Preparation method and application of ionizable cationic lipid

Abstract

The invention belongs to the technical field of biological medicine, and discloses a preparation method of ionizable cationic lipid. The preparation method comprises the following steps: reacting 7-oxoheptyl caprate with hydroxylamine or hydroxylamine salt in the presence of alkali in the presence of a reducing agent and a cobalt-ruthenium alloy catalyst; (9Z, 12Z)-octadecane-9, 12-dienal is added into the reaction product, the reaction continues in the presence of a reducing agent, and a hydroxylamine derivative intermediate is obtained; and performing amidation reaction with 4-dimethylaminobutyric acid or hydrochloride thereof in the presence of a condensing agent and a catalyst to obtain the ionizable cationic lipid. The invention also discloses an application of the ionizable cationic lipid, the lipid nanoparticle composition or the mRNA-loaded lipid nanoparticles obtained by the preparation method in preparation of a pharmaceutical composition or a kit for mRNA delivery. The LNP provided by the invention has good physicochemical properties, biocompatibility and efficient mRNA encapsulation and intracellular delivery capabilities, and can be applied to preparation of pharmaceutical compositions or kits for mRNA delivery.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a preparation method of ionizable cationic lipids and application thereof. BACKGROUND

[0002] Messenger RNA (mRNA) as a new form of biological macromolecular drugs has shown great potential in the fields of infectious disease prevention (vaccine), tumor immunotherapy and protein replacement therapy. However, the mRNA molecule itself has challenges in physical and chemical properties, including large molecular weight, carrying negative charge, and being easily degraded by nucleases widely existing in the body, which limits its direct application in the body in terms of efficiency and effect. Therefore, developing a safe and efficient delivery system to deliver mRNA into the cytoplasm of target cells is a key link to realize its biological function and therapeutic value.

[0003] At present, various mRNA delivery strategies have been explored, such as direct injection of naked mRNA, virus vector-based delivery, polymer-based delivery and lipid-based delivery system. Among them, naked mRNA has poor stability and low delivery efficiency; although the virus vector has high efficiency, it faces problems such as immunogenicity, potential genomic integration risk and complex production process; the polymer carrier may be accompanied by biological toxicity and challenges of in vivo clearance mechanism.

[0004] Among non-viral vectors, lipid nanoparticles (LNP) have become the most successful mRNA delivery platform in current clinical transformation. A typical LNP is usually composed of four key components: (i) ionizable cationic lipids; (ii) auxiliary lipids such as phospholipids (e.g. DSPC); (iii) structural lipids such as cholesterol; and (iv) PEGylated lipids (PEG-lipids). Among them, ionizable cationic lipids are the core functional components of LNP. This kind of lipids is usually designed to be approximately electrically neutral or weakly positively charged at physiological pH (about 7.4, blood circulation environment) to reduce non-specific interactions and potential toxicity; while in acidic environment (pH about 5.0-6.5, endosome / lysosome environment after cellular internalization), the ionizable groups (such as tertiary amines) on the molecules will be protonated and carry positive charges. This pH-responsive property enables LNP to effectively bind and encapsulate negatively charged mRNA through electrostatic interaction at lower pH, remain stable in circulation, and promote the interaction of LNP with endosome membrane after cellular internalization, ultimately helping mRNA escape to the cytoplasm to exert its function.

[0005] Although ionizable lipids such as ALC-0315, SM-102 have been successfully applied to marketed mRNA vaccines, the exploration of new, efficient, and low-toxicity ionizable lipids is still ongoing. Developing ionizable lipids with novel chemical structures that can further improve mRNA delivery efficiency, improve biological safety (e.g., reduce potential liver toxicity or immunogenicity), increase drug loading capacity, or achieve more optimal in vivo distribution characteristics is crucial for the development of the next generation of mRNA drugs. The structural diversity of the existing lipid library still has room for expansion, and the structure-activity relationship (SAR) between lipid structure and its delivery performance and safety still needs to be further studied.

