Lipid nanoparticle for muscle in-situ delivery as well as preparation method and application of lipid nanoparticle

Abstract

The invention belongs to the technical field of biological medicines, and discloses lipid nanoparticles for muscle in-situ delivery as well as a preparation method and application of the lipid nanoparticles. According to the invention, four components Ugi of aldehyde, isonitrile, amine and carboxylic acid are subjected to a reaction to synthesize the ionizable cationic lipid. The ionizable cation lipid formed by introducing an ionizable cation head group into an isonitrile component has muscle in-situ nucleic acid delivery performance. The unsaturation degree of a lipid hydrophobic tail chain, the number of methylene spacers of an ionizable cation head group and LNP formula composition and process are further optimized, muscle in-situ selective mRNA delivery performance is enhanced, off-target liver expression is avoided, safety can be improved, and different application requirements of mRNA vaccines, nucleic acid drugs, gene editing and the like can be met.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a lipid nanoparticle for in situ delivery to muscle, its preparation method, and its application. Background Technology

[0002] In recent years, messenger RNA (mRNA) vaccine technology has made breakthrough progress, greatly promoting the development of gene therapy drugs. Examples include the mRNA vaccines Spikevax and Comirnaty for preventing COVID-19 infection, and the mRNA vaccine mRESVIA for preventing respiratory syncytial virus (RSV) infection. Currently, mRNA-based preventative vaccines, therapeutic cancer vaccines, protein replacement therapies, and gene-editing drugs are advancing into clinical trials globally.

[0003] The application of mRNA drugs heavily relies on safe and efficient nucleic acid delivery systems. Lipid nanoparticles (LNPs) are one of the most mature and widely used delivery platforms, and have become a core technology for mRNA drug development. After intramuscular injection, mRNA vaccines exert their effects by locally inducing antigen expression and activating the immune response. However, traditional LNP delivery technologies exhibit a significant tendency for liver enrichment, which limits their application in extrahepatic targeted delivery. Furthermore, intramuscular injection often results in side effects such as off-target antigen expression and induced inflammatory responses, which restrict the widespread clinical application of mRNA vaccines.

[0004] Current LNPs typically consist of four components: ionizable cationic lipids, accessory lipids (phospholipids), cholesterol, and polyethylene glycol (PEG)-modified lipids. Among these, the ionizable cationic lipids play a crucial role in enhancing the mRNA encapsulation efficiency and lysosomal escape of LNPs. Furthermore, the four-component LNP process requires rapid mixing of the lipid-ethanol phase and an acidic mRNA buffer solution, utilizing the electrostatic interaction between the ionizable cationic lipids and mRNA for drug encapsulation, followed by dialysis with neutral PBS to prepare the formulation, making the process relatively complex. A more significant challenge is the current difficulty in achieving in-situ specific mRNA delivery to muscle using LNPs. Therefore, there is an urgent need to develop novel ionizable cationic lipids to obtain safe, efficient, and precise LNP systems, as well as LNP formulations with simpler processes, to meet the broad clinical application needs in the field of mRNA drugs. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a lipid nanoparticle for in situ delivery to muscle, its preparation method and application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention synthesizes an ionizable cationic lipid having a structure as shown in formula (I): ——Formula (I); In the formula, n = 0~3; R1 and R2 are independently selected from methyl or ethyl; M1 is independently selected , , , , , , ; M2 is selected independently , , , ; M3 is selected independently , , , , , , , .

[0007] In a preferred embodiment of the ionizable cationic lipid of the present invention, n = 1 or 2; And / or, the M1 is independently selected from , , , ; And / or, the M2 is independently selected from , , ; And / or, the M3 is independently selected from , , , , , , .

[0008] In a further preferred embodiment of the ionizable cationic lipid of the present invention, n = 2; And / or, M1 is ; And / or, the M2 is ; The M3 is independently selected from , , , , .

[0009] In a second aspect, the present invention provides lipid nanoparticles comprising the ionizable cationic lipids, cofactor phospholipids, sterols, and polymer-modified lipids described in the first aspect.

