A lipid nanoparticle based on centella asiatica acid and a preparation method and application thereof

Abstract

The present application relates to the technical field of nucleic acid drug preparation, and discloses a lipid nanoparticle based on asiatic acid and a preparation method and application thereof, the lipid nanoparticle is composed of ionizable cationic lipids, structural lipids, neutral lipids, PEG lipids and asiatic acid with a molar ratio of 10-85:20-60:1-30:0.1-10:0.1-50, and is used as a delivery carrier of nucleic acid drugs for delivering nucleic acid drugs. The present application innovatively uses natural product asiatic acid to partially replace the structural lipids (such as cholesterol) in the traditional LNP formula, significantly optimizes the micro morphology, and greatly enhances the transfection and expression efficiency of mRNA in vitro and in vivo. The mRNA vaccine based on the carrier can induce the body to produce high-titer specific antibodies and significantly enhance the cytotoxic T lymphocyte (CTL) killing activity. The LNP system performs outstandingly in the field of tumor immunoprevention and treatment, and shows superior conversion application potential and broad market prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations, in particular to a lipid nanoparticle based on asiatic acid and a preparation method and application thereof. BACKGROUND

[0002] mRNA therapy has been extensively studied in the fields of infectious disease vaccines and cancer treatment. In cancer treatment, mRNA sequences can be flexibly designed to encode various types of proteins, including tumor-associated antigens, inflammatory cytokines, and tumor suppressors, and even multiple target proteins at the same time. Compared with traditional methods, mRNA tumor vaccines have multiple advantages: antigen specificity, sustained immune response, small side effects, rapid production, and potential for personalized treatment. However, mRNA molecules themselves have large single-stranded structures and negative charges, resulting in short half-lives in blood and difficulty in effectively penetrating cell membranes to mediate protein expression. Therefore, developing efficient delivery systems is crucial for the clinical application of mRNA therapy.

[0003] Lipid nanoparticles (LNP) are the most advanced mRNA delivery system, and three RNA-based LNPs have been approved by the US Food and Drug Administration (FDA): Onpattro®, Comirnaty®, and Spikevax®. The successful application of LNP is attributed to the following advantages: protecting mRNA from nucleases, promoting tissue targeting and cellular internalization, and facilitating the escape of mRNA from endosomes to the cytosol, where mRNA is translated into encoded proteins. After being taken up by antigen-presenting cells (APCs), part of the proteins activate cytotoxic T cells (CTLs) and helper T cells (Ths) via the MHC-I and MHC-II pathways, respectively, inducing anti-tumor immunity. After B cells recognize antigens, they are activated and differentiated into plasma cells with the help of Ths, producing neutralizing antibodies. LNP components are usually biocompatible and degradable, and some also have adjuvant activity, which can enhance cellular and humoral immunity, and can also be used to deliver immunomodulators to further enhance immune response effects.

[0004] Typical LNP formulations contain ionizable cationic lipids, neutral lipids, structural lipids, and PEG lipids, each of which has a specific role. Ionizable cationic lipids are an essential component of LNP, which contains ionizable amino groups (usually tertiary amines) in the head group. The pH responsiveness of ionizable lipids enables LNP to maintain a neutral charge in the physiological environment, thereby avoiding charge-driven clearance by the reticuloendothelial system (RES) and prolonging the circulation time, while becoming positively charged in the weakly acidic environment, thereby disrupting the endosome membrane and facilitating endosome escape. PEG lipids are another key component of LNP, which can stabilize LNP, prevent LNP aggregation and fusion, and prolong circulation time. Neutral lipids can maintain LNP stability, adjust LNP size, and improve LNP biocompatibility. Structural lipids, such as cholesterol, are present in high amounts in LNP, filling the gap in the lipid bilayer, adjusting the membrane integrity and rigidity, and increasing the stability of LNP. It is essential to optimize the composition of LNP in consideration of solving the unique requirements of specific applications.

