Ionizable cationic lipid compound, lipid nanoparticles prepared from ionizable cationic lipid compound, and nano preparation prepared from ionizable cationic lipid compound

Abstract

The invention provides an ionizable cationic lipid compound, a lipid nanoparticle prepared from the ionizable cationic lipid compound and a nano preparation. The ionizable cationic lipid compound has a structure as shown in a formula I. The invention further provides a preparation method of the ionizable cationic lipid compound. According to the ionizable cationic lipid compound, through a specific structural design, lipid nanoparticles prepared from the ionizable cationic lipid compound are high in loading capacity, high in delivery efficiency and good in safety, and when the ionizable cationic lipid compound is used for delivering nucleic acid drugs, the in-vivo translation expression level of the nucleic acid drugs can be remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an ionizable cationic lipid compound, a lipid nanoparticle prepared therefrom, and a nanoformulation. Background Art

[0002] In recent years, technological innovations in the fields of gene therapy and nucleic acid drugs have provided new strategies for the precise intervention of various refractory diseases. Nucleic acid molecules represented by mRNA and siRNA can regulate target gene expression or repair abnormal protein functions, showing significant potential in the treatment of monogenic genetic diseases, tumor immunotherapy, and the prevention and treatment of infectious diseases. However, the clinical application of nucleic acid drugs still faces core challenges: their physical and chemical properties of high molecular weight, strong negative charge, and easy degradation by nucleases make it difficult for them to penetrate cell membranes and be stably delivered into target cells to exert their functions. Therefore, the development of safe and efficient delivery carrier technologies is the key to promoting the clinical transformation of nucleic acid drugs.

[0003] Currently, gene delivery carriers are mainly divided into two categories: viral carriers and non-viral carriers. Although viral carriers (such as recombinant adeno-associated viruses) have high transfection efficiency, their inherent defects are significant: viral structural proteins are prone to trigger host immune responses, resulting in accelerated carrier clearance and failure of repeated dosing; in addition, their large-scale production requires complex processes, high costs, and there is gene integration, which may pose clinical safety risks. Non-viral delivery systems (such as lipid nanoparticles, LNP) have the advantages of low immunogenicity and high design flexibility. By chemically synthesizing lipid components, the nucleic acid encapsulation efficiency, release kinetics, and targeting properties can be precisely regulated (such as polyethylene glycol modification to extend the circulation time), while avoiding the immune clearance problems caused by viral proteins and supporting repeated dosing. In addition, LNP can deliver various nucleic acid types (such as mRNA, siRNA, etc.), with less loading capacity limitation, and the production process is standardized, making it easy for large-scale production.

[0004] LNP encapsulates nucleic acids through electrostatic interactions, and its core functional component - cationic / ionizable lipids play a role throughout the entire process of nucleic acid drug encapsulation, delivery, and release. By optimizing the design of the cationic lipid structure, the electrostatic interaction with negatively charged nucleic acids (such as mRNA) can be enhanced, forming a tight complex, thereby improving the drug loading efficiency and delivery efficiency.

[0005] Currently, cationic lipid compounds have achieved great success in nucleic acid delivery, but it is still necessary to improve the cationic lipids and LNP nanoformulations for nucleic acid delivery to further improve the nucleic acid delivery efficiency and make them suitable for systemic or local delivery. And ensure the clinical drug safety in the treatment of patients with an effective dose of LNP formulations.

[0006] Therefore, it is an urgent problem to be solved in this field to develop an ionizable cationic lipid compound that can improve the loading capacity, delivery efficiency and safety of lipid drug carriers. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide an ionizable cationic lipid compound, lipid nanoparticles prepared therefrom and their applications. The lipid nanoparticles prepared from the ionizable cationic lipid compound have strong loading capacity, high delivery efficiency and good safety as drug carriers.

[0008] To achieve this purpose, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides an ionizable cationic lipid compound, and the ionizable cationic lipid compound has the structure shown in Formula I.

[0010]

[0011] In Formula I, R1 is selected from any one of substituted or unsubstituted C1-C12 straight-chain or branched-chain alkylene groups, substituted or unsubstituted C3-C6 cycloalkylene groups, and at least one C in the C1-C12 straight-chain or branched-chain alkylene group and C3-C6 cycloalkylene group can be independently replaced by a heteroatom.

