A DNA and RNA lipid delivery carrier

Abstract

The application provides a high-efficiency and low-toxicity DNA and RNA lipid delivery carrier. In order to enrich the types of nucleic acid drug delivery carriers, improve the in-vivo delivery and expression effect of nucleic acid drugs, and reduce toxic side effects, the application provides a new ionizable lipid compound with a special structure, which has a good combination effect with a negatively charged nucleic acid, effectively prevents the premature degradation of the nucleic acid in cells by nucleases, is beneficial to the transmembrane of the nucleic acid-loaded liposome, can realize effective degradation, rapid in-vivo clearance, reduces the toxic side effects of nucleic acid drugs, and can form a nucleic acid drug delivery carrier which truly considers high transfection efficiency, high expression effect and low toxicity.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid drug delivery vector technology, specifically relating to a highly efficient and low-toxicity DNA and RNA lipid delivery vector. Background Technology

[0002] Currently, lipid delivery carriers are widely used for drug delivery, especially with the rapid expansion of their application in nucleic acid drugs. Because nucleic acid molecules carry a negative charge, they are not conducive to cell membrane interaction and have poor cell permeability. Furthermore, nucleic acids in a naked state once inside tissues or cells are easily degraded by nucleases. Nucleic acid transfection not only requires the successful transport of biologically functional nucleic acids into cells but also ensures that these nucleic acids can maintain their biological functions within the cell. Commonly used carriers in research and clinical applications include viral and non-viral vectors. Liposomes, being non-viral vectors, offer higher nucleic acid transfection efficiency, lower cytotoxicity, and more convenient preparation processes compared to viral vectors, making them more practical and attracting increasing attention.

[0003] As research progresses, researchers have actively developed various liposome carriers, continuously improving their safety and efficiency. Early carriers, most commonly formed from cationic lipid compounds, work by forming a liposome-nucleic acid drug complex with a negatively charged nucleic acid. The surface of this complex is positively charged and adsorbs onto the negatively charged cell surface via electrostatic interactions. It then enters the cell via endocytosis, forming an endosome. The cationic lipids in the liposome interact electrostatically with the negatively charged lipids in the endosome, causing the negatively charged lipids to flip from outside the endosome lumen to inside, forming neutral ion pairs with the positively charged lipids, thus releasing the nucleic acid drug from the cationic lipids. In 2018, the FDA approved the first siRNA drug (patisiran [Onpattro]), which used Dlin-MC3-DMA liposomes as its delivery carrier. More recently, research has revealed that ionizable lipids can change their electrical charge in response to environmental pH, attracting significant attention.

[0004] Despite recent advancements in the use of ionizable lipids for drug delivery, demonstrating significant advantages over viral vectors and other non-viral vectors in terms of encapsulation efficiency, nucleic acid expression, and cytotoxicity, the number of commercially available ionizable lipid molecules remains limited. Furthermore, many of these molecules tend to distribute into the liver, increasing its metabolic burden and potentially causing toxic side effects. Therefore, it is still necessary to explore more ionizable lipid compounds suitable for nucleic acid drug applications and develop nucleic acid drug delivery vectors that truly balance high transfection efficiency, high expression efficacy, and low toxicity. Summary of the Invention

[0005] The purpose of this invention is to provide an ionizable lipid compound that is simple to prepare, easily binds to nucleic acids, and is easily degraded, thereby enriching the variety of ionizable lipid compounds and providing more options for nucleic acid drug delivery.

