Fluorine-containing ionizable lipid compound, preparation method and application thereof, and LNP composition

Abstract

The invention relates to a fluorine-containing ionizable lipid compound, a preparation method and application thereof, and an LNP composition, and relates to the technical field of medical biologication.The fluorine-containing ionizable lipid compound is synthesized by taking an aldehyde compound, an amine compound, a carboxylic acid compound and an isonitrile compound as raw materials through a Ugi reaction; the obtained fluorine-containing ionizable lipid compound, sterol, auxiliary lipid and a polyethylene glycol lipid derivative form a lipid nanoparticle-like composition, and the composition can be used for drug delivery, including nucleic acid drugs such as small molecule drugs, mRNA, DNA and the like, protein / polypeptide drugs, and gene editing complexes such as mRNA / sgRNA, Cas9 / sgRNA and the like; the mRNA expression of different organs in an animal body is realized through different administration modes, and the application requirements of mRNA nucleic acid therapy, nucleic acid vaccines, gene editing and the like can be met.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical biotechnology, and in particular to a fluorine-containing ionizable lipid compound, its preparation method and application, and LNP compositions. Background Technology

[0002] Messenger RNA (mRNA) therapy has shown great potential in treating infectious diseases, cancer, and genetic disorders. The development of mRNA-based drugs has long been a subject of interest. However, the physicochemical and biological properties of mRNA present significant challenges to its translation. Due to its negative charge, hydrophilicity, and large molecular weight, mRNA cannot cross cell membranes. Furthermore, naked mRNA is rapidly degraded by enzymes in biological media. Therefore, mRNA requires delivery vehicles to transport it into cells and enhance its stability.

[0003] To realize the therapeutic potential of RNA more safely and efficiently, researchers have developed lipid nanoparticles (LNPs) as advanced delivery carriers. The LNP system, through precise formulation design, consists of key components such as ionizable lipids, cofactor lipids, cholesterol, and polyethylene glycol (PEG)-modified lipids, enabling effective encapsulation and targeted delivery of RNA to target tissues or cells. Among these core components, the molecular structure design of the ionizable lipids is particularly crucial, directly determining key delivery parameters such as mRNA encapsulation efficiency, cellular uptake rate, and endosome escape capability.

[0004] Compared to traditional cationic lipids, modern ionizable lipids have achieved revolutionary improvements through pH-responsive design. They remain neutral under physiological pH conditions to reduce non-specific interactions, while protonating in an acidic endosomal environment to promote mRNA release. This property not only significantly reduces cytotoxicity but also avoids negative interactions with blood components, greatly improving biocompatibility. Currently, the research community has successfully developed several generations of ionizable lipids, including DLin-MC3-DMA, SM-102, and ALC-0315, which have demonstrated excellent delivery performance in clinical applications such as COVID-19 mRNA vaccines. Despite these advances, there remains a strong need for novel ionizable lipids with higher delivery efficiency to reduce toxicity and immunogenicity associated with mRNA and LNPs.

[0005] Fluorinated lipids, polymers, and dendritic macromolecules exhibit significantly higher delivery efficiency than non-fluorinated materials in delivering biopharmaceuticals such as DNA, RNA, and proteins. This enhancement primarily stems from the unique physicochemical properties of fluorinated carbon chains: compared to conventional hydrocarbons, fluorinated structures possess both hydrophobic and lipophobic properties, exhibiting stronger surface activity, higher chemical stability, and superior self-assembly capabilities. Studies have shown that fluorinated lipids or polymers can form more structurally stable nanoparticles, which not only prolongs their half-life in biological cycles but also significantly improves the delivery efficiency of LNP carriers for nucleic acid drugs such as mRNA. Based on this, this application was developed. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a fluorinated ionizable lipid compound with high delivery efficiency and good safety, its preparation method and application, and LNP composition.

