Method for preparing lipid nanoparticles and complexes thereof

Abstract

The application provides a preparation method of a lipid nanoparticle and a complex thereof, wherein the lipid nanoparticle comprises an amino lipid compound, and the amino lipid compound comprises at least one of the following steps: compound (I), compound (II) and compound (III). The amino lipid compound has hydrophobic characteristics due to containing long non-polar residues, and has hydrophilic characteristics due to containing amino groups. Due to the amphiphilic characteristics, the amino lipid compound can be used to form the lipid nanoparticle, the hydrophobic part of the amino lipid compound helps to stabilize the structure of the lipid nanoparticle, and the amino group can produce strong hydrophilicity in the body, so that the water solubility and biocompatibility of the lipid nanoparticle are increased, the loading capacity and the encapsulation efficiency of the lipid nanoparticle are improved, the drug or gene delivery is more efficient, and the required administration dose is reduced and the side effects are reduced.

Description

Technical Field

[0001] This invention belongs to the field of synthetic chemistry, and particularly relates to a method for preparing lipid nanoparticles and their complexes. Background Technology

[0002] Lipid nanoparticles (LNPs) are nanoscale particles composed of lipid materials, commonly used to encapsulate and deliver bioactive substances such as nucleic acids (e.g., mRNA, siRNA), proteins, and drugs. These particles exhibit good biocompatibility and biodegradability, and can effectively penetrate cell membranes, thereby delivering active ingredients into the cell. They are widely used in gene therapy, vaccine development, and drug delivery. In recent years, with the rapid development of nucleic acid drugs and mRNA vaccines, the research and application of LNPs have received considerable attention.

[0003] Many liquid nitrogen nanoparticles (LNPs) degrade during storage, especially in liquid form, potentially altering encapsulation efficiency and particle size, thus affecting drug efficacy and bioavailability. Overcoming this issue typically requires cryogenic storage or freeze-drying, but this increases storage costs and transportation difficulties. Particularly in nucleic acid drug delivery, existing LNP technologies suffer from insufficient loading and encapsulation efficiency, leading to larger dosages and increased side effects. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing lipid nanoparticles and their complexes, which aims to solve the problems of high storage difficulty, low loading capacity and low encapsulation efficiency of lipid nanoparticles.

[0005] To address the aforementioned technical problems, this invention proposes a lipid nanoparticle, wherein the lipid nanoparticle comprises an aminolipid compound, and the aminolipid compound comprises at least one of compound (I), compound (II), and compound (III); wherein,

[0006] The structural formula of compound (Ⅰ) is as follows: The structural formula of compound (II) is as follows: The structural formula of compound (Ⅲ) is as follows:

[0007] In the above structural formula, Z 1 Z 2 Z 3 Z 4 and Z 5 Whether they are the same or different, Z 1 Z 2 Z 3 Z 4 Z 5All include -CH(OR) a )-, -C=C-, -C≡C-, -O-, -NH-, -C(=O)O-, -C(=O)-, -OC(=O)-, -N(R a )C(=O)-、-C(=O)N(R a )-、-N(R a )C(=O)N(R a )-、-OC(=O)N(R a )-、-N(R a Z is at least one of C(=O)O-, -C(=O)S-, -SC(=O)-, -SC=C-, and -SS-. 1 Z 2 Z 3 Z 4 Z 5 R in any one a Including H, C1-C 18 C2-C groups with optional substitution of hydrocarbon group, -OH, or -O- 18 Hydrocarbon group, -C=C-, -C≡C- optional substitution of C4-C 18 Hydrocarbon group or C1-C 18 At least one of the heteroalkyl groups;

[0008] A 1 and A 2 Whether they are the same or different, A 1 and A 2 All include C1-C 18 Hydroxyl group, C3-C 18 At least one of the following: cyclic hydrocarbon group, phenyl group, benzyl group, and 4-7 membered heterocycle; R 1 R 2 R 3 R 4 All include H, C1-C 18 alkane group, C1-C 18 olefinic, C1-C 18 Alkyne group, C3-C 18 At least one of cycloalkyl, phenyl, benzyl, and 4-7 membered heterocycles, wherein the heterocycle in the 4-7 membered heterocycle includes at least one of C1-C8 alkyl, haloalkyl, -O-C1-C8 alkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C6 alkyl), and N(C1-C6 alkyl)2 substituents;

[0009] m and n may be the same or different from each other, and each is an independent integer from 1 to 12;

[0010] G 1 and G 2 Whether they are the same or different, G1 and G 2 All of these include -C=C-, -C≡C-, -NH-, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, and -S(O). p -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-、-C(=O)NR a -、-NR a C(=O)NR a -、-OC(=O)NR a -NR a At least one of C(=O)O-, p=0, 1 or 2, G 1 and G 2 R in a Including H, C1-C 12 At least one of the hydrocarbon groups,

[0011] L 1 L 2 L 3 and L 4 Whether they are the same or different, L 1 L 2 L 3 L 4 All include C1-C 24 Alkylene, C2-C 24 alkenyl, C3-C8 cycloalkylene, C3-C8 cycloalkenyl, optionally substituted 4- to 10-membered heterocycles selected from nitrogen, sulfur, and oxygen, wherein the C1-C 24 Alkylene, the C2-C 24 At least one of the alkenyl group, the C3-C8 cycloalkyl group, and the C3-C8 cycloalkenyl group is substituted by one or more substituents selected from hydrocarbon groups, carboxyl groups, acyl groups, and alkoxy groups;

[0012] R 1 Including H, OR 1a CN, -C(=O)OR 1a -OC(=O)R 1a -NR 1b C(=O)R 1a -NR 1a R 1b At least one of them, R 1a and R 1b Including H, C1-C 12 At least one of the hydrocarbon groups, R 2 Including branched C6-C 24 Alkyl, branched C6-C 24 Alkenyl, branched C6-C24 At least one of the alkynyl groups.

[0013] On the other hand, the present invention proposes a preparation method for preparing an mRNA-lipid nanoparticle complex comprising lipid nanoparticles, wherein the preparation method includes the following steps:

[0014] S1, Synthesizing amino lipid compounds;

[0015] S2. Dissolve the auxiliary lipid, structural lipid, polyethylene glycol-lipid, and amino lipid compound in an organic solvent to obtain an auxiliary lipid solution, a structural lipid solution, a polyethylene glycol-lipid solution, and an amino lipid compound solution, respectively. Mix the auxiliary lipid solution, the structural lipid solution, the polyethylene glycol-lipid solution, and the amino lipid compound solution to obtain an alcohol phase, wherein the organic solvent includes at least one of anhydrous ethanol, chloroform, and ethyl acetate.

[0016] S3. Mix the mRNA stock solution, pH adjuster and solvent to obtain an aqueous phase, wherein the pH adjuster includes at least one of phosphate buffer, sodium acetate buffer and sodium bicarbonate buffer, and the solvent includes at least one of anhydrous ethanol, pure water and dichloromethane.

[0017] S4. First, mix the alcohol phase and the aqueous phase, then add buffer solution to obtain the mRNA-lipid nanoparticle complex.

[0018] The method for preparing lipid nanoparticles and their complexes proposed in this invention has the following advantages:

[0019] Amino lipid compounds, containing long, nonpolar residues, are entirely hydrophobic, while the amino groups are simultaneously hydrophilic. This amphiphilic nature allows amino lipid compounds to be used to form lipid nanoparticles. The hydrophobic portion of the amino lipid compound helps stabilize the structure of the lipid nanoparticles, while the amino groups generate strong hydrophilicity in vivo, increasing the water solubility and biocompatibility of the lipid nanoparticles. This, in turn, improves the loading capacity and encapsulation efficiency of the lipid nanoparticles, making drug or gene delivery more efficient, helping to reduce the required dosage and decrease side effects. Attached Figure Description

[0020] Figure 1 This is a graph showing the effect of different types of lipid nanoparticles on the release of cellular lactate dehydrogenase in one embodiment of the present invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0022] This invention proposes a lipid nanoparticle, which comprises an aminolipid compound, wherein the aminolipid compound comprises at least one of compound (I), compound (II), and compound (III); wherein,

[0023] The structural formula of compound (Ⅰ) is: The structural formula of compound (II) is: The structural formula of compound (Ⅲ) is

[0024] In the above structural formula, Z 1 Z 2 Z 3 Z 4 and Z 5 Whether they are the same or different, Z 1 Z 2 Z 3 Z 4 Z 5 All include -CH(OR) a )-, -C=C-, -C≡C-, -O-, -NH-, -C(=O)O-, -C(=O)-, -OC(=O)-, -N(R a )C(=O)-、-C(=O)N(R a )-、-N(R a )C(=O)N(R a )-、-OC(=O)N(R a )-、-N(R a Z is at least one of C(=O)O-, -C(=O)S-, -SC(=O)-, -SC=C-, and -SS-. 1 Z 2 Z 3 Z 4 Z 5 R in any one a Including H, C1-C 18 C2-C groups with optional substitution of hydrocarbon group, -OH, or -O- 18 Hydrocarbon group, -C=C-, -C≡C- optional substitution of C4-C 18 Hydrocarbon group or C1-C 18 At least one of the heteroalkyl groups;

[0025] A 1 and A 2 Whether they are the same or different, A1 and A 2 All include C1-C 18 Hydroxyl group, C3-C 18 At least one of the following: cyclic hydrocarbon group, phenyl group, benzyl group, and 4-7 membered heterocycle; R 1 R 2 R 3 R 4 All include H, C1-C 18 alkane group, C1-C 18 olefinic, C1-C 18 Alkyne group, C3-C 18 At least one of cycloalkyl, phenyl, benzyl, and 4-7 membered heterocycles, wherein the heterocycles in the 4-7 membered heterocycles include at least one of C1-C8 alkyl, haloalkyl, -O-C1-C8 alkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C6 alkyl), and N(C1-C6 alkyl)2 substituents;