[0005] LNP utilizes the hydrophobic similarity with the cell membrane to enhance their nucleic acid delivery capacity. In addition, mRNA encapsulated in LNP exhibits higher stability compared to naked mRNA, minimizing degradation by nucleases. Nucleotide modifications pioneered by Kariko and Weissman further improve stability. Despite these significant developments, the delivery efficiency of LNP is still not ideal in some cases, especially in the acidic environment of endosomes, where the efficiency of LNP to release mRNA is insufficient, limiting its effective delivery within cells. Zerial et al. confirmed that only less than 2% of siRNA or mRNA delivered by LNPs can enter the cytoplasm. Therefore, how to optimize the LNP formulation to improve the transfection efficiency of mRNA has become a key problem in the current mRNA vaccine industrialization process. SUMMARY

[0006] In view of the technical problems of the existing LNP system, such as insufficient mRNA release efficiency in vivo and insufficient transfection efficiency, the present application provides a lipid nanoparticle based on asclepinic acid replacing part of the structural lipid and a preparation method and application thereof.

[0007] To achieve the above-mentioned purpose, in a first aspect, the present application provides a lipid nanoparticle based on asclepinic acid, which is prepared from ionizable cationic lipids, structural lipids, neutral lipids, PEG lipids, and asclepinic acid in a molar ratio of 10-85:20-60:1-30:0.1-10:0.1-50, wherein the structural lipids are selected from one or more of cholesterols, ergosterols, campesterols, oxysterols, desmosterols, sitosterols, and stigmasterols. The lipid nanoparticle can be used as a delivery carrier for nucleic acid drugs for delivering nucleic acid drugs.

[0008] As a further preferred technical solution of the present application, the molar ratio of the ionizable cationic lipid, the structural lipid, the neutral lipid, the PEG lipid and asiatic acid is 45-55:20-50:5-15:0.1-5:0.1-20.

[0009] As a further preferred technical solution of the present application, the molar ratio of the ionizable cationic lipid, the structural lipid, the neutral lipid, the PEG lipid and asiatic acid is 50:34.65:10:1.5:3.85.

[0010] As a further preferred technical solution of the present application, the ionizable cationic lipid is selected from one or more of ((4-hydroxybutyl)azaniumyl dialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), octadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), 4-(N,N-dimethylamino)butanoic acid, dilinoleyl methyl ester (DLin-MC3-DMA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioctadecenyl- oxy-3-methylammonium propane (chloride salt) (DOTMA), 1,2-dilinoleyl- oxy-3-dimethylaminopropane (DLin-DMA), and 1,2-dioleoyl-3-dimethylamino- propane (DODMA), (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP);

[0011] As a further preferred technical solution of the present application, the neutral lipid is selected from one or more of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC), egg phosphatidylcholine (EPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phospho-rac-(glycerol sodium salt) (DOPG), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-3-phosphocholine (DMPC), distearoylphosphatidylethanolamine (DSPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC);

[0012] And / or, the PEG lipid is selected from one or more of 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DSG-PEG2000), 1,2-distearoyloxypropyl-3-ammonium-N-[methoxy(polyethylene glycol)-2000] (DSA-PEG2000), and 2-[(polyethylene glycol)-2000]-N,N-tetracosanoylacetamide (ALC-0159).

[0013] According to a second aspect of the present application, the present application further provides a preparation method of a lipid nanoparticle system based on asiatic acid, which comprises: dissolving ionizable cationic lipids, structural lipids, neutral lipids, PEG lipids, and asiatic acid in ethanol to obtain an ethanol solution, and form a LNP suspension; then diluting with an aqueous medium, and purifying and concentrating by dialysis or ultrafiltration to obtain the lipid nanoparticle system.

[0014] As a further preferred technical solution of the present application, nucleic acid drugs are further added in the system, specifically: the ethanol solution obtained by dissolving ionizable cationic lipids, structural lipids, neutral lipids, PEG lipids, and asiatic acid in ethanol is mixed with a sodium citrate buffer solution with pH=4.0±0.2 in which the nucleic acid drugs are dissolved to form a LNP suspension, then diluted with an aqueous medium, and purified and concentrated by dialysis or ultrafiltration to obtain a nucleic acid-lipid nanoparticle system in which the nucleic acid drugs are loaded by the lipid nanoparticle.

