A preparation method of anionic liposomes for in situ capturing of antigens and lymph node targeting delivery and application thereof

Abstract

The present application relates to an anionic liposome and a preparation method and application thereof. The anionic liposome of the present application has a structure shown in formula (I), wherein R1 and R2 are each independently C 10‑20 alkyl; R3 is an aspartic acid residue, a glutamic acid residue, a tyrosine residue, or a serine residue. The anionic liposome of the present application is biodegradable, non-toxic, environmentally friendly, easy to obtain, and has good safety and biocompatibility. (I)

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a method for preparing anionic liposomes for in-situ capture of antigens and targeted delivery to lymph nodes, and their applications. Background Technology

[0002] Therapeutic cancer vaccines have become an important tool in cancer treatment. These vaccines exert their anti-tumor effects by initiating an immune response among tumor antigens, antigen-presenting cells (APCs), and cytotoxic T lymphocytes (CTLs), and include dendritic cell-based vaccines, mRNA vaccines, and personalized vaccines.

[0003] Meanwhile, nanocarriers hold great potential in the development of therapeutic tumor vaccines. Liposomes, in particular, are a safe and efficient drug delivery carrier capable of loading vaccine components such as proteins, DNA, and mRNA. Furthermore, research has shown that liposomes exhibit a certain adjuvant effect. Immune adjuvants enhance immune responses by prolonging antigen retention time, improving internalization, and activating pattern recognition receptors (PRRs).

[0004] Therefore, this invention develops a biocompatible vector with good in-situ antigen capture and lymph node targeted delivery effects, which can be used as a delivery vector for therapeutic tumor vaccines. Summary of the Invention

[0005] The purpose of this invention is to provide anionic liposomes with good biocompatibility, excellent in-situ antigen capture and lymph node targeted delivery effects, as well as their preparation method and application.

[0006] In a first aspect, an anionic lipid compound of formula (I), its stereoisomers, tautomers, or pharmaceutically acceptable salts are provided. (I) In the formula, R1 and R2 are each independently C 10-20 alkyl; R3 is an aspartic acid residue, a glutamic acid residue, a tyrosine residue, or a serine residue.

[0007] In another preferred embodiment, R1 and R2 are each independently C 14-18 alkyl.

[0008] In another preferred embodiment, both R1 and R2 are C 14-18 alkyl.

[0009] In another preferred embodiment, the anionic lipid compound is selected from the group consisting of: , , , .

[0010] In another preferred embodiment, the anionic lipid compound is used to prepare a lipid composition.

[0011] In another preferred embodiment, the lipid composition is a drug delivery carrier.

[0012] In another preferred embodiment, the drug delivery carrier is a lipid nanoparticle.

[0013] In another preferred embodiment, the drug is a small molecule, nucleic acid, protein, or a combination thereof.

[0014] In a second aspect of the present invention, a method for preparing anionic lipid compounds as described in the first aspect of the present invention is provided, comprising the steps of: (s1) In the presence of an organic solvent and a basic catalyst, 3,4-dihydroxybenzaldehyde reacts with R1Br / R2Br to generate intermediate compound A; (s2) In the presence of a catalyst, intermediate compound A reacts with dimethyl phosphite and an amino acid selected from the group consisting of aspartic acid, glutamic acid, tyrosine, or serine to obtain an anionic lipid compound as described in the first aspect of the present invention. The definitions of R1 and R2 are as described in the first aspect of this invention.

[0015] In another preferred embodiment, in step (s1), the organic solvent comprises N,N-dimethylformamide.

[0016] In another preferred embodiment, in step (s1), the alkaline catalyst is selected from the group consisting of potassium carbonate, potassium iodide, or combinations thereof.

[0017] In another preferred embodiment, in step (s1), the temperature of the reaction is 70-100°C, more preferably 70-90°C, more preferably 75-85°C, for example about 80°C.

[0018] In another preferred embodiment, in step (s1), the reaction time is 8-16 hours, more preferably 10-14 hours, for example about 12 hours.

[0019] In another preferred embodiment, in step (s2), the temperature of the reaction is 70-100°C, more preferably 70-90°C, more preferably 75-85°C, for example about 80°C.

[0020] In another preferred embodiment, in step (s2), the reaction time is 0.1-5h, more preferably 0.3-3h, more preferably 0.3-1h, for example about 0.5h.

[0021] In another preferred embodiment, in step (s2), the catalyst comprises magnesium perchlorate.