[0026] m and n may be the same or different from each other, and each is an independent integer from 1 to 12;

[0027] G 1 and G 2 Whether they are the same or different, G 1 and G 2 All of these include -C=C-, -C≡C-, -NH-, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, and -S(O). p -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-、-C(=O)NR a -、-NR a C(=O)NR a -、-OC(=O)NR a -NR a At least one of C(=O)O-, p=0, 1 or 2, G 1 and G 2 R in a Including H, C1-C 12 At least one of the hydrocarbon groups,

[0028] L 1 L 2 L 3 and L 4 Whether they are the same or different, L 1 L 2 L 3 L 4 All include C1-C 24 Alkylene, C2-C 24C1-C1 hemi-olefin, C3-C8 cycloalkylene, C3-C8 cycloalkylene, optional substituted 4- to 10-membered heterocycles selected from nitrogen, sulfur, and oxygen, C1-C 24 Alkylene, C2-C 24 At least one of the following groups—alkenyl, C3-C8 cycloalkyl, and C3-C8 cycloalkenyl—is substituted by one or more substituents selected from hydrocarbon, carboxyl, acyl, and alkoxy groups;

[0029] R 1 Including H, OR 1a CN, -C(=O)OR 1a -OC(=O)R 1a -NR 1b C(=O)R 1a -NR 1a R 1b At least one of them, R 1a and R 1b Including H, C1-C 12 At least one of the hydrocarbon groups, R 2 Including branched C6-C 24 Alkyl, branched C6-C 24 Alkenyl, branched C6-C 24 At least one of the alkynyl groups.

[0030] Amino lipid compounds are entirely hydrophobic due to their long, nonpolar residues, while the amino groups also possess hydrophilic properties. This amphiphilic nature allows amino lipid compounds to be used to form lipid nanoparticles, such as lipid bilayers, micelles, and liposomes. The hydrophobic portion of amino lipid compounds helps stabilize the structure of lipid nanoparticles, while the amino groups generate strong hydrophilicity in vivo, increasing the water solubility and biocompatibility of lipid nanoparticles.

[0031] Within the scope of this invention, the term "lipid nanoparticles" refers to nanoscale substances prepared by placing an amino-lipid compound in an aqueous solution. These particles are, in particular, lipid nanoparticles, lipid bilayer vesicles (liposomes), multilayer vesicles, or micelles. In some preferred embodiments, lipid nanoparticles are liposomes containing the amino-lipid compound of this disclosure, and liposomes are microvesicles composed of a bilayer of amphiphilic lipid molecules encapsulating aqueous compartments. Liposome formation is not a spontaneous process. When lipids are placed in water, lipid vesicles are first formed, thus forming a bilayer or a series of bilayers, each separated by water molecules. Liposomes can be formed by sonicating lipid vesicles in water. Within the scope of this invention, the term "lipid bilayer" refers to a thin film formed by two layers of lipid molecules. The term "micelle" refers to an aggregate of surfactant molecules dispersed in a liquid colloid. Typical micelles in aqueous solutions aggregate with the hydrophilic head region upon contact with water, integrating the hydrophobic single-tail region of the micelle center.

[0032] Therefore, lipid nanoparticles possess excellent encapsulation properties for bioactive ingredients, and lipid nanoparticles containing bioactive ingredients can be used to deliver any of a variety of therapeutic agents into cells. This invention discloses the use of lipid nanoparticles, as described above, for delivering bioactive ingredients into cells. This invention also provides methods for delivering bioactive ingredients into cells, tissues, or organs, comprising contacting lipid nanoparticles containing bioactive ingredients with the cells, tissues, or organs, providing novel therapeutic possibilities for organisms.

[0033] The amphiphilic nature of lipid nanoparticles enables them to effectively encapsulate nucleic acid molecules (such as DNA or RNA) and enter cells through the cell membrane. This highly efficient cellular uptake significantly improves the efficiency of nucleic acid transport. Lipid nanoparticles can form a protective coating, preventing nucleic acid degradation in the in vivo environment and enhancing its stability. By reducing contact with external enzymes and other reactive molecules, lipid nanoparticles ensure the preservation of nucleic acid activity.

[0034] Based on the design of lipid nanoparticles, targeted release can be achieved by modulating their surface properties. By modifying the affinity ligands of lipid molecules, nanoparticles can bind more selectively to specific cell types, thereby achieving targeted transport. The construction of lipid nanoparticles can be designed to have controllable release properties, ensuring the release of nucleic acids under appropriate time and environmental conditions. This tunability can improve therapeutic efficacy and reduce side effects. The interaction between the formation of lipid nanoparticles and the cell membrane can promote their entry into specific subcellular structures within the cell, enhancing the biological effects of nucleic acids. For example, they can help transport nucleic acids into the cell nucleus, thereby improving transcription and translation efficiency.

[0035] Lipid nanoparticles, based on natural lipids or their derivatives, typically exhibit good biocompatibility, reducing toxic reactions in organisms and contributing to improved drug safety. Beyond nucleic acid transport, lipid nanoparticles can also be surface-modified to incorporate other drug components, enabling combination therapy. This versatility gives them significant potential for treating various diseases, such as cancer and genetic defects.

[0036] In one embodiment, the present invention discloses the use of amino-lipid compounds for preparing carriers of active ingredients. In some embodiments, the carrier is in the form of lipid nanoparticles, such as lipid bilayers, micelles, or liposomes.

[0037] In one embodiment, the lipid nanoparticles contain an amino lipid compound and a pharmaceutically acceptable carrier, diluent, or excipient, all of which work together to ensure the stability, delivery efficiency, and biocompatibility of the nanoparticles.

[0038] Carriers can be phospholipids, cholesterol, polyethylene glycol (PEG), lactose, etc. Carriers provide support, stabilizing the structure of lipid nanoparticles and preventing aggregation or degradation in vivo or in vitro. Certain carriers (such as PEG) can prolong drug circulation time in vivo, increasing the probability of the drug reaching the target tissue. Carriers can also optimize the biocompatibility of nanoparticles, reducing recognition and clearance by the immune system and improving efficacy.

[0039] Diluents can be water, sterile saline, glucose solution, etc. They are used to adjust the concentration of the lipid nanoparticle dispersion to an acceptable level for easy injection or administration. Appropriate diluents can reduce interparticle interactions, prolong the stability of the dispersion system, and prevent particle aggregation. Diluents also help ensure uniform distribution of nanoparticles in the solution, guaranteeing consistent dosage and facilitating precise delivery.

[0040] Excipients can be glycerol, polysorbate 80 (such as Tween 80), propylene glycol, sorbitol, etc. Excipients can impart better physical properties to lipid nanoparticles, such as increasing their flowability, lubricity, and palatability, making the dosage form more suitable for clinical use. Excipients can protect the active pharmaceutical ingredient in the nanoparticles from degradation or oxidation; for example, antioxidant excipients can prolong drug stability. Some excipients can interact with the outer layer of nanoparticles, regulating the drug release rate and thus prolonging the duration of drug action.

[0041] In some embodiments of the present invention, the lipid nanoparticles further include at least one of auxiliary lipids, structural lipids, and polyethylene glycol-lipids, wherein the molar ratio of aminolipid compound: auxiliary lipid: structural lipid: polyethylene glycol-lipid is 28-60:5-42:15.5-53.5:0.5-3.5. For example, it can be 45:10:42.5:2.5, 45:11:41.5:2.5, 42.0:10.5:45.0:2.5, 42.0:16.0:39.5:2.5, 40.0:16.0:41.5:2.5, 40.0:18.0:39.5:2.5, 35.0:16.0:46.5:2.5, 35.0:25.0: 36.5:3.5, 28.0:33.5:35.0:3.5, 32.0:37.0:40.5:0.5, 35.0:40.0:22.5:2.5, 40.0:42.0:15.5:25, 40.0:20.0:38.5:1.5, 45.0:15.0:38.5:1.5, 55.0:5.0:38.5:1.5 60.0:5.0:33.5:1.5, 45.0:20.0:33.5:1.5, 50.0:20.0:28.5:1.5, 55.0:20.0:23.5:1.5, 60.0:20.0:18.5:1.5, 40.0:15.0:43.5:1.5, 50.0:15.0:33.5:1.5, 55.0:15. 0:28.5:1.5, 60.0:15.0:23.5:1.5, 40.0:10.0:48.5:15, 45.0:10.0:43.5:1.5, 55.0:10.0:33.5:1.5, 40.0:5.0:53.5:1.5, 45.0:5.0:48.5:1.5, 50.0:5.0:43.5:1.5.

[0042] Optimizing the molar ratio helps obtain lipid nanoparticles of appropriate size, ensuring a uniform particle size distribution and avoiding overly large or small particles. This facilitates in vivo delivery and improves drug loading and delivery efficiency. By adjusting the proportions of lipid components, lipid nanoparticles suitable for different production scales can be obtained, thus making the technology highly scalable and suitable for large-scale industrial production. Optimizing the composition of lipid nanoparticles by adjusting the molar ratios of different lipids can improve the cell membrane penetration and endocytosis efficiency of liposomes, ensuring that mRNA can effectively enter target cells for expression.

[0043] By adjusting the molar ratio of aminolipid compounds, auxiliary lipids, structural lipids, and PEG-lipids, the particle size, morphology, and surface properties of lipid nanoparticles can be further optimized, thereby enhancing their uptake and release efficiency in specific cells or tissues.