[0015] As a further preferred technical solution of the present application, the average particle size of the lipid nanoparticle system is 50-150 nm, and the system has an ellipsoidal core-shell structure. In the core-shell structure, the external shell is formed by the neutral lipids, the PEG lipids, part of the ionizable cationic lipids, part of the structural lipids, and part of the asiatic acid, and the nucleic acid drugs are wrapped by part of the ionizable cationic lipids, part of the structural lipids, and part of the asiatic acid to form the core inside the shell.

[0016] As a further preferred technical solution of the present application, the nucleic acid drugs are selected from any one of RNA drugs, DNA drugs, and plasmids, preferably RNA drugs, and further preferably siRNA and mRNA; and / or, the nitrogen-phosphorus ratio of the lipid nanoparticle and the nucleic acid drugs is 1:10-10:1, and more preferably 3:1-8:1.

[0017] As a further preferred technical solution of the present application, the LNP suspension is prepared by using a rapid mixing method or a microfluidic synthesis method.

[0018] According to a third aspect of the present application, the present application also provides a nucleic acid-lipid nanoparticle system for use in the preparation of a medicament for preventing or treating an infectious disease, cancer, diabetes.

[0019] Compared with the prior art, the present application can achieve the following beneficial effects:

[0020] The present application innovatively uses natural product asiatic acid to replace the structural lipid (such as cholesterol) in the traditional LNP formula, significantly optimizes the micro morphology, and greatly enhances the transfection and expression efficiency of mRNA in vitro and in vivo. The mRNA vaccine constructed based on the carrier can induce the body to produce high titer of specific antibodies, and significantly enhance the cytotoxic T lymphocyte (CTL) killing activity. The LNP system performs outstandingly in the field of tumor immunoprevention and treatment, and shows superior transformation application potential and broad market prospect. BRIEF DESCRIPTION OF DRAWINGS

[0021] The present application will be further described in detail below in combination with the drawings and specific embodiments.

[0022] Figure 1 Schematic diagram of CHOL / AA-LNP structure for encapsulating mRNA prepared in Example 1.

[0023] Figure 2 Particle size diagram of CHOL-LNP and CHOL / AA-LNP for encapsulating mRNA prepared in Example 1 and Comparative Example 1, wherein A is a representative particle size distribution diagram of CHOL-LNP, and B is a representative particle size distribution diagram of CHOL / AA-LNP.

[0024] Figure 3 Electron micrograph of CHOL-LNP and CHOL / AA-LNP for encapsulating mRNA prepared in Example 1 and Comparative Example 1, wherein A is an electron micrograph of CHOL-LNP, and B is an electron micrograph of CHOL / AA-LNP.

[0025] Figure 4 Investigation of encapsulation efficiency of CHOL-LNP and CHOL / AA-LNP for encapsulating mRNA prepared in Example 1 and Comparative Example 1.

[0026] Figure 5 Cell transfection level of CHOL-LNP and CHOL / AA-LNP for encapsulating mRNA prepared in Example 1 and Comparative Example 1, wherein A is a bioluminescence diagram, and B is a semi-quantitative result of bioluminescence.

[0027] Figure 6Animal transfection levels of mRNA-encapsulated CHOL-LNP and CHOL / AA-LNP prepared for Example 1 and Comparative Example 1, wherein A is live imaging graph, B is front semi-quantitative result, and C is back semi-quantitative result.

[0028] Figure 7 Levels of antigen-specific antibody production in mice induced by mRNA-encapsulated CHOL-LNP and CHOL / AA-LNP prepared for Example 2 and Comparative Example 2, wherein A is anti-OVA IgG antibody titer in serum of mice in each group on day 21, B is anti-OVA IgG2a antibody titer in serum of mice in each group on day 21, and C is anti-OVA IgG1 antibody titer in serum of mice in each group on day 21.

[0029] Figure 8 Specific killing efficiency of mRNA-encapsulated CHOL-LNP and CHOL / AA-LNP prepared for Example 2 and Comparative Example 2, wherein A is relative specific killing efficiency, and B is a representative flow detection graph.

[0030] Figure 9 Tumor prevention effect evaluation of mRNA-encapsulated CHOL-LNP and CHOL / AA-LNP prepared for Example 2 and Comparative Example 2, wherein A is tumor growth curve of the PBS group, B is tumor growth curve of the CHOL-LNP group, and C is tumor growth curve of the CHOL / AA-LNP group.

