Gene vector capable of ionizing lipid and organ selective regulation and control method

Abstract

The invention relates to an organ selective regulation and control method of an ionizable lipid gene vector, which comprises the following steps: firstly, synthesizing uncoordinated ionizable lipid containing a ligand unit (such as cyclic amine), and forming ionizable lipid with different structural characteristics by selecting different tail chains, so that the ionizable lipid can directionally select liver or spleen; and metal ions are introduced into the non-coordinated ionizable lipid structure for coordination, so that the lung can be directionally selected. According to the method, nucleic acid molecules can be controllably and efficiently delivered to the liver, spleen or lung of a mouse, the method has the advantages of being simple, easy to implement and wide in universality, metal ion-ligand interaction is introduced into an uncoordinated ionizable lipid structure, charge distribution and apparent pKa of lipid nano particles are changed, and therefore the nucleic acid molecules can be efficiently and controllably delivered to the liver, spleen or lung of the mouse. The lipid nanoparticle organ is selectively converted from the liver or spleen to the lung, and ultrahigh nucleic acid molecule delivery efficiency is shown.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, and in particular relates to an ionizable lipid gene carrier and an organ-selective regulation method. Background Technology

[0002] Messenger RNA (mRNA) is considered a promising therapeutic tool due to its ability to produce any functional protein, and it has wide applications in various fields such as vaccines, gene editing, and protein replacement therapy. Unlike DNA, mRNA only needs to enter the cytoplasm to produce target proteins, reducing the risk of gene insertion mutations. However, mRNA alone is easily degraded by nucleases in the blood and cannot enter cells, making the development of safe and efficient nucleic acid vectors extremely important. Lipid nanoparticles (LNPs) are currently the most promising non-viral nucleic acid vectors in clinical practice, typically comprising four components: ionizable lipids, steroidal lipids, helper phospholipids, and polyethylene glycol-modified lipids. Among these, ionizable lipids are one of the most important components. They are positively charged at acidic pH, which can be used to aggregate and encapsulate negatively charged mRNA; while they are electrically neutral at physiological pH, thereby reducing potential cytotoxicity. Ionizable lipids typically consist of an amino head, a linker, and a hydrophobic tail. However, most developed lipid nanoparticles currently exhibit liver selectivity, making selective delivery to extrahepatic organs (e.g., lungs or spleen) difficult. Furthermore, methods for screening large libraries of ionizable lipids with ideal selectivity and delivery efficiency are highly complex and time-consuming. Therefore, there is an urgent need to develop strategies for regulating organ selectivity in lipid nanoparticles. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides an ionizable lipid gene vector and an organ-selective regulation method.

[0004] The technical solution adopted in this invention is: an ionizable lipid gene vector, comprising uncoordinated ionizable lipids with the structure shown in Formula 1;

[0005]

[0006] Where An is any one of the formulas A1-A5;

[0007]

[0008] y is an integer selected from 1 to 6;

[0009] Bm is selected from the tail chain containing 1 to 6 straight-chain alkyl thiols or branched alkyl thiols, with an unsaturation degree of 0-6. Preferably, Bm is any structure shown in B1-B27.

[0010]

[0011] or,

[0012] Including metal-coordinated ionized lipids; metal coordination is performed on the uncoordinated ionizable lipids shown in Formula 1 to obtain metal-coordinated ionized lipids.

[0013] Preferably, the metal ion is Co. 3+ Gd 3+ Zn 2+ Fe 3+ Fe 2+ Ca 2+ Mg 2+ Al 3+ and Cu 2+ One or more of the following plasmas.

[0014] Preferably, uncoordinated ionizable lipids or metal-coordinated ionizable lipids are mixed with auxiliary lipids, steroidal lipids and polyethylene glycol lipids, and then target nucleic acid molecules are added to prepare nanoparticles for use as gene carriers.

[0015] Preferably, the molar ratio of uncoordinated ionizable lipids or metal-coordinated ionizable lipids, auxiliary lipids, steroidal lipids and polyethylene glycol lipids is 5-45:15:30:0-5;

[0016] The molar ratio of uncoordinated ionizable lipids or metal-coordinated ionizable lipids to nucleic acid molecules is 2000-15000:1.

[0017] Preferably, the auxiliary lipid is one or more of the following: 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE).

[0018] Steroidal lipids are one or more combinations of dihydrocholesterol, physcosterol, sitosterol, cholic acid, alfalfa sterol, glycocholic acid, deoxycholic acid, cholesterol, brassosterol, ergosterol, ergocalciferol and hydroxycholesterol.

[0019] The polyethylene glycol lipids are one or more combinations of distearyl-rac-glycerol-polyethylene glycol 1000 (DSG-PEG1000), distearyl-rac-glycerol-polyethylene glycol 2000 (DSG-PEG2000), myristoyl-sn-glycerol-methoxy polyethylene glycol 1000 (DMG-PEG1000), myristoyl-sn-glycerol-methoxy polyethylene glycol 2000 (DMG-PEG2000), myristoyl-sn-glycerol-methoxy polyethylene glycol 5000 (DMG-PEG5000), PEG-distearate glycerol, dipalmitoylphosphatidylcholine polyethylene glycol, dipalmitoylphosphatidylethanolamine-polyethylene glycol, PEG-distearate group, and PEG-diacylglycerol amide.

[0020] Preferably, the nucleic acid molecule is one or more of the following: deoxyribonucleic acid (DNA), antisense nucleic acid (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), circular RNA (circRNA), messenger RNA (mRNA), aptamer, ribozyme, and antibody-nucleic acid conjugate (ARC).

[0021] The preparation method of ionizable lipid gene vectors, specifically the steps for uncoordinated ionizable lipids, is as follows:

[0022] Step 1: Acryloyl chloride is linked to one end of the hydroxyl group of 2-hydroxyethyl disulfide through a condensation reaction to generate an asymmetric acrylate, which is a disulfide linker.

[0023] The compound Bm thiol was prepared into a hydrophobic tail chain precursor by sequential addition, hydrolysis and chloroacylation reactions.

[0024] Step 2: The hydrophobic tail chain precursor is condensed with the disulfide linker to obtain the hydrophobic tail chain;

[0025] Step 3: The hydrophobic tail chain is reacted with compound An via an aza-McCl addition reaction to obtain an uncoordinated ionizable lipid;

[0026] Preferably, in step three, the catalyst dibutylhydroxytoluene is added, and the reaction temperature is 20-60℃.

[0027] Preferably, uncoordinated ionizable lipids and coordinated metal ions are mixed and reacted at 20-60°C to obtain metal-coordinated ionizable lipids.

[0028] An organ-selective regulation method for ionizable lipid gene vectors, wherein uncoordinated ionizable lipid nanoparticles constructed using Bm with a total number of carbon atoms less than 14 exhibit liver selectivity; uncoordinated ionizable lipid nanoparticles constructed using Bm with a carbon atom number greater than or equal to 14 exhibit spleen selectivity; and metal-coordinated ionizable lipid nanoparticles exhibit lung selectivity.