[0044] By using an appropriate molar ratio, lipid nanoparticles can better avoid recognition and clearance by the immune system in vivo, thereby reducing immune responses and side effects. Furthermore, a suitable lipid ratio helps prevent premature degradation of lipid particles in vivo, enhancing their long-term stability.

[0045] In summary, the composition and molar ratio of these lipid nanoparticles provide an optimized delivery system that can effectively improve the stability, delivery efficiency, cell penetration, and biocompatibility of mRNA, thus providing a more efficient and safer delivery platform for mRNA vaccines or gene therapy applications.

[0046] The auxiliary lipids include phospholipids, including at least one of distearylphosphatidylcholine, oleoylphosphatidylethanolamine, dioleoyllecithin, dioleoylphosphatidylserine, 1,2-octacosacyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, 1,2-dipalmitoyl-sn-glycerol-3'-O-4'-(N,N,N-trimethyl)homoserine, and phospholipids.

[0047] As auxiliary components in lipid systems, helper lipids can increase the stability of lipid particles, improve their morphology and size distribution, and further enhance mRNA delivery efficiency. Simultaneously, helper lipids can also help improve the formation of lipid bilayers, preventing premature degradation of lipid particles in vivo.

[0048] Structural lipids include sterols, which include at least one of cholesterol, cholesterol esters, sterol hormones, sterol vitamins, bile acids, cholesterol, ergosterol, β-sitosterol, and oxidized cholesterol derivatives.

[0049] Structural lipids provide core support and stability for lipid particles, ensuring the integrity and functionality of lipid nanoparticles, thereby effectively encapsulating mRNA and preventing its degradation during transport.

[0050] Polyethylene glycol-lipids include at least one of polyethylene glycol-distearate phosphatidylethanolamine and polyethylene glycol derivatives of 1,2-dimyristic acid glyceride, wherein the average molecular weight of the polyethylene glycol in the polyethylene glycol-lipid is 2000 to 5000. For example, it can be 2000, 3000, 4000, 5000, etc.

[0051] Polyethylene glycol-lipids (PEG-lipids) are commonly used to increase the blood half-life of lipid nanoparticles and reduce their clearance by the immune system. The hydrophilic portion of PEG gives lipid particles greater stability and reduces non-specific adsorption of lipid nanoparticles to cell surfaces, thereby improving drug targeting and delivery efficiency.

[0052] The role of PEG-lipids: PEG-modified lipids can effectively reduce the interaction between lipid nanoparticles and proteins and immune cells in the blood, thereby prolonging the circulation time of lipid nanoparticles and reducing their distribution in non-target tissues. By optimizing the ratio of PEG-lipids, the accumulation of lipid particles in target tissues (such as liver and muscle) can be improved, thus increasing delivery efficiency.

[0053] The molecular weight of PEG has a significant impact on the surface properties and particle size of lipid particles. An appropriate PEG molecular weight can regulate the size and surface properties of lipid particles, leading to better distribution and delivery in vivo. Too small a PEG may fail to effectively reduce the immune response, while too large a PEG may result in excessively large particles that hinder endocytosis. By optimizing the PEG molecular weight and lipid ratio, it is possible to ensure the stability and size uniformity of liposomes while avoiding cellular rejection of the particles, thus enhancing their delivery efficiency.

[0054] In one embodiment of the present invention, when the aminolipid compound includes compound (Ⅰ), step S1 includes:

[0055] S1.1. Add a phosphorus source reagent to compounds M11 and M12 to carry out a substitution reaction to obtain compound M13, wherein the phosphorus source reagent includes phosphorus oxychloride.

[0056] Phosphorus source reagents are used to initiate substitution reactions, typically by substituting the position of functional groups or functional groups. The selectivity of the phosphorus source reagent and the reaction conditions can affect the selectivity and yield of the reaction, and are therefore crucial in optimizing synthetic routes. This step of the reaction helps to introduce phosphate groups, endowing compounds with higher hydrophilicity or biological activity, and is widely used, especially in drug development.

[0057] S1.2. Compounds M13 and M14 are placed in an alkaline reagent and subjected to an amidation reaction to obtain compound M15. The alkaline reagent includes at least one of triethylamine, pyridine, N-methylmorpholine, N,N-diisopropylethylamine, potassium carbonate, and potassium hydroxide.

[0058] Amide fusion is a common bonding reaction in chemical synthesis, enabling the efficient connection of amino and acyl groups to form biologically active amide bonds. Basic reagents typically facilitate the deprotonation of the amine group, increasing its nucleophilicity and thus promoting the amidation reaction. Different basic reagents can affect the reaction rate and selectivity. By precisely controlling the type and concentration of the basic reagent, the desired amidated product can be obtained in the synthesis, avoiding side reactions.

[0059] S1.3. Compound M16 and compound M17a are placed in an alkaline reagent to undergo a condensation reaction to obtain compound M18; or, compound M16 and compound M17b are placed in an alkaline reagent to undergo a substitution reaction to obtain compound M18.

[0060] This step generates new chemical bonds through condensation reactions, typically involving the removal of small molecules (such as water and alcohols), which is crucial for the formation of large molecules or complex structures. Different reagents or reaction conditions can selectively introduce new functional groups, further altering the properties or biological activity of the molecule. For example, substitution reactions can introduce different alkyl, aryl, or other functional groups, providing greater diversity for subsequent synthesis. By choosing different reaction pathways (condensation or substitution), structurally different but functionally similar products can be obtained. This provides greater flexibility for subsequent steps.

[0061] S1.4. Compounds M15 and M18 were placed in a basic reagent and subjected to a nucleophilic substitution reaction to obtain compound (Ⅰ).

[0062] Nucleophilic substitution reactions involve a nucleophile reacting with a carbon atom or other electron-deficient center in the reactant to form a new chemical bond. This reaction is commonly used to construct complex organic molecules and exhibits high selectivity. Nucleophilic substitution reactions facilitate the introduction of specific functional groups into synthetic routes, thereby optimizing the functional properties of the final product, such as improving the water solubility, stability, or bioactivity of drugs. Precise control of reaction conditions can improve the yield and purity of the reaction, ensuring the quality of the synthesized target compound, which is especially crucial in the synthesis of complex molecules.

[0063] The structural formula of the above compound is:

[0064]

[0065] The reaction process is as follows:

[0066]

[0067] In one embodiment of the present invention, when the aminolipid compound includes compound (II), step S1 includes:

[0068] S1.1. A phosphorus source reagent is added to compounds M21 and M22 to carry out a substitution reaction, yielding compound M23. The phosphorus source reagent includes phosphorus trichloride. The use of this phosphorus source reagent stably introduces phosphorus, resulting in an active phosphorus group in the compound structure. This facilitates further modification in subsequent reactions, improves the reactivity of the target compound and the efficiency of introducing specific functional groups, thereby enabling the substitution reaction between the hydroxyl groups and chlorine atoms in compounds M21 and M22.

[0069] S1.2 Compounds M23 and M24 are placed in an alkaline reagent to undergo a substitution reaction, yielding compound M25. The alkaline reagent includes at least one selected from triethylamine, pyridine, N-methylmorpholine, N,N-diisopropylethylamine, potassium carbonate, and potassium hydroxide. The alkaline environment in this step effectively promotes the substitution reaction between the chlorine atom in compound M23 and the terminal hydrogen group in compound M24, generating the desired functional group and ensuring high selectivity. Furthermore, the alkaline environment inhibits side reactions, improving product purity and reaction yield. This endows the final compound with better structural stability.

[0070] The bromine atom in compound M24 can also be replaced with an iodine atom, or with a sulfide group (-SH). When the sulfide group reacts with a strong reducing agent (such as hydrogen) or other reducing chemicals, a reduction reaction occurs to produce hydrogen sulfide.

[0071] S1.3. Compounds M26 and M27 are placed in an acidic reagent for iminolation to yield compound M28. The acidic reagent includes at least one of acetic acid, hydrochloric acid, and p-toluenesulfonic acid. Under acidic conditions (such as acetic acid or hydrochloric acid), the aldehyde group of compound M26 reacts with the amino group of compound M27 to generate the imine intermediate M28. The iminolation reaction introduces a double bond and nitrogen element into the target molecule by forming an imine group containing C=N. The technical advantage of this step is that the introduction of the imine group increases the functional group diversity of the compound, enhances intermolecular interactions, and creates conditions for further reduction reactions.

[0072] S1.4. Add a reducing agent to compound M28 to carry out a reduction reaction to obtain compound M29, wherein the reducing agent includes at least one of sodium borohydride, lithium aluminum hydride, and sodium cyanoborohydride.

[0073] In this step, imine intermediate M28 is reduced to amino intermediate M29 by adding a reducing agent (such as sodium cyanoborohydride). The reduction reaction ensures the complete conversion of the imine group, resulting in a more stable amino structure. The technical advantage of this reduction step is that it improves the chemical stability of the target compound, avoids the reactivity of the C=N double bond, thereby enhancing the controllability of subsequent reactions and the biocompatibility of the product.

[0074] S1.5. Compounds M25 and M29 were placed in a basic reagent and subjected to a nucleophilic substitution reaction to obtain compound (II). The selection of the nucleophile helps optimize the reaction rate and selectivity, making the reaction more efficient. This reaction endows compound (II) with a unique arrangement of active functional groups and stability, while ensuring the efficient formation of the final product. The technical advantage of this substitution reaction is that it enhances the compound's biological activity, solubility, and versatility of applications.

[0075] The structural formula of the above compound is:

[0076]

[0077] The reaction process is as follows:

[0078]

[0079]

[0080] In one embodiment of the present invention, when the aminolipid compound includes compound (Ⅲ), step S1 includes:

[0081] S1.1 Add an activator to compounds M31 and M32 to carry out an acyl chloride reaction, and then place them in an alkaline reagent to obtain M33. The activator includes at least one of thionyl chloride, oxalyl chloride, thionyl chloride, and cyanogen chloride, and the alkaline reagent includes at least one of 4-dimethylaminopyridine, triethylamine, N,N-diisopropylethylamine, pyridine, potassium carbonate, potassium hydroxide, sodium carbonate, and N-methylmorpholine.

