics of good biocompatibility and good degradability, can be used as the new nucleic acid drug delivery method, and can promote the development of the nucleic acid drug.

Description

Technical Field

[0001] The present invention relates to the technical field of drug delivery, and in particular to a degradable ionizable lipid compound 2N12B and an application and a preparation method thereof. Background Art

[0002] Nucleic acid drugs replace, block, correct or compensate specific genes by introducing exogenous nucleic acid molecules, thereby achieving the purpose of preventing and treating diseases. Nucleic acid drugs have broken through the target limitations of small molecule drugs, have a wide range of applications and are simple in design, and have become one of the main development directions of modern pharmaceuticals. Although nucleic acid drugs have obvious advantages over traditional drugs, the presence of nucleases in the extracellular space will lead to rapid degradation of nucleic acids, and the negative charge of nucleic acid molecules also makes it difficult for them to cross the cell membrane, which is also negatively charged, and it is difficult to reach the cell to exert therapeutic effects. Therefore, it is necessary to develop delivery vectors to promote the clinical application of nucleic acid drugs. Among the delivery vectors currently developed, lipid nanoparticles are more advanced and effective. However, there are very few products on the market and they have large side effects. Continuous design and screening are needed to explore better delivery efficiency and biosafety. Summary of the invention

[0003] In order to solve the defects and shortcomings of the prior art, the present invention provides a degradable ionizable lipid compound 2N12B and its application and preparation method.

[0004] The technical solution adopted by the present invention is: a degradable ionizable lipid compound 2N12B, wherein the amine head and hydrocarbon chain tail of the lipid of the degradable ionizable lipid compound 2N12B respectively have an ionizable tertiary amine and a reduction-responsive disulfide bond, realizing dual intelligent response to ensure the biosafety and drug delivery efficiency of the compound. The ionizable amine head makes it negatively charged under physiological conditions, does not produce cationic toxicity, and does not bind to plasma proteins to cause degradation. It is protonated under acidic conditions, which is conducive to targeted accumulation in low pH environments and lysosomal escape in cells. Both ester bonds and disulfide bonds can respond to the microenvironment in the cytoplasm (esterases and high concentrations of glutathione in the cytoplasm) to achieve biodegradation. At the same time, when introduced, the cleavage of the disulfide bond drives the nucleophilic attack on the ester bond, accelerates the degradation of lipids, increases the cytoplasmic clearance rate of the carrier, and reduces chronic toxicity.

[0005] The structural formula of the degradable ionizable lipid compound 2N12B is:

[0007] Application of a degradable ionizable lipid compound 2N12B in the preparation of a drug carrier.

[0008] The drug carrier is a nucleic acid drug carrier.

[0009] A method for preparing the degradable ionizable lipid compound 2N12B comprises the following steps: (1) synthesis of intermediate P1: weighing dithiodipyridine and dissolving it in methanol, adding acetic acid, dropping a solution of octanethiol dissolved in dichloromethane into the mixture, stirring the mixture at room temperature for 2h-6h, monitoring the progress of the reaction to ensure that the reaction is complete, and after the reaction is completed, rotary evaporating the solvent, purifying the crude product with ethyl acetate and petroleum ether in a volume ratio of 1:9 as eluent, and rotary evaporating again to obtain a yellow-green oily substance P1 with a pungent odor; (2) Synthesis of intermediate P2: Compound P1 was dissolved in methanol, acetic acid was added, and β-mercaptoethanol dissolved in methanol was added dropwise to the solution. The reaction mixture was stirred at room temperature. After the reaction was completed, the solvent was evaporated, and the crude product was purified by column chromatography using ethyl acetate / n-hexane in a volume ratio of 1:4 as the eluent to obtain a light yellow-green liquid P2. (3) Synthesis of intermediate P3: Compound P2 was dissolved in DCM, triethylamine was added as an acid-binding agent, the solution was cooled to 0°C, acryloyl chloride was dissolved in DCM, and the solution was slowly added dropwise to the reaction solution. The reaction was stirred at room temperature. After the reaction was complete, it was extracted with 5% NaOH. The aqueous phase obtained by the two extractions was extracted with DCM, the organic phase was collected, and the aqueous phase was extracted with DCM, saturated aqueous NaHCO3 solution, NaCl, and water in sequence to wash away impurities. The organic phases were combined, anhydrous magnesium sulfate was added to dry overnight, and then filtered, rotary evaporated, and column chromatographed to obtain a yellow-green liquid P3. (4) Synthesis of lipid compound 2N12B: P3 was mixed with N-methylethylenediamine and reacted at 70°C without solvent. After the reaction was completed, the mixture was cooled to room temperature and purified by column chromatography (ethyl acetate: n-hexane = 4:1) to obtain a yellow-brown viscous liquid lipid compound 2N12B.