[0029] Application of ionizable lipid gene vectors or organ-selective regulation methods of ionizable lipid gene vectors in the preparation of nucleic acid drugs and gene vaccine drugs.

[0030] The advantages and positive effects of this invention are: in the design of the ionizable lipid structure, the disulfide bond of the linker portion serves as a biodegradable stimulating unit, effectively improving the escape efficiency and biosafety of lipid nanoparticles within the integument; the use of S atoms to connect the hydrophobic tail chain portion increases the conformational flexibility of the hydrophobic tail chain to a certain extent, thereby improving the delivery efficiency of nucleic acid molecules.

[0031] By selecting different types of tail chain structures or coordinating with metal ions, the characteristics of ionizable lipid structures can be changed, thereby altering the charge distribution and apparent pKa of lipid nanoparticles, and achieving organ-selective conversion of lipid nanoparticles from the liver or spleen to the lungs.

[0032] The organ-selective regulation method using ionizable lipid gene vectors can deliver nucleic acid molecules to the liver, spleen, or lungs of mice in a controllable and efficient manner, and has the advantages of being simple, easy to implement, and widely applicable. Attached Figure Description

[0033] Figure 1 A schematic diagram of the organ-selective regulation strategy of ionizable lipid gene vectors;

[0034] Figure 2 400MHz NMR spectrum of hydrophobic tail chain SCC-B4;

[0035] Figure 3 400MHz NMR spectrum of uncoordinated ionizable lipid A2-SCC-B4;

[0036] Figure 4 Particle size and zeta potential of uncoordinated lipid nanoparticles A2-SCC-B4 LNPs;

[0037] Figure 5 Particle size and zeta potential of zinc ion coordinated lipid nanoparticles Zn-A2-SCC-B4 LNPs;

[0038] Figure 6 Image showing the effect of transfecting Zn-A2-SCC-B4 LNPs with mCherry mRNA in IGROV1 cells in vitro;

[0039] Figure 7 A comparison of the efficiency of Zn-A2-SCC-B4 LNPs in transfecting luciferase mRNA in IGROWV1 cells in vitro with that of commercially available ionizable lipids.

[0040] Figure 8 Transfection efficiency of liver-selective A2-SCC-B4 LNPs in BALB / c mice;

[0041] Figure 9 Transfection efficiency of lung-selective Zn-A2-SCC-B4 LNPs in BALB / c mice;

[0042] Figure 10 Zn-A2-SCC-B4 LNPs enable tumor imaging in a mouse tumor model;

[0043] Figure 11 Transfection efficiency of spleen-selective A1-SCC-B13 LNPs in BALB / c mice;

[0044] Figure 12 Transfection efficiency of lung-selective Zn-A1-SCC-B13 LNPs in BALB / c mice;

[0045] Figure 13 1H NMR spectrum of uncoordinated ionizable lipid A1-SCC-B3;

[0046] Figure 14 1H NMR spectrum of uncoordinated ionizable lipid A1-SCC-B5;

[0047] Figure 15 1H NMR spectrum of uncoordinated ionizable lipid A1-SCC-B11;

[0048] Figure 16 1H NMR spectrum of uncoordinated ionizable lipid A1-SCC-B12;

[0049] Figure 17 1H NMR spectrum of uncoordinated ionizable lipid A2-SCC-B3;

[0050] Figure 18 1H NMR spectrum of uncoordinated ionizable lipid A2-SCC-B5;

[0051] Figure 19 1H NMR spectrum of uncoordinated ionizable lipid A2-SCC-B11;

[0052] Figure 20 1H NMR spectrum of uncoordinated ionizable lipid A2-SCC-B12;

[0053] Figure 211H NMR spectrum of uncoordinated ionizable lipid A2-SCC-B13;

[0054] Figure 22 1H NMR spectrum of uncoordinated ionizable lipid A3-SCC-B3;

[0055] Figure 23 1H NMR spectrum of uncoordinated ionizable lipid A3-SCC-B5;

[0056] Figure 24 1H NMR spectrum of uncoordinated ionizable lipid A3-SCC-B11;

[0057] Figure 25 1H NMR spectrum of uncoordinated ionizable lipid A3-SCC-B12;

[0058] Figure 26 1H NMR spectrum of uncoordinated ionizable lipid A3-SCC-B13. Detailed Implementation

[0059] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0060] This invention relates to a method for organ-selective regulation of an ionizable lipid gene vector, as well as lipid nanoparticles capable of targeting different organs and their preparation methods. First, an uncoordinated ionizable lipid containing a ligand unit (such as a cyclic amine) is synthesized. By selecting different tail chains, it can be directed to the liver or spleen. Then, by introducing metal ion ligands into the uncoordinated ionizable lipid structure, it can be directed to the lung.

[0061] The general formula for uncoordinated ionizable lipid structures is shown in Formula 1.

[0062]

[0063] Where An is any one of the formulas A1-A5;

[0064]

[0065] y is an integer selected from 1 to 6;

[0066] Bm is selected from the tail chain of 1 to 6 straight-chain alkyl thiols or branched alkyl thiols, with an unsaturation degree of 0-6, and Bm is attached to the secondary amine in An; preferably, Bm is any structure shown in B1-B27.

[0067]

[0068] The preparation method of uncoordinated ionizable lipids is as follows:

[0069]

[0070] Step 1: Prepare a linker containing a disulfide bond stimulus-response unit. The linker precursor 2-hydroxyethyl disulfide reacts with acryloyl chloride at one end to generate an asymmetric acrylate, which is called a disulfide bond linker.

[0071] Step 2: Bm thiol was successively added, hydrolyzed and chloroacylated to prepare a hydrophobic tail chain precursor; the hydrophobic tail chain precursor and disulfide linker were reacted in dichloromethane solvent by condensation reaction for 12 hours, and the crude product was purified by column chromatography to obtain the hydrophobic tail chain.

[0072] Step 3: The hydrophobic tail chain is reacted with at least one An structure at a certain molar ratio (related to the number of secondary amines in An) at 20-60°C for 24-72 hours with 2-10 mol% butylated hydroxytoluene (BHT). The crude product is purified by column chromatography to obtain uncoordinated ionizable lipids.

[0073] Introducing metal ion-ligand interactions into the uncoordinated ionizable lipid structure alters the charge distribution and apparent pKa of lipid nanoparticles, enabling organ-selective transfer of lipid nanoparticles from the liver or spleen to the lungs. The coordinating metal can specifically be Co. 3+ Gd 3+ Zn 2+ Fe 3+ Fe 2+ Ca 2+ Mg 2+ Al 3+ Cu 2+ Metal ions, etc.

[0074] The preparation method of metal-coordinated ionizable lipid nanoparticles is as follows:

[0075] Step 4: Dissolve at least one uncoordinated ionizable lipid and at least one coordinated metal ion salt in methanol solvent, mix them in a molar ratio of 1:1, react at a temperature of 20-60℃ for 12-72 hours, remove the solvent by rotary evaporation, and obtain the corresponding metal coordinated ionizable lipid.