[0082] Acyl chloride reactions enhance the reactivity of molecules by introducing acyl chloride functional groups, making them more selective and efficient in subsequent amidation reactions. Furthermore, acyl chloride treatment effectively reduces the formation of unwanted byproducts, thereby improving the overall reaction purity.

[0083] S1.2. Compounds M33 and M34 are placed in an alkaline reagent and subjected to an amidation reaction to obtain M35.

[0084] Amide reactions convert acyl chloride intermediates into amide structures, improving molecular stability and increasing the number of polar and hydrogen bond donors in the molecule, thus enhancing the solubility of the final product in water or other polar solvents. Simultaneously, the introduction of amide bonds enhances the biocompatibility of the compound, contributing to its performance in pharmaceutical or biological applications.

[0085] S1.3. Compounds M36 and M37 were placed in an alkaline reagent to undergo a substitution reaction, yielding M38.

[0086] Substitution reactions introduce specific functional groups, enhancing intermolecular interactions and potential functionality by altering the chemical structure of the compound. This process helps improve the chemical stability and durability of the compound, making it more stable in different environments and thus broadening its application range.

[0087] S1.4. Compounds M35 and M38 were placed in an alkaline reagent to carry out a cross-linking reaction, yielding compound (Ⅲ).

[0088] The cross-linking reaction forms a multifunctional network structure, giving compound (III) higher mechanical strength and thermal stability, especially exhibiting superior tensile strength and hydrolysis resistance in materials science. The cross-linking structure also endows the compound with better chemical corrosion resistance, making it suitable for use under high-intensity or special environmental conditions.

[0089] The structural formula of the above compound is:

[0090]

[0091] The reaction process is as follows:

[0092]

[0093]

[0094] Specifically, some structural formulas of compound (I) are as follows:

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106] Specifically, the steps for synthesizing I-1 are as follows:

[0107]

[0108]

[0109] Tridecanool (1-1, 2.00 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stir bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour, then stirred at 50 °C under nitrogen for 1 hour. After completion, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 mL) was added to a colorless liquid, and DIPEA (2.68 mL, 15 mmol) and 2-(2-aminoethyl)aminoethanol (0.52 g, 5 mmol) were added under nitrogen. The solution was stirred at room temperature under nitrogen for 3 hours. Then it was diluted with DCM (10 mL) and washed twice with water. The organic phase was separated and dried over anhydrous MgSO4. The desired products were purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give products 1-3 as a colorless oil (2.46 g, 90%).

[0110] 6-Bromohexanoyl chloride (1-4, 2.12 g, 10 mmol), n-undecanool (1-5, 1.72 g, 10 mmol), and dry DCM (10 mL) were added to a 50 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added under nitrogen atmosphere. The mixture was stirred at room temperature for 3 hours. The solution was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure to give a colorless oily compound (3.10 g, 90% yield).

[0111] Di-tetrazyl(2-((2-hydroxyethyl)amino)ethyl)phosphamide (1-3, 0.245 g, 0.5 mmol), undecyl 6-bromohexanoate (1-6, 0.174 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in 5 ml vials. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-1 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 80%, 0.60 g. MS (ES) m / z: 817 (M+H) + ).

[0112] The steps for synthesizing I-2:

[0113]

[0114] 6-Bromohexanoyl chloride (2-1, 2.12 g, 10 mmol), n-tetranol (2-2, 2.01 g, 10 mmol), and dry DCM (10 mL) were added to a 50 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added under nitrogen atmosphere. The mixture was stirred at room temperature for 3 hours. The solution was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure to give a colorless oily compound (3.21 g, 90% yield).

[0115] Di-tetrazyl(2-((2-hydroxyethyl)amino)ethyl)phosphamide (1-3, 0.245 g, 0.5 mmol), tridecyl 6-bromohexanoate (2-4, 0.174 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in 5 ml vials. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-2 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 80%, 0.63 g. MS (ES) m / z: 845 (M+H) + ).

[0116] The steps for synthesizing I-3:

[0117]

[0118]

[0119] Undecanool (3-1, 1.72 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stir bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour, then stirred at 50 °C under nitrogen for 1 hour. After completion, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 mL) was added to a colorless liquid, and DIPEA (2.68 mL, 15 mmol) and 2-(2-aminoethyl)aminoethanol (0.52 g, 5 mmol) were added under nitrogen. The solution was stirred at room temperature under nitrogen for 3 hours. Then it was diluted with DCM (10 mL) and washed twice with water. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give product 3-3 as a colorless oil (2.46 g, 90%).

[0120] Di-undecyl(2-((2-hydroxyethyl)amino)ethyl)phosphoramide (3-3, 0.256 g, 0.5 mmol), undecyl 6-bromohexanoate (1-6, 0.174 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in 5 ml vials. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-3 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 80%, 0.60 g. MS (ES) m / z: 761 (M+H + ).

[0121] The steps for synthesizing I-4:

[0122]

[0123] I-1 (0.016 g, 0.2 mmol), KOH (0.001 g, 0.2 mmol), and EtOH (5 mL) were added to a 5 mL round-bottom flask. The mixture was stirred at room temperature for 10 hours, and the organic solvent was removed under reduced pressure. The solution was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to obtain the desired product I-4; Yield: 90%, 0.011 g. MS (ES) m / z: 691 (M+H+).

[0124] The steps for synthesizing I-6:

[0125]

[0126] Undecanoyl chloride (6-2, 2.05 g, 10 mmol), 6-bromohexanol (6-1, 1.81 g, 10 mmol), and dry DCM (10 mL) were added to a 50 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was then added, and the mixture was stirred at room temperature for 3 hours. The solution was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to give a colorless oil (3.06 g, 90%).

[0127] Di-tetrazyl(2-((2-hydroxyethyl)amino)ethyl)phosphoramide (1-3, 0.245 g, 0.5 mmol), undecyl 6-bromohexyl undecanoate (6-3, 0.174 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in 5 ml vials. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-6 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 80%, 0.60 g. MS (ES) m / z: 817 (M+H) + ).

[0128] The steps for synthesizing I-7:

[0129]

[0130]

[0131] 6-Bromohexanoyl chloride (7-1, 2.12 g, 10 mmol), dec-2-yn-1-ol (7-2, 2.01 g, 10 mmol), and dry DCM (10 mL) were added to a 50 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added under nitrogen. The reaction mixture was stirred at room temperature for 3 hours. The solution was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to give a colorless oil (3.21 g, 90%).

[0132] Di-tetrazyl(2-((2-hydroxyethyl)amino)ethyl)phosphoramide (1-3, 0.245 g, 0.5 mmol), dec-2-ynyl-1-yl 6-bromohexanoate (7-3, 0.18874 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in 5 ml vials. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-7 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 64%, 0.53 g. MS (ES) m / z: 799 (M+H) + ).

[0133] The steps for synthesizing I-8:

[0134]

[0135] 6-Bromohexanol (8-1, 1.81 g, 10 mmol) and thionyl chloride (0.73 mL, 10 mmol) were added to a 50 mL round-bottom flask containing a nitrogen balloon and stirred at room temperature for 1 hour. The generated HCl gas was removed under reduced pressure and the nitrogen in the balloon was replaced. Under nitrogen protection, dry dichloromethane (10 mL), diisopropylethylamine (2.68 mL, 15 mmol), and 2-hexyldecanoic acid (8-2, 2.56 g, 10 mmol) were added to the reaction flask, and the mixture was stirred at room temperature for 3 hours. The mixture was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to give a colorless oil (3.97 g, 95%).

[0136] Di-tetrazyl(2-((2-hydroxyethyl)amino)ethyl)phosphamide (1-3, 0.245 g, 0.5 mmol), 6-bromohexyl-2-decanoate (8-3, 0.21 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-8 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 52%, 0.24 g. MS (ES) m / z: 887 (M+H) + ).

[0137] The steps for synthesizing I-9:

[0138]

[0139] 6-Bromohexanoyl chloride (9-1, 2.12 g, 10 mmol), 2-hexyldecyl-1-ol (9-2, 2.01 g, 10 mmol), and dry dichloromethane (10 mL) were added to a 50 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added under nitrogen atmosphere. The mixture was stirred at room temperature for 3 hours. The solution was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure to give a colorless oily compound (4.08 g, 96% yield).

[0140] Di-tetrazyl(2-((2-hydroxyethyl)amino)ethyl)phosphamide (1-3, 0.245 g, 0.5 mmol), 2-hexyldecyl 6-bromohexanoate (9-3, 0.21 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-9 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 52%, 0.24 g. MS (ES) m / z: 887 (M+H + ).

[0141] The steps for synthesizing I-10:

[0142]

[0143] Dec-2-yn-1-ol (10⁻¹, 1.72 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stir bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour, then stirred at 50 °C under nitrogen for 1 hour. After this, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 mL) was added to a colorless liquid, followed by the addition of DIPEA (2.68 mL, 15 mmol) and 2-(3-aminopropyl)aminoethanol (0.52 g, 5 mmol) under nitrogen. The solution was stirred at room temperature under nitrogen for 3 hours. It was then diluted with DCM (10 mL) and washed twice with water. The organic phase was separated and dried over anhydrous MgSO₄. The desired product was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give a colorless oil (2.46 g, 90%).