[0006] Another object of the present invention is to provide a delivery carrier, which is a highly efficient and low-toxicity lipid delivery carrier for delivering DNA and RNA. This delivery carrier, along with the nucleic acid molecules encapsulated within it, constitutes a drug composition that ensures the activity of the nucleic acid drug and high expression efficiency of the nucleic acid molecules, while significantly reducing drug distribution in the liver.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] The ionizable lipid compounds represented by general formula (I) and general formula (II)

[0009]

[0010] in,

[0011] In general formula (I), G1 is C1-C 10 Straight-chain alkylene groups, where G2 is -OC(=O)R, -C(=O)OR, or -C(=O)NHR, and R is C5-C. 20 straight-chain alkyl or R1 is hydrogen, methyl, ethyl or isopropyl, m is an integer between 1 and 10, n is an integer between 1 and 5, and f is an integer between 1 and 5;

[0012] In general formula (II), G3 is C1-C 10 Straight-chain alkylene groups, G4 is -OC(=O)R', -C(=O)OR' or -C(=O)NHR', R' is R2 and R3 are independently hydrogen, methyl, ethyl, or isopropyl, and q is an integer between 1 and 3.

[0013] Preferably, G1 is a C2-C8 straight-chain alkylene group.

[0014] Preferably, G3 is C5-C. 10 Straight-chain alkylene groups.

[0015] Preferably, R1 is hydrogen.

[0016] Preferably, m is an integer between 3 and 8, and more preferably an integer between 4 and 6.

[0017] Preferably, f is an integer between 1 and 4, and more preferably 2 or 3.

[0018] Preferably, R is C5-C 15 straight-chain alkyl or

[0019] Preferably, G2 is -C(=O)OR.

[0020] Preferably, G4 is -C(=O)OR'.

[0021] Preferably, one of R2 and R3 is hydrogen, and the other is methyl, ethyl, or isopropyl.

[0022] More preferably, one of R2 and R3 is hydrogen and the other is methyl.

[0023] In a further preferred embodiment, R2 is methyl and R3 is hydrogen.

[0024] According to some specific and preferred embodiments, the ionizable lipid compound is one or more of the following compounds:

[0025]

[0026] The present invention also provides a delivery carrier comprising one or more of the ionizable lipid compounds represented by general formula (I) and general formula (II).

[0027] Preferably, the delivery carrier further includes auxiliary molecules.

[0028] More preferably, the molar ratio of the ionizable lipid compound to the auxiliary molecule is (0.1-1):(0.1-1), and more preferably (0.5-1):(0.5-1).

[0029] The auxiliary molecule may be a commonly used auxiliary molecule in the art.

[0030] Preferably, the auxiliary molecules include one or more of the following: synthetic or naturally derived auxiliary lipids or lipid molecules, animal-derived molecules of any species, and cells or vesicles (including exosomes) or components thereof, polypeptide molecules, polymer molecules, carbohydrate molecules, or inorganic substances.

[0031] More preferably, the auxiliary molecules include one or more of cholesterol, calcipotriol, stigmasterol, β-sitosterol, lupeol, betulinol, ursolic acid, oleanolic acid, dioleoylphosphatidylcholine, distearylphosphatidylcholine, 1-stearoyl-2-oleoyllecithin, dioleoylphosphatidylethanolamine, (1,2-dioleoyloxypropyl)trimethylammonium chloride, didecyldimethylammonium bromide, 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine, dipalmitoylphosphatidylethanolamine-methoxy polyethylene glycol 5000, distearylphosphatidylethanolamine-polyethylene glycol 2000, activated carbon, silicon dioxide, and calcium phosphate.

[0032] Preferably, the ionizable lipid compound and / or the auxiliary molecule are modified with a targeting substance.

[0033] More preferably, the targeting substance includes one or more of folic acid, single-chain antibodies, or targeting peptides.

[0034] Preferably, the delivery carrier is a nanolipid particle.

[0035] More preferably, the average size of the nanoparticle formulation is 50 nm to 200 nm.

[0036] More preferably, the average size of the nanoparticle formulation is 50 nm to 150 nm.

[0037] Furthermore, the polydispersity index of the nanoparticle formulation is ≤0.4.

[0038] Furthermore, the polydispersity index of the nanoparticle formulation is ≤0.3.

[0039] The delivery carrier described in this invention, together with the nucleic acid molecules encapsulated in the delivery carrier, constitutes a nucleic acid drug composition.

[0040] Preferably, the nucleic acid molecule is one or more of pDNA, siRNA, ASO, or mRNA.

