CN117327018A - STING activated type ionizable lipid, lipid nanoparticle and application - Google Patents

Abstract

The invention discloses STING activated type ionizable lipid, a lipid nanoparticle and application thereof, wherein a lipid structure comprises a six-membered nitrogen-containing piperazine heterocycle, a dihydroimidazole ring, an alkyl chain for connecting the two, a long alkyl side chain and the like; the alkyl chain connecting the two and the long alkyl side chain attached to the dihydroimidazole ring, including but not limited to saturated or unsaturated alkyl chains of from one to ten carbons; in the preparation process of LNPs, the auxiliary lipid materials used include but are not limited to lipid materials such as dimyristoyl phosphatidylcholine and the like; the nucleic acid medicine at least comprises at least one of siRNA, mRNA and the like. The STING activated type ionizable heterocyclic lipid can be used as a vaccine adjuvant, has the capability of loading nucleic acid medicaments and endowing the prepared LNPs with the chemotaxis of a lymphatic immune system, enhances the accumulation of the LNPs in immune organs, activates STING channels and enhances the treatment effect of tumor vaccines.

Description

A STING-activated ionizable lipid, lipid nanoparticles and applications

Technical field

The present invention relates to an ionizable lipid, lipid nanoparticles and applications, and in particular to a STING-activated ionizable lipid, lipid nanoparticles and applications.

Background technique

mRNA technology can generate specific tumor neoantigens through the protein synthesis system of human cells to induce de novo immune responses against cancer-specific neoantigens, thereby specifically attacking tumor cells and preventing tumor recurrence, becoming an important strategy for personalized immunotherapy. one.

Although nanomaterials can promote the residence of mRNA vaccines in lymphoid organs, most vaccines cannot effectively reach antigen-presenting cells in lymphoid organs, which becomes the main rate-limiting step in inducing immune responses. Tumor diseases can reshape the body's lymphatic system, leading to systemic immune system mobilization disorders and immune tolerance. In addition, the deposition of adjuvants and vaccines at the injection site, lymphatic drainage, antigen presentation barrier, and cascade signal amplification between antigen-presenting cells and T cells are all important factors affecting the efficacy of nanovaccines. Therefore, the development of lipid nanovaccines that precisely target lymphoid organs can significantly limit tumor metastasis and promote the body's immune response. It can also effectively solve the comprehensive cellular response signal cascade amplification of mRNA and adjuvants. It is an important step to promote the application of nanovaccines in tumors. Key to clinical application in personalized therapy.

Based on the laboratory's preliminary research findings, specific proteins can be combined with LNP technology to form a protein corona, which can then target specific receptors, cells and organs. Ionizable lipid materials can adsorb nucleic acid drugs through electrostatic interactions. Their head groups are usually positively charged and can interact with cell membranes to improve the lysosomal escape and transfection efficiency of mRNA. As a six-membered nitrogen-containing heterocycle, piperazine has efficient ionizability and non-destructive penetration into epithelial cells. Therefore, if the piperazine ring is introduced as a scaffold structure for ionizable lipids, not only can the ionizable lipids be ionized, The transformation of "liver tropism" into "lymphatic system tropism" can also enhance the system's penetration ability in the lymphatic system and improve its biomedical application potential.

Stimulator of interferon genes (STING) signal agonists are currently one of the most promising immune adjuvants. They stimulate the activation of STING signals in antigen-presenting cells and can assist mRNA nanovaccines in inducing type I interferon (IFN-I). ) secretion, thereby promoting the cross-presentation of tumor antigens, the proliferation and activation of T lymphocytes, and direct killing of tumors. However, when the free cyclic dinucleotide and aminobenzimidazole compound dimer STING agonist is used as an immune adjuvant to participate in the design of tumor vaccines, it still has poor lymphoid organ delivery efficiency and cannot improve the cytoplasmic release of antigen. Problems such as these are one of the key reasons for the poor clinical efficacy of current tumor vaccines. Therefore, developing a new generation of STING vaccine adjuvants and effectively combining them with the organ-targeted delivery technology of LNP-mRNA to synergistically promote the cytoplasmic delivery of tumor antigens in lymphoid organs and the efficient activation of STING signals is of great significance for improving tumor vaccine treatment.

