Lipid nanoparticles based on asiatic acid as well as preparation method and application of lipid nanoparticles

Abstract

The invention relates to the technical field of nucleic acid medicine preparations, and discloses lipid nanoparticles based on asiatic acid and a preparation method and application thereof.The lipid nanoparticles are composed of ionizable cationic lipid, structural lipid, neutral lipid, PEG lipid and asiatic acid according to the molar ratio of (10-85): (20-60): (1-30): (0.1-10): (0.1-50) and serve as delivery carriers of nucleic acid medicine. For delivery of nucleic acid drugs. According to the invention, a natural product asiatic acid is creatively adopted to partially replace a structural lipid (such as cholesterol) in a traditional LNP formula, and the micromorphology of the LNP is obviously optimized, so that the in-vivo and in-vitro transfection and expression efficiency of mRNA is greatly enhanced. The mRNA vaccine constructed based on the vector can induce an organism to generate a high-titer specific antibody, and the killing activity of cytotoxic T lymphocytes (CTL) is remarkably enhanced. The LNP system has outstanding performance in the field of tumor immunoprophylaxis and treatment, and shows excellent transformation application potential and wide market prospect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, specifically to a lipid nanoparticle based on asiatic acid, its preparation method, and its application. Background Technology

[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 suppressor factors, and can even simultaneously encode multiple target proteins. Compared with traditional methods, mRNA cancer vaccines have multiple advantages: antigen specificity, sustained immune response, fewer side effects, rapid production, and potential for personalized treatment. However, the large single-stranded structure and negative charge of mRNA molecules themselves result in a short half-life in the 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 (LNPs) represent state-of-the-art mRNA delivery systems, with three RNA-based LNPs already approved by the U.S. Food and Drug Administration (FDA): Onpattro®, Comirnaty®, and Spikevax®. The successful application of LNPs is attributed to their advantages: protection of mRNA from nucleases, promotion of tissue targeting and cellular internalization, and facilitation of mRNA escape from endosomes into the cytosol, where it is translated into encoded proteins. Some of these proteins are taken up by antigen-presenting cells (APCs) and activate cytotoxic T cells (CTLs) and helper T cells (Th cells) via the MHC-I and MHC-II pathways, respectively, inducing anti-tumor immunity. B cells, upon recognizing the antigen, are activated and differentiate into plasma cells with the help of Th cells, producing neutralizing antibodies. LNP components are typically biocompatible and degradable, and some possess adjuvant activity, enhancing cellular and humoral immunity. They can also be used to deliver immunomodulators to further enhance the immune response.

[0004] Typical LNP formulations contain ionizable cationic lipids, neutral lipids, structural lipids, and PEG lipids, each with a specific function. Ionizable cationic lipids are a crucial component of LNPs, containing ionizable amino groups (typically tertiary amines) in their head groups. The pH-responsiveness of ionizable lipids allows LNPs to maintain a neutral charge in physiological environments, thus circumventing charge-driven clearance by the reticuloendothelial system (RES) and prolonging circulation time. Simultaneously, they carry a positive charge in weakly acidic environments, thereby disrupting endosome membranes and promoting endosome escape. PEG lipids are another key component of LNPs, stabilizing them, preventing aggregation and fusion, and prolonging circulation time. Neutral lipids maintain LNP stability, regulate LNP size, and improve LNP biocompatibility. Structural lipids, such as cholesterol, are abundant in LNPs, filling gaps in the lipid bilayer, regulating membrane integrity and rigidity, and increasing LNP stability. Optimizing the composition of LNPs is crucial to addressing the unique requirements of specific applications.

[0005] LNPs enhance their nucleic acid delivery capabilities by leveraging their hydrophobic similarity to cell membranes. Furthermore, compared to naked mRNA, mRNA encapsulated in LNPs exhibits greater stability, minimizing nuclease degradation. Nucleotide modifications pioneered by Kariko and Weissman further improve stability. Despite these significant advancements, LNP delivery efficiency remains less than ideal in certain situations, particularly in the acidic environment of the endosomal environment, where insufficient mRNA release from LNPs limits their effective intracellular delivery. Zerial et al. demonstrated that less than 2% of the siRNA or mRNA delivered by LNPs enters the cytoplasm. Therefore, optimizing LNP formulations to improve mRNA transfection efficiency has become a key issue in the current industrialization of mRNA vaccines. Summary of the Invention

[0006] To address the technical problems of insufficient mRNA release efficiency and transfection efficiency of existing LNP systems in vivo, this invention provides lipid nanoparticles based on asiatic acid to replace part of the structural lipids, as well as their preparation method and application.