[0010] A lipid nano drug comprises the drug carrier, auxiliary lipids and nucleic acid drugs.

[0011] A method for preparing the lipid nanomedicine comprises the following steps: (1) Preparation of lipid nanoparticles: The lipid compound 2N12B and the auxiliary lipid DOPE, cholesterol and DMG-PEG2000 were dissolved in a chloroform solution and mixed evenly. The mixture was rotary evaporated to obtain a uniform lipid thin layer. The residual chloroform was evaporated by vacuum drying. Sodium acetate buffer was added to ultrasonically hydrate the lipid thin layer. After complete hydration, a milky white liquid was obtained. After extrusion, the lipid nanoparticles were dialyzed in water overnight to remove free molecules. (2) Preparation of lipid nanomedicine: Nanoparticles and nucleic acid drugs are mixed and dissolved in sodium acetate buffer at room temperature according to the nitrogen-phosphorus ratio to form lipid-drug complexes.

[0012] A method for preparing the lipid nanomedicine comprises the following steps: (1) Preparation of lipid nanodrugs: The lipid compound 2N12B and the auxiliary lipids DOPE, cholesterol and DMG-PEG2000 were dissolved in ethanol; the nucleic acid drug was dissolved in sodium acetate buffer; the nucleic acid drug and lipid solution were vortexed to form a lipid nanodrug complex, and the product was dialyzed in PBS overnight.

[0013] The beneficial effects of the present invention are as follows: the present invention provides a degradable ionizable lipid compound 2N12B and its application and preparation method; the nanoparticles prepared from the compound have a pKa of 6.50, are neutral under physiological conditions, have good in vivo stability and biocompatibility, are protonated under a slightly acidic environment, are conducive to targeted accumulation in a low pH environment and intracellular lysosomal escape, and the ester bonds and disulfide bonds break in response to the microenvironment in the cytoplasm, and the cleavage of the disulfide bonds accelerates the cleavage of the ester bonds, thereby increasing the cytoplasmic clearance rate of the compound and reducing chronic toxicity; the nanomedicine prepared by the present invention has good biocompatibility and degradability, can be used as a new method for nucleic acid drug delivery, and promotes the development of nucleic acid drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the hydrogen NMR spectrum of compound P1.

[0015] Figure 2 This is the hydrogen NMR spectrum of compound P2.

[0016] Figure 3 This is the P3 H NMR spectrum.

[0017] Figure 4 This is the hydrogen nuclear magnetic resonance spectrum of compound 2N12B.

[0018] Figure 5 This is the carbon NMR spectrum of compound 2N12B.

[0019] Figure 6 This is the mass spectrum of compound 2N12B.

[0020] Figure 7 This is an electron micrograph of lipid nanoparticles.

[0021] Figure 8 The fluorescence intensity diagram of lipid nanoparticles at different pH values.

[0022] Fig. 9 Electron micrographs of 2N12H and 2N12H+glutathione.

[0023] Fig.10 The electrophoresis diagram of 2N12H and total RNA at different nitrogen-phosphorus ratios.

[0024] Fig.11 Agarose gel electrophoresis of lipid nanoparticles.