[0010] In a preferred embodiment of the lipid nanoparticles of the present invention, the auxiliary phospholipid is at least one of distearylphosphatidylcholine (DSPC), dilauryl lecithin (DLPC), dioleoyl lecithin (DOPC), dimyristoyl lecithin (DMPC), 1-palmitoyl-2-oleoyl lecithin (POPC), dipalmitoyl lecithin (DPPC), dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylethanolamine (DMPE), distearylphosphatidylethanolamine (DSPE), and dipalmitoylphosphatidylethanolamine (DPPE); the sterol is at least one of cholesterol, sitosterol, stigmasterol, and cholesterol derivatives; the polymer-modified lipid is at least one of polyethylene glycol-modified lipid, polysarcosine-modified lipid, or polyzwitterionic lipid and its derivatives; the molar ratio of the ionizable cationic lipid, sterol, auxiliary phospholipid, and polymer-modified lipid is (25~75):(20~50):(5~25):(0.5~10).

[0011] Preferably, the molar ratio of the ionizable cationic lipid, cholesterol, cofactor phospholipid, and polymer-modified lipid is 50:38.5:10:1.5.

[0012] Thirdly, the present invention provides another type of lipid nanoparticle, comprising the ionizable cationic lipids, sterols and polymer-modified lipids described in the first aspect.

[0013] As a preferred embodiment of the lipid nanoparticles of the present invention, the sterol is at least one of cholesterol, sitosterol, stigmasterol, and cholesterol derivatives; the polymer-modified lipid is at least one of polyethylene glycol-modified lipid, polysarcosine-modified lipid, or zwitterionic lipid and its derivatives; the molar ratio of the ionizable cationic lipid, sterol and polymer-modified lipid is (90~10):(88~8):(0.5~10).

[0014] Preferably, the molar ratio of the ionizable cationic lipid, sterol, and polymer-modified lipid is 45:53.5:1.5.

[0015] Fourthly, the present invention provides a drug-loaded nanoparticle, characterized in that it comprises the lipid nanoparticles and the drug described in the second or third aspect.

[0016] As a preferred embodiment of the drug-loaded nanoparticles of the present invention, the drug includes at least one of small molecule compounds, nucleic acid molecules, protein or polypeptide molecules, and gene editing complexes.

[0017] As a further preferred embodiment of the drug-loaded nanoparticles of the present invention, the nucleic acid molecule is at least one of messenger RNA, circular RNA, transfer RNA, dsRNA, shRNA, DNA, plasmid DNA, siRNA, antisense oligonucleotide, aiRNA, and miRNA; the gene editing complex is mRNA / sgRNA or Cas9 / sgRNA.

[0018] As a further preferred embodiment of the drug-loaded nanoparticles of the present invention, the mass ratio of the ionizable cationic lipid to the nucleic acid molecule is (2~50):1.

[0019] Fifthly, the present invention provides a method for preparing the lipid nanoparticles described in the fourth aspect, comprising the following steps: (1) Dissolve the ionizable cationic lipids, sterols, auxiliary phospholipids and polymer-modified lipids in an organic solution to obtain an organic phase; (2) Dissolve the drug in a buffer solution to obtain an aqueous phase; The volume ratio of the aqueous phase to the organic phase is (1~6):1; (3) The aqueous phase and the organic phase are rapidly mixed evenly, and then dialyzed to obtain the final product; In a sixth aspect, the present invention provides another method for preparing the lipid nanoparticles described in the fourth aspect, comprising the following steps: (1) Dissolve the ionizable cationic lipid, sterol and polymer-modified lipid in an organic solution to obtain an organic phase; (2) Dissolve the drug in nuclease-free water or neutral pH buffer (preferably PBS buffer) to obtain an aqueous phase; The volume ratio of the aqueous phase to the organic phase is (1~6):1; (3) The aqueous phase and the organic phase are quickly mixed evenly and dialyzed to obtain the product.

[0020] In a seventh aspect, the present invention applies the ionizable cationic lipids described in the first aspect, the lipid nanoparticles described in the second or third aspect, and the lipid nanoparticles described in the fourth aspect in the preparation, delivery, or transport of molecular drugs or nucleic acid vaccines.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention utilizes a four-component Ugi reaction of aldehydes, isonitriles, amines, and carboxylic acids to synthesize an ionizable cationic lipid. The ionizable cationic lipid, formed by introducing ionizable cationic head groups through the isonitrile component, exhibits in-situ muscle nucleic acid delivery performance. Further optimization of the unsaturation degree of the lipid's hydrophobic tail chain, the number of methylene spacers in the ionizable cationic head group, and the LNP formulation and process significantly enhances the selective in-situ muscle mRNA delivery performance and avoids off-target liver expression issues.

