Glycerophosphorylethanolamine series compounds and lipid nanomedicine carriers including the same

Abstract

The application belongs to the field of nucleic acid drug delivery, and mainly relates to a glycerophosphoryl ethanolamine skeleton compound and a lipid nano drug carrier comprising the same. The glycerophosphoryl ethanolamine skeleton compound has a structure as shown in formula (I): The application modifies a dithiolane structure at the phosphate hydrophilic end of a glycerophosphoryl ethanolamine lipid, so that the dithiolane structure is more fully exposed on the surface of LNP, so that more dithiolane is exposed on the surface of the prepared LNP, and the endocytosis of mucosal cells to LNP can be further promoted. The glycerophosphoryl ethanolamine skeleton compound has a wide adjustment range in the LNP prescription, and can increase the content of dithiolane in LNP. The nucleic acid lipid nanoparticle composition and the pharmaceutical preparation provided by the application can be used for ocular delivery, pulmonary inhalation delivery and nasal spray of nucleic acid drugs, and provide a new choice for extrahepatic delivery of nucleic acid drugs.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid drug delivery, and mainly relates to a glycerol phosphoryl ethanolamine backbone compound and a lipid nanomedicine carrier including the compound. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Currently, lipid nanoparticles (LNPs) used for nucleic acid drug delivery mainly consist of four components: cationic / ionizable lipids, neutral cofactor phospholipids, cholesterol, and PEGylated lipids. These four components can be used to encapsulate nucleic acid drugs using microfluidic devices to obtain lipid nanoparticles. Three LNP-based nucleic acid drugs are currently on the market. The first LNP-based nucleic acid drug, Patisiran, was successfully launched in 2018. This drug is the first siRNA drug for treating hereditary thyroxine-mediated amyloidosis polyneuropathy. The other two are conditionally approved mRNA vaccines during the COVID-19 pandemic. In recent years, billions of doses of LNP-based mRNA drugs have been widely administered, preventing the harm of COVID-19 to healthy individuals, demonstrating the good safety and excellent immunogenicity of LNP technology in mRNA drug delivery.

[0004] LNPs exhibit high encapsulation rates for nucleic acid drugs, typically exceeding 80%, and can protect nucleic acids from degradation by various enzymes in the body. However, LNP-based nucleic acid drugs are primarily concentrated in the fields of liver disease and vaccines. Although a large number of nucleic acid drugs are currently in different stages of clinical trials, no nucleic acid drugs targeting other organs have yet entered clinical research.

[0005] Existing patent CN116212031A discloses a mucosal adhesive lipid material (DSPE-PEG-LA) composed of three parts: lipoic acid, polyethylene glycol, and phosphoryl ethanolamine phospholipid, and the chemical bonds linking these three parts. While this material can be constructed into a lipid nanoparticle (LNP) and further prepared into a nucleic acid drug with mucosal adhesion properties, the PEG chain segments are highly flexible. During LNP assembly, the hydrophobic lipoic acid is easily trapped within the hydrophobic core of the LNP, making it difficult for the dithiopental ring to be exposed on the LNP surface, thus affecting its function in promoting endocytosis. Furthermore, the preferred dosage of DSPE-PEG-LA in LNP formulations is generally between 1% and 5%, which significantly limits the content range of the dithiopental ring in the LNP. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a glycerol phosphoryl ethanolamine (LNP) backbone compound and a lipid nanoparticle carrier comprising the same. This invention designs a novel compound for nucleic acid drug delivery. This compound contains a dithiopentane structure, which can chemically react with thiol groups on the cell surface, thereby promoting LNP endocytosis and enhancing the ability of nucleic acid drugs to enter cells. This invention modifies the glycerol phosphoryl ethanolamine lipid with the dithiopentane structure at the hydrophilic phosphate end. Compared to DSPE-PEG-LA, the glycerol phosphoryl ethanolamine lipid material with the dithiopentane structure designed in this invention allows for more complete exposure of the dithiopentane structure on the LNP surface. LNPs prepared using the lipid material of this invention will have a more exposed dithiopentane surface, further promoting LNP endocytosis by mucosal cells. The glycerol phosphoryl ethanolamine backbone compound of this invention has a wide range of adjustment in LNP formulations, which can increase the content of dithiopentane in LNPs. The nucleic acid lipid nanoparticle composition and pharmaceutical formulation of this invention can be used for ocular delivery, pulmonary inhalation delivery, and nasal spray of nucleic acid drugs, providing a new option for extrahepatic delivery of nucleic acid drugs.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] In a first aspect, the present invention provides a glycerol phosphoryl ethanolamine skeleton compound having a structure as shown in formula (I):

[0009]

[0010] Where R1 and R2 are each independently C 1-24 Alkyl or C 1-24 One of the alkenyl groups, where A1 is methylene, 1,2-ethylenediyl, 1,3-propadiyl, 1-methyl-1,2-ethylenediyl, 1,2-isopropyl, 1-methyl-1,3-propadiyl, 2-methyl-1,3-propadiyl, 1,4-butadiyl, 1,5-pentadiyl, 1-methyl-1,4-butadiyl, 2-methyl-1,4-butadiyl, 3-methyl-1,4-butadiyl, 2,2-dimethyl-1,3-butadiyl, 1,6-hexadiyl, 1-methyl-1,5-pentadiyl, 2-methyl-1,5-pentadiyl, 3-methyl-1,5-pentadiyl, 1-ethyl-1,4-butadiyl, or 2-ethyl-1,4-butadiyl.