[0076] Nanoparticles prepared by mixing uncoordinated or metal-coordinated ionizable lipids with auxiliary lipids, steroidal lipids, and polyethylene glycol lipids can serve as carriers for nucleic acid molecules, transferring target nucleic acid molecules to their target locations. The regulatory strategies are as follows... Figure 1 As shown.

[0077] The accessory lipids are one or more combinations of 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE). The steroidal lipids are one or more combinations of dihydrocholesterol, phycosterol, sitosterol, cholic acid, alfalfa sterol, glycocholic acid, deoxycholic acid, cholesterol, brassosterol, ergosterol, ergocalciferol, and hydroxycholesterol. The polyethylene glycol lipids are one or more combinations of distearyl-rac-glycerol-polyethylene glycol 1000 (DSG-PEG1000), distearyl-rac-glycerol-polyethylene glycol 2000 (DSG-PEG2000), myristoyl-sn-glycerol-methoxy polyethylene glycol 1000 (DMG-PEG1000), myristoyl-sn-glycerol-methoxy polyethylene glycol 2000 (DMG-PEG2000), myristoyl-sn-glycerol-methoxy polyethylene glycol 5000 (DMG-PEG5000), PEG-distearate glycerol, dipalmitoylphosphatidylcholine polyethylene glycol, dipalmitoylphosphatidylethanolamine-polyethylene glycol, PEG-distearate group, and PEG-diacylglycerol amide.

[0078] Nanoparticles can serve as carriers for a variety of nucleic acid molecules, which can be one or more of the following: deoxyribonucleic acid (DNA), antisense nucleic acid (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), circular RNA (circRNA), messenger RNA (mRNA), aptamers, ribozymes, and antibody-nucleic acid conjugates (ARC).

[0079] At least one metal-coordinated ionizable lipid or uncoordinated ionizable lipid and at least one auxiliary lipid, steroid lipid, or polyethylene glycol lipid are dissolved in an organic solvent at a specific molar ratio to form a mixture; at least one nucleic acid molecule is dissolved in an acetic acid / sodium acetate solution (pH 5.2); the mixture (organic phase) is mixed with the nucleic acid molecule solution (aqueous phase) at a volume ratio of 1:3, with the molar ratio of uncoordinated ionizable lipid or metal-coordinated lipid to nucleic acid molecule being 2000-15000:1, rapidly mixed, and allowed to stand for 10-30 minutes to obtain uncoordinated lipid nanoparticles or metal-coordinated lipid nanoparticles.

[0080] Uncoordinated lipid nanoparticles are composed of a mixture of uncoordinated ionizable lipids, auxiliary lipids, steroidal lipids, and polyethylene glycol lipids, with a molar ratio of 5-45:15:30:0-5. When uncoordinated lipid nanoparticles carry nucleic acid molecules, the molar ratio of uncoordinated ionizable lipids to nucleic acid molecules is 2000-15000:1. Metal-coordinated lipid nanoparticles are also composed of a mixture of metal-coordinated ionizable lipids, auxiliary lipids, steroidal lipids, and polyethylene glycol lipids, with a molar ratio of 5-45:15:30:0-5. When metal-coordinated lipid nanoparticles carry nucleic acid molecules, the molar ratio of metal-coordinated ionizable lipids to nucleic acid molecules is 2000-15000:1.

[0081] Uncoordinated lipid nanoparticles or metal-coordinated lipid nanoparticles can be used as non-viral gene carriers, enabling the delivery of mixed nucleic acid molecules to in vitro cells. This regulatory method allows for the controllable and efficient delivery of nucleic acid molecules to the liver, spleen, or lungs of mice, offering advantages such as simplicity, ease of implementation, and broad applicability. By introducing metal ion-ligand interactions into the uncoordinated ionizable lipid structure, the charge distribution and apparent pKa of the lipid nanoparticles are altered, achieving organ selectivity from the liver or spleen to the lungs, and exhibiting ultra-high nucleic acid molecule delivery efficiency.

[0082] Uncoordinated ionizable lipids can be localized to the liver or spleen. The structure of the nanoparticles prepared from these uncoordinated ionizable lipids varies depending on the length or structure of their tail chains, thus achieving selective modification of the liver or spleen. In some embodiments of this invention, longer tail chains facilitate localization to the spleen, while shorter tail chains facilitate localization to the liver. However, when uncoordinated ionizable lipids are coordinated with metal ions, regardless of the tail chain structure, they will be localized to the lungs.

[0083] Uncoordinated lipid nanoparticles were injected intravenously into mice. When the total number of carbon atoms in the Bm tail chain was less than 14, the resulting uncoordinated ionizable lipid nanoparticle nucleic acid molecules functioned in the liver. When the number of carbon atoms in the Bm tail chain was greater than or equal to 14, the resulting uncoordinated ionizable lipid nanoparticle nucleic acid molecules functioned in the liver. Metal-coordinated lipid nanoparticles were injected intravenously into mice, and the nucleic acid molecules functioned accordingly in the lungs.

[0084] The nanoparticle structure formed by ionizable lipids contains disulfide bond linkers. The glutathione content in tumors is higher than that in normal tissues, so the material has a higher mRNA delivery efficiency in tumors. In addition, tumor cells are more likely to internalize nanoparticles than normal tissues, so lipid nanoparticles will preferentially locate in tumors during delivery.

[0085] In some embodiments of the present invention, the uncoordinated ionizable lipid is selected from the following structures;

[0086]

[0087]

[0088] Compared to previously reported work, this strategy eliminates the need for additional cationic lipid molecules, improving biocompatibility. The amino head containing the ligand unit can coordinate with metal ions, making the organ selectivity of the material predictable. In the design of the ionizable lipid structure, the disulfide bond in the linker portion serves as a biodegradable stimulating unit, effectively enhancing the endosome escape efficiency and biocompatibility of the lipid nanoparticles. Using sulfur atoms to connect the hydrophobic tail chain increases the conformational flexibility of the hydrophobic tail chain to some extent, improving nucleic acid molecule delivery efficiency. This organ-selectivity regulation strategy and related nanoparticle materials have broad application prospects as non-viral nucleic acid carriers. Based on the properties of uncoordinated or metal-coordinated lipid nanoparticles, they can be used to prepare small molecule drugs such as nucleic acid drugs and gene vaccines; organ-selectivity regulation methods based on ionizable lipid gene carriers can control the site of action of corresponding drugs.

