A novel ionizable cationic lipid compound, nanoparticles, and methods of making and using the same

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a novel ionizable cationic lipid compound, nanoparticles, their preparation method, and applications. The structure of the ionizable cationic lipid compound is shown in formula (Ⅰ), wherein the substituents R1, R2, G1, G2, G3, and G4 are defined as described herein. Using the novel ionizable cationic lipid provided by this invention, lipid nanoparticles composed of neutral auxiliary lipids, cholesterol, and polyethylene glycol-modified lipids are efficiently encapsulated with the mRNA of therapeutic proteins via microfluidic technology. This system features high encapsulation efficiency, excellent delivery efficiency, and good biocompatibility, exhibiting superior protein expression or gene editing effects compared to the benchmark lipid DLin-MC3-DMA in vivo, for example, achieving significant protein knockdown in TTR gene editing therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a novel ionizable cationic lipid compound, nanoparticles, their preparation methods, and applications. Background Technology

[0002] Messenger RNA (mRNA) is a class of single-stranded RNA molecules transcribed from a DNA template. It carries genetic information and guides protein synthesis within cells. Based on its coding function, mRNA is considered to have broad therapeutic potential, including expressing vaccine antigens, replacing missing or dysfunctional proteins, and gene editing. However, mRNA molecules themselves have significant limitations in drug development: they are unstable in physiological environments and easily degraded by nucleases; furthermore, due to their large molecular weight, strong hydrophilicity, and negative charge, they struggle to autonomously cross cell membrane barriers, resulting in low cytoplasmic delivery efficiency. Therefore, developing safe and efficient delivery systems is crucial for the clinical translation of mRNA.

[0003] Lipid nanoparticles (LNPs) are among the most promising mRNA delivery carriers, typically composed of ionizable cationic lipids, cholesterol, polyethylene glycol-modified lipids, and neutral auxiliary lipids. These components work synergistically to achieve efficient encapsulation, stable in vivo transport, cellular uptake, and intracellular release of mRNA. Ionizable cationic lipids, as the core functional material of LNPs, can protonate under acidic conditions and complex mRNA via electrostatic interactions to form a stable complex. After entering the cell, they further promote membrane fusion and content release in the acidic environment of the endosomal environment, thereby completing the cytoplasmic delivery of mRNA.

[0004] While existing LNP technology has achieved success in some applications, its core component—ionizable cationic lipids—still faces the challenge of balancing delivery efficiency with biosafety. Many cationic lipids, while enhancing transfection efficiency, can induce significant cytotoxicity or in vivo immune responses, limiting their clinical applications. Therefore, there is an urgent need for the systematic design and optimization of the chemical structure of ionizable cationic lipids to significantly reduce their toxic side effects while maintaining efficient mRNA delivery, thereby driving the development of mRNA drugs towards safer and broader indications. Summary of the Invention

[0005] To address the aforementioned shortcomings, this invention aims to provide a novel type of ionizable cationic lipid with novel structure, high delivery efficiency, and good biocompatibility, as well as lipid nanoparticle (LNP) compositions and mRNA formulations containing the lipid, in order to overcome the above-mentioned defects in existing delivery systems.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a novel ionizable lipid compound, wherein the ionizable cationic lipid compound has the structure shown in formula (I): , in: R1 is selected from nitrogen-containing alkyl groups containing substituents or nitrogen-containing five- or six-membered heterocyclic alkyl groups containing one or more substituents; G1, G2, G3 and G4 may be the same or different, and each is independently selected from unsubstituted C1-C4 alkylene groups; R2 is selected from (i) substituted or unsubstituted C6-C 23 (ii) One of straight-chain or branched alkyl groups; (iii) One of branched alkyl groups containing an ester group.

[0008] Preferably, the heterocyclic alkyl group in R1 refers to a cycloalkyl group that includes at least one heteroatom selected from O, N and S.

[0009] Preferably, R1 is selected from nitrogen-containing alkyl groups containing substituents, or nitrogen-containing five- or six-membered heterocyclic alkyl groups containing one or more substituents, such as any one of the following: .

[0010] Preferably, R2 is selected from (i) substituted or unsubstituted C6-C. 23 (ii) One of straight-chain or branched alkyl groups; such as any of the following: .

[0011] Preferably, the ionizable lipid compound has the structure shown in formula (II): , The definitions of R1 and R2 are the same as those in the previous text.