Contents of the invention

Purpose of the invention: The present invention aims to provide an immune adjuvant STING-activated ionizable lipid, which has the functions of STING activator, nucleic acid delivery and immune system tropism; the second purpose of the present invention is to provide an STING-activated ionizable lipid. The application of type ionizable lipids in the preparation of lipid nanoparticles and nucleic acid drug delivery with STING activation effect and immune system tropism; the third purpose of the present invention is to provide a method containing the STING activation type ionizable lipids Lipid nanoparticles.

Technical solution: The structural formula of the STING-activated ionizable lipid of the present invention is as follows:

Among them, R ₁ to R ₆ are selected from a saturated or unsaturated alkyl chain with 1 to 10 carbon atoms.

Preferably, R ₁ , R ₂ , R ₅ , and R ₆ are selected from saturated or unsaturated linear alkyl groups with 1 to 10 carbon atoms, and R ₃ and R ₄ are selected from 1 to 10 carbon atoms. Saturated linear alkyl group. More preferably, R ₃ and R ₄ are selected from saturated linear alkyl groups with 1 to 5 carbon atoms.

The STING-activated ionizable lipid can be used in the preparation of lipid nanoparticles and nucleic acid drug delivery with STING activation effect and immune system tropism.

The lipid nanoparticles containing STING-activated ionizable lipids include lipid materials and nucleic acid drugs; the lipid materials include STING-activated ionizable heterocyclic lipids and other lipid materials.

Preferably, the mass ratio of STING-activated ionizable lipids to other lipid materials is 1:5 to 5:1.

The other lipid materials are selected from dioleoyl lecithin (DOPC), hydrogenated soybean phosphatidylglycerol (HSPG), lecithin glycerol (EPG), lecithin phosphatidylinositol (EPI), hydrogenated soybean phosphatidylethanolamine (HSPE) ), phosphatidylethanolamine (EPE), soy phosphatidylcholine (SPC), soy phosphatidylglycerol (SPG), soy phosphatidylserine (SPS), soy phosphatidyl inositol (SPI), hydrogenated soy phosphatidyl serine (HSPS) ), soy phosphatidylethanolamine (SPE), soy phosphatidic acid (SPA), hydrogenated egg phosphatidylcholine (HEPC), hydrogenated egg phosphatidylglycerol (HEPG), egg phosphatidylserine (EPS), hydrogenated egg phosphatidyl inositol (HEPI), hydrogenated lecithin phosphatidylserine (HEPS), hydrogenated phosphatidylethanolamine (HEPE), hydrogenated phosphatidic acid (HEPA), hydrogenated soy phosphatidylcholine (HSPC), hydrogenated soy phosphatidylinositol (HSPI), hydrogenated soybean Phosphatidic acid (HSPA), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPI), egg phosphatidylcholine (EPC), dimyristoylphosphatidylcholine (DMPC), Myristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), phosphatidic acid (EPA), dioleic acid Alkenyl phosphatidyl-ethanolamine (DOPE), palmitoylstearoylphosphatidylcholine (PSPC), dipalmitoylphosphatidic acid (DPPA), palmitoylstearoylphosphatidylglycerol (PSPG), monooleoyl-phosphatidyl Ethanolamine (MOPE), tocopherol, ammonium salts of fatty acids, ammonium salts of phospholipids, ammonium salts of glycerides, dilauroyl ethylphosphocholine (DLEP), distearoylphosphatidylserine (DSPS), dimyristoyl Ethyl phosphocholine (DMEP), dipalmitoyl ethyl phosphocholine (DPEP) and distearoyl ethyl phosphocholine (DSEP), N-(2,3-di-(9-(Z)- Octadecenyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-(trimethylammonium ) Propane (DOTAP), Distearoyl Phosphatidylglycerol (DSPG), Dimyristoyl Phosphatidic Acid (DMPA), Distearoyl Phosphatidic Acid (DSPA), Dimyristoyl Phosphatidylinositol (DMPI), Dipalmitoyl Phosphatidylinositol (DPPI), Dimyristoyl Phosphatidyl Serine (DMPS), Dipalmitoyl Phosphatidyl Serine (DPPS), Myristoyl Lysolecithin (M-LysoPC), Palmitoyl Lysolecithin (P-LysoPC) , Stearoyl lysolecithin (S-lysoPC), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), phosphatidylcholine-polyethylene glycol (PC-PEG), phosphatidylethanolamine- Polyethylene glycol (PE-PEG), distearoylphosphatidylcholine-polyethylene glycol (DSPC-PEG), cholesterol, lanosterol, sitosterol, stigmasterol, ergosterol and other functionalized phospholipids and one or more of water-soluble derivatives of sterols.

