Esterase-responsive ionizable cationic lipid and application thereof in improving delivery efficiency of nucleic acid drugs

Abstract

The invention provides esterase-responsive ionizable cationic lipid and application thereof in improving the delivery efficiency of nucleic acid drugs. The lipid provided by the invention is electrically neutral under physiological conditions, has excellent biological safety, is positively charged in a meta-acidic endosome / lysosome environment, promotes escape of a carrier and a nucleic acid drug through a proton sponge effect, is hydrolyzed under the action of intracellular esterase and then is converted into an electrically neutral or electronegative compound, and is used for preparing a drug carrier. Dissociation with nucleic acid drugs with negative charges is completed, and the biological functions of the nucleic acid drugs are played to the maximum extent. Therefore, the lipid provided by the invention can be used for efficient delivery of in-vivo and in-vitro nucleic acid drugs, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterial preparation technology, specifically relating to an esterase-responsive ionizable cationic lipid and its application in improving the efficiency of nucleic acid drug delivery. Background Technology

[0002] Nucleic acid drugs (also known as nucleotide drugs) are a class of ribonucleic acids with different functions. They primarily regulate protein expression at the gene level to achieve therapeutic goals. Common nucleic acid drugs include mRNA, siRNA, microRNA, ASOs, and DNA. Their application has expanded from initially treating rare diseases to covering multiple fields such as chronic diseases, oncology, and viral infections, demonstrating revolutionary therapeutic potential. For example, mRNA vaccines... and The approval of mRNA and its significant clinical efficacy in combating novel coronavirus infection have made mRNA one of the most watched research hotspots in the biomedical field. However, due to the problems of nucleic acid drugs being easily degraded by nucleases, their large molecular weight and negative charge making it difficult for them to penetrate cell membranes, resulting in low delivery efficiency, they cannot be directly used for disease treatment and need to rely on vector delivery.

[0003] Lipid nanoparticles (LNPs) are currently the primary carriers for nucleic acid drug delivery, composed of cationic lipids, polyethylene glycol lipids, cofactor phospholipids, and cholesterol. Cationic lipids are the most important energy-providing excipients in LNPs, binding to negatively charged mRNA and efficiently encapsulating nucleic acid drugs. These lipids are generally classified into permanently positively charged cationic lipids and ionizable cationic lipids. Permanently positively charged cationic lipids exhibit strong cytotoxicity and immunogenicity, activating Toll-like receptors and inducing inflammatory responses. Furthermore, under physiological conditions, they readily bind to negatively charged serum proteins, leading to the degradation and clearance of LNPs containing these lipids, hindering the delivery of nucleic acid drugs to their target sites. In contrast, ionizable cationic lipids are electrically neutral under physiological conditions, significantly reducing their cytotoxicity and off-target effects. They protonate only at pH values ​​below their pKa. + It is continuously pumped into the endosome / lysosome; in order to maintain electrolyte balance, Cl - It also pumps large amounts of endosomes / lysosomes, leading to increased osmotic pressure, known as the "proton sponge effect," which ultimately causes endosomes / lysosomes to rupture, releasing nucleic acid drugs and delivery systems. This is the core mechanism of lysosomal escape from nucleic acid drugs and delivery systems. Permanently positively charged cationic lipids, however, do not undergo a proton change process, significantly reducing their efficiency in delivering nucleic acid drugs.

[0004] However, existing research results indicate that prolonged retention of ionizable cationic lipids can lead to additional inflammatory reactions at the injection site. For example, Chinese patent document CN110638759A discloses an mRNA preparation for in vitro transfection and in vivo delivery. In this invention, the lipid molecules used to bind mRNA are ionizable cationic lipids and permanently positively charged cationic lipids. Permanently positively charged cationic lipids have strong cytotoxicity and immunogenicity. Furthermore, the ionizable cationic lipids contained in this patent are not degradable in vivo, which may also cause potential toxicity.

[0005] Therefore, developing biodegradable ionizable cationic lipids is of paramount importance in the subsequent development of cationic lipids. Summary of the Invention

[0006] To address the aforementioned technical challenges, the esterase-responsive ionizable cationic lipids provided by this invention exhibit excellent biocompatibility under physiological conditions. This is achieved through a strategy that hydrolyzes these lipids into negatively charged compounds via intracellular esterases, enabling their dissociation from negatively charged nucleic acid drugs. Compared to commercially available ionizable cationic lipids, these lipids further enhance nucleic acid drug delivery efficiency, maximizing their biological functions.

[0007] To achieve the above-mentioned technical effects, the technical solution adopted by the present invention is as follows:

[0008] An esterase-responsive cationic lipid is present, which is electrically neutral under physiological conditions and positively charged in a slightly acidic endosome / lysosomal environment. It is hydrolyzed by intracellular esterases into an electrically neutral or negatively charged compound. The structural formula of the compound is as follows:

[0009]

[0010] Wherein, n1 and n2 represent the number of CH2, and n is a natural number; n1 is 1-2, and n2 is 1-4.

[0011] X is an alkylamine or a hydroxyalkylamine; if it is an alkylamine, it is selected from... One of them;

[0012] If it is a hydroxyalkylamine, then the structure is: The n represents the number of CH2, where n is a natural number, n4 is 2-5, and n5 is 0-2.

[0013] R represents a hydrocarbon chain, and N represents the number of hydrocarbon chains. N is a natural number, and N can be 1 or 2.

[0014] Preferably, the R structure of the compound contains 0-2 -C=C- or -C≡C-.

[0015] Preferably, when both N values ​​are 1, the length of each chain is 6-16 C; when one N value is 2 and the other N value is 1 or 2, the length of each chain is 4-10 C.