[0041] Preferably, the mass ratio of the nucleic acid molecule to the delivery vector is 1:(5-50); further preferably 1:(5-40); and even more preferably 1:(5-30).

[0042] Preferably, the nucleic acid drug composition includes a drug-grade additive, which includes one or more excipients, stabilizers, or diluents.

[0043] Furthermore, the additives include, but are not limited to, sucrose, trehalose, or other stabilizers.

[0044] Specifically, the amount of the additive added is 1% to 20% of the total mass of the pharmaceutical composition.

[0045] Preferably, the nucleic acid drug composition can be a lyophilized powder or an injection, wherein the injection is administered locally via intramuscular, subcutaneous, endothelial, or intratumoral administration using microneedles, injection, or perfusion, or administered via intravenous injection.

[0046] In this invention, the delivery carrier or the nucleic acid drug composition is used to deliver nucleic acid molecules into mammalian cells.

[0047] Preferably, the mammal is a human.

[0048] The present invention also provides a method for in vivo delivery of nucleic acid molecules, wherein the delivery carrier is used to deliver the nucleic acid molecules into the body of a subject.

[0049] Preferably, the subject is a mammal.

[0050] More preferably, the subject is a human being.

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

[0052] This invention provides a novel ionizable lipid compound, enriching the variety of ionizable lipid compounds. The delivery carrier formed by this compound has the advantages of high encapsulation efficiency and low toxicity, enabling efficient delivery and expression of nucleic acid drugs in vivo. This provides more options for nucleic acid drug delivery and is of great significance to the development and application of nucleic acid drugs. Attached Figure Description

[0053] Figure 1 The hydrogen spectrum of compound a is shown below.

[0054] Figure 2 The hydrogen spectrum of compound b is shown below.

[0055] Figure 3 The hydrogen spectrum of compound 1-1;

[0056] Figure 4 The hydrogen spectrum of compound 2-1;

[0057] Figure 5 The hydrogen spectrum of compound 3-1;

[0058] Figure 6 The hydrogen spectrum of compound 1 is shown below.

[0059] Figure 7 This is the high-resolution mass spectrum of compound 1;

[0060] Figure 8 The hydrogen spectrum of compound 2 is shown below.

[0061] Figure 9 This is the high-resolution mass spectrum of compound 2;

[0062] Figure 10 The hydrogen spectrum of compound 3 is shown below.

[0063] Figure 11 This is the high-resolution mass spectrum of compound 3;

[0064] Figure 12 The particle size distribution diagram is shown for the nanolipid particles prepared in Example 4.

[0065] Figure 13 The in vivo delivery effect of Lipid-01 liposomes in mice in Example 5;

[0066] Figure 14 The in vivo delivery effect of Lipid-02 liposomes in mice in Example 5;

[0067] Figure 15 The in vivo delivery effect of Lipid-03 liposomes in mice in Example 5 is shown. Detailed Implementation

[0068] The present invention will be further described below with reference to embodiments. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0069] To reduce the toxic side effects caused by the accumulation of drug components in the liver, lower the production cost of nucleic acid drug delivery carriers, and improve the in vivo delivery and expression effects of nucleic acid drugs, the inventors conducted extensive research and experimental verification, and developed a new ionizable lipid compound that can form a nucleic acid drug delivery carrier that truly balances high transfection efficiency, high expression effect, and low toxicity.

[0070] Specifically, in this invention, the ionizable lipid compound is a compound represented by general formula (I) or general formula (II):

[0071]

[0072] in,

[0073] In general formula (I), G1 is C1-C 10 Straight-chain alkylene groups, where G2 is -OC(=O)R, -C(=O)OR, or -C(=O)NHR, and R is C5-C. 20 straight-chain alkyl or R1 is hydrogen, methyl, ethyl or isopropyl, m is an integer between 1 and 10, n is an integer between 1 and 5, and f is an integer between 1 and 5;

[0074] In general formula (II), G3 is C1-C 10 Straight-chain alkylene groups, G4 is -OC(=O)R', -C(=O)OR' or -C(=O)NHR', R' is R2 and R3 are independently hydrogen, methyl, ethyl, or isopropyl, and q is an integer between 1 and 3.