[0031] Figure 10 Tumor treatment effect evaluation of mRNA-encapsulated CHOL-LNP and CHOL / AA-LNP prepared for Example 2 and Comparative Example 2, wherein A is tumor growth curve of the PBS group, B is tumor growth curve of the CHOL-LNP group, and C is tumor growth curve of the CHOL / AA-LNP group.

[0032] The purposes, functional features and advantages of the present application will be further explained with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0033] The specific embodiments of the present application will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely intended to illustrate and explain the present application, and are not intended to limit the present application.

[0034] Unless otherwise defined, the technical terms used in the following examples have the same meanings as generally understood by those skilled in the art to which the present application pertains. The experimental reagents used in the following examples are all conventional biochemical reagents unless otherwise specified; and the experimental methods described are all conventional methods unless otherwise specified.

[0035] The nucleic acid-lipid nanoparticle system based on the replacement of part of the structural lipid by asiatic acid of the present application is abbreviated as CHOL / AA-LNP (NC), wherein NC is a nucleic acid drug, CHOL is cholesterol, AA is asiatic acid, and LNP is a lipid nanoparticle. The nucleic acid-lipid nanoparticle system based on the traditional cholesterol lipid nanoparticle is abbreviated as CHOL-LNP (NC).

[0036] Example 1: The molar ratio of CHOL to AA is 9:1 (10% of CHOL is replaced by AA)

[0037] This example provides a microfluidic synthesis method for preparing CHOL / AA-LNP (mRNA), as follows:

[0038] S1, preparation of carrier material ethanol solution: first, accurately prepare SM-102, asiatic acid (AA), cholesterol (CHOL), DMG-PEG2000 and DSPC into mother liquor with a certain concentration, mix the materials according to the volume shown in Table 1, use anhydrous ethanol as the solvent, and fully dissolve under the condition of water bath ultrasonic.

[0039] S2, preparation of mRNA aqueous solution: dissolve 222 μg of Luciferase-mRNA (purchased from Shanghai Hesince Biotechnology Co., Ltd., name: FLUC-L-Cap1AG(N1Ψ), product number: 110209-100) in 3 mL of 50 mM sodium citrate buffer solution with pH = 4.0, configure it to 74 μg / mL, and place it on ice for standby.

[0040] S3, preparation of nucleic acid-lipid nanoparticle system: use a microfluidic synthesis instrument and its matching microfluidic chip to prepare LNP, inject 3 mL of mRNA aqueous solution and 1 mL of carrier material ethanol solution into the left and right injectors respectively, and set the total liquid flow rate to 9 mL / min. The flow rate ratio of the left and right sides is 3:1, the initial discard volume is 0.1 mL, and the end discard volume is 0.05 mL. Collect the prepared nucleic acid-lipid nanoparticle system CHOL / AA-LNP (mRNA). Finally, dilute it with PBS solution so that the ethanol ratio is not more than 0.5%, and ultrafilter the CHOL / AA-LNP (mRNA) to reach an appropriate concentration. Refer to Figure 1 which is a core-shell structure, and can protect the nucleic acid in the structural core.

[0041] Table 1

[0042]

[0043] Comparative Example 1

[0044] As a control experiment of Example 1, the difference is only that: the mother liquor of asiatic acid (AA) is omitted in the system, and the mother liquor of cholesterol is the sum (volume) of the mother liquor of AA and CHOL in Example 1. The nucleic acid-lipid nanoparticle system CHOL-LNP(mRNA) without asiatic acid is finally prepared.

[0045] Example 2

[0046] The difference from Example 1 is only that: the nucleic acid drug Luciferase-mRNA is replaced by OVA-mRNA (purchased from Jiangsu Shenji Biological Technology Co., Ltd., name: OVA mRNA with N1-Me-pUTP(5'CAP), product number: 11016-CAP-1), and the nucleic acid-lipid nanoparticle system containing asiatic acid is finally prepared. The configuration of the carrier material ethanol solution is shown in Table 2.

