Ionizable cationic lipid and use thereof
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2026-02-11
- Publication Date
- 2026-08-13
Smart Images
Figure CN2026078670_13082026_PF_FP_ABST
Abstract
Description
A class of ionizable cationic lipids and their applications Technical Field
[0001] This application relates to the field of drug delivery based on lipid nanoparticles, and in particular to a class of ionizable cationic lipids and their applications. Background Technology
[0002] Biopharmaceuticals have evolved from small-molecule chemical drugs to biopharmaceuticals based on biomolecules and cells, including a wide range of protein drugs and, in recent years, nucleic acid drugs and cell gene therapy (RCG). Drug delivery is a key technology for biopharmaceuticals to exert their effects. Nanobiopharmaceuticals combine nanotechnology and biopharmaceutical technology to develop effective drugs. Nanoparticles are widely used for the delivery of biomolecules to prepare vaccines and biopharmaceuticals for disease prevention and treatment. Biomolecules such as protein and nucleic acid drugs have more defined therapeutic mechanisms and efficacy, and have seen faster development in recent years. However, protein and nucleic acid drugs, especially nucleic acid drugs, have poor stability, requiring effective delivery technologies to protect the drug and enhance efficacy.
[0003] Lipid nanoparticles (LNPs) have been developed in recent years for delivering nucleic acid drugs, including mRNA, siRNA, and ASO. mRNA-LNP technology has been widely used in the development of vaccines, protein replacement drugs, gene editing, and cell therapy, including several COVID-19 mRNA vaccines approved by the FDA between 2020 and 2023, and the RSV mRNA vaccine approved in 2024. Lipid nanoparticles, also known as lipid nanoparticles, are nanoparticles assembled from various lipids using technologies such as microfluidic chips to encapsulate and deliver biopharmaceuticals such as mRNA. As shown in Figure 1, the lipid composition typically includes four types of lipids: ionizable cationic lipids, structural lipids, accessory lipids, and PEGylated lipids. The number of lipids can be reduced, such as by removing accessory lipids, and even three types of lipids can form lipid nanoparticles; other lipids, such as a fifth type, can be added to regulate charge and other properties, acting as a guide. Regardless of the composition of the lipids, ionizable lipids are indispensable.
[0004] In lipid nanoparticles, ionizable lipids play a crucial functional role. Cationic lipids encapsulate RNA by binding to its anions, but their cytotoxicity limits drug delivery. Ionizable lipids, or ionizable cationic lipids, effectively solve this problem. In an acidic pH range, ionizable lipids become positively charged, binding and encapsulating RNA to form nanoparticles. After replacing the buffer solution to neutral, the nanoparticles can be safely delivered to target organs and cells in a neutral in vivo environment. After being engulfed by cells, the nanoparticles form endosomes. In the acidic intracellular environment, the nanoparticles re-protonate and become positively charged, thereby disrupting the outer membrane structure of the endosomes and releasing them into the cytoplasm. Then, ribosomes are activated to translate mRNA into target proteins, thereby exerting disease prevention and treatment effects.
[0005] Ionizable lipids typically consist of three structural units: an ionizable head, a linker, and a hydrophobic carbon chain. The head usually contains an amino functional group, such as primary, secondary, tertiary, or quaternary amines. Tertiary amines are commonly used; they protonate at acidic pH to carry a positive charge and deprotonate at neutral pH, leaving them uncharged. Therefore, pKa control is crucial. The head can carry hydroxyl groups to form hydrogen bonds with nucleic acids to improve transfection efficiency, or carry other genes to play a stabilizing role or reduce toxicity. The linker connects the head and tail and can use degradable groups such as amino groups, amide groups, and disulfide bonds to accelerate in vivo degradation and clearance, reduce toxicity, and increase dosage and frequency. The tail structure is typically a carbon chain.
[0006] For lipid nanoparticles to effectively deliver RNA drugs, they need to overcome multiple biological barriers. First, the RNA drug must be protected to reach the target organ cells in the human body. Second, the nanoparticles must fuse into the cells. Third, and more importantly, the nanoparticles that have entered the cells must be effectively released from the endosomes. It has been reported that the endosome release efficiency of existing lipid nanoparticles is low, generally below 10%; therefore, improving the release efficiency is crucial to enhancing the therapeutic efficacy of lipid nanoparticles. In addition, the in vitro and in vivo stability of nanoparticles, especially temperature stability, is a key issue. Existing mRNA nanoparticles require ultra-low temperature transportation and storage at -20°C or -70°C to ensure product quality, which not only increases the cost of transportation and use but also significantly impacts product accessibility.
[0007] In summary, improving the endosome release efficiency and temperature stability of lipid nanoparticles remains a key research focus and challenge in the field of lipid nanoparticles. Summary of the Invention
[0008] The purpose of this application is to provide a new class of ionizable cationic lipids and their applications.
[0009] To achieve the above objectives, this application adopts the following technical solution:
[0010] The first aspect of this application discloses an ionizable cationic ester having the structure shown in Formula 1;
[0011] Formula 1
[0012] In this configuration, any three of R1, R2, R3, and R4 are saturated or unsaturated aliphatic chains, and the other is H. At least two of the three saturated or unsaturated aliphatic chains are unsaturated aliphatic chains; alternatively, all of R1, R2, R3, and R4 are saturated or unsaturated aliphatic chains, with at least two being unsaturated aliphatic chains. All saturated or unsaturated aliphatic chains have a carbon chain length of 2-18. For example, saturated aliphatic chains are straight-chain alkyl groups with a carbon chain length of 2-18, and unsaturated aliphatic chains are straight-chain olefins with a carbon chain length of 2-18. R5 is -C.1-3 OH, piperidine ring, or -C 1-3 An ester formed by the condensation of OH with piperidine carboxylic acid; x takes values of 2-8, and y takes values of 2-8.