[0016] Preferably, the esterase-responsive cationic lipid molecule is a compound selected from one of the following structural formulas:

[0017]

[0018] The esterase-responsive ionizable cationic lipids of the present invention, after protonation under acidic conditions (e.g., acidic conditions of endosomes / lysosomes), generate negatively charged carboxylic acid groups through autocatalytic hydrolysis of ester bonds. Taking the above compound (7) as an example, its charge reversal process is shown in the following formula:

[0019]

[0020] The present invention also provides a method for preparing the esterase-responsive ionizable cationic lipid, comprising the following steps:

[0021] S1. Dissolve the fatty acid or its corresponding fatty acid acyl chloride in an organic solvent, react with p-hydroxybenzyl alcohol to form intermediate 1, and then purify it; if the N value of one hydrocarbon chain R is 2 and the other is 1 (such as compounds 2, 5, 8, 11), they need to be prepared separately.

[0022] S2. Dissolve the purified intermediate product 1 in an organic solvent, then dissolve the bromoalkyl acyl chloride compound in an organic solvent and add it to the reaction system to react. After purification, the purified intermediate product 2 is obtained. If the N value of one hydrocarbon chain R is 2 and the other is 1, the intermediate products 2 prepared separately need to be mixed in a molar ratio of 1:1 before the reaction.

[0023] S3. Dissolve 3,4-dihydroxy aromatic aldehyde or 3,4-dihydroxy aromatic aliphatic aldehyde in an organic solvent, simultaneously alkalize the reaction system, add iodine compound as a catalyst, and organic solvent containing intermediate product 2 in sequence. Stir the reaction at 10-60℃ for 2-24h, and obtain intermediate product 3 after purification.

[0024] S4. Dissolve the compound containing the secondary amine in an organic solvent, add an organic solvent containing intermediate product 3 obtained in step S3, protect with N2, stir at room temperature for 1-8 h, then adjust the pH of the reaction system, add a reducing agent, and stir the reaction at 0-60℃ for 2-24 h to obtain the final product.

[0025] Preferably, the compound containing a secondary amine in step S4 includes: dimethylamine, N-ethylmethylamine, ethylmethylamine, N-methyl-2-hydroxyethylamine, 2-(ethylamino)ethanol, N-propylethanolamine, 3-(methylamino)-1-propanol, 4-(methylamino)-1-butanol, 5-(methylamino)-1-pentanol, 3-(ethylamino)-1-propanol, 4-(ethylamino)-1-butanol, 5-(ethylamino)-1-pentanol, 3-(propylamino)-1-propanol, 4-(propylamino)-1-butanol, and 5-(propylamino)-1-pentanol.

[0026] Preferably, the fatty acids or their corresponding fatty acid acyl chlorides mentioned in steps S1, S2, S3, and S4 include: 2-butyloctanoic acid, 2-hexyloctanoic acid, 2-hexylnonanoic acid, 2-hexyldecanoic acid, 2-heptylnonanoic acid, 2-octyldecanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, undecanoic acid, 2-hexenoic acid, 2-tridecenoic acid, cis-9-hexadecenoic acid, linoleic acid, oleic acid, octadecyl-9-alkynic acid; and the acyl chlorides corresponding to the above acids.

[0027] Preferably, the organic solvent mentioned in steps S1, S2, S3, and S4 is one of methanol, dichloromethane, N,N-dimethylformamide (DMF), tetrahydrofuran, and dimethyl sulfoxide. The bromoalkyl acyl chloride compound mentioned in step S2 is one of bromoacetyl chloride, 3-bromopropionyl chloride, 4-bromobutyryl chloride, and 5-bromopentanoyl chloride; the 3,4-dihydroxy aromatic aldehyde or 3,4-dihydroxy aromatic aliphatic aldehyde mentioned in step S3 includes: 3,4-dihydroxybenzaldehyde, 2-(3,4-dihydroxyphenyl)acetaldehyde, and 3-(3,4-dihydroxyphenyl)propionaldehyde. The reducing agent mentioned in step S4 is one of sodium borohydride, sodium triacetoxyborohydride, and sodium cyanoborohydride.

[0028] The present invention also provides the application of the esterase-responsive ionizable cationic lipid as described in claim 1 in the preparation of nucleic acid drug carriers.

[0029] The present invention also provides a nucleic acid drug delivery system, comprising lipid nanoparticle carriers (LNPs) and a nucleic acid drug loaded within the carriers, wherein the lipid nanoparticle carriers are formed by the esterase-responsive ionizable cationic lipids, auxiliary lipids and PEG lipids; the molar ratio of esterase-responsive ionizable cationic lipids: auxiliary lipids: PEG lipid molecules is 30-80: 10-70: 0.5-2.

[0030] Preferably, the nucleic acid drug is one of DNA, siRNA, mRNA, microRNA, shRNA, and ASOs.

[0031] In this invention, the esterase-responsive ionizable cationic lipids are protonated under acidic preparation conditions (pH = 3.0-6.0), becoming positively charged and capable of efficiently encapsulating negatively charged nucleic acid drugs. The delivery system composed of these lipids and other lipids is dialyzed to a pH of approximately 7, becoming electrically neutral or negatively charged. Under physiological conditions, this delivery system also remains electrically neutral or negatively charged. After endocytosis and escape from endosomes / lysosomes, it can be gradually degraded by esterases, resulting in complete dissociation of the nucleic acid drug from the ionizable cationic lipids and improving its delivery efficiency.

[0032] Compared with existing technologies, the technical advantages of this invention are as follows: The lipids provided by this invention exhibit excellent biocompatibility under physiological conditions. In endosomes / lysosomes, they facilitate the escape of carriers and nucleic acid drugs through the "proton sponge effect," and subsequently undergo charge changes under esterase hydrolysis, thereby dissociating from the nucleic acid drugs and maximizing their biological functions. Simultaneously, the biodegradable nature of these lipids avoids the potential toxicity caused by the accumulation of ionizable cationic lipids. Therefore, this delivery system not only solves the biocompatibility problem of nucleic acid drug delivery systems but also significantly improves gene transfection efficiency and enhances the efficacy of treatments for related diseases. Attached Figure Description

[0033] Figure 1 This is a graph showing the transfection data from the orthogonal experimental ratio screening in Example 4.