[0047] Table 2

[0048]

[0049] Comparative Example 2

[0050] As a control experiment of Example 2, the difference is only that: the mother liquor of asiatic acid (AA) is omitted in the system, and the mother liquor of cholesterol is the sum (volume) of the mother liquor of AA and CHOL in Example 2. The nucleic acid-lipid nanoparticle system without asiatic acid is finally prepared.

[0051] Example 3

[0052] The difference from Example 1 is only that: SM-102 is replaced by DLin-MC3-DMA, cholesterol is replaced by sitosterol, and DMG-PEG2000 is replaced by ALC-0159. The configuration of the carrier material ethanol solution is shown in Table 3.

[0053] Table 3

[0054]

[0055] Example 4

[0056] The difference from Example 1 is only that: SM-102 is replaced by ALC-0315, and cholesterol is replaced by ergosterol, oxysterol, and chain sterol with a molar ratio of 1:1:1. The configuration of the carrier material ethanol solution is shown in Table 4.

[0057] Table 4

[0058]

[0059] The following comparative tests were performed on the nucleic acid-lipid nanoparticle system samples of the above examples and comparative examples:

[0060] (1) Particle size and morphology investigation: CHOL-LNP and CHOL / AA-LNP encapsulating Luciferase-mRNA were prepared as in Example 1 and Comparative Example 1. The hydrodynamic size of the lipid nanoparticles was measured using Malvern Zetasizer Nano. The morphological features of the lipid nanoparticles were observed by JEM2100Plus transmission electron microscope. The sample treatment method was as follows: 0.1 mg / mL of the lipid nanoparticles was taken up with a capillary tube, dropped onto a copper grid covered with carbon film, after a while the excess liquid was absorbed with filter paper, then 2% phosphotungstic acid was added for negative staining for 30 s, then the excess staining solution was absorbed with filter paper, and dried under an infrared lamp. The test results are shown in Figure 2 and Figure 3 The average particle size of CHOL-LNP (mRNA) was 127 ± 6.9 nm, and the shape was spherical. The average particle size of CHOL / AA-LNP (mRNA) was 124.4 ± 2.8 nm, and the shape was ellipsoidal.

[0061] In addition, the particle size and morphology of the samples of Examples 2, 3 and 4 were investigated by the same test method as Example 1, and the results were basically the same as Example 1, and the shape was ellipsoidal.

[0062] (2) Encapsulation efficiency was measured by agarose gel method: CHOL-LNP and CHOL / AA-LNP encapsulating Luciferase-mRNA were prepared as in Example 1 and Comparative Example 1. 300 mg of agarose was weighed and dissolved in 30 mL of 1 × TAE buffer, placed in a microwave oven and heated with medium-high fire, heated to micro-boiling, then turned off, and repeated twice. When it cools down to about 60°C, add 3 μL of nucleic acid dye, shake gently to make the color uniform. Then pour it into the gel mold immediately, insert the comb, and after the agarose cools and solidifies, place it in the electrophoresis tank, pour in 1 × TAE solution, and gently pull out the comb. Prepare the sample during the cooling process of the agarose gel. First, adjust the RNA concentration in the LNP to 10 μg / mL with DEPC water, then take 10 μL and add 2 μL of loading buffer as the sample without emulsion breaking. Take another 10 μL and add 2 μL of loading buffer containing Triton X-100 as the sample after emulsion breaking. Each sample was vortexed, then centrifuged to the bottom of the EP tube. After the sample was prepared, the comb was pulled out, and care should be taken not to let the sample float outside the well. After all the samples were loaded, run the gel at a voltage of 120 V for 5 min. After the end, place the agarose gel in a box containing 1 × TAE buffer, and take a picture using a gel imager. The results are shown in Figure 4As shown, if the nucleic acid is completely wrapped in the LNP, no band can be detected, and only after the structure of the LNP is destroyed by Triton X-100 as a demulsifier, the nucleic acid is released, and the band can be detected. If the nucleic acid cannot be completely wrapped in the LNP, the band can be detected without demulsification. Based on this, we found that the replacement of part of the cholesterol with asiatic acid did not affect the encapsulation efficiency of the LNP.

[0063] In addition, the samples of Examples 2, 3 and 4 were subjected to encapsulation rate investigation by the same test method as Example 1, and the results were the same as Example 1, and also did not affect the encapsulation efficiency of the corresponding LNP.