[0013] It should be noted that the ionizable cationic lipid of this application improves encapsulation efficiency by forming charge, hydrogen bonds, and hydrophobic adsorption with biopharmaceuticals such as nucleic acids through the head design of the R5 end; it uses ester bonds as linkers for R1, R2, R3, and R4 to facilitate in vivo degradation and reduce toxicity; furthermore, the design of unsaturated fatty acid chains in R1, R2, R3, and R4 increases membrane fluidity during endosome release, effectively improving the release efficiency of biopharmaceuticals, and effectively enhancing protein expression when the biopharmaceutical is a protein; in addition, the ionizable cationic lipid of this application has good temperature stability, which not only improves product quality but also expands product accessibility.
[0014] It is understood that in this application, the carbon chain length of the saturated or unsaturated aliphatic chain, as well as the values of x and y, directly affect the hydrophobicity and pKa and other physicochemical properties of the ionizable cationic lipid. In one implementation of this application, a saturated or unsaturated aliphatic chain with a carbon chain length of 2-12 is preferred, and a saturated or unsaturated aliphatic chain with a carbon chain length of 8-10 is more preferred. At the same time, x is preferably 5-7 and y is preferably 5-7.
[0015] In one implementation of this application, any three of R1, R2, R3, and R4 are unsaturated fatty acid chains, and the other is H; or, all of R1, R2, R3, and R4 are unsaturated fatty acid chains.
[0016] It should be noted that this application research found that adding an unsaturated fatty bond to each branch in the three-branched and four-branched structures can further improve the release efficiency of the biopharmaceutical; therefore, in the preferred scheme, three or four of R1, R2, R3 and R4 are designed to be unsaturated fatty chains.
[0017] In one implementation of this application, the ionizable cationic ester has the structure shown in Formula 2.
[0018] Formula 2
[0019] In one implementation of this application, the ionizable cationic ester is at least one of VB219, VB220, VB221, VB222, VB223, VB224, VB230, VB231, VB232, VB233, VB234, VB235, VB236, VB237 and VB238;
[0020] VB219
[0021] VB220
[0022] VB221
[0023] VB222
[0024] VB223
[0025] VB224
[0026] VB230
[0027] VB231
[0028] VB232
[0029] VB233
[0030] VB234
[0031] VB235
[0032] VB236
[0033] VB237
[0034] VB238
[0035] In one implementation of this application, the ionizable cationic ester is a combination of at least one of VB219, VB220, VB230, VB233, and VB236 and at least one of VB221, VB222, VB223, VB224, VB231, VB232, VB234, VB235, VB237, and VB238; for example, in one implementation of this application, it is a combination of VB219 and VB222, VB219 and VB224, VB220 and VB222, or VB220 and VB224.
[0036] Another aspect of this application discloses a lipid nanoparticle comprising an encapsulating material and a biopharmaceutical encapsulated within the encapsulating material, wherein the encapsulating material comprises the ionizable cationic lipid of this application.
[0037] It should be noted that the key to this application lies in the improvement and optimization of ionizable cationic lipids. As for other components in lipid nanoparticles, such as structural lipids, auxiliary lipids, PEG lipids, or other functional lipids that regulate charge properties, they can be referred to in the prior art and are not specifically limited here.
[0038] In one implementation of this application, the biopharmaceutical is at least one of DNA, RNA, protein, and small molecule drugs.
[0039] In one implementation of this application, DNA is at least one of deoxyribonucleic acid fragments, plasmids, antisense DNA, and DNA aptamers.
[0040] In one implementation of this application, the RNA is at least one of mRNA, circRNA, saRNA, siRNA, miRNA, antisense RNA, RNA aptamer, shRNA, and small activating RNA.
[0041] In one implementation of this application, the mRNA is at least one of ordinary linear mRNA, self-replicating mRNA, and circular mRNA.
[0042] In one implementation of this application, the protein is at least one of an antibody, an enzyme, a protein drug, or a functional polypeptide.
[0043] It should be noted that the key to this application lies in the improvement and optimization of ionizable cationic lipids. As for the specific encapsulated biopharmaceuticals, existing technologies can be referenced, including but not limited to various DNA, RNA, protein and small molecule drugs.
[0044] In one implementation of this application, the lipid nanoparticles have a particle size of 30-500 nm.
[0045] Preferably, the lipid nanoparticles have a particle size of 60-150 nm, more preferably 70-90 nm.
[0046] It should be noted that, in one implementation of this application, lipid nanoparticles with a diameter of 70-90 nm were specifically prepared, and their distribution range is relatively small, indicating that the prepared lipid nanoparticles have good uniformity and stability.
[0047] Another aspect of this application discloses a drug delivery kit comprising at least one of the following components:
[0048] (a) The ionizable cationic ester of this application;
[0049] (b) The lipid nanoparticles of this application.
[0050] It should be noted that the drug delivery kit of this application can be a kit pre-encapsulated with a specific biological drug, or it can be the cationic lipid of this application. Users can design and encapsulate the corresponding biological drug according to their needs. It is understood that the key to this kit is the presence of the ionizable cationic lipid or lipid nanoparticles of this application. Other conventional reagents required for drug delivery can be obtained by referring to existing technologies or purchasing commercially available reagents. Of course, for ease of use, some reagents can also be combined into the kit of this application; no specific limitations are made here.
[0051] Another aspect of this application discloses the use of the ionizable cationic lipids, lipid nanoparticles, or reagent kits of this application in the preparation of preventive vaccines, therapeutic vaccines, protein replacement drugs, gene editing drugs, or cell therapy drugs.
[0052] It should be noted that lipid nanoparticle drugs prepared using the ionizable cationic lipids, lipid nanoparticles or kits of this application have advantages such as high encapsulation efficiency, low toxicity, high endosome release efficiency and good temperature stability, which can improve the quality and therapeutic effect of biopharmaceuticals.