[0011] In a second aspect, the present invention provides a method for preparing the glycerol phosphoryl ethanolamine skeleton compound as described in the first aspect, comprising the following steps:

[0012] The compound shown in formula (II) was dissolved in chloroform by stirring. Carbonyl diimidazole was added and stirred in the dark. Then the compound shown in formula (III) was added and reacted in the dark. After the reaction was completed, the chloroform was removed by rotary evaporation and the glycerol phosphoryl ethanolamine skeleton compound was obtained by column chromatography.

[0013]

[0014]

[0015] Preferably, the molar ratio of the compound shown in formula (II), carbonyl diimidazole and the compound shown in formula (III) is 1:0.75-0.85:1.05-1.15.

[0016] Preferably, the reaction temperature in the dark is 29–31°C, and the reaction time is 22–26 h.

[0017] Thirdly, the present invention provides a lipid nanomedicine carrier comprising a first lipid compound and a second lipid compound, wherein the first lipid compound comprises one or more of the glycerophosphorylethanolamine skeleton compound as described in the first aspect or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, and non-covalent complex, and the second lipid compound comprises cationic or ionizable lipids, sterols, and amphiphilic lipids.

[0018] Preferably, the cationic or ionizable lipid includes one or more of DLinDMA, DODMA, DLin-MC2-MPZ, DLin-KC2-DMA, DOTAP, C12-200, SM-102 and ALC-0315;

[0019] The amphiphilic lipids include one or more of PEG-DSPE, PEG-PE, PEG-DMG, PEG-C14, PEG-c-DMA, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, Tween-20, Tween-80, PEG-DPG, PEG-s-DMG, PEG-c-DOMG, and GalNAc-PEG-DSG.

[0020] Preferably, the molar ratio of the first lipid compound, the cationic or ionizable lipid, the sterol, and the amphiphilic lipid is 2–20:10–65:20–55:0.2–20.

[0021] Preferably, the molar ratio of the first lipid compound, the cationic or ionizable lipid, the sterol, and the amphiphilic lipid is 5–10:30–50:35–55:0.5–10.

[0022] Fourthly, the present invention provides a nucleic acid lipid nanoparticle composition with a particle size of 30-300 nm, comprising a lipid nanomedicine carrier and a nucleic acid drug as described in the third aspect, wherein the mass ratio of the lipid nanomedicine carrier to the nucleic acid drug is 1:1 to 20.

[0023] Preferably, the nucleic acid drug comprises one or more of DNA, siRNA, mRNA, dsRNA, antisense nucleic acid, microRNA, antisense microRNA, antagomir, microRNA inhibitor, microRNA activator, and immunostimulatory nucleic acid.

[0024] Fifthly, the present invention provides a pharmaceutical formulation comprising the nucleic acid lipid nanoparticle composition as described in the fourth aspect, a pharmaceutically acceptable buffer salt, an osmotic pressure regulator, and a suspending agent.

[0025] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0026] In the glycerol phosphoryl ethanolamine skeleton compound of the present invention, the dithiopentane is located at the hydrophilic phosphate end. During the assembly of LNP, the dithiopentane is exposed to a large amount on the surface of LNP, making it easier to interact with the thiol groups on the cell surface.

[0027] The glycerol phosphoryl ethanolamine skeleton compound of the present invention has a wide range of regulation in LNP formulations, and can regulate the content of dithiopentane in LNP within a large range to promote the endocytosis of LNP by mucosal cells.

[0028] Using eGFP plasmids as model nucleic acid drugs, nucleic acid lipid nanoparticle compositions based on glycerophosphorylethanolamine backbone compounds exhibit excellent transfection efficacy. Drug formulations based on these compositions show broad application prospects in the pulmonary inhalation, ocular, and nasal administration of nucleic acids.

[0029] The glycerol phosphoryl ethanolamine skeleton compound provided by this invention has a simple synthetic route, a convenient purification process, and excellent market prospects. Attached Figure Description

[0030] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0031] Figure 1 The particle size distribution diagram of the nucleic acid lipid nanoparticle composition loaded with siPLK1 small interfering RNA in Example 14 is shown.

[0032] Figure 2 The particle size distribution diagram of the nucleic acid lipid nanoparticle formulation loaded with siPLK1 small interfering RNA in Example 16 is shown.

[0033] Figure 3 The image shows the distribution of Cy3-labeled lipid nanoparticles in A549 cells in Example 18. Blue represents DAPI-stained cell nuclei, red represents Cy3-labeled LNPs, and green represents Lysotracker green-labeled lysosomes. The left image shows lipid nanoparticles containing compound 7, and the right image shows LNP lipid nanoparticles without the dithiopentane structure.

[0034] Figure 4 The image shows a fluorescence microscope image of A549 cells transfected with the nucleic acid lipid nanoparticle composition containing eGFP plasmid in Example 19.

[0035] Figure 5 This is a particle size distribution diagram of the nucleic acid lipid nanoparticle drug formulation loaded with siPLK1 small interfering RNA, as shown in Example 20.