[0064] (3) In vitro transfection efficiency investigation: CHOL-LNP and CHOL / AA-LNP encapsulating Luciferase-mRNA were prepared as in Example 1 and Comparative Example 1. DC2.4 cells were inoculated in a 96-well plate at a concentration of 6x10 3 cells / well and cultured overnight in a cell incubator at 37°C with 5% CO2. When the cells grew to about 80%, the culture medium was carefully aspirated and discarded with a 1 mL syringe. The mRNA concentration of CHOL-LNP (mRNA) and CHOL / AA-LNP (mRNA) was adjusted to 1 μg / mL with complete culture medium, and added to the 96-well plate at a volume of 100 μL / well. After all the addition was completed, the plate was placed in a 37°C incubator. After 12 h of incubation, the plate was removed, the medium was carefully aspirated and discarded with a 1 mL syringe, and then 100 μL of complete culture medium was added and the plate was placed in the incubator for continuous culture. After 12 h, 10 μL of 1.5 mg / mL D-luciferin potassium salt solution was added to each well, and the cell bioluminescence was investigated using a live imaging instrument, and a semi-quantitative analysis was performed. The results are shown in Figure 5 Figure 2, which shows that the transfection level of CHOL / AA-LNP (mRNA) on DC2.4 cells is higher than that of CHOL-LNP (mRNA).

[0065] (4) In vivo transfection efficiency investigation: CHOL-LNP and CHOL / AA-LNP encapsulating Luciferase-mRNA were prepared as in Example 1 and Comparative Example 1. Using an insulin syringe, 100 μL of CHOL-LNP (mRNA) or CHOL / AA-LNP (mRNA) was injected into the thigh muscle of 6-8 week old C57BL / 6 female mice, and the injection was recorded as 0 h. At specific time points (6 h, 24 h and 48 h), the front and back of the mice were photographed by a live imaging instrument. Fifteen minutes before each photographing, 200 μL of D-luciferin potassium salt (15 mg / mL) solution was injected into the mice by intraperitoneal injection. The results are shown in Figure 6As shown, at each time point, the transfection efficiency of CHOL / AA-LNP (mRNA) at the injection site was higher than that of CHOL-LNP (mRNA).

[0066] In addition, the samples of Examples 3 and 4 were subjected to in vivo / in vitro transfection efficiency tests by the same test method as Example 1, and the results were basically the same as Example 1, that is, the in vivo / in vitro transfection efficiency of the nucleic acid-lipid nanoparticle system by replacing part of the structural lipid with asiatic acid was higher than that of the nucleic acid-lipid nanoparticle system without asiatic acid replacement.

[0067] (5) Antigen-specific antibody detection in mouse serum: CHOL-LNP and CHOL / AA-LNP encapsulating OVA-mRNA were prepared as in Example 2 and Comparative Example 2. 6-8 week old female C57BL / 6 mice were randomly divided into 3 groups, 5 in each group, namely PBS group (blank control), CHOL-LNP (mRNA) group and CHOL / AA-LNP (mRNA) group. The mice were immunized by intramuscular injection of the thigh on day 0 and day 14. On day 21, the levels of IgG, IgG2a and IgG1 in the mouse serum were investigated using enzyme-linked immunosorbent assay. The results are shown in Figure 7 As shown, CHOL / AA-LNP (mRNA) not only produced IgG1 antibodies comparable to CHOL-LNP (mRNA), but also significantly increased the secretion of IgG and IgG2a antibodies.