[0022] In another preferred embodiment, in step (s1), the molar ratio of 3,4-dihydroxybenzaldehyde to R1Br / R2Br is 1-3:1-5, more preferably 1:1-3, and even more preferably 1:2; In step (s2), the molar ratio of intermediate compound A to amino acid is 1-3:1-5, preferably 1-3:1-3, more preferably 1:1-3, for example about 1:1; The molar ratio of intermediate compound A to dimethyl phosphite is 1-3:1-5, preferably 1-3:1-3, more preferably 1:1-3, for example about 1:1.

[0023] In a third aspect of the invention, a lipid nanoparticle is provided, the lipid nanoparticle comprising anionic lipid compounds as described in the first aspect of the invention.

[0024] In another preferred embodiment, the lipid nanoparticles have an average particle size of 80-220 nm, more preferably 90-150 nm, and even more preferably 120-150 nm.

[0025] In another preferred embodiment, the lipid nanoparticles have an average zeta potential of -60 to -10 mV.

[0026] In another preferred embodiment, the lipid nanoparticles comprise anionic lipid compounds of formula (I), DMG-PEG, CHO-HP, and DOPE.

[0027] In another preferred embodiment, the lipid nanoparticles further include other lipids; The other lipids are selected from the group consisting of phospholipids, sterol lipids, PEG lipids, or combinations thereof.

[0028] In another preferred embodiment, the molar ratio of the anionic lipid compound, DMG-PEG, CHO-HP and DOPE is 5-55:1-40:1-40:5-55; more preferably 5-50:1-10:30-40:9-54; and even more preferably 5-50:1:40:9-54.

[0029] In another preferred embodiment, the molar ratio of DMG-PEG to CHO-HP is 1:40.

[0030] In another preferred embodiment, the molar ratio of the anionic lipid compound to DOPE is 5-50:9-54.

[0031] In another preferred embodiment, the lipid nanoparticles are prepared by the following steps: (1) Anionic lipid compounds, DMG-PEG, CHO-HP and DOPE were dissolved in an organic solvent, sonicated and rotary evaporated to obtain anionic liposome films; (2) Add ultrapure water to the anionic liposome membrane for hydration, stir, and sonicate to obtain the lipid nanoparticles.

[0032] In another preferred embodiment, in step (1), the organic solvent includes methanol, chloroform, or a mixture thereof.

[0033] In another preferred embodiment, in step (1), an anionic liposome film is obtained by removing the organic solvent by rotary evaporation at 200-400 mbar for 5-15 min, followed by rotary evaporation at 0 mbar for 5-15 min.

[0034] In another preferred embodiment, in step (2), the stirring is carried out at 50-70°C, more preferably 55-65°C, and even more preferably 60°C.

[0035] In another preferred embodiment, in step (2), the power of the ultrasound is 250-350W, more preferably 280-320W, for example about 300W.

[0036] In another preferred embodiment, in step (2), the ultrasound conditions are: ultrasound for 25-35 seconds, with an interval of 25-35 seconds, and 8-15 times; more preferably, ultrasound for 28-32 seconds, with an interval of 28-32 seconds, and 8-12 times; and even more preferably, ultrasound for 30 seconds, with an interval of 30 seconds, and 10 times.

[0037] In a fourth aspect of the invention, the use of an anionic lipid compound, its stereoisomers, tautomers, or pharmaceutically acceptable salts as described in the first aspect of the invention, or lipid nanoparticles as described in the third aspect of the invention, for: (1) Encapsulated therapeutic and / or preventative agents; (2) Preparation of in vivo delivery systems for therapeutic agents and / or preventative agents; and / or (3) Therapeutic tumor vaccines.

[0038] In a fifth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising lipid nanoparticles as described in the third aspect of the invention, a bioactive agent encapsulated in the lipid nanoparticles, and a pharmaceutically acceptable carrier, excipients, and excipients.

[0039] In a sixth aspect of the invention, there is provided the use of a pharmaceutical composition as described in the fifth aspect of the invention for preparing a medicament for the prevention and / or treatment of inflammatory, infectious, cancer, proliferative, genetic, autoimmune, or metabolic diseases.

[0040] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0041] Figure 1 The 1H NMR spectrum of 3,4-bis(hexadecyloxy)benzaldehyde is shown.

[0042] Figure 2 The proton NMR spectrum of the anionic lipid B16-Asp is shown.

[0043] Figure 3 The proton NMR spectrum of the anionic lipid B16-Glu is shown.

[0044] Figure 4 The proton NMR spectrum of the anionic lipid B16-Ser is shown.

[0045] Figure 5 The proton NMR spectrum of the anionic lipid B16-Tyr is shown.

[0046] Figure 6 The dynamic light scattering (DLS) particle size distribution of anionic liposomes is shown.