[0007] To achieve the above objectives, the present invention mainly provides the following technical solutions:

[0008] In a first aspect, the present invention provides a fluorinated ionizable lipid compound (the fluorinated ionizable lipid compound can be used to prepare an LNP composition, the LNP composition can be used to deliver mRNA or DNA), wherein the fluorinated ionizable lipid compound has a structure as shown in either formula (I) or formula (II):

[0009]

[0010] In the formula, n1 is at least one group independently selected from hydrogen atom, straight-chain alkyl, branched-chain alkyl, straight-chain alkenyl, branched-chain alkenyl, substituted alkynyl, cycloalkyl, phenyl, and heteroatom-containing aromatic group;

[0011] n2 is independently selected from at least one group selected from straight-chain alkyl, straight-chain alkenyl, heteroatom-containing straight-chain alkyl, heteroatom-containing straight-chain alkylene, heteroatom-containing branched alkyl, heteroatom-containing branched alkylene, heteroatom-containing cyclic alkyl, heteroatom-containing cyclic alkylene, ester-containing alkyl, ether-containing alkyl.

[0012] n3 is a group independently selected from at least one of the following: straight-chain alkyl, straight-chain alkenyl, cycloalkyl, heteroatom-containing straight-chain alkyl, heteroatom-containing branched alkyl, heteroatom-containing branched alkylene, heteroatom-containing cyclic alkyl, heteroatom-containing and aromatic alkyl, hydroxyl-containing alkyl, ester-containing alkyl, ether-containing alkyl, and sulfonate-containing alkyl.

[0013] n4 is a group independently selected from at least one of the following: straight-chain alkyl, branched alkyl, straight-chain alkenyl, branched alkenyl, straight-chain alkynyl, heteroatom-containing straight-chain alkyl, heteroatom-containing branched alkyl, and heteroatom-containing cyclic alkyl.

[0014] Specifically, in the aforementioned fluorinated ionizable lipid compounds, n1 is independently selected from any one of the following groups; wherein, Represents the location of the group connection;

[0015]

[0016] n2 is independently selected from any of the following groups; wherein, Represents the location of the group connection;

[0017]

[0018] n3 is independently selected from any of the following groups; wherein, Represents the location of the group connection;

[0019]

[0020] n4 is independently selected from any of the following groups; wherein, Represents the location of the group connection;

[0021]

[0022] As a preferred embodiment, the fluorinated ionizable lipid compound includes at least one of the following compounds:

[0023]

[0024]

[0025]

[0026]

[0027] Secondly, the present invention provides a method for preparing the fluorine-containing ionizable lipid compound, specifically comprising the following steps:

[0028] Aldehyde compounds and amine compounds are added to an organic solvent, preferably methanol, and reacted at -20 to 120°C for 1 to 15 hours. Then, carboxylic acid compounds and isonitrile compounds are added, and the reaction is carried out at -20 to 120°C (preferably -20 to 60°C) for 1 to 72 hours. The product is then separated and purified.

[0029] Specifically, the molar ratio of the aldehyde compound, amine compound, carboxylic acid compound and isonitrile compound can be (0.1-1.5):(0.1-1.5):(0.1-1.5):(0.1-1.5); more preferably, the molar ratio is 1.1:1.1:1:1.

[0030] The fluorine-containing ionizable lipid compounds of this invention have mild reaction conditions, simple synthesis process, and good stability.

[0031] Preferably, the aldehyde compound is any one of the following compounds:

[0032]

[0033] Preferably, the amine compound is any one of the following compounds:

[0034]

[0035] Preferably, the carboxylic acid compound is any one of the following compounds:

[0036]

[0037] Preferably, the isonitrile compound is any one of the following compounds:

[0038]

[0039] This invention also provides the application of the above-mentioned fluorinated ionizable lipid compounds in the preparation of drug carriers.

[0040] Thirdly, the present invention provides a lipid nanoparticle (LNP) composition carrier, which includes one or more of the fluorinated ionizable lipid compounds, polyethylene glycol lipids, cholesterol, and neutral phospholipids, wherein the neutral phospholipid is preferably at least one of distearylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), and lecithin (ESM).

[0041] Furthermore, the lipid nanoparticle LNP composition carrier comprises, by molar percentage: 10%–70% fluorinated ionizable lipid compound, 0.1%–25% polyethylene glycol lipid, 10%–50% cholesterol, and 5%–50% neutral phospholipid.