[0075] Ionizable lipid compounds with special structures can enhance their binding ability to negatively charged nucleic acids, preventing premature degradation of nucleic acids by nucleases within cells. This facilitates the passage of nucleic acid-loaded liposomes across cell membranes, enabling effective degradation and rapid clearance in vivo, and reducing the toxic side effects of nucleic acid drugs.

[0076] According to the present invention, the delivery carrier comprises one or more of the ionizable lipid compounds represented by general formula (I) and general formula (II), and selectively includes auxiliary molecules.

[0077] According to the present invention, the delivery carrier is lipid nanoparticles with a particle size of 50 nm to 200 nm.

[0078] According to the present invention, the delivery vector can be used for delivery of one or more of pDNA, siRNA, ASO or mRNA.

[0079] According to the present invention, a delivery vector and a nucleic acid molecule encapsulated in the delivery vector constitute a nucleic acid drug composition, wherein the nucleic acid molecule is one or more of pDNA, siRNA, ASO, or mRNA.

[0080] According to the present invention, the nucleic acid drug composition may be a lyophilized powder or an injection, wherein the injection is administered locally via intramuscular, subcutaneous, endothelial, or intratumoral administration by microneedle, injection, or perfusion, or administered via intravenous injection.

[0081] In this invention, the delivery carrier or the nucleic acid drug composition is used to deliver nucleic acid molecules into mammalian cells, preferably human mammals.

[0082] The technical solution and technical effects of the present invention will be further described below with reference to specific embodiments.

[0083] Unless otherwise specified, all methods used in the following examples are conventional; and all experimental materials used, unless otherwise specified, were purchased from conventional biochemical reagent manufacturers.

[0084] Example 1

[0085] Synthetic route of compound 1:

[0086]

[0087] Step 1: Synthesis of Compound 1-1

[0088] 8-Bromooctanoic acid (1.139 g, 5.13 mmol) and citronellol (1.599 g, 10.25 mmol) were dissolved in dichloromethane (60 mL). After complete dissolution, EDC hydrochloride (0.98 g, 5.13 mmol) and DMAP (0.125 g, 1.03 mmol) were added. The mixture was stirred at room temperature for 18 hours. After the reaction was complete, it was diluted with DCM (200 mL) and washed with saturated NaHCO3 (100 mL) and brine (100 mL). The combined organic layers were dried over anhydrous Na2SO4, and the solvent was removed under vacuum to obtain the crude product. The crude product was purified by chromatography (silica gel column, eluent: petroleum ether containing 0.5% EA (v / v)). The purified product was evaporated to remove the eluent, giving a pale yellow oily compound (0.648 g, 35%). The 1H NMR spectrum of compound 1-1 is shown below. Figure 3 .

[0089] 1H NMR (400MHz, CDCl3) δ: 5.09 (s, 1H), 4.18-4.01 (m, 2H), 3.40 (t, J = 6.8Hz, 2H), 2.29 (t, J = 7.4Hz, 2H), 1.98 (s,2H),1.84(dd,J=14.3,7.0Hz,2H),1.70-1.60(m,9H),1.38(d,J=37.7Hz,9H),0.89(t,J=12.9Hz,4H).

[0090] Step 2: Synthesis of Compound 1

[0091] 2-(bis(2-aminoethyl)amino)ethyl-1-ol) (compound a, 0.044 g, 0.30 mmol, 1H NMR spectrum shown) Figure 13,7-dimethyl-6-enyl 6-bromohexanoate (0.398 g, 1.2 mmol) was added to a reaction flask and dissolved in THF / CH3CN (1:1, 6 mL), followed by the addition of DIPEA (0.155 g, 1.20 mmol). The reaction mixture was stirred at 63 °C for 72 h. After cooling to room temperature, the solvent was removed under vacuum. The crude product was extracted with ethyl acetate and saturated NaHCO3. The combined organic layers were dried over anhydrous Na2SO4, and the solvent was removed under vacuum to obtain the crude product. The crude product was purified by chromatography (silica gel column, eluent: dichloromethane containing 2% methanol (v / v)). The purified product was evaporated to remove the eluent, yielding a yellow oily compound 1 (25.7 mg, yield 3.6%). The 1H NMR spectrum of compound 1 is shown below. Figure 6 Mass spectrometry Figure 7 .