[0025] Fig.12 The effect of 2N12H on cell viability was detected by CCK8.

[0026] Fig.13 The biosafety of 2N12H was demonstrated for ring inoculation of RPE cells.

[0027] Fig.14 Flow cytometry was used to detect the RNA uptake level of RPE cells.

[0028] Fig.15 The levels of VEGFA were detected by immunoblotting after different drugs were administered to OIR model mice.

[0029] Fig.16 Fundus fluorescence angiography images of OIR model mice after administration of different drugs.

[0030] Fig.17 The synthetic route of the degradable ionizable lipid compound 2N12B. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with the accompanying drawings and the following embodiments. It should be understood that the accompanying drawings and the following embodiments are only used to illustrate the present invention rather than to limit the present invention.

[0032] Example 1: Synthesis of Degradable Ionizable Lipid Compound 2N12B Step 1: Synthesis of P1: In a 250mL round-bottom flask, weigh 10.0g of dithiodipyridine and dissolve it in 60mL of methanol (MeOH), and add 670mL of acetic acid (pH4-5). 3.63ml of octanethiol solution dissolved in 20mL of dichloromethane (DCM) was added dropwise to the mixture. Stir the reaction at 240r for 2h-6h at room temperature, and monitor the progress of the reaction by tapping the plate to ensure that the reaction is complete. The product P1 has a greater polarity than the reactant heptylthiol (completely consumed). After the reaction, the solvent was rotary evaporated, and the crude product was purified with ethyl acetate and petroleum ether (volume ratio of 1:9) as eluents. The separated liquid was monitored by tapping the plate, and rotary evaporated again to obtain a yellow-green oil P1 with a pungent odor (5.5g, yield 93.4%). The hydrogen nuclear magnetic resonance spectrum of compound P1 is shown in Figure 1. 1 H NMR (400 MHz, CDCl 3) δ 8.46 (d, J = 4.8Hz, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.68 – 7.59 (m, 1H), 7.10 – 7.04 (m, 1H), 2.79 (t, J = 7.3 Hz, 2H), 1.69 (dt, J = 14.9, 7.4 Hz, 2H), 1.45 – 1.16 (m,10H), 0.87 (t, J = 6.6 Hz, 3H).

[0033] Step 2: Synthesis of P2: Compound P1 (3.00 g) was dissolved in 40 mL of methanol, and 536 μL of acetic acid was added in a catalytic amount. β-mercaptoethanol (1.9 mL) dissolved in 10 mL of methanol was added dropwise to the solution, and the reaction mixture was stirred at room temperature for 6 h. After the reaction was completed, the solvent was evaporated, and the crude product was purified by column chromatography using ethyl acetate / n-hexane (volume ratio of 1 / 4) as the eluent to obtain a light yellow-green liquid P2 (2.63 g, yield of 65%). The hydrogen nuclear magnetic resonance spectrum of compound P2 is shown in Figure 2. 1 H NMR (400 MHz, CDCl3) δ 3.93(d, J = 5.9 Hz, 2H), 2.89 (t, J = 5.8 Hz, 2H), 2.78 – 2.70 (m, 2H), 2.01 (t, J =6.3 Hz, 1H), 1.72 (s, 2H), 1.48 – 1.39 (m, 2H), 1.33 (d, J = 10.7 Hz, 8H), 0.94– 0.89 (m, 3H).