[0042] The present invention also provides the application of the above-mentioned lipid nanoparticle (LNP) composition carrier in the preparation of nucleic acid-loaded drugs.

[0043] Fourthly, the present invention provides a method for preparing a nanoparticle composition encapsulating a nucleic acid drug, specifically comprising the following steps:

[0044] 1) The fluorinated ionizable lipid compound, neutral phospholipid, polyethylene glycol lipid and cholesterol are dissolved and mixed in a water-miscible solvent to obtain an organic phase liposome solution, wherein the concentration of the fluorinated ionizable lipid compound is 1-100 mg / mL, the concentration of the neutral phospholipid is 1-100 mg / mL, the concentration of the polyethylene glycol lipid is 1-100 mg / mL, and the concentration of cholesterol is 1-100 mg / mL;

[0045] 2) Dissolve the nucleic acid drug in acidic buffer solution to obtain an aqueous nucleic acid drug solution;

[0046] 3) The organic phase liposome solution and the aqueous phase nucleic acid drug solution are mixed and dialyzed using a dialysis bag with a molecular weight cutoff of 1000 to obtain a nanoparticle composition loaded with nucleic acid drugs.

[0047] Specifically, the water-miscible solvent is preferably ethanol, and the acidic buffer solution is preferably a citrate / sodium citrate buffer solution with a pH of 1.0-6.9.

[0048] Furthermore, in the aqueous nucleic acid drug solution, the concentration of the nucleic acid drug is 0.01-1.00 mg / mL. Preferably, the concentration of the nucleic acid drug is 0.8 mg / mL.

[0049] Preferably, the volume ratio of the organic phase liposome solution to the aqueous phase nucleic acid drug solution during mixing is 1:2 to 1:5, and more preferably 1:3.

[0050] Fifthly, the present invention also provides the application of the above-mentioned nanoparticle composition containing nucleic acid drugs as an antitumor drug.

[0051] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0052] This invention provides a novel type of fluorinated ionizable lipid compound, and further synthesizes the fluorinated ionizable lipid compound via the Ugi reaction using aldehydes, amines, carboxylic acids, and isonitriles as raw materials. The obtained fluorinated ionizable lipid compound forms a lipid nanoparticle-like composition with sterols, auxiliary lipids, and polyethylene glycol lipid derivatives. This composition can be used for drug delivery, including small molecule drugs, nucleic acid drugs such as mRNA and DNA, protein / peptide drugs, and gene editing complexes such as mRNA / sgRNA and Cas9 / sgRNA. Through different administration methods, mRNA expression in different organs of animals can be achieved, which can meet the application needs of mRNA nucleic acid therapy, nucleic acid vaccines, and gene editing. Attached Figure Description

[0053] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0054] Figure 1 This is the hydrogen NMR spectrum of A4R2I18F1 in Example 1 of this invention;

[0055] Figure 2 This is the mass spectrum of A4R2I18F1 in Example 1 of the present invention;

[0056] Figure 3 This is the hydrogen NMR spectrum of A4R2I18F5 in Example 2 of this invention;

[0057] Figure 4 This is the mass spectrum of A4R2I18F5 in Example 2 of the present invention;

[0058] Figure 5 This is the hydrogen NMR spectrum of A4R2I18F7 in Example 3 of this invention;

[0059] Figure 6 This is the mass spectrum of A4R2I18F7 in Example 3 of the present invention;

[0060] Figure 7 The hydrogen NMR spectrum of A4R2I18F11 in Example 4 of this invention;

[0061] Figure 8 This is the mass spectrum of A4R2I18F11 in Example 4 of the present invention;

[0062] Figure 9 The image shows an in vivo imaging study of A4R2I18F1 administered via intramuscular injection in Example 1 of this invention; (left) shows the bioluminescence intensity of A4R2I18F1 administered via intramuscular injection in Example 1, and (right) shows the bioluminescence intensity of each organ in vitro.