[0036] Figure 6 This is a transmission electron microscope image of lipid nanoparticle 1 in Example 21;

[0037] Figure 7 This is a transmission electron microscope image of lipid nanoparticle 2 in Example 21. Detailed Implementation

[0038] Unless otherwise stated, the following terms have the following meanings:

[0039] The term "pharmaceutically acceptable salt" refers to a salt of a compound that is substantially non-toxic to humans. Pharmaceutically acceptable salts generally include (but are not limited to) salts formed by the reaction of the compounds of this invention with pharmaceutically acceptable inorganic / organic acids or inorganic / organic bases; such salts are also known as acid addition salts or base addition salts. Common inorganic acids include (but are not limited to) hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc.; common organic acids include (but are not limited to) trifluoroacetic acid, citric acid, maleic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, acetic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.; common inorganic bases include (but are not limited to) sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, etc.; and common organic bases include (but are not limited to) diethylamine, triethylamine, ethylaminobutanol, etc.

[0040] The term "stereoisomer" (or "optical isomer") refers to a stable isomer that has a perpendicular asymmetric plane due to having at least one chiral element (including a chiral center, chiral axis, chiral plane, etc.), thereby enabling the rotation of plane-polarized light. Since the compounds of this invention contain asymmetric centers and other chemical structures that may lead to stereoisomerism, this invention also includes these stereoisomers and mixtures thereof. Because the compounds of this invention and their salts include asymmetric carbon atoms, they can exist as single stereoisomers, racemates, or mixtures of enantiomers and diastereomers. Typically, these compounds can be prepared as racemic mixtures. However, if desired, such compounds can be prepared or isolated to obtain pure stereoisomers, i.e., single enantiomers or diastereomers, or mixtures enriched with single stereoisomers (purity ≥98%, ≥95%, ≥93%, ≥90%, ≥88%, ≥85%, or ≥80%). The single stereoisomer of a compound is synthesized from an optically active starting material containing the desired chiral center, or obtained by preparing a mixture of enantiomers followed by separation or resolution, for example, by converting it into a mixture of diastereomers followed by separation or recrystallization, chromatographic treatment, using chiral resolving reagents, or by direct separation of the enantiomers on a chiral chromatographic column. Starting compounds with specific stereochemistry are commercially available or can be prepared according to the methods described below and then resolved by methods well known in the art.

[0041] The term "tautomer" (or "tautomer form") refers to structural isomers with different energies that can interconvert through a low energy barrier. If tautomerism is possible (e.g., in solution), chemical equilibrium can be achieved in the tautomer. For example, proton tautomers (or proton transfer tautomers) include (but are not limited to) interconversions via proton transfer, such as keto-enol isomerization, imine-enamine isomerization, amide-imine alcohol isomerization, etc. Unless otherwise stated, all tautomer forms of the compounds of this invention are within the scope of this invention.

[0042] The term "solvent" refers to a substance formed by the combination of a compound of the present invention or a pharmaceutically acceptable salt thereof with at least one solvent molecule through non-covalent intermolecular forces. Common solvates include (but are not limited to) hydrates, ethanol compounds, acetone compounds, etc.

[0043] The term "chelate" refers to a complex with a cyclic structure, obtained through the chelation of two or more ligands with the same metal ion to form a chelate ring.

[0044] The term "non-covalent complex" refers to a complex formed through the interaction of a compound with another molecule, where no covalent bond is formed between the two molecules. Complexation can occur, for example, through van der Waals interactions, hydrogen bonding, and electrostatic interactions (also known as ionic bonding).

[0045] The term "independently" means that at least two groups (or ring systems) in a structure with the same or similar value ranges can have the same or different meanings under specific circumstances. For example, substituent R1 and substituent R2 can be dodecane-1-yl, octadecane-1-yl, or tetradecane-1-yl independently. For instance, when substituent R1 is dodecane-1-yl, substituent R2 can be dodecane-1-yl, octadecane-1-yl, or tetradecane-1-yl; similarly, when substituent R2 is dodecane-1-yl, substituent R1 can also be dodecane-1-yl, octadecane-1-yl, or tetradecane-1-yl.

[0046] The term “optional” or “optionally” means that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0047] The terms “contain” and “include” are used in their open, non-restrictive sense.

[0048] The term "alkyl" refers to a monovalent, straight-chain or branched alkane group consisting only of carbon and hydrogen atoms, without unsaturation, and connected to other segments by a single bond, including (but not limited to) methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, and tert-butyl. For example, "C1-24 alkyl" refers to a saturated monovalent, straight-chain or branched hydrocarbon group containing 1 to 24 carbon atoms.

[0049] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0050] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0051] In this invention, the "equivalent (eq)" ratio refers to the molar ratio of the solvent or the drug.

[0052] In this invention, "appropriate amount" means that the amount of solvent or reagent added can be adjusted within a large range and has little impact on the synthesis result, and no specific limitation is required.

[0053] In the following examples, all solvents and reagents used were of analytical or chemical purity; all solvents were redistilled before use; and all anhydrous solvents were processed according to standard or literature methods.