[0007] To achieve the above objectives, in a first aspect, the present invention provides lipid nanoparticles based on asiatic acid. These lipid nanoparticles are prepared from ionizable cationic lipids, structural lipids, neutral lipids, PEG lipids, and asiatic acid in a molar ratio of 10-85:20-60:1-30:0.1-10:0.1-50. The structural lipids are selected from one or more of cholesterol, ergosterol, campesterol, oxostosterol, sterol, sitosterol, and stigmasterol. These lipid nanoparticles can serve as delivery carriers for nucleic acid drugs.

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

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

[0010] As a further preferred embodiment of the present invention, the ionizable cationic lipid is selected from one or more of the following: ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), octadecano-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl) (DLin-MC3-DMA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-bisoctadecenoxy-3-methylammonium propane (chloride) (DOTMA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 1,2-dioleoyl-3-dimethylaminopropane (DODMA), and (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP). And / or, the neutral lipid is selected from 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC), lecithinylcholine (EPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-glycerol sodium salt (DOPG), 1,2-dilauroyl-sn-glycerol-3-phosphate choline (DLPC), 1 One or more of the following: 2-dimyristoyl-sn-3-phosphocholine (DMPC), distearate phosphatidylethanolamine (DSPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycerol-3-phospho-L-serine (DOPS), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC); And / or, the PEG lipid is selected from one or more of 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), 1,2-distearyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (DSG-PEG2000), 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (DSA-PEG2000), and 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159).

[0011] According to a second aspect of the present invention, the present invention also provides a method for preparing lipid nanoparticles based on asiatic acid, wherein an ethanol solution is obtained by dissolving ionizable cationic lipids, structural lipids, neutral lipids, PEG lipids and asiatic acid in ethanol, and then preparing an LNP suspension, which is then diluted with an aqueous medium and purified and concentrated by dialysis or ultrafiltration to obtain lipid nanoparticles.

[0012] As a further preferred technical solution of the present invention, nucleic acid drugs are also added to the system. Specifically, an 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 containing nucleic acid drugs at pH=4.0±0.2 to form an LNP suspension. The suspension is then diluted with an aqueous medium and purified and concentrated by dialysis or ultrafiltration to obtain a nucleic acid-lipid nanoparticle system in which nucleic acid drugs are encapsulated by lipid nanoparticles.

[0013] As a further preferred embodiment of the present invention, the lipid nanoparticle system has an average particle size of 50-150 nm and exhibits an ellipsoidal core-shell structure. In this core-shell structure, the outer shell is formed by neutral lipids, PEG lipids, partially ionizable cationic lipids, partially structural lipids, and partially asiatic acid. Inside the shell, the nucleic acid drug is encapsulated by partially ionizable cationic lipids, partially structural lipids, and partially asiatic acid to form a core.

[0014] As a further preferred technical solution of the present invention, the nucleic acid drug is selected from any one of RNA drugs, DNA drugs, and plasmids, preferably RNA drugs, and more preferably siRNA and mRNA; and / or, the nitrogen-phosphorus ratio of lipid nanoparticles and nucleic acid drugs is 1:10-10:1, and even more preferably 3:1-8:1.

[0015] As a further preferred technical solution of the present invention, the LNP suspension is prepared by rapid mixing method or microfluidic synthesis method.

[0016] According to a third aspect of the present invention, the present invention also provides the application of a nucleic acid-lipid nanoparticle system in the preparation of medicaments for the prevention or treatment of infectious diseases, cancer, and diabetes.