[0022] 2. The ionizable cationic lipids of this invention have mild reaction conditions, simple synthesis process, and good stability. The synthesized ionizable cationic lipids can be assembled with sterols, helper phospholipids, polyethylene glycol-modified lipids, etc., to form lipid nanoparticles, which can be used to significantly enhance in-situ drug delivery in muscle, including small molecule compounds, nucleic acid molecules such as mRNA, DNA, siRNA, and circular RNA, protein / peptide molecules, and gene editing complexes such as mRNA / sgRNA and Cas9 / sgRNA. Efficient mRNA transfection and high protein expression levels can be achieved in local muscle tissue of animals via intramuscular injection, avoiding off-target expression problems in the liver, improving safety, and meeting the needs of different applications such as mRNA vaccines, nucleic acid drugs, and gene editing. Attached Figure Description

[0023] Figure 1 The mass spectrometry characterization results are for the ionizable cationic lipid A4B2C5D2 (theoretical molecular weight is 770.33).

[0024] Figure 2 The mass spectrometry characterization results are for the ionizable cationic lipid A4B2C6D2 (theoretical molecular weight is 768.31).

[0025] Figure 3 The mass spectrometry characterization results are for the ionizable cationic lipid A4B2C2D3 (theoretical molecular weight is 786.77).

[0026] Figure 4 Molecular dynamics simulations of the interactions between ionizable cationic lipids A4B2C8D9, A4B4C2D9, and A4B2C2D2 and mRNA are shown. In the figure, (a) is a snapshot of the molecular dynamics simulation, taken at 0 ps and 100,000 ps. RMSD (root mean square deviation) during the simulation is shown in (b); Coulomb interaction (c) and van der Waals interaction (d).

[0027] Figure 5 To assess the number of hydrogen bond interactions between ionizable cationic lipids A4B2C8D9, A4B4C2D9, and A4B2C2D2 and mRNA.

[0028] Figure 6This study compares the ability of ionizable cationic lipids A4B2C8D9, A4B4C2D9, and A4B2C2D2 to bind mRNA under neutral PBS conditions.

[0029] Figure 7 Comparison of relative mRNA expression efficiency in in situ muscle after intramuscular injection of four components of LNP with different ionizable cationic lipids (compared to A4B2C2D2).

[0030] Figure 8 The ratio of mRNA expression efficiency (MLR) in the in situ muscle and liver sites after intramuscular injection of four components of different ionizable cationic lipids.

[0031] Figure 9 Bioluminescence imaging of animals and isolated organs after intramuscular injection of four components of LNP with different ionizable cationic lipids.

[0032] Figure 10 Bioluminescence imaging of animals and isolated organs after intramuscular injection of three components of LNP with different ionizable cationic lipids. Detailed Implementation

[0033] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0034] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0035] Example 1: Synthesis of ionizable cationic lipids Aldehydes, amines, carboxylic acids, and isonitriles were synthesized via the Ugi four-component reaction (Ugi-4CR) to create different ionizable cationic lipids. The synthetic route is as follows: Among them, aldehyde compounds The M1 group is derived from: , amine compounds The M2 group is derived from: , Carboxylic acid compounds The M3 group is derived from: , Isonitrile compounds n is 0~3, and R1 and R2 are independently selected from methyl or ethyl; or the structure of the isonitrile compound is D9: .