[0063] Figure 10 The images show in vivo imaging after intraperitoneal injection of A4R2I18F11 in Example 4 of this invention; (left) shows the bioluminescence intensity of in vivo after intraperitoneal injection of A4R2I18F11 in Example 4; (right) shows the bioluminescence intensity of each organ in vitro. Detailed Implementation

[0064] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments, but the scope of protection of this invention is not limited thereto.

[0065] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods in the art, and the materials and reagents used are commercially available unless otherwise specified.

[0066] The general formula for synthesizing fluorine-containing ionizable lipid compounds using chemical reaction formula (I) is as follows:

[0067]

[0068] The specific preparation method is as follows:

[0069] At room temperature, 1.1 mmol of an aldehyde compound and 1.1 mmol of an amine compound were added to 400 μL of methanol, respectively, and reacted at 30 °C for 6–12 h. Then, 1.0 mmol of a carboxylic acid compound was added, and the reaction was continued at 30 °C for 30 min. Finally, 1.0 mmol of an isonitrile compound was added, and the reaction was continued at 45 °C for 12 h. After the reaction was complete, the product was purified by silica gel chromatography, using a mixture of methanol and dichloromethane as the eluent. The solvent was removed by rotary evaporation, and the product was dried under vacuum overnight at room temperature. 1 Chemical structure characterization was performed using 1H NMR and mass spectrometry.

[0070] The general formula for the synthesis of Equation I is:

[0071]

[0072] Among them, the aldehyde compounds are selected as A1, A2, A3, A4, and A36; the amine compounds are selected as R1, R2, R3, R5, R10, and R23; the isonitrile compounds are selected as I12, I16, and I18; and the carboxylic acid compounds are selected as F1, F2, F5, F7, and F11.

[0073] The general structural formula of the isonitrile compound is:

[0074] n2——NC

[0075] The isonitrile compound in this invention is synthesized by the following method:

[0076] 50 mmol of amine was refluxed in 1300 mmol of ethyl formate for 20 h, and then concentrated to dryness under reduced pressure. The resulting product was dissolved in 60 mL of dry dichloromethane, and then 43 mmol of triethylamine was added, denoted as solvent A. 150 mmol of phosphorus oxychloride was dissolved in 40 mL of dichloromethane to prepare solution B, which was then added dropwise to solution A over 30 min at -20 °C. The resulting reaction mixture was slowly heated to room temperature and stirred overnight. Thin-layer chromatography was used to confirm the completion of the reaction. The resulting mixture was poured into 200 mL of cold water and extracted twice with 100 mL of dichloromethane. The organic layer was washed with 100 mL of saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. Subsequently, it was purified by column chromatography (petroleum ether:ethyl acetate = 30:1, v / v) to obtain the isonitrile product.

[0077] In the above synthesis process, the amine is selected according to n3. For example, if dodecylamine is selected as the raw material, the corresponding isonitrile product is dodecylisocyanate (I12); if hexadecylamine is selected as the raw material, the corresponding isonitrile product is hexadecylisocyanate (I16); if octadecylamine is selected as the raw material, the corresponding isonitrile product is octadecylisocyanate (I18).

[0078] In a preferred embodiment, the fluorinated ionizable lipid compound includes any one of the following compounds:

[0079]

[0080]

[0081] Example 1

[0082] This embodiment provides a fluorine-containing ionizable lipid compound, the structural formula of which is shown below: A4R2I18F1.

[0083]

[0084] Preparation of A4R2I18F1:

[0085] 3-Dimethylaminopropylamine (112.4 mg, 1.1 mmol) and 2-ethylhexanal (141.0 mg, 1.1 mmol) were dissolved in methanol (400 μL) and reacted at 30 °C for 12 h with stirring. Trifluoroacetic acid (114.0 mg, 1 mmol) was added and the mixture was stirred for another 30 min. Octadecylisocyanate (279.5 mg, 1 mmol) was then added, and the reaction was carried out at 45 °C for 12 h. After the reaction was complete, the product was purified by silica gel chromatography (eluent: dichloromethane:methanol = 20:1, v / v). Excess solvent was removed by rotary evaporation, yielding product A4R2I18F1, which was dried under vacuum overnight at room temperature (H NMR spectrum shown below). Figure 1 As shown, the mass spectrum is as follows Figure 2 (As shown).

