Sulfonic zwitterionic lipid molecules, nanoparticle compositions, and methods of making and using the same

Abstract

The application discloses a sulfonic zwitterionic lipid molecule, a nanoparticle composition and a preparation method and application thereof, belongs to the technical field of biological medicine, and provides a sulfonic zwitterionic lipid molecule with a specific general structure; the compound is prepared through ring-opening reaction of an amine compound and a sulfolactone, the synthesis process is simple, and a series of lipid molecule libraries can be constructed. Meanwhile, a lipid nanoparticle containing the lipid molecule, a nucleic acid-loaded lipid nanoparticle composition and a corresponding preparation method are disclosed. The sulfonic zwitterionic lipid molecule, the nanoparticle composition and the preparation method and application thereof are used, the sulfonic zwitterionic lipid molecule can be used as an auxiliary lipid, is used in combination with various main stream ionizable cationic lipids, the obtained lipid nanoparticle is stable in physical and chemical properties, has faster cell uptake efficiency and stronger endosome membrane disruption capacity. The system can realize spleen-specific transfection through intraperitoneal administration, and greatly reduces liver transfection level.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to sulfonic acid zwitterionic lipid molecules, nanoparticle compositions, their preparation methods and applications. Background Technology

[0002] Lipid nanoparticles (LNPs) are among the most widely used carriers for nucleic acid delivery, typically comprising four components: ionizable cationic lipids, helper lipids, cholesterol, and polyethylene glycol-modified lipids. Ionizable cationic lipids primarily bind to nucleic acids and undergo protonation in an acidic environment to facilitate nucleic acid delivery. Helper lipids play a crucial regulatory role in particle assembly stability, membrane interactions, cellular uptake, and intracellular transport.

[0003] In existing LNP systems, the auxiliary lipids are mostly zwitterionic lipids. Although their amount usually accounts for only about 10% to 20% of the total lipid molarity, they have a significant impact on the physicochemical properties and delivery performance of LNPs. Commonly used auxiliary lipids are mainly phospholipid zwitterionic lipids, such as distearylphosphatidylcholine (DSPC) and dioleoylphosphatidylcholine (DOPC) with phosphatidylcholine (PC) as the hydrophilic head group, and dioleoylphosphatidylethanolamine (DOPE) with phosphatidylethanolamine (PE) as the hydrophilic head group.

[0004] Different types of helper lipids possess different characteristics. DSPCs have a high phase transition temperature, which is generally beneficial for improving the compactness of particle membrane structures and system stability; DOPCs, due to their unsaturated fatty acid chains, exhibit high membrane fluidity; and DOPEs are often used to improve intracellular delivery performance in some systems. However, existing helper lipids still struggle to simultaneously meet multiple performance requirements in practical applications. For example, helper lipids that prioritize membrane stability often exhibit weaker interactions with both cell and endosomal membranes, hindering the simultaneous enhancement of cellular uptake and intracellular release; helper lipids that prioritize membrane fusion or transfection efficiency are limited in terms of particle stability and in vivo delivery performance. Furthermore, the tunable structural sites of the natural phospholipid backbone are relatively limited, making it difficult to design systems around head-base structures, hydrophobic chain structures, and their interfacial behaviors.

[0005] Therefore, providing a novel auxiliary lipid suitable for LNP systems, which can maintain particle assembly and stability while taking into account cellular uptake, intracellular release and extrahepatic organ delivery performance, is a technical problem that needs to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a sulfonic acid zwitterionic lipid molecule, nanoparticle composition, its preparation method, and its application to solve the aforementioned problems. This lipid molecule can serve as an auxiliary lipid in LNPs, co-assembling with ionizable cationic lipids, cholesterol, and polyethylene glycol-modified lipids to form lipid nanoparticles for nucleic acid delivery. LNPs containing this lipid molecule, compared to control LNPs containing DSPC, exhibit higher cellular uptake, stronger endosome membrane-breaking ability, and higher mRNA transfection efficiency, and demonstrate spleen-specific transfection under intraperitoneal injection conditions.

[0007] This invention provides a sulfonic acid zwitterionic lipid molecule with the structure shown in formula (I): (I) R1, R2, and R3 are each independently hydrogen or one of C1-C20 alkyl groups; X is one of halogen, C1-C6 alkyl, or aralkyl.

[0008] Preferably, R1, R2, R3, and X satisfy any of the following conditions: (1) R1 and R3 are both hydrogen, R2 is an aliphatic alkyl group with 1-20 carbon atoms, and X is selected from halogen, C1-C6 alkyl or aralkyl; (2) R1 is hydrogen, R2 and R3 are both aliphatic alkyl groups with 1-20 carbon atoms, and X is selected from halogen, C1-C6 alkyl or aralkyl; (3) R1, R2, and R3 are all aliphatic alkyl groups with 1-20 carbon atoms, and X is selected from one of halogen, C1-C6 alkyl or aralkyl; (4) R1 and R3 are both methyl groups, R2 is an aliphatic alkyl group with 1-20 carbon atoms, and X is selected from halogen, C1-C6 alkyl or aralkyl. (5) R1 is methyl, R2 and R3 are aliphatic alkyl groups with 1-20 carbon atoms, and X is selected from halogen, C1-C6 alkyl or aralkyl; The structure of the zwitterionic sulfonic acid lipid molecule corresponds to any one of the structures shown in formula (II): (II).