[0053] Due to the adoption of the above technical solutions, the beneficial effects of this application are as follows:
[0054] The ionizable cationic lipid of this application has advantages such as high encapsulation efficiency, low toxicity, high endosome release efficiency, and good temperature stability, which can improve the quality and therapeutic effect of lipid nanoparticle drugs and provide a new solution and option for biopharmaceutical delivery. Attached Figure Description
[0055] Figure 1 is a schematic diagram of the structure of the LNP in this application;
[0056] Figure 2 is a statistical result of the pKa and cpKa of some ionizable lipids in the embodiments of this application;
[0057] Figure 3 is a statistical chart of the LNP relative effectiveness of VB219 in the embodiments of this application;
[0058] Figure 4 shows the stability test results of VB219 at -80℃ in the embodiments of this application;
[0059] Figure 5 shows the stability test results of VB219 at -20℃ in the embodiments of this application;
[0060] Figure 6 shows the stability test results of VB219 at 4℃ in the embodiments of this application;
[0061] Figure 7 shows the stability test results of VB219 at 25°C in the embodiments of this application;
[0062] Figure 8 is a statistical result of the LNP relative effectiveness of VB220 in the embodiments of this application;
[0063] Figure 9 is a statistical result of the LNP relative effectiveness of VB222 in the embodiments of this application. Detailed Implementation
[0064] To address the issues of generally low endosome release efficiency and poor temperature stability of existing lipid nanoparticles, this application creatively develops a new ionizable cationic lipid, namely the compound with the structure shown in Formula 1.
[0065] The design of the ionizable cationic lipid in this application takes into account the following factors:
[0066] 1) Molecular toxicity: This is a fundamental requirement that needs to be considered first.
[0067] 2) Physicochemical properties of molecules: including pKa and LogP lipophilicity, which affect the physicochemical properties, encapsulation efficiency, in vivo release and therapeutic effects of nanoparticles.
[0068] 3) Molecular structure and function: The head is considered to have an amino group structure, using linear and cyclic amino groups; the linker uses ester bonds to promote intracellular biodegradation; the tail is considered to have carbon chain length and branched chains to form an inverted cone structure to disrupt endosome membrane stability, and unsaturated bonds are used to increase fluidity and endosome release.
[0069] Taking all the above factors into consideration, this application first designed a three-branched VB219 structure. VB219 uses a linear quaternary ammonium as its head, which forms a charge adsorption with nucleic acids. A hydroxyl group is added two carbons away in the head, increasing the interaction with nucleic acids through hydrogen bonds. Ester bonds are used as linkers between the two main chains to facilitate in vivo degradation and reduce toxicity. Each of the three branches adds an unsaturated fatty acid bond to increase membrane fluidity during endosome release, thereby improving release efficiency and protein expression. Actual functional testing results demonstrate that using VB219 to deliver mRNA-expressed proteins significantly enhances their expression.
[0070] Further optimization and improvement were made based on VB219. The hydroxyl group at the head of VB219 was changed to a cyclic piperidine structure, which yielded VB221, thus improving thermal stability. By utilizing the hydroxyl group of VB219 to form an ester bond and simultaneously linking the piperidine structure, VB222 was obtained, which also improved thermal stability and promoted intracellular degradation to reduce toxicity.
[0071] Based on VB219, an additional branch was added, forming a four-branch structure, namely VB220. Each branch of VB220 adds one unsaturated aliphatic bond, and the rest of the design is similar to VB219. Based on VB220, the designs of VB223 and VB224 correspond to VB221 and VB222, respectively. Functional testing showed that VB219 significantly outperformed SM102, currently the most potent on the market. Therefore, based on VB219, by adjusting the carbon chain length and the position of the unsaturated aliphatic bonds, VB230, VB233, and VB236, as well as their corresponding piperidine derivatives VB231, VB232, VB234, VB235, VB237, and VB238, were designed.
[0072] This application provides a variety of novel ionizable lipids for preparing nanoparticles to deliver nanomedicines. These ionizable lipids are mixed with other lipids and drugs to form drug-encapsulated lipid nanoparticles, providing high efficiency and stability to protect and deliver drugs to specific organs and cells, thereby effectively preventing and treating diseases.
[0073] The present application will be further described in detail below through specific embodiments. The following embodiments are only for further illustration of the present application and should not be construed as limiting the present application.
[0074] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.
[0075] Example
[0076] I. Synthesis of Ionizable Cationic Lipids
[0077] We first synthesized the important intermediate VB200, which introduces unsaturated aliphatic bonds on both branches. Based on this, we synthesized VB219 and VB220. Then, by introducing piperidine structures into VB219 and VB220, we further synthesized VB221, VB222, VB223, and VB224. Details are as follows.
[0078] Synthesis of VB200 and VB219:
[0079] Compound 1a
[0080] cis-3-octen-1-ol (50 g, 390 mmol) was dissolved in dichloromethane. Triphenylphosphine (123 g, 468 mmol) was added at 0-10 °C, followed by fractional addition of carbon tetrabromide (142 g, 429 mmol). After the addition was complete, the mixture was stirred for 1-2 hours. The reaction solution was diluted with n-hexane, resulting in the precipitation of a large amount of solid. The solution was filtered, and the filtrate was concentrated and purified by column chromatography to give compound 1a (68 g, colorless oily liquid), yield: 91.3%.
[0081] Compound 1b (VB200)
[0082] Magnesium shavings were added to anhydrous THF, and compound 1a (60 g, 314 mmol) was added dropwise to initiate the Grignard reaction. After the addition was complete, the mixture was kept at 60 °C for 2 hours, then cooled to 0-10 °C, and ethyl formate (23.2 g, 314 mmol) was added dropwise with stirring overnight. Post-treatment: The reaction was quenched dropwise with water, and the mixture was separated. The aqueous phase was extracted once with DCM, and the combined organic phases were concentrated. The concentrate was added to concentrated sodium hydroxide aqueous solution and stirred for 2 hours. The pH was adjusted to acidic with hydrochloric acid, and the mixture was extracted twice with DCM. The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and then purified by column chromatography to obtain compound 1b (20 g, pale yellow oily liquid), yield: 50.6%.