[0092] 1H NMR (400MHz, CDCl3) δ5.09 (s, 4H), 4.10 (dd, J=12.6, 6.5Hz, 8H), 3.61 (d, J= 21.6Hz,2H),3.19(s,1H),3.03(s,1H),2.98-2.92(m,1H),2.82(s,4H),2.67 (s,4H),2.28(t,J=7.5Hz,8H),2.03-1.93(m,8H),1.74-1.50(m,49H),1.36( ddd,J=44.4,23.9,10.0Hz,39H),1.23-1.13(m,4H),0.91(d,J=6.5Hz,12H).

[0093] Example 2

[0094] Synthetic route of compound 2

[0095]

[0096] Step 1: Synthesis of Compound 2-1

[0097] Linolenic acid (0.267 g, 1 mmol) and triethylamine (0.133 g, 1.3 mmol) were added to a reaction flask and placed in an ice-water bath. Dichloromethane (6 mL) was added, and acryloyl chloride (0.11 g, 1.2 mmol) dissolved in dichloromethane (2.2 mL) was slowly added dropwise to the reaction flask. The reaction was continued for 10 minutes, maintained below 10 °C. Finally, the ice bath was removed, and the reaction mixture was allowed to react at room temperature for 2 hours. The product was washed with saturated brine to obtain a crude product. The crude product was purified by chromatography (silica gel column, eluent: petroleum ether containing 0.5% EA (v / v)). The purified product was evaporated to remove the eluent, yielding a pale yellow oily compound 2-1 (0.173 g, yield: 50%). The 1H NMR spectrum of compound 2-1 is shown below. Figure 4 .

[0098] 1H NMR (400MHz, CDCl3) δ: 6.41 (dd, J=17.3, 1.5Hz, 1H), 6.13 (dd, J=17.3, 10.4Hz, 1H), 5.82 (dd, J=10.4, 1.5Hz, 1H), 5.47-5.26 (m, 4H), 4.16 ( t,J=6.7Hz,2H),2.78(t,J=6.5Hz,2H),2.06(dd,J=13.6,6.7Hz,4H),1.75-1.60(m,2H),1.39–1.17(m,16H),0.88(dt,J=10.4,5.3Hz,3H).

[0099] Step 2: Synthesis of Compound 2

[0100] 1,3-Diamino-2-propanol (compound b, 0.04504 g, 0.50 mmol, 1H NMR spectrum shown) was used. Figure 2 2-Allyl acid (9Z, 12Z)-octadecadienyl ester (0.64 g, 2 mmol) was added to a reaction flask and dissolved in THF / CH3CN (1:1, 6 mL), followed by the addition of DIPEA (0.155 g, 1.20 mmol). The reaction was carried out at 80 °C for 48 hours. After cooling to room temperature, the solvent was removed under vacuum to obtain the crude product. The crude product was purified by chromatography (silica gel column, eluent: dichloromethane containing 0.3% methanol (v / v)). The purified product was evaporated to remove the eluent, yielding a pale yellow oily compound 2 (20.58 mg, yield 3%). The proton NMR spectrum of compound 2 is shown below. Figure 8 Mass spectrometry Figure 9 .

[0101] 1H NMR (400MHz, CDCl3) δ5.45 -5.32(m,16H),4.09(t,J=6.8Hz,8H),3.78(d,J=20.7Hz,1H),3.51(d,J=27.2Hz,1H),2.81(t,J=6.4Hz,1 6H), 2.49 (s, 10H), 2.09 (q, J = 6.8Hz, 16H), 1.69-1.60 (m, 8H), 1.41-1.30 (m, 66H), 0.93 (t, J = 6.8Hz, 12H).