[0034] Figure 2 Particle size and zeta potential diagrams of the nanoparticles prepared in Examples 1-5;

[0035] Figure 3 The graph shows the changes in Zeta potential during the hydrolysis of nanoparticles prepared in Examples 1 and 4 by esterase.

[0036] Figure 4 Electron micrographs of the morphological changes of the nanoparticles prepared in Examples 1 and 4 during esterase hydrolysis;

[0037] Figure 5 The graph shows the cytotoxicity results of the nanoparticles prepared in Examples 1-5;

[0038] Figure 6 The graph shows the in vitro transfection efficiency of the nanoparticles prepared in Examples 1, 2 and 4 in B16F10 cells.

[0039] Figure 7 The results of the escape of nucleic acid drugs delivered by nanoparticles prepared in Examples 1, 2 and 4 from endosomes / lysosomes;

[0040] Figure 8 Gel electrophoresis images to simulate the dissociation process of nanoparticles and mRNA prepared in Examples 1 and 4 by esterases in the cytoplasmic environment;

[0041] Figure 9 Flow cytometry results of DC2.4 cells maturated with nanoparticles prepared in Examples 1 and 4;

[0042] Figure 10 In vivo distribution of the nanoparticles prepared in Examples 1 and 4 in mice;

[0043] Figure 11 The transfection efficiency of the nanoparticles prepared in Example 4 in mice is shown in the graph.

[0044] Figure 12 This is a diagram showing the results of the nanoparticles prepared in Example 4 activating the maturation of lymph node DCs cells;

[0045] Figure 13 Nanoparticles prepared in Example 4 activate CD8 in tumors + Results of T cell analysis;

[0046] Figure 14 The image shows the antitumor effect of the nanoparticles prepared in Example 4. Detailed Implementation

[0047] The present invention will be further explained below with reference to specific embodiments. However, it should be noted that the following embodiments are only used to explain the present invention and cannot be used to limit the present invention. All technical solutions that are the same as or similar to the present invention are within the protection scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

[0048] Representative examples of cofactor lipids include, but are not limited to: phosphatidylcholine (PC), hydrogenated soybean phosphatidylcholine (HSPC), dilauroyl phosphatidylcholine (DLPC), disqualyl phosphatidylcholine (DEPC), dioleoyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), palmitoyl oleoyl phosphatidylcholine (POPC), distearyl phosphatidylcholine (DSPC), dipalmitoyl oleoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylethanolamine (DPPE), dioleoyl phosphatidylethanolamine (DOPE), dimyristoyl phosphatidylethanolamine (DMPE), and distearyl phosphatidylethanolamine (...). The following lipids are used: DSPE, phosphatidylserine (PS), dipalmitoylphosphatidylserine (DPPS), phosphatidylinositol (PI), dioleoylphosphatidylglycerol (DOPG), palmitoyloleoylphosphatidylglycerol (POPG), distearylphosphatidylglycerol (DSPG), dipalmitoyloleoylphosphatidylglycerol (DPPG), trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP-Br), trimethyl-2,3-dioleenoyloxypropylammonium chloride (DOTAP-Cl), 3β-[N-(N',N'-dimethylaminoethyl)aminoformyl]cholesterol (DC-CHOL), sphingomyelin (SM), and cholesterol (CHOL). One of these lipids can be used alone or in combination.

[0049] Representative examples of PEG lipids include, but are not limited to: dipalmitoylphosphatidylethanolamine-methoxy polyethylene glycol 2000 (DPPE-PEG2000), distearate-phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000), and PEG2000-Ceramide-C14.

[0050] Example 1: Synthesis of Compound 2

[0051] S1. Dissolve 0.769 g of 2-hexyldecanoic acid in 10 mL of dichloromethane, add 237 μL of thionyl chloride and react for 2 h to prepare 2-hexyldecanoic acid acyl chloride. Remove the solvent and redissolve in 5 mL of tetrahydrofuran for later use. Dissolve 0.420 g of p-hydroxybenzyl alcohol in 5 mL of tetrahydrofuran, add 0.55 mL of triethylamine, and then add the prepared 2-hexyldecanoic acid acyl chloride solution in tetrahydrofuran. After the reaction, intermediate 1-1 is formed. Remove the solvent and purify by column chromatography (petroleum ether: ethyl acetate = 4:1). Take 0.475 g of nonanoic acid to prepare acyl chloride in the same way and react with p-hydroxybenzyl alcohol to form intermediate 1-2.

[0052] S2. Redissolve 0.634 g of purified intermediate 1-1 in 5 mL of dichloromethane and set aside. Add 350 μL of triethylamine. Then react with a solution containing 212 μL of 3-bromopropionyl chloride in dichloromethane (total 5 mL) for 2 h to form intermediate 2-1. Remove the solvent and purify by column chromatography (petroleum ether: ethyl acetate = 4:1). Take 0.463 g of intermediate 1-2 and react it in the same way to obtain intermediate 2-2. Take 0.627 g of intermediate 2-1 and 0.503 g of intermediate 2-2 and redissolve them in 10 mL of DMF (molar ratio = 1:1) to prepare a mixed solution of intermediate 2.

[0053] S3. Dissolve 0.166 g of 3,4-dihydroxybenzaldehyde in 10 mL of DMF, add 0.522 g of potassium carbonate and stir. Then add 0.100 g of potassium iodide and the intermediate product 2 solution obtained in step S2. Stir the reaction at 40 °C for 8 h. After quenching the reaction, extract with ethyl acetate and purify by column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain intermediate product 3.