[0047] Figure 7 The dynamic light scattering (DLS) potential map of anionic liposomes is shown.

[0048] Figure 8 The distribution of fluorescence intensity in various organs after administration of different anionic liposomes is shown.

[0049] Figure 9 The graph shows the quantitative analysis of the average fluorescence intensity of inguinal lymph nodes after administration of different anionic liposomes.

[0050] Figure 10 The graphs show the effects of different anionic liposome peptide delivery.

[0051] Figure 11 The diagram shows the stimulatory effects of different anionic liposomes on the maturation of BMDCs. Detailed Implementation

[0052] Through extensive and in-depth research, and after numerous experiments and screenings, the inventors unexpectedly discovered for the first time anionic liposomes, their preparation method, and applications. The anionic liposomes of this invention are biodegradable, non-toxic, environmentally friendly, and made from readily available materials, exhibiting excellent safety and biocompatibility. These anionic liposomes are transported to lymph nodes via the endothelial cells of the skin's lymphatic vessels, where they are phagocytosed by immune cells and enhance their function, thereby effectively improving drug efficacy and enhancing the immune response to therapeutic tumor vaccines. This invention was completed based on these findings.

[0053] the term To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0054] As used herein, the term “comprising” or its variations such as “including” or “comprises” are understood to include the said element or component without excluding other elements or other components.

[0055] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0056] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0057] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0058] As used herein, the term "alkyl" refers to a monovalent, straight-chain or branched saturated hydrocarbon group consisting of carbon and hydrogen atoms, for example, "C 1-6"Alkyl" means an alkyl group having 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5, or 6). Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. In this application, alkyl is also intended to include deuterated alkyl groups, examples of which include, but are not limited to, CD3, CD2CD3, and CD2CD2CD3.

[0059] Anionic lipid compounds As used herein, the term "anionic lipid compound" refers to a compound having the structure of formula (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. The term "anionic liposome" refers to a vesicular aggregate obtained from said anionic lipid compound. Although the two differ in physical form, in the context of this invention, references to "anionic lipid-related substances" or "substances described in this invention" should be understood to encompass both the said anionic lipid compound and the anionic liposomes formed therefrom, unless otherwise specified.

[0060] Unless otherwise specified, the structural formulas described in this invention are intended to include all stereoisomers (such as cis-trans isomers, enantiomers, diastereomers, and conformational isomers): R and S configurations containing an asymmetric center, (Z) and (E) isomers of double bonds, cis-trans isomers of cycloalkanes, etc. Therefore, any single stereochemical isomer of the compounds of this invention, or a mixture of its enantiomers, diastereomers, or conformational isomers, is within the scope of this invention.

[0061] The compounds of this invention may contain cis-trans isomers, one or more chiral carbon atoms, and thus can produce cis-trans isomers, chiral isomers, enantiomers, diastereomers, and other combinations of stereoisomers. Cis-trans isomerism refers to the diastereomeric phenomenon in which different functional groups in a compound molecule are arranged differently in space due to a restrictive factor that allows for free rotation. This restrictive factor is generally caused by non-rotating functional groups in the structure of organic compounds, such as C=C double bonds, C=N double bonds, C=S double bonds, N=N double bonds, heterocycles, or cycloalkanes. Organic molecules containing such isomers, such as alkenes, azo compounds, and cycloalkanes, are considered cis-trans isomers. Cis refers to the same ligands being in adjacent positions, generally denoted by "cis" or "cis-"; trans refers to the same ligands being in diagonal positions, generally denoted by "trans" or "trans-". Each chiral carbon atom can be defined as (R)- or (S)- based on stereochemistry. This invention aims to include all possible isomers, their racemic and optically pure forms. The compounds of this invention can be prepared using racemic, cis-trans, chiral, diastereomer, or enantiomers as starting materials or intermediates. Optically active isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as crystallization and chiral chromatography.

[0062] Conventional techniques for preparing / separating individual optical isomers (i.e., cis-trans isomers and chiral isomers) include chiral synthesis from suitable cis-trans precursors or optically pure precursors, or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high-performance liquid chromatography.

[0063] To design the synthesis of a specific stereoisomer of the compound of this invention, it can be prepared asymmetrically or derivatized with a chiral auxiliary. The resulting stereo mixture is then separated, and the chiral auxiliary is removed to obtain pure cis-trans monomers, chiral monomers, or mixed stereoisomers. If the molecule contains a cis-trans isomer center, it can be purified by column chromatography (normal-phase silica gel column or reverse-phase high-performance liquid chromatography) to obtain pure cis or trans products. Alternatively, if the molecule contains a basic functional group, such as an amino acid, or an acidic functional group, such as a carboxyl group, it can be formed with a suitable optically active acid or base to form a diastereomeric salt, which is then separated by conventional methods such as separation crystallization or chromatography to obtain pure enantiomers.