[0083] H1-NMR(CDCl3)5.31-5.43(m,4H),5.03(m,1H),2.00-2.13(m,8H),1.59-1.68(m,4H),1.28-1.37(m,8H),0.89(m,6H)
[0084] Compound 1c
[0085] Compound 1b (5 g, 19.8 mmol) was dissolved in dichloromethane, and 8-bromooctanoic acid (4.4 g, 19.8 mmol) and DMAP (0.49 g, 4 mmol) were added. EDC (4.5 g, 23.8 mmol) was added dropwise at 0-10 °C. After the addition was complete, the mixture was brought to room temperature and stirred for 3-5 hours. The reaction solution was washed once with water and once with dilute sodium bisulfate. The organic phase was dried over anhydrous sodium sulfate and concentrated to give compound 1c (7.2 g, a pale yellow oily liquid), which was used directly in the next step. Yield: 79.5%.
[0086] Compound 1d
[0087] Compound 1c (7 g, 15.3 mmol) was dissolved in a mixture of acetonitrile and THF, and ethanolamine (14 g, 230 mmol) and potassium carbonate (4.2 g, 30.6 mmol) were added. The mixture was stirred at 50 °C for 1–2 h. The reaction solution was filtered and concentrated. The concentrate was diluted with dichloromethane, washed twice with water, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give compound 1d (4.4 g, pale yellow oily liquid), in 65.7% yield.
[0088] Compound 1e
[0089] (Z)-4-decen-1-ol (10 g, 64 mmol) was dissolved in dichloromethane, and 7-bromoheptanoic acid (13.4 g, 64 mmol) and DMAP (1.6 g, 12.8 mmol) were added. EDC (14.7 g, 76.8 mmol) was added dropwise at 0-10 °C. After the addition was complete, the mixture was brought to room temperature and stirred for 3-5 h. The reaction solution was washed once with water and once with dilute sodium bisulfate. The organic phase was dried over anhydrous sodium sulfate and concentrated to give compound 1e (17.3 g, colorless oily liquid), which was used directly in the next step. Yield: 77.8%.
[0090] Compound VB219
[0091] English name: (5Z,12Z)-heptadeca-5,12-dien-9-yl 8-((7-(((Z)-dec-4-en-1-yl)oxy)-7-oxoheptyl)(2-hydroxyethyl)amino)octanoate
[0092] Chinese name: (5Z,12Z)-heptadec-5,12-dien-9-yl 8-(7-((((Z)-dec-4-en-1-yl)oxy)-7-oxohepyl)(2-hydroxyethyl)amino)octanoate
[0093] Compound 1d (2.5 g, 5.7 mmol) and compound 1e (2.0 g, 5.7 mmol) were dissolved in a mixed solution of acetonitrile and THF. Anhydrous potassium carbonate (1.6 g, 11.4 mmol) and potassium iodide (0.95 g, 5.7 mmol) were added, and the mixture was heated to 70-80 °C and stirred overnight. The reaction solution was filtered and concentrated. The concentrate was diluted with dichloromethane, washed twice with water, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give compound VB219 (2.3 g, pale yellow oily liquid), in 57.2% yield.
[0094] H1-NMR(CDCl3)5.34-5.40(m,6H),4.92(m,1H),4.06(t,2H),3.52(t,2H),2.57(t,2H),2.43(m ,4H),2.31(m,4H),1.98-2.09(m,12H),1.57-1.68(m,10H),1.26-1.43(m,28H),0.89(m,9H);MS m / z 704(M+H)
[0095] Synthesis of VB220:
[0096] Compound 1f
[0097] Compound 1b (3 g, 11.9 mmol) was dissolved in dichloromethane, and 7-bromoheptanoic acid (2.5 g, 11.9 mmol) and DMAP (0.3 g, 2.4 mmol) were added. EDC (2.7 g, 14.3 mmol) was added dropwise at 0-10 °C. After the addition was complete, the mixture was brought to room temperature and stirred for 3-5 hours. The reaction solution was washed once with water and once with dilute sodium bisulfate. The organic phase was dried over anhydrous sodium sulfate and concentrated to give compound 1f (3.8 g, a pale yellow oily liquid), which was used directly in the next step. Yield: 72.1%.
[0098] Compound VB220
[0099] English name: (5Z,12Z)-heptadeca-5,12-dien-9-yl 8-((7-(((5Z,12Z)-heptadeca-5,12-dien-9-yl)oxy)-7-oxoheptyl)(2-hydroxyethyl)amino)octanoate
[0100] Chinese name: (5Z, 12Z)-heptadec-5,12-dien-9-yl 8-(7-(((5Z, 12Z)-5-heptadec-9-yl)oxy)-7-oxohepyl)(2-hydroxyethyl)amino)octanoate
[0101] Compound 1d (1.9 g, 4.3 mmol) and compound 1f (1.9 g, 4.3 mmol) were dissolved in a mixed solution of acetonitrile and THF. Anhydrous potassium carbonate (1.2 g, 8.6 mmol) and potassium iodide (0.7 g, 4.3 mmol) were added, and the mixture was heated to 70-80 °C and stirred overnight. The reaction solution was filtered and concentrated. The concentrate was diluted with dichloromethane, washed twice with water, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give compound VB220 (1.6 g, pale yellow oily liquid), in a yield of 46.1%.
[0102] H1-NMR(CDCl3)5.32-5.41(m,8H),4.92(m,2H),3.58(t,2H),2.63(t,2H),2.51(m,4H),2.31(m, 4H),1.99-2.09(m,16H),1.59-1.67(m,12H),1.48(m,4H),1.31-1.34(m,26H),0.90(m,12H);MS m / z 801(M+H)
[0103] Synthesis of VB221:
[0104] 1g of compound
[0105] Compound 1c (13 g, 28.4 mmol) was dissolved in a mixture of acetonitrile and THF. 4-Amino-1-methylpiperidine (1.6 g, 142 mmol) and potassium carbonate (7.8 g, 56.8 mmol) were added, and the mixture was stirred at 50 °C for 1–2 h. The reaction solution was filtered and concentrated. The concentrate was diluted with dichloromethane, washed twice with water, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give compound 1 g (4.7 g, yellow oily liquid), in a yield of 33.7%.