Preferably, the nucleic acid drug is selected from one or more of siRNA, miRNA, mRNA, ASO, ssRNA, ssDNA, etc.

Preferably, the method for preparing the above-mentioned lipid nanoparticles is a liposome extrusion method, a film hydration method, a nanoprecipitation method, a microfluidic process or an impact jet mixing method.

The above-mentioned lipid nanoparticles can also be used in the preparation of nucleic acid vaccines for nucleic acid drug loading, STING activation and immune system tropism.

Beneficial effects: Compared with the existing technology, the present invention has the following significant advantages: the STING-activated ionizable heterocyclic lipid and lipid nanoparticles, on the one hand, have efficient nucleic acid drug loading capacity and protective effect; on the other hand, they have The STING signal activates, thereby inducing the expression of type I IFN and initiating the interferon immune response. IFNs will stimulate the proliferation of anti-tumor T cells, penetrate into tumor tissue, and directly kill. And the signaling downstream of STING will lead to the activation of antigen-presenting cells (APCs) and the production of inflammatory cytokines, thereby promoting the initiation and recruitment of T cells; on the other hand, enhancing the tropism of the immune system can not only reduce the off-target effects of nucleic acid vaccines Side effects such as liver damage can also increase the accumulation of adjuvants and vaccines in lymphatic sites, enhance the translation and presentation of intracellular antigens, and enable the immune system to form a normal system of antigen presentation/interferon signaling/CD8+ T cells. The feedback loop then changes the tumor immune pattern, triggers the immune infiltration of inflammatory macrophages, neutrophils and natural killer (NK) populations, and improves the durability of the immune response.

Description of drawings

Figure 1 is the synthetic route of STING-activated ionizable heterocyclic lipid in Example 1, and Figure 2 is its hydrogen nuclear magnetic resonance spectrum;

Figure 3 is the synthetic route of STING-activated ionizable heterocyclic lipid in Example 2;

Figure 4 is the molecular docking result of lipids and STING protein in Example 1;

Figure 5 is the isothermal titration calorimetry detection results of lipids and STING protein in Example 1;

Figure 6 is a characteristic particle size diagram of the preparation in Example 3;

Figure 7 is a scanning electron microscope image of the preparation in Example 3;

Figure 8 shows the nuclease stability and serum stability of the preparation in Example 3;

Figure 9, Figure 10 and Figure 11 are the investigation of the activation, antigen presentation enhancing effect and OVA-specific T cell proliferation effect of the preparation in Example 4 on BMDC;

Figure 12 shows the BrdU method used to detect the proliferation effect of the preparation on T cells in Example 4;

Figures 13 and 14 show the distribution of the preparation prepared according to the method of Example 1 in vivo and in vitro tissues of mice using a small animal in vivo imaging system.

Detailed ways

The technical solution of the present invention will be further described below with reference to the accompanying drawings.

Example 1

The preparation process of a STING-activated ionizable heterocyclic lipid is as follows (the synthesis route is shown in Figure 1):

Precisely measure the prescribed amounts of 1,4-bis(3-aminopropyl)piperazine (0.25mmol), 8-15alkanone (1mmol) and ethyl isocyanoacetate (0.75mmol) and add 5mL of trichloro In a volumetric flask of methane, the entire reaction system was heated and stirred in an oil bath at 60°C and protected from light for 48 hours. After the reaction is completed, the target product is obtained through silica gel column separation, and the product structure is determined by the hydrogen nuclear magnetic resonance spectrum shown in Figure 2.

Example 2

The preparation process of another STING-activated ionizable heterocyclic lipid is as follows (synthetic route shown in Figure 3):

Precisely measure the prescribed amounts of 1,4-piperazinedibutylamine (0.25mmol), 2,13-pentadecen-8-one (1mmol) and ethyl isocyanoacetate (0.75mmol) and add 5mL In a volumetric flask of chloroform, the entire reaction system was heated and stirred in an oil bath at 60°C in the dark for 48 hours. After the reaction is completed, the target product is obtained through silica gel column separation.