[0009] A method for preparing the above-mentioned sulfonic acid zwitterionic lipid molecule is provided, comprising the following steps: (1) Dissolve the amine compound shown in formula (III) and the sulfonyl lactone compound shown in formula (IV) in an organic solvent and carry out a ring-opening reaction under heating conditions; (III) (IV) (2) After the reaction is complete, remove the solvent, precipitate with a medium- to low-polarity organic solvent, separate and dry to obtain the zwitterionic sulfonic acid lipid molecule; The molar ratio of the amine compound to the sulfonyl lactone compound is 1:(1.2-1.5), preferably 1:1.2; the organic solvent is acetonitrile, and the ring-opening reaction is carried out at 70°C for 12-24 hours; the medium-to-low polar organic solvent is glacial ether or tetrahydrofuran.

[0010] This preparation method features mild reaction conditions, simple operation steps, and easy product purification, making it suitable for rapidly constructing a diverse library of sulfonic acid zwitterionic lipid molecules.

[0011] A lipid nanoparticle is provided, comprising the above-mentioned zwitterionic sulfonic acid lipid molecules, ionizable cationic lipids, cholesterol, and polyethylene glycol-modified lipids, wherein the components are self-assembled to form the lipid nanoparticle.

[0012] Preferably, the ionizable cationic lipid is selected from one or more of DLin-MC3-DMA, SM-102, ALC-0315, C12-200, C14-4, CKK-E12, DODAP, BAMEA-O16B, Lipid 5, and Lipid 14, with DLin-MC3-DMA being the most preferred; the PEGylated lipid is ALC-0159, DSPE-PEG, or DMG-PEG. 2k One or more of the following; the molar ratio of the ionizable cationic lipid, sulfonic acid zwitterionic lipid molecule, cholesterol and polyethylene glycol-modified lipid is 50:(10-40):(8.5-38.5):1.5, preferably 50:20:28.5:1.5.

[0013] A lipid nanoparticle composition is provided, comprising the above-described lipid nanoparticles and nucleic acid encapsulated therein; wherein the nitrogen-to-phosphorus ratio of the ionizable cationic lipid to the nucleic acid is 5-50, preferably 10.

[0014] A method for preparing the lipid nanoparticle composition as described above is provided, comprising the following steps: (1) Dissolve each component of the lipid nanoparticles in the ethanol phase; (2) Dissolve the nucleic acid in an acidic buffer solution to form an aqueous phase; (3) Mix the ethanol phase with the water phase and let it stand at room temperature to allow it to self-assemble into lipid nanoparticles carrying nucleic acids; (4) Optionally, dialysis or purification is performed to obtain the target composition.

[0015] Preferably, the acidic buffer includes, but is not limited to, 10 mM sodium citrate buffer at pH 4.2 or 10 mM HEPES buffer at pH 4.4, preferably 10 mM HEPES buffer at pH 4.4; the volume ratio of the ethanol phase to the aqueous phase is 1:3; and the incubation time at room temperature is 20 minutes.

[0016] Preferably, the lipid nanoparticle composition is used to prepare a nucleic acid delivery formulation, wherein the nucleic acid is mRNA, and the formulation is a spleen-specific transfection formulation that, after intraperitoneal administration, has spleen-specific transfection function. More preferably, the formulation is used for spleen-specific transfection via intraperitoneal administration.

[0017] Therefore, the present invention, employing the above-mentioned zwitterionic sulfonic acid lipid molecules, nanoparticle compositions, and their preparation and application, has the following beneficial effects: (1) The sulfonic acid zwitterionic lipid molecules provided are easy to synthesize and easy to post-process. By changing the amine raw materials and adjusting the sulfonyl lactone substituents, a series of sulfonic acid zwitterionic lipid molecules with well-defined structures and tunable physicochemical properties can be constructed, which facilitates structural optimization, performance screening and subsequent application research.

[0018] (2) Existing commonly used phospholipid-based auxiliary lipids are difficult to balance particle stability, cellular uptake and endosome escape capabilities simultaneously. The zwitterionic sulfonic acid lipid molecule of the present invention, when applied as an auxiliary lipid to lipid nanoparticles, can ensure normal particle assembly and physicochemical stability, while significantly improving the cellular uptake efficiency of lipid nanoparticles, enhancing the ability to disrupt endosome membranes, effectively promoting the release of nucleic acids into cells, and greatly improving the in vitro transfection efficiency of mRNA.

[0019] (3) The provided sulfonic acid zwitterionic lipids can not only be combined with DLin-MC3-DMA to form LNPs, but also can be used in combination with different ionizable cationic lipids such as SM-102 and ALC-0315. The LNPs containing the sulfonic acid zwitterionic lipids all showed better in vitro transfection levels, indicating that this type of auxiliary lipid has good formulation adaptability and application potential.