[0036] Examples of methods for synthesizing isonitrile compounds are as follows: Isocyanates can be prepared by formylation of their corresponding amine compounds followed by dehydration. Taking the synthesis of D3 isonitriles as an example, firstly... N,N Dimethyl-1,4-butanediamine (1.0 eq.) was dissolved in 10 molar equivalents of ethyl formate, and the mixture was heated to 70 °C and refluxed with stirring for 24 h to obtain formamide. Formamide was dissolved in dichloromethane and the reaction concentration was controlled at 1 M. When phosphorus oxychloride was chosen as the dehydrating agent, 2 molar equivalents of triethylamine were added, followed by the slow addition of 1.5 molar equivalents of phosphorus oxychloride under nitrogen protection in an ice bath. When p-toluenesulfonyl chloride was chosen as the dehydrating agent, 3 molar equivalents of pyridine were added to the solution, followed by the slow addition of 1.5 molar equivalents of p-toluenesulfonyl chloride under nitrogen protection in an ice bath. The reaction was brought back to room temperature and continued for 3 h. The reaction solution was slowly poured into an ice bath of potassium carbonate aqueous solution (20% wt) and stirred for 1 h to quench the reaction. The aqueous phase was extracted three times with dichloromethane, and the organic phase was collected. The organic phase was washed three times each with an aqueous potassium carbonate solution (20% wt) and a saturated sodium chloride solution. After drying the organic phase with anhydrous sodium sulfate, the crude product was enriched using a rotary evaporator. The crude product was purified by silica gel column chromatography with ethyl acetate / n-hexane as the eluent (1 / 20). The eluent was removed by rotary evaporation, and the product was dried in a vacuum oven to obtain a yellow liquid.

[0037] Examples of the synthesis of ionizable cationic lipids are as follows: Using the Ugi-4CR reaction, 1.0 mmol of aldehyde and 1.0 mmol of amine were added to 0.5 mL of methanol solution in a one-pot reaction at room temperature. After reacting at room temperature for 1 h, 1.0 mmol of carboxylic acid was added, followed by 1 h of reaction at room temperature, and then 1.0 mmol of isonitrile was added. The reaction was carried out at 40 °C for 24 h. After the reaction, the products were purified by column chromatography using a mixture of methanol and dichloromethane as the mobile phase. The purified products were then dried by rotary evaporation to obtain ionizable cationic lipid products. The obtained ionizable cationic lipids were named AxByCmDn, where x (0~6), y (0~4), m (0~8), and n (1~9) are the corresponding numbers of aldehyde (A), amine (B), carboxylic acid (C), and isonitrile (D). By chemically combining different aldehydes, amines, carboxylic acids, and isonitriles, different ionizable cationic lipids can be obtained through similar synthetic methods. The representative products and their structures are shown in Table 1. Table 1. Names and structural formulas of ionizable cationic lipids The structures of the above ionizable cationic lipids were characterized by mass spectrometry. Data for three representative ionizable cationic lipids, A4B2C5D2, A4B2C6D2, and A4B2C2D3, are shown below. Figure 1 , Figure 2 and Figure 3 As shown, this indicates that the corresponding lipid product was successfully prepared.

[0038] Example 2: Preparation and characterization of drug-loaded four-component lipid nanoparticles (LNPs) Taking drug-loaded LNPs (Fluc mRNA LNPs) encoding luciferase mRNA as an example, four LNP components were prepared: First, an organic phase was prepared by dissolving ionizable cationic lipids, cholesterol, distearate phosphatidylcholine (DSPC), and 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000) in ethanol at a molar ratio of 50:38.5:10:1.5. An aqueous phase was prepared by diluting Fluc mRNA to a corresponding mass ratio in citrate buffer (10 mM, pH = 4). The aqueous and organic phases were then rapidly mixed at a volume ratio of 3:1 using microfluidics, with the aqueous phase flow rate controlled at 9 mL / min and the organic phase flow rate at 3 mL / min (adjustable from 1 to 10 mL / min). After standing for 10 minutes, the solution was placed in a 1000 Da dialysis bag and dialyzed in 1×PBS solution at 4℃ for 2 hours to obtain Fluc mRNA LNP, in which the mass ratio of ionizable cationic lipids to nucleic acids was ionizable cationic lipids: nucleic acids = 11:1.