Example 3

Prescription composition:

Preparation process: Microfluidic process is used to prepare lipid nanoparticles. Precisely weigh the prescribed amount of ionizable lipids, add 300 μL of methanol: dioxane (1:1) and sonicate until completely dissolved; accurately weigh the prescribed amount of cholesterol, add 100 μL of ethanol and ultrasonic until completely dissolved; accurately weigh the prescription Measure DSPE-PEG6000 and DMPC, add 100 μL of methanol and sonicate until completely dissolved; mix the above solutions to obtain the organic phase. Take about 8OD of siRNA, add 255 μL DEPC water to dissolve, and then add 1.245 μL pH 4.0 HEPES buffer as the water phase. Add the corresponding solution to the two-phase injection port of the microfluidic, mix the aqueous phase and the organic phase evenly through the program, dilute the preparation obtained at the collection port 40 times with PBS, and remove the organic phase through ultrafiltration.

Example 4

Prescription composition:

Preparation process; using nanoprecipitation method to prepare lipid nanoparticles. Precisely weigh the prescribed amount of ionizable lipids, DSPE-PEG6000, cholesterol and DMPC, add 100 μL of methanol:tetrahydrofuran (1:1) and sonicate until completely dissolved as the organic phase; add 1 mL of HEPES with pH 3.0 into a 10 mL volumetric flask. Buffer serves as the aqueous phase. After stirring for 3-5 minutes under vigorous magnetic stirring, add 0.05 mg of OVA mRNA and continue stirring for 3-5 minutes. Then add 100 μL of organic phase and continue stirring for 5 minutes. Place the preparation in a dialysis bag (MW3500), dialyze it with enzyme-free HEPES Buffer with a pH of 7.4 for 2 hours to remove the organic phase and adjust the pH value of the preparation to 7.4.

Example 5

Prescription composition:

Preparation process; using nanoprecipitation method to prepare lipid nanoparticles. Precisely weigh the prescribed amount of ionizable lipids, DSPE-PEG6000, cholesterol and DMPC, add 100 μL of methanol:tetrahydrofuran (1:1) and sonicate until completely dissolved as the organic phase; add 1 mL of HEPES with pH 3.0 into a 10 mL volumetric flask. Buffer serves as the aqueous phase. After stirring for 3-5 minutes under vigorous magnetic stirring, add 0.05 mg of mRNA and continue stirring for 3-5 minutes. Then add 100 μL of organic phase and continue stirring for 5 minutes. Use a rotary evaporation device to remove the residual organic phase in the preparation, and confirm that the organic phase is completely removed by weighing the volumetric flask before and after rotary evaporation.

The STING-activated ionizable heterocyclic lipid prepared in Example 1 was used to simulate the binding of lipid small molecules and STING protein through molecular docking. Figure 4 shows the simulated binding situation and STING with different crystal forms. Simulated binding capacity of protein; use Malvern Panalytical Isothermal Titration Microcalorimeter (MicroCal PEAQ-ITC) to detect the binding ability of lipid small molecules and human STING protein through isothermal calorimetric titration (ITC), as shown in Figure 5 Shown is the titration curve and the resulting binding parameters. It can be seen that the binding force between the lipid small molecule and the STING protein is at the level of μM close to nM, and the binding energy ΔG and enthalpy change ΔH are negative values, indicating that the reaction system is exothermic. The reaction can proceed spontaneously, and the affinity between the lipid material and the STING protein mainly comes from specific binding abilities such as hydrogen bonds and van der Waals forces.

The preparation prepared in Example 3 was used to detect the particle size using a Malven Zetasizer. As shown in Figure 6, lipid nanoparticles with smaller particle size and uniform PDI were prepared, with a particle size of approximately 162 nm and a PDI of 0.18. Figure 7 is a TEM scanning electron microscope image of the prepared preparation.

The NC siRNA-containing preparation prepared in Example 3 was incubated with nuclease and serum for a certain period of time. SDS and heparin were added to extract the nucleic acid drug, and the stability of the preparation was detected by nucleic acid gel electrophoresis.

Figure 8 shows the results of nucleic acid gel electrophoresis. It can be seen that when the preparation is incubated with nuclease for 0h to 4h, and the serum is incubated for 0h to 24h, the preparation has good stability and has a strong protective effect on the loaded nucleic acid drugs.