[0006] Reductive amination reactions are mainly catalyzed by metal-based catalysts (Ru, Co, Ni, Rh, Pt, Ir, etc.). Among these metal-based catalysts, Ru-based and Co-based catalysts (e.g., special-shaped Ru nanoparticles, Ru nanoparticles supported on metal oxides, Ru SACs, MOF-derived Co nanoparticles, and Co nanoparticles supported on nitrogen-doped carbon materials or metal oxides) exhibit excellent catalytic performance in selectively producing primary amines and have attracted great attention. However, these catalysts usually require harsh reaction conditions (high temperature or longer reaction time), and only a few catalysts can perform reductive amination reactions under mild conditions. Therefore, based on the inherent performance advantages of Ru and Co-based catalysts, it is crucial to develop more efficient catalysts.

[0007] Therefore, there is an urgent need in the art to design, synthesize, and evaluate ionizable cationic lipids with novel structures, and to construct LNP delivery systems with better performance based on this, to meet the needs of mRNA vaccine and therapy development. SUMMARY

[0008] The present application aims to provide an ionizable cationic lipid, further provide a lipid nanoparticle (LNP) comprising the lipid, which has good physicochemical properties, biocompatibility, and efficient mRNA encapsulation and intracellular delivery capacity; the present application also provides the use of the lipid or LNP in the preparation of a pharmaceutical composition or kit for mRNA delivery.

[0009] To achieve the above-mentioned purposes, the present application provides the following technical solutions:

[0010] The first aspect of the present application provides a preparation method of an ionizable cationic lipid, the preparation method provided by the present application is realized by esterification, twice reductive amination, and final amidation steps, and specifically comprises the following steps:

[0011] (a) reacting 7-oxoheptyl decanoate with hydroxylamine or a salt thereof in the presence of a base, in the presence of a first reducing agent and a first catalyst, the first catalyst being a cobalt-ruthenium alloy catalyst Co-Ru@zeolite;

[0012] (b) adding (9Z,12Z)-octadeca-9,12-dienal to the reaction product of step (a) and continuing the reaction in the presence of a second reducing agent to obtain a hydroxylamine derivative intermediate represented by formula (I):

[0013]

[0014] (c) amidating the hydroxylamine derivative intermediate obtained in step (b) with 4-dimethylaminobutyric acid or its hydrochloride salt in the presence of a condensing agent and a second catalyst to obtain an ionizable cationic lipid represented by formula (II):

[0015]

[0016] The ionizable cationic lipid provided by the present application comprises an ionizable tertiary amine head, a saturated C10 hydrophobic chain connected by an ester bond, and a (9Z,12Z)-octadecadienyl hydrophobic chain connected by a hydroxylamine linker, which is named as Lipid 16. The ionizable cationic lipid provided by the present application has certain physicochemical properties, for example:

[0017] The nuclear magnetic resonance hydrogen spectrum (1H NMR, 400MHz, CDCl3) thereof shows the following characteristic chemical shifts (δ): about 5.46-5.27 ppm (m, 4H, olefinic hydrogen), about 4.04 ppm (t, J≈6.7 Hz, 2H, -CH2-O-C=O), about 2.78 ppm (t, J≈7.9 Hz, 4H, allylic hydrogen), about 2.77 ppm (t, J≈6.4 Hz, 2H, -N-CH2-), about 2.22 ppm (s, 6H, -N(CH3)2), etc.

[0018] The liquid chromatography-mass spectrometry (LC-MS) analysis thereof shows quasi-molecular ion peaks: m / z about 663.6±0.2 ([M+H] + ) and / or m / z about 685.6±0.2 ([M+Na] + ).

[0019] In step (a) and step (b), the first reducing agent and the second reducing agent are independently selected from sodium triacetoxyborohydride, sodium cyanoborohydride or sodium borohydride.