[0012] R2 is selected from R 21 is selected from substituted or unsubstituted C1-C20 straight-chain or branched-chain alkylene groups; R 22 , R 23 are each independently selected from any one of H, substituted or unsubstituted C1-C20 straight-chain or branched-chain alkyl groups;

[0013] The substituents of the substitution include any one of -COOH, C1-C6 straight-chain or branched-chain alkylene groups, C1-C6 straight-chain or branched-chain alkyl groups, -COO-, -NH2 or -OH; the heteroatom includes an N atom; the wavy line represents the connection site.

[0014] In the present invention, through specific structural design, the lipid nanoparticles prepared from the ionizable cationic lipid compound have strong loading capacity, high delivery efficiency and good safety, and are used to deliver nucleic acid drugs, which can significantly improve the translation and expression level of nucleic acid drugs in vivo.

[0015] In the present invention, C1~C12 straight chain or branched alkylene groups may be, for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12 straight chain or branched alkylene groups; exemplary groups include but are not limited to methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, tert-butylene, n-pentylene, n-hexylene, n-heptylene, n-octylene, n-nonylene, n-decylene, n-undecylene, n-dodecylene, and the like.

[0016] In the present invention, C3-C6 cycloalkylene groups may be, for example, C3, C4, C5, or C6 alkylene groups; exemplary groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, and cyclohexylene.

[0017] In the present invention, C1-C20 straight chain or branched alkylene groups may be, for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20 straight chain or branched alkylene groups; exemplary groups include, but are not limited to, methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, tert-butylene, n-pentylene, n-hexylene, n-heptylene, n-octylene, n-nonylene, n-decylene, n-dodecylene, n-tetradecylene, n-hexadecylene, n-octadecylene, n-docosylene, n-tetracosylene, and the like.

[0018] In the present invention, the C1-C20 straight chain or branched alkyl group may be, for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20 straight chain or branched alkyl group; exemplary groups include but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, n-eicosyl, and the like.

[0019] In the present invention, C1~C6 straight chain or branched alkylene groups may be, for example, C1, C2, C3, C4, C5, C6 straight chain or branched alkylene groups; exemplary groups include but are not limited to methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, tert-butylene, n-pentylene, n-hexylene, and the like.

[0020] In the present invention, the C1~C6 straight chain or branched alkyl group may be, for example, a C1, C2, C3, C4, C5, C6 straight chain or branched alkyl group; exemplary examples include but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, etc.

[0021] Preferably, R1 is selected from any one of the following structures; wherein, n is the same or different and is independently selected from an integer of 1 to 3 (for example, it can be 1, 2, 3); the wavy line represents the connection site; more preferably, R1 is selected from

[0022] Preferably, the R 22 and R 23 at least one of them is selected from substituted or unsubstituted C1-C20 linear or branched alkyl; the total number of carbon atoms in the R 21 and R 22 and R 23 is 12 to 28, for example, it can be 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, etc.; more preferably, the total number of carbon atoms is 18 to 24.

[0023] Preferably, the R 21 is selected from a linear alkylene group of C3-C10; at least one of R 22 and R 23 is selected from a linear alkyl group of C6-C18.

[0024] Preferably, R2 is selected from any one of the following structures.

[0025]

[0026] Preferably, the ionizable cationic lipid compound is selected from any one of compounds HX1006, HX1008, HX1017, HX1017-1, HX1017-2, HX1017-3, HX1017-4.

[0027]

[0028]

[0029] In the present invention, the ionizable cationic lipid compound can be prepared by a conventional method. Exemplarily, the synthesis route of the ionizable cationic lipid compound is as follows:

[0030]

[0031] In the present invention, the raw materials for preparing the ionizable cationic lipid compound, namely and Br-R2 can be commercially available or can be prepared by a conventional method; for example, Br-R3 can be prepared by an esterification reaction.

[0032] Second aspect, the present invention provides a lipid nanoparticle, and the raw materials for preparing the lipid nanoparticle include a cationic lipid, a phospholipid, a sterol lipid, and a polyethylene glycolylated lipid; the cationic lipid includes at least one ionizable cationic lipid compound as described in the first aspect.