The OVA mRNA-containing preparation prepared in Example 4 was used to evaluate and examine the BMDC activation and antigen presentation enhancing effects. The thigh femur of C57BL/6J mice was taken, bone marrow cells were extracted, and cultured in RPMI 1640 medium containing GM-CSF. On the 7th day, suspended and poorly adherent cells were collected and the CD11c positive rate was measured. After administration, cells were collected in groups 24 hours later, flow cytometric staining and detection were performed with CD11c, CD80, CD86, MHC II and MHC I-OVA257-264 (SIINFEKL) monoclonal antibodies, and the proportion of positive cells and average fluorescence intensity were evaluated. BMDC activation and antigen presentation enhancement effects; take the spleens of C57BL/6J mice and extract spleen cells, then administer them in groups, collect the cells 24 hours later, and use CD3, CD4, CD8 and MHC I-OVA257-264 (SIINFEKL) monoclonal The antibodies were stained and detected by flow cytometry, and the activation effect of OVA-specific T cells was evaluated by the proportion of positive cells.

Figure 9 shows the proportion of CD80 and CD86 positive cells, indicating that the preparation group can stimulate the maturation of DC cells more effectively than the PBS group; Figure 10 shows CD80, MHC II and MHC I-OVA257-264 ( SIINFEKL); Figure 11 shows the proliferation of OVA-specific CD3 ⁺ CD8 ⁺ T cells and OVA-specific CD3 ⁺ CD4 ⁺ T cells in different groups. It shows that compared with the control group, the preparation group can promote the cross-presentation of antigens by stimulating the activation of STING signals in DC cells, and can further promote the proliferation of OVA-specific T cells after combined use with siSTAT3.

STAT3 siRNA was selected as the model siRNA, and the preparation containing STAT3 siRNA prepared according to the preparation protocol in Example 3 was used to detect the proliferation of T cells using the BrdU method. The spleens of C57BL/6J mice were taken and spleen cells were extracted. After labeling with Brdu for 1 hour, they were plated in groups and administered. Cells were collected after 36 h, and flow cytometric staining was performed with CD3, CD8 and BrdU antibodies to detect the proliferation level of T cells. Figure 12 shows that compared with the PBS group, the preparation group significantly increased the proliferation levels of cytotoxic T lymphocytes (CD3 ⁺ CD8 ⁺ ), Th1 cells (CD3 ⁺ CD183 ⁺ ) and NK cells (CD49b ⁺ ), and was closely related to siSTAT3 After combined use, the proliferation effect was significantly improved.

Cy5-siRNA was selected as the model siRNA, and the preparation containing Cy5-siRNA prepared according to the preparation protocol in Example 3 was evaluated for in vivo distribution in mice. ICR mice were selected as model mice, keeping the formulation ratio unchanged, and replacing the ionizable lipid with the cationic lipid DOTAP to prepare the formulation as a control. LNPs-Cy5-siRNA/DOTAP-LNPs-Cy5-siRNA Two groups of mice were injected with the corresponding group preparations through the tail vein. The fluorescence was observed through the small animal in vivo imaging system 1h, 2h, 3h, 4h, 6h and 24h after administration. After that, the mice were euthanized, and the heart, liver, spleen, lung, kidney and lymph node were taken to observe the distribution of the fluorescent preparation in the isolated tissues.

Figure 13 shows the distribution of each group of fluorescent preparations in mice at different time points. At the time point from 1h to 6h, compared with the DOTAP-LNPs control group, the preparations in the LNPs group had more obvious uptake and accumulation in the mice, and a small amount was still present at 24h, indicating that the preparations have long-lasting effects in the body. The ability to cycle. Figure 14 shows the distribution of each group of fluorescent preparations in the isolated mouse tissue heart, liver, spleen, lung, kidney and lymph node at different time points. At each time point, the accumulation of preparations in the spleen and lymph nodes of the LNPs group was significantly higher than that of the DOTAP-LNPs group, indicating that the synthesized ionizable lipids endow lipid nanoparticles with immune system tropism and increase their presence in immune organs. Accumulation in spleen and lymph nodes.