[0086] 1H NMR (400MHz, CDCl3) δ6.60 (s, 1H), 4.25-4.14 (m, 1H), 3.48 (t, J = 8.2Hz, 2H), 3.22 (dq, J = 12.7, 6.6Hz, 2H), 2.38 (d, J = 10.3Hz, 1H), 2.30-2.26 (m,3H),2.21(s,5H),1.85(s,3H),1.52-1.39(m,4H),1.25(s,45H),0.88(tt,J=6.9,3.9Hz,10H),0.80(t,J=7.4Hz,1H).MS:m / z:[M+H]+calcd for C34H66N3O2F3,605.51; found,605.62.

[0087] Example 2

[0088] This embodiment provides a fluorine-containing ionizable lipid compound, the structural formula of which is shown below: A4R2I18F5.

[0089]

[0090] Preparation of A4R2I18F5:

[0091] 3-Dimethylaminopropylamine (112.4 mg, 1.1 mmol) and 2-ethylhexanal (141.0 mg, 1.1 mmol) were dissolved in methanol (400 μL), and the mixture was stirred at 30 °C for 12 h. Pentafluoropropionic acid (164.1 mg, 1 mmol) was added, and the mixture was stirred for another 30 min. Octadecylisocyanate (279.5 mg, 1 mmol) was then added, and the mixture was reacted at 45 °C for 12 h. After the reaction was complete, the product was purified by silica gel chromatography (eluent: dichloromethane:methanol = 10:1, v / v). Excess solvent was removed by rotary evaporation, yielding product A4R2I18F5, which was dried under vacuum overnight at room temperature (H NMR spectrum shown below). Figure 3 As shown, the mass spectrum is as follows Figure 4 (As shown).

[0092] 1H NMR (400MHz, CDCl3) δ6.55(s,1H),4.23(s,1H),3.53(t,J=15.2Hz,2H),3.28-3.12(m,2H),2.37(d,J=8.0Hz,1H),2.26(t,J=6.7Hz,2H),2. 21(d,J=7.3Hz,6H),1.87(s,2H),1.52-1.41(m,3H),1.25(d,J=3.2Hz,41H),0.93-0.83(m,8H),0.78(t,J=7.4Hz,1H).MS:m / z:[M+H]+calcd for C35H66N3O2F5,655.51; found,655.51.

[0093] Example 3

[0094] This embodiment provides a fluorine-containing ionizable lipid compound, the structural formula of which is shown below: A4R2I18F7.

[0095]

[0096] Preparation of A4R2I18F7:

[0097] 3-Dimethylaminopropylamine (112.4 mg, 1.1 mmol) and 2-ethylhexanal (141.0 mg, 1.1 mmol) were dissolved in methanol (400 μL), and the mixture was stirred at 30 °C for 12 h. Heptafluorobutyric acid (213.1 mg, 1 mmol) was added, and the mixture was stirred for another 30 min. Octadecylisocyanate (279.5 mg, 1 mmol) was then added, and the mixture was reacted at 45 °C for 12 h. After the reaction was complete, the product was purified by silica gel chromatography (eluent: dichloromethane:methanol = 20:1, v / v). Excess solvent was removed by rotary evaporation, yielding product A4R2I18F7, which was dried under vacuum overnight at room temperature (H NMR spectrum shown below). Figure 5 As shown, the mass spectrum is as follows Figure 6 (As shown).

[0098] 1H NMR (400MHz, CDCl3) δ6.50(s,1H),4.30(s,1H),3.64-3.40(m,2H),3.29-3.14(m,2H),2.41-2.31(m,1H),2.28-2.17(m,8H),1.91(s,3H) ),1.46(dtd,J=16.9,9.5,3.9Hz,4H),1.25(d,J=2.9Hz,41H),0.88(tt,J=7.3,4.2Hz,8H),0.78(t,J=7.4Hz,1H).MS:m / z:[M+H]+calcd for C36H66N3O2F7,705.50; found,705.51.