[0020] In step (a), the Co-Ru@zeolite catalyst is prepared by mixing a zeolite carrier with a cobalt salt and a ruthenium salt, and then adding NaBH4 to reduce the metals to obtain Co-Ru@zeolite.

[0021] In step (c), the condensing agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and the second catalyst is 4-dimethylaminopyridine (DMAP).

[0022] As an optional step, the crude product obtained in step (c) is purified (e.g., column chromatography).

[0023] The second aspect of the present application provides a lipid nanoparticle (LNP) composition comprising: (a) the above-mentioned ionizable cationic lipid (lipid 16); (b) at least one helper lipid selected from phospholipids and / or cholesterol and derivatives thereof; (c) at least one PEGylated lipid (PEG-lipid).

[0024] The helper lipid comprises a phospholipid and cholesterol or a derivative thereof. The phospholipid is preferably distearoylphosphatidylcholine (DSPC).

[0025] The cholesterol and derivatives thereof are preferably cholesterol.

[0026] The PEG-lipid is a lipid comprising a polyethylene glycol segment and a hydrophobic anchor moiety, and the PEG-lipid is preferably dimethyl glutarate-polyethylene glycol (DMG-PEG).

[0027] The weight ratio of the ionizable cationic lipid (lipid 16), DSPC, cholesterol, and DMG-PEG is preferably about 20-60:5-20:3-16:1-10.

[0028] The above-mentioned LNP is prepared by standard LNP preparation techniques including the thin film dispersion method or the microfluidic mixing method, and has at least one of the following physicochemical properties:

[0029] The average particle size (e.g., determined by dynamic light scattering DLS) is in the range of 50 nm to 150 nm, for example, about 95 nm;

[0030] The morphology structure is approximately spherical as observed by transmission electron microscopy (TEM).

[0031] The third aspect of the present application provides an mRNA-loaded lipid nanoparticle (mRNA-LNP) comprising the above-mentioned lipid nanoparticle composition and mRNA molecules encapsulated therein.

[0032] The mRNA can be mRNA encoding a therapeutic protein, a vaccine antigen or a gene editing tool such as Cas9; the mRNA can be unmodified mRNA or chemically modified mRNA; the mRNA-LNP has an encapsulation efficiency (EE%) of mRNA of preferably more than 60%, more preferably more than 70% (e.g. determined using the RiboGreen method).

[0033] The fourth aspect of the present application provides the use of the above ionizable cationic lipid (lipid 16), LNP composition or mRNA-LNP in the preparation of a pharmaceutical composition or a kit for delivering mRNA to target cells (such as mammalian cells) or tissues.

[0034] Preferably, the use is for the preparation of an mRNA vaccine for the prevention or treatment of an infectious disease or cancer.

[0035] Preferably, the use is for the preparation of an mRNA therapeutic drug for protein replacement therapy or gene therapy.

[0036] Preferably, the use is for in vitro cell transfection or in vivo administration.

[0037] Compared with the prior art, the present application has the following beneficial effects:

[0038] 1. Synthesis accessibility: a detailed synthetic route and method for preparing lipid 16 is provided, the starting material is readily available, the reaction steps are clear, and it is easy to realize large-scale preparation by standard organic synthesis techniques; by adding a cobalt-ruthenium alloy catalyst Co-Ru@zeolite, the reaction time is effectively shortened, and the yield is effectively improved.