[0064] As used herein, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0065] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionates, hexanoates, octanoates, decanoates, undecenoates, glycolates, gluconates, lactates, sebates, adipates, glutarate, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbic acid salts, salicylates, 4-aminosalicylic acid salts, and naphthalene disulfonates. These salts can be prepared using methods known in this field.

[0066] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0067] Preparation methods of anionic lipid compounds The method for preparing the anionic lipid compound of the present invention includes the following steps: (s1) In the presence of an organic solvent and a basic catalyst, 3,4-dihydroxybenzaldehyde reacts with R1Br / R2Br to generate intermediate compound A; (s2) In the presence of a catalyst, intermediate compound A reacts with dimethyl phosphite and an amino acid selected from the group consisting of aspartic acid, glutamic acid, tyrosine, or serine to obtain an anionic lipid compound as described in the first aspect of the present invention. The definitions of R1 and R2 are as described in the first aspect of this invention.

[0068] In one specific implementation, the method includes the following steps: (s1) 3,4-Dihydroxybenzaldehyde and hexadecane bromide were dissolved in organic solvent A in a molar ratio of 1:2, and then an alkaline catalyst was added. The mixture was heated to 80°C and reacted for 12 hours. The reaction solution was purified to obtain 3,4-di(hexadecyloxy)benzaldehyde. The molecular formula of 3,4-di(hexadecyloxy)benzaldehyde is C 39 H 70 O3, the structural formula is as follows: (s2) Mix 3,4-bis(hexadecyloxy)benzaldehyde and magnesium perchlorate at a molar ratio of 2:1 until homogeneous, then add amino acids and dimethyl phosphite and heat to 80°C; after the mixture melts, stir and react for 0.5 h under solvent-free conditions; separate and purify to obtain anionic lipid compounds.

[0069] In one specific embodiment, in step (1), the reaction is carried out under the conditions of organic solvent A and alkaline catalyst; the organic solvent A is selected from N,N-dimethylformamide; the alkaline catalyst is selected from potassium carbonate and potassium iodide; the reaction temperature is 80°C, and the reaction time is 10-16 hours, preferably 12 hours.

[0070] In one specific embodiment, in step (2), the reaction is carried out in the absence of solvent and catalyst; the amount of amino acid and dimethyl phosphite added is 1.0 mmol; the catalyst is selected from magnesium perchlorate; the reaction temperature is 80°C, and the reaction time is 0.5-6 hours, preferably 1 hour.

[0071] In one exemplary implementation, the method includes the steps of: 3,4-Dihydroxybenzaldehyde (0.4144 g, 3 mmol) and hexadecane (1.8320 g, 6 mmol) were dissolved in anhydrous N,N-dimethylformamide (10 ml), followed by the addition of potassium carbonate (0.9951 g, 7.2 mmol) and potassium iodide (0.17 g, 1.0 mmol). After the addition was complete, the mixture was heated to 80 °C and reacted for 12 h. After TLC indicated that the reactants had completely reacted, N,N-dimethylformamide was removed, 20 ml of dichloromethane was added, and the mixture was washed three times with 5 ml of deionized water. The organic phase was collected, dried with anhydrous sodium sulfate, filtered, and the liquid was concentrated to obtain 3,4-di(hexadecyloxy)benzaldehyde. 3,4-Di(hexadecyloxy)benzaldehyde (0.2524 g, 0.5 mmol) and magnesium perchlorate (0.0112 g, 0.025 mmol) were mixed evenly, and then aspartic acid (0.1331 g, 1.0 mmol) and dimethyl phosphite (0.1541 g, 1.0 mmol) were added. The mixture was heated to 80 °C. After the mixture melted, it was stirred for 0.5 h under solvent-free conditions. Heating was stopped after TLC indicated that the reactants had reacted completely. 50 ml of deionized water was added, and the mixture was sonicated to disperse it evenly in the water to form stable micelles. The mixture was then transferred to a 3.5 kD dialysis bag and dialyzed for 12 h to remove unreacted water-soluble reactants. After dialysis, the mixture was transferred to a 500 ml round-bottom flask, and the solvent water was removed by rotary evaporation to obtain the anionic lipid compound described in the first aspect of this invention.

[0072] Applications of anionic lipid compounds In another aspect, the present invention provides the use of anionic lipid compounds as described in any embodiment herein in the preparation of lipid compositions.