[0099] Example 4

[0100] This embodiment provides a fluorine-containing ionizable lipid compound, the structural formula of which is shown below: A4R2I18F11.

[0101]

[0102] Preparation of A4R2I18F11:

[0103] 3-Dimethylaminopropylamine (112.4 mg, 1.1 mmol) and 2-ethylhexanal (141.0 mg, 1.1 mmol) were dissolved in methanol (400 μL), and the mixture was stirred at 30 °C for 12 h. Perfluorohexanoic acid (314.1 mg, 1 mmol) was added, and the mixture was stirred for another 30 min. Octadecylisocyanate (279.5 mg, 1 mmol) was then added, and the mixture was reacted at 45 °C for 12 h. After the reaction was complete, the product was purified by silica gel chromatography (eluent: dichloromethane:methanol = 20:1, v / v). Excess solvent was removed by rotary evaporation, yielding product A4R2I18F11, which was dried under vacuum overnight at room temperature (H NMR spectrum shown below). Figure 7 As shown, the mass spectrum is as follows Figure 8 (As shown).

[0104] 1H NMR (400MHz, CDCl3) δ6.49(s,1H),4.31(s,1H),3.64-3.41(m,2H),3.29-3.13(m,2H),2.41-2.32(m,1H),2.24(d,J=24.0Hz,8H),1 .95(s,3H),1.45(dqd,J=15.7,8.0,3.7Hz,4H),1.35-1.07(m,42H),0.94-0.82(m,9H),0.77(t,J=7.4Hz,2H).MS:m / z:[M+H]+calcd for C38H66N3O2F11,805.95; found,805.51.

[0105] Application Example 1

[0106] This application example provides a nanoparticle composition encapsulating a nucleic acid drug, which is prepared by the following steps:

[0107] 1) Fluorine-containing ionizable lipid compound A4R2I18F1, cholesterol, neutral phospholipid (DSPC), and polyethylene glycol (PEG2000) were dissolved in ethanol to prepare 10 mg / mL ethanol solutions. These solutions were then mixed in a molar ratio of 50%:10%:38.5%:1.5%, and a certain amount of ethanol was added to bring the volume to 80 μL to obtain an organic phase liposome mixed solution. At this point, the concentration of the fluorine-containing lipid compound was 5.5 mg / mL, the cholesterol concentration was 4.24 mg / mL, the neutral phospholipid concentration was 5.63 mg / mL, and the polyethylene glycol lipid concentration was 5.36 mg / mL.

[0108] 2) The reporter gene encoding the firefly luciferase protein was selected (Firefly Luciferase-mRNA, catalog number: 17101ES, purchased from Yisheng Biotechnology Co., Ltd.), and dissolved and diluted with citric acid / sodium citrate buffer at pH 4 to obtain an aqueous solution with an mRNA concentration of 5 μg / 60 μL.

[0109] 3) The organic phase liposome mixture was added dropwise to the aqueous phase solution at a volume ratio of 1:3. The mass ratio of the fluorinated ionizable lipid compound to mRNA in the resulting system was 11:1 (wherein, the amount of mRNA was 5 μg). Subsequently, the mixture was dialyzed at 4°C for 2 h using a dialysis bag with a molecular weight cutoff of 1000 to remove ethanol, yielding a fluorinated lipid nanoparticle composition encapsulating mRNA.

[0110] Application Example 2

[0111] This application example provides a nanoparticle composition encapsulating a nucleic acid drug, which differs from Application Example 1 only in that A4R2I18F1 is replaced with an equal concentration of A4R2I18F5, while the other steps are completely consistent with Example 1.

[0112] Application Example 3

[0113] This application example provides a nanoparticle composition encapsulating a nucleic acid drug, which differs from Application Example 1 only in that A4R2I18F1 is replaced with an equal concentration of A4R2I18F7, while the other steps are completely consistent with Example 1.

[0114] Application Example 4

[0115] This application example provides a nanoparticle composition encapsulating a nucleic acid drug. The only difference from Application Example 1 is that A4R2I18F1 is replaced with an equal concentration of A4R2I18F11. All other steps are exactly the same as in Example 1.