[0054] Example 1: Synthesis of Compound 1

[0055] Lipoic acid (1.0 eq) was dissolved in an appropriate amount of chloroform and stirred until dissolved. Carbonyl diimidazole (1.1 eq) was added and stirred at room temperature in the dark for 30 minutes. Then, 1,2-heptaylphosphatidylethanolamine (0.8 eq) was added, and the mixture was reacted at 30°C in the dark for 24 hours. After the reaction was complete, chloroform was removed by rotary evaporation, and column chromatography yielded compound 1.

[0056]

[0057] 1 H NMR (400MHz, CDCl3): δ0.86-0.90 (t, 6H), 1.27-1.30 (d, 14H), 1.51-1.66 (m, 8H), 1.73 (s, 1H), 1.97 (s, 1H), 2.01-2.36 (m, 8H), 2.59 (s, 1H), 3.46 (t, 2H), 4.17-4.38 (m, 6H), 5.27 (m, 1H), 6.26 (s, 1H).

[0058] Example 2: Synthesis of Compound 2

[0059] Lipoic acid (1.0 eq) was dissolved in an appropriate amount of chloroform and stirred until dissolved. Carbonyl diimidazole (1.1 eq) was added, and the mixture was stirred at room temperature in the dark for 30 minutes. Then, 1,2-octanoylphosphatidylethanolamine (0.8 eq) was added, and the mixture was reacted at 30°C in the dark for 24 hours. After the reaction was complete, chloroform was removed by rotary evaporation, and column chromatography yielded compound 2.

[0060]

[0061] 1 H NMR (400MHz, CDCl3): δ0.83-0.92 (t, 6H), 1.25-1.31 (d, 18H), 1.46-1.62 (m, 8H), 1.70 (s, 1H), 1.93 (s, 1H), 2.05-2.32 (m, 8H), 2.66 (s, 1H), 3.36 (t, 2H), 4.15-4.31 (m, 6H), 5.37 (m, 1H), 6.86 (s, 1H).

[0062] Example 3: Synthesis of Compound 3

[0063] Lipoic acid (1.0 eq) was dissolved in an appropriate amount of chloroform and stirred until dissolved. Carbonyl diimidazole (1.1 eq) was added and stirred at room temperature in the dark for 30 minutes. Then, 1,2-nonanoylphosphatidylethanolamine (0.8 eq) was added, and the mixture was reacted at 30°C in the dark for 24 hours. After the reaction was complete, chloroform was removed by rotary evaporation, and column chromatography yielded compound 3.

[0064]

[0065] 1 H NMR (400MHz, CDCl3): δ0.82-0.96 (t, 6H), 1.22-1.36 (d, 22H), 1.45-1.58 (m, 8H), 1.67 (s, 1H), 1.83 (s, 1H), 2.12-2.37 (m, 8H), 2.62 (s, 1H), 3.41 (t, 2H), 4.21-4.37 (m, 6H), 5.27 (m, 1H), 5.86 (s, 1H).

[0066] Example 4: Synthesis of Compound 4

[0067] Lipoic acid (1.0 eq) was dissolved in an appropriate amount of chloroform and stirred until dissolved. Carbonyl diimidazole (1.1 eq) was added and stirred at room temperature in the dark for 30 minutes. Then, 1,2-dodecanoylphosphatidylethanolamine (0.8 eq) was added, and the mixture was reacted at 30°C in the dark for 24 hours. After the reaction was complete, chloroform was removed by rotary evaporation, and column chromatography yielded compound 4.

[0068]

[0069] 1 H NMR (400MHz, CDCl3): δ0.85-0.96 (t, 6H), 1.22-1.36 (d, 34H), 1.45-1.58 (m, 8H), 1.67 (s, 1H), 1.83 (s, 1H), 2.12-2.37 (m, 8H), 2.62 (s, 1H), 3.41 (t, 2H), 4.21-4.37 (m, 6H), 5.27 (m, 1H), 5.86 (s, 1H).

[0070] Example 5: Synthesis of Compound 5

[0071] Lipoic acid (1.0 eq) was dissolved in an appropriate amount of chloroform and stirred until dissolved. Carbonyl diimidazole (1.1 eq) was added and stirred at room temperature in the dark for 30 minutes. Then, 1,2-tetradecanoylphosphatidylethanolamine (0.8 eq) was added, and the mixture was reacted at 30°C in the dark for 24 hours. After the reaction was complete, chloroform was removed by rotary evaporation, and column chromatography yielded compound 5.

[0072]

[0073] 1 H NMR (400MHz, CDCl3): δ0.87-0.93 (t, 6H), 1.24-1.32 (d, 42H), 1.40-1.52 (m, 8H), 1.63 (s, 1H), 1.73 (s, 1H), 2.16-2.31 (m, 8H), 2.52 (s, 1H), 3.46 (t, 2H), 4.26-4.32 (m, 6H), 5.24 (m, 1H), 5.66 (s, 1H).

[0074] Example 6: Synthesis of Compound 6

[0075] Lipoic acid (1.0 eq) was dissolved in an appropriate amount of chloroform and stirred until dissolved. Carbonyl diimidazole (1.1 eq) was added and stirred at room temperature in the dark for 30 minutes. Then, dipalmitoylphosphatidylethanolamine (0.8 eq) was added, and the mixture was reacted at 30°C in the dark for 24 hours. After the reaction was complete, chloroform was removed by rotary evaporation, and column chromatography yielded compound 6.