[0073] In one embodiment, the lipid composition is used as a drug delivery carrier, preferably, the drug delivery carrier is a lipid nanoparticle (LNP).

[0074] In one embodiment, the drug is a small molecule drug, a polypeptide, or a protein. More preferably, the drug is a nucleic acid, which is DNA or RNA. More preferably, the DNA is linear DNA or circular DNA. More preferably, the circular DNA is a plasmid. More preferably, the RNA is siRNA, ASO, mRNA, saRNA, dsRNA, or shRNA. More preferably, the RNA is siRNA or mRNA.

[0075] In another aspect, the present invention provides the use of the anionic lipid as described above or the lipid composition as described above, said use being selected from the group consisting of: (1) Encapsulated therapeutic and / or preventative agents; (2) Preparation of in vivo delivery systems for therapeutic agents and / or preventive agents and / or (3) Therapeutic tumor vaccines.

[0076] In another aspect, the present invention provides a pharmaceutical composition comprising a lipid composition, a therapeutic and / or preventive agent, and a pharmaceutically acceptable carrier, excipient, or excipient as described in any embodiment herein.

[0077] In one embodiment, the therapeutic agent and / or preventive agent is selected from one or more combinations of small molecule drugs, nucleic acids, and proteins.

[0078] In another aspect, the present invention provides the use of the pharmaceutical composition described above in the preparation of a medicament for the prevention or treatment of inflammation, infectious diseases, cancer, proliferative diseases, genetic diseases, autoimmune diseases, or metabolic diseases.

[0079] lipid nanoparticles The lipid nanoparticles of the present invention include the ionizable lipid compounds of the present invention as described in formula (I) and other lipids, wherein the other lipids are selected from the group consisting of phospholipids, sterol lipids, PEG lipids, or combinations thereof.

[0080] Cholesterol plays a crucial role in the self-assembly of membrane structures. It has been shown to be an effective substance for increasing lipid membrane fluidity while maintaining its stability. Cholesterol analogues include: vitamin D2, vitamin D3, β-sitosterol, brassosterol, and ergosterol.

[0081] Cholesterol is known to be (3β)-cholesterol-5-en-3-ol, an essential component of cell membranes in higher animals. As used herein, "cholesterol compounds" include cholesterol, cholesterol derivatives, and cholesterol analogs. Cholesterol derivatives refer to natural or synthetic cholesterol derivatives, such as acyl cholesterol as an ester body with a fatty acid partially bonded to the hydroxyl group. Cholesterol analogs refer to natural or synthetic cholesterol analogs, such as phytosterols derived from plant cells (e.g., sitosterol, stigmasterol, fucosterol, spinasterol, campesterol, and brassicerol) and ergosterol derived from fungi.

[0082] Cholesterol, cholesterol derivatives, and cholesterol analogs are all classified as steroids, belonging to the subgroup known as sterols (steroidal alcohols).

[0083] The PEG-based lipids described in this invention generally refer to conjugates formed by chemically linking PEG (polyethylene glycol) and lipid molecules. This includes, but is not limited to, PEG-modified phospholipids and derived lipids, exemplified by one or more combinations of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and methoxy-polyethylene glycol bis(tetradecylacetamide).

[0084] In one embodiment, the PEG lipids include, but are not limited to, PEG-C-DMG, PEG-C-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, PEG-DPPC, PEG-distearate phosphatidylethanolamine (PEG-DSPE), PEG-DS, Chol (cholesterol)-PEG, 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol (PEG-DMG), PEG-S-DMG, polyethylene glycol phosphatidylethanolamine, polyethylene glycol ceramide, and polyethylene glycol diglycerol. The following are combinations of one or more of the following: PEG-DMA, PEG distearate glycerol, PEG dipalmitoyl, PEG dioleyl, PEG distearate, PEG diacyl glycine, PEG dipalmitoyl phosphatidylethanolamine, PEG phosphatidylethanol, PEG phosphatidyl ethylenedimyristyloxypropyl-3-amine, PEG oxypropyl alcoholamine, 1,2-distearate oxypropyl-3-amine-N[methoxy(polyethylene glycol)] (PEG-DSA), methoxypolyethylene glycol lauric acid, and methoxypolyethylene glycol bis(tetradecyl acetamide) (ALC0159).

[0085] In one embodiment, the PEG lipid is DMG PEG.

[0086] In one embodiment, the weight-average molecular weight of PEG in the PEG lipid is 1000 to 10000, for example, 1000 to 2000, 2000 to 4000, 4000 to 6000, 6000 to 8000, 8000 to 10000, preferably 2000.