[0089] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0090] Example 1: Preparation of uncoordinated ionizable lipid A2-SCC-B4

[0091]

[0092] Preparation of disulfide linkers: 2-hydroxyethyl disulfide (2.0 eq) and triethylamine (1.2 eq) were dissolved in a suitable amount of tetrahydrofuran solvent, and the mixture was cooled and stirred to 0°C. Acryloyl chloride (1.0 eq) was dissolved in a suitable amount of tetrahydrofuran and added dropwise to the above cooled solution, and the mixture was stirred at room temperature for 12 hours. After the reaction was completed, the solvent was removed by rotary evaporation, and the product was purified by rapid column chromatography to obtain the disulfide linkers. 1 H NMR (400MHz, CDCl3) δ6.43 (d, J=17.2Hz, 1H), 6.12 (dd, J=17.2, 10.4Hz, 1H), 5.86 (d, J=10.4Hz, 1H), 4.43 (t, J = 6.8Hz, 2H), 3.88 (t, J = 6.0Hz, 2H), 2.96 (t, J = 6.8Hz, 2H), 2.88 (t, J = 5.6Hz, 2H).

[0093]

[0094] B4 (1.0 eq), methyl methacrylate (1.2 eq), and 2% dimethylphenylphosphine were added to a 250 mL flask and reacted at 60 °C for 12 hours to obtain intermediate 1-1. Subsequently, sodium hydroxide (2.0 eq) was added and dissolved in an appropriate amount of methanol and deionized water, and the mixture was reacted at room temperature for 12 hours. After the reaction was completed, methanol was removed by rotary evaporation, and the pH of the system was adjusted to <7 by adding an appropriate amount of hydrochloric acid. The mixture was extracted with ethyl acetate, and the organic phase was retained. After drying with anhydrous magnesium sulfate for 2 hours, the mixture was filtered and concentrated to obtain intermediate 1-2.

[0095] Intermediate 1-2 (1.0 eq) was dissolved in an appropriate amount of dichloromethane, and oxaloyl chloride (4.0 eq) and 2 drops of N,N-dimethylformamide were added as catalysts. The mixture was stirred at room temperature for 12 hours, and the reaction was stopped. The solvent and excess oxaloyl chloride were removed by rotary evaporation to obtain intermediate 1-3.

[0096] Intermediates 1-3 (1.2 eq) and the disulfide linker (1.0 eq) were dissolved in a suitable amount of dichloromethane, and triethylamine (1.5 eq) was added. The mixture was stirred at room temperature for 12 hours. The reaction was stopped, the solvent was removed by rotary evaporation, and the product was purified by rapid column chromatography to obtain the hydrophobic tail chain SCC-B4. The 400 MHz NMR spectrum of the hydrophobic tail chain SCC-B4 is shown below. Figure 2 As shown. 1 H NMR(400MHz, CDCl3) δ6.44(d,J=17.2,1H),6.13(dd,J=17.2,10.4Hz,1H),5.86 (d,J=10.4Hz),4.42(t,J=6.8Hz,2H),4.37(t,J=6.4Hz,2H),2.97(t,J=6.4Hz, 2H),2.94(t,J=4.8Hz,2H),2.83(dd,J=12.8,7.2Hz,1H),2.73-2.64(m,1H),2. 60-2.49(m,3H),1.60-1.53(m,4H),1.37-1.25(m,15H),0.88(t,J=6.8Hz,3H).

[0097] The hydrophobic tail chain SCC-B4 (4.4 eq) and the amino head A2 (1.0 eq) were added to a reaction flask and reacted at 60°C for 48 hours. The reaction was then stopped, and the product was purified by rapid chromatography to obtain the biodegradable uncoordinated lipid A2-SCC-B4. Figure 3 The image shows the 400MHz NMR spectrum of the uncoordinated ionizable lipid A2-SCC-B4. 1HNMR(400MHz, CDCl3)δ4.38-4.35(m,16H),3.22-3.00(m,24H),2.96-2.92(m,16H),2.85-2.80(m,4H), 2.71-2.66(m,8H),2.80-2.49(m,16H),1.60-1.52(m,8H),1.37-1.25(m,68H),0.88(t,J=6.0Hz,12H).

[0098] Example 2: Preparation of uncoordinated ionizable lipid nanoparticles A2-SCC-B4 LNPs

[0099] The nanoparticles are composed of four components: uncoordinated ionizable lipid A2-SCC-B4 prepared in Example 1, auxiliary lipid (DOPE), steroidal lipid (Chol), and polyethylene glycol lipid (DMG-PEG2000). The prepared nanoparticles can be used to deliver nucleic acid molecules (luciferase mRNA). The specific steps are as follows:

[0100] Lipids A2-SCC-B4, DOPE, Chol, and DMG-PEG2000 were dissolved in an ethanol / N,N-dimethylformamide mixed solvent at a molar ratio of 15:15:30:3. An appropriate amount of luciferase mRNA was dissolved in a citrate / sodium citrate buffer solution, with a molar ratio of A2-SCC-B4 to mRNA of 6000:1. The above lipid mixture and mRNA solution were rapidly mixed at a volume ratio of 1:3, allowed to stand for 30 min, and then dialyzed for 2 hours to remove the organic solvent and acidic buffer, yielding A2-SCC-B4 LNPs.

[0101] The obtained A2-SCC-B4 LNPs were diluted to 0.2 mg / mL with PBS, and the particle size and zeta potential were determined using a Zetasizer Nano Series Nano-ZS. Figure 4 The image shows the particle size and potential of uncoordinated lipid nanoparticles A2-SCC-B4 LNPs. The prepared uncoordinated lipid nanoparticles A2-SCC-B4 LNPs are negatively charged.

[0102] Example 3: Preparation of metal-coordinated lipid nanoparticles Zn-A2-SCC-B4 LNPs

[0103] Organ-selective regulation strategy for ionizable lipid gene vectors with coordinating groups, by introducing Zn into the uncoordinated ionizable lipid A2-SCC-B4 structure. 2+ — Ligand interactions enable organ-selective switching from the liver to the lungs.

[0104] Introducing Zn into the uncoordinated ionizable lipid A2-SCC-B4 structure 2+ —Ligand interactions yield Zn 2+ The coordinated ionizable lipid Zn-A2-SCC-B4 comprises the following steps: dissolving 0.02 mmol A2-SCC-B4 and 0.02 mmol Zn(NO3)2 in chromatographically pure methanol, reacting at 40 °C for 24 h, and removing the solvent by rotary evaporation to obtain the product Zn-A2-SCC-B4.

[0105] The Zn-A2-SCC-B4, DOPE, Chol, and DMG-PEG2000 were dissolved in an ethanol / N,N-dimethylformamide mixed solvent at a molar ratio of 15:15:30:3. An appropriate amount of the luciferase mRNA was dissolved in an acetate / sodium acetate buffer solution, with a molar ratio of A2-SCC-B4 to mRNA of 6000:1. The lipid mixture and mRNA solution were rapidly mixed at a volume ratio of 1:3, allowed to stand for 30 min, and then dialyzed for 2 hours to remove the organic solvent and acidic buffer, yielding Zn-A2-SCC-B4 LNPs.

[0106] The obtained Zn-A2-SCC-B4 LNPs were diluted to 0.2 mg / mL with PBS, and the particle size and zeta potential were determined using a Zetasizer NanoSeries Nano-ZS. Figure 5 The image shows the particle size and potential of metal-coordinated lipid nanoparticles Zn-A2-SCC-B4LNPs. The metal-coordinated lipid nanoparticles Zn-A2-SCC-B4LNPs are positively charged.