[0020] (4) The lipid nanoparticles constructed using the sulfonic acid zwitterionic lipid molecules of the present invention can reduce the level of liver transfection after intraperitoneal administration and achieve spleen-targeted specific transfection, which solves the problem of traditional lipid nanoparticles being biased towards liver transfection and provides a new material and technical solution for targeted nucleic acid delivery to extrahepatic organs.

[0021] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0022] Figure 1 The particle size and distribution characterization results of the lipid nanoparticles prepared in Example 3 of the present invention are shown in the sulfonic acid zwitterionic lipid molecule, nanoparticle composition, preparation method and application thereof. Figure 2 The transfection results of LNP-Luci mRNA in HeLa cells in Example 4 of the present invention are shown in the sulfonic acid zwitterionic lipid molecule, nanoparticle composition, preparation method and application thereof. Figure 3 This is the transfection result of LNP-eGFP mRNA in HeLa cells in Example 5 of the present invention, which describes the sulfonic acid zwitterionic lipid molecule, nanoparticle composition, its preparation method and application. In the example, a is the MFI thermogram of average fluorescence intensity; b is the eGFP... + Heatmap of positive cell percentage; Figure 4 This is the transfection result of LNP-eGFP mRNA in DC2.4 and HepG2 cells in Example 6 of the present invention, which describes the sulfonic acid zwitterionic lipid molecule, nanoparticle composition, and its preparation and application. In this example, a represents DC2.4 cells and b represents HepG2 cells. Figure 5 The results of LNP-eGFP mRNA transfection in HeLa cells under different ionizable lipid conditions in Example 7 of the present invention are shown in the sulfonic acid zwitterionic lipid molecule, nanoparticle composition, preparation method and application thereof. Figure 6 This is the uptake result of LNP-Luci(Cy5) mRNA in HeLa cells in Example 8 of the present invention, which describes the sulfonic acid zwitterionic lipid molecules, nanoparticle compositions, their preparation and application, and the results. In this example, a is the flow cytometry histogram for different incubation times; b is the fluorescence intensity statistics for different cell subpopulations. Figure 7 The results of the detection of LNP-Luci (Cy5) mRNA on the intracellular membrane disruption of HeLa cells in Example 9 of the present invention are presented in the sulfonic acid zwitterionic lipid molecule, nanoparticle composition, preparation method and application thereof. Figure 8 The distribution of LNP-Luci(Cy5) mRNA in mice after intraperitoneal injection in Example 10 of the present invention is shown in the sulfonic acid zwitterionic lipid molecule, nanoparticle composition, preparation method and application thereof. Figure 9 This is the result of transfection of LNP-Luci mRNA in mice after intraperitoneal injection in Example 11 of the present invention, which describes the sulfonic acid zwitterionic lipid molecule, nanoparticle composition, its preparation and application, and the method thereof. Detailed Implementation

[0023] To better understand the above technical solutions, a detailed description of the solutions will be provided below in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0024] The terminology used in the embodiments of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The singular forms “a,” “the,” and “the” as used in the embodiments of this invention and the appended claims are also intended to include the plural forms, and “multiple” generally includes at least two unless the context clearly indicates otherwise.

[0025] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that an article or device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such an article or device. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the article or device that includes said element.

[0026] Unless otherwise specified, all reagents and materials used are commercially available. Key compounds in the examples are described below: Ionizable lipid: DLin-MC3-DMA, CAS No.: 1224606-06-7, structural formula as follows: SM-102, CAS No.: 2089251-47-6, has the following structural formula: ALC-0315, CAS No.: 2036272-55-4, structural formula as follows: Cholesterol, CAS No.: 57-88-5, structural formula as follows: Polyethylene glycol-modified lipids: DMG-PEG 2k CAS No.: 160743-62-4, structural formula as follows: DSPC lipid, CAS No.: 816-94-4, has the following structural formula: .