[0116] Test Example 1

[0117] Evaluation of the in vivo delivery performance of firefly luciferase mRNA.

[0118] Test sample: The lipid nanoparticle composition carrying nucleic acid drugs provided in the above application example.

[0119] Test method: as follows.

[0120] Intramuscular injection administration: The lipid nanoparticle composition prepared by intramuscular injection was administered at a dose of 5 μg mRNA per mouse. The molar ratio of fluorinated ionizable lipid compound, cholesterol, DSPC, and DMG-PEG2000 was 50%:10%:38.5%:1.5%; the mass ratio of fluorinated ionizable lipid compound to mRNA was 11:1. Six hours later, 200 μL of D-firefly luciferase substrate at a concentration of 15 mg / ml was injected intraperitoneally into each mouse for subsequent in vivo imaging. Fifteen minutes later, the mice were placed under the in vivo imaging system to observe and photograph the bioluminescence intensity of the mouse body and isolated organs.

[0121] Intraperitoneal administration: The lipid nanoparticle composition prepared for intraperitoneal injection was administered at a dose of 5 μg mRNA per mouse. The molar ratio of fluorinated ionizable lipid compound, cholesterol, DSPC, and DMG-PEG2000 was 50%:10%:38.5%:1.5%; the mass ratio of fluorinated lipid to mRNA was 11:1. Six hours later, 200 μL of D-firefly luciferase substrate at a concentration of 15 mg / ml was injected intraperitoneally into each mouse for subsequent in vivo imaging. Fifteen minutes later, the mice were placed under an in vivo imaging system to observe and photograph the bioluminescence intensity of the mouse body and isolated organs.

[0122] The test results are shown in Table 1 below. Figure 9 and Figure 10 As shown.

[0123] Table 1. In vivo luminescence intensity of different lipid nanoparticle compositions under different injection methods

[0124] sample muscle abdominal cavity A4R2I18F1 <![CDATA[1.069×10 6 ]]> <![CDATA[2.800×10 5 ]]> A4R2I18F5 <![CDATA[3.930×10 5 ]]> <![CDATA[5.000×10 4 ]]> A4R2I18F7 <![CDATA[3.014×10 5 ]]> <![CDATA[6.400×10 4 ]]> A4R2I18F11 <![CDATA[2.595×10 6 ]]> <![CDATA[7.400×10 4 ]]>

[0125] As shown in Table 1, different injection methods produce different delivery effects. Among them, A4R2I18F1 has a good delivery effect in both intramuscular and intraperitoneal injections.

[0126] Figure 9 The left image shows the in vivo bioluminescence intensity of A4R2I18F1 injected intramuscularly in Application Example 1, while the right image shows the in vitro bioluminescence intensity of various organs. Figure 9 It can be seen that A4R2I18F1 is highly expressed in the injection site after intramuscular injection.

[0127] Figure 10 The left image shows the bioluminescence intensity of A4R2I18F11 injected intraperitoneally in application example 4; the right image shows the bioluminescence intensity of various organs in vitro. Figure 10 It can be seen that intraperitoneal injection of A4R2I18F11 can efficiently deliver it to the spleen.

[0128] In summary, the fluorinated lipids provided by this invention, when used as delivery carriers for nucleic acid drugs, enable highly efficient in situ expression of mRNA at the injection site. This technology exhibits significant local targeting, effectively restricting protein expression to muscle tissue, thereby minimizing potential impacts on other organs, and combining high efficiency with safety.