[0107] Example 4: Application of metal-coordinated lipid nanoparticles Zn-A2-SCC-B4 LNPs

[0108] IGROWV1 cells were distributed at a density of 1 x 10⁻⁶ cells per well. 4 The cells were seeded into 96-well plates and cultured for 24 hours. Zn-A2-SCC-B4 LNPs prepared according to the method described in Example 3 were added, and mCherry mRNA was used as the nucleic acid molecule to prepare nanoparticles. The amount of mRNA used was 50 nanograms / well, and the cells were cultured for another 24 hours.

[0109] The transfection of nanoparticles containing mCherry mRNA was observed using an inverted microscope; the results are as follows: Figure 6 The image shows the effect of transfecting Zn-A2-SCC-B4 LNPs with mCherry mRNA in IGROV1 cells in vitro; it can be seen from the image that the mcherry protein (with red fluorescence) was successfully expressed in the cells.

[0110] Example 5: Comparison of transfection efficiency between metal-coordinated lipid nanoparticles Zn-A2-SCC-B4 LNPs and Dlin-Mc3-DMA nanoparticles

[0111] IGROWV1 cells were distributed at a density of 1 x 10⁻⁶ cells per well. 4 Cells were seeded into 96-well plates and cultured for 24 hours. Zn-A2-SCC-B4 LNPs prepared according to the method described in Example 3 were added, and nanoparticles were prepared using luciferase mRNA as the nucleic acid molecule, with an mRNA concentration of 50 nanograms per well. The cells were then cultured for another 24 hours. The control group used Dlin-Mc3-DMA (a commercially standardized reagent), with equal amounts of luciferase mRNA mixed with Dlin-Mc3-DMA to prepare nanoparticles. Both types of nanoparticles were used for in vitro IGROWV1 cell transfection. When transfected with luciferase mRNA, the transfection efficiency was assessed using a luciferase assay kit. Transfection efficiency comparisons are shown below. Figure 7 As shown in the figure, data comparison reveals that the metal-coordinated lipid nanoparticles prepared in the embodiments of the present invention have a higher transfection effect.

[0112] Example 6: Delivery of nucleic acid molecules to mouse liver using uncoordinated lipid nanoparticles A2-SCC-B4 LNPs

[0113] The nucleic acid molecule was luciferase mRNA; uncoordinated lipid nanoparticles A2-SCC-B4 LNPs were prepared according to the method described in Example 2, with an mRNA concentration of 0.1 mg / kg. The nanoparticles were dialyzed in PBS for 2 hours to remove organic solvents and acidic buffer. The nanoparticles were injected into BALB / c mice via tail vein, and bioluminescence imaging was performed 6 hours later using a small animal in vivo fluorescence imaging system. The results are as follows: Figure 8 As shown, uncoordinated lipid nanoparticles A2-SCC-B4 LNPs can selectively carry luciferase mRNA into the mouse liver.

[0114] Example 7: Delivery of nucleic acid molecules to mouse lungs using metal-coordinated lipid nanoparticles Zn-A2-SCC-B4 LNPs

[0115] The nucleic acid molecule was luciferase mRNA, prepared as Zn-A2-SCC-B4LNPs according to the method in Example 3. The amount of mRNA used was 0.2 mg / kg. The mRNA was dialyzed in PBS for 2 hours to remove organic solvents and acidic buffer. It was then injected into BALB / c mice via tail vein. Six hours later, bioluminescence imaging was performed using a small animal in vivo fluorescence imaging system. The results are as follows: Figure 9As shown, metal-coordinated lipid nanoparticles Zn-A2-SCC-B4 LNPs can carry luciferase mRNA into the mouse lungs. By introducing metal-ligand interactions into the structure of the uncoordinated ionizable lipid A2-SCC-B4, A2-SCC-B4 is converted into Zn-A2-SCC-B4, thus transforming the liver-selective A2-SCC-B4 LNPs into lung-selective Zn-A2-SCC-B4 LNPs, achieving organ-selective conversion from the liver to the lungs in mice.

[0116] Example 8: Delivery of nucleic acid molecules to mouse tumors by metal-coordinated lipid nanoparticles Zn-A2-SCC-B4 LNPs

[0117] The nucleic acid molecule was luciferase mRNA; Zn-A2-SCC-B4LNPs were prepared according to the method described in Example 6, with an mRNA dosage of 0.2 mg / kg. The mRNA was dialyzed in PBS for 2 hours to remove organic solvents and acidic buffer. The mRNA was injected intravenously into a mouse tumor model. Six hours later, bioluminescence imaging was performed using a small animal in vivo fluorescence imaging system to achieve tumor luminescence imaging. Results are as follows... Figure 10 As shown, there is a significant luminescent signal at the tumor site and a partial luminescent signal in the lung, demonstrating that the metal-coordinated lipid nanoparticles Zn-A2-SCC-B4 LNPs can preferentially select the tumor site and deliver drugs to the tumor.

[0118] Example 9: Preparation of uncoordinated ionizable lipid A1-SCC-B4

[0119] Includes the following steps:

[0120]

[0121] The hydrophobic tail chain SCC-B4 was prepared according to the steps in Example 1. The hydrophobic tail chain SCC-B4 (3.3 eq) and the amino head A1 (1.0 eq) were added to a reaction flask and reacted at 60°C for 48 hours. The reaction was then stopped, and the product was purified by rapid chromatography to obtain the biodegradable uncoordinated lipid A1-SCC-B4. 1 H NMR (400MHz, CDCl3) δ4.38-4.35(m,12H),3.15-3.07(m,12H),2.96-2.93(m,12H),2.85-2.80(m,6H), 2.73-2.64(m,9H),2.60-2.49(m,12H),1.60-1.52(m,6H),1.37-1.25(m,51H),0.88(t,J=6.4Hz,9H).

[0122] Example 10: Preparation of uncoordinated ionizable lipid A3-SCC-B4

[0123] Includes the following steps:

[0124]

[0125] Hydrophobic tail chain SCC-B4 was prepared according to Example 1. The hydrophobic tail chain SCC-B4 (4.4 eq) and the amino head A3 (1.0 eq) were added to a reaction flask and reacted at 60°C for 48 hours. The reaction was then stopped, and the product was purified by rapid chromatography to obtain biodegradable uncoordinated lipid A3-SCC-B4. 1 H NMR (400MHz, CDCl3) δ4.38-4.32(m,16H),3.03-2.92(m,24H),2.87-2.77(m,8H),2.71-2.68(m,4H),2 .60-2.46(m,32H),1.80-1.71(m,4H),1.60-1.52(m,8H),1.38-1.25(m,68H),0.88(t,J=6.4Hz,12H).