[0054] S4. Dissolve 0.090 g of N-methyl-2-hydroxyethylamine in 10 mL of DMF, add 0.349 g of intermediate 3 in 5 mL of DMF solution, stir at room temperature for 2 h under N2 protection, then adjust the pH of the reaction system to 5-6 with glacial acetic acid, add 0.127 g of sodium triacetoxyborohydride, and stir at room temperature for 4 h. After extraction with ethyl acetate, purify by column chromatography (eluting first with petroleum ether:ethyl acetate = 2:1, then with dichloromethane:methanol = 10:1) to obtain compound MAE-3, with the following structure:

[0055]

[0056] Example 2 Synthesis of Compound 3

[0057] S1. Dissolve 1.54 g of 2-hexyldecanoic acid in 10 mL of dichloromethane, add 237 μL of thionyl chloride and react for 2 h to prepare 2-hexyldecanoic acid acyl chloride. Remove the solvent and redissolve in 10 mL of tetrahydrofuran for later use. Dissolve 0.819 g of p-hydroxybenzyl alcohol in 10 mL of tetrahydrofuran, add 1.1 mL of triethylamine, and then add the tetrahydrofuran solution of 2-hexyldecanoic acid acyl chloride. After the reaction, intermediate 1 is formed. Remove the solvent and purify by column chromatography (petroleum ether: ethyl acetate = 4:1).

[0058] S2. The purified intermediate 1 was dissolved in 10 mL of dichloromethane with 700 μL of triethylamine, and reacted with a solution of bromoacetyl chloride (350 μL) in dichloromethane (10 mL) for 2 h to form intermediate 2. The solvent was removed and purified by column chromatography (petroleum ether: ethyl acetate = 4:1).

[0059] S3. Dissolve 0.182 g of 2-(3,4-dihydroxyphenyl)acetaldehyde in 10 mL of DMF, add 0.522 g of potassium carbonate and stir. Then add 0.090 g of sodium iodide and a 10 mL solution of DMF containing 1.281 g of intermediate 2. Stir the reaction at 40 °C for 10 h. After extraction with ethyl acetate, purify by column chromatography (petroleum ether: ethyl acetate = 2:1).

[0060] S4. Dissolve 0.107 g of 3-(methylamino)-1-propanol in 10 mL of tetrahydrofuran, add 0.383 g of a tetrahydrofuran solution of intermediate 3 (5 mL), and stir at room temperature for 2 h under N2 protection. Then, adjust the pH of the reaction system to 5-6 with glacial acetic acid, add 0.038 g of sodium cyanoborohydride, and stir at room temperature for 10 h. After quenching the reaction, extract with ethyl acetate and purify by column chromatography (eluting first with petroleum ether:ethyl acetate = 2:1, then with dichloromethane:methanol = 10:1) to obtain compound EAE-4, with the following structure:

[0061]

[0062] Example 3 Synthesis of Compound 5

[0063] S1. Dissolve 0.685 g of 2-hexyloctanoic acid in 10 mL of dichloromethane, add 237 μL of thionyl chloride and react for 2 h to prepare 2-hexyloctanoic acid chloride. Remove the solvent and redissolve in 5 mL of tetrahydrofuran for later use. Dissolve 0.420 g of p-hydroxybenzyl alcohol in 5 mL of tetrahydrofuran, add 0.55 mL of triethylamine, and then add the tetrahydrofuran solution of 2-hexyloctanoic acid chloride. After the reaction, intermediate 1-1 is formed. Remove the solvent and purify by column chromatography (petroleum ether: ethyl acetate = 4:1). Take 0.348 g of hexanoic acid to prepare the acyl chloride in the same way and react with p-hydroxybenzyl alcohol to form intermediate 1-2.

[0064] S2. Redissolve 0.585g of purified intermediate 1-1 in 5mL of dichloromethane and set aside. Add 350μL of triethylamine. Then react with 212μL of 3-bromopropionyl chloride in 5mL of dichloromethane for 4h to form intermediate 2-1. Remove the solvent and purify by column chromatography (petroleum ether: ethyl acetate = 4:1). Take 0.389g of intermediate 1-2 and react it in the same way to obtain intermediate 2-2. Take 0.592g of intermediate 2-1 and 0.450g of intermediate 2-2 and redissolve them in 10mL of DMF (molar ratio = 1:1) to prepare a mixed solution of intermediate 2.

[0065] S3. Dissolve 0.182 g of 2-(3,4-dihydroxyphenyl)acetaldehyde in 10 mL of DMF, add 0.522 g of potassium carbonate and stir, then add 0.100 g of potassium iodide and a mixed solution containing intermediate 2 successively. Stir the reaction at 40 °C for 12 h. Extract with ethyl acetate and purify by column chromatography (petroleum ether: ethyl acetate = 2:1).

[0066] S4. Dissolve 0.115 g of N-methylethylamine hydrochloride and 250 μL of triethylamine in 10 mL of tetrahydrofuran. Add 0.327 g of a tetrahydrofuran solution of intermediate 3 (5 mL). Under N2 protection, stir at room temperature for 2 h. Then, adjust the pH of the reaction system to 5-6 with glacial acetic acid, add 0.038 g of sodium cyanoborohydride, and stir at room temperature for 4 h. After quenching the reaction, extract with ethyl acetate and purify by column chromatography (eluting first with petroleum ether:ethyl acetate = 2:1, then with dichloromethane:methanol = 10:1) to obtain compound EMA-3, with the following structure:

[0067]

[0068] Example 4 Synthesis of Compound 7

[0069] S1. Dissolve 1.68 g of linoleic acid in 10 mL of dichloromethane, add 660 μL of oxaloyl chloride and N,N-dimethylformamide (DMF, 185 μL), and react for 1 h to prepare linoleic acid acyl chloride. Remove the solvent and redissolve in 10 mL of tetrahydrofuran for later use. Dissolve 0.819 g of p-hydroxybenzyl alcohol in 10 mL of tetrahydrofuran, add 1.1 mL of triethylamine, and then add the tetrahydrofuran solution of linoleic acid acyl chloride. After the reaction, intermediate 1 is formed. Remove the solvent and purify by column chromatography (petroleum ether: ethyl acetate = 4:1).