[0034] Step 3: Synthesis of P3: Compound P2 (2g) was dissolved in 30mL DCM, 1.54mL triethylamine was added as an acid binding agent, and the solution was cooled to 0°C. 755μL acryloyl chloride was dissolved in 20mL DCM and slowly added dropwise to the reaction solution. The reaction was stirred at room temperature for 4h. After the reaction was complete, it was extracted with 5% NaOH. The aqueous phase obtained by the two extractions was extracted with 5mL DCM, the organic phase was collected, and the aqueous phase obtained was added with 5mL DCM, and extracted with a saturated aqueous solution of NaHCO3 (unsaturated 0.1g / mL), NaCl (0.1g / mL), and water in turn to wash away impurities and combine the organic phases. About 10g of anhydrous magnesium sulfate was added and dried overnight, filtered, rotary evaporated, and column chromatographed (ethyl acetate: n-hexane = 1:4) to obtain a yellow-green liquid P3 with a yield of 0.82g (34.3%). The hydrogen nuclear magnetic resonance spectrum of compound P3 is shown in Figure 3. 1 H NMR (400 MHz, CDCl3) δ 6.47 (dd, J = 17.3, 1.4 Hz, 1H), 6.17 (dd, J = 17.3, 10.4 Hz, 1H), 5.89(dd, J = 10.4, 1.4 Hz, 1H), 4.46 (t, J = 6.7 Hz, 2H), 2.98 (t, J = 6.7 Hz, 2H),2.79 – 2.69 (m, 2H), 1.71 (p, J = 7.3 Hz, 2H), 1.46 – 1.37 (m, 2H), 1.37 – 1.23(m, 8H), 0.92 (t, J = 6.8 Hz, 3H).

[0035] Step 4: Synthesis of 2N12B: In a 5 mL glass bottle, 0.82 g of P3 and 0.072 g of N-methylethylenediamine were mixed and reacted at 70 ° C without solvent for 36 hours. After the reaction was completed, it was cooled to room temperature and purified by column chromatography (ethyl acetate: n-hexane = 4:1) to obtain a yellow-brown viscous liquid (0.76 g, yield 85%). The H NMR spectrum of compound 2N12B is as follows: See Figure 4. 1 H NMR (400 MHz, CDCl3) δ4.35 (td, J = 6.7, 4.2 Hz, 6H), 2.91 (td, J = 6.7, 1.4 Hz, 6H), 2.81 (t, J = 7.2Hz, 4H), 2.72 (td,J = 7.3, 4.8 Hz, 8H), 2.57 (dd, J = 8.4, 5.1 Hz, 2H), 2.48(dq, J = 8.8, 6.5 Hz, 8H), 2.26 (s, 3H), 1.69 (p, J = 7.2 Hz, 6H), 1.38 (d, J = 6.5Hz, 6H), 1.36 – 1.21 (m, 24H), 0.97 – 0.85 (m, 9H). Carbon spectrum is attached Figure 5 ,13C NMR (101MHz, CDCl3) δ 172.33, 172.29, 62.54, 55.53, 53.16, 51.85, 49.62, 42.43,39.25, 37.08, 37.03, 32.60, 32.48, 31.81, 29.20, 29.18, 29.15, 28.52, 22.66,14.12. Mass spectrum is attached Figure 6 , Theoretical molecular weight C42H82N2O6S6 (MH+): 903.4570, Measured molecular weight: 903.4564. Example 2: Preparation of lipid nanoparticles Thin film hydration method Step 1: Preparation of lipid nanoparticles The lipid compound 2N12H was dissolved in chloroform solution with DOPE, cholesterol and DMG-PEG2000 at a molar ratio of 50:10:39:1 and mixed evenly. Slow low-temperature rotary evaporation for 15 minutes was performed to obtain a uniform lipid thin layer, and the residual chloroform was evaporated by vacuum drying. An appropriate amount of sodium acetate buffer (10 mM, pH = 5.2) was added and ultrasonicated for 80 minutes to hydrate the lipid thin layer. After complete hydration, a milky white liquid was obtained; after 200nm extrusion, dialyzed in water overnight to remove free molecules, and lipid nanoparticles (final concentration 10mM) were obtained. Observation under a transmission electron microscope revealed that the lipid nanoparticles were spherical vesicles, as shown in the attached figure. Figure 7 The average hydrated particle size of lipid nanoparticles in solution was measured by dynamic light scattering to be 143nm±3nm. The surface potential gradually became positive as the pH decreased. It was nearly neutral under physiological conditions (pH 7.4), which was beneficial to the stability of the drug in vivo and its high biosafety. It showed a positive charge of +8mV at pH 7.0 and a high positive charge (+27mV) when the pH dropped to 5.2, showing ionizability (see Table 1).