[0033] Preferably, the molar ratio of the cationic lipid, the phospholipid, the sterol lipid, and the polyethylene glycolylated lipid is (20-70):(10-60):(2-40):(0.1-5), wherein the specific values in (20-70) can be, for example, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, etc.; the specific values in (10-60) can be, for example, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, etc.; the specific values in (2-40) can be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, etc.; the specific values in (0.1-5) can be, for example, 0.1, 0.2, 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, etc.

[0034] In the present invention, the phospholipid includes but is not limited to at least one of dioleoyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC); the sterol lipid includes but is not limited to at least one of cholesterol, sitosterol, lanosterol, stigmasterol, ergosterol; the polyethylene glycol compound includes but is not limited to at least one of dimyristoyl glycerol-rac-methoxypolyethylene glycol 2000 (DMG-PEG2000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DMPE-PEG2000), distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG), distearoyl glycerol-polyethylene glycol (DSG-PEG2000).

[0035] Third aspect, the present invention provides a nanoformulation, and the nanoformulation includes the lipid nanoparticle as described in the second aspect and a drug loaded in the lipid nanoparticle.

[0036] Preferably, the drug includes a nucleic acid drug.

[0037] In the present invention, the nucleic acid drug includes, but is not limited to, siRNA, mRNA, self-amplifying mRNA, circular mRNA, etc.

[0038] In the present invention, the preparation methods of the lipid nanoparticles and the nanoformulations include, but are not limited to, the microfluidic synthesis method; taking the nanoformulation, that is, the lipid nanoparticles loaded with nucleic acid drugs, as an example, the preparation method includes the following steps:

[0039] (1) According to the formula amount, mix the cationic lipid, phospholipid, sterol lipid, polyethylene glycolated lipid with solvent A to obtain a lipid phase solution with a concentration of 1-10 mg / mL; mix the nucleic acid drug with solvent B to obtain a nucleic acid solution; the solvent A includes absolute ethanol; the solvent B includes a buffer solution, such as a citric acid buffer solution with pH = 4;

[0040] (2) Add the lipid phase solution to the nucleic acid solution, shake for 20-40 s, then add PBS buffer solution thereto, and centrifuge using an EMD Millipore MWCO 30 kDa ultrafiltration device to remove organic solvents and free compounds; then after washing with PBS, collect the washed nanoformulation and disperse it in PBS buffer solution with pH = 7.4 for further use or store it in a 4°C refrigerator, that is, obtain the nanoformulation. If preparing lipid nanoparticles alone, that is, preparing empty lipid nanoparticles, replace the nucleic acid solution with a buffer solution without nucleic acid drugs.

[0041] The numerical ranges described in the present invention not only include the above-listed point values, but also include any point values between the above numerical ranges not listed. Due to space limitations and for the sake of simplicity, the present invention does not exhaustively list the specific point values included in the range.

[0042] Compared with the prior art, the beneficial effects of the present invention are:

[0043] The present invention provides an ionizable cationic lipid compound. Through specific structural design, the lipid nanoparticles prepared therefrom have strong loading capacity, high delivery efficiency and good safety; when used for delivering nucleic acid drugs, it can significantly improve the translation and expression level of nucleic acid drugs in vivo. Description of the Drawings

[0044] Figure 1 It is the particle size distribution diagram of the nanoformulation provided in Application Example 3 of the present invention.

[0045] Figure 2 It is the particle size distribution diagram of the nanoformulation provided in Application Example 3 of the present invention after being stored at 4°C for 24 days.

[0046] Figure 3Potential map of the nano - preparation provided in Application Example 3 of the present invention.

[0047] Figure 4 Delivery effect diagrams of the nano - preparation provided in Application Example 3 of the present invention and the nano - preparation provided in Comparative Application Example 1 at different times after in - vivo injection.

[0048] Figure 5 Column chart of delivery efficiency of the nano - preparation provided in Application Example 3 of the present invention and the nano - preparation provided in Comparative Application Example 1 24 hours after in - vivo injection.

[0049] Figure 6 Delivery effect diagram of the nano - preparation provided in Application Example 3 of the present invention 24 hours after in - vivo injection after being stored at 4°C for 24 days.

[0050] Figure 7 Data diagram of delivery efficiency of the nano - preparation provided in Application Example 3 of the present invention 24 hours after in - vivo injection after being stored at 4°C for 24 days.