[0017] Compared with the prior art, the present invention can achieve the following beneficial effects: This invention innovatively uses asiatic acid, a natural product, to partially replace structural lipids (such as cholesterol) in traditional LNP formulations, significantly optimizing its microstructure and thereby greatly enhancing the transfection and expression efficiency of mRNA in vivo and in vitro. The mRNA vaccine constructed based on this vector can induce the body to produce high-titer specific antibodies and significantly enhance the cytotoxic T lymphocyte (CTL) killing activity. This LNP system demonstrates outstanding performance in the field of tumor immunoprophylaxis and therapy, showing excellent translational application potential and broad market prospects. Attached Figure Description

[0018] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0019] Figure 1 This is a schematic diagram of the CHOL / AA-LNP structure containing mRNA prepared in Example 1.

[0020] Figure 2 The particle size distributions of CHOL-LNP and CHOL / AA-LNP, which are loaded with mRNA and prepared in Example 1 and Comparative Example 1, are shown in Figure A, which is a representative particle size distribution of CHOL-LNP, and Figure B is a representative particle size distribution of CHOL / AA-LNP.

[0021] Figure 3 Electron micrographs of CHOL-LNP and CHOL / AA-LNP containing mRNA prepared in Example 1 and Comparative Example 1 are shown, where A is an electron micrograph of CHOL-LNP and B is an electron micrograph of CHOL / AA-LNP.

[0022] Figure 4 The encapsulation efficiency of CHOL-LNP and CHOL / AA-LNP containing mRNA prepared for Example 1 and Comparative Example 1 was investigated.

[0023] Figure 5 Cellular transfection levels of CHOL-LNP and CHOL / AA-LNP loaded with mRNA prepared in Example 1 and Comparative Example 1, where A is a bioluminescence graph and B is a semi-quantitative bioluminescence result.

[0024] Figure 6Animal transfection levels of CHOL-LNP and CHOL / AA-LNP carrying mRNA prepared in Example 1 and Comparative Example 1, where A is an in vivo imaging image, B is a semi-quantitative result from the front, and C is a semi-quantitative result from the back.

[0025] Figure 7 The levels of antigen-specific antibodies induced in mice by CHOL-LNP and CHOL / AA-LNP loaded with mRNA prepared in Example 2 and Comparative Example 2 are shown in Figure 2. A represents the anti-OVA IgG antibody titer in the serum of mice in each group on day 21, B represents the anti-OVA IgG2a antibody titer in the serum of mice in each group on day 21, and C represents the anti-OVA IgG1 antibody titer in the serum of mice in each group on day 21.

[0026] Figure 8 The specific killing efficiency of CHOL-LNP and CHOL / AA-LNP loaded with mRNA prepared in Example 2 and Comparative Example 2 was investigated, where A is the relative specific killing efficiency and B is a representative flow cytometry chromatogram.

[0027] Figure 9 The tumor prevention efficacy of CHOL-LNP and CHOL / AA-LNP, which are loaded with mRNA and prepared in Example 2 and Comparative Example 2, was evaluated. In this example, A is the tumor growth curve of the PBS group, B is the tumor growth curve of the CHOL-LNP group, and C is the tumor growth curve of the CHOL / AA-LNP group.

[0028] Figure 10 The efficacy of CHOL-LNP and CHOL / AA-LNP, which are loaded with mRNA and prepared in Example 2 and Comparative Example 2, in treating tumors was evaluated. In this example, A is the tumor growth curve of the PBS group, B is the tumor growth curve of the CHOL-LNP group, and C is the tumor growth curve of the CHOL / AA-LNP group.

[0029] The objectives, features, and advantages of this invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0030] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0031] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which this invention pertains. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0032] The nucleic acid-lipid nanoparticle system of the present invention, based on the substitution of part of the structural lipids with asiatic acid, is abbreviated as CHOL / AA-LNP (NC), where NC is the nucleic acid drug, CHOL is cholesterol, AA is asiatic acid, and LNP is the lipid nanoparticle. The nucleic acid-lipid nanoparticle system based on traditional cholesterol lipid nanoparticles is abbreviated as CHOL-LNP (NC).