[0027] Example 1 Preparation of Lipid I-1: First, dissolve 0.5 mmol of butylamine in 10 mL of acetonitrile. Separately, dissolve 0.6 mmol of 2,4-butanesulfonyl lactone in 5 mL of acetonitrile, and then slowly add this solution to the aforementioned mixture. Incubate the reaction system at 70°C for 12 hours. After the reaction, remove the solvent by rotary evaporation, precipitate the product using icy diethyl ether, and after separation and vacuum drying, obtain the target product, named I-1. The reaction formula is as follows: Proton NMR characterization of I-1: 1 H NMR (400 MHz, D2O) δ 3.13 (dd, J = 8.9, 7.2 Hz, 2H), 3.04 – 2.83(m, 3H), 2.18 – 2.06 (m, 1H), 1.89 – 1.76 (m, 1H), 1.68 – 1.49 (m, 2H), 1.32(dt, J = 15.0, 7.4 Hz, 2H), 1.25 (t, J = 6.3 Hz, 3H), 0.85 (t, J = 7.4 Hz, 3H). According to the preparation method of this embodiment, Lipid Ⅰ-2~18 can be obtained, and the corresponding reaction formula is as follows: Example 2 Preparation of Lipid II: First, dissolve 0.5 mmol of butylamine in 10 mL of acetonitrile. Separately, dissolve 0.6 mmol of 3-benzyl-1,2-oxothiacyclopentane 2,2-dioxide in 5 mL of acetonitrile, and then slowly add this solution to the aforementioned mixture. Incubate the reaction system at 70°C for 12 hours. After the reaction, remove the solvent by rotary evaporation, precipitate the product using icy diethyl ether, and after separation and vacuum drying, obtain the target product, named II-1. The reaction formula is as follows: Proton NMR characterization of II-1: 1H NMR (400 MHz, D2O) δ 7.28 – 7.20 (m, 2H), 7.17 (d, J = 7.2 Hz, 3H), 3.23 (dd, J = 13.8, 3.9 Hz, 1H), 3.02 (ddt, J = 11.1, 7.1, 4.2 Hz, 1H), 2.92 (ddd, J = 12.7, 10.1, 5.6 Hz, 1H), 2.69 (ddd, J = 15.6, 9.5, 6.3 Hz, 3H), 2.55 (dd, J = 13.8, 10.8 Hz, 1H), 1.90 (dddd, J = 15.6, 10.1, 7.2, 5.7Hz, 1H), 1.82 – 1.69 (m, 1H), 1.41 – 1.27 (m, 2H), 1.13 (h, J = 7.3 Hz, 2H), 0.71 (t, J = 7.4 Hz, 3H). According to the preparation method of this embodiment, Lipid II-2~18 can be obtained, and the corresponding reaction formula is as follows: Example 3 Preparation and characterization of lipid nanoparticles: In this embodiment, the zwitterionic sulfonic acid lipid molecules prepared in Examples 1-2 are used as auxiliary lipids in lipid nanoparticles instead of DSPC. Ionizable cationic lipids, auxiliary lipids, cholesterol, and DMG-PEG are used in a molar ratio of 50:10:38.5:1.5. 2k All lipid components were dissolved together in 20 μL of ethanol, with DLin-MC3-DMA (34 μg) being the ionizable cationic lipid. Separately, 0.5 μg of Luciferase-mRNA was dissolved in 60 μL of 10 mM HEPES buffer at pH 4.4, with a nitrogen-to-phosphorus molar ratio of 35. The nucleic acid-containing buffer was then rapidly injected into the ethanol-lipid solution, vigorously mixed by pipetting for 5 seconds, and incubated at room temperature for 20 minutes to obtain lipid nanoparticles encapsulating Luciferase-mRNA, denoted as LNP-Luci mRNA.

[0028] The lipid nanoparticles prepared were characterized using dynamic light scattering. The results showed that the prepared lipid nanoparticles had a particle size of less than 200 nm, a low polydispersity index, and a generally neutral zeta potential. Relevant test results are shown in [link to relevant data]. Figure 1 .

[0029] Example 4 Transfection experiment of LNP-Luci mRNA in HeLa cells: HeLa cells were seeded into 48-well culture plates, with 2 × 10⁶ cells per well. 4 The culture system used MEM medium supplemented with a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin at a volume fraction, and was continuously cultured for 24 hours at 37°C and 5% carbon dioxide. After culture, the medium was replaced with serum-free medium, and 20 μL of LNP-Luci mRNA prepared in Example 3 was added to each well, with a total mRNA content of 125 ng per well. After incubation for 4 hours, the medium was replaced again with complete medium, and cultured for another 36 hours.

[0030] After the entire culture process was completed, the expression level of Luciferase inside the cells was measured using a luciferase assay kit. The results were normalized based on protein content to evaluate the transfection efficiency of the vector. The experimental results showed that, among the lipid molecules screened, lipid nanoparticles prepared from lipid molecules with a benzyl group at the hydrophilic head and a double-stranded hydrophobic tail with a carbon chain length in the C10-C14 range (i.e., the products corresponding to Lipid II-12~14) achieved high transfection levels in HeLa cells. Lipid nanoparticles constructed from other types of lipid molecules also exhibited excellent mRNA transfection effects; relevant experimental results can be found in [link to relevant results]. Figure 2 .

[0031] Example 5 Transfection experiment of LNP-eGFP mRNA in HeLa cells: First, lipid nanoparticles encapsulating eGFP-mRNA were prepared, and this product was designated LNP-eGFP mRNA. Ionizable cationic lipids, cofactor lipids, cholesterol, and DMG-PEG were then mixed in a molar ratio of 50:10:38.5:1.5. 2k All lipid components were dissolved together in 20 μL of ethanol, with DLin-MC3-DMA (34 μg) being the ionizable cationic lipid. Separately, 0.5 μg of eGFP-mRNA was dissolved in 60 μL of 10 mM HEPES buffer (pH 4.4). The buffer solution was then rapidly injected into the ethanol lipid solution, and the mixture was vigorously aspirated and pipetted for 5 seconds. After standing at room temperature for 20 minutes, LNP-eGFP mRNA was obtained.

[0032] HeLa cells were seeded into 48-well culture plates, with 2 × 10⁶ cells per well. 4Cells were cultured in MEM medium supplemented with a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% carbon dioxide for 24 hours. After switching to serum-free medium, 40 μL of prepared LNP-eGFP mRNA was added to each well, resulting in a total mRNA content of 250 ng per well. Cells were incubated for 4 hours, then replaced with complete medium and cultured for another 24 hours.