[0068] (6) Specificity CTL killing efficiency investigation: CHOL-LNP and CHOL / AA-LNP encapsulating OVA-mRNA were prepared as in Example 2 and Comparative Example 2. A certain number of blank mice (blank mice: experimental mice = 1:3) were sacrificed, sterilized with 75% ethanol, and then transferred to a cell room super-clean bench. The mouse spleen was dissected and the adherent fur tissue was washed off with PBS. Then the mouse spleen was placed in a corresponding six-well plate (3 mL PBS per well). The mouse spleen was placed on a 70 μm sieve, and the piston rubber head of a 10 mL syringe was used to rapidly grind the spleen under light shielding conditions and pass it through the sieve. After grinding, all the cell suspensions of the mouse spleen were collected in a 50 mL EP tube, centrifuged at 400 g for 4 min, and the supernatant was discarded. A certain amount of RBC lysis solution was added, and after lysis at room temperature for 10 min, the sample was centrifuged at 400 g for 4 min. After centrifugation, the cells were washed once with PBS, and then the cell pellet was resuspended with 20 mL complete medium. Half of the cell suspension was added with SIINFEKL peptide (final concentration 2 μg / mL), and then transferred to a culture dish as target cells. The remaining 10 mL of cell suspension was added with an equal amount of PBS and transferred to another culture dish as internal reference cells. Then the two culture dishes were placed in a 37°C cell incubator with 5% CO2, and after 2 h of incubation, the target cells and internal reference cells were gently blown off and transferred to 50 mL EP tubes, which were centrifuged at 400 g for 4 min, washed once with PBS, and resuspended with 15 mL PBS. The cells were fluorescently labeled with hydroxyfluorescein diacetate succinimidyl ester (CFSE), and the target cells were added with CFSE at a final concentration of 4 μM, and the internal reference cells were added with CFSE at a final concentration of 0.4 μM. The cells were placed in a 37°C cell incubator with 5% CO2 for 20 min, and the EP tube was inverted once during the process. After staining, 8 mL of serum was added to stop the staining. Then the cells were centrifuged at 400 g for 4 min, washed with PBS, and resuspended in 1 mL of PBS. The two types of cells were counted using a cell counter, and their concentrations were adjusted to 5x10 7 6 / mL. They were mixed in equal amounts and injected into mice via the tail vein, with 200 μL injected per mouse. After 20 h, the mouse spleen was removed and prepared into a single cell suspension. The fluorescence intensity of the cells was detected under the FITC channel using a flow cytometer, and the specific killing rate was calculated. The results are shown in Figure 8 Table 2. The number of target cells in the body of unimmunized mice (blank group Control) was basically the same as that of internal reference cells, while the number of target cells in the body of mice immunized with the two types of LNP was greatly reduced, and the specific killing efficiency induced by CHOL / AA-LNP(mRNA) was significantly higher than that by CHOL-LNP(mRNA).

[0069] (7) CHOL / AA-LNP(mRNA) prevents tumor growth effect investigation: CHOL-LNP and CHOL / AA-LNP encapsulating OVA-mRNA were prepared as in Example 2 and Comparative Example 2. 6-8 weeks old female C57BL / 6 mice were randomly divided into 3 groups, 5 mice in each group, namely PBS group (blank group Control), CHOL-LNP(mRNA) group and CHOL / AA-LNP(mRNA) group. The mice were immunized by intramuscular injection of the thigh on day 0 and day 14. The E.G7-OVA cells in logarithmic growth phase were resuspended in sterile PBS, counted by a cell counter, and the concentration was adjusted to 7×10 6 / mL. On day 21, the mice were subcutaneously inoculated with E.G7-OVA cells, and the long diameter L and short diameter W of the mouse tumor were measured using a vernier caliper on the 6th day after inoculation, once every other day, the tumor volume V was calculated, and the tumor growth curve was drawn. The results are shown in Figure 9 CHOL / AA-LNP(mRNA) has a better effect on preventing tumor growth than CHOL-LNP(mRNA).

[0070] (8) CHOL / AA-LNP(mRNA) inhibits tumor growth effect investigation: CHOL-LNP and CHOL / AA-LNP encapsulating OVA-mRNA were prepared as in Example 2 and Comparative Example 2. 15 6-8 weeks old female C57BL / 6 mice were randomly divided into 3 groups, namely PBS group (blank group Control), CHOL-LNP(mRNA) group and CHOL / AA-LNP(mRNA) group. The E.G7-OVA cells in logarithmic growth phase were resuspended in sterile PBS, counted by a cell counter, and the concentration was adjusted to 7×10 6 / mL, the mice were subcutaneously inoculated with E.G7-OVA cells on the back, and the inoculation day was recorded as day 0, and the drug was administered by intramuscular injection on day 3, 7 and 11. The long diameter L and short diameter W of the tumor were recorded during the treatment, the tumor volume V was calculated, and the tumor growth curve was drawn. The results are shown in Figure 10 CHOL / AA-LNP(mRNA) has a better effect on inhibiting tumor growth than CHOL-LNP(mRNA).