[0039] 2. Optimized LNP performance: the LNP constructed based on lipid 16 exhibits suitable physicochemical properties, such as a uniform particle size at the nanoscale (e.g. about 95 nm determined by DLS) and a regular spherical morphology (observed by TEM), which are beneficial to its in vivo behavior and cellular uptake as a drug carrier;

[0040] 3. Good biological safety: preliminary in vitro cytotoxicity evaluation (CCK-8 method) shows that the LNP does not show obvious toxicity to the tested cell lines (such as 97H, LM3) at a concentration of up to 1000 μg / ml, indicating that it has good biocompatibility potential;

[0041] 4. High efficiency of mRNA encapsulation: the LNP can effectively encapsulate mRNA molecules, and the encapsulation efficiency can reach about 70%, which is crucial for ensuring sufficient drug dose delivery;

[0042] 5. Effective intracellular delivery function: preliminary experiments of in vitro cell transfection confirmed that LNP loaded with reporter mRNA (such as GFP-mRNA) can mediate mRNA into cells (such as RAW 264.7) and successfully express target proteins, proving the functionality of the delivery system;

[0043] 6. Wide application prospect: in combination with the above-mentioned properties, the ionizable cationic lipid (lipid 16) prepared by the present application and the LNP system constructed therefrom have important application value and potential in the development of new mRNA vaccines, gene therapy drugs, protein replacement therapy drugs and other biological and pharmaceutical fields; BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 The reaction scheme is a reaction scheme for synthesizing 7-octyl heptyl decanoate in the embodiments of the present application.

[0045] Figure 2 The reaction scheme is a reaction scheme for synthesizing ionizable cationic lipid (lipid 16) in the embodiments of the present application.

[0046] Figure 3 The 1H NMR spectrum of 7-octyl heptyl decanoate prepared in Example 1 of the present application is shown in the figure.

[0047] Figure 4 The GC-MS spectrum of 7-octyl heptyl decanoate prepared in Example 1 of the present application is shown in the figure.

[0048] Figure 5 The GC-MS spectrum of 7-octyl heptyl decanoate prepared in Example 1 of the present application is shown in the figure.

[0049] Figure 6 The 1H NMR spectrum of lipid 16 prepared in Example 2 of the present application is shown in the figure.

[0050] Figure 7 The high resolution mass spectrum of lipid 16 prepared in Example 2 of the present application is shown in the figure.

[0051] Figure 8 The transmission electron microscopy (TEM) photograph of empty LNP prepared in Example 3 of the present application is shown in the figure.

[0052] Figure 9 The particle size distribution diagram determined by dynamic light scattering (DLS) of empty LNP prepared in Example 3 of the present application is shown in the figure.

[0053] Figure 10 The in vitro cytotoxicity test results (cell survival rate percentage diagram) of empty LNP on 97H cells and LM3 cells in Example 4 of the present application are shown in the figure.

[0054] Figure 11Figure 1 shows the standard curve for the RiboGreen assay used to determine mRNA encapsulation efficiency in Example 5 of the present application.

[0055] Figure 12 Figure 4 shows the confocal microscopy images of GFP-mRNA loaded LNP transfected RAW 264.7 cells (showing GFP expression) in Example 6 of the present application. DETAILED DESCRIPTION

[0056] The application will be further described in the following by specific examples. It should be understood that these examples are only intended to illustrate the present application, and not to limit the scope of the present application in any way. If no specific techniques or conditions are specified, the routine techniques and conditions in the art or according to the product instructions are used.

[0057] Example 1: Synthesis and characterization of intermediate 7-oxoheptyl decanoate

[0058] The reaction route for the synthesis of intermediate 7-oxoheptyl decanoate is shown in Figure 1 and specifically comprises:

[0059] (1) Preparation of 7-hydroxyheptanal: 1,7-heptanediol (5.0 g, 37.0 mmol) was taken in a 100 ml flask, dissolved in dry dichloromethane (DCM), and then PCC (8.9 g, 41.6 mmol) was added slowly dropwise over a period of 15 minutes. The reaction mixture was then stirred at room temperature for 2 hours. The crude reaction mixture was filtered through a silica gel column and washed with dichloromethane (2 x 50 ml). The organic solvent was dried over Na2SO4, removed by evaporation under reduced pressure, and the crude hydroxyaldehyde obtained without further purification was used directly in the next step;