[0126] Example 11: Preparation of uncoordinated ionizable lipid A1-SCC-B13

[0127] Includes the following steps:

[0128]

[0129] Disulfide linkers were prepared according to Example 1;

[0130] B13 (1.0 eq), methyl methacrylate (1.2 eq), and 2% dimethylphenylphosphine were added to a 250 mL flask and reacted at 60 °C for 12 hours to give intermediate 2-1. Subsequently, sodium hydroxide (2.0 eq) was added and dissolved in an appropriate amount of methanol and deionized water, and the mixture was reacted at room temperature for 12 hours. After the reaction was completed, methanol was removed by rotary evaporation, and the pH of the system was adjusted to <7 by adding an appropriate amount of hydrochloric acid. The mixture was extracted with ethyl acetate, and the organic phase was retained. After drying with anhydrous magnesium sulfate for 2 hours, the mixture was filtered and concentrated to give intermediate 2-2.

[0131] Intermediate 2-2 (1.0 eq) was dissolved in an appropriate amount of dichloromethane, and oxaloyl chloride (4.0 eq) and 2 drops of N,N-dimethylformamide were added as catalysts. The mixture was stirred at room temperature for 12 hours, and the reaction was stopped. The solvent and excess oxaloyl chloride were removed by rotary evaporation to obtain intermediate 2-3.

[0132] Intermediate 2-3 (1.2 eq) and the disulfide linker obtained in step 1) (1.0 eq) were dissolved in an appropriate amount of dichloromethane, and triethylamine (1.5 eq) was added. The mixture was stirred at room temperature for 12 hours. The reaction was stopped, the solvent was removed by rotary evaporation, and the product was purified by rapid column chromatography to obtain the hydrophobic tail chain SCC-B3.

[0133] The hydrophobic tail chain SCC-B13 (3.3 eq) and the amino head A1 (1.0 eq) were added to a reaction flask and reacted at 60 °C for 48 hours. The reaction was then stopped, and the product was purified by rapid chromatography to obtain the biodegradable uncoordinated lipid A1-SCC-B13. 1 H NMR (400MHz, CDCl3) δ4.39-4.35(m,12H),3.13-3.10(m,12H),2.96-2.93(m,15H),2.83-2. 78(m,6H),2.71-2.48(m,18H),1.45-1.40(m,3H),1.31-1.26(m,57H),0.91-0.87(m,18H).

[0134] Example 12: Delivery of nucleic acid molecules to mouse spleen by uncoordinated lipid nanoparticles A1-SCC-B13 LNPs

[0135] The nucleic acid molecule was luciferase mRNA; A1-SCC-B13LNPs were prepared according to the method described in Example 2, with an mRNA concentration of 0.1 mg / kg. The mRNA was dialyzed in PBS for 2 hours to remove organic solvents and acidic buffer. The mRNA was injected intravenously into BALB / c mice, and bioluminescence imaging was performed using a small animal in vivo fluorescence imaging system 6 hours later. The results are as follows: Figure 11 As shown, uncoordinated lipid nanoparticles A1-SCC-B13LNPs can carry luciferase mRNA into the mouse spleen. Organ selectivity was altered by changing the hydrophobic tail chain of the lipid nanoparticles, specifically from the liver to the spleen.

[0136] Example 13: Delivery of nucleic acid molecules to mouse lungs by metal-coordinated lipid nanoparticles Zn-A1-SCC-B13

[0137] The nucleic acid molecule was luciferase mRNA, A1-SCC-B13 prepared according to the method described in Example 10; subsequently, Zn-A1-SCC-B13 LNPs were prepared from A1-SCC-B13 according to Example 3, with an mRNA concentration of 0.2 mg / kg. The mRNA was dialyzed in PBS for 2 hours to remove organic solvents and acidic buffer. The mice were then injected via tail vein into BALB / c mice, and bioluminescence imaging was performed using a small animal in vivo fluorescence imaging system 6 hours later. The results are as follows: Figure 12As shown, luciferase mRNA is delivered to the mouse lungs under the action of Zn-A1-SCC-B13 LNPs.

[0138] It is evident that the introduction of metal-ligand interactions into the structure of the uncoordinated ionizable lipid A1-SCC-B13 transforms A1-SCC-B13 into Zn-A1-SCC-B13, thereby converting spleen-selective A1-SCC-B13 LNPs into lung-selective Zn-A1-SCC-B13 LNPs, achieving organ selectivity in mice from spleen to lung.

[0139] Example 14: Effect of metal ion-ligand interactions on the apparent pKa of lipid nanoparticles

[0140] Following the steps described in Examples 2 and 3, uncoordinated lipid nanoparticles and corresponding metal ion-coordinated lipid nanoparticles were prepared. The apparent pKa of the nanoparticles was then determined using potassium 2-(p-toluidine)-6-naphthalenesulfonate (TNS method). The specific steps are as follows:

[0141] Uncoordinated lipid nanoparticles were prepared according to the steps described in Example 2, and metal ion-coordinated lipid nanoparticles were prepared according to the steps described in Example 3. Subsequently, the total lipid concentration was diluted to 1.5 mM using PBS. 96 μL of pKa test buffer, 2 μL of nanoparticle solution, and 2 μL of stock TNS (1 mM) were added to each well of a 96-well plate; the final TNS concentration used was 2 μM. The plate was incubated with shaking for 30 min, and the fluorescence intensity of each well was read using a microplate reader (Ex / Em = 321 / 445 nm). The acquired fluorescence data were analyzed using S-fit, with the pH value corresponding to a pKa of half the maximum fluorescence intensity.

[0142] Information on the preparation of uncoordinated lipid nanoparticles and corresponding metal ion-coordinated lipid nanoparticles, as well as the apparent pKa of the nanoparticles determined using potassium 2-(p-toluamide)-6-naphthalenesulfonate (TNS method), is shown in Table 1. It is evident that lipid nanoparticles constructed with different tail chains exhibit different pKa values, leading to selectivity of the nanoparticles for the liver or spleen.

[0143] Table 1

[0144] Uncoordinated lipid nanoparticles / Metal-coordinated lipid nanoparticles pKa A1-SCC-B3 7.91 A1-SCC-B4 6.42 A1-SCC-B5 7.23 A1-SCC-B11 6.49 A1-SCC-B12 5.76 A1-SCC-B13 5.79 Zn-A1-SCC-B3 7.88 Zn-A1-SCC-B4 7.85 Zn-A1-SCC-B5 8.29 Zn-A1-SCC-B11 7.15 Zn-A1-SCC-B12 8.15 Zn-A1-SCC-B13 8.62

[0145] Example 15: Preparation and NMR characterization of uncoordinated ionizable lipid A1-SCC-B3

[0146]

[0147] Following the method described in Example 1, uncoordinated ionizable lipid A1-SCC-B3 was synthesized using starting materials A1 and B3. The biodegradable uncoordinated lipid A1-SCC-B3 was obtained by rapid column chromatography purification. 1 H NMR (400MHz, CDCl3) δ4.38-4.34(m,12H),3.15-3.09(m,12H),2.96-2.92(m,15H),2.85-2.80(m,6H), 2.71-2.49(m,18H),1.60-1.53(m,6H),1.38-1.36(m,3H),1.34-1.25(m,36H),0.88(t,J=6.4Hz,9H).