[0051] Figure 8 Graph of the content change of immune factor IL6 24 hours after in - vivo delivery of the nano - preparation provided in Application Example 3 of the present invention and the nano - preparation provided in Comparative Application Example 1.

[0052] Figure 9 Graph of the content change of immune factor TNFα 24 hours after in - vivo delivery of the nano - preparation provided in Application Example 3 of the present invention and the nano - preparation provided in Comparative Application Example 1.

[0053] Figure 10 Graph of the high - resolution mass spectrometry test results of the ionizable cationic lipid compound HX1017 provided in Example 3 of the present invention.

[0054] Figure 11 Liquid chromatography - mass spectrometry map of the ionizable cationic lipid compound HX1006 provided in Example 1 of the present invention. Detailed implementation manners

[0055] The technical solution of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the described embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0056] The experimental materials used in the present invention can be obtained from conventional biochemical reagent companies without special instructions.

[0057] The following examples exemplarily provide the synthesis methods of a series of specific compounds. For compounds without specific synthesis methods mentioned, they can be synthesized by similar methods or other existing methods, and the present invention does not make specific limitations on this.

[0058] Example 1

[0059] This example provides an ionizable cationic lipid compound HX1006, the structural formula of which is The synthetic route of the ionizable cationic lipid compound is as follows:

[0060]

[0061] The specific steps include:

[0062] (1) At room temperature, 4-dimethylaminopyridine (DMAP, 24 mg, 0.2 mmol) was added to a dichloromethane (DCM) solution of 4-bromobutyric acid (381 mg, 2.2 mmol) and n-decanol (316 mg, 2.0 mmol). Then, under ice bath conditions, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 468 mg, 2.4 mmol) was added thereto, and the mixture was stirred overnight at room temperature. After the reaction was completed, it was washed with saturated Na2CO3 solution and NaCl solution, the organic phase was collected, dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography using a n-hexane solution of 3 wt% ethyl acetate as the eluent to obtain the target compound HX1006-1 (382 mg, 1.25 mmol, yield 62%).

[0063] (2) NaOH (48 mg, 1.2 mmol) was added to a DMF (1.5 ml) solution of L-lysine (44 mg, 0.3 mmol), and HX1006-1 (214 mg, 0.7 mmol) was added dropwise thereto, and the mixture was stirred overnight at 30 °C. After the reaction was completed, the reaction solution was washed with NaCl solution, extracted with ethyl acetate, and the organic phase was collected; the organic phase was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography using a dichloromethane solution of 4 wt% methanol as the eluent to obtain the ionizable cationic lipid compound (35 mg, 0.058 mmol, yield 19%).

[0064] The ionizable cationic lipid compound was characterized by a liquid chromatography-mass spectrometer, and the results are as Figure 11 shown.

[0065] Example 2

[0066] This example provides an ionizable cationic lipid compound HX1008, the structural formula of which is The synthetic route of the ionizable cationic lipid compound is as follows:

[0067]

[0068] Specifically, the following steps are included:

[0069] (1) At room temperature, 4-dimethylaminopyridine (DMAP, 24 mg, 0.2 mmol) was added to a dichloromethane (DCM) solution of 8-bromooctanoic acid (492 mg, 2.2 mmol) and 7-tridecanol (400 mg, 2.0 mmol). Then, under an ice bath condition, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 468 mg, 2.4 mmol) was added thereto, and the mixture was stirred overnight at room temperature. After the reaction was completed, it was washed with saturated Na2CO3 solution and NaCl solution, the organic phase was collected, dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography using a n-hexane solution of 3 wt% ethyl acetate as the eluent to obtain the target compound HX1008-1 (420 mg, 1.04 mmol, yield 52%).

[0070] (2) NaOH (48 mg, 1.2 mmol) was added to a DMF (1.5 ml) solution of L-lysine (44 mg, 0.3 mmol), and HX1008-1 (284 mg, 0.7 mmol) was added dropwise, and the mixture was stirred overnight at 30 °C. After the reaction was completed, the reaction solution was washed with NaCl solution, extracted with ethyl acetate, and the organic phase was collected; the organic phase was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography using a dichloromethane solution of 4 wt% methanol as the eluent to obtain the ionizable cationic lipid compound (35 mg, 0.044 mol, yield 15%).