[0144] Di(decyl-2-yn-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (10⁻², 0.256 g, 0.5 mmol), decyl-2-yn-1-yl 6-bromohexanoate (7⁻³, 0.18874 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in 5 ml vials. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-10 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 45%, 0.16 g. MS (ES) m / z: 721 (M+H + ).

[0145] The steps for synthesizing I-11:

[0146]

[0147] Di(decyl-2-yn-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (10⁻², 0.256 g, 0.5 mmol), undecyl 6-bromohexanoate (1-6, 0.174 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-11 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 45%, 0.18 g. MS (ES) m / z: 739 (M+H) + ).

[0148] The steps for synthesizing I-12:

[0149]

[0150] 6-Bromohexanoyl chloride (12-1, 2.12 g, 10 mmol), undecyl-10-enyl-1-ol (12-2, 2.01 g, 10 mmol), and dry dichloromethane (10 mL) were added to a 50 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added under nitrogen. The mixture was stirred at room temperature for 3 hours. The solution was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure to give a colorless oily compound (3.22 g, 90% yield).

[0151] Undecyl-1-ol (12-2, 2.01 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stir bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour, then stirred at 50 °C under nitrogen for 1 hour. After this, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 mL) was added to a colorless liquid, followed by the addition of DIPEA (2.68 mL, 15 mmol) and 2-(3-aminopropyl)aminoethanol (0.52 g, 5 mmol) under nitrogen. The solution was stirred at room temperature under nitrogen for 3 hours. It was then diluted with DCM (10 mL) and washed twice with water. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give a colorless oil (2.46 g, 90%).

[0152] Di(undecyl-1-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (12-4, 0.25 g, 0.5 mmol), undecyl-1-en-1-yl 6-bromohexanoate (12-3, 0.173 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-12 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 50%, 0.19 g. MS (ES) m / z: 769 (M+H) + ).

[0153] The steps for synthesizing I-13:

[0154]

[0155] Di(undecyl-1-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (12-4, 0.25 g, 0.5 mmol), tridecyl 6-bromohexanoate (2-4, 0.18 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-13 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 50%, 0.21 g. MS (ES) m / z: 799 (M+H) + ).

[0156] The steps for synthesizing I-14:

[0157]

[0158] Undecanool (14-1, 1.72 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stir bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour, then stirred at 50 °C under nitrogen for 1 hour. After completion, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 mL) was added to a colorless liquid, and DIPEA (2.68 mL, 15 mmol) and 3-((4-aminobutyl)amino)propyl-1-ol (0.52 g, 5 mmol) were added under nitrogen. The solution was stirred at room temperature under nitrogen for 3 hours. Then it was diluted with DCM (10 mL) and washed twice with water. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give a colorless oil (2.46 g, 90%).

[0159] Di-undecyl(4-((3-hydroxypropyl)amino)butyl)phosphamide (14-2, 70.26 g, 0.5 mmol), undecyl 6-bromohexanoate (1-6, 0.174 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-14 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 45%, 0.21 g. MS (ES) m / z: 803 (M+H) + ).

[0160] The steps for synthesizing I-15:

[0161]

[0162] Di-tetrazyl(2-((2-hydroxyethyl)amino)ethyl)phosphoramide (1-3, 0.245 g, 0.5 mmol), undecyl-10-en-1-yl-6-bromohexanoate (12-3, 0.173 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in 5 ml vials. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-15 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to yield a yield of 45%, 0.19 g. MS (ES) m / z: 815 (M+H + ).

[0163] The steps for synthesizing I-23:

[0164]

[0165] Di(undecyl-10-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (12-4, 0.25 g, 0.5 mmol), decyl-2-ynyl-1-yl 6-bromohexanoate (7-3, 0.18874 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in 5 ml vials. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-23 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 50%, 0.19 g. MS (ES) m / z: 753 (M+H) + ).

[0166] The steps for synthesizing I-24:

[0167]

[0168] 6-Bromohexanoyl chloride (24-1, 2.12 g, 10 mmol), (E)-undecyl-2-en-1-ol (24-2, 2.01 g, 10 mmol), and dry dichloromethane (10 mL) were added to a 50 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added under nitrogen atmosphere. The mixture was stirred at room temperature for 3 hours. The solution was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure to give a colorless oily compound (3.22 g, 90% yield).

[0169] Di(undecyl-10-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (12-4, 0.25 g, 0.5 mmol), (E)-undecyl-2-en-1-yl 6-bromohexanoate (24-3, 0.173 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-24 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 45%, 0.18 g. MS (ES) m / z: 769 (M+H) + ).

[0170] The steps for synthesizing I-25:

[0171]

[0172] 8-Bromooctanoyl chloride (25-1, 2.40 g, 10 mmol), heptadecano-9-ol (25-2, 2.56 g, 10 mmol), and dry dichloromethane (10 mL) were added to a 50 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added under nitrogen atmosphere. The mixture was stirred at room temperature for 3 hours. The solution was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure to give a colorless oily compound (3.63 g, 90% yield).

[0173] Di(undecyl-1-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (12-4, 0.25 g, 0.5 mmol), heptadecan-9-yl 8-bromooctanoate (25-3, 0.23 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-25 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 45%, 0.23 g. MS (ES) m / z: 883 (M+H) + ).

[0174] The steps for synthesizing I-26:

[0175]

[0176] 6-Bromohexanoyl chloride (26-1, 2.12 g, 10 mmol), 2-pentyldecyl-1-ol (26-2, 2.28 g, 10 mmol), and dry dichloromethane (10 mL) were added to a 50 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added under nitrogen atmosphere. The mixture was stirred at room temperature for 3 hours. The solution was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure to give a colorless oily compound (3.56 g, 90% yield).

[0177] Di(undecyl-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (12-4, 0.25 g, 0.5 mmol), 2-pentyldecyl-1-yl 6-bromohexanoate (26-3, 0.23 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-26 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 45%, 0.31 g. MS (ES) m / z: 827 (M+H) + ).

[0178] The steps for synthesizing I-28:

[0179]

[0180] 6-Bromohexanoyl chloride (28-1, 2.12 g, 10 mmol), (Z)-octadecyl-9-en-1-ol (28-2, 2.68 g, 10 mmol), and dry dichloromethane (10 mL) were added to a 50 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added under nitrogen atmosphere. The mixture was stirred at room temperature for 3 hours. The solution was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure to give a colorless oily compound (3.63 g, 90% yield).

[0181] Di(undecyl-1-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (12-4, 0.25 g, 0.5 mmol), (Z)-octadecyl-9-en-1-yl 6-bromohexanoate (28-3, 0.22 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-28 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 45%, 0.20 g. MS (ES) m / z: 867 (M+H) + ).

[0182] The steps for synthesizing I-29:

[0183]

[0184] Dodeca-11-ynyl-1-ol (29-1, 1.82 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stir bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour, then stirred at 50 °C under nitrogen for 1 hour. After this, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 mL) was added to a colorless liquid, followed by the addition of DIPEA (2.68 mL, 15 mmol) and 2-(3-aminopropyl)aminoethanol (0.52 g, 5 mmol) under nitrogen. The solution was stirred at room temperature under nitrogen for 3 hours. It was then diluted with DCM (10 mL) and washed twice with water. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give a colorless oil (2.03 g, 80%).

[0185] Di(dodecyl-11-ynyl-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (29-2, 0.26 g, 0.5 mmol), tridecyl 6-bromohexanoate (2-4, 0.174 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-29 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 50%, 0.19 g. MS (ES) m / z: 823 (M+H) + ).

[0186] The steps for synthesizing I-30:

[0187]

[0188] Di(dodecyl-1-ynyl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (29-2, 0.26 g, 0.5 mmol), undecyl 6-bromohexanoate (1-6, 0.16 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-30 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 50%, 0.19 g. MS (ES) m / z: 795 (M+H) + ).

[0189] The steps for synthesizing I-31:

[0190]

[0191] 6-Bromohexanoyl chloride (31-1, 2.12 g, 10 mmol), dodeca-11-ynyl-1-ol (31-2, 1.82 g, 10 mmol), and dry dichloromethane (10 mL) were added to a 50 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added under nitrogen atmosphere. The mixture was stirred at room temperature for 3 hours. It was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure to give a colorless oily compound (3.12 g, 90% yield).

[0192] Di(undecyl-1-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (12-4, 0.25 g, 0.5 mmol), dodeca-11-ynyl-1-yl 6-bromohexanoate (31-3, 0.18 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-31 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to obtain a yield of 45% (0.16 g). MS (ES) m / z: 781 (M+H) + ).

[0193] The steps for synthesizing I-32:

[0194]

[0195] (Z)-octadecyl-9-en-1-ol (32-1, 2.68 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stir bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour, then stirred at 50 °C under nitrogen for 1 hour. After completion, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 mL) was added to a colorless liquid, and DIPEA (2.68 mL, 15 mmol) and 2-(2-aminoethyl)aminoethanol (0.52 g, 5 mmol) were added under nitrogen. The solution was stirred at room temperature under nitrogen for 3 hours. Then it was diluted with DCM (10 mL) and washed twice with water. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give a colorless oily product (6.12 g, 90%).

[0196] Di((Z)-octadecyl-9-en-1-yl)(2-((2-hydroxyethyl)amino)ethyl)phosphoramide (32-2, 0.34 g, 0.5 mmol), (Z)-octadecyl-9-en-1-yl 6-bromohexanoate (28-3, 0.22 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in 5 ml vials. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-32 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 45%, 0.31 g. MS (ES) m / z: 1077 (M+H) + ).