[0060] (2) Synthesis of 7-oxoheptyl decanoate: The crude hydroxyaldehyde prepared above (1.3 g, 10 mmol), decanoic acid (2.0 g, 12 mmol) and EDC (2.8 g, 15.0 mmol) were taken in a 100 ml flask, dissolved in dry dichloromethane, and DMAP (catalyst) was added at 0 °C. The reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was quenched with saturated NaHCO3, then extracted with dichloromethane (3 times) and washed with brine, dried over anhydrous Na2SO4. The crude mixture was purified by column chromatography (ethyl acetate: n-hexane = 5:95) to obtain the product as a colorless oil;

[0061] (3) Characterization: 1H NMR (400 MHz, CDC13): δ 9.76 (t, J = 1.9 Hz, 1H), 4.06 (t, J = 6.7 Hz, 2H), 2.45 (dt, J = 7.3, 5.4 Hz, 2H), 2.29 (t, J = 7.5 Hz, 2H), 1.78 - 1.52 (m, 8H), 1.42 - 1.33 (m, 4H), 1.32 - 1.20 (m, 12H), 0.88 (t, J = 6.8 Hz, 3H). (See Figure 3 ) GC-MS: m / z 287.3 [M+H] + (See Figure 4 and Figure 5 );

[0062] The results confirmed the successful synthesis of the target intermediate, 7-oxoheptyl decanoate.

[0063] Example 2: Preparation of cobalt-ruthenium alloy catalyst Co-Ru@zeolite

[0064] The cobalt-ruthenium alloy catalyst is an effective catalyst for shortening the preparation of liposome 16, and the specific synthesis method is as follows:

[0065] 0.5 g of porous zeolite carrier was mixed with an aqueous solution of cobalt nitrate and ruthenium (III) chloride in 50 mL of deionized water. After stirring the mixed solution for 1 hour, 0.3 g of NaBH4 was added to reduce the metal. After stirring for another 1 hour, the catalyst was separated, washed with deionized water three times, and then dried under vacuum for 12 hours.

[0066] Example 2: Synthesis and characterization of ionizable cationic lipid (lipid 16)

[0067] The reaction route of the ionizable cationic lipid (lipid 16) is shown in Figure 2 , which is specifically:

[0068] (1) Synthesis of hydroxylamine derivative containing amphiphilic chains: A suspension of hydroxylamine hydrochloride (1.12 mmol, 1.0 eq) was added to dry dichloromethane (5 ml) followed by triethylamine (1.12 mmol, 1.0 eq) under nitrogen atmosphere and stirred for 5 min at room temperature. Thereafter, a solution of 7-oxoheptyl decanoate (1.12 mmol, 1.0 eq) in dry dichloromethane (15 ml) was added dropwise, followed by Co-Ru@zeolite catalyst and stirred for 2 h. Subsequently, sodium cyanoborohydride (1.5 eq) was added portion wise slowly and stirred for 10 min. Thereafter, a solution of linoleic aldehyde (1.12 mmol, 1.0 eq) in dry dichloromethane (10 ml) was added dropwise and stirred for another 10 min. Subsequently, another portion of sodium cyanoborohydride was added in the same manner portion wise slowly and stirred for 1.5 h under nitrogen atmosphere. The reaction was quenched with saturated sodium bicarbonate solution and extracted with DCM (3 times). The organic phase was washed with brine solution and dried over anhydrous sodium sulfate. The solvent was evaporated and the resulting mixture was purified by column chromatography using 5% ethyl acetate in hexane to obtain the desired hydroxylamine as a pale yellow liquid; yield 83%.