[0148] Example 16: Preparation and NMR characterization of uncoordinated ionizable lipid A1-SCC-B5

[0149]

[0150] Following the method described in Example 1, uncoordinated ionizable lipid A1-SCC-B5 was synthesized using starting materials A1 and B5. The biodegradable uncoordinated lipid A1-SCC-B5 was obtained by rapid column chromatography purification. 1 H NMR (400MHz, CDCl3) δ4.43-4.35(m,12H),3.14-3.09(m,12H),2.96-2.92(m,15H),2.85-2.80(m,6H),2.73-2.6 6(m,6H),2.61-2.49(m,12H),1.60-1.52(m,6H),1.38-1.34(m,6H),1.27-1.25(m,57H),0.88(t,J=6.4Hz,9H).

[0151] Example 17: Preparation and NMR characterization of uncoordinated ionizable lipid A1-SCC-B11

[0152]

[0153] Following the method described in Example 11, uncoordinated ionizable lipid A1-SCC-B11 was synthesized using raw materials A1 and B11. The biodegradable uncoordinated lipid A1-SCC-B11 was obtained by rapid column chromatography purification. 1HNMR (400MHz, CDCl3) δ4.39-4.32(m,12H),2.96-2.89(m,12H),2.83-2.78(m,12H),2.73-2.66(m,9H ),2.58-2.49(m,18H),1.47-1.41(m,6H),1.33-1.32(m,3H),1.28-1.25(m,27H),0.91-0.85(m,18H).

[0154] Example 18: Preparation and NMR characterization of uncoordinated ionizable lipid A1-SCC-B12

[0155]

[0156] Following the method described in Example 11, uncoordinated ionizable lipid A1-SCC-B12 was synthesized using raw materials A1 and B12. The biodegradable uncoordinated lipid A1-SCC-B12 was obtained by rapid column chromatography purification. 1 HNMR (400MHz, CDCl3) δ4.41-4.34(m,12H),3.18-3.09(m,12H),2.98-2.92(m,15H),2.83-2 .78(m,6H),2.71-2.48(m,18H),1.55-1.50(m,3H),1.33-1.25(m,45H),0.91-0.87(m,18H).

[0157] Example 19: Preparation and NMR characterization of uncoordinated ionizable lipid A2-SCC-B3

[0158]

[0159] Following the method described in Example 1, uncoordinated ionizable lipid A2-SCC-B3 was synthesized using starting materials A2 and B3. The biodegradable uncoordinated lipid A2-SCC-B3 was obtained by rapid column chromatography purification. 1 H NMR (400MHz, CDCl3) δ4.38-4.33(m,16H),2.99-2.92(m,24H),2.88-2.79(m,12H),2 .73-2.37(m,32H),1.60-1.54(m,16H),1.34-1.25(m,44H),0.88(t,J=6.0Hz,12H).

[0160] Example 20: Preparation and NMR characterization of uncoordinated ionizable lipid A2-SCC-B5

[0161]

[0162] Following the method described in Example 1, uncoordinated ionizable lipid A2-SCC-B5 was synthesized using starting materials A2 and B5. The biodegradable uncoordinated lipid A2-SCC-B5 was obtained by rapid column chromatography purification. 1 H NMR (400MHz, CDCl3) δ4.42-4.33(m,16H),3.01-2.92(m,24H),2.89-2.80(m,12H),2.71-2.66 (m,16H),2.60-2.49(m,16H),1.60-1.53(m,16H),1.33-1.25(m,76H),0.88(t,J=6.4Hz,12H).

[0163] Example 21: Preparation and NMR characterization of uncoordinated ionizable lipid A2-SCC-B11

[0164]

[0165] Following the method described in Example 11, uncoordinated ionizable lipid A2-SCC-B11 was synthesized using raw materials A2 and B11. The biodegradable uncoordinated lipid A2-SCC-B11 was obtained by rapid column chromatography purification. 1 HNMR (400MHz, CDCl3) δ4.44-4.35(m,16H),3.18-2.92(m,24H),2.83-2.76(m,12H),2.71-2.48(m,32H),1.48-1.25(m,48H),0.91-0.85(m,24H).

[0166] Example 22: Preparation and NMR characterization of uncoordinated ionizable lipid A2-SCC-B12

[0167]

[0168] Following the method described in Example 11, uncoordinated ionizable lipid A2-SCC-B12 was synthesized using A2 and B12 starting materials. The biodegradable uncoordinated lipid A2-SCC-B12 was obtained by rapid column chromatography purification. 1 HNMR(400MHz, CDCl3)δ4.44-4.33(m,16H),3.01-2.88(m,24H),2.83-2.78(m,12H ),2.71-2.35(m,32H),1.55-1.52(m,4H),1.35-1.25(m,60H),0.91-0.87(m,24H).

[0169] Example 23: Preparation and NMR characterization of uncoordinated ionizable lipid A2-SCC-B13

[0170]

[0171] Following the method described in Example 11, uncoordinated ionizable lipid A2-SCC-B13 was synthesized using starting materials A2 and B13. The biodegradable uncoordinated lipid A2-SCC-B13 was obtained by rapid column chromatography purification. 1 HNMR (400MHz, CDCl3) δ4.44-4.33(m,16H),3.01-2.92(m,24H),2.88-2.78(m,12H),2.73-2. 60(m,16H),2.57-2.40(m,16H),1.53-1.48(m,4H),1.33-1.25(m,76H),0.91-0.87(m,24H).

[0172] Example 24: Preparation and NMR characterization of uncoordinated ionizable lipid A3-SCC-B3

[0173]

[0174] Following the method described in Example 1, uncoordinated ionizable lipid A3-SCC-B3 was synthesized using starting materials A3 and B3. The biodegradable uncoordinated lipid A3-SCC-B3 was obtained by rapid column chromatography purification. 1 H NMR (400MHz, CDCl3) δ4.38-4.32(m,16H),2.97-2.92(m,24H),2.85-2.79(m,8H),2.71-2.66(m,8H),2.60-2.46 (m,28H),1.77-1.75(m,4H),1.60-1.53(m,8H),1.38-1.34(m,8H),1.29-1.25(m,44H),0.88(t,J=6.4Hz,12H).

[0175] Example 25: Preparation and NMR characterization of uncoordinated ionizable lipid A3-SCC-B5

[0176]

[0177] Following the method described in Example 1, uncoordinated ionizable lipid A3-SCC-B5 was synthesized using starting materials A3 and B5. The biodegradable uncoordinated lipid A3-SCC-B5 was obtained by rapid column chromatography purification. 1H NMR (400MHz, CDCl3) δ4.46-4.32(m,16H),3.00-2.92(m,24H),2.85-2.80(m,8H),2.71-2.66(m,8H),2 .60-2.46(m,28H),1.77-1.75(m,4H),1.60-1.52(m,8H),1.42-1.25(m,76H),0.88(t,J=6.4Hz,12H).