[0197] The steps for synthesizing I-36:

[0198]

[0199] (Z)-Non-3-enyl-1-ol (36-1, 1.42 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stir bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour, then stirred at 50 °C under nitrogen for 1 hour. After this, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 mL) was added to a colorless liquid, followed by the addition of DIPEA (2.68 mL, 15 mmol) and 2-(3-aminopropyl)aminoethanol (0.52 g, 5 mmol) under nitrogen. The solution was stirred at room temperature under nitrogen for 3 hours. It was then diluted with DCM (10 mL) and washed twice with water. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give a colorless oil (2.03 g, 80%).

[0200] Di((Z)-non-3-enyl-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (36-2, 0.23 g, 0.5 mmol), (Z)-octadecyl-9-enyl-1-bromohexanoate (28-3, 0.22 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in 5 ml vials. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-36 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 50%, 0.19 g. MS (ES) m / z: 811 (M+H + ).

[0201] The steps for synthesizing I-38:

[0202]

[0203] 6-Bromohexanoyl chloride (38-1, 2.12 g, 10 mmol), dodecyl-3-enyl-1-ol (38-2, 1.82 g, 10 mmol), and dry dichloromethane (10 mL) were added to a 50 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added under nitrogen atmosphere. The mixture was stirred at room temperature for 3 hours. The solution was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure to give a colorless oily compound (3.12 g, 90% yield).

[0204] Di(undecyl-1-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (12-4, 0.25 g, 0.5 mmol), dodeca-3-enyl-1-yl 6-bromohexanoate (38-3, 0.18 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-38 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to yield a yield of 45%, 0.16 g. MS (ES) m / z: 783 (M+H) + ).

[0205] The steps for synthesizing I-40:

[0206]

[0207] (Z)-dodecyl-3-enyl-1-ol (40-1, 1.78 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stir bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour, then stirred at 50 °C under nitrogen for 1 hour. After this, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 mL) was added to a colorless liquid, followed by the addition of DIPEA (2.68 mL, 15 mmol) and 2-(3-aminopropyl)aminoethanol (0.52 g, 5 mmol) under nitrogen. The solution was stirred at room temperature under nitrogen for 3 hours. It was then diluted with DCM (10 mL) and washed twice with water. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give a colorless oil (2.03 g, 80%).

[0208] Di((Z)-dodecyl-3-enyl-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (40-2, 0.23 g, 0.5 mmol), (Z)-octadecyl-9-enyl-1-bromohexanoate (28-3, 0.22 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in 5 ml vials. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-40 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to yield a yield of 50%, 0.21 g. MS (ES) m / z: 895 (M+H + ).

[0209] The steps for synthesizing I-43:

[0210]

[0211] Undecyl-1-ol (43-1, 2.01 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stir bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour, then stirred at 50 °C under nitrogen for 1 hour. After this, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 mL) was added to a colorless liquid, followed by the addition of DIPEA (2.68 mL, 15 mmol) and 2-(4-aminobutyl)aminoethanol (0.53 g, 5 mmol) under nitrogen. The solution was stirred at room temperature under nitrogen for 3 hours. It was then diluted with DCM (10 mL) and washed twice with water. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give a colorless oil (2.46 g, 90%).

[0212] Di(undecyl-1-en-1-yl)(4-((2-hydroxyethyl)amino)butyl)phosphamide (43-2, 0.24 g, 0.5 mmol), (Z)-octadecyl-9-en-1-yl 6-bromohexanoate (28-3, 0.22 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-43 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 50%, 0.22 g. MS (ES) m / z: 881 (M+H + ).

[0213] The steps for synthesizing I-44:

[0214]

[0215] Undecyl-1-ol (43-1, 2.01 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stir bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour, then stirred at 50 °C under nitrogen for 1 hour. After this, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 mL) was added to a colorless liquid, followed by the addition of DIPEA (2.68 mL, 15 mmol) and 3-(4-aminobutyl)aminopropanol (0.54 g, 5 mmol) under nitrogen. The solution was stirred at room temperature under nitrogen for 3 hours. It was then diluted with DCM (10 mL) and washed twice with water. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give a colorless oil (2.46 g, 90%).

[0216] Di(undecyl-1-en-1-yl)(4-((3-hydroxypropyl)amino)butyl)phosphamide (44-2, 0.24 g, 0.5 mmol), (Z)-octadecyl-9-en-1-yl 6-bromohexanoate (28-3, 0.22 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-44 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 50%, 0.22 g. MS (ES) m / z: 895 (M+H + ).

[0217] The steps for synthesizing I-45:

[0218]

[0219] 8-Bromooctanoyl chloride (45-1, 2.40 g, 10 mmol), (Z)-octadecyl-9-en-1-ol (45-2, 2.68 g, 10 mmol), and dry dichloromethane (10 mL) were added to a 50 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added under nitrogen atmosphere. The mixture was stirred at room temperature for 3 hours. The solution was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure to give a colorless oily compound (3.65 g, 90% yield).

[0220] Di(undecyl-1-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (12-4, 0.25 g, 0.5 mmol), (Z)-octadecyl-9-en-1-yl 8-bromooctanoate (45-3, 0.24 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-45 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to obtain a yield of 45% (Yield: 45%), 0.21 g. MS (ES) m / z: 895 (M+H) + ).

[0221] The steps for synthesizing I-46:

[0222]

[0223] 6-Bromohexanoyl chloride (46-1, 2.12 g, 10 mmol), (9Z, 12Z)-octadecyl-9,12-dien-1-ol (46-2, 2.68 g, 10 mmol), and dry dichloromethane (10 mL) were added to a 50 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added under nitrogen. The mixture was stirred at room temperature for 3 hours. The solution was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure to give a colorless oily compound (3.63 g, 90% yield).

[0224] Di(undecyl-1-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (12-4, 0.25 g, 0.5 mmol), (9Z, 12Z)-octadecyl-9,12-dien-1-yl 6-bromohexanoate (46-3, 0.22 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in 5 ml vials. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-46 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to obtain a yield of 45% (0.20 g). MS (ES) m / z: 865 (M+H) + ).

[0225] The steps for synthesizing I-47:

[0226]

[0227] Undecyl-1-ol (47-1, 1.78 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stir bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour, then stirred at 50 °C under nitrogen for 1 hour. After this, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 mL) was added to a colorless liquid, followed by the addition of DIPEA (2.68 mL, 15 mmol) and 2-(3-aminopropyl)aminoethanol (0.52 g, 5 mmol) under nitrogen. The solution was stirred at room temperature under nitrogen for 3 hours. It was then diluted with DCM (10 mL) and washed twice with water. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give a colorless oil (2.03 g, 80%).

[0228] Di(undecyl-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (47-2, 0.23 g, 0.5 mmol), (Z)-octadecyl-9-en-1-yl 6-bromohexanoate (28-3, 0.22 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-47 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 50%, 0.21 g. MS (ES) m / z: 863 (M+H) + ).

[0229] The steps for synthesizing I-48:

[0230]

[0231] Dodeca-11-ynyl-1-ol (48-1, 1.92 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stir bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour, then stirred at 50 °C under nitrogen for 1 hour. After this, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 mL) was added to a colorless liquid, followed by the addition of DIPEA (2.68 mL, 15 mmol) and 2-(3-aminopropyl)aminoethanol (0.52 g, 5 mmol) under nitrogen. The solution was stirred at room temperature under nitrogen for 3 hours. It was then diluted with DCM (10 mL) and washed twice with water. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give a colorless oil (2.03 g, 80%).

[0232] Di(dodecyl-1-ynyl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (48-2, 0.24 g, 0.5 mmol), (Z)-octadecyl-9-en-1-yl 6-bromohexanoate (28-3, 0.22 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in 5 ml vials. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-48 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to obtain a yield of 48; Yield: 48%, 0.21 g. MS (ES) m / z: 891 (M+H) + )

[0233] The steps for synthesizing I-50:

[0234]

[0235] 8-Bromooctanoyl chloride (50-1, 2.40 g, 10 mmol), dodeca-11-yn-1-ol (50-2, 2.24 g, 10 mmol), and dry dichloromethane (10 mL) were added to a 50 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added under nitrogen atmosphere. The mixture was stirred at room temperature for 3 hours. It was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure to give a colorless oily compound (3.65 g, 90% yield).

[0236] Di(undecyl-1-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (12-4, 0.25 g, 0.5 mmol), dodeca-11-yn-1-yl 8-bromooctanoate (50-3, 0.21 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-50 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 42%, 0.21 g. MS (ES) m / z: 809 (M+H) + ).

[0237] The steps for synthesizing I-51:

[0238]

[0239] Di((Z)-dodecyl-3-enyl-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphamide (40-2, 0.23 g, 0.5 mmol), (9Z, 12Z)-octadecyl-9,12-dien-1-yl 6-bromohexanoate (46-3, 0.22 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were placed in 5 ml vials. The mixture was stirred at 60 °C for 24 hours. The solvent was removed under reduced pressure. The product I-51 was purified by silica gel column chromatography (DCM / MeOH = 20 / 1); Yield: 50%, 0.21 g. MS (ES) m / z: 893 (M+H + ).

[0240] The synthesis steps of I-34:

[0241]

[0242]

[0243] 8-Bromooctanoic acid (34-1, 2.23 g, 10 mmol) and thionyl chloride (1.3 g, 0.79 mL, 11 mmol) were added to a 50 mL round-bottom flask containing a nitrogen balloon and stirred at room temperature for 1 hour. The byproduct hydrogen chloride gas was removed and replaced with nitrogen. Dry dichloromethane (10 mL), triethylamine (2.02 g, 2.84 mL, 20 mmol), and heptadecanol (34-2, 2.56 g, 10 mmol) were added to the flask and stirred at room temperature for 1 hour. The mixture was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to give a colorless oil (3.71 g, 80% yield).