[0012] The novel ionizable cationic lipid compounds of this invention include compounds with the following structural formulas:

[0013] Secondly, the present invention provides a method for synthesizing novel ionizable cationic lipid compounds, the preparation method comprising the following steps: (1) Reaction of bis(ethylene sulfone)methane (≥2 equivalents) with an organic amine compound (1 equivalent) in an appropriate amount of dichloromethane at room temperature; (2) Add a mercapto compound (≥2 equivalents) to the reaction system of step (1) and continue the reaction until the reaction is complete; An exemplary synthesis path is shown below: , The definitions of R1 and R2 are the same as those in the previous text.

[0014] Preferably, the solvent is selected from dichloromethane, tetrahydrofuran, methanol, or ethanol.

[0015] Preferably, the reaction temperature is 15-35℃ and the reaction time is 4-15h.

[0016] More preferably, the solvent is selected from dichloromethane.

[0017] More preferably, the reaction temperature is selected from one of 15℃, 17.5℃, 20℃, 22.5℃, 25℃, 27.5℃, 30℃, 32.5℃, and 35℃.

[0018] More preferably, the reaction time is selected from one of 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, and 15h.

[0019] Thirdly, the present invention provides lipid nanoparticles based on novel ionizable cationic lipids, wherein the lipid nanoparticles comprise: (1) Ionizable cationic lipids as described in the first aspect, which are core functional materials; (2) Neutral auxiliary lipids used to stabilize lipid bilayer structures; (3) Cholesterol used to regulate membrane fluidity; (4) Polyethylene glycol-modified lipids used to improve particle stability and in vivo circulation time.

[0020] Preferably, the molar ratio of the ionizable cationic lipid, neutral auxiliary lipid, cholesterol and polyethylene glycol lipid is (30-50):16:(5-50):2.5.

[0021] More preferably, the molar ratio of the ionizable cationic lipid, neutral auxiliary lipid, cholesterol and polyethylene glycol lipid is (30-45):16:(30-50):2.5.

[0022] More preferably, the molar ratio of the ionizable cationic lipid, neutral auxiliary lipid, cholesterol and polyethylene glycol lipid is 35:16:46.5:2.5.

[0023] Preferably, the novel ionizable cationic lipid compound is selected from the constructed library.

[0024] Preferably, the neutral auxiliary lipid is selected from at least one of DOPE, DSPC, or DPPC.

[0025] Preferably, the polyethylene glycol lipid is selected from at least one of DMG-PEG 2000, DSPE-PEG 2000 or DSPE-PEG 5000.

[0026] Specifically, the sequence of the mRNA is shown in SEQ ID NO.1.

[0027] A fourth aspect of the present invention relates to a method for preparing the aforementioned lipid nanoparticles, comprising the following steps: Step 1, Preparation of organic phase: Dissolve the ionizable cationic lipids, neutral auxiliary lipids, cholesterol and polyethylene glycol-modified lipids in a polar organic solvent at a preset molar ratio to obtain an organic phase solution; Step 2, Preparation of aqueous phase: Dissolve the therapeutic or functional mRNA in a buffer solution with a suitable pH to prepare an aqueous mRNA solution; Step 3, Mixing and Self-Assembly: In the microfluidic chip, the organic phase solution and the mRNA aqueous phase solution are mixed at a constant flow rate ratio to promote the spontaneous assembly of lipids and mRNA to form an encapsulated complex, and the resulting crude product is collected.

[0028] Step 4, purification: The crude product is dialyzed or tangentially filtered to remove organic solvents and unencapsulated components, thereby obtaining a purified aqueous dispersion of lipid nanoparticles.

[0029] Preferably, the polar organic solvent in step one is anhydrous ethanol, and the concentration of each component in the organic phase solution is 4-6 mg / mL.

[0030] Preferably, the concentration of each component in the organic phase in step one is 5 mg / mL.

[0031] Preferably, in step two, the mRNA is dissolved in an acidic buffer solution with a pH of 3.8-4.2.

[0032] Preferably, in step two, the concentration of mRNA in the aqueous mRNA solution is 30-80 μg / mL.

[0033] Preferably, the citrate-sodium citrate buffer has a pH of 4.0 and an mRNA concentration of 60 μg / mL.

[0034] Preferably, in step three, the volume ratio of the organic phase solution to the mRNA aqueous phase solution can be 1:(2.5-3.5), and the total flow rate can be 10-14 mL / min.