[0039] The particle size, polydispersity index (PDI), and zeta potential of diluted Flux mRNA LNPs were determined using a Malvern particle size analyzer, and the mRNA encapsulation efficiency was detected using the RiboGreen RNA assay. The specific results are shown in Table 2. Table 2 Physicochemical properties of four-component Flux mRNA-LNPs prepared by microfluidic method (different LNPs are named according to their corresponding ionizable cationic lipids). Example 3: Characterization of intermolecular forces between ionizable cationic lipids and mRNA molecules Molecular dynamics simulations: The ionizable cationic lipid molecule structure was constructed using GaussView 5.0.9, followed by structure optimization over 6–31 G(d), and RESP charge calculations were performed using Multiwfn. An 18-nucleotide polyadenylated single-stranded RNA structure was generated using the online tool RNAComposer, and the RNA was parameterized using amber14sb_parmbsc1. Then, the Gaff force field topological parameters of the ionizable cationic lipid molecule were generated using Sobtop software and modified for application to the amber14sb_parmbsc1 force field.

[0040] All simulations were performed in Gromacs 2023.02. First, RNA and different ionizable cationic lipid molecules were placed into separate boxes, one RNA and 18 lipid molecules per box. The boxes were filled with water using the TIP3P water model, ensuring a neutral system without the need for ionization. The steepest descent method was then used to minimize the energy of the system, followed by a 0.5 ns NVT and a 1 ns NPT simulation. RNA position was constrained for 5 ns to allow contact between the ionizable cationic lipid chain and the RNA bases. Finally, RNA was released for a 200 ns production simulation. All bonds containing hydrogen atoms were handled using the default linear constraint (LINCS). A V-rescale thermostat and a Parrinello-rahman pressure regulator were used, with the temperature set to 298.15 K and the pressure to 1.0 bar. The Particle Mesh Ewald (PME) method was used to handle long-range electrostatic interactions, including van der Waals interactions, with a cutoff radius of 1.0 nm. The time step was 2 fs, with a snapshot taken every 5.0 ps. RMSD (root mean square deviation), hydrogen bond analysis, and interaction energies of RNA and ionizable cationic lipid molecules were performed using Gromacs commands, and hydrogen bond structure snapshots were plotted using PyMOL 3.0.0.

[0041] The interaction between ionizable cationic lipid isomers with ionizable cationic head groups (isocyanate head group, amine head group, and carboxylic acid head group) introduced by different reaction components and mRNA molecules was evaluated using molecular dynamics simulations. Figure 4 a shows the results of simulating mRNA with three ionizable cationic lipids (A4B2C8D9, A4B4C2D9, and A4B2C2D2) for 100,000 ps. All three ionizable cationic lipids can form aggregates with mRNA molecules from their initial free state, indicating an interaction between the ionizable cationic lipids and mRNA molecules. Furthermore, the simulation system tends to stabilize after 20,000 ps. Figure 4b). Further statistical analysis was conducted on the electrostatic interactions between ionizable cationic lipids and mRNA during the simulation. It was found that different head isoforms of ionizable cationic lipids had a significant impact on coulombic interactions. For example... Figure 4 As shown in c, the isonitrile-headed ionizable cationic lipid A4B2C2D2 exhibits a stronger Coulombic interaction with mRNA (-9284 kJ / mol), which is higher than that of the other two carboxylic acid-headed or amine-headed ionizable cationic lipids (A4B2C8D9 and A4B4C2D9) (-5800 ~ -6400 kJ / mol). Regarding van der Waals interactions ( Figure 4 d), the three ionizable cationic lipids are all quite similar (-550 ~ -820 kJ / mol).

[0042] Further analysis was conducted on the ability of ionizable cationic lipids to form hydrogen bonds with the phosphate, ribose, and base backbone of mRNA. The results are as follows: Figure 5 As shown, for the phosphate backbone of mRNA, the ionizable cationic lipid A4B2C2D2 with the isonitrile head group exhibits stronger hydrogen bonding (average number 4.9), higher than A4B2C8D9 or A4B4C2D9 (average number 1.3–2.2). For the ribosome backbone of mRNA, A4B2C2D2 has slightly stronger hydrogen bonding (average number 2.7), higher than A4B2C8D9 or A4B4C2D9 (average number 1.7–1.8). For the base backbone of mRNA, A4B2C2D2 and A4B4C2D9 exhibit stronger hydrogen bonding (average number 3.5–3.8), higher than A4B2C8D9.