[0244] Heptadecan-9-yl-8-bromooctanoate (34-3, 2.3 g, 5 mmol), ethanolamine (1.22 g, 1.22 mL, 20 mmol), and MeCN (10 mL) were added to a 50 mL round-bottom flask and stirred at 70 °C for 12 hours. After cooling to room temperature, the solvent was removed under reduced pressure. The solution was purified by silica gel column chromatography (DCM / MeOH = 20 / 1) to give a colorless liquid (1.62 g, 85% yield).

[0245] 6-Bromohexanoyl chloride (34-5, 1.06 g, 5 mmol), dodeca-11-ynyl-1-ol (34-6, 0.91 g, 5 mmol), and dry dichloromethane (10 mL) were added to a 25 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. The mixture was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to give a colorless oil (1.03 g, 80% yield).

[0246] Heptadecanyl-9-yl-8-((2-hydroxyethyl)amino)octanoate (34-4, 0.22 g, 0.5 mmol), dodeca-11-yn-1-yl-6-bromohexanoate (34-7, 0.18 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were added to a 10 ml reaction flask. The reaction was stirred at 70 °C for 24 hours and then cooled to room temperature. The solvent was removed under reduced pressure. Purification by silica gel column chromatography (DCM / MeOH = 20 / 1) yielded a colorless liquid I-34; Yield: 50%, 0.18 g. MS (ES) m / z: 720 (M+H) + ).

[0247] The synthesis steps of I-39:

[0248]

[0249] 6-Bromohexanoyl chloride (39-1, 1.06 g, 5 mmol), undecyl-10-ynyl-1-ol (39-2, 0.91 g, 5 mmol), and dry dichloromethane (10 mL) were added to a 25 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. The mixture was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to give a colorless oil (1.03 g, 80% yield).

[0250] Heptadecanyl-9-yl-8-((2-hydroxyethyl)amino)octanoate (34-4, 0.22 g, 0.5 mmol), undecyl-10-yn-1-yl-6-bromohexanoate (39-3, 0.18 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 mL) were added to a 10 mL reaction flask. The reaction was stirred at 70 °C for 24 hours, then cooled to room temperature. The solvent was removed under reduced pressure. Purification by silica gel column chromatography (DCM / MeOH = 20 / 1) yielded a colorless liquid I-39; Yield: 50%, 0.18 g. MS (ES) m / z: 706 (M+H) + ).

[0251] The synthesis steps of I-41:

[0252]

[0253] 8-Bromooctanoyl chloride (41-1, 1.20 g, 5 mmol), dodeca-11-ynyl-1-ol (41-2, 0.91 g, 5 mmol), and dry dichloromethane (10 mL) were added to a 25 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. The mixture was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to give a colorless oil (1.83 g, 80% yield).

[0254] Heptadecanyl-9-yl-8-((2-hydroxyethyl)amino)octanoate (34-4, 0.22 g, 0.5 mmol), dodeca-11-yn-1-yl-8-bromooctanoate (41-3, 0.19 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were added to a 10 ml reaction flask. The reaction was stirred at 70 °C for 24 hours and then cooled to room temperature. The solvent was removed under reduced pressure. Purification by silica gel column chromatography (DCM / MeOH = 20 / 1) yielded a colorless liquid I-41; Yield: 48%, 0.18 g. MS (ES) m / z: 748 (M+H) + ).

[0255] The synthesis steps of I-42:

[0256]

[0257] 8-Bromooctanoyl chloride (42-1, 1.20 g, 5 mmol), hexano-5-ynyl-1-ol (42-2, 0.49 g, 5 mmol), and dry dichloromethane (10 mL) were added to a 25 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. The mixture was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to give a colorless oil (1.21 g, 80% yield).

[0258] Heptadecanyl-9-yl-8-((2-hydroxyethyl)amino)octanoate (34-4, 0.22 g, 0.5 mmol), hex-5-ynyl-1-yl-8-bromooctanoate (42-3, 0.15 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were added to a 10 ml reaction flask. The reaction was stirred at 70 °C for 24 hours, then cooled to room temperature. The solvent was removed under reduced pressure. Purification by silica gel column chromatography (DCM / MeOH = 20 / 1) yielded a colorless liquid I-42; Yield: 48%, 0.14 g. MS (ES) m / z: 664 (M+H) + ).

[0259] The synthesis steps of I-49:

[0260]

[0261] 8-Bromooctanoyl chloride (49-1, 1.20 g, 5 mmol), non-8-ynyl-1-ol (49-2, 0.70 g, 5 mmol), and dry dichloromethane (10 mL) were added to a 25 mL round-bottom flask containing a nitrogen balloon. Triethylamine (1.66 mL, 12 mmol) was slowly added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. The mixture was then diluted with DCM (10 mL) and washed twice with water. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to give a colorless oil (1.41 g, 80% yield).

[0262] Heptadecanyl-9-yl-8-((2-hydroxyethyl)amino)octanoate (34-4, 0.22 g, 0.5 mmol), nonyl-8-ynyl-1-8-bromooctanoate (42-3, 0.15 g, 0.5 mmol), K₂CO₃ (0.138 g, 1 mmol), KI (0.0032 g, 0.02 mmol), and acetonitrile (4 ml) were added to a 10 ml reaction flask. The reaction was stirred at 70 °C for 24 hours and then cooled to room temperature. The solvent was removed under reduced pressure. Purification by silica gel column chromatography (DCM / MeOH = 20 / 1) yielded a colorless liquid I-49; Yield: 48%, 0.18 g. MS (ES) m / z: 706 (M+H) + ).

[0263] In one embodiment of the present invention, the preparation steps of lipid nanoparticles encapsulating luciferase mRNA (FucmRNA) include:

[0264] Add 3 μL of CleanCapFluc mRNA stock solution (1 mg / mL), 5.63 μL of 0.1 M sodium acetate buffer, and 13.87 μL to a container and mix well to obtain the aqueous phase.

[0265] The amino lipid compound, auxiliary lipid, structural lipid, and PEG-lipid of this disclosure were dissolved in anhydrous ethanol to prepare solutions with concentrations of 20 mg / mL, 10 mg / mL, 20 mg / mL, and 25 mg / mL, respectively. Using a molar ratio of Lipid:DSPC:Cholesterol:DMG-PEG2000 of 48:10:40.5:1.5, the four solutions were transferred to prepare an alcohol phase with a Lipid concentration of 7.3 mmol / L.

[0266] Take 7.5 μL of the above ethanol solution and aqueous solution, mix them quickly, and then add 570 μL of 25 mM Tris buffer-10% sucrose (pH 7.0, filtered) solution and mix rapidly to obtain the mRNA-LNP complex sample.

[0267] Furthermore, the in vitro mRNA-LNP delivery efficiency assay includes:

[0268] The cell line used was HEK-293 (Procell, CL-0001); the culture medium was DMEM (Invitrogen) supplemented with 10% fetal bovine serum; and the selection method was 24-well plate cell transfection.

[0269] Detection (readout): Absolute value of reporter gene firefly luciferase activity relative to total cellular protein expression (total protein content per well was measured using a BCA protein concentration assay kit, Beyotime, P0012) (firefly luciferase activity was indirectly determined by measuring biofluorescence intensity using a Luciferase Assay Kit, Promega E1483). The TransIT-mRNA Transfection Kit (Mirusbio, MIR2225) was used as a positive control according to the manufacturer's instructions.

[0270] The steps include:

[0271] 1. One day before mRNA transfection, HEK293 cells were seeded into 24-well plates at 25,000 cells / ml, 0.4 ml per well, and cultured overnight at 37°C and 5% CO2 to allow the cells to adhere.

[0272] 2. On the day of transfection, prepare the TransIT-mRNAReagent / mRNABoost / RNA complex and the mRNA-LNP complex, respectively.

[0273] a) Take 240 μL of preheated Opti-MEM serum-reduced medium and put it into a 1.5 ml centrifuge tube. First, add 1.2 μg mRNA and gently mix by pipetting. Then, add 2.4 μL mRNABoost and gently mix by pipetting. Finally, add 2.4 μL mRNAReagent and gently mix by pipetting. Let it stand at room temperature for 5 min to form a complex. Take 100 μL of the complex and add it evenly to each well of cells. Add it to 2 wells in total.

[0274] b) Take 100 μL of the prepared mRNA-LNP complex and add it evenly to each well of cells. Add 2 wells for each LNP sample.

[0275] c) Gently shake the 24-well plate and incubate it overnight in an incubator at 37°C and 5% CO2.

[0276] 3. On the second day after transfection, remove the 24-well plate from the incubator, aspirate the cell supernatant, add 150 μL of cell lysis buffer (Passivelysis 5X buffer, Promega, E1941) to each well, repeatedly pipette the cells, collect them into a new 1.5 ml centrifuge tube, centrifuge at 14000 rpm for 30 s, take 20 μL of cell lysis supernatant for BCA assay, and take 2 μL of cell lysis supernatant for luciferase assay. Use a Varioskan LUX multi-mode microplate reader (Thermo, VLBL00D1) to perform absorbance and chemiluminescence assays at 562 nm, respectively.

[0277]

[0278] The test results are shown in Table 1:

[0279] Table 1:

[0280]

[0281]

[0282] The TransIT control group showed high luciferase activity (around 1.65E+06), indicating high transfection efficiency. Therefore, it can be used as a positive control to assess the relative efficiency of other LNP samples. The numerical differences between different LNP samples (e.g., I-1, I-2, etc.) in the table indicate varying mRNA delivery efficiencies in HEK-293 cells based on different formulations. For example, some samples showed high luciferase activity (e.g., I-29, I-37, etc.), while others showed low activity (e.g., I-6, I-12, etc.), indicating significant differences in delivery efficiency among these samples.