[0070] S2. The purified intermediate 1 (1.114 g) was dissolved in 10 mL of dichloromethane with 700 μL of triethylamine. The solution was then reacted with a solution of bromoacetyl chloride (350 μL) in dichloromethane (10 mL) for 2 h to form intermediate 2. The solvent was removed and the product was purified by column chromatography (petroleum ether: ethyl acetate = 4:1).

[0071] S3. Dissolve 0.166 g of 3,4-dihydroxybenzaldehyde in 10 mL of DMF, add 0.522 g of potassium carbonate and stir. Then add a 10 mL solution of DMF containing 0.100 g of potassium iodide and 1.107 g of intermediate 2. Stir the reaction at 40 °C for 4 h. After extraction with ethyl acetate, purify by column chromatography (petroleum ether: ethyl acetate = 2:1).

[0072] S4. Dissolve 0.098 g of dimethylamine hydrochloride and 250 μL of triethylamine in 10 mL of DMF, add 0.397 g of intermediate 3 in 5 mL of DMF solution, stir at room temperature for 2 h under N2 protection, then adjust the pH of the reaction system to 5-6 with glacial acetic acid, add 0.127 g of sodium triacetoxyborohydride, and stir at room temperature for 4 h. After quenching the reaction, extract with ethyl acetate and purify by column chromatography (eluting first with petroleum ether:ethyl acetate = 2:1, then with dichloromethane:methanol = 10:1) to obtain compound DMA-2, with the following structure:

[0073]

[0074] Example 5 Synthesis of Compound 10

[0075] S1. Dissolve 1.27 g of 2-tridecenoic acid in 10 mL of dichloromethane, add 660 μL of oxaloyl chloride and N,N-dimethylformamide (DMF, 185 μL), and react for 1 h to prepare 2-tridecenoic acid acyl chloride. Remove the solvent and redissolve in 10 mL of tetrahydrofuran for later use. Dissolve 0.819 g of p-hydroxybenzyl alcohol in 10 mL of tetrahydrofuran, add 1.1 mL of triethylamine, and then add a tetrahydrofuran solution of linoleic acid acyl chloride. After the reaction, intermediate 1 is formed. Remove the solvent and purify by column chromatography (petroleum ether: ethyl acetate = 4:1).

[0076] S2. The purified intermediate 1 (1.114 g) was dissolved in 10 mL of dichloromethane with 700 μL of triethylamine. The solution was then reacted with 4-bromobutyryl chloride (487 μL) in dichloromethane (10 mL) for 2 h to form intermediate 2. The solvent was removed and the product was purified by column chromatography (petroleum ether: ethyl acetate = 4:1).

[0077] S3. Dissolve 0.199 g of 3-(3,4-dihydroxyphenyl)propionaldehyde in 10 mL of DMF, add 0.522 g of potassium carbonate and stir. Then add 0.245 g of diphenyliodonium iodide and a 10 mL solution of DMF containing 1.107 g of intermediate 2. Stir the reaction at 40 °C for 16 h. After extraction with ethyl acetate, purify by column chromatography (petroleum ether: ethyl acetate = 2:1).

[0078] S4. Dissolve 0.132 g of diethylamine hydrochloride and 250 μL of triethylamine in 10 mL of methanol, and add 0.364 g of intermediate 3 in 10 mL of methanol solution. Under N2 protection, stir at room temperature for 2 h. Then add 0.023 g of sodium borohydride and stir at room temperature for 2 h. Remove the solvent and purify by column chromatography (eluting first with petroleum ether:ethyl acetate = 2:1, then with dichloromethane:methanol = 10:1) to obtain compound DEA-2, with the following structure:

[0079]

[0080] Unless otherwise specified, the following experimental examples all use commercially available ionizable cationic lipid Dlin-MC3-DMA (MC3) nanoparticles prepared according to the same formulation as the control group. Unless otherwise specified, the culture medium used in the cell experiments was RPMI 1640 serum-free medium.

[0081] Experimental Example 1: Preparation of lipid nanoparticles containing esterase-responsive cationic lipids

[0082] Experimental Procedure: Nanoparticles were prepared according to Examples 1-5: Esterase-responsive cationic lipids, auxiliary lipids, and PEG lipids were dissolved in ethanol according to the prescribed amounts to obtain a lipid-containing alcohol phase. mRNA was dissolved in citrate-sodium citrate buffer (pH=4, final concentration 50mM, RNase-free) to obtain an aqueous phase containing 0.1 mg / mL mRNA. Microfluidic mixing was used for preparation: the above lipid-containing alcohol and aqueous phases were mixed at a volume ratio of 1:3, with the alcohol phase flow rate:water phase flow rate set at 1:3, in a microfluidic nanoparticle preparation system. The mixture was then dialyzed with phosphate-buffered saline (PBS, pH=7.4) until the pH reached approximately 7 to obtain a lipid nanoparticle solution.