[0043] Furthermore, the ability of ionizable cationic lipids to bind mRNA under neutral PBS conditions (10 mM, pH 7.2) was investigated. Under these conditions, the effects of tertiary amine protonation are eliminated, and charge interactions are minimized. The ability of ionizable cationic lipids to bind mRNA via hydrogen bonding can be assessed by detecting mRNA encapsulation efficiency (RiboGreen RNA assay). Figure 6As shown, under neutral pH conditions, the isonitrile-headed ionizable cationic lipid A4B2C2D2 still provides a high mRNA binding efficiency (>70%), while the carboxylic acid and amine-headed ionizable cationic lipids (A4B2C8D9, A4B4C2D9) completely lose their ability to bind mRNA. Furthermore, under neutral pH conditions, four-component LNPs corresponding to A4B2C8D9, A4B4C2D9, and A4B2C2D2 were also prepared (method of Example 2). Similarly, the LNP based on the isonitrile-headed ionizable cationic lipid A4B2C2D2 provides a high mRNA binding efficiency (>75%), while the LNP based on the carboxylic acid and amine-headed ionizable cationic lipids (A4B2C8D9, A4B4C2D9) completely lose their ability to bind mRNA. Therefore, it can be concluded that isonitrile-headed ionizable cationic lipids have a stronger interaction with mRNA, especially under neutral conditions with strong hydrogen bonding, which can achieve highly efficient mRNA binding.

[0044] Example 4: Preparation and characterization of drug-loaded three-component lipid nanoparticles (LNPs) By utilizing the strong hydrogen bonding between the ionizable cationic lipids with isonitrile head groups and mRNA under neutral conditions, drug-loaded LNPs (Fluc mRNA LNPs) encoding luciferase mRNA (Fluc mRNA) can be directly mixed and prepared under nuclease-free water conditions, simplifying the traditional four-component LNP preparation process.

[0045] An organic phase was prepared by dissolving three lipid components—ionizable cationic lipids (A4B2C2D2), cholesterol, and DMG-PEG2000 (polyethylene glycol-modified lipids)—in ethanol at a specific molar ratio. An aqueous phase was prepared by diluting Fluc mRNA of the corresponding mass ratio in a nuclease-free aqueous solution (without requiring an acidic buffer). The aqueous and organic phases were then manually and rapidly mixed at a volume ratio of 3:1 using a pipette. After standing for 10 minutes, the mixture was placed in a 1000 Da dialysis bag and dialyzed in 1×PBS solution at 4°C for 2 hours to obtain Fluc mRNA LNPs, in which the mass ratio of ionizable cationic lipids to nucleic acids was ionizable cationic lipids:nucleic acid = 10:1.

[0046] The particle size, polydispersity index (PDI), and zeta potential of diluted Flux mRNA LNPs were determined using a Malvern particle size analyzer, and the mRNA encapsulation efficiency was detected using the RiboGreen RNA assay. The specific results are shown in Table 3. Table 3 Physicochemical properties of Fluc mRNA-LNP prepared by manual pipetting method (different LNPs are named according to the corresponding molar ratio of ionizable cationic lipid / cholesterol / DMG-PEG2000). Furthermore, by fixing the molar ratio of the three lipid components—ionizable cationic lipids, cholesterol, and DMG-PEG2000 (polyethylene glycol-modified lipids)—at 45:53.5:1.5, drug-loaded three-component Fluc mRNA LNPs based on ionizable cationic lipids A4B2C5D3 or A4B2C6D3 were prepared. The specific results are shown in Table 4. Table 4 Physicochemical properties of Fluc mRNA-LNP prepared by manual pipetting method (different LNPs are named according to their corresponding ionizable cationic lipids). Furthermore, with the molar ratio of the three lipid components—ionizable cationic lipids, cholesterol, and DMG-PEG2000 (polyethylene glycol modified lipids)—fixed at 45:53.5:1.5, the aqueous and organic phases were rapidly and uniformly mixed using a microfluidic method at a volume ratio of 3:1 to prepare drug-loaded three-component Fluc mRNA LNPs based on ionizable cationic lipids A4B2C2D2, A4B2C5D3, or A4B2C6D3. Specific results are shown in Table 5. Table 5 Physicochemical properties of Fluc mRNA-LNP prepared by microfluidic method (different LNPs are named according to their corresponding ionizable cationic lipids). As experimental controls, three-component drug-loaded LNPs (A4B2C2D2 / DSPC / DMG-PEG2000 and A4B2C2D2 / cholesterol / DSPC) were prepared by manual rapid mixing with a pipette. The specific results are shown in Table 6 below: Table 6 Physicochemical properties of Fluc mRNA-LNPs obtained by manual pipetting method (different LNPs are named according to the corresponding lipid molar ratios in the three LNP components). Example 5: Animal experiment to detect mRNA expression level after intramuscular injection (1) Different drug-loaded LNPs prepared in Example 2 or Example 4 were used to detect the in vivo protein expression efficiency of different LNPs after intramuscular injection in animal experiments. The Flux mRNA reporter gene was used as a drug model.