[0033] Cells were washed with phosphate-buffered saline (PFS), digested with trypsin, and resuspended. The suspension was then filtered through a 45 μm nylon mesh, and cell fluorescence signals were detected by flow cytometry. The relevant results are shown below. Figure 3 Experimental results showed that among the series of structural products screened, the eGFP fluorescence intensity and the proportion of positive cells expressed by the lipid nanoparticles corresponding to Lipid II-12~14 both showed a gradual increasing trend, with the system constructed using Lipid II-14 exhibiting the best overall performance. It was selected as the auxiliary lipid in subsequent examples.

[0034] Example 6 Transfection experiments of LNP-eGFP mRNA in DC2.4 and HepG2 cells: DC2.4 cells and HepG2 cells were seeded into 48-well culture plates, with 2 × 10⁶ cells per well. 4 The culture system used DMEM medium supplemented with a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin, and was continuously cultured for 24 hours at 37°C and 5% carbon dioxide. After replacing the medium with serum-free medium, 40 μL of LNP-eGFP mRNA prepared in Example 4 was added to each well, with a total mRNA content of 250 ng per well. After incubating the cells for 4 hours, the medium was replaced again with complete medium, and the cells were cultured for another 24 hours.

[0035] Cells were collected according to the procedure described in Example 5, and cell fluorescence signals were detected using flow cytometry. The relevant detection results are shown below. Figure 4 The experimental results showed that the transfection effect of lipid nanoparticles loaded with Lipid II-14 in DC2.4 cells was better than that of the control group sample using DSPC as an auxiliary lipid, while the transfection effect of this type of lipid nanoparticle in HepG2 cells was basically the same as that of the control group sample.

[0036] Example 7 Transfection experiments of LNP-eGFP mRNA with different ionizable cationic lipids in HeLa cells: This embodiment is used to verify the compatibility of the sulfonic acid zwitterionic lipid molecule of the present invention as an auxiliary lipid with different types of ionizable cationic lipids. Three types of ionizable cationic lipids, DLin-MC3-DMA, SM-102, and ALC-0315, were selected for the experiment and labeled M, S, and A, respectively. The nitrogen-to-phosphorus ratio of the LNP-eGFP mRNA system was uniformly set to 10. The lipid components were prepared according to the molar ratios shown in Table 1, and the mixed lipid raw materials were dissolved in 10 μL of ethanol. Separately, 1 μg of eGFP-mRNA was dissolved in 30 μL of 10 mM HEPES buffer with a pH of 4.4. The aqueous buffer was then rapidly added to the ethanol lipid solution, mixed thoroughly for 5 seconds, and allowed to stand at room temperature for 20 minutes to obtain the corresponding LNP-eGFP mRNA for each group.

[0037] HeLa cells were seeded into 48-well culture plates, with 2 × 10⁶ cells per well. 4 Cells were cultured in MEM medium supplemented with a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% carbon dioxide for 24 hours. After replacing the medium with serum-free MEM, 10 μL of the prepared LNP-eGFP mRNA was added to each well, resulting in a total mRNA content of 250 ng per well. After 4 hours of incubation, the medium was replaced again with complete medium, and the cells were cultured for another 24 hours. Cells were collected according to the procedure described in Example 5, and cell fluorescence signals were detected using flow cytometry.

[0038] Table 1. Molar proportions of lipid components and nitrogen-phosphorus ratio parameters for each group.

[0039] Within the above experimental groups, the auxiliary lipid used in groups M0, S0, and A0 was DSPC, while the auxiliary lipid used in all other experimental groups was Lipid II-14.

[0040] Table 2. Particle size and polydispersity index of LNPs in each group

[0041] The ionizable lipid C12-200 is labeled as C.

[0042] The results of the transfection experiment are shown below. Figure 5Experimental results showed that, with the substitution of different types of ionizable cationic lipids, the transfection efficiency of lipid nanoparticles loaded with Lipid II-14 was superior to that of the control samples in the same group. Among them, the four groups M2, S2 and A2 were allocated in a molar ratio of 50:20:28.5:1.5, and the lipid nanoparticles under this ratio exhibited better mRNA transfection efficiency.

[0043] Example 8 Uptake experiment of LNP-Luci (Cy5) mRNA in HeLa cells: This experiment used the three formulations M0, M1 and M2 described in Example 7 for testing, and the nucleic acid used in the experiment was Luci mRNA labeled with Cy5 fluorescence.

[0044] HeLa cells were seeded into 24-well culture plates, with 5 × 10⁶ cells per well. 4 The culture system used MEM medium supplemented with a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin at a volume fraction, and was continuously cultured for 24 hours at 37°C and 5% carbon dioxide. After replacing the medium with serum-free MEM, 20 μL of LNP-Luci (Cy5) mRNA was added to each well, with a total mRNA content of 500 ng per well. Samples were taken at four time points: 0.5 hours, 1 hour, 2 hours, and 4 hours after the reaction.