[0083] Experimental Example 2: Screening of Optimal Formulation

[0084] Experimental Procedure: Enhanced green fluorescent protein (EGFP) mRNA is a common reporter mRNA. After cell transfection, it expresses EGFP and emits bright green fluorescence upon excitation. Therefore, observing the fluorescence intensity of transfected cells can reflect the transfection efficiency of nanoparticles at the cellular level. Taking Example 4 (compound 7) as an example, a three-factor, four-level orthogonal experiment was set up to screen the optimal ratio for B16F10 cell transfection experiments. The three factors included: esterase-responsive ionizable cationic lipids, auxiliary lipids (taking the combination of DSPC and CHOL as an example, examining the change in the amount of DSPC, with CHOL as a supplement, to make up to a total molar ratio of 100), and PEG lipids (taking DMG-PEG2000 as an example), with values ​​representing molar ratios. Simultaneously, a blank nanoparticle solution without EGFP mRNA was used as the control group.

[0085]

[0086]

[0087] Results analysis is as follows: Figure 1As shown, based on the mean main effect analysis, the optimal ratio for this orthogonal experiment is esterase-responsive ionizable cationic lipid: auxiliary lipid (DSPC+CHOL): DMG-PEG2000 molar ratio = 60:38.5(10+28.5):1.5. This ratio is used for the proportion of nanoparticle components in other application examples.

[0088] Characterization of the charge and morphology of nanoparticles in Experiment Example 3

[0089] Experimental Procedure: Lipid nanoparticles were prepared using the formulations from Examples 1-5 and MC3, with a molar ratio of 60:38.5 (10+28.5):1.5 for esterase-responsive ionizable cationic lipids, auxiliary lipids (DSPC+CHOL), and DMG-PEG2000. Particle size and zeta potential were measured. The particle size and zeta potential of the lipid nanoparticles were determined using a dynamic light scattering instrument at 25°C. Each measurement was repeated three times, and the average value was taken.

[0090] like Figure 2 As shown, the nanoparticles have a particle size of 80-260 nm and an electric potential of -1.5 to -9 mV.

[0091] Example 4: Characterization of charge and morphological changes after esterase response

[0092] Experimental Procedure: Nanoparticles containing ovalbumin (OVA) mRNA were prepared according to Example 2, using Compound 2 (Compound 2) and Compound 7 (Compound 4), with MC3-DMA as a control. The nanoparticles were mixed thoroughly with 100 U / mL porcine liver esterase (ES; Sigma-Aldrich, catalog number: E3019-3.5KU) solution at a ratio of 3:1 (v:v). The samples were placed in a 37℃ constant-temperature shaker according to the hydrolysis time, and removed simultaneously after 0 h, 2 h, 4 h, and 10 h of hydrolysis. Particle size and zeta potential were immediately measured. Transmission electron microscopy (TEM) characterization (4 h hydrolysis) was then performed.

[0093] like Figure 3 As shown, under the action of esterase, the potential of the nanoparticles gradually decreased over time, and the negative charge decreased to varying degrees after 10 hours. (Results from transmission electron microscopy) Figure 4 The results showed that after hydrolysis by esterase, the nanoparticles increased in size to varying degrees, and their morphology changed from a spherical structure with uniform electron density and smooth edges to a non-uniform electron density structure with numerous "depressions" and "cracks" (indicated by white arrows). This indicates that the charge decreased and the carrier structure was destroyed after hydrolysis by esterase, which facilitates the dissociation of nucleic acid drugs and reduces carrier toxicity. In contrast, the MC3 nanoparticles in the control group did not show any changes in charge or morphology.

[0094] Experimental Example 5: MTT assay for the cytotoxicity of nanoparticles

[0095] Experimental procedure: B16F10 cells were cultured at a rate of 4 × 10⁻⁶. 3 Cells were seeded per well in 96-well plates and incubated in a CO2 incubator for 24 h. The lipid nanoparticles from Examples 1-5 were diluted with culture medium to final concentrations of 75 nmol / mL, 150 nmol / mL, and 300 nmol / mL (based on the concentration of ionizable cationic lipids) to form working solutions, which served as experimental groups. An equal volume of culture medium was added to the control group. After incubation for another 24 h, the culture medium was replaced, and 10 μL of MTT solution (MTT concentration 5 mg / mL) was added to each well. The plates were then incubated in a CO2 incubator for another 4 h. The original culture medium was discarded, and 150 μL of DMSO was added to each well. The plates were shaken to dissolve all crystals, and the absorbance at 490 nm was read using a microplate reader. Six replicates were performed for each sample group, and the average value was taken. Cell viability (%) was expressed as the absorbance value of the experimental group divided by the absorbance value of the control group.

[0096] Figure 5 The data provided are for the cytotoxicity of the nanoparticles composed in Examples 1-5. Within the normal concentration range for use of nanoparticles, the cytotoxicity is low.

[0097] Experiment Example 6: In vitro transfection experiment

[0098] 1. Experimental samples: Nanoparticles mainly prepared according to Examples 1, 2 and 4, and control MC3 nanoparticles;

[0099] 2. Experimental Procedure: B16F10 cells (mouse skin melanoma cells) were cultured at a rate of 1×10⁻⁶. 5 Cells were seeded per well in 24-well cell culture plates containing cell culture slides. After 24 hours of incubation, the medium was replaced with medium containing EGFP mRNA nanoparticles (100 nmol / mL), and incubated in a CO2 incubator for 4 hours. The original medium was discarded, and the cells were washed twice with PBS, then incubated for another 24 hours with medium containing 10% fetal bovine serum. After incubation, the cells were washed twice with PBS, and then fixed with 4% paraformaldehyde at room temperature for 15 minutes. The fixative was discarded, and the cells were washed with PBS and observed under a fluorescence microscope. Cells from six random fields of view were selected from all samples, and the fluorescence intensity was semi-quantitatively analyzed using ImageJ software.

[0100] 3. Experimental results: such as Figure 6 As shown. The nanoparticles composed of Compound 1 (Compound 2), Compound 2 (Compound 3), and Compound 4 (Compound 7) showed higher fluorescence intensity in B16F10 cells when expressing EGFP protein than the control group containing MC3 nanoparticles, indicating that these nanoparticles have higher in vitro transfection efficiency.