[0047] The specific operating method is as follows: 6-8 week old C57BL / 6 mice were injected intramuscularly with 100 μL of different groups of Fluc mRNA-LNP (see Tables 2 or 4 for specific LNP groups). The mRNA dose was 0.125 mg / kg per mouse. Six hours later, the mice were injected intraperitoneally with 200 μL of the luciferase substrate D-Luciferin potassium salt (150 mg / kg per mouse). After 6 minutes, the mice were anesthetized and placed in a small animal imaging system for bioluminescence imaging. If necessary, the mice were sacrificed, and organs were collected for in vitro bioluminescence imaging. The in vivo mRNA delivery efficiency of LNP was assessed by the luminescence intensity of firefly luciferase in muscle tissue or organs.

[0048] The results are as follows Figure 7 As shown, A4B2C2D2 and A4B4C2D9 exhibited the strongest mRNA expression efficiency at the intramuscular injection site. However, further analysis of the mRNA expression ratio (MLR, where a higher value indicates stronger muscle selectivity and lower off-target expression in the liver) revealed significant differences. Figure 8 The results show that LNPs with isonitrile head groups that are ionizable cationic lipids have higher muscle selectivity (MLR>10), among which A4B2C2D2 has the highest efficiency in muscle-selective mRNA delivery and expression.

[0049] Furthermore, using A4B2C2D2 as a baseline, the unsaturation degree of the hydrophobic tail chain of the isonitrile-headed ionizable cationic lipid and the number of methylene spacers in the ionizable cationic head group were optimized to obtain bioluminescence imaging results of animals and isolated organs after intramuscular injection of four-component drug-loaded LNPs, as shown in the figure. Figure 9 As shown in Table 7 below, the statistical results of the corresponding liver and muscle expression are presented.

[0050] Table 7. In vivo expression of drug-loaded LNPs in different groups after intramuscular injection. Furthermore, using A4B2C2D2 as a baseline, the LNP formulation and process were optimized to prepare a three-component drug-loaded LNP. The bioluminescence imaging results of the LNP after intramuscular injection in animals and isolated organs are as follows: Figure 10As shown in Table 7, the statistical results of expression in the corresponding liver and muscle are presented. The results indicate that, among the four-component LNPs, the isonitrile-headed ionizable cationic lipid-based LNP exhibits higher muscle selectivity compared to the commercial lipid SM102 (MLR=1.79), with a significantly higher MLR value. Among the three-component LNPs, the ionizable cationic lipid / DSPC / DMG-PEG2000 and ionizable cationic lipid / cholesterol / DSPC formulations showed poor performance, while the LNP based on ionizable cationic lipid / cholesterol / DMG-PEG2000 demonstrated excellent in-situ expression performance in muscle and a higher MLR value (greater than 350), indicating better muscle-selective mRNA delivery and significantly avoiding safety risks caused by off-target expression in the liver.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An ionizable cationic lipid, characterized in that, It has the structure shown in equation (I): --Formula (I); In the formula, n = 0~3; R1 and R2 are independently selected from methyl or ethyl; M1 is independently selected , , , , , , ; M2 is selected independently , , , ; M3 is selected independently , , , , , , , .

2. The ionizable cationic lipid according to claim 1, characterized in that, The n = 1 or 2; And / or, the M1 is independently selected from , , , ; And / or, the M2 is independently selected from , , ; And / or, the M3 is independently selected from , , , , , , .