[0106] Compound VB221
[0107] English name: (5Z,12Z)-heptadeca-5,12-dien-9-yl 8-((7-(((Z)-dec-4-en-1-yl)oxy)-7-oxoheptyl)(1-methylpiperidin-4-yl)amino)octanoate
[0108] Chinese name: (5Z,12Z)-heptadec-5,12-dien-9-yl 8-(7-((((Z)-dec-4-en-1-yl)oxy)-7-oxohepyl)(1-methylpiperidin-4-yl)amino)octanoate
[0109] Compound 1 g (3 g, 73.5 mmol) and compound 1e (2.6 g, 73.5 mmol) were dissolved in a mixed solution of acetonitrile and THF. Anhydrous potassium carbonate (2.1 g, 147 mmol) and potassium iodide (1.2 g, 73.5 mmol) were added, and the mixture was heated to 70-80 °C and stirred overnight. The reaction solution was filtered and concentrated. The concentrate was diluted with dichloromethane, washed twice with water, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give 0.7 g of compound VB221 (0.7 g, yellow oily liquid), in a yield of 15.1%.
[0110] H1-NMR(CDCl3)5.30-5.40(m,6H),4.88(m,1H),4.05(m,5H),3.80(m,2H),3.65(m,2H),3.33(s,3H),2.89(m ,4H),2.54(m,2H),2.28(m,4H),1.97-2.11(m,12H),1.54-1.70(m,14H),1.26-1.44(m,24H),0.89(m,9H); MS m / z 758(M+H)
[0111] Synthesis of VB222:
[0112] Compound VB222
[0113] English name:
[0114] 2-((7-(((Z)-dec-4-en-1-yl)oxy)-7-oxoheptyl)(8-(((5Z,12Z)-heptadeca-5,12-dien-9-yl)oxy)-8-oxooctyl)amino)ethyl 1-methylpiperidine-4-carboxylate
[0115] Chinese name: 2-(7-((((Z)-dec-4-en-1-yl)oxy)-7-oxohepyl)(8-(((5Z,12Z)-heptadecyl-5,12-dien-9-yl)oxy)-8-oxooctyl)amino)1-methylpiperidin-4-carboxylic acid ethyl ester
[0116] VB219 (1.3 g, 1.8 mmol) was dissolved in dichloromethane, and 1-methylpiperidin-4-carboxylic acid (0.27 g, 1.9 mmol) and DMAP (50 mg, 0.4 mmol) were added. EDC (0.42 g, 2.2 mmol) was added dropwise at 0-10 °C. After the addition was complete, the mixture was brought to room temperature and stirred overnight. The reaction solution was washed once with water and once with dilute sodium bisulfate. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain an oily substance. The oily substance was diluted with n-hexane, washed twice with acetonitrile, and the n-hexane phase was concentrated to dryness to obtain compound VB222 (1.1 g, pale yellow oily liquid). Yield: 71.8%.
[0117] H1-NMR(CDCl3)5.33-5.39(m,6H),4.92(m,1H),4.08(m,4H),2.88(m,2H),2.66(t,2H),2.42(m ,4H),2.30(m,8H),1.99-2.09(m,16H),1.57-1.67(m,12H),1.26-1.41(m,28H),0.89(m,9H);MS m / z 830(M+H)
[0118] Synthesis of VB223:
[0119] Compound 1h
[0120] cis-3-nonen-1-ol (50 g, 352 mmol) was dissolved in dichloromethane (10 V). Triphenylphosphine (111 g, 422 mmol) was added at 0-10 °C, followed by fractional addition of carbon tetrabromide (128 g, 387 mmol). After the addition was complete, the mixture was stirred for 1-2 hours. The reaction solution was diluted with n-hexane (10 V), resulting in the precipitation of a large amount of solid. The solid was filtered, and the filtrate was concentrated and purified by column chromatography to give compound 1h (60 g, colorless oily liquid). Yield: 83.2%.
[0121] Compound 1i
[0122] Magnesium shavings were added to anhydrous THF, and compound 1 h (60 g, 292 mmol) was added dropwise to initiate the Grignard reaction. After the addition was complete, the mixture was kept at 60 °C for 2 h, then cooled to 0-10 °C, and ethyl formate (21 g, 292 mmol) was added dropwise, followed by stirring overnight. Post-treatment: The reaction was quenched dropwise with water, and the mixture was separated. The aqueous phase was extracted once with DCM, and the combined organic phases were concentrated. The concentrate was added to concentrated sodium hydroxide aqueous solution and stirred for 2 h. The pH was adjusted to acidic with hydrochloric acid, and the mixture was extracted twice with DCM. The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and then purified by column chromatography to obtain compound 1i (13 g, pale yellow oily liquid), yield: 31.7%.
[0123] Compound 1j
[0124] Compound 1i (10 g, 35.7 mmol) was dissolved in dichloromethane, and 7-bromoheptanoic acid (7.5 g, 35.7 mmol) and DMAP (0.87 g, 7.1 mmol) were added. EDC (8.2 g, 42.8 mmol) was added dropwise at 0-10 °C. After the addition was complete, the mixture was brought to room temperature and stirred for 3-5 hours. The reaction solution was washed once with water and once with dilute sodium bisulfate. The organic phase was dried over anhydrous sodium sulfate and concentrated to give compound 1j (13 g, pale yellow oily liquid), which was used directly in the next step. Yield: 59.5%.