[0076]

[0077] 1 H NMR (400MHz, CDCl3): δ0.82-0.87 (t, 6H), 1.27-1.36 (d, 50H), 1.46-1.57 (m, 8H), 1.66 (s, 1H), 1.75 (s, 1H), 2.23-2.37 (m, 8H), 2.58 (s, 1H), 3.44 (t, 2H), 4.23-4.37 (m, 6H), 5.26 (m, 1H), 5.54 (s, 1H).

[0078] Example 7: Synthesis of Compound 7

[0079] Lipoic acid (1.0 eq) was dissolved in an appropriate amount of chloroform and stirred until dissolved. Carbonyl diimidazole (1.1 eq) was added and stirred at room temperature in the dark for 30 minutes. Then, 1-palmitoyl-2-oleoylethanolamine (0.8 eq) was added, and the mixture was reacted at 30°C in the dark for 24 hours. After the reaction was complete, chloroform was removed by rotary evaporation, and column chromatography yielded compound 7.

[0080]

[0081] 1H NMR (400MHz, CDCl3): δ0.83-0.87 (t, 6H), 1.21-1.36 (d, 48H), 1.43-1.58 (m, 8H), 1.66 (s, 1H), 1.78 (s, 1H) ), 2.19-2.40(m, 8H), 2.56(s, 1H), 3.49(t, 2H), 4.23-4.30(m, 6H), 5.24(m, 1H), 5.45(m, 2H), 5.67(s, 1H).

[0082] Example 8: Synthesis of Compound 8

[0083] Lipoic acid (1.0 eq) was dissolved in an appropriate amount of chloroform and stirred until dissolved. Carbonyl diimidazole (1.1 eq) was added and stirred at room temperature in the dark for 30 minutes. Then, 1,2-dioleoyl-SN-glycerol-3-phosphorylethanolamine (0.8 eq) was added, and the mixture was reacted at 30°C in the dark for 24 hours. After the reaction was complete, chloroform was removed by rotary evaporation, and column chromatography yielded compound 8.

[0084]

[0085] 1 H NMR (400MHz, CDCl3): δ0.86-0.89 (t, 6H), 1.22-1.36 (d, 48H), 1.40-1.52 (m, 8H), 1.63 (s, 1H), 1.73 (s, 1H) ), 2.16-2.31(m, 8H), 2.52(s, 1H), 3.46(t, 2H), 4.26-4.32(m, 6H), 5.29(m, 1H), 5.65(m, 4H), 5.86(s, 1H).

[0086] Example 9: Synthesis of Compound 9

[0087] Lipoic acid (1.0 eq) was dissolved in an appropriate amount of chloroform and stirred until dissolved. Carbonyl diimidazole (1.1 eq) was added, and the mixture was stirred at room temperature in the dark for 30 minutes. Then, (0.8 eq) of 1-stearoyl-2-linoleic acid-sn-glycerol-3-phosphoethanolamine was added, and the mixture was reacted at 30°C in the dark for 24 hours. After the reaction was complete, chloroform was removed by rotary evaporation, and column chromatography yielded compound 9.

[0088]

[0089] 1H NMR (400MHz, CDCl3): δ0.83-0.87(t, 6H), 1.24-1.32(d, 50H), 1.44-1.56(m, 8H), 1.67(s, 1H), 1.72(s, 1H ), 2.14-2.36(m, 8H), 2.53(s, 1H), 3.42(t, 2H), 4.24-4.36(m, 6H), 5.16(m, 1H), 5.62(m, 2H), 5.72(s, 1H).

[0090] Example 10: Synthesis of Compound 10

[0091] 1.0 eq of 2-(1,2-dithio-3-yl)acetic acid was dissolved in an appropriate amount of chloroform and stirred until dissolved. 1.1 eq of carbonyl diimidazole was added and stirred at room temperature in the dark for 30 minutes. Then, 0.8 eq of 1,2-heptanylphosphatidylethanolamine was added, and the reaction was carried out at 30°C in the dark for 24 hours. After the reaction was complete, the chloroform was removed by rotary evaporation, and column chromatography yielded compound 10.

[0092]

[0093] 1 H NMR (400MHz, CDCl3): δ0.82-0.88 (t, 6H), 1.31-1.33 (d, 24H), 1.54-1.66 (m, 4H), 1.74 (s, 1H), 1.9 6(s, 1H), 2.33-2.54(m, 5H), 3.46-3.48(s, 2H), 4.12-4.36(m, 6H), 5.26-5.28(m, 1H), 5.62(m, 2H).

[0094] Example 11: Synthesis of Compound 11

[0095] 2-(1,2-dithio-3-yl)propionic acid (1.0 eq) was dissolved in an appropriate amount of chloroform and stirred until dissolved. Carbonyl diimidazole (1.1 eq) was added, and the mixture was stirred at room temperature in the dark for 30 minutes. Then, 1-stearoyl-2-linoleic acid-sn-glycerol-3-phosphate ethanolamine (0.8 eq) was added, and the mixture was reacted at 30°C in the dark for 24 hours. After the reaction was complete, the chloroform was removed by rotary evaporation, and column chromatography yielded compound 11.