[0035] Preferably, the volume ratio of the organic phase to the aqueous phase is 1:3, and the total flow rate is 12 mL / min.

[0036] Preferably, in step four, dialysis is performed using a dialysis membrane with a molecular weight cutoff of 18-22 kDa, with the external aqueous phase being a neutral buffer solution with pH = 7.2-7.6, and dialysis is performed at 23-27°C for 5-7 hours; or ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 100-300 kDa, concentrating the lipid nanoparticles to a concentration of 0.1-0.3 mg / mL.

[0037] Compared with the prior art, the present invention has the following significant advantages: (1) High encapsulation and delivery efficiency: The novel ionizable cationic lipid used in this invention, through innovative design of its chemical structure, can be efficiently protonated in an acidic environment to encapsulate and protect mRNA, and precisely trigger release in the cell. This design optimizes lipophilicity and charge distribution at the molecular level, which can significantly reduce cell membrane damage and immunogenicity caused by the lipid itself while achieving high transfection efficiency, fundamentally overcoming the technical bottleneck of traditional cationic lipids in achieving both delivery efficiency and biosafety. (2) Simple preparation process: The preparation method clearly optimizes the molar ratio of each component, phase mixing conditions and purification process; in particular, the precise and rapid mixing of the two phases is achieved through microfluidic technology, which ensures that the LNP particle size is uniform and the dispersion is low, and achieves high encapsulation rate of mRNA; (3) Wide range of applications and great therapeutic potential: Based on the excellent delivery performance and safety of this novel LNP system, it can be widely used in many fields such as preventive vaccines, protein replacement therapy, tumor immunotherapy and gene editing. Attached Figure Description

[0038] Figure 1 Morphological image of the lipid nanoparticles prepared in Example 2 under a transmission electron microscope; Figure 2 The pKa result diagram is shown for the lipid nanoparticles prepared in Example 2; Figure 3 The image shows the cellular-level mRNA delivery effect of the lipid nanoparticles prepared in Experiment Example 4. Figure 4 This image shows the animal-level delivery effect of the prepared lipid nanoparticles. Figure 5 The image shows the effect of the prepared lipid nanoparticles used in gene editing therapy. Detailed Implementation

[0039] To make the objectives, technical solutions, and beneficial effects of this invention clearer, detailed explanations are provided below through specific embodiments. It should be noted that these embodiments are for illustrative purposes only and do not constitute any limitation on the scope of the invention. Unless otherwise stated, the experimental methods described in the embodiments are conventional techniques in the art, and the materials and reagents used are commercially available.

[0040] The firefly luciferase mRNA drug described in this embodiment of the invention was purchased from Myannah.

[0041] The mice used in this embodiment of the invention are wild-type C57BL / 6 mice, 6-8 weeks old, female, weighing about 18-20g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0042] Compound 36 represents a novel ionizable cationic lipid, with the structural formula shown in Formula 1; DLin-MC3-DMA represents an ionizable cationic lipid, with the structural formula shown in Formula 2; DOPE represents a neutral auxiliary lipid, with the structural formula shown in Formula 3; Cholesterol represents a sterol lipid, with the structural formula shown in Formula 4; and DMG-PEG 2000 represents a polyethylene glycol lipid, with the structural formula shown in Formula 5.

[0043]

[0044]

[0045] Example 1 Preparation of compound 36

[0046] 1-(2-aminoethyl)piperidine (100 mg, 779.92 µmol) was dissolved in 5 mL of dichloromethane, and bis(vinylsulfone)methane (306.09 mg, 1.56 mmol) was added. The mixture was stirred at room temperature for 4 h. After the reaction was complete, 5-methylheptyl-3-mercaptopropionate (318.71 mg, 1.56 mmol) was added to the reaction solution from the first step, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the product was finally purified by column chromatography to obtain approximately 600 mg of product (yield 82.8%; high-resolution mass spectrometry data 928.5).

[0047] Compound 36 detection data: 1 H NMR (500MHz, DMSO) δ5.53(s, 4H), 4.13(d, 4H), 3.78-3.51(d ,8H),3.0–2.92(d, 8H), 2.83(d, 4H), 2.58(d, 4H), 2.42-2.37(t ,8H), 1.62-1.49(m ,10H), 1.37-1.19(m ,10H), 0.91(d ,12H) MS (ESI): m / z of [M+H] + = 928.5, (calc.929.35).