[0071] Example 3

[0072] This example provides an ionizable cationic lipid compound HX1017, and the structural formula is The synthetic route of the ionizable cationic lipid compound is as follows:

[0073]

[0074] Specifically, it includes the following steps:

[0075] NaOH (48 mg, 1.2 mmol) was added to a DMF (1.5 ml) solution of ethylenediamine hydrochloride (40 mg, 0.3 mmol), and HX1008-1 (286 mg, 0.7 mmol) was added dropwise, and the mixture was stirred overnight at 30 °C. After the reaction was completed, it was washed with NaCl solution, extracted with ethyl acetate, and the organic phase was collected. The organic phase was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography using a dichloromethane solution of 4 wt% methanol as the eluent to obtain the ionizable cationic lipid compound (62 mg, 0.087 mmol, yield 29.1%).

[0076] The ionizable cationic lipid compound was characterized using a high-resolution mass spectrometer, and the results are as Figure 10 shown.

[0077] The preparation methods of the compounds of the present invention used in the specific embodiments are all similar to the above methods, and will not be elaborated one by one. Only the characterization results are provided. The mass spectrometry analysis results of different ionizable cationic lipid compounds, that is, the actually measured molecular weights, are shown in Table 1.

[0078] Table 1

[0079]

[0080] Application Example 1

[0081] This application example provides a nanoformulation, including lipid nanoparticles and mRNA loaded in the lipid nanoparticles; the raw materials for preparing the lipid nanoparticles include an ionizable cationic lipid compound (HX1006), DSPC, cholesterol, and DMG-PEG with a molar ratio of 50:10:38.5:1.5.

[0082] The preparation method of the nanoformulation includes:

[0083] (1) The cationic lipid compound, DSPC, cholesterol, and DMG-PEG were respectively dissolved in absolute ethanol to form uniform solutions with concentrations of 5 mg / ml, 10 mg / ml, 10 mg / ml, and 10 mg / ml, respectively; then, according to the formulated amounts, the cationic lipid solution, DSPC solution, cholesterol solution, and DMG-PEG solution were mixed to prepare a lipid phase solution with a total lipid concentration of 7.5 mg / mL; 5 μg of luciferace mRNA was mixed with a citric acid buffer solution with pH = 4 to obtain an mRNA solution (concentration: 50 μg / ml);

[0084] (2) On an oscillator at 800 revolutions per minute, the lipid phase solution was rapidly added to the mRNA solution, shaken for 30 s, and then 200 μL of PBS buffer solution was added drop by drop. After the addition was completed, centrifugation was performed using an EMD Millipore MWCO 30 kDa ultrafiltration device to remove the organic solvents and free compounds in the formed nanoformulation dispersion; after washing 3 times with PBS, the washed nanoformulation was collected and dispersed in a PBS buffer solution with pH = 7.4 to obtain the nanoformulation, denoted as mRNA-LNP.

[0085] The preparation method of luciferace mRNA includes:

[0086] (1) PCR amplify the open reading frame plasmid of the luciferace gene carrying the T7 promoter and T7 terminator, and purify the PCR product to form linearized DNA as an in vitro transcription template;

[0087] (2) In vitro transcription: Mix the Novoprotein T7 transcription kit evenly with 1 - 2 μg of the in vitro transcription template, 7.5 mM guanosine triphosphate, 7.5 mM 5-methyl-cytidine triphosphate, 7.5 mM adenosine triphosphate, and 7.5 mM pseudouridine-5′-triphosphate. The reaction is carried out at 37 °C for 2 hours, followed by deoxyribonuclease treatment to obtain luciferace mRNA; the luciferace mRNA is purified by high performance liquid chromatography, and the salts in the buffer are removed by centrifugation using an EMD Millipore MWCO 30 kDa ultrafiltration device; after washing 3 times with enzyme-free water, the luciferace mRNA is collected in enzyme-free water for further use or stored in an -80 °C refrigerator.

[0088] Application Examples 2 - 7

[0089] Application Examples 2 - 7 respectively provide a nanoformulation, which is only different from Application Example 1 in that the ionizable cationic lipid compounds are those provided in Examples 2 - 7; other components, dosages, and preparation methods are the same as those in Application Example 1.