[0045] Cells were washed with phosphate-buffered saline (PBS) and then digested with trypsin. The cell suspension was resuspended in PBS and filtered. Cy5 fluorescence signal was then detected by flow cytometry. The results are shown below. Figure 6 Experimental results showed that the uptake of lipid nanoparticles in HeLa cells increased with prolonged reaction time in all groups, exhibiting a clear time-dependent characteristic. Compared with the control group using DSPC as an auxiliary lipid, the lipid nanoparticles loaded with Lipid II-14 showed a higher overall level of cellular endocytosis. The M2 group exhibited the fastest cellular internalization rate, with a Cy5 positivity rate of 63.5% after 0.5 hours of reaction. This was significantly higher than the 17.2% in the M1 group and the 3.52% in the M0 group, demonstrating that increasing the proportion of Lipid II-14 in the system effectively promotes the uptake of lipid nanoparticles by cells. At 4 hours of reaction, the average Cy5 fluorescence intensity of the M1 and M2 groups remained essentially the same, with both groups showing values ​​approximately five times that of the M0 group.

[0046] Example 9 Experiment on the disruption of the endosomal membrane of HeLa cells by LNP-Luci (Cy5) mRNA: This experiment used the three formulations M0, M1 and M2 described in Example 7 for testing, and the nucleic acid used in the experiment was Luci mRNA labeled with Cy5 fluorescence.

[0047] HeLa cells were seeded into confocal culture dishes, with 1 × 10⁶ cells seeded per dish. 5 Cells were cultured in MEM medium supplemented with a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% carbon dioxide for 24 hours. After switching to serum-free MEM medium, 40 μL of LNP-Luci (Cy5) mRNA was added to each culture dish, resulting in a total mRNA content of 1 μg per dish. Cells were incubated for 4 hours before being replaced with complete medium again.

[0048] Nuclear dye was added to the system, and the mixture was incubated at 37°C for 15 minutes. The culture medium was then discarded, and the system was washed with phosphate buffer. Acridine orange dye was then added to the system, and the mixture was incubated at 37°C for another 15 minutes. The samples were observed using a confocal laser scanning microscope, maintaining consistent imaging parameters throughout the process. ImageJ software was used to quantitatively analyze the red and green fluorescence signals within the cells. The ratio of the average red fluorescence intensity to the average green fluorescence intensity was used to characterize the integrity of the endosomes. Acridine orange dye protonates upon entering the acidic endosomes, resulting in red fluorescence. Once the endosome membrane structure is disrupted, acridine orange dye is released into the cytoplasm and deprotonates, resulting in diffuse green fluorescence. Therefore, a lower ratio of the average red fluorescence intensity to the average green fluorescence intensity indicates a lower degree of endosome membrane integrity.

[0049] The relevant experimental results are shown in Figure 7 Experimental results showed that as the proportion of Lipid II-14 added to the system increased, the ratio of the average intensity of red fluorescence to the average intensity of green fluorescence gradually decreased. The ratio in group M2 was significantly lower than that in group M0, demonstrating that lipid nanoparticles with a higher proportion of Lipid II-14 had a more pronounced destructive effect on endosomal membrane structures. Combining the experimental conclusions of Examples 8 and 9, it can be concluded that the lipid nanoparticles corresponding to the formulation in group M2 possess both the characteristic of rapid cell entry and the ability to effectively facilitate the release of nucleic acids within cells. This performance characteristic is corroborated by the experimental results showing that this group of samples exhibited higher mRNA transfection efficiency.

[0050] Example 10 Distribution of LNP-Luci (Cy5) mRNA in mice after intraperitoneal injection: This experiment used the M0 and M2 formulations described in Example 7 for in vivo animal experiments. Taking the preparation process of the M2 group sample as an example, ionizable cationic lipids, auxiliary lipids, cholesterol, and DMG-PEG were taken in a molar ratio of 50:20:28.5:1.5. 2k All lipid components were dissolved together in 25 μL of ethanol, with 97 μg of DLin-MC3-DMA selected as the ionizable cationic lipid. Separately, 5 μg of Cy5-labeled Luciferase-mRNA was dissolved in 75 μL of 10 mM HEPES buffer (pH 4.4). The aqueous buffer was then rapidly added to the ethanol-lipid solution, mixed thoroughly for 5 seconds, and incubated at room temperature for 10 minutes.

[0051] A dialysis apparatus with a molecular weight cutoff of 3.5K was used to process the mixed system. The system was dialyzed against phosphate buffer (pH 7.4) for 1 hour to completely remove ethanol, ultimately yielding LNP-Luci(Cy5) mRNA suitable for in vivo administration to mice. Samples in group M0 were prepared using the same procedure. All LNP-Luci(Cy5) mRNA was administered to mice via intraperitoneal injection. Six hours after administration, mice were dissected, and in vivo imaging was used to detect Cy5 fluorescence signals. The imaging results are shown below. Figure 8 The experimental results showed that 6 hours after administration, the samples from both the M0 and M2 groups were mainly enriched and distributed in the liver and spleen tissues of mice.