[0048] The ionizable lipid compounds described in this invention are all chemically synthesized using the methods described above, with yields all greater than 60%. Based on the compounds prepared in Example 1 above, an mRNA delivery system, mRNA-LNPs, was constructed. The preparation method of the ionizable lipid nanoparticles and their physicochemical properties, such as particle size, potential, encapsulation efficiency, and pKa, were investigated to evaluate their formulation characteristics.

[0049] Example 2 This embodiment provides a novel ionizable lipid nanoparticle, which is prepared by the following steps: (1) Preparation of organic phase: The novel ionizable cationic lipid (compound 36 prepared in Example 1), DOPE, cholesterol, and DMG-PEG 2000 were respectively prepared into single-component stock solutions of 5 mg / mL with anhydrous ethanol. Subsequently, the corresponding volumes of each component stock solution were transferred in sequence according to the predetermined molar ratio (compound 36:DOPE:Cholesterol:DMG-PEG 2000=35:16:46.5:2.5) and mixed thoroughly to obtain a homogeneous organic phase; (2) Preparation of aqueous phase: The mRNA encoding the target protein (the sequence of which is shown in SEQ ID NO.1) was dissolved in citrate-sodium citrate buffer at pH=4.0 to prepare an aqueous solution with a concentration of 60 μg / mL; (3) Microfluidic mixing and particle shaping: The microfluidic chip tubing was pre-cleaned and wetted with citrate buffer (pH 4.0) and anhydrous ethanol. 200 μL of organic phase and 600 μL of aqueous phase were respectively loaded into a 1 mL syringe and fixed to a dual-channel syringe pump. The flow rates of the aqueous phase and the organic phase were set to 9 mL / min and 3 mL / min respectively (total flow rate 12 mL / min, volume ratio of the two phases 3:1), the program was started to mix, and the outflowing crude product suspension was collected. (4) Dialysis purification: The crude product suspension was transferred into a dialysis bag with a molecular weight cutoff of 20 kDa, and dialysis was performed at 25°C for 6 hours using 1×PBS buffer (pH=7.4) as the external dialysis solution. After completion, the purified lipid nanoparticle (LNP) final formulation was obtained.

[0050] The mRNA sequence is shown in SEQ ID NO.1:

[0051] Experimental Example 1: Particle size, PDI, and Zeta potential tests of the LNPs prepared in Example 2. The testing method is as follows: The particle size, polydispersity index (PDI), and zeta potential of LNP were characterized using a Malvern Zetasizer Nano. Before testing, the LNP stock solution was diluted with ultrapure water (10 μL of stock solution added to 1 mL of ultrapure water). Particle size and PDI were determined by dynamic light scattering (DLS): an appropriate amount of diluted sample was added to a cuvette, equilibrated at 25°C for 1 minute, and then measured at 5-second intervals. Zeta potential was measured using a dedicated electrode cell under the same instrument and conditions.

[0052] Measurement Results Table 1 Figure 1 As shown, the prepared lipid nanoparticles have a particle size of 93.47 nm and a PDI value of 0.1965. Transmission electron microscopy also shows that the LNPs exhibit a regular spherical structure. In addition, the lipid nanoparticles carry a certain negative charge, with a Zeta potential of 5.59 mV.

[0053] Table 1

[0054] Experimental Example 2: pKa value test of LNP prepared in Example 2 The testing method is as follows: The apparent pKa of the LNP was determined using the TNS fluorescent probe method. The determination was performed using the following buffer system: 150 mM NaCl, 20 mM sodium phosphate, 25 mM sodium citrate, and 20 mM sodium acetate. The pH of the sample environment was gradually adjusted from 2.0 to 12.0 using this buffer (adjustment step size of 0.5 pH units). At each set pH, the fluorescence intensity of the TNS at an excitation wavelength of 325 nm and an emission wavelength of 435 nm was recorded. The apparent pKa of the sample was defined as the pH value corresponding to 50% maximum fluorescence intensity (i.e., half-protonation). The results are shown in Table 1. Figure 2 As shown, the pKa value of this lipid nanoparticle is 6.72, indicating that it will protonate and become positively charged in a weakly acidic environment, which is conducive to its encapsulation of negatively charged nucleic acids (such as mRNA) through electrostatic interaction.