[0125] Compound VB223
[0126] English name: (5Z,12Z)-heptadeca-5,12-dien-9-yl 8-((1-methylpiperidin-4-yl)(7-(((6Z,13Z)-nonadeca-6,13-dien-10-yl)oxy)-7-oxoheptyl)amino)octanoate
[0127] Chinese name: (5Z, 12Z)-heptadec-5,12-dien-9-yl 8-((1-methylpiperidin-4-yl)(7-(((6Z, 13Z)-nonadecan-6,13-dien-10-yl)oxy)-7-oxoheptyl)amino)octanoate
[0128] Compound 1j (1.6 g, 3.3 mmol) and intermediate 7 (1.6 g, 3.3 mmol) were dissolved in a mixed solution of acetonitrile and THF. Anhydrous potassium carbonate (0.9 g, 6.6 mmol) and potassium iodide (0.55 g, 3.3 mmol) were added, and the mixture was heated to 70-80 °C and stirred overnight. The reaction solution was filtered and concentrated. The concentrate was diluted with dichloromethane, washed twice with water, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give 0.3 g of VB223 (0.3 g, yellow oily liquid), in a yield of 9.3%.
[0129] H1-NMR(CDCl3)5.30-5.40(m,8H),4.88(m,2H),4.11(m,2H),3.92(m,2H),3.71(m,2H),3.33(s,3H),2.89(m, 6H),2.50(m,2H),2.31(m,4H),1.85-2.01(m,16H),1.57-1.63(m,14H),1.26-1.46(m,32H),0.89(m,12H); m / z 882(M+H)
[0130] Synthesis of VB224:
[0131] Compound 1k
[0132] Compound 1d (3 g, 6.9 mmol) and compound 1j (3.3 g, 6.9 mmol) were dissolved in a mixed solution of acetonitrile and THF. Anhydrous potassium carbonate (1.9 g, 13.8 mmol) and potassium iodide (1.1 g, 6.9 mmol) were added, and the mixture was heated to 70-80 °C and stirred overnight. The reaction solution was filtered and concentrated. The concentrate was diluted with dichloromethane, washed twice with water, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give 1.5 g of compound 1k (1.5 g, yellow oily liquid), in a yield of 26.4%.
[0133] Compound VB224
[0134] English name:
[0135] 2-((8-(((5Z,12Z)-heptadeca-5,12-dien-9-yl)oxy)-8-oxooctyl)(7-(((6Z,13Z)-nonadeca-6,13-dien-10-yl)oxy)-7-oxoheptyl)amino)ethyl 1-methylpiperidine-4-carboxylate
[0136] Chinese name: 2-((8-(((5Z,12Z)-heptadec-5,12-dien-9-yl)oxy)-8-oxooctyl)(7-(((6Z,13Z)-nonadecan-6,13-dien-10-yl)oxy)-7-oxoheptyl)amino)1-methylpiperidin-4-carboxylic acid ethyl ester
[0137] Compound 1k (1.5 g, 1.8 mmol) was dissolved in dichloromethane, and 1-methylpiperidin-4-carboxylic acid (0.26 g, 1.8 mmol) and DMAP (0.44 g, 3.6 mmol) were added. EDC (4.2 g, 2.2 mmol) was added dropwise at 0-10 °C. After the addition was complete, the mixture was brought to room temperature and stirred overnight. The reaction solution was washed once with water and once with dilute sodium bisulfate. The organic phase was dried over anhydrous sodium sulfate and concentrated to an oil. The oil was diluted with n-hexane, washed twice with acetonitrile, and the n-hexane phase was concentrated to dryness to give VB224 (1.0 g, yellow oily liquid). Yield: 57.9%.
[0138] H1-NMR(CDCl3)5.32-5.39(m,8H),4.88(m,2H),4.11(t,2H),2.87(m,2H),2.66(t,2H),2 .26(m,8H),1.98-2.05(m,18H),1.54-1.67(m,18H),1.25-1.41(m,36H),0.89(m,12H);MS m / z 954(M+H)
[0139] Based on VB219, VB230, VB233 and VB236, as well as their corresponding piperidine derivatives VB231, VB232, VB234, VB235, VB237 and VB238, were designed by adjusting the carbon chain length and the position of the unsaturated aliphatic bonds.
[0140] Synthesis of VB230: The synthetic route is analogous to that of VB219. The unbranched side chain is one carbon longer than that of VB219.
[0141] Synthesis of VB231: The molecular structure was designed based on VB230, and the synthetic route was analogous to that of VB221.
[0142] Synthesis of VB232: The molecular structure was designed based on VB230, and the synthetic route was analogous to that of VB222.
[0143] Synthesis of VB233: The synthetic route is analogous to that of VB219. The unbranched side chain is one carbon shorter than that of VB219.
[0144] Synthesis of VB234: The molecular structure was designed based on VB233, and the synthetic route was analogous to that of VB221.
[0145] Synthesis of VB235: The molecular structure was designed based on VB233, and the synthetic route was analogous to that of VB222.
[0146] Synthesis of VB236: The synthetic route is similar to that of VB233. They have the same molecular formula, but the positions of the unsaturated aliphatic bonds in the unbranched side chains are different, and the distance between the VB236 and the ester bond is reduced from 3 carbons to 1 carbon.
[0147] Synthesis of VB237: The molecular structure was designed based on VB236, and the synthetic route was analogous to that of VB221.
[0148] Synthesis of VB238: The molecular structure was designed based on VB236, and the synthetic route was analogous to that of VB222.
[0149] II. Preparation of Lipid Nanoparticles
[0150] As shown in Figure 1, typical lipid nanoparticles consist of four types of lipids. Ionizable cationic lipids, DSPC, mPEG-DMG-2K (PEG lipids), and cholesterol were dissolved in ethanol at a molar ratio of 50:10:1.5:38.5 to generate a 20 mM lipid mixture. The mRNA solution contained approximately 0.1 mg / ml mRNA in 25 mM pH 5.0 sodium acetate buffer. The lipid mixture and mRNA solution were mixed in a microfluidic chip at a flow rate of 1:3 and a total flow rate of 12 ml / min, as detailed in patent application 202410628754.5. This experiment used respiratory syncytial virus (RSVF) fusion protein mRNA as the target product.