[0129] 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 the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fluorine-containing ionizable lipid compound, characterized by, The fluorine-containing ionizable lipid compound has a structure as shown in any one of formula (I) and formula (II): In the formula, n1 is at least one group independently selected from a hydrogen atom, a linear alkyl group, a branched alkyl group, a linear alkenyl group, a branched alkenyl group, a substituted alkynyl group, a cycloalkyl group, a phenyl group, and a heteroatom-containing aromatic group; n2 is at least one group independently selected from a linear alkyl group, a linear alkenyl group, a heteroatom-containing linear alkyl group, a heteroatom-containing linear alkylene group, a heteroatom-containing branched alkyl group, a heteroatom-containing branched alkylene group, a heteroatom-containing cyclic alkyl group, a heteroatom-containing cyclic alkylene group, an ester-containing alkyl group, and an ether-containing alkyl group; n3 is at least one group independently selected from a linear alkyl group, a linear alkenyl group, a cycloalkyl group, a heteroatom-containing linear alkyl group, a heteroatom-containing branched alkyl group, a heteroatom-containing branched alkylene group, a heteroatom-containing cyclic alkyl group, a heteroatom-containing and aromatic cyclic alkyl group, a hydroxyl-containing alkyl group, an ester-containing alkyl group, an ether-containing alkyl group, and a sulfonate-containing alkyl group; n4 is at least one group independently selected from a linear alkyl group, a branched alkyl group, a linear alkenyl group, a branched alkenyl group, a linear alkynyl group, a heteroatom-containing linear alkyl group, and a heteroatom-containing branched alkyl group.

2. The fluorolipid compound of claim 1, wherein, n1is independently selected from any one of the following groups; wherein, represents the position of attachment of the group. n2 is any one independently selected from the group consisting of: wherein: represents the position of the group attachment; n3 is any one independently selected from the group consisting of: represents the position of the group attachment; n4 is any one independently selected from the group consisting of: represents the position of the group attachment; 。 3. The fluorolipid compound of claim 1, wherein The fluorine-containing ionizable lipid compound includes at least one of the following compounds:

4. A process for the preparation of the fluorine-containing ionizable lipid compound according to any one of claims 1 to 3, characterized in that, The method includes the following steps: The aldehyde compound and the amine compound are added to an organic solvent, preferably methanol as the reaction solvent, after 1-15 h of reaction, the carboxylic acid compound and the isonitrile compound are added, and then the reaction is carried out at -20-120℃ for 1-72 h, the product is separated and purified, and then the fluorine-containing ionizable lipid compound is obtained. The aldehyde compound is any one of the following compounds: ； The amine compound is any one of the following compounds: ； The carboxylic acid compound is any one of the following compounds: ； The isonitrile compound is any one of the following compounds: 。 5. The method for preparing the fluorine-containing ionizable lipid compound according to claim 4, characterized in that, The molar ratio of the aldehyde compound, the amine compound, the carboxylic acid compound, and the isonitrile compound is (0.1-1.5):(0.1-1.5):(0.1-1.5):(0.1-1.5).

6. A lipid nanoparticle (LNP) composition carrier, characterized in that, The lipid nanoparticle composition carrier includes one or more than two combinations of the fluorine-containing ionizable lipid compound, the polyethylene glycol lipid, the cholesterol, and the neutral phospholipid according to any one of claims 1-3.

7. The lipid nanoparticle (LNP) composition carrier of claim 6, wherein, The lipid nanoparticle composition includes the following components in terms of molar percentage: 10%-70% of the fluorine-containing ionizable lipid compound, 0.1%-25% of the polyethylene glycol lipid, 10%-50% of the cholesterol, and 5%-50% of the neutral phospholipid.

8. Use of the lipid nanoparticle LNP composition carrier according to claim 6 or 7 in the preparation of a nucleic acid drug carrier.

9. A method of making a nanoparticle composition encapsulating a nucleic acid drug, characterized by The method includes the following steps: The fluorine-containing ionizable lipid compound, the neutral phospholipid, the polyethylene glycol lipid, and the cholesterol according to any one of claims 1-3 are dissolved and mixed with a water-miscible organic solvent, preferably ethanol as the organic solvent, to obtain an organic phase liposome solution; The nucleic acid drug is dissolved in an acidic buffer solution with pH = 1.0-6.9, preferably a citric acid / sodium citrate buffer solution, to obtain an aqueous phase nucleic acid drug solution; The organic phase liposome solution and the aqueous phase nucleic acid drug solution are mixed and dialyzed to obtain the nucleic acid drug-loaded nanoparticle composition.

10. The nucleic acid drug-loaded nanoparticle composition prepared by the preparation method of claim 9.

Images