[0096]

[0097] 1H NMR (400MHz, CDCl3): δ0.82-0.85 (t, 6H), 1.22-1.30 (d, 46H), 1.41-1.52 (m, 8H), 1.66 (s, 1H), 1.70 (s, 1H) ), 2.11-2.32(m, 8H), 2.51(s, 1H), 3.46(t, 2H), 4.27-4.34(m, 6H), 5.18(m, 1H), 5.62(m, 2H), 5.81(s, 1H).

[0098] Example 12: Synthesis of Compound 12

[0099] 2-(1,2-dithio-3-yl)valeric acid (1.0 eq) was dissolved in an appropriate amount of chloroform and stirred until dissolved. Carbonyl diimidazole (1.1 eq) was added and stirred at room temperature in the dark for 30 minutes. Then, (0.8 eq) dioleoylphosphatidyl-glycerol-3-phosphate ethanolamine was added, and the reaction was carried out at 30°C in the dark for 24 hours. After the reaction was completed, chloroform was removed by rotary evaporation, and column chromatography yielded compound 12.

[0100]

[0101] 1 H NMR (400MHz, CDCl3): δ0.85-0.88 (t, 6H), 1.21-1.37 (d, 46H), 1.38-1.53 ​​(m, 8H), 1.61 (s, 1H), 1.70 (s, 1H) ), 2.15-2.33(m, 8H), 2.50(s, 1H), 3.41(t, 2H), 4.20-4.27(m, 6H), 5.25(m, 1H), 5.65(m, 4H), 5.76(s, 1H).

[0102] Example 13: Preparation of lipid nanoparticles

[0103] Compounds 7, 8, 9, and 10 were dissolved in ethanol at a molar ratio of 10:38:49:3 (to obtain a lipid solution with a concentration of 20 mg / mL based on the total weight of lipids). PBS was used as the aqueous phase, with a volume ratio of 1:3 between the two solutions (where the equivalent volume of the ethanol solution was 1 and the equivalent volume of the aqueous solution was 3). The two phases were rapidly mixed using microfluidic technology, and the ethanol was removed by dialyzing to obtain lipid nanoparticles.

[0104] Example 14: Preparation of Nucleic Acid Lipid Nanoparticle Composition

[0105] Compounds 7, 8, 9, and 10 were dissolved in ethanol at a molar ratio of 10:38:49:3 (to obtain a lipid solution with a concentration of 30 mg / mL based on the total weight of lipids). siPLK1 small interfering RNA was dissolved in a 10 mM citrate buffer solution at pH 4.0 at a volume ratio of 1:3 (where the ethanol equivalent was 1 and the aqueous solution equivalent was 3). The two phases were rapidly mixed using microfluidic technology, and the buffer environment was replaced with PBS at pH 7.4 by dialysis to remove ethanol, yielding the nucleic acid lipid nanoparticle composition. Figure 1 As shown, the nucleic acid lipid nanoparticle compositions prepared based on compounds 7, 8, 9, and 10 have similar particle size distributions.

[0106] Example 15: Preparation of Nucleic Acid Lipid Nanoparticle Drug Formulation

[0107] Compounds 7, 8, 9, and 10 were dissolved in ethanol at a molar ratio of 10:38:49:3 (to obtain a lipid solution with a concentration of 20 mg / mL based on the total weight of lipids). The eGFP plasmid was dissolved in a 10 mM citrate buffer solution at pH 4.0 at a volume ratio of 1:3 (where the ethanol solution had an equivalent volume of 1 and the aqueous solution had an equivalent volume of 3). The two phases were rapidly mixed using a collision mixer, and the buffer environment was replaced with PBS at pH 7.4 by dialysis. Sodium chloride was added to adjust the osmotic pressure, and ethyl cellulose was added as a suspending agent to obtain the nucleic acid lipid nanoparticle drug formulation.

[0108] Example 16: Preparation of Nucleic Acid Lipid Nanoparticle Drug Formulation

[0109] Compounds 7, 8, 9, and 10 were dissolved in ethanol at a molar ratio of 10:38:49:3 (to obtain a lipid solution with a concentration of 20 mg / mL based on the total weight of lipids). siPLK1 small interfering RNA was dissolved in a 10 mM citrate buffer solution at pH 4.0 at a volume ratio of 1:3 (where the ethanol equivalent was 1 and the aqueous solution equivalent was 3). The two phases were rapidly mixed using microfluidic technology, and the buffer environment was replaced with PBS at pH 7.4 by dialysis. Glycerol was added to adjust the osmotic pressure, and hydroxypropyl methylcellulose was added as a suspending agent to obtain the nucleic acid lipid nanoparticle drug formulation. The particle size distribution is shown in the figure. Figure 2 As shown.

[0110] Example 17: Preparation of Nucleic Acid Lipid Nanoparticle Drug Formulation

[0111] Compounds 7, 8, 9, and 10 were dissolved in ethanol at a molar ratio of 10:38:49:3 (to obtain a lipid solution with a concentration of 20 mg / mL based on the total weight of lipids). The eGFP plasmid was dissolved in a 10 mM citrate buffer solution at pH 4.0, with a volume ratio of 1:3 (where the ethanol equivalent was 1 and the aqueous solution equivalent was 3). The two phases were rapidly mixed using a microfluidic jet, and the buffer environment was replaced with a HEPES buffer solution at pH 7.4 using dialysis. Glucose was added to adjust the osmotic pressure, and polyvinyl alcohol was added as a suspending agent to obtain the nucleic acid lipid nanoparticle drug formulation.