[0151] III. Performance Testing
[0152] 1. Toxicity testing
[0153] We used the OSIRIS Property Explorer (https: / / www.cheminfo.org / flavor / cheminformatics / Utility / Property_explorer / index.html) to analyze the toxicity, cpKa (acidity coefficient), cLogP (lipophilicity), and LogS (solubility) of the newly designed lipid molecules and compared them with ionizable lipids used in FDA-approved nucleic acid drugs and vaccines.
[0154] 2. Ionizable cationic lipid pKa
[0155] pKa detection uses a fluorescent probe method, specifically including:
[0156] Apparent pKa is a key parameter for measuring the ionization degree and surface charge characteristics of lipid nanoparticles (LNPs), playing a crucial role in improving RNA encapsulation efficiency, promoting endosome escape, and cytoplasmic RNA release. We employed a fluorescent probe method to determine the pKa value of LNPs. This method involves binding LNPs to a pH-sensitive probe, measuring fluorescence intensity under different pH conditions, and plotting corresponding curves to determine the pKa. By detecting and optimizing the pKa value, we can effectively evaluate LNP performance, thereby improving the delivery efficiency of mRNA drugs.
[0157] 3. mRNA-LNP preparation and cell expression
[0158] The nitrogen-to-phosphorus ratio was obtained by calculating the ratio of LNP to mRNA concentrations. LNP particle size and PDI were measured using ZETASIZER (MALVERN PANALYTICAL, UK).
[0159] Encapsulation efficiency was measured using the Ribogreen assay.
[0160] The Ribogreen assay is a highly sensitive method for determining mRNA encapsulation efficiency. Based on the significant increase in fluorescence intensity after Ribogreen dye binds to nucleic acids, this method calculates encapsulation efficiency by comparing the fluorescence signals of mRNA before and after encapsulation. This method is simple to operate and highly sensitive, making it an important tool for evaluating the performance of LNP delivery systems and helping to optimize the preparation process and delivery efficiency of mRNA drugs.
[0161] Emax / Dose uses Hep3B cell ELISA detection method
[0162] The Hep3B cell ELISA assay is used to assess the expression level of the target protein after mRNA-LNP transfection. After Hep3B cells are transfected with mRNA-LNP and cultured for a period of time, the expression level of the target protein in the cells is detected using the principle of antigen-specific antibody binding in ELISA, thereby assessing the transfection efficiency and functional effect of mRNA-LNP in a real cell environment. The Emax and corresponding Dose are obtained through ELISA detection. Emax / Dose represents the efficacy produced per unit dose of the drug.
[0163] 4. Temperature stability test
[0164] LNPs were stored at four temperatures (-80℃, -20℃, 4℃, 25℃), and their particle size, PDI, encapsulation efficiency, and mRNA concentration were continuously monitored to assess their stability.
[0165] IV. Results and Analysis
[0166] 1. Toxicity test results
[0167] The toxicity and physicochemical properties of ionizable lipids are very important. We used the OSIRIS Property Explorer to analyze the toxicity, cpKa (acidity coefficient), cLogP (lipophilicity), and LogS (solubility) of the newly designed lipid molecules and compared them with ionizable lipids approved by the FDA for use in nucleic acid drugs and vaccines. The results are shown in Table 1.
[0168] Table 1. Results of four toxicity assessments: T1-T4, cpKa, cLogP, and LogS.
[0169] The toxicity analysis results (T1-T4) show that our newly designed VB219-VB224, VB230-VB235, and the two approved lipids MC3 and SM102 have no teratogenic, tumorigenic, irritant, or reproductive impact risks. VB236-VB238 have no teratogenic, tumorigenic, or reproductive impact risks, but have some irritant risk. ALC0315, approved for use in a nucleic acid vaccine, has no teratogenic, irritant, or reproductive impact risks, but has some tumorigenic risk.
[0170] The newly designed VB219-VB224, VB230-VB235 have similar cpKa (8-10), cLogP (14-17), and LogS (-8 to -11) to the ionized lipids of three approved nucleic acid drugs.
[0171] 2. Results of pKa and cpKa of ionizable cationic lipids
[0172] The pKa and cpKa results of ionizable cationic lipids are shown in Figure 2. The detected pKa is usually 2-3 pH units lower than the cpKa. The detected pKa of VB220, VB222, VB224, MC3, SM102, and ALC0315 is between 6.6 and 6.9, while VB219 reaches 7.02, which is higher than other ionizable cationic lipids.
[0173] The results indicate that the newly designed cationic lipid has similar charge characteristics to FDA-approved cationic lipids, with a slightly higher pKa. This results in VB219 carrying a stronger positive charge in acidic buffer systems, which is beneficial for binding mRNA and improving encapsulation efficiency and stability. Simultaneously, it remains uncharged in neutral physiological buffer systems, thus reducing cytotoxicity.
[0174] 3. mRNA-LNP cell expression results
[0175] The results of mRNA-LNP cell expression are shown in Tables 2, 3, 4, Figures 3, 8, and 9.
[0176] Table 2 Physicochemical properties and cell expression efficacy of VB219 LNP
[0177] Table 3 Physicochemical properties and cell expression efficacy of VB220 LNP
[0178] Table 4 Physicochemical properties and cell expression efficacy of VB222 LNP
[0179] In Table 2, F1 to F11 all use VB219, Ref is SM102; in Table 3, F1 to F4 all use VB220, Ref is SM102; in Table 3, F1 to F11 all use VB222, Ref is SM102.
[0180] The results in Table 2 and Figure 3 show that, using SM102 as the reference, which currently exhibits the best performance, VB219 can achieve 0.7-4.4 times the efficacy when expressed with different parameters. Using the optimized process parameters approved by the FDA for SM102, VB219 achieved higher expression efficacy at pH 4.0 and a nitrogen-to-phosphorus ratio of 8:1, being 3.3 times (45% and 50% IL) and 4.4 times (55% IL) higher than SM102. These process optimization results indicate that different cationic lipids require specific optimization of process parameters to achieve optimal efficacy.