[0036] Table 1 Hydration particle size and surface potential of lipid nanoparticles obtained by different preparation methods

[0037] Step 2: Preparation of lipid nanomedicine 2N12H / RNA The nanoparticles and the nucleic acid drug were mixed and dissolved in sodium acetate buffer (10 mM, pH = 5.2) at room temperature for 10 min to form lipid drug complexes according to the nitrogen-phosphorus ratio (N / P). The average hydrated particle size of the 2N12H / RNA complex was 175 nm (see Table 1).

[0038] The ethanol injection method dissolves lipid compounds with DOPE, cholesterol and DMG-PEG2000 in a molar ratio of 50:10:39:1 in 90% ethanol to self-assemble into LNPs; RNA is dissolved in sodium acetate buffer (10mM pH 5.2); mRNA and lipid solution are vortexed at a volume ratio of 9:1 to form drug complexes. The product is dialyzed in PBS (pH 7.4 10mM) overnight. The average hydrated particle size of the lipid nanodrug 2N12H / RNA obtained by the ethanol injection method is about 155nm, showing weak ionization performance.

[0039] Since the thin film hydration method showed better ionizability and dispersibility, the product of the thin film hydration method was used in subsequent experiments.

[0040] Performance testing of lipid nanoparticles Example 3: Detection of ionizability of lipid nanoparticles In addition to dynamic light scattering to determine the surface potential of lipid nanoparticles at different pH, TNS titration was also used to detect pKa to verify the ionizability of lipid nanoparticles. Lipid nanoparticles were dialyzed overnight in PBS (pH 7.4 10mM) to prepare samples for testing. A series of buffers with pH 3-11.5 (intervals of 0.5) were prepared by adjusting with 0.1M NaOH and 0.1M HCl, which contained 20 mM sodium phosphate, 20 mM citric acid, 20 mM ammonium acetate and 150mM NaCl. 2.5μL of drug complex sample (final concentration 250μM) and 10μL of TNS stock solution (final concentration 2μM) were added to a black 96-well plate containing 87.5μL of buffer. The fluorescence intensity at different pH was measured using a Multi-Mode Microplate Reader (excitation wavelength 321 nm, emission wavelength 445 nm). Each point was repeated three times. Fluorescence data were subjected to S-shaped best fit analysis, and the pH corresponding to the half-maximum fluorescence intensity was the pKa value. The pKa of 2N12H lipid nanoparticles was measured to be 6.496. Figure 8 shown.

[0041] Example 4: Reduction-responsive detection of lipid nanoparticles 17.75 μL of 6.6 mM 2N12H was diluted in 80 μL of 10 mM AcB buffer and incubated directly or with glutathione (final concentration of 10 mM) at 37 °C for 10 min before phosphotungstic acid staining. The morphology was observed by transmission electron microscopy. Fig. 9 As shown, lipid nanoparticles can be degraded in response to glutathione at the same concentration as in the cytoplasm.

[0042] Example 5: Detection of RNA encapsulation effect of lipid nanoparticles In order to evaluate the ability of 2N12H vector to encapsulate RNA and protect RNA from degradation by nucleases, gel electrophoresis experiments were performed. 2N12H and total RNA (0.5 μg) were mixed in sodium acetate buffer solution (10mM, pH = 5.2) at different nitrogen-phosphorus ratios for 10 minutes to prepare 2N12H / RNA nanocomplexes, which were electrophoresed in 1% (w / v) agarose gel. The results showed that total RNA could be completely encapsulated when the nitrogen-phosphorus ratio reached 5:1, which met the design requirements. After incubation with 0.1µg RNase A at 37°C for 3 hours, the siRNA encapsulated by 2N12H was still not degraded, while the naked siRNA had been completely degraded, indicating that 2N12H can effectively encapsulate RNA to resist degradation by nucleases.