[0101] Experiment 7: Observation of endosome / lysosomal escape of nucleic acid drugs using laser confocal microscopy

[0102] Experimental Procedure: Compounds 1 (Compound 2), 2 (Compound 3), and 4 (Compound 7) were selected. FAM-N (French-N) fluorescently labeled nucleic acid sequences were used to replace mRNA, and FAM-N-loaded nanoparticles were prepared following the procedures in Example 2. DC2.4 cells (dendritic cells) were used at a concentration of 1 × 10⁻⁶. 5 Each well was seeded with 100 nanoparticles (100 nmol / mL) into a glass dish. After 24 h of incubation, the medium was replaced with one well of FAM-N nanoparticles (100 nmol / mL), and the dishes were incubated in a CO2 incubator for 4 h. LysoTracker was added 1 h before the end of the incubation period. TM Deep Red (final concentration 50 nM) was added 15 min before the end of incubation, followed by Hoechst 33342 staining reagent. After incubation, the samples were washed with PBS, replaced with culture medium, and observed under a laser confocal microscope. Nanoparticles containing Dlin-MC3-DMA (a commercially available ionizable cationic lipid) were prepared according to the same formulation as a control group. FAM was excited at 488 nm and exhibited green fluorescence; the endosome / lysosomal dye LysoTracker was used. TM Deep Red is excited at 647 nm and exhibits red fluorescence; if the green fluorescence of FAM coincides with the red fluorescence of the endosome / lysosomal dye, it appears yellow. The nuclear dye Hoechst is excited at 405 nm and exhibits blue fluorescence.

[0103] like Figure 7 As shown, the group containing MC3 nanoparticles loaded with FAM-N (nucleic acid) (control group) exhibited numerous yellow spots, indicating that FAM-N was mainly delivered to endosomes / lysosomes (red) by the MC3 nanoparticles, with weak endosome / lysosome escape ability. In contrast, the nanoparticle groups prepared in Examples 1 (compound 2), 2 (compound 3), and 4 (compound 7) showed numerous free green spots and a small number of yellow spots, indicating that most of the FAM-N delivered by the nanoparticles prepared from the above three lipids had escaped from the endosomes / lysosomes.

[0104] Experiment Example 8 simulates the effect of esterases in the cytoplasmic environment on the dissociation of nanoparticles from mRNA.

[0105] Experimental Procedure: Nanoparticles were prepared using Compound 3 (Example 1) and Compound 7 (Example 4). The nanoparticles were dialyzed to approximately pH 7.0 (simulating the state of nanoparticles in a neutral cytoplasmic environment). One part of OVA mRNA (0.2 μg / μL) was added and mixed with three parts of nanoparticles to form a complex. 10 μL of the complex was added to 2.5 μL of esterase solution according to Example 4 and treated for 1 h, 2 h, and 4 h, respectively. The complex was then loaded onto a 0.8% agarose gel containing Gel-Red nucleic acid dye after being mixed with 5×RNA loading buffer. Pure mRNA was used as a control group. Electrophoresis was performed at 50 mA for 15 min.

[0106] like Figure 8 As shown, under simulated cytoplasmic neutral conditions (pH approximately 7.0), the negative charge generated by esterase hydrolysis of lipids repels the equally negatively charged nucleic acid, causing the nucleic acid to dissociate, which is beneficial for nucleic acid drugs to exert their biological functions.

[0107] Experimental Example 9: In vitro immune activation

[0108] Examples 1 (compound 2) and 4 (compound 7) were selected to inoculate DC2.4 cells at a rate of 2.5 × 10⁻⁶. 5 Cells were seeded per well in 24-well cell culture plates. After 24 h of incubation, different nanoparticles were added. PBS was used as the negative control, and 200 ng / mL lipopolysaccharide (LPS) was used as the positive control. All groups were incubated in a CO2 incubator for 10 h. Cells were washed twice with PBS, digested with trypsin, resuspended in PBS, washed twice, blocked with medium containing 4% FBS, and then stained on ice with fluorescently labeled anti-CD11c, CD80, and CD86 antibodies. Flow cytometry was used for quantitative analysis of the labeled cells. Fluorescence intensity was measured using a flow cytometer.

[0109] like Figure 9 As shown, the lipid nanoparticles prepared by the esterase-responsive ionizable cationic lipid molecules in Examples 2 and 4 according to the optimal ratio have a higher ability to stimulate DC2.4 cell maturation than the group containing MC3 nanoparticles. This indicates that the lipid nanoparticles can effectively transfect DC2.4 cells and play an immune-activating role.

[0110] Experimental Example 10: Distribution of Organs in the Body

[0111] Experimental procedure: DiR is a fluorescent dye commonly used in in vivo deep tissue imaging and dynamic tracing studies. Examples 1 (Compound 2) and 4 (Compound 7) were selected to prepare nanoparticles containing DiR dye for in vivo distribution studies. B16F10 cell-bearing C57BL / 6 mice (weighing approximately 20 ± 2 g, purchased from the Guangdong Provincial Medical Experimental Animal Center, license number: SCXK(Yue)2022-0002) were intradermally injected with 50 μL of DiR solution and DiR-labeled nanoparticles respectively. After 24 h of administration, the mice were sacrificed, and the heart, liver, spleen, lung, kidney, draining lymph nodes, and tumor tissues were dissected and imaged using an imager.

[0112] As Figure 10 shown, the nanoparticles prepared from Example 1 (Compound 2) and 4 (Compound 7) were enriched at the draining lymph nodes rich in immune cells.