[0052] Example 11 In vivo transfection experiment of LNP-Luci mRNA in mice after intraperitoneal injection: This experiment used the M0 and M2 formulations described in Example 7 for in vivo animal experiments. Taking the preparation process of the M2 group sample as an example, ionizable cationic lipids, auxiliary lipids, cholesterol, and DMG-PEG were taken in a molar ratio of 50:20:28.5:1.5. 2k All lipid components were dissolved together in 25 μL of ethanol, with DLin-MC3-DMA (97 μg) being the ionizable cationic lipid. Separately, 5 μg of Luciferase-mRNA was dissolved in 75 μL of 10 mM HEPES buffer (pH 4.4). The aqueous buffer was then rapidly added to the ethanol-lipid solution, mixed thoroughly for 5 seconds, and allowed to stand at room temperature for 10 minutes.

[0053] A dialysis apparatus with a molecular weight cutoff of 3.5K was used to process the mixed system. Dialysis was performed for 1 hour using phosphate buffer (pH 7.4) to completely remove ethanol, ultimately yielding LNP-Luci mRNA suitable for in vivo administration to mice. Samples in group M0 were prepared using the same procedure. All LNP-Luci mRNAs were administered to mice via intraperitoneal injection. Six hours after administration, 100 μL of a 30 mg / mL D-luciferase potassium solution was injected intraperitoneally into the mice. The mice were then dissected, and in vivo imaging was used to detect Luciferase luminescence signals. The relevant imaging results are shown below. Figure 9 The experimental results showed that, 6 hours after administration, compared with the M0 group sample, the M2 group sample showed a significantly weakened transfection signal in mouse liver tissue, while exhibiting an extremely strong transfection signal in spleen tissue, demonstrating that the lipid nanoparticles possess spleen-specific transfection capabilities.

[0054] Eleven sets of examples were set up to complete the full-chain experiment in sequence, including the synthesis of zwitterionic sulfonic acid lipid molecules, preparation and physicochemical characterization of lipid nanoparticles, evaluation of in vitro transfection of multiple cell lines, formulation suitability testing, analysis of cell action mechanism, and in vivo distribution and targeted transfection verification in mice.

[0055] Examples 1-2 demonstrate the preparation of Lipid I-II. This synthetic route uses amine compounds and sulfonyl lactone compounds as raw materials via a ring-opening reaction. The reaction conditions are mild, the operation is simple, and the product purification is convenient. Furthermore, this preparation method is applicable to all sulfonic acid zwitterionic lipid molecules covered by formula (II), enabling the rapid construction of diverse lipid molecule libraries, facilitating subsequent structure optimization and performance screening. The preparation method possesses good practicality and scalability.

[0056] Example 3 characterized the lipid nanoparticles prepared using some Lipid I-II as auxiliary lipids. The results showed that the obtained particles had a particle size of less than 200 nm, a small polydispersity index, and a near-neutral potential. The overall dispersibility and stability of the particles were good, meeting the physicochemical requirements of nucleic acid delivery carriers and possessing a basis for practical application.

[0057] Examples 4 to 6 were conducted in various cell lines including HeLa, DC2.4, and HepG2. The results showed that a series of zwitterionic sulfonic acid molecules with a hydrophilic head containing a benzyl group and a hydrophobic double-stranded carbon chain length of C10-C14 could all confer excellent transfection ability to lipid nanoparticles, with Lipid II-14 showing the best overall effect. Compared with the traditional helper lipid DSPC, lipid nanoparticles containing Lipid II-14 significantly improved transfection efficiency in DC2.4 immune cells and achieved a transfection level comparable to the control group in HepG2 cells, demonstrating a wide range of in vitro cell applications.

[0058] Example 7 used three mainstream ionizable cationic lipids currently used in nucleic acid delivery: DLin-MC3-DMA, SM-102, and ALC-0315, to conduct compatibility experiments. Under different lipid ratios, the transfection efficiency of lipid nanoparticles with added Lipid II-14 was significantly higher than that of the DSPC control group; the formulation with a four-component molar ratio of 50:20:28.5:1.5 showed the best performance. This demonstrates that zwitterionic sulfonic acid lipid molecules are not limited by a single lipid system, exhibiting strong formulation compatibility and high application flexibility.

[0059] Examples 8 and 9 reveal the underlying mechanism of improved transfection efficiency: on the one hand, Lipid II-14 can significantly accelerate the cellular uptake rate of lipid nanoparticles and increase the total endocytosis, and increasing its addition ratio can further enhance this effect; on the other hand, this lipid can effectively disrupt the integrity of the endosome membrane, facilitating the release of nucleic acids from the endosome into the cytoplasm. This invention overcomes the technical bottleneck of existing phospholipid-based assistive lipids, which struggle to simultaneously achieve cellular uptake and endosome escape, optimizing carrier performance throughout the entire intracellular delivery process.

[0060] Examples 10 and 11 conducted in vivo experiments involving intraperitoneal administration in mice. In vivo distribution results showed that lipid nanoparticles in both the experimental and control groups were primarily enriched in the liver and spleen. In vivo transfection results further demonstrated that lipid nanoparticles containing Lipid II-14 significantly reduced liver transfection levels and produced a significant and specific transfection effect in the spleen. This alters the inherent characteristic of traditional lipid nanoparticles that tend to target the liver, providing a novel technical solution for nucleic acid delivery to extrahepatic organs such as the spleen.