[0178] Example 26: Preparation and NMR characterization of uncoordinated ionizable lipid A3-SCC-B11

[0179]

[0180] Following the method described in Example 11, uncoordinated ionizable lipid A3-SCC-B11 was synthesized using A3 and B11 starting materials. The biodegradable uncoordinated lipid A3-SCC-B11 was obtained by rapid column chromatography purification. 1 HNMR (400MHz, CDCl3) δ4.38-4.33(m,16H),2.97-2.92(m,24H),2.83-2.80(m,8H),2.78-2.66(m,8H),2.58-2.4 5(m,28H),1.47-1.43(m,4H),1.40-1.35(m,12H),1.33-1.29(m,12H),1.28-1.25(m,24H),0.91-0.85(m,24H).

[0181] Example 27: Preparation and NMR characterization of uncoordinated ionizable lipid A3-SCC-B12

[0182]

[0183] Following the method described in Example 11, uncoordinated ionizable lipid A3-SCC-B12 was synthesized using A3 and B12 starting materials. The biodegradable uncoordinated lipid A3-SCC-B12 was obtained by rapid column chromatography purification. 1 HNMR (400MHz, CDCl3) δ4.42-4.32(m,16H),2.99-2.91(m,24H),2.83-2.78(m,8H),2.71-2. 60(m,8H),2.57-2.45(m,28H),1.54-1.52(m,4H),1.30-1.25(m,64H),0.90-0.87(m,24H).

[0184] Example 28: Preparation and NMR characterization of uncoordinated ionizable lipid A3-SCC-B13

[0185]

[0186] Following the method described in Example 11, uncoordinated ionizable lipid A3-SCC-B13 was synthesized using A3 and B13 starting materials. The biodegradable uncoordinated lipid A3-SCC-B13 was obtained by rapid column chromatography purification. 1 HNMR (400MHz, CDCl3) δ4.42-4.32(m,16H),2.99-2.92(m,24H),2.83-2.78(m,8H),2.73-2. 66(m,8H),2.61-2.48(m,28H),1.53-1.48(m,4H),1.37-1.25(m,80H),0.91-0.87(m,24H).

[0187] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. An ionizable lipid gene vector, characterized in that: This includes uncoordinated ionizable lipids with structures as shown in Formula 1; Where An is any one of the formulas A1-A5; Bm is selected from the tail chain containing 1 to 6 straight-chain alkyl thiols or branched alkyl thiols, with an unsaturation degree of 0-6. Preferably, Bm is any structure shown in B1-B27. or, Including metal-coordinated ionized lipids; metal coordination is performed on the uncoordinated ionizable lipids shown in Formula 1 to obtain metal-coordinated ionized lipids.

2. The ionizable lipid gene vector according to claim 1, characterized in that: The coordinating metal ion is Co 3+ Gd 3+ Zn 2+ Fe 3+ Fe 2+ Ca 2+ Mg 2+ Al 3+ and Cu 2+ One of them.

3. The ionizable lipid gene vector according to claim 1, characterized in that: Uncoordinated or metal-coordinated ionizable lipids are mixed with auxiliary lipids, steroidal lipids, and polyethylene glycol lipids, and then target nucleic acid molecules are added to prepare nanoparticles for use as gene carriers.

4. The ionizable lipid gene vector according to claim 3, characterized in that: The molar ratio of uncoordinated ionizable lipids or metal-coordinated ionizable lipids, auxiliary lipids, steroidal lipids and polyethylene glycol lipids is 5-45:15:30:0-5; the molar ratio of uncoordinated ionizable lipids or metal-coordinated ionizable lipids to nucleic acid molecules is 2000-15000:

1.

5. The ionizable lipid gene vector according to claim 3, characterized in that: The auxiliary lipid is one or more of the following: 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE); Steroidal lipids are one or more combinations of dihydrocholesterol, physcosterol, sitosterol, cholic acid, alfalfa sterol, glycocholic acid, deoxycholic acid, cholesterol, brassosterol, ergosterol, ergocalciferol and hydroxycholesterol. The polyethylene glycol lipids are one or more combinations of distearyl-rac-glycerol-polyethylene glycol 1000 (DSG-PEG1000), distearyl-rac-glycerol-polyethylene glycol 2000 (DSG-PEG2000), myristoyl-sn-glycerol-methoxy polyethylene glycol 1000 (DMG-PEG1000), myristoyl-sn-glycerol-methoxy polyethylene glycol 2000 (DMG-PEG2000), myristoyl-sn-glycerol-methoxy polyethylene glycol 5000 (DMG-PEG5000), PEG-distearate glycerol, dipalmitoylphosphatidylcholine polyethylene glycol, dipalmitoylphosphatidylethanolamine-polyethylene glycol, PEG-distearate group, and PEG-diacylglycerol amide.

6. The ionizable lipid gene vector according to claim 3, characterized in that: The nucleic acid molecule is one or more of the following: deoxyribonucleic acid (DNA), antisense nucleic acid (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), circular RNA (circRNA), messenger RNA (mRNA), aptamer, ribozyme, and antibody-nucleic acid conjugate (ARC).

7. The method for preparing the ionizable lipid gene vector according to any one of claims 1-6, characterized in that: The specific steps for uncoordinated ionizable lipids are as follows: Step 1: Acryloyl chloride is linked to one end of the hydroxyl group of 2-hydroxyethyl disulfide through a condensation reaction to generate an asymmetric acrylate, which is a disulfide linker. The compound Bm thiol was prepared into a hydrophobic tail chain precursor by sequential addition, hydrolysis and chloroacylation reactions. Step 2: The hydrophobic tail chain precursor is condensed with the disulfide linker to obtain the hydrophobic tail chain; Step 3: The hydrophobic tail chain is reacted with compound An via an aza-McCl addition reaction to obtain an uncoordinated ionizable lipid; Preferably, in step three, the catalyst dibutylhydroxytoluene is added, and the reaction temperature is 20-60℃.

8. The method for preparing the ionizable lipid gene vector according to claim 7, characterized in that: Uncoordinated ionizable lipids and coordinated metal ions are mixed and reacted at 20-60℃ to obtain metal-coordinated ionizable lipids.

9. A method for organ-selective regulation of ionizable lipid gene vectors, characterized in that: Nanoparticles of uncoordinated ionizable lipids constructed from Bm atoms with a total number of carbon atoms less than 14 exhibit liver selectivity; nanoparticles of uncoordinated ionizable lipids constructed from Bm atoms with a total number of carbon atoms greater than or equal to 14 exhibit spleen selectivity; and nanoparticles of metal-coordinated ionizable lipids exhibit lung selectivity.

10. The application of the organ-selective regulation method of the ionizable lipid gene vector according to any one of claims 1-6 or the ionizable lipid gene vector according to claim 9 in the preparation of nucleic acid drugs and gene vaccine drugs.

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