[0283] The two repeated measurements for each sample were generally close, indicating good experimental reproducibility. Larger differences between the two measurements for some samples may suggest poor sample stability or homogeneity, warranting further optimization. The delivery efficiency of different LNP samples could be evaluated by comparing the luciferase activity of the TransIT control group and LNP samples. The higher value of the control group provided a benchmark for LNP samples; if the sample's value was close to or higher than the control group, the delivery effect was more ideal; conversely, the delivery effect needed improvement.

[0284] In one embodiment, the in vitro toxicity detection device for LNP nanoparticles includes:

[0285] (1) Ingredients:

[0286] A specified amount of RNase-free purified water and sodium acetate buffer were added to a container and mixed to prepare a 0.25 M sodium acetate solution. The amino lipid compound, auxiliary lipid, structural lipid, and PEG-lipid of this disclosure were dissolved in anhydrous ethanol to prepare solutions with concentrations of 20 mg / mL, 10 mg / mL, 20 mg / mL, and 25 mg / mL, respectively. Using a molar ratio of Lipid:DSPC:CHO-HPM-DMG-2000 of 48:10:40.5:1.5, the four solutions were transferred to prepare an alcohol phase with a Lipid concentration of 7.3 mmol / L.

[0287] (2) Microfluidic control equipment:

[0288] A blank lipid nanoparticle sample was obtained by injecting the aqueous phase into the microfluidic chip at a flow rate of 7.5 mL / min and the alcohol phase at 4 mL / min using an automated syringe pump.

[0289] (3) Dialysis: The product from step (2) was loaded into a dialysis bag and placed in a TrisBuffer-10% sucrose solution for replacement to remove residual ethanol, unassembled lipids, and other components. Dialysis was performed for 24 hours at room temperature in the dark with magnetic stirring (the dialysis solution was changed every 12 hours).

[0290] (4) Concentration:

[0291] The product from step (3) was centrifuged and concentrated 5 times to obtain the sample to be tested.

[0292] (5) Particle size test

[0293] The particle size of some samples was measured using a Malvern Panalytical Zetasizer Pro instrument, and the particle size data are shown in Table 2.

[0294] Table 2:

[0295] sample Particle size (nM) I-29 183 I-39 220 SM-102 (Control) 210

[0296] In one embodiment, the apparatus for assessing cytotoxicity includes:

[0297] Cell line: HEK-293 cells (Procell, CL-0001)

[0298] Culture medium: DMEM (Invitrogen) supplemented with 10% fetal bovine serum.

[0299] Screening method: 96-well cell culture plate

[0300] Detection (readout): Using the CyQUANT cytotoxicity assay kit. TMThe LDHCytotoxicityAssayKit (Invitrogen, C20300) converts lactate dehydrogenase (LDH) in cell culture supernatant into a red formazan product via a coupled enzymatic reaction. This product can be measured spectrophotometrically at 490 nm. The level of formazan formation is directly proportional to the amount of LDH released into the culture medium, thereby assessing the degree of cytotoxicity.

[0301] The steps involved in cytotoxicity assessment include:

[0302] 1. On the first day, HEK293 cells were seeded at 100,000 cells / ml into 96-well plates, 0.1 ml per well, and cultured overnight at 37°C in a 5% CO2 incubator to allow the cells to adhere.

[0303] 2. On the second day, the prepared blank LNP lipid nanoparticles were diluted to three concentrations (62.5 μg / ml, 125 μg / ml, and 250 μg / ml) and added to the cells, 10 μL per well, with 3 wells containing cells for each concentration. For the spontaneous LDH release control wells, 10 μL of sterile water was added, with a total of 3 wells containing cells. The maximum LDH release control wells were left untreated. The 96-well cell culture plates were incubated overnight at 37°C with 5% CO2.

[0304] 3. On the third day, remove the 96-well plate from the incubator and add 10 μL of cell lysis buffer to the large LDH release control well, adding cells to a total of 3 wells. Incubate again at 37°C and 5% CO2 for 45 minutes. After this time, remove the 96-well plate and transfer 50 μL of cell culture supernatant from each well to a new 96-well plate. Add 50 μL of reaction mixture to each well, mix well, and incubate at room temperature in the dark for 30 minutes. Then, add 50 μL of stop solution to each well and immediately measure the absorbance at 490 nm and 680 nm (Varioskan LUX multi-functional microplate reader, Thermo, VLBL00D1). Subtract the 680 nm absorbance value (background) from the 490 nm absorbance. Calculate cytotoxicity using the following formula:

[0305]

[0306] Figure 1 This study aimed to measure the effects of three different types of lipid nanoparticles (LNPs) on cellular lactate dehydrogenase (LDH) release. LDH release is commonly used to evaluate cell membrane damage and cytotoxicity; therefore, the results of this experiment can reflect the cytotoxic effects of different concentrations of LNPs. Experimental groups: The figure shows three different types of LNPs—I-29, I-39, and SM-102. In the legend, I-29 is labeled blue, I-39 is orange, and SM-102 is gray.

[0307] The experiment used three different concentrations of LNP: 62.5 μg / mL, 125 μg / mL, and 250 μg / mL. The percentage change in LDH release was measured at each concentration. The percentage data shown in the figure indicate the LDH release level of different LNPs at each concentration. All LDH release values ​​were negative (e.g., -7.51%, -4.01%, etc.), which generally indicates a reduction (inhibition) in LDH release. Lower values ​​indicate a smaller effect on LDH release, or lower cytotoxicity.

[0308] All experimental groups showed negative LDH release values ​​at all concentrations, indicating that these LNPs had little or no significant effect on LDH release in cells, suggesting low cytotoxicity. The three LNPs (I-29, I-39, and SM-102) showed slightly different effects on LDH release at different concentrations, but the overall differences were small. This may indicate that the cytotoxicity of these LNPs was not significantly different. Furthermore, the negative values ​​of LDH release did not change much with increasing concentration (from 62.5 μg / mL to 250 μg / mL), indicating that these LNPs did not significantly increase cytotoxicity within the measured concentration range.

[0309] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A lipid nanoparticle characterized in that, The lipid nanoparticle comprises an amino lipid compound, the amino lipid compound is compound 1-39, and the structure of compound 1-39 is as follows: 。 2. Lipid nanoparticle according to claim 1, characterized in that, The lipid nanoparticle further comprises an auxiliary lipid, a structural lipid, a polyethylene glycol-lipid, and a molar ratio of the amino lipid compound, the auxiliary lipid, the structural lipid, and the polyethylene glycol-lipid is 28-60:5-42:15.5-53.5:0.5-3.

5.

3. Lipid nanoparticle according to claim 2, characterized in that, The auxiliary lipid comprises a phospholipid, and the phospholipid comprises at least one of distearoylphosphatidylcholine, oleoylphosphatidylethanolamine, dioleoylphosphatidylcholine, dioleoylphosphatidylserine, 1,2-dioctadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, 1.2-dipalmitoyl-sn-glycero-3'-O-4'-(N,N,N-trimethyl)homoserine, and hemophospholipid.

4. The lipid nanoparticle according to claim 2, characterized in that, The structural lipid comprises a sterol, and the sterol comprises at least one of cholesterol, cholesteryl ester, a sterol hormone, a sterol vitamin, a bile acid, cholestanol, ergosterol, and beta-sitosterol.

5. The lipid nanoparticle according to claim 2, characterized in that, The polyethylene glycol-lipid is polyethylene glycol-distearoylphosphatidylethanolamine, and an average molecular weight of polyethylene glycol in the polyethylene glycol-lipid is 2000-5000.

6. A method of manufacture characterised by, A method for preparing an mRNA-lipid nanoparticle complex comprising the lipid nanoparticle according to any one of claims 1-5, and steps of the method comprise: S1, synthesizing an amino lipid compound; S2, respectively dissolving an auxiliary lipid, a structural lipid, a polyethylene glycol-lipid, and the amino lipid compound in an organic solvent to obtain an auxiliary lipid solution, a structural lipid solution, a polyethylene glycol-lipid solution, and an amino lipid compound solution, and mixing the auxiliary lipid solution, the structural lipid solution, the polyethylene glycol-lipid solution, and the amino lipid compound solution to obtain an alcohol phase, wherein the organic solvent comprises at least one of anhydrous ethanol, chloroform, and ethyl acetate; S3, mixing an mRNA stock solution, a pH regulator, and a solvent to obtain an aqueous phase, wherein the pH regulator comprises at least one of a phosphate buffer, a sodium acetate buffer, and a sodium bicarbonate buffer, and the solvent comprises at least one of anhydrous ethanol, pure water, and dichloromethane; S4, mixing the alcohol phase and the aqueous phase first, and then adding a buffer to obtain an mRNA-lipid nanoparticle complex.

7. The production method according to claim 6, wherein The amino lipid is compound 1-39, and the step S1 comprises: S1.1, adding an activating agent to compound 34-1 and compound 34-2 to perform an acyl chloride reaction, and then placing in an alkaline reagent to obtain compound 34-3, wherein the activating agent comprises at least one of dichloro sulfoxide, oxalyl chloride, sulfenyl chloride, and cyanogen chloride, and the alkaline reagent comprises at least one of 4-dimethylaminopyridine, triethylamine, N,N-diisopropylethylamine, pyridine, potassium carbonate, potassium hydroxide, sodium carbonate, and N-methylmorpholine. S1.2, the compound 34-3 and the compound M34 are placed in the basic reagent to perform an amidation reaction to obtain a compound 34-4; S1.3, the compound 39-1 and the compound 39-2 are placed in the basic reagent to perform a substitution reaction to obtain 39-3; S1.4, the compound 34-4 and the compound 39-3 are placed in the basic reagent to perform a cross-linking reaction to obtain a compound 1-39; The structural formula of the compound is as follows: 、 、 、 、 、 、 、 。

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