[0033] Example 1: The molar ratio of CHOL to AA is 9:1 (10% of CHOL is replaced with AA). This embodiment provides a microfluidic synthesis method for preparing CHOL / AA-LNP (mRNA), as detailed below: S1. Prepare the carrier material ethanol solution: First, take appropriate amounts of SM-102, asiatic acid (AA), cholesterol (CHOL), DMG-PEG2000 and DSPC and accurately prepare them into stock solutions with a certain concentration with anhydrous ethanol. Mix the materials according to the volume shown in Table 1, use anhydrous ethanol as solvent, and fully dissolve them under water bath ultrasonic conditions.

[0034] S2, Prepare mRNA aqueous solution: Dissolve 222 μg of Luciferase-mRNA (purchased from Shanghai Hexincheng Biotechnology Co., Ltd., name: FLUC-L-Cap1AG(N1Ψ), catalog number: 110209-100) in 3 mL of 50 mM sodium citrate buffer solution at pH=4.0 to prepare a solution of 74 μg / mL, and keep it on ice for later use.

[0035] S3, Preparation of the Nucleic Acid-Lipid Nanoparticle System: LNPs were prepared using a microfluidic synthesis instrument and its matching microfluidic chip. 3 mL of mRNA aqueous solution and 1 mL of carrier material ethanol solution were added to the left and right syringes, respectively, with a total flow rate of 9 mL / min. The flow rate ratio between the left and right sides was 3:1. The initial discard volume was 0.1 mL, and the final discard volume was 0.05 mL. The prepared nucleic acid-lipid nanoparticle system CHOL / AA-LNP (mRNA) was collected. Finally, it was diluted with PBS solution to a concentration not exceeding 0.5% ethanol, and ultrafiltration was performed to achieve an appropriate concentration of CHOL / AA-LNP (mRNA). See also... Figure 1 It has a core-shell structure, which can protect nucleic acids in the structural core.

[0036] Table 1

[0037] Comparative Example 1 As a control experiment of Example 1, the only difference is that the asiatic acid (AA) stock solution was omitted in the system, and the cholesterol stock solution was the sum of the total amount (volume) of the AA and CHOL stock solutions in Example 1. Finally, a nucleic acid-lipid nanoparticle system CHOL-LNP (mRNA) without asiatic acid was prepared.

[0038] Example 2 The only difference from Example 1 is that the nucleic acid drug Luciferase-mRNA was replaced with OVA-mRNA (purchased from Jiangsu Shenji Biotechnology Co., Ltd., name: OVA mRNA with N1-Me-pUTP(5'CAP), catalog number: 11016-CAP-1), and the final preparation of a nucleic acid-lipid nanoparticle system containing asiatic acid was obtained. The preparation of the ethanol solution of the carrier material is shown in Table 2.

[0039] Table 2

[0040] Comparative Example 2 As a control experiment of Example 2, the only difference is that the asiatic acid (AA) stock solution was omitted in the system, and the cholesterol stock solution was the sum of the total amount (volume) of the AA and CHOL stock solutions in Example 2. Finally, a nucleic acid-lipid nanoparticle system without asiatic acid was prepared.

[0041] Example 3 The only differences from Example 1 are: SM-102 is replaced with DLin-MC3-DMA, cholesterol is replaced with sitosterol, and DMG-PEG2000 is replaced with ALC-0159. The preparation of the ethanol solution of the carrier material is shown in Table 3.

[0042] Table 3

[0043] Example 4 The only difference from Example 1 is that SM-102 is replaced with ALC-0315, and cholesterol is replaced with ergosterol, oxosteroside, and sterol in a molar ratio of 1:1:1. The preparation of the ethanol solution of the carrier material is shown in Table 4.

[0044] Table 4

[0045] The following comparative tests were performed on the nucleic acid-lipid nanoparticle system samples from the above embodiments and comparative examples: (1) Particle size and morphology: CHOL-LNP and CHOL / AA-LNP loaded with Luciferase-mRNA were prepared as in Example 1 and Comparative Example 1. The hydrodynamic size of the lipid nanoparticles was measured using a Malvern Zetasizer Nano. The morphological characteristics of the lipid nanoparticles were observed using a JEM2100Plus transmission electron microscope. The sample preparation method was as follows: 0.1 mg / mL of lipid nanoparticles were aspirated with a capillary tube and dropped onto a copper sieve coated with a carbon film. After a short while, the excess liquid was blotted off with filter paper. Then, 2% phosphotungstic acid was added for negative staining for 30 s. The excess staining solution was then blotted off with filter paper and dried under an infrared lamp. The test results are shown in [reference]. Figure 2 and Figure 3 The average particle size of CHOL-LNP (mRNA) is 127 ± 6.9 nm, and its shape is spherical. The average particle size of CHOL / AA-LNP (mRNA) is 124.4 ± 2.8 nm, and its shape is ellipsoidal.