3. The ionizable cationic lipid according to claim 2, characterized in that, The n = 2; And / or, M1 is ; And / or, the M2 is ; And / or, the M3 is independently selected from , , , , .

4. A lipid nanoparticle, characterized in that, It includes the ionizable cationic lipids, cofactor phospholipids, sterols and polymer-modified lipids as described in any one of claims 1 to 3.

5. The lipid nanoparticles according to claim 4, characterized in that, The auxiliary phospholipid is at least one of distearylphosphatidylcholine (DSPC), dilauryl lecithin (DLPC), dioleoyl lecithin (DOPC), dimyristoyl lecithin (DMPC), 1-palmitoyl-2-oleoyl lecithin (POPC), dipalmitoyl lecithin (DPPC), dioleoyl phosphatidylethanolamine (DOPE), dimyristoyl phosphatidylethanolamine (DMPE), distearylphosphatidylethanolamine (DSPE), and dipalmitoyl phosphatidylethanolamine (DPPE); the sterol is at least one of cholesterol, sitosterol, stigmasterol, and cholesterol derivatives; the polymer-modified lipid is at least one of polyethylene glycol-modified lipid, polysarcosine-modified lipid, or polyzwitterionic lipid and its derivatives; the molar ratio of the ionizable cationic lipid, sterol, auxiliary phospholipid, and polymer-modified lipid is (25~75):(20~50):(5~25):(0.5~10).

6. A lipid nanoparticle, characterized in that, Includes the ionizable cationic lipids, sterols, and polymer-modified lipids as described in any one of claims 1 to 3.

7. The lipid nanoparticles according to claim 6, characterized in that, The sterol is at least one of cholesterol, sitosterol, stigmasterol, and cholesterol derivatives; the polymer-modified lipid is at least one of polyethylene glycol-modified lipid, polysarcosine-modified lipid, or zwitterionic lipid and its derivatives; the molar ratio of the ionizable cationic lipid, sterol, and polymer-modified lipid is (90~10):(88~8):(0.5~10).

8. A drug-loaded nanoparticle, characterized in that, Includes the lipid nanoparticles and drugs described in any one of claims 4 to 7.

9. The drug-loaded nanoparticles according to claim 8, characterized in that, The drug comprises at least one of small molecule compounds, nucleic acid molecules, protein or polypeptide molecules, and gene editing complexes.

10. The drug-loaded nanoparticles according to claim 9, characterized in that, The nucleic acid molecule is at least one of messenger RNA, circular RNA, transfer RNA, dsRNA, shRNA, DNA, plasmid DNA, siRNA, antisense oligonucleotide, aiRNA, and miRNA; the gene editing complex is mRNA / sgRNA or Cas9 / sgRNA.

11. The drug-loaded nanoparticles according to claim 10, characterized in that, The mass ratio of the ionizable cationic lipid to the nucleic acid molecule is (2~50):

1.

12. A method for preparing lipid nanoparticles according to any one of claims 8 to 11, characterized in that, Includes the following steps: (1) Dissolve the ionizable cationic lipids, sterols, auxiliary phospholipids and polymer-modified lipids in an organic solution to obtain an organic phase; (2) Dissolve the drug in a buffer solution to obtain an aqueous phase; The volume ratio of the aqueous phase to the organic phase is (1~6):1; (3) The aqueous phase and the organic phase are quickly mixed evenly and dialyzed to obtain the product.

13. A method for preparing lipid nanoparticles according to any one of claims 8 to 11, characterized in that, Includes the following steps: (1) Dissolve the ionizable cationic lipid, sterol and polymer-modified lipid in an organic solution to obtain an organic phase; (2) Dissolve the drug in a buffer solution, wherein the buffer solution is nuclease-free water or PBS solution, to obtain an aqueous phase; The volume ratio of the aqueous phase to the organic phase is (1~6):1; (3) The aqueous phase and the organic phase are quickly mixed evenly and dialyzed to obtain the product.

14. The use of the ionizable cationic lipids of any one of claims 1 to 3, the lipid nanoparticles of any one of claims 4 to 7, and the lipid nanoparticles of any one of claims 8 to 11 in the preparation, delivery, or transport of molecular drugs or nucleic acid vaccines.

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