[0113] Experimental Example 11 In vivo transfection efficiency and mRNA encoding co-stimulatory antigens activate the body's immune system to treat tumors

[0114] Example 4 (Compound 7) was selected to prepare nanoparticles containing OVA mRNA and blank nanoparticles. B16F10 cell-bearing C57BL / 6 mice were intradermally administered once every 3 days for 3 consecutive times, with a dosing dose of 10 μg OVA mRNA / mouse. Injection of an equal volume of PBS was used as a negative control. The group containing MC3 nanoparticles and OVA mRNA were used as control groups. Blank nanoparticles without OVA mRNA were injected simultaneously. Blood samples were taken to detect in vivo transfection efficiency (OVA-specific antibody IgG), lymph nodes and tumors were taken to detect immunity (CD11c, CD80, CD86), and anti-tumor indicators (CD8).

[0115] As Figure 11 shown, the nanoparticles containing OVA mRNA prepared from Example 4 (Compound 7) produced more IgG than the nanoparticles containing MC3, further demonstrating its high transfection efficiency in vivo.

[0116] As Figure 12-14 shown, both the blank nanoparticles and the nanoparticles containing OVA mRNA prepared from Example 4 (Compound 7) could stimulate the maturation of DCs (expressing markers CD80, CD86) in the draining lymph nodes in vivo. The OVA mRNA nanoparticles containing this lipid promoted the infiltration of CD8 + T (expressing marker CD8) cells in the tumor and significantly inhibited tumor growth. The nanoparticles containing MC3 only induced weak immune activation, and the ability of OVA mRNA nanoparticles to inhibit tumors was poor.

[0117] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. An esterase-responsive ionizable cationic lipid, characterized in that, It is electrically neutral under physiological conditions, positively charged in a slightly acidic endosome / lysosomal environment, and hydrolyzed by intracellular esterases into electrically neutral or negatively charged compounds. The structural formula of the compound is: Wherein, n1 and n2 represent the number of CH2, n1 is 1-2, and n2 is 1-4; X is an alkylamine or a hydroxyalkylamine; if it is an alkylamine, it is selected from... One of them; If it is a hydroxyalkylamine, then the structure is: The n4 and n5 represent the number of CH2, where n4 is 2-5 and n5 is 0-2; R represents a hydrocarbon chain, and N represents the number of hydrocarbon chains, where N is 1 or 2.

2. The lipid according to claim 1, characterized in that, The R structure of the compound contains 0-2 -C=C- or -C≡C-.

3. The lipid according to claim 1, characterized in that, When both N atoms in the structure of compound R are 1, each chain has a length of 6-16 C atoms; when one N atom is 2 and the other N atom is 1 or 2, each chain has a length of 4-10 C atoms.

4. The lipid according to claim 1, characterized in that, The esterase-responsive cationic lipid is selected from compounds having one of the following structural formulas:

5. A method for preparing an esterase-responsive ionizable cationic lipid as described in claim 1, characterized in that, The process includes the following: S1. Dissolve the fatty acid or its corresponding fatty acid acyl chloride in an organic solvent, react with p-hydroxybenzyl alcohol to form intermediate 1, and purify to obtain intermediate 1. S2. Dissolve the purified intermediate product 1 in an organic solvent, then dissolve the bromoalkyl acyl chloride compound in an organic solvent and add it to the reaction system to react. After purification, intermediate product 2 is obtained. S3. Dissolve 3,4-dihydroxy aromatic aldehyde or 3,4-dihydroxy aromatic aliphatic aldehyde in an organic solvent, simultaneously alkalize the reaction system, add iodine compound as a catalyst, and an organic solvent containing intermediate product 2 obtained in step S2. Stir the reaction at 10-60℃ for 2-24 hours, and purify to obtain intermediate product 3. S4. Dissolve the compound containing the secondary amine in an organic solvent, add an organic solvent containing intermediate product 3 obtained in step S3, protect with N2, stir at room temperature for 1-8 h, then adjust the pH of the reaction system, add a reducing agent, and stir the reaction at 0-60℃ for 2-24 h to obtain the final product.

6. The preparation method according to claim 5, characterized in that, The compounds containing secondary amines mentioned in step S4 include: dimethylamine, N-ethylmethylamine, ethylmethylamine, N-methyl-2-hydroxyethylamine, 2-(ethylamino)ethanol, N-propylethanolamine, 3-(methylamino)-1-propanol, 4-(methylamino)-1-butanol, 5-(methylamino)-1-pentanol, 3-(ethylamino)-1-propanol, 4-(ethylamino)-1-butanol, 5-(ethylamino)-1-pentanol, 3-(propylamino)-1-propanol, 4-(propylamino)-1-butanol, and 5-(propylamino)-1-pentanol.

7. The preparation method according to claim 5, characterized in that, The fatty acids or their corresponding fatty acid acyl chlorides mentioned in step S1 include: 2-butyloctanoic acid, 2-hexyloctanoic acid, 2-hexylnonanoic acid, 2-hexyldecanoic acid, 2-heptylnonanoic acid, 2-octyldecanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, undecanoic acid, 2-hexenoic acid, 2-tridecenoic acid, 9-hexadecenoic acid, linoleic acid, oleic acid, octadecyl-9-alkynic acid; and the acyl chlorides corresponding to the above acids.

8. The use of the esterase-responsive ionizable cationic lipid as described in claim 1 in the preparation of nucleic acid drug carriers.

9. A nucleic acid drug delivery system, characterized in that, The invention includes a lipid nanoparticle carrier and a nucleic acid drug loaded on the carrier. The lipid nanoparticle carrier is formed by the esterase-responsive ionizable cationic lipid, auxiliary lipid and PEG lipid as described in claim 1. The molar ratio of esterase-responsive ionizable cationic lipid: auxiliary lipid: PEG lipid molecules is 30-80: 10-70: 0.5-2.

10. The nucleic acid drug delivery system as described in claim 9, characterized in that, The nucleic acid drug is one of DNA, siRNA, mRNA, microRNA, shRNA, and ASOs.

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