[0090] Application Example 8

[0091] This application example provides a nanoformulation, which is only different from Application Example 1 in that the total molar amounts of the ionizable cationic lipid compound (HX1006), DSPC, cholesterol, and DMG-PEG remain unchanged, and the molar ratio is 30:50:15.5:4.5; other components, dosages, and preparation methods are the same as those in Application Example 1.

[0092] Application Example 9

[0093] This application example provides a nanoformulation, which is only different from Application Example 1 in that the total molar amounts of the ionizable cationic lipid compound (HX1006), DSPC, cholesterol, and DMG-PEG remain unchanged, and the molar ratio is 40:28:28.5:3.5; other components, dosages, and preparation methods are the same as those in Application Example 1.

[0094] Comparative Application Examples 1 - 2

[0095] Comparative Application Examples 1 - 2 respectively provide a nanoformulation, which is only different from Application Example 1 in that the ionizable cationic lipid compounds are those provided in Comparative Examples 1 and 2; other components, dosages, and preparation methods are the same as those in Application Example 1.

[0096] Result Characterization and Performance Testing

[0097] (1) Testing of Particle Size and Polydispersity Index (PDI)

[0098] Taking HX1017 as an example, using a Malvern Zetasier Lab particle size and zeta potential analyzer, dynamic light scattering measurement was carried out in the lateral scattering detection mode to test the particle size and potential of the nanopreparations prepared therefrom. The particle size test results are as Figure 1 shown. It can be seen from Figure 1 that the particle size of the nanopreparation is about 126.7 nm and the PDI is 0.123; after storing the nanopreparation at 4 °C for 24 days, its particle size was tested again, and the results are as Figure 2 shown. The particle size of the nanopreparation is about 140 nm and the PDI is 0.136; it indicates that the nanopreparation has good stability. The potential diagram of the nanopreparation is as Figure 3 shown.

[0099] (2) Testing of In Vivo Delivery Efficiency

[0100] According to the guidelines formulated by the National Science and Technology Commission, male BALB / C mice about 6 weeks old were selected and injected with 1 μg of nanopreparation intramuscularly or via the tail vein respectively. Small animal in vivo imaging was used to detect the signal intensity (i.e., total flux Total Flux) at different time periods.

[0101] Among them, the delivery effect diagrams of the nanopreparations provided in Application Example 3 and Comparative Application Example 1 at different times after in vivo injection are as Figure 4 shown; the bar chart after 24H injection is as Figure 5 shown ( Figure 5 uses the compound abbreviations to represent different nanopreparations).

[0102] It can be seen from Figure 4 and 5 that after giving the substrate fluorescein sodium, the results of small animal in vivo imaging show that signals can be detected after intramuscular and tail vein injection of the nanopreparations provided by the present invention, and the signals after tail vein injection are mainly concentrated in the liver region. The LNP prepared from the cationic lipid compound provided by the present invention has excellent delivery effect in vivo. And it can be seen from Figure 5 that after intramuscular injection of the nanopreparation prepared from the ionizable cationic lipid compound provided by the present invention for 24H, the signal intensity is higher, indicating that the nanopreparation prepared from the ionizable cationic lipid compound provided by the present invention has higher delivery efficiency after intramuscular injection for 24H.

[0103] After storing the nanopreparation provided in Application Example 3 at 4 °C for 24 days, the in vivo delivery effect after 24 hours of injection is as Figure 6 shown; the bar chart of in vivo delivery efficiency after 24 hours of injection is asFigure 7 As shown, it indicates that the nano - formulation prepared from the ionizable cationic lipid compound provided by the present invention has good stability and still has a high delivery efficiency after long - term storage.

[0104] (3) Safety assessment

[0105] The nano - formulations provided in Application Example 3 (HX1017) and Comparative Application Example 1 (MC3) were respectively injected via the tail vein at a dose of 1 μg. The blank group (NC) was injected with the same volume of normal saline. After 24 hours, serum was collected, and the changes in the immune factors IL6 and TNFα were detected by ELISA. The results are as Figure 8 (IL6) and Figure 9 (TNFα) shown.

[0106] From Figure 8 and Figure 9 it can be seen that there is no significant difference between the nano - formulation provided by the present invention and the nano - formulation prepared from MC3 and normal saline. The levels of IL6 and TNFα did not show a significant increase, indicating that the nano - formulation provided by the present invention has good safety in vivo.