[0055] Experimental Example 3: Determination of mRNA drug encapsulation efficiency.

[0056] The testing method is as follows: The encapsulation efficiency of mRNA was determined by fluorescence assay using the Quant-iT RiboGreen RNA reagent. The fluorescence intensity of this reagent is directly proportional to the RNA content. Total RNA and free RNA were measured separately: 1 μL of LNP-mRNA sample (prepared in Example 2) was mixed with 99 μL of Triton X-100 (2%, v / v) to completely lyse the particles, and the fluorescence signal of total RNA was measured; separately, 1 μL of the same sample was mixed with 99 μL of TE buffer, and the fluorescence signal of unencapsulated free RNA was measured.

[0057] The formula for calculating encapsulation efficiency (EE%) is: EE% = (1 (Afree / Atotal) × 100%. Where Afree refers to the amount of free mRNA, and Atotal refers to the total amount of all mRNA in Triton X-100.

[0058] The results are shown in Table 1, with an encapsulation efficiency of 85% for the lipid nanoparticles. This result indicates that the prepared lipid nanoparticles can efficiently encapsulate mRNA drugs within the particles.

[0059] Experiment Example 4: Screening of cellular-level mRNA delivery efficiency of prepared lipid nanoparticles To evaluate the delivery efficiency of novel ionizable lipid nanoparticles, chemiluminescence experiments were conducted in HeLa cells.

[0060] The preparation method of lipid nanoparticles loaded with firefly luciferase mRNA is the same as in Example 2, except that 90 novel ionizable cationic lipids prepared in this invention were tested in the preparation of the organic phase to prepare 90 lipid nanoparticles loaded with firefly luciferase mRNA, numbered 1-90. Meanwhile, the commercially available ionizable cationic lipid DLin-MC3-DMA was used as a control. Specifically, in the preparation of lipid nanoparticles loaded with firefly luciferase mRNA, the novel ionizable cationic lipid was replaced with DLin-MC3-DMA in the preparation of the organic phase according to the method in Example 2, thus obtaining DLin-MC3-DMA LNPs.

[0061] Lipid nanoparticles loaded with firefly luciferase mRNA (20 ng / well) (novel ionizable cationic lipid nanoparticles 1-90, DLin-MC3-DMA LNP) were sequentially added to 96-well plates containing HeLa cells. After incubation for 24 h, the cells were washed once with PBS, and 20 μL of cell lysis buffer and 50 μL of luciferase substrate were added. Chemiluminescence values ​​were measured. Four parallel wells were set up for each sample, and the final chemiluminescence value was the average of the four samples.

[0062] The data is presented in the form of a heatmap. Figure 3 The chemiluminescence values ​​of the novel ionizable cationic lipid nanoparticles were calculated relative to the chemiluminescence values ​​of DLin-MC3-DMA LNPs. The results demonstrated that the prepared novel ionizable cationic lipid nanoparticles exhibited high mRNA delivery efficiency, with the lipid nanoparticles assembled from compound 36 showing the best delivery performance.

[0063] Experiment Example 5: Screening of mRNA delivery efficiency at the animal level Screening of the mRNA delivery efficiency of the prepared lipid nanoparticles at the animal level. The test method is as follows: The lipid nanoparticles assembled from compound 36 (prepared in Example 4) selected at the cell level were compared with the DLin-MC3-DMA LNPs (prepared in Example 4) obtained from the baseline lipid DLin-MC3-DMA at the animal level for mRNA delivery. 2 μg of encapsulated mRNA-LNP was injected via the tail vein, followed by intraperitoneal injection of 15 mg / mL luciferase substrate 6 hours later. Imaging was performed using a small animal in vivo imaging system 5 minutes later.

[0064] Imaging results as follows Figure 4 As shown in the figure, 36 represents lipid nanoparticles assembled from compound 36, and MC3 represents DLin-MC3-DMA LNP. Figure 4 The results showed that the lipid nanoparticles assembled with compound 36 had a higher animal-level delivery efficiency than DLin-MC3-DMA LNP. This indicates that a novel lipid nanoparticle has been successfully developed.