[0046] Furthermore, the particle size and morphology of the samples from Examples 2, 3, and 4 were examined using the same testing methods as in Example 1, and the results were basically the same as in Example 1, with all samples showing an ellipsoidal shape.

[0047] (2) Encapsulation efficiency was determined by agarose gel electrophoresis: CHOL-LNP and CHOL / AA-LNP encapsulated with 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. The solution was placed in a microwave oven and heated on medium-high heat until it just began to boil. The heat was then turned off and the heating was repeated twice. When the solution cooled to about 60°C, 3 μL of nucleic acid dye was added and the mixture was gently shaken to ensure uniform color. The solution was then quickly poured into a gel mold, and a comb was immediately inserted. After the agarose solidified, the solution was placed in an electrophoresis tank, 1×TAE solution was poured in, and the comb was gently removed. While waiting for the agarose gel to cool, the sample was prepared. First, the RNA concentration in the LNP was adjusted to 10 μg / mL with DEPC water. Then, 10 μL of the solution was added to 2 μL of loading buffer as the unbroken sample. Take another 10 μL and add 2 μL of loading buffer containing Triton X-100 as the demulsified sample. Vortex each sample to mix well, then centrifuge to the bottom of the EP tube. After the samples are prepared, remove the comb, taking care not to let the sample float outside the wells during loading. After all samples have been loaded, run the gel at 120 V for 5 min. After completion, place the agarose gel in a box containing 1×TAE buffer and image it using a gel imaging system. The results are as follows. Figure 4As shown, if the nucleic acid is completely encapsulated in the LNP, no band can be detected. Only after using Triton X-100 as a demulsifier to disrupt the structure of the LNP and release the nucleic acid can the band be detected. If the nucleic acid is not completely encapsulated in the LNP, the band can be detected without demulsification. Based on this, to determine the encapsulation of nucleic acid by the LNP, we found that replacing part of the cholesterol with asiatic acid does not affect the encapsulation efficiency of the LNP.

[0048] Furthermore, the encapsulation efficiency of the samples from Examples 2, 3, and 4 was examined using the same test method as in Example 1, and the results were the same as in Example 1, showing that the encapsulation efficiency of the corresponding LNPs was not affected.

[0049] (3) In vitro transfection efficiency assessment: CHOL-LNP and CHOL / AA-LNP loaded with Luciferase-mRNA were prepared as in Example 1 and Comparative Example 1. DC2.4 cells were transfected at a rate of 6 × 10⁻⁶ mRNAs. 3 Cells were seeded at a rate of 1 cell / well in 96-well plates and cultured overnight at 37°C in a cell culture incubator containing 5% CO2. When the cells reached approximately 80% confluence, the culture medium was carefully aspirated and discarded using a 1 mL syringe. The mRNA concentrations of CHOL-LNP (mRNA) and CHOL / AA-LNP (mRNA) were adjusted to 1 μg / mL using complete culture medium, and 100 μL was added to each well of the 96-well plate. After all the medium was added, the plates were placed in a 37°C incubator. After 12 h of incubation, the plates were removed, the culture medium was carefully aspirated and discarded using a 1 mL syringe, and 100 μL of complete culture medium was added again. The plates were then placed back in the incubator. After 12 h, 10 μL of 1.5 mg / mL D-fluorescein potassium solution was added to each well, and the bioluminescence of the cells was observed using a live imaging system, followed by semi-quantitative analysis. The results are as follows: Figure 5 As shown, the transfection level of CHOL / AA-LNP (mRNA) in DC2.4 cells was higher than that of CHOL-LNP (mRNA).