[0107] In addition, the in - vivo delivery effects of Application Examples 1 - 9 and Comparative Application Example 2 are shown in Table 2.

[0108] Table 2

[0109] Delivery efficiency [p / s] Application Example 1 2.76E+06 Application Example 2 1.70E+06 Application Example 3 1.13E+07 Application Example 4 1.02E+06 Application Example 5 4.91E+05 Application Example 6 8.56E+05 Application Example 7 9.25E+04 Application Example 8 3.44E+05 Application Example 9 1.12E+05 Comparative Application Example 2 7.10E+05

[0110] Note: In Table 2, the delivery efficiency is the total signal value data of the small animal in - vivo imaging system 24 hours after intramuscular injection of 1 μg of the nano - formulation provided by the application example and the comparative application example.

[0111] As can be seen from Table 2, the ionizable cationic lipid compound provided by the present invention, through specific structural design, the lipid nanoparticles prepared therefrom have strong loading capacity, high delivery efficiency and good safety; when used to deliver nucleic acid drugs, it can significantly improve the translation and expression level of nucleic acid drugs in vivo; the total signal value of the nano - formulation prepared from the ionizable cationic lipid compound in the small animal in - vivo imaging system after 24 hours ≥ 9.25E+04, and the delivery efficiency is high.

[0112] The applicant declares that the above - mentioned is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. An ionizable cationic lipid compound, characterized in that, The ionizable cationic lipid compound has the structure shown in Formula I: In Formula I, R1 is selected from any one of substituted or unsubstituted C1-C12 straight-chain or branched-chain alkylene groups and substituted or unsubstituted C3-C6 cycloalkylene groups, and at least one C in the C1-C12 straight-chain or branched-chain alkylene group and C3-C6 cycloalkylene group can be independently replaced by a heteroatom; R2 is selected from R 21 selected from substituted or unsubstituted C1-C20 linear or branched alkylene; R 22 , R 23 each independently selected from any one of H, substituted or unsubstituted C1-C20 linear or branched alkyl; The substituents of the substitution include any one of -COOH, C1-C6 straight-chain or branched-chain alkylene groups, C1-C6 straight-chain or branched-chain alkyl groups, -COO-, -NH2 or -OH; The heteroatom includes an N atom; The wavy line represents the connection site.

2. The ionizable cationic lipid compound according to claim 1, wherein The R1 is selected from any one of the following structures; Wherein, n is the same or different, and is independently selected from an integer of 1-3; the wavy line represents the connection site.

3. The ionizable cationic lipid compound according to claim 1, wherein Said R 22 , R 23 At least one of them is selected from substituted or unsubstituted C1-C20 straight-chain or branched-chain alkyl groups; The said R 21 , R 22 , R 23 The total number of carbon atoms is 12 to 28.

4. The ionizable cationic lipid compound according to claim 1, wherein The R 21 is selected from linear alkylene groups having 3 to 10 carbon atoms; R 22 and R 23 at least one of which is selected from linear alkyl groups having 6 to 18 carbon atoms.

5. The ionizable cationic lipid compound according to claim 1, wherein The R2 is selected from any one of the following structures; 6. The ionizable cationic lipid compound according to claim 1, wherein The ionizable cationic lipid compound is selected from any one of compounds HX1006, HX1008, HX1017, HX1017-1, HX1017-2, HX1017-3, HX1017-4:

7. A lipid nanoparticle, characterized in that, The raw materials for preparing the lipid nanoparticles include cationic lipids, phospholipids, sterol lipids and polyethylene glycolated lipids; the cationic lipid includes at least one ionizable cationic lipid compound described in any one of claims 1-6.

8. The lipid nanoparticle according to claim 7, wherein, The molar ratio of the cationic lipid, phospholipid, sterol lipid and polyethylene glycolated lipid is (20-70):(10-60):(2-40):(0.1-5).

9. A nano - preparation, characterized in that, The nanoformulation includes the lipid nanoparticles described in claim 7 or 8 and a drug loaded in the lipid nanoparticles.

10. The nano - preparation according to claim 9, characterized in that, The drug includes a nucleic acid drug.

Images