[0102] Example 3

[0103] Synthetic route of compound 3

[0104]

[0105] Step 1: Synthesis of Compound 3-1

[0106] 6-Bromhexanoic acid (1.0 g, 5.13 mmol) and undecyl alcohol (1.77 g, 10.25 mmol) were dissolved in dichloromethane (60 mL), and EDC hydrochloride (0.98 g, 5.13 mmol) and DMAP (0.125 g, 1.03 mmol) were added. The mixture was stirred at room temperature for 18 hours. After the reaction was complete, the mixture was diluted with DCM (200 mL) and washed with saturated NaHCO3 (100 mL) and brine (100 mL). The combined organic layers were dried over anhydrous Na2SO4, and the solvent was removed under vacuum to obtain the crude product. The crude product was purified by chromatography (silica gel column, eluent: petroleum ether containing 0.5% EA (v / v)). The purified product was evaporated to remove the eluent, yielding a pale yellow oily compound 3-1 (0.69 g, yield 38.6%). The 1H NMR spectrum of compound 3-1 is shown below. Figure 5 .

[0107] 1H NMR(400MHz, CDCl3)δ:4.10(t,J=6.6Hz,2H),3.45(t,J=6.7Hz,2H),2.36(t,J=7.3Hz,2H),1.97-1.88(m,2H) ,1.68(tt,J=14.5,7.3Hz,4H),1.53(dd,J=15.1,7.9Hz,2H),1.33(d,J=16.9Hz,16H),0.92(t,J=6.5Hz,3H).

[0108] Step 2: Synthesis of Compound 3

[0109] 1,3-Diamino-2-propanol (0.027 g, 0.30 mmol) and undecanoate 6-bromohexanoate (0.417 g, 1.20 mmol) were dissolved in THF / CH3CN (1:1, 6 mL), followed by the addition of DIPEA (0.155 g, 1.20 mmol). The reaction mixture was stirred at 63 °C for 72 h. After cooling to room temperature, the solvent was removed under vacuum. The crude product was extracted with ethyl acetate and saturated NaHCO3. The combined organic layers were dried over anhydrous Na2SO4, and the solvent was removed under vacuum to obtain the crude product. The crude product was purified by chromatography (silica gel column, eluent: dichloromethane containing 1% methanol (v / v)). The purified product was evaporated to remove the eluent, yielding a pale yellow oily compound 3 (47.72 mg, yield 4.1%). The proton NMR spectrum of compound 3 is shown below. Figure 10 Mass spectrometry Figure 11 .

[0110] 1H NMR (400MHz, CDCl3) δ5.30(s,1H),4.05(t,J=6.8Hz,8H),3.68(s,1H),2.47(s,8H),2.30(t,J=7.5H z,8H),1.62(dd,J=15.1,7.7Hz,16H),1.47(s,6H),1.28(d,J=16.8Hz,78H),0.88(t,J=6.8Hz,12H).

[0111] Example 4:

[0112] Prepare lipid nanoparticles and test their particle size and potential.

[0113] Preparation method of lipid nanoparticles:

[0114] (1) According to the ionizable compound: DSPC: DMG-PEG2000: cholesterol in a molar ratio of 50:10:1.5:38.5, anhydrous ethanol was used as the solvent to prepare the liposome solution. The sum of the concentrations of each component was controlled to be 50mM. After dissolving and mixing, the solution was stored at -20℃ for later use.

[0115] (2) Dissolve the mRNA in 25mM sodium acetate buffer at a pH of about 5.2 to prepare a nucleic acid preparation with a final concentration of about 0.1mg / mL.