[0043] Example 6: Biosafety of Lipid Nanoparticles CCK-8 was used to evaluate the viability of cells treated with nanoparticles. When HUVEC cells were in the logarithmic growth phase, the cells were plated at 1×10 4 Cells were inoculated in a 96-well plate for 12 hours. Then the cells were transfected with 2N12H / siRNA NPs, and the final siRNA concentration was 50 nM. Since 2N12H contains a reducing agent, a simple drug group without cells was set as a background control. After 24 hours, 10μL Cell CountingKit-8 (CCK-8; CK04, DOJINDO) was added and incubated at 37°C for 1-3 hours. The absorbance was read at 450 nm using a Multi-Mode Microplate Reader, and the cell viability was the relative value of the experimental group to the control group. The experiment found that the cell viability of the 2N12H-treated group was about 100%, and it did not affect the cell viability, so we believe that 2N12H has no obvious cytotoxicity. A circle of cells was inoculated around the periphery of the well plate. After transfection with Lipo 2000 or 2N12H, it was found that Lipo 2000 had obvious cell debris in the middle of the well plate without cells, while 2N12H was the same as the blank group and did not have this phenomenon. Fig.13As shown. This indicates that 2N12H has a higher safety than Lipo 2000. Example 7: Drug delivery effect of lipid nanoparticles When HUVEC cells were cultured to a confluence of 60%, the cells were transfected with TPPA / siVEGFA NPs, and Lipo 2000 was used as a positive control. RT-qPCR samples were collected 24 hours after transfection in 24-well plates, and WB samples were collected 48 hours after transfection in 6-well plates. In order to study the subcellular localization of TPPA / siRNA loaded with siRNA, HCEC cells were cultured at 2 × 10 5 The cells were inoculated into 35 mm culture dishes at a density of 100 cells / well. HUVEC cells were cultured in 6-well plates to a confluence of 60% and then transfected with 2N12H / siRNA-Cy5 or Lipo 2000 / siRNA. After 24 hours, 100 μL trypsin / well was used for complete digestion, and the supernatant was discarded after centrifugation at 400 g and resuspended in 500 μL PBS for detection by flow cytometry (C6 Plus, BD Accuri™). The experiment found that there was no statistical difference in the fluorescence intensity of intracellular Cy5 signals between 2N12H and Lipo 2000, indicating that the cellular uptake capacity of the two was comparable. Example 8: Therapeutic effect of lipid nanoparticles in animals C57BL / 6J mother mice at gestational days 16 to 18 (E16-E18) were purchased from JieSiJie (Shanghai, China) and fed by themselves. The offspring were placed in a 75% high oxygen incubator for 5 days 7 days after birth, and the nursing mother mice were replaced every day to reduce the impact on the mother mice. After leaving the chamber on P12, we selected healthy mice weighing more than 4g. After anesthesia and dilation of the pupils, we randomly divided the mice into four groups: solvent control group (PBS), positive control group (Ranibizumab, 1ug), vehicle control group (Lipo 2000 / siVEGFA, siRNA 2μg), and drug group (2N12H / siVEGFA, N / P ratio 5:1, siRNA 2μg). We administered the drug into the vitreous cavity (0.5μL). We kept the mice in a normal environment for 5 days after drug administration. Fundus fluorescein angiography was performed on P17. Fig.16 As shown, similar to ranibizumab, the fundus leakage in the 2N12H-treated group was alleviated.

[0044] Technical personnel should note that although the present invention has been described according to the above specific implementation methods, the inventive concept of the present invention is not limited to this invention, and any modification using the inventive concept will be included in the scope of protection of this patent.

[0045] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A degradable ionizable lipid compound 2N12B, characterized in that , the amino head and hydrocarbon chain tail of the lipid of the degradable ionizable lipid compound 2N12B have an ionizable tertiary amine and a reduction-responsive disulfide bond, respectively.