[0050] (4) In vivo transfection efficiency assessment: CHOL-LNP and CHOL / AA-LNP loaded with Luciferase-mRNA were prepared as in Example 1 and Comparative Example 1. 100 μL of CHOL-LNP (mRNA) or CHOL / AA-LNP (mRNA) was injected into the thigh muscles of 6-8 week old female C57BL / 6 mice using an insulin injector. The end of the injection was recorded as 0 h. Frontal and back images of the mice were taken at specific time points (6 h, 24 h, and 48 h) using a live imaging system. 15 min before each image was taken, 200 μL of D-fluorescein potassium salt (15 mg / mL) solution was injected intraperitoneally into the mice. Results are as follows: 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).

[0051] In addition, the in vivo / in vitro transfection efficiency of the samples in Examples 3 and 4 was tested using the same test method as in Example 1. The results were basically the same as in Example 1, that is, the in vivo / in vitro transfection efficiency of the nucleic acid-lipid nanoparticle system with partial replacement of structural lipids by asiatic acid was higher than that of the nucleic acid-lipid nanoparticle system without replacement of structural lipids by asiatic acid.

[0052] (5) Detection of antigen-specific antibodies in mouse serum: CHOL-LNP and CHOL / AA-LNP loaded with OVA-mRNA were prepared as in Example 2 and Comparative Example 2. Six- to eight-week-old female C57BL / 6 mice were randomly divided into three groups of five mice each: PBS group (Control group), CHOL-LNP (mRNA) group, and CHOL / AA-LNP (mRNA) group. Mice were immunized by intramuscular injection in the thigh on days 0 and 14. On day 21, enzyme-linked immunosorbent assay (ELISA) was used to examine the levels of IgG, IgG2a, and IgG1 in mouse serum. Results are as follows: Figure 7 As shown, CHOL / AA-LNP (mRNA) can not only produce IgG1 antibodies comparable to CHOL-LNP (mRNA), but also significantly enhance the secretion of IgG and IgG2a antibodies.

[0053] (6) Specific CTL killing efficiency: CHOL-LNP and CHOL / AA-LNP loaded with 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 taken, sacrificed, and transferred to a clean bench in the cell culture room after disinfection with 75% ethanol. The spleens of the mice were dissected and washed with PBS to remove the adhering fur tissue. Then, they were placed in six-well plates with corresponding numbers (3 mL PBS per well). The mouse spleens were placed on a 70 μm sieve and rapidly ground under light-protected conditions using a 10 mL syringe plunger rubber tip, allowing them to pass through the sieve. After grinding, the cell suspensions of all mouse spleens were collected into a 50 mL EP tube, centrifuged at 400 g for 4 min, and the supernatant was discarded. A certain amount of RBC lysis buffer was added, and the cells were lysed at room temperature for 10 min, followed by centrifugation at 400 g for 4 min. After centrifugation, the cells were washed once with PBS, then resuspended in 20 mL of complete culture medium. Half of the cell suspension was added to SIINFEKL peptide (final concentration 2 μg / mL) and transferred to a culture dish as target cells. The remaining 10 mL of cell suspension was added to an equal volume of PBS and transferred to another culture dish as internal control cells. Both culture dishes were then placed in a cell culture incubator at 37°C with 5% CO2 and incubated for 2 h. After incubation, the target cells and internal control cells were gently aspirated and transferred to 50 mL EP tubes, centrifuged at 400g for 4 min, washed once with PBS, and resuspended in 15 mL of PBS. Cells were fluorescently labeled with fluorescein diacetate succinimide (CFSE). Target cells were added to a final concentration of 4 μM CFSE, and internal control cells were added to a final concentration of 0.4 μM CFSE. Cells were stained in a cell culture incubator at 37°C with 5% CO2 for 20 min, inverting the EP tube once during staining. After staining, 8 mL of serum was added to stop the staining process. Then, the cells were centrifuged at 400 g for 4 min, the cell pellet was collected, washed with PBS, and resuspended in 1 mL of PBS. The cell counts of both cell types were performed using a cell counter, and their concentrations were adjusted to 5 × 10⁻⁶ cells / mL. 7 Cells / mL. Equal volumes were mixed and injected into mice via the tail vein, 200 μL per mouse. After 20 h, the mouse spleens were harvested, and a single-cell suspension was prepared. The fluorescence intensity of the cells was detected using flow cytometry in the FITC channel, and the specific killing rate was calculated. Results are as follows: Figure 8 As shown, in unimmunized mice (Control group), the number of target cells and internal control cells in vivo was basically the same; while in mice immunized with the two LNPs, the number of target cells in vivo was greatly reduced, and the specific killing efficiency induced by CHOL / AA-LNP (mRNA) was significantly higher than that of CHOL-LNP (mRNA).