[0181] VB220 achieved 0.6-1.4 times the efficacy compared to SM102, as shown in Table 3 and Figure 8; VB222 achieved 0.2-1.6 times the efficacy compared to SM102, as shown in Table 4 and Figure 9.
[0182] 4. Temperature stability test
[0183] The stability of VB219 is shown in Figures 4 to 7. Figure 4 shows the stability test results at -80℃, Figure 5 shows the stability test results at -20℃, Figure 6 shows the stability test results at 4℃, and Figure 7 shows the stability test results at 25℃.
[0184] Figures 4 through 7 show that LNPs were stored at four temperatures (-80℃, -20℃, 4℃, and 25℃), and the particle size, PDI, encapsulation efficiency, and mRNA concentration of LNPs were continuously monitored. During the one-month storage period, the LNP particle size remained at 70-90 nm, the PDI < 0.1, the encapsulation efficiency remained at 70%-100%, and the mRNA concentration remained at 50-60 μg / ml, demonstrating good temperature stability.
[0185] VB220 showed 0.6–1.4 times the potency of SM102, as shown in Table 3 and Figure 8; VB222 showed 0.2–1.6 times the potency of SM102, as shown in Table 4 and Figure 9; VB224 showed lower potency than SM102. Both VB222 and VB224 incorporate piperidine ring structures to further increase temperature stability, but the ring structure may affect the spatial binding of the amino group to RNA, thus resulting in lower potency than VB219.
[0186] To simultaneously improve efficacy and stability, combining VB219 / VB220 and VB222 / VB224 can generate new ionizable combinations including VB219-VB222, VB219-VB224, VB220-VB222, and VB220-VB224. Further assaying and optimization of other novel molecules can uncover more effective and stable ionizable lipids for efficient drug delivery.
[0187] The newly designed ionizable lipids can be widely used to prepare nanoparticles (LNPs) for delivering nanomedicines, including DNA (plasmids, aptamers), RNA (siRNA, ASO, mRNA), proteins (antibodies, enzymes, various protein drugs), and small molecules. mRNA-LNPs (including ordinary linear mRNA, self-replicating mRNA, and circular mRNA) can be widely used in various biomedical fields, including preventative vaccines, therapeutic vaccines, protein replacement, gene editing, and in vivo cell therapy, among other advanced medicines and therapies.
[0188] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.
Claims
1. A class of ionizable cationic lipids, characterized in that: It has the structure shown in Equation 1; Set 1 In this case, any three of R1, R2, R3, and R4 are saturated or unsaturated fatty chains, and the other is H. Among the three saturated or unsaturated fatty chains, at least two are unsaturated fatty chains; or, R1, R2, R3, and R4 are all saturated or unsaturated fatty chains, among which at least two are unsaturated fatty chains; the carbon chain length of all saturated or unsaturated fatty chains is 2-18. R5 is -C 1-3 OH, piperidine ring, or -C 1-3 Ester formed by the condensation of OH with piperidine carboxylic acid; The values of x and y are both 2-8.
2. The ionizable cationic lipid according to claim 1, characterized in that: Any three of R1, R2, R3, and R4 are unsaturated fatty acid chains, and the other is H; or, all of R1, R2, R3, and R4 are unsaturated fatty acid chains. Preferably, the ionizable cationic lipid has the structure shown in Formula 2. Formula 2 3. The ionizable cationic lipid according to claim 1, characterized in that: It is at least one of VB219, VB220, VB221, VB222, VB223, VB224, VB230, VB231, VB232, VB233, VB234, VB235, VB236, VB237 and VB238; VB219 VB220 VB221 VB222 VB223 VB224 VB230 VB231 VB232 VB233 VB234 VB235 VB236 VB237 VB238 4. The ionizable cationic lipid according to claim 1, characterized in that: It is a combination of at least one of VB219, VB220, VB230, VB233, and VB236 and at least one of VB221, VB222, VB223, VB224, VB231, VB232, VB234, VB235, VB237, and VB238; Preferably, it is a combination of VB219 and VB222, a combination of VB219 and VB224, a combination of VB220 and VB222, or a combination of VB220 and VB224.
5. A lipid nanoparticle, characterized in that: The invention includes an encapsulation material and a biopharmaceutical encapsulated within the encapsulation material, wherein the encapsulation material comprises an ionizable cationic lipid as described in any one of claims 1-4.
6. The lipid nanoparticles according to claim 5, characterized in that: The biopharmaceutical is at least one of DNA, RNA, protein, and small molecule drugs.
7. The lipid nanoparticles according to claim 6, characterized in that: The DNA is at least one of deoxyribonucleic acid fragments, plasmids, antisense DNA, and DNA aptamers; Preferably, the RNA is at least one of mRNA, circRNA, saRNA, siRNA, miRNA, antisense RNA, RNA aptamer, shRNA, and small activating RNA; Preferably, the mRNA is at least one of ordinary linear mRNA, self-replicating mRNA, and circular mRNA; Preferably, the protein is at least one of an antibody, an enzyme, a protein drug, or a functional polypeptide.
8. The lipid nanoparticles according to any one of claims 5-7, characterized in that: The lipid nanoparticles have a particle size of 30-500 nm.
9. A drug delivery kit, characterized in that: Includes at least one of the following components, (a) The ionizable cationic lipid according to any one of claims 1-4; (b) The lipid nanoparticles according to any one of claims 5-8.
10. The use of the ionizable cationic lipid according to any one of claims 1-4, the lipid nanoparticles according to any one of claims 5-8, or the kit according to claim 9 in the preparation of preventive vaccines, therapeutic vaccines, protein replacement drugs, gene editing drugs, or cell therapy drugs.