[0065] Experimental Example 6: The prepared lipid nanoparticles were used for TTR gene editing therapy. The lipid nanoparticles assembled from compound 36 are prepared by the following method: (1) Preparation of organic phase: Compound 36, DOPE, cholesterol, and DMG-PEG 2000 were prepared into single-component stock solutions of 5 mg / mL using anhydrous ethanol. Subsequently, the corresponding volumes of each component stock solution were transferred in sequence according to the predetermined molar ratio (compound 36:DOPE:Cholesterol:DMG-PEG 2000=35:16:46.5:2.5) and mixed thoroughly to obtain a homogeneous organic phase.

[0066] (2) Preparation of aqueous phase: Cas9 mRNA and sg RNA were mixed at a mass ratio of 1:4 and dissolved in citrate-sodium citrate buffer at pH 4.0 to prepare an aqueous phase solution with a total concentration of 60 μg / mL.

[0067] (3) Microfluidic mixing and particle shaping: The microfluidic chip tubing was pre-cleaned and moistened with citrate buffer (pH 4.0) and anhydrous ethanol. 200 μL of organic phase and 600 μL of aqueous phase were respectively loaded into a 1 mL syringe and fixed to a dual-channel syringe pump. The flow rates of the aqueous phase and the organic phase were set to 9 mL / min and 3 mL / min respectively (total flow rate 12 mL / min, volume ratio of the two phases 3:1), the program was started to mix, and the crude product suspension was collected.

[0068] (4) Dialysis purification: The crude product suspension was transferred into a dialysis bag with a molecular weight cutoff of 20 kDa, and dialysis was performed at 25°C for 6 hours using 1×PBS buffer (pH=7.4) as the external dialysis solution. After completion, lipid nanoparticles of compound 36 were obtained.

[0069] The preparation method of commercial lipid nanoparticles DLin-MC3-DMA LNP is as follows: (1) Preparation of organic phase: DLin-MC3-DMA, DOPE, cholesterol (Cholesterol), and DMG-PEG2000 were prepared into single-component stock solutions of 5 mg / mL using anhydrous ethanol. Subsequently, the corresponding volumes of each component stock solution were transferred sequentially according to the predetermined molar ratio (compound 36:DOPE:Cholesterol:DMG-PEG 2000=35:16:46.5:2.5) and mixed thoroughly to obtain a homogeneous organic phase.

[0070] (2) Preparation of aqueous phase: Cas9 mRNA and sg RNA were mixed at a mass ratio of 1:4 and dissolved in citrate-sodium citrate buffer at pH 4.0 to prepare an aqueous phase solution with a total concentration of 60 μg / mL.

[0071] (3) Microfluidic mixing and particle shaping: The microfluidic chip tubing was pre-cleaned and moistened with citrate buffer (pH 4.0) and anhydrous ethanol. 200 μL of organic phase and 600 μL of aqueous phase were respectively loaded into a 1 mL syringe and fixed to a dual-channel syringe pump. The flow rates of the aqueous phase and the organic phase were set to 9 mL / min and 3 mL / min respectively (total flow rate 12 mL / min, volume ratio of the two phases 3:1), the program was started to mix, and the crude product suspension was collected.

[0072] (4) Dialysis purification: The crude product suspension was transferred into a dialysis bag with a molecular weight cutoff of 20 kDa, and dialysis was performed at 25°C for 6 hours using 1×PBS buffer (pH=7.4) as the external dialysis solution. After completion, lipid nanoparticles DLin-MC3-DMA LNP were obtained.

[0073] The two types of lipid nanoparticles prepared above were administered to mice via tail vein injection, with 5 μg mRNA each time, for a total of three administrations, each three days apart. Two days after the last administration, blood was collected via orbital sampling, and TTR protein levels were quantified using an ELISA kit.

[0074] ELISA results as follows Figure 5 As shown in the figure: Compound LNP represents the lipid nanoparticles assembled from compound 36, and MC3 LNP represents DLin-MC3-DMA LNP. Figure 5 The results showed that the lipid nanoparticles assembled with compound 36 exhibited superior TTR editing efficiency compared to DLin-MC3-DMA LNP. This demonstrates its powerful editing capability in gene editing therapy.

[0075] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A novel ionizable cationic lipid compound having the structure shown in formula (I): in: R1 is selected from nitrogen-containing alkyl groups containing substituents, or nitrogen-containing five- or six-membered heterocyclic alkyl groups containing one or more substituents; G1, G2, G3 and G4 may be the same or different, and each is independently selected from unsubstituted C1-C4 alkylene groups; R2 is selected from (i) substituted or unsubstituted C6-C 23 (ii) One of straight-chain or branched alkyl groups; (iii) One of branched alkyl groups containing an ester group.