2. The degradable ionizable lipid compound 2N12B according to claim 1, characterized in that: The structural formula of the degradable ionizable lipid compound 2N12B is: 。 3. Use of the degradable ionizable lipid compound 2N12B according to claim 1 in preparing a drug carrier.

4. The use according to claim 3, characterized in that: The drug carrier is a nucleic acid drug carrier.

5. A method for preparing the degradable ionizable lipid compound 2N12B according to claim 1, characterized in that: The following steps are involved: (1) Synthesis of intermediate P1: Weigh dithiodipyridine and dissolve it in methanol, add acetic acid, and dropwise add octanethiol solution dissolved in dichloromethane to the mixture. Stir the mixture at room temperature for 2 h to 6 h, monitor the progress of the reaction to ensure that the reaction is complete. After the reaction is completed, rotary evaporate the solvent, and purify the crude product with ethyl acetate and petroleum ether in a volume ratio of 1:9 v / v as eluent. Rotary evaporate again to obtain a yellow-green oil P1 with a pungent odor. (2) Synthesis of intermediate P2: Compound P1 was dissolved in methanol, acetic acid was added, and β-mercaptoethanol dissolved in methanol was added dropwise to the solution. The reaction mixture was stirred at room temperature. After the reaction was completed, the solvent was evaporated, and the crude product was purified by column chromatography using ethyl acetate / n-hexane in a volume ratio of 1:4 v / v as the eluent to obtain a light yellow-green liquid P2. (3) Synthesis of intermediate P3: Compound P2 was dissolved in DCM, triethylamine was added as an acid-binding agent, the solution was cooled to 0°C, acryloyl chloride was dissolved in DCM, and the solution was slowly added dropwise to the reaction solution. The reaction was stirred at room temperature. After the reaction was completed, it was extracted with 5% NaOH. The aqueous phase obtained by the two extractions was extracted with DCM, the organic phase was collected, and the aqueous phase was extracted with DCM, saturated aqueous NaHCO3 solution, NaCl, and water in sequence to wash away impurities. The organic phases were combined, anhydrous magnesium sulfate was added to dry overnight, and then filtered, rotary evaporated, and column chromatographed to obtain a yellow-green liquid P3. (4) Synthesis of lipid compound 2N12B: P3 was mixed with N-methylethylenediamine and reacted at 70°C without solvent. After the reaction was completed, the mixture was cooled to room temperature to obtain a yellow-brown viscous liquid lipid compound 2N12B.

6. A lipid nanomedicine, characterized in that: The lipid nanomedicine comprises the drug carrier, auxiliary lipid and nucleic acid drug according to claim 3.

7. A method for preparing the lipid nano drug according to claim 6, characterized in that: The following steps are involved: (1) Preparation of lipid nanoparticles: The lipid compound 2N12B and the auxiliary lipid DOPE, cholesterol and DMG-PEG2000 were dissolved in a chloroform solution and mixed evenly. The mixture was rotary evaporated to obtain a uniform lipid thin layer. The residual chloroform was evaporated by vacuum drying. Sodium acetate buffer was added to ultrasonically hydrate the lipid thin layer. After complete hydration, a milky white liquid was obtained. After extrusion, the lipid nanoparticles were dialyzed in water overnight to remove free molecules. (2) Preparation of lipid nanomedicine: Nanoparticles and nucleic acid drugs are mixed and dissolved in sodium acetate buffer at room temperature according to the nitrogen-phosphorus ratio to form lipid-drug complexes.

8. A method for preparing the lipid nano drug according to claim 6, characterized in that: The following steps are involved: (1) Preparation of lipid nanodrugs: The lipid compound 2N12B and the auxiliary lipids DOPE, cholesterol and DMG-PEG2000 were dissolved in ethanol; the nucleic acid drug was dissolved in sodium acetate buffer; the nucleic acid drug and lipid solution were vortexed to form a lipid nanodrug complex, and the product was dialyzed in PBS overnight.

Images