[0069] (2) Synthesis of lipid 16: The above hydroxylamine derivative (395 mg, 0.72 mmol, 1.0 eq), N,N-dimethylamino butyric acid hydrochloride (240 mg, 1.44 mmol, 2.0 eq), EDC.HC1 (275 mg, 1.44 mmol, 2.0 eq) and DMAP (17 mg, 0.14 mmol, 0.2 eq) were dissolved in dry dichloromethane (20 ml) under nitrogen atmosphere and stirred for 6 h at room temperature. Thereafter, the reaction was quenched with saturated sodium bicarbonate solution followed by extraction with DCM 3 times. The organic phase was washed with saturated brine solution and dried over anhydrous magnesium sulfate. The solvent was evaporated and the resulting mixture was purified by gradient elution using 3% chloroform in isopropyl alcohol to obtain lipid 16 as a pale yellow oil; yield 75%.

[0070] (3) Characterization: 1H NMR (400 MHz, CDC13): δ 5.46-5.27 (m, 4H), 4.04 (t, J = 6.7 Hz, 2H), 2.78 (t, J = 7.9 Hz, 4H), 2.77 (t, J = 6.4 Hz, 2H), 2.32 (t, J = 7.6 Hz, 2H), 2.30 (t, J = 6.8 Hz, 2H), 2.28 (t, J = 7.6 Hz, 2H), 2.22 (s, 6H), 2.09-1.99 (m, 4H), 1.80 (m, 2H), 1.70-1.59 (m, 4H), 1.56-1.43 (m, 4H), 1.40-1.19 (m, 34H), 0.89 (t, J = 6.8 Hz, 3H), 0.88 (t, J = 7.2 Hz, 3H). (See Figure 6 );

[0071] High resolution mass: m / z 663.6033 [M+H] + , 685.5855 [M+Na] + . (See Figure 7 );

[0072] The results confirmed the successful synthesis of the target ionizable cationic lipid (lipid 16).

[0073] Example 3: Preparation and characterization of lipid nanoparticles (LNPs)

[0074] (1) Preparation of LNPs (film dispersion method example): Take 650 mg of lipid 16, 15 mg of DSPC, 5 mg of DMG-PEG, and 11 mg of cholesterol into a 20 ml seed bottle, add 1-2 ml of anhydrous ethanol, heat slightly and stir until completely dissolved, then rotate the small beaker to form a film of ethanol liquid on the wall, and gently blow with flowing nitrogen until the ethanol evaporates, leaving a thin film on the wall;

[0075] Take 8 ml of enzyme-free water and add it to the small beaker containing the film, stir the water in a water bath manually for 10 minutes, then place the small beaker on a magnetic stirrer and stir at room temperature for 30 minutes, add the RNA solution, mix well and stir for another 30 minutes to obtain the LNP solution (10 mg / ml);

[0076] (2) Characterization of LNPs: Transmission electron microscopy (TEM): Dilute the LNPs to a solution of 1 mg / ml, take 20 μL of the solution with a pipette, drop it on a copper mesh, dry it, then use uranyl acetate as a negative stain, and after the sample is dried, observe it with a transmission electron microscope. The results show that the LNPs are approximately spherical and relatively uniformly distributed (see Figure 8 );

[0077] Dynamic light scattering (DLS): Dynamic light scattering test was performed after the sample was ultrasonically agitated for 1 min, and repeated three times. The results showed that the average hydration particle size (Z-average) of the LNP was about 95 nm, and the PDI value was low, indicating that the particle size distribution was narrow (see Figure 9 );

[0078] The results showed that LNP with required morphology and particle size was successfully prepared.

[0079] Example 4: In vitro cytotoxicity test of LNP

[0080] The 97H and LM3 cell lines were selected to prepare cell suspensions. The cells were inoculated into 96-well plates: according to the appropriate plating cell number (about ≥5×104 cells per well), about 200ul of cell suspension per well, and 5 replicates were set. The prepared plate was placed in a 37°C incubator: the cells adhered after inoculation, and this step needed to be observed under a microscope for adhesion, which required about 24 hours of culture. The 96-well plate was changed, different concentrations of toxic substances were added, and the final concentration of LNP was set to 0, 15.625, 31.25, 62.5, 125, 250, 500 and 1000ug / mL, and each well was made up to 200uL. Incubate in a 37°C incubator for 24 hours. Add 100uL of 10% CCK8 complete medium solution to each well of the 96-well plate, and add 5 wells as a blank control group. After incubation in a 37°C incubator for 2 hours, the absorbance at 450nm was measured by a microplate reader, and the cytotoxicity of different concentrations was compared.