[0116] (3) The liposome solution and nucleic acid preparation were mixed by manual vortexing at a volume ratio of approximately 4:1 and a total flow rate of 12 mL / min to form a nanoliposome particle solution. Immediately after mixing, the solution was diluted 20 times with PBS buffer (pH 7.2) or sodium acetate buffer (pH 7.4). The solution was then concentrated using a 10 kDa ultrafiltration tube. The centrifuge speed should not exceed the maximum speed limit of the ultrafiltration tube. After 2-3 liquid changes, the solution environment of the nanoliposome particles changed from pH 5.2 to pH 7.2. Finally, the nanoliposome particle solution was concentrated to a final concentration of approximately 200 mM and stored at 4°C for later use.

[0117] The nanoliposome particle solution was diluted 50 times with 1×PBS, and the particle size of the nanoliposome particles was detected by Zetasizer Nano ZS (Malvern, Worcestershire, UK).

[0118] The zeta potential was measured by diluting the nanoliposome particles in 15 mM PBS.

[0119] Encapsulation efficiency was determined using the Quant-It RiboGreen RNA Quantitative Detection Kit on a Modulus microwell multifunction analyzer.

[0120] The results of particle size, PDI, encapsulation efficiency, and potential are shown in Table 1 and... Figure 12 As shown.

[0121] Table 1

[0122]

[0123] Example 5: In vivo transfection experiment with liposomes in animals:

[0124] Nanolipid particles were prepared using mRNA expressing Luciferase fluorescent protein according to the preparation method in Example 4, wherein the amount of mRNA used was 120 μg, and the total amount of ionizable liposome compound, DSPC, DMG-PEG2000 and cholesterol was 1200 μg. The liposome environment was rapidly converted using 400 μL of neutral PBS buffer.

[0125] The prepared nanolipid particles were rapidly injected intramuscularly (IM) into the medial hind limb muscles of 6-8 week old female Babl / c mice, with 30 μg of mRNA injected into each hind limb. The expression of luciferase in the mice was observed at different time points post-injection using a small animal imaging system.

[0126] The prepared nanolipid particles were rapidly injected intravenously (IV) into 6-8 week old female Babl / c mice, with an mRNA injection dose of 60 μg. The expression of luciferase in the mice was observed at different time points after injection using a small animal imaging system.

[0127] Four hours later, the heart, liver, spleen, lungs, and kidneys of the mice were subjected to fluorescence imaging.

[0128] The in vivo delivery efficacy of Lipid-01 liposomes in mice is shown in [reference needed]. Figure 13 The results showed that the fluorescence expression reached 7×10⁻⁶ four hours after intramuscular injection. 7 Imaging of various organs in mice showed that after intramuscular injection, the fluorescence expression in the organs was mainly concentrated in the spleen (80%), while after intravenous injection, the fluorescence was distributed in the liver (63%), spleen (31%), and lungs (6%), indicating that Lipid-01 intramuscular injection has good spleen targeting.

[0129] The in vivo delivery effect of Lipid-02 liposomes in mice is shown in [see details]. Figure 14 The results showed that the fluorescence expression was approximately 10 ppm 6 hours after intramuscular injection. 7Imaging of various organs in mice showed that after intravenous injection, fluorescence was mainly distributed in the liver, while after intramuscular injection, the fluorescence expression in the liver of mice was significantly lower, indicating that Lipid-02 is more suitable for intramuscular injection.

[0130] The in vivo delivery efficacy of Lipid-03 liposomes in mice is shown in [see details]. Figure 15 The results showed that the fluorescence expression was approximately 10 ppm 4 hours after intramuscular injection. 7 Imaging of various organs in mice showed that fluorescence was concentrated in the spleen after both intramuscular and intravenous injections, indicating that Lipid-03 has good spleen targeting properties after both intramuscular and intravenous injections.

[0131] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand and implement the invention. However, this description should not be construed as limiting the scope of protection of the invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be included within the scope of protection of the invention.