[0087] Pharmaceutical Composition The lipid compositions of the present invention comprise, as described above, a bioactive agent encapsulated within the lipid nanoparticles, and a pharmaceutically acceptable carrier, excipient, or excipient.

[0088] In one embodiment, the bioactive agent is selected from the group consisting of peptides, proteins, and small molecules, or combinations thereof.

[0089] The term "small molecule" as used in this invention refers to a compound that is not a protein or nucleic acid molecule. Small molecules can be small molecules of therapeutic and / or preventative agents, such as antibiotics, anti-inflammatory drugs, anticancer drugs, antiviral drugs, immunosuppressants, analgesics, antifungal drugs, antiparasitic drugs, anticonvulsants, antidepressants, anti-anxiety drugs, antipsychotics, lipid-lowering drugs, hypoglycemic drugs, weight-loss drugs, etc.

[0090] The term "protein" as used in this invention refers to a molecule or complex comprising one or more polypeptides having secondary, tertiary, and / or quaternary structures. The secondary, tertiary, and / or quaternary structures of proteins are typically stabilized using non-covalent bonds such as ionic bonds, hydrogen bonds, hydrophobic interactions, and / or van der Waals interactions. Additionally, or alternatively, proteins may include disulfide bonds, for example, between thiol groups of cysteine ​​residues. Exemplary proteins include, but are not limited to, antibodies, antigens or fragments thereof, fusion proteins, recombinant proteins, polypeptides, short peptides, enzymes, glycoproteins, lipoproteins, ribosomal proteins, chemically modified proteins, etc.

[0091] The term "pharmaceutical acceptable" as used in this invention means that when the drug is properly administered to animals or humans, it does not produce adverse, allergic, or other adverse reactions.

[0092] "Pharmaceuticalally acceptable excipients" should be compatible with the active ingredient, meaning they can be miscible with it without significantly reducing the drug's efficacy under normal circumstances. Specific examples of substances that can be used as pharmaceutically acceptable excipients include, but are not limited to, alcohols such as ethanol, propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; surfactants; lyophilization protectants; colorants; flavoring agents; tableting agents; stabilizers; diluents; excipients; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; buffer solutions, etc., and combinations thereof. These substances are used, as needed, to improve the stability of the formulation or to help improve its activity or bioavailability, or to produce an acceptable taste or odor when taken orally.

[0093] "Pharmaceutically acceptable carriers" refer to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween®), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0094] In one embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: injections, lyophilized formulations, nebulized inhalers, and topical medications.

[0095] The administration methods of the pharmaceutical compositions of the present invention include, but are not limited to, injection, inhalation, and transdermal administration.

[0096] In one embodiment, the pharmaceutical composition is administered by injection, i.e., intravenous, intramuscular, intradermal, subcutaneous, intrathecal, duodenal, or intraperitoneal injection.

[0097] In one embodiment, the pharmaceutical composition is administered by inhalation, such as intranasal administration.

[0098] In one embodiment, the pharmaceutical composition is administered transdermally, such as by topical application or electrode delivery.

[0099] In one embodiment, the pharmaceutical composition is delivered to target cells; preferably, the target cells are eukaryotic cells, and more preferably, the target cells are one or more of immune cells, stem cells, or nerve cells.

[0100] In one embodiment, the immune cells include, but are not limited to, one or more of T cells, macrophages, dendritic cells, natural killer cells, monocytes, bone marrow cells, B cells, lymphocytes, and tumor-infiltrating lymphocytes; and / or, the stem cells include, but are not limited to, one or more of induced pluripotent stem cells (iPSCs), human embryonic stem cells, adult stem cells, mesenchymal stem cells, and hematopoietic stem cells; and / or, the nerve cells include, but are not limited to, one or more of neurons, astrocytes, oligodendrocytes, microglia, Schwann cells, and neuroblasts.

[0101] In one embodiment, the pharmaceutical composition is delivered to target cells for cell therapy to treat cancer.

[0102] The cell therapies include, but are not limited to, one or more combinations of chimeric antigen receptor T-cell (CAR-T cell) therapy, T-cell receptor engineered T-cell (TCR-T) therapy, chimeric antigen receptor natural killer (CAR-NK cell) therapy, chimeric antigen receptor natural killer (CAR-NKT cell) therapy, chimeric antigen receptor macrophage (CAR-M cell) therapy, and tumor-infiltrating lymphocyte (TIL) cell therapy. The cancers include, but are not limited to, one or more of leukemia, lymphoma, melanoma, cervical cancer, and sarcoma. The leukemias include, but are not limited to, one or more of childhood acute lymphoblastic leukemia and chronic lymphocytic leukemia. The lymphomas include, but are not limited to, one or more of adult advanced lymphoma and relapsed or refractory non-Hodgkin's lymphoma.