[0081] The results (see Figure 10 ) showed that the lipid 16 LNP had low toxicity to 97H and LM3 cells within the test concentration range (up to 1000ug / ml), and the cell survival rate remained at a high level, showing good in vitro biocompatibility.

[0082] Example 5: mRNA encapsulation rate test

[0083] ①Preparation of Buffer

[0084] 1X TE Buffer: Use 20X TE Buffer in the kit for dilution.

[0085] 2% Triton-TE buffer: Triton-100 is used with 1X TE buffer at a volume ratio of 1:50.

[0086] ②Add RNA-LNP sample to a full black 96-well plate

[0087] According to the amount of RNA prepared, the dilution factor can be roughly calculated. According to the final volume after dilution, ultrafiltration, etc., the total RNA concentration can be estimated. At the same time, assuming a 90% encapsulation rate, the free RNA concentration can be estimated. Then calculate the dilution factor needed according to the highest concentration of the standard curve.

[0088] Dilute the RNA-LNP by the appropriate factor using 1X TE buffer to detect the free RNA concentration outside the LNP, and dilute the RNA-LNP by the appropriate factor using 2% Triton-TE buffer to detect the total RNA concentration.

[0089] Finally, 100 μL of the diluted sample was removed and added to a full black 96-well plate.

[0090] ③Standard addition

[0091] The standard curve was established directly using the 2 μg / mL RNA standard provided in the kit. Prepare the standard samples according to Table 1 to draw the standard curve.

[0092] Table 1 Reagents required for standard sample preparation

[0093]

[0094] Remove 200 μL of each concentration of standard and add to a full black 96-well plate. When the RNA-LNP sample and the standard sample of a certain concentration are added to the 96-well plate, incubate at 37°C in the dark for 10 min to allow the RNA-LNP to fully dissolve in the Triton buffer.

[0095] ④Enzyme detector detection

[0096] Turn on the enzyme detector, select the fluorescence mode, excitation light 485 nm, emission light 528 nm, read, and record the data as shown in Table 2.

[0097] ⑤Data processing and analysis

[0098] Subtract the blank fluorescence value from the measured fluorescence value of each sample to obtain the actual fluorescence value. According to the fluorescence value and concentration gradient of the standard sample, draw a standard curve to obtain the regression equation. Substitute the sample fluorescence value into the regression equation and multiply by the dilution factor to obtain the RNA concentration of the sample. Calculate the encapsulation efficiency according to the encapsulation efficiency formula.

[0099] Encapsulation efficiency (EE%) = 1 - C1 / C0*100%

[0100] C1: Free RNA concentration.

[0101] C0: Concentration of all RNA in the RNA-LNP solution system.

[0102] Table 2 Cell fluorescence intensity record table

[0103]

[0104] The established concentration standard curve is shown in Figure 11 The relationship concentration = 2.9122 * fluorescence intensity + 14.187 is obtained.

[0105] The detected fluorescence intensity is shown in Table 3:

[0106] Table 3 Fluorescence intensity record before and after LNP lysis

[0107]

[0108] It is calculated that the encapsulation rate of mRNA of the LNP prepared in this embodiment is about 69.89%.

[0109] Example 6: In vitro mRNA transfection test

[0110] Tumor-related RAW 264.7 cells (2.5 x 10 5 The cells were seeded on sterile glass-bottomed culture dishes and incubated with LNP containing CAR mRNA and Siglec-GΔITIMs mRNA (CAR and Siglec-GΔITIMs LNPs) for 12 hours after overnight. After incubation with primary anti-myc antibody and anti-His antibody at 4°C overnight, the samples were then co-incubated with secondary antibody labeled with Cy3 or FITC for 1 hour. The nuclei were stained with DAPI (10 μg / mL). Immunofluorescence images were obtained by confocal laser scanning microscope (LSM 900 with AiryScan2).