Claims (10)

1. A STING-activated ionizable lipid, characterized by: the structural formula is as follows: Among them, R ₁ to R ₆ are selected from a saturated or unsaturated alkyl chain with 1 to 10 carbon atoms. 2. The STING-activated ionizable lipid according to claim 1, characterized in that: the R ₁ , R ₂ , R ₅ , and R ₆ are selected from saturated or unsaturated linear chains with 1 to 10 carbon atoms. Alkyl group, R ₃ and R ₄ are selected from saturated linear alkyl groups with 1 to 10 carbon atoms. 3. The STING-activated ionizable lipid according to claim 1, characterized in that: R ₃ and R ₄ are selected from saturated linear alkyl groups with 1 to 5 carbon atoms. 4. Application of the STING-activated ionizable lipid described in any one of claims 1 to 3 in the preparation of lipid nanoparticles and nucleic acid drug delivery with STING activation effect and immune system tropism. 5. A lipid nanoparticle containing the STING-activated ionizable lipid according to any one of claims 1 to 3, characterized in that it includes lipid materials and nucleic acid drugs. 6. Lipid nanoparticles according to claim 5, characterized in that: the lipid material includes STING-activated ionizable heterocyclic lipids and other lipid materials. 7. The lipid nanoparticle according to claim 6, characterized in that: the mass ratio of the STING-activated ionizable lipid to other lipid materials is 1:5 to 5:1. 8. Lipid nanoparticles according to claim 5, characterized in that: the other lipid materials are selected from the group consisting of dioleoyl lecithin, hydrogenated soybean phosphatidylglycerol, lecithin glycerol, lecithin, and hydrogenated soybean. Phosphatidylethanolamine, phosphatidylethanolamine, soy phosphatidylcholine, soy phosphatidylglycerol, soy phosphatidylserine, soy phosphatidyl inositol, hydrogenated soy phosphatidyl serine, soy phosphatidylethanolamine, soy phosphatidic acid, hydrogenated lecithin choline Alkali, hydrogenated lecithin, phosphatidylserine, hydrogenated lecithin, hydrogenated lecithin, hydrogenated phosphatidyl ethanolamine, hydrogenated phosphatidic acid, hydrogenated soybean phosphatidylcholine, hydrogenated soybean phosphatidylinositol, hydrogenated Soy phosphatidic acid, dipalmitoylphosphatidylcholine, distearoylphosphatidylinositol, egg phosphatidylcholine, dimyristoylphosphatidylcholine, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, di Stearoylphosphatidylcholine, distearoylphosphatidylglycerol, phosphatidic acid, dioleylphosphatidyl-ethanolamine, palmitoylstearoylphosphatidylcholine, dipalmitoylphosphatidic acid, palmitoylstearoylphospholipid Acylglycerol, monooleoyl-phosphatidylethanolamine, tocopherol, ammonium salt of fatty acid, ammonium salt of phospholipid, ammonium salt of glyceride, dilauroyl ethyl phosphocholine, distearoylphosphatidylserine, dimyristoyl Ethyl phosphocholine, dipalmitoyl ethyl phosphocholine and distearoyl ethyl phosphocholine, N-(2,3-bis-(9-(Z)-octadecenyloxy)- Propan-1-yl-N,N,N-trimethylammonium chloride, 1,2-bis(oleoyloxy)-3-(trimethylammonium)propane, distearoylphosphatidylglycerol, di Myristoyl Phosphatidic Acid, Distearoyl Phosphatidic Acid, Dimyristoyl Phosphatidylinositol, Dipalmitoyl Phosphatidylinositol, Dimyristoyl Phosphatidyl Serine, Dipalmitoyl Phosphatidyl Serine, Myristoyl Lysolecithin, Palm Acyl lysolecithin, stearoyl lysolecithin, distearoylphosphatidylethanolamine-polyethylene glycol, phosphatidylcholine-polyethylene glycol, phosphatidylethanolamine-polyethylene glycol, distearoylphosphatidyl Choline - one or more of polyethylene glycol, cholesterol, lanosterol, sitosterol, stigmasterol, and ergosterol. 9. The lipid nanoparticle according to claim 5, characterized in that: the nucleic acid drug is selected from one or more of siRNA, miRNA, mRNA, ASO, ssRNA, and ssDNA. 10. Lipid nanoparticles according to claim 5, characterized in that: the method for preparing the lipid nanoparticles includes liposome extrusion, film hydration, nanoprecipitation, microfluidics or impact jet. Mixed method.