[0103] The main advantages of this invention include: 1. The anionic liposomes of the present invention are made from readily available, non-toxic, environmentally friendly, and biodegradable materials, and have excellent safety and biocompatibility.

[0104] 2. The anionic liposomes of the present invention can be transported to lymph nodes through the endothelial cells of the skin lymphatic vessels, where they are phagocytosed by immune cells and enhance the function of immune cells, thereby effectively improving the efficacy of drugs.

[0105] 3. The anionic liposomes of the present invention can load hydrophilic and hydrophobic drugs through a hydrophobic shell or a hydrophilic core, which greatly improves the universality of drug loading.

[0106] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0107] Preparation Example Synthesis of 3,4-bis(hexadecyloxy)benzaldehyde 3,4-Dihydroxybenzaldehyde (0.4144 g, 3 mmol) and hexadecane bromide (1.8320 g, 6 mmol) were dissolved in anhydrous N,N-dimethylformamide (10 ml), followed by the addition of potassium carbonate (0.9951 g, 7.2 mmol) and potassium iodide (0.17 g, 1.0 mmol). After the addition was complete, the mixture was heated to 80 °C and reacted for 12 h. Once TLC indicated that the reactants had completely reacted, the N,N-dimethylformamide was removed, and 20 ml of dichloromethane was added. The mixture was washed three times with 5 ml of deionized water, and the organic phase was collected. After drying with anhydrous sodium sulfate, the liquid was filtered, and the filtrate was concentrated. The crude product was purified by silica gel column chromatography (100% dichloromethane) to obtain a white powdery compound.

[0108] 3,4-Di(hexadecyloxy)benzaldehyde, for example Figure 1 As shown, its 1H NMR characteristics are as follows: 1H NMR (400MHz, Chloroform-d) 9.82 (s, 1H), 7.42-7.38 (m, 2H), 6.94 (d, J=8.1Hz, 1H) 4.06 (dt, J=11.1, 6.6Hz, 4H), 1.85 (q, J=7.6Hz, 4H), 1.51-1.42 (m, 4H), 1.26 (s, 48H), 0.90-0.85 (m, 6H). Synthesis of (3,4-bis(hexadecoxy)phenyl)(dimethoxyphosphoryl)methyl-D-aspartic acid 3,4-Di(hexadecyloxy)benzaldehyde (0.2524 g, 0.5 mmol) and magnesium perchlorate (0.0112 g, 0.025 mmol) were mixed thoroughly, and then aspartic acid (0.1331 g, 1.0 mmol) and dimethyl phosphite (0.1541 g, 1.0 mmol) were added. The mixture was heated to 80 °C. After the mixture melted, it was stirred for 0.5 h under solvent-free conditions. Heating was stopped after TLC indicated that the reactants had reacted completely. 50 mL of deionized water was added, and the mixture was sonicated to disperse it evenly in the water. After forming stable micelles, the mixture was transferred to a 3.5 kD dialysis bag and dialyzed for 12 h to remove unreacted water-soluble reactants. After dialysis, the mixture was transferred to a 500 mL round-bottom flask, and the solvent water was removed by rotary evaporation to obtain a yellow, viscous crude product. After purification by silica gel column chromatography (dichloromethane / methanol = 10:1, v / v), a pale yellow powder (denoted as B16-Asp) was obtained. The 1H NMR spectrum of B16-Asp is shown below. Figure 2 As shown.

[0109] Synthesis of (3,4-bis(hexadecyloxy)phenyl)(dimethoxyphosphoryl)methyl-glutamic acid 3,4-Di(hexadecyloxy)benzaldehyde (0.2524 g, 0.5 mmol) and magnesium perchlorate (0.0112 g, 0.025 mmol) were mixed thoroughly, and then glutamic acid (0.1471 g, 1.0 mmol) and dimethyl phosphite (0.1541 g, 1.0 mmol) were added. The mixture was heated to 80 °C. After the mixture melted, it was stirred for 0.5 h under solvent-free conditions. Heating was stopped after TLC indicated that the reactants had reacted completely. 50 mL of deionized water was added, and the mixture was sonicated to disperse it evenly in the water. After forming stable micelles, the mixture was transferred to a 3.5 kD dialysis bag and dialyzed for 12 h to remove unreacted water-soluble reactants. After dialysis, the mixture was transferred to a 500 mL round-bottom flask, and the solvent water was removed by rotary evaporation to obtain a yellow, viscous crude product. After purification by silica gel column chromatography (dichloromethane / methanol = 10:1, v / v), a white powder (denoted as B16-Glu) was obtained. The 1H NMR spectrum of B16-Glu is shown below. Figure 3 As shown.