2. The ionizable cationic lipid compound of claim 1, wherein, The heterocyclic alkyl group in R1 refers to a cycloalkyl group that includes at least one heteroatom selected from O, N, and S.

3. The ionizable cationic lipid compound of claim 1, wherein, The structure of R1 is selected from any of the following: 。 4. The ionizable cationic lipid compound of claim 1, wherein The R2 structure is selected from any of the following: 。 5. The ionizable cationic lipid compound of claim 1, wherein, The ionizable cationic lipid compound has the structure shown in formula (II): The definitions of R1 and R2 are the same as those in the previous text.

6. The ionizable cationic lipid compound of claim 5, wherein, The structure of the ionizable cationic lipid compound is selected from any of the following: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 7. A method of preparing an ionizable cationic lipid compound as claimed in any one of claims 5-6, characterized in that, The preparation method includes the following steps: (1) Reaction of bis(ethylene sulfone)methane (≥2 equivalents) with an organic amine compound (1 equivalent) in an appropriate amount of dichloromethane at room temperature; (2) Add a mercapto compound (≥2 equivalents) to the reaction system of step (1) and continue the reaction until the reaction is complete; An example reaction route is as follows: The definitions of R1 and R2 are the same as those in the previous text.

8. The preparation method according to claim 7, characterized in that, The solvent mentioned in step (1) is selected from any one of dichloromethane, tetrahydrofuran, methanol and ethanol.

9. The preparation method according to claim 7, characterized in that, The reaction temperatures for steps (1) and (2) are independently selected from 15-35℃, and the reaction times are independently selected from 4-15h.

10. The preparation method according to claim 9, characterized in that, The reaction temperature is independently selected from 15℃, 17.5℃, 20℃, 22.5℃, 25℃, 27.5℃, 30℃, 32.5℃ or 35℃, and the reaction time is independently selected from 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h or 15h.

11. A lipid nanoparticle comprising the novel ionizable cationic lipid compound according to any one of claims 1-6, characterized in that, The lipid nanoparticles also include components of neutral auxiliary lipids, cholesterol, and polyethylene glycol lipids, wherein the molar ratio of the ionizable cationic lipids, neutral auxiliary lipids, cholesterol, and polyethylene glycol lipids is (30-50):16:(5-50):2.

5.

12. The lipid nanoparticles according to claim 11, characterized in that, The neutral auxiliary lipid is selected from at least one of DOPE, DSPC or DPPC; the polyethylene glycol lipid is selected from at least one of DMG-PEG 2000, DSPE-PEG 2000 or DSPE-PEG 5000.

13. A method for preparing lipid nanoparticles according to any one of claims 11-12, characterized in that, The preparation method includes the following steps: (1) Dissolve the ionizable cationic lipids, neutral auxiliary lipids, cholesterol and polyethylene glycol-modified lipids in a polar organic solvent at a preset molar ratio to obtain an organic phase solution; (2) Dissolve the mRNA in a buffer solution to prepare an aqueous mRNA solution; (3) The organic phase solution and the mRNA aqueous phase solution are mixed at a constant flow rate ratio, and spontaneously assembled and encapsulated to obtain the crude product; (4) The crude product is filtered to obtain a purified lipid nanoparticle aqueous dispersion.

14. The preparation method according to claim 13, characterized in that, The organic solvent mentioned in step (1) is anhydrous ethanol, and the concentration of each component in the organic phase solution is 4-6 mg / mL.

15. The preparation method according to claim 13, characterized in that, The buffer solution mentioned in step (2) is an acidic buffer solution with pH=3.8-4.2, and the concentration of mRNA in the aqueous mRNA solution is 30-80 μg / mL.

16. The preparation method according to claim 13, characterized in that, The volume ratio of the organic phase solution to the mRNA aqueous phase solution in step (3) is 1:(2.5-3.5), and the flow rate is 10-14 mL / min.

17. The preparation method according to claim 13, characterized in that, The filtration in step (4) uses a dialysis membrane with a molecular weight cutoff of 18-22 kDa, with a neutral buffer solution of pH 7.2-7.6 in the external aqueous phase, and dialysis at 23-27℃ for 5-7 hours; or ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 100-300 kDa, and concentrates the lipid nanoparticles to a concentration of 0.1-0.3 mg / mL.

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