[0112] Example 18: Laser confocal experiment on lipid nanoparticles

[0113] Compound 7 was dissolved in ethanol at a molar ratio of 10:38:49:3 (to obtain a lipid solution with a concentration of 20 mg / mL based on the total weight of lipids). PBS was used as the aqueous phase, with a volume ratio of 1:3 for both solutions (where the ethanol equivalent was 1 and the aqueous solution equivalent was 3). The two phases were rapidly mixed using microfluidic technology, and the ethanol was removed by dialyzing to obtain lipid nanoparticle 1. Lipid nanoparticle 2 was prepared by replacing compound 7 with DSPC using the same formulation and process. Two types of lipid nanoparticles were co-incubated with A549 cells for 0.5, 1, and 2 hours, respectively. The samples were then washed with PBS, and lysosomes were stained with Lystracker-Green for 1 hour. Cells were then fixed with 4% paraformaldehyde, and finally, the nuclei were stained with DAPI. Bright-field microscopy was used to observe the cells, nuclei, lysosomes, and samples. Confocal microscopy revealed that the lipids containing the dithiopentane structure designed in this invention promoted the endocytosis of lipid nanoparticles by A549 cells and exhibited lysosomal escape effects. See the laser confocal images below. Figure 3 Lipid nanoparticles were labeled with Cy3 to observe their distribution in A549 cells. DAPI staining was used to stain the cells, and Lysotracker green-labeled lysosomes were used as a reference. The left side shows lipid nanoparticle 1, and the right side shows lipid nanoparticle 2. Figure 3 As shown, blue represents DAPI-stained cell nuclei, red represents Cy3-labeled LNPs, and green represents Lysotracker green-labeled lysosomes. LNPs with dithiopental rings can significantly enhance the ability of LNPs to enter cells. Confocal results show that LNPs are distributed around the nuclei of A549 cells, indicating that A549 cells can effectively internalize LNPs and have a certain ability to escape from lysosomes.

[0114] Example 19: eGFP lipid nanoparticle transfection experiment

[0115] Compound 7: cholesterol: ALC-0315 (((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)): Cy3-PEG-DSPE (polyethylene glycol-distearate phosphoryl ethanolamine) was dissolved in ethanol at a molar ratio of 10:38:49:3 (resulting in a lipid solution with a concentration of 20 mg / mL based on the total weight of lipids). Citric acid at pH 4.0 and a concentration of 100 mM was used as the aqueous phase, with an eGFP concentration of 0.2 mg / mL. The volume ratio of the two solutions was 1:3 (where the ethanol solution had an equivalent volume of 1 and the aqueous solution had an equivalent volume of 3). The two phases were rapidly mixed using microfluidic technology, and the ethanol was removed by dialyzing to obtain lipid nanoparticles loaded with eGFP. After incubating A549 cells with lipid nanoparticles loaded with eGFP for 1 hour, the samples were washed with PBS to remove the residues. Intracellular fluorescence was observed under a fluorescence microscope. The fluorescence microscopy showed that almost all A549 cells were successfully transfected with the eGFP plasmid. (See fluorescence images below.) Figure 4 .

[0116] Example 20: siPLK1 lipid nanoparticle protein inhibition experiment

[0117] Compound 7 was dissolved in ethanol at a molar ratio of 10:38:49:3 (to obtain a lipid solution with a concentration of 20 mg / mL based on the total weight of lipids). A 100 mM citric acid solution at pH 4.0 was used as the aqueous phase, with a siPLK1 concentration of 0.2 mg / mL. The volume ratio of the two solutions was 1:3 (where the ethanol equivalent was 1 and the aqueous equivalent was 3). The two phases were rapidly mixed using microfluidic technology, and the ethanol was removed by dialyzing to obtain lipid nanoparticles loaded with siPLK1. Lipid nanoparticle 2 was prepared by replacing compound 7 with DSPC using the same formulation and process. Two types of lipid nanoparticles encapsulating siPLK1 were incubated with A549 cells for 1 hour, followed by washing with PBS to remove the samples. Using β-actin protein as a reference, intracellular PLK1 protein levels were detected by protein electrophoresis. The electrophoresis results showed that the siPLK1-encapsulated lipid nanoparticles inhibited PLK1 protein expression in A549 cells. (See the electrophoresis images below.) Figure 5 .