[0111] The results (see Figure 12 ) show that a significant green fluorescent signal is observed in RAW 264.7 cells treated with GFP-mRNA-LNP, indicating that LNP successfully mediates mRNA delivery and expression of GFP protein, compared with the control group.

[0112] A novel ionizable cationic lipid (Lipid 16) was successfully synthesized by a systematic chemical synthesis. The LNP formulated with Lipid 16 and other helper lipids (DSPC, cholesterol, DMG-PEG) exhibited suitable particle size (~95 nm), regular morphology, good in vitro biocompatibility, satisfactory mRNA encapsulation efficiency (~70%), and efficient in vitro mRNA delivery and expression function. These results fully demonstrated the potential of Lipid 16 as a novel ionizable lipid for the construction of mRNA delivery systems, laying a foundation for subsequent in vivo evaluation and application in the development of mRNA vaccines and therapies.

Claims

1. A method for preparing an ionizable cationic lipid, characterized in that: The preparation method comprises the following steps: (a) reacting 7-oxoheptyldecanoate with hydroxylamine or a salt thereof in the presence of a base, a first reducing agent and a first catalyst, wherein the first catalyst is a cobalt-ruthenium alloy catalyst Co-Ru@zeolite; (b) adding (9Z,12Z)-octadeca-9,12-dienal to the reaction product of step (a), and continuing the reaction in the presence of a second reducing agent to obtain a hydroxylamine derivative intermediate represented by formula (I): (c) subjecting the hydroxylamine derivative intermediate obtained in step (b) to an amidation reaction with 4-dimethylaminobutyric acid or its hydrochloride in the presence of a condensing agent and a second catalyst to obtain an ionizable cationic lipid represented by formula (II):

2. The method for preparing an ionizable cationic lipid according to claim 1, wherein In step (a) and step (b), the first reducing agent and the second reducing agent are independently selected from sodium triacetoxyborohydride, sodium cyanoborohydride or sodium borohydride.

3. The method for preparing an ionizable cationic lipid according to claim 1, wherein In step (a), the cobalt-ruthenium alloy catalyst Co-Ru@zeolite is prepared by mixing a zeolite carrier with a cobalt salt and a ruthenium salt, and then adding NaBH4 to reduce the metal to obtain Co-Ru@zeolite.

4. The method for preparing an ionizable cationic lipid according to claim 1, wherein In step (c), the condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and the second catalyst is 4-dimethylaminopyridine.

5. A lipid nanoparticle composition, characterized in that The lipid nanoparticle composition comprises an ionizable cationic lipid, an auxiliary lipid and a PEG-lipid obtained by any one of the preparation methods of claims 1 to 4.

6. The lipid nanoparticle composition according to claim 5, characterized in that The helper lipid includes phospholipid, cholesterol or its derivatives; the PEG-lipid is a lipid containing a polyethylene glycol segment and a hydrophobic anchoring part.

7. The lipid nanoparticle composition according to claim 5, characterized in that The Z-average hydrated particle size of the lipid nanoparticle composition is between 50 nm and 150 nm.

8. A lipid nanoparticle loaded with mRNA, characterized in that The mRNA-loaded lipid nanoparticles include the lipid nanoparticle composition according to claim 5 and mRNA molecules encapsulated therein.

9. Use of the ionizable cationic lipid obtained by the preparation method of any one of claims 1 to 4, or the lipid nanoparticle composition according to claim 5, or the mRNA-loaded lipid nanoparticle according to claim 8 in preparing a pharmaceutical composition or kit for mRNA delivery.

10. The use according to claim 9, characterized in that The pharmaceutical composition is an mRNA vaccine or an mRNA therapeutic drug.

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