[0054] (7) Investigation of the anti-tumor growth effect of CHOL / AA-LNP (mRNA): CHOL-LNP and CHOL / AA-LNP loaded with OVA-mRNA were prepared as in Example 2 and Comparative Example 2. Six- to eight-week-old female C57BL / 6 mice were randomly divided into three groups of five mice each: PBS group (Control group), CHOL-LNP (mRNA) group, and CHOL / AA-LNP (mRNA) group. Mice were immunized by intramuscular injection in the thigh on days 0 and 14. E.G7-OVA cells in the logarithmic growth phase were taken, resuspended in sterile PBS, counted using a cell counter, and their concentration was adjusted to 7 × 10⁻⁶. 6 / mL. On day 21, E.G7-OVA cells were subcutaneously injected into the backs of mice. On day 6 after inoculation, the major axis (L) and minor axis (W) of the mouse tumors were measured using calipers. Measurements were taken every other day, and the tumor volume (V) was calculated. Tumor growth curves were then plotted. The results are as follows: Figure 9 As shown, CHOL / AA-LNP (mRNA) has a better effect in preventing tumor growth compared with CHOL-LNP (mRNA).

[0055] (8) Investigation of the inhibitory effect of CHOL / AA-LNP (mRNA) on tumor growth: CHOL-LNP and CHOL / AA-LNP loaded with OVA-mRNA were prepared as in Example 2 and Comparative Example 2. Fifteen 6-8 week old female C57BL / 6 mice were randomly divided into three groups: PBS group (Control group), CHOL-LNP (mRNA) group, and CHOL / AA-LNP (mRNA) group. E.G7-OVA cells in the logarithmic growth phase were taken, resuspended in sterile PBS, counted using a cell counter, and their concentration was adjusted to 7×10⁻⁶. 6 / mL, E.G7-OVA cells were subcutaneously injected into the back of mice, with the day of injection recorded as day 0. The cells were administered intramuscularly on days 3, 7, and 11. During treatment, the long axis (L) and short axis (W) of the tumor were recorded, the tumor volume (V) was calculated, and tumor growth curves were plotted. Results are as follows: Figure 10 As shown, CHOL / AA-LNP (mRNA) is more effective at inhibiting tumor growth than CHOL-LNP (mRNA).

[0056] To further demonstrate the beneficial technical effects of the present invention, based on the preparation method of Example 1, a series of nucleic acid-lipid nanoparticle systems were prepared by partially replacing cholesterol (CHOL) with different amounts 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) for comparative testing: 293T cells were prepared at a ratio of 6 × 10⁻⁶...3 Cells were seeded at a rate of 1 cell per well in 96-well plates and cultured overnight at 37 °C in a cell culture incubator containing 5% CO2. When the cells reached approximately 80% confluence, the culture medium was carefully aspirated and discarded using a 1 mL syringe. Nucleic acid-lipid nanoparticle systems with different AA / cholesterol ratios (molar ratios, cholesterol:AA = 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 0:10) were diluted with serum-containing medium, with the concentration of Luciferase-mRNA adjusted to 1 μg / mL. 100 μL of this diluted solution was added to each well of the 96-well plate. After all the cells were added, the plates were incubated at 37 °C in a cell culture incubator containing 5% CO2. After 12 h, the culture medium was aspirated and discarded using a 1 mL syringe. 100 μL of complete culture medium was then added to each well for further incubation. After another 12 h, 10 μL of 1.5 mg / mL D-fluorescein potassium solution was added to each well, and the bioluminescence of the cells was observed using a live imaging system. The experimental results showed that the luminescence intensity first increased and then decreased. The luminescence intensity was the highest when asiatic acid replaced 10% of cholesterol (cholesterol:AA=9:1), indicating that the prepared nucleic acid-lipid nanoparticle system had the highest transfection efficiency on 293T cells. Moreover, the luminescence intensity of asiatic acid replacing 100% of cholesterol (cholesterol:AA=0:10) was still higher than that of not using asiatic acid to replace cholesterol (cholesterol:AA=10:0).