Claims

1. A nucleic acid drug composition comprising a delivery carrier and a nucleic acid molecule, characterized in that, The delivery carrier includes an ionizable lipid compound, wherein the ionizable lipid compound is an ionizable lipid compound of general formula (I), an ionizable lipid compound of general formula (II), or a lipid compound of general formula (II). One or more of them, in, In general formula (I), G1 is C1-C 10 Straight-chain alkylene groups, G2 is -C(=O)OR, R is C5-C 20 straight-chain alkyl or R1 is hydrogen, methyl, ethyl or isopropyl, m is an integer between 1 and 10, n is an integer between 1 and 5, and f is an integer between 1 and 5; In general formula (II), G3 is C1-C 10 Straight-chain alkylene groups, G4 is -C(=O)OR', R' is... In the case of R2 and R3, one is hydrogen and the other is methyl, ethyl or isopropyl, and q is an integer between 1 and 3.

2. The nucleic acid drug composition according to claim 1, characterized in that, The G1 mentioned is a C2-C8 straight-chain alkylene group; And / or, the G3 is C5-C 10 Straight-chain alkylene; And / or, R1 is hydrogen; And / or, m is an integer between 3 and 8; And / or, where f is an integer between 1 and 4; And / or, the R is C5-C 15 straight-chain alkyl or 3. The nucleic acid drug composition according to claim 2, characterized in that, The m mentioned is an integer between 4 and 6; And / or, f is 2 or 3.

4. The nucleic acid drug composition according to claim 1, characterized in that, R2 is methyl and R3 is hydrogen.

5. The nucleic acid drug composition according to claim 1, characterized in that, The delivery carrier further includes an auxiliary molecule, and the molar ratio of the ionizable lipid compound to the auxiliary molecule is (0.1–1): (0.1～1)。 6. The nucleic acid drug composition according to claim 5, characterized in that, The accessory molecules include one or more of the following: synthetic or naturally derived accessory lipids or lipid molecules, animal-derived molecules of any species, and cells or vesicles of any kind, polypeptide molecules, polymer molecules, carbohydrate molecules, or inorganic substances.

7. The nucleic acid drug composition according to claim 6, characterized in that, The auxiliary molecules include one or more of the following: cholesterol, calcipotriol, stigmasterol, lupeol, β-sitosterol, betulinol, ursolic acid, oleanolic acid, dioleoylphosphatidylcholine, distearylphosphatidylcholine, 1-stearoyl-2-oleoyllecithin, dioleoylphosphatidylethanolamine, (1,2-dioleoyloxypropyl)trimethylammonium chloride, didecyldimethylammonium bromide, 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine, dipalmitoylphosphatidylethanolamine-methoxy polyethylene glycol 5000, distearylphosphatidylethanolamine-polyethylene glycol 2000, activated carbon, silicon dioxide, and calcium phosphate.

8. The nucleic acid drug composition according to claim 5, characterized in that, The ionizable lipid compound and / or the auxiliary molecule are modified with a targeting substance, which includes one or more of folic acid, single-chain antibodies, or targeting peptides.

9. The nucleic acid drug composition according to claim 1, characterized in that, The delivery carrier is a nanolipid particle with a particle size of 50 nm to 200 nm; and / or, the polydispersity index of the nanoparticle formulation is ≤0.

4.

10. The nucleic acid drug composition according to claim 9, characterized in that, The nucleic acid molecule includes one or more of plasmid DNA (pDNA), siRNA, ASO, or mRNA; and / or, the mass ratio of the nucleic acid molecule to the delivery vector is 1:(5-50).

11. The nucleic acid drug composition according to claim 1, characterized in that, The nucleic acid drug composition further includes drug-grade additives, which include one or more excipients, stabilizers, or diluents.

12. The nucleic acid drug composition according to claim 11, characterized in that, The amount of the additive added is 1% to 20% of the total mass of the nucleic acid drug composition.

13. The nucleic acid drug composition according to claim 1, characterized in that, The nucleic acid drug composition is a lyophilized powder or injection, which is administered locally via intramuscular, subcutaneous, endothelial, or intratumoral administration using microneedles, injection, or perfusion, or via intravenous injection.

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