[0071] To further prove the beneficial technical effects of the present application, based on the preparation method of Example 1, only by replacing cholesterol (CHOL) with different contents of asiatic acid (AA) (specifically, the molar ratio of cholesterol to asiatic acid = 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 0:10), a series of nucleic acid-lipid nanoparticle systems were prepared for comparative testing: 293T cells were plated at 6×103 The nucleic acid-lipid nanoparticle systems with different AA / cholesterol ratios (molar ratio, cholesterin:AA=10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 0:10) were diluted with the serum-containing medium, in which the concentration of Luciferase-mRNA was adjusted to 1 μg / mL, and added to the 96-well plate in an amount of 100 μL / well. After all the addition, the plate was placed in a cell incubator at 37 °C containing 5% CO2for culture. After 12 h, the medium was aspirated and discarded with a 1 mL syringe, and then 100 μL of complete medium was added to each well for continued culture. After 12 h, 10 μL of 1.5 mg / mL D-luciferin potassium salt solution was added to each well, and the cell bioluminescence was observed using a live imaging instrument. The experimental results showed that the luminescence intensity presented a trend of first increasing and then decreasing, and the luminescence intensity was the highest when 10% of the cholesterin was replaced by asiatic acid (cholesterin:AA=9:1), indicating that the prepared nucleic acid-lipid nanoparticle system had the highest transfection efficiency on 293T cells, and the luminescence intensity of the system in which 100% of the cholesterin was replaced by asiatic acid (cholesterin:AA=0:10) was still higher than that of the system without asiatic acid replacing cholesterin (cholesterin:AA=10:0).

[0072] Although the specific embodiments of the present application are described above, those skilled in the art should understand that these are only examples, and various changes or modifications can be made to the embodiments without departing from the principles and essence of the present application, and the protection scope of the present application is only defined by the appended claims.

Claims

1. Use of a nucleic acid-lipid nanoparticle system in the manufacture of a medicament for the prevention or treatment of cancer, characterized in that, The nucleic acid-lipid nanoparticle system has an ellipsoidal core-shell structure, and is composed of a nucleic acid drug encapsulated by a lipid nanoparticle; The lipid nanoparticle is prepared from ionizable cationic lipids, structural lipids, neutral lipids, PEG lipids and asiatic acid at a molar ratio of 10:1.5:3.85:50:34.

65. 10:1.5:3.85:50:34.

65. The structural lipids are selected from cholesterols, the cationic lipids are octadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, the neutral lipids are 1,2-distearoyl-sn-glycerol-3-phosphocholine, and the PEG lipids are 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000; the nucleic acid drug is selected from OVA-mRNA; and the cancer is T lymphoma.

2. Use according to claim 1, characterized in that, The preparation method of the lipid nanoparticle comprises: dissolving ionizable cationic lipids, structural lipids, neutral lipids, PEG lipids and asiatic acid in ethanol to obtain an ethanol solution, and formulating an LNP suspension, then diluting with an aqueous medium, and purifying and concentrating by dialysis or ultrafiltration to obtain asiatic acid-based lipid nanoparticles.

3. Use according to claim 2, characterized in that, The preparation method of the nucleic acid-lipid nanoparticle system comprises: mixing an ethanol solution obtained by dissolving ionizable cationic lipids, structural lipids, neutral lipids, PEG lipids and asiatic acid in ethanol with a sodium citrate buffer solution with pH=4.0±0.2 in which a nucleic acid drug is dissolved to form an LNP suspension, then diluting with an aqueous medium, and purifying and concentrating by dialysis or ultrafiltration to obtain a nucleic acid-lipid nanoparticle system in which a nucleic acid drug is encapsulated by a lipid nanoparticle.

4. Use according to claim 3, characterized in that, The nitrogen-phosphorus ratio of the lipid nanoparticle and the nucleic acid drug is 1:10-10:

1.

5. Use according to claim 2 or 3, characterized in that, The LNP suspension is formulated by a rapid mixing method or a microfluidic synthesis method.

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