[0061] The sulfonic acid zwitterionic lipid molecule provided by this invention is simple to synthesize and has strong structural designability. When used as an auxiliary lipid in lipid nanoparticles, it can simultaneously improve cellular uptake capacity and endosomal escape efficiency while ensuring the physicochemical stability of the particles. It has excellent in vitro transfection effect and good formulation versatility. Animal experiments have confirmed that this system can achieve spleen-specific transfection, effectively making up for many shortcomings of existing phospholipid auxiliary lipids. It has high research and development value and industrialization prospects in the field of mRNA and other nucleic acid drug delivery.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. 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 still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A sulfonic acid zwitterionic lipid molecule, characterized in that, The structure is shown in equation (I): （I） R1, R2, and R3 are each independently hydrogen or one of C1-C20 alkyl groups; X is one of halogen, C1-C6 alkyl, or aralkyl.

2. The zwitterionic sulfonic acid lipid molecule according to claim 1, characterized in that, R1, R2, R3, and X satisfy any of the following conditions: (1) R1 and R3 are both hydrogen, R2 is an aliphatic alkyl group with 1-20 carbon atoms, and X is one of halogen, C1-C6 alkyl or aralkyl; (2) R1 is hydrogen, R2 and R3 are both aliphatic alkyl groups with 1-20 carbon atoms, and X is one of halogen, C1-C6 alkyl or aralkyl; (3) R1, R2, and R3 are all aliphatic alkyl groups with 1-20 carbon atoms, and X is one of halogen, C1-C6 alkyl, or aralkyl. (4) R1 and R3 are both methyl, R2 is an aliphatic alkyl group with 1-20 carbon atoms, and X is one of halogen, C1-C6 alkyl or aralkyl; (5) R1 is methyl, R2 and R3 are aliphatic alkyl groups with 1-20 carbon atoms, and X is one of halogen, C1-C6 alkyl or aralkyl; The structure of the zwitterionic sulfonic acid lipid molecule corresponds to any one of the structures shown in formula (II): （Ⅱ）。 3. A method for preparing the zwitterionic sulfonic acid lipid molecule according to claim 1, characterized in that, Includes the following steps: (1) Dissolve the amine compound shown in formula (III) and the sulfonyl lactone compound shown in formula (IV) in an organic solvent and carry out a ring-opening reaction under heating conditions; （Ⅲ） （Ⅳ） (2) After the reaction is complete, remove the solvent, precipitate with a medium- to low-polarity organic solvent, separate and dry to obtain the zwitterionic sulfonic acid lipid molecule; The molar ratio of the amine compound to the sulfonyl lactone compound is 1:(1.2-1.5), the organic solvent is acetonitrile, and the ring-opening reaction is carried out at 70°C for 12-24 hours; the medium-to-low polar organic solvent is glacial ether or tetrahydrofuran.

4. A lipid nanoparticle, characterized in that, The lipid nanoparticles are composed of zwitterionic sulfonic acid lipid molecules as described in claim 1, ionizable cationic lipids, cholesterol, and polyethylene glycol-modified lipids, and are formed by self-assembly of the components.

5. The lipid nanoparticles according to claim 4, characterized in that, The ionizable cationic lipid is selected from one or more of DLin-MC3-DMA, SM-102, ALC-0315, C12-200, C14-4, CKK-E12, DODAP, BAMEA-O16B, Lipid 5, and Lipid 14; the PEGylated lipid is ALC-0159, DSPE-PEG, or DMG-PEG. 2k One or more of the following; the molar ratio of the ionizable cationic lipid, sulfonic acid zwitterionic lipid molecules, cholesterol and polyethylene glycol-modified lipid is 50:(10-40):(8.5-38.5):1.

5.

6. A lipid nanoparticle composition, characterized in that, It includes the lipid nanoparticles as described in claim 4 and nucleic acids loaded therein; the nitrogen-to-phosphorus ratio of the ionizable cationic lipid to the nucleic acid is 5-50.

7. A method for preparing the lipid nanoparticle composition as described in claim 6, characterized in that, Includes the following steps: (1) Dissolve each component of the lipid nanoparticles in the ethanol phase; (2) Dissolve the nucleic acid in an acidic buffer solution to form an aqueous phase; (3) Mix the ethanol phase with the water phase and let it stand at room temperature to allow it to self-assemble into lipid nanoparticles carrying nucleic acids; (4) Perform dialysis or purification to obtain the target composition.

8. The method for preparing the lipid nanoparticle composition according to claim 7, characterized in that, The volume ratio of the ethanol phase to the water phase is 1:3; the standing time at room temperature is 20 minutes.

9. The application of the lipid nanoparticle composition as described in claim 6, characterized in that, This is used to prepare nucleic acid delivery formulations, wherein the nucleic acid is mRNA, and the formulation is a spleen-specific transfection formulation that, after intraperitoneal administration, has a specific transfection function.

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