[0118] Example 21: Preparation of siPLK1 lipid nanoparticle transmission electron microscopy samples

[0119] Compound 7 was dissolved in ethanol at a molar ratio of 10:38:49:3 (to obtain a lipid solution with a concentration of 20 mg / mL based on the total weight of lipids). A 100 mM citric acid solution at pH 4.0 was used as the aqueous phase, with a siPLK1 concentration of 0.2 mg / mL. The volume ratio of the two solutions was 1:3 (where the ethanol equivalent was 1 and the aqueous equivalent was 3). The two phases were rapidly mixed using microfluidic technology, and the ethanol was removed by dialyzing to obtain lipid nanoparticles loaded with siPLK1. Lipid nanoparticle 2 was prepared by replacing compound 7 with DSPC using the same formulation and process. The two types of lipid nanoparticles were respectively dropped onto two copper electron microscope (TEM) grids. After standing for 3 minutes, the liquid was absorbed from the back of the grid using filter paper. Then, a drop of phosphotungstic acid staining solution was added, and the grids were left to stand for 3 minutes. The liquid was then absorbed from the back of the grid using filter paper. Finally, a drop of purified water was added, and the liquid was absorbed from the back of the grid using filter paper to clean it. The prepared TEM samples were stored in the dark for observation. TEM images are shown below. Figure 4 , Figure 5 The dithiopentane structure readily complexes with heavy metal ions. Phosphotungstic acid was used as a staining agent for transmission electron microscopy (TEM). After staining with phosphotungstic acid, both the nucleic acid-lipid nanoparticle composition prepared in this example and the nucleic acid-lipid nanoparticle composition prepared using DSPE-PEG-LA material were photographed under TEM. Figure 6 As shown, the LNP in this embodiment exhibits clear staining in the electron microscope images due to the complexation with phosphotungstic acid. Figure 7 As shown, the LNP prepared using DSPE-PEG-LA material has a darker electron microscope image because the amount of dithiopentane exposed on the LNP surface is relatively small.

[0120] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A glycerol phosphoryl ethanolamine skeleton compound, characterized in that, It has a structure as shown in equation (I): Equation (I); Where R1 and R2 are each independently C 1-24 Alkyl or C 1-24 One of the alkenyl groups, where A1 is methylene, 1,2-ethylenediyl, 1,3-propanediyl, 1-methyl-1,2-ethylenediyl, 1-methyl-1,3-propanediyl, 2-methyl-1,3-propanediyl, 1,4-butanediyl, 1,5-pentanediyl, 1-methyl-1,4-butanediyl, 2-methyl-1,4-butanediyl, 3-methyl-1,4-butanediyl, 1,6-hexanediyl, 1-methyl-1,5-pentanediyl, 2-methyl-1,5-pentanediyl, 3-methyl-1,5-pentanediyl, 1-ethyl-1,4-butanediyl, or 2-ethyl-1,4-butanediyl.

2. A method for preparing the glycerol phosphoryl ethanolamine skeleton compound as described in claim 1, characterized in that, Includes the following steps: The compound shown in formula (II) was dissolved in chloroform by stirring. Carbonyl diimidazole was added and stirred in the dark. Then the compound shown in formula (III) was added and reacted in the dark. After the reaction was completed, the chloroform was removed by rotary evaporation and the glycerol phosphoryl ethanolamine skeleton compound was obtained by column chromatography. Equation (II); Formula (III).

3. The preparation method according to claim 2, characterized in that, The molar ratio of the compound shown in formula (II), carbonyl diimidazole, and the compound shown in formula (III) is 1:0.75~0.85:1.05~1.

15.

4. The preparation method according to claim 2, characterized in that, The reaction temperature is 29~31 ℃ and the reaction time is 22~26 h, which is protected from light.

5. A lipid nanomedicine carrier, characterized in that, It includes a first lipid compound and a second lipid compound, wherein the first lipid compound includes the glycerophosphorylethanolamine skeleton compound as described in claim 1 or a pharmaceutically acceptable salt thereof, and the second lipid compound includes cationic or ionizable lipids, sterols and amphiphilic lipids.

6. The lipid nanomedicine carrier as described in claim 5, characterized in that, The cationic or ionizable lipids include one or more of DLinDMA, DODMA, DLin-MC2-MPZ, DLin-KC2-DMA, DOTAP, C12-200, SM-102 and ALC-0315; The amphiphilic lipids include one or more of PEG-DSPE, PEG-PE, PEG-DMG, PEG-C14, PEG-c-DMA, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, Tween-20, Tween-80, PEG-DPG, PEG-s-DMG, PEG-c-DOMG, and GalNAc-PEG-DSG.

7. The lipid nanomedicine carrier as described in claim 5, characterized in that, The molar ratio of the first lipid compound, the cationic or ionizable lipid, the sterol, and the amphiphilic lipid is 2–20:10–65:20–55:0.2–20.

8. The lipid nanomedicine carrier according to claim 7, characterized in that, The molar ratio of the first lipid compound, the cationic or ionizable lipid, the sterol, and the amphiphilic lipid is 5–10:30–50:35–55:0.5–10.

9. A nucleic acid lipid nanoparticle composition, characterized in that, Its particle size is 30-300 nm, including the lipid nanomedicine carrier and nucleic acid drug as described in any one of claims 5-8, wherein the mass ratio of the lipid nanomedicine carrier to the nucleic acid drug is 1:1 to 20.

10. The nucleic acid lipid nanoparticle composition according to claim 9, characterized in that, The nucleic acid drugs include one or more of DNA, siRNA, mRNA, dsRNA, antisense nucleic acid, microRNA, antisense microRNA, antagomir, microRNA inhibitors, microRNA activators, and immunostimulatory nucleic acids.

11. A pharmaceutical preparation, characterized in that, Includes the nucleic acid lipid nanoparticle composition as described in claim 9 or 10, pharmaceutically acceptable buffer salts, osmotic pressure regulators, and suspending agents.

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