[0057] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. The scope of protection of the present invention is defined only by the appended claims.

Claims

1. A lipid nanoparticle based on asiatic acid, characterized in that, The lipid nanoparticles were prepared from ionizable cationic lipids, structural lipids, neutral lipids, PEG lipids and asiatic acid in a molar ratio of 10-85:20-60:1-30:0.1-10:0.1-50. The structural lipids are selected from one or more of cholesterol, ergosterol, campesterol, oxysterol, sterol, sitosterol, and stigmasterol.

2. The lipid nanoparticles based on asiatic acid according to claim 1, characterized in that, The molar ratio of the ionizable cationic lipids, structural lipids, neutral lipids, PEG lipids and asiatic acid is 45-55:20-50:5-15:0.1-5:0.1-20.

3. The lipid nanoparticles based on asiatic acid according to claim 1, characterized in that, The molar ratio of the ionizable cationic lipid, structural lipid, neutral lipid, PEG lipid, and asiatic acid is 50:34.65: 10：1.5：3.85。 4. The lipid nanoparticles based on asiatic acid according to any one of claims 1-3, characterized in that, The ionizable cationic lipid is selected from one or more of the following: ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), octadecano-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester, 1,2-dioleoyl-3-dimethylammonium-propane, 1,2-bisoctadecenooxy-3-methylammonium propane (chloride), 1,2-dilinoleyloxy-3-dimethylaminopropane, 1,2-dioleoyl-3-dimethylaminopropane, and (2,3-dioleoyl-propyl)-trimethylammonium chloride; And / or, the neutral lipid is selected from one or more of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-distearyl-sn-glycerol-3-phosphate choline, lecithinylcholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-glycerol sodium salt, 1,2-dilauroyl-sn-glycerol-3-phosphate choline, 1,2-dimyristoyl-sn-3-phosphate choline, distearylphosphatidylethanolamine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine, 1,2-dioleoyl-sn-glycerol-3-phosphate choline, and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline; And / or, the PEG lipid is selected from one or more of 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000, 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000], 1,2-distearyl-rac-glycerol-3-methoxy polyethylene glycol 2000, 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000], and 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide.

5. A method for preparing lipid nanoparticles based on asiatic acid according to any one of claims 1-4, characterized in that, Ionizable cationic lipids, structural lipids, neutral lipids, PEG lipids, and asiatic acid were dissolved in ethanol to obtain an ethanol solution, which was then prepared into an LNP suspension. The suspension was then diluted with an aqueous medium and purified and concentrated by dialysis or ultrafiltration to obtain lipid nanoparticles.

6. The preparation method according to claim 5, characterized in that, An ethanol solution containing ionizable cationic lipids, structural lipids, neutral lipids, PEG lipids, and asiatic acid was mixed with a sodium citrate buffer solution containing nucleic acid drugs at pH 4.0 ± 0.2 to prepare an LNP suspension. The suspension was then diluted with an aqueous medium and purified and concentrated by dialysis or ultrafiltration to obtain a nucleic acid-lipid nanoparticle system in which nucleic acid drugs are encapsulated by lipid nanoparticles.

7. The preparation method according to claim 6, characterized in that, The nucleic acid-lipid nanoparticle system has an ellipsoidal core-shell structure with an average particle size of 50-150 nm.

8. The preparation method according to claim 6, characterized in that, The nucleic acid drug is selected from any one of RNA drugs, DNA drugs, and plasmids, and the nitrogen-to-phosphorus ratio of the lipid nanoparticles and the nucleic acid drug is 1:10-10:

1.

9. The preparation method according to claim 6, characterized in that, LNP suspensions were prepared using either rapid mixing or microfluidic synthesis methods.

10. The use of the nucleic acid-lipid nanoparticle system according to any one of claims 6-9 in the preparation of medicaments for the prevention or treatment of infectious diseases, cancer, and diabetes.

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