[0110] Synthesis of (3,4-bis(hexadecoxy)phenyl)(dimethoxyphosphoryl)methyl-serine 3,4-Di(hexadecyloxy)benzaldehyde (0.2524 g, 0.5 mmol) and magnesium perchlorate (0.0112 g, 0.025 mmol) were mixed thoroughly, and then serine (0.1051 g, 1.0 mmol) and dimethyl phosphite (0.1541 g, 1.0 mmol) were added. The mixture was heated to 80 °C. After the mixture melted, it was stirred for 0.5 h under solvent-free conditions. Heating was stopped after TLC indicated that the reactants had reacted completely. 50 mL of deionized water was added, and the mixture was sonicated to disperse it evenly in the water. After forming stable micelles, the mixture was transferred to a 3.5 kD dialysis bag and dialyzed for 12 h to remove unreacted water-soluble reactants. After dialysis, the mixture was transferred to a 500 mL round-bottom flask, and the solvent water was removed by rotary evaporation to obtain a yellow, viscous crude product. After purification by silica gel column chromatography (dichloromethane / methanol = 10:1, v / v), a white powder (denoted as B16-Ser) was obtained. The 1H NMR spectrum of B16-Ser is shown below. Figure 4 As shown.

[0111] Synthesis of (3,4-bis(hexadecyloxy)phenyl)(dimethoxyphosphoryl)methyl-tyrosine 3,4-Di(hexadecyloxy)benzaldehyde (0.2524 g, 0.5 mmol) and magnesium perchlorate (0.0112 g, 0.025 mmol) were mixed thoroughly, and then tyrosine (0.1882 g, 1.0 mmol) and dimethyl phosphite (0.1541 g, 1.0 mmol) were added. The mixture was heated to 80 °C. After the mixture melted, it was stirred for 0.5 h under solvent-free conditions. Heating was stopped after TLC indicated that the reactants had reacted completely. 50 mL of deionized water was added, and the mixture was sonicated to disperse it evenly in the water. After forming stable micelles, the mixture was transferred to a 3.5 kD dialysis bag and dialyzed for 12 h to remove unreacted water-soluble reactants. After dialysis, the mixture was transferred to a 500 mL round-bottom flask, and the solvent water was removed by rotary evaporation to obtain a yellow, viscous crude product. After purification by silica gel column chromatography (dichloromethane / methanol = 10:1, v / v), a pale yellow powder (denoted as B16-Tyr) was obtained. The 1H NMR spectrum of B16-Tyr is shown below. Figure 5 As shown.

[0112] Preparation of anionic lipids The anionic liposome raw materials DMG-PEG, CHO-HP, DOPE, and anionic lipids were dissolved in 2 mL of methanol-chloroform mixed solution (methanol:chloroform = 3:1) according to the molar ratio in Table 1, and then added to a round-bottom flask and ultrasonically dispersed in a water bath for 2 minutes. Table 1 Anionic liposome films were prepared by using a thin film dispersion method. The solution was uniformly dispersed in a round-bottom flask and the organic solvent was removed by rotary evaporation at 60°C and 300 mbar for 10 min. Then the temperature was reduced to 0 mbar and the solution was rotary evaporated for 10 min to keep the round-bottom flask dry. Remove the round-bottom flask from which the film has been successfully formed, add 2 mL of ultrapure water to hydrate it at 60°C, stir for 2 hours to ensure complete hydration, and then transfer the product to a 5 mL EP tube. Place 5 mL EP tubes on ice and use sonication to homogenize liposomes. The sonication power of the ice bath probe is 300 W, the sonication time is 30 s, the interval is 30 s, and the number of times is 10, to obtain 12 anionic liposome formulations.

[0113] Example 1: Determination of Particle Size and Surface Potential of Liposomes by Dynamic Light Scattering The particle size of formulations 1-1 to 4-3 prepared by the method described in the preparation section was determined by dynamic light scattering, and the results are as follows: Figure 6 As shown. The prepared anionic lipids were diluted 5-fold with 20 mM HEPES solution, and the surface potentials of formulations 1-1 to 4-3 were measured using dynamic light scattering. The results are shown below. Figure 7 As shown.

[0114] Example 2 Lymphatic drainage experiment with different anionic liposomes Liposomes loaded with the fluorescent dye DiR were prepared using a method similar to the preparation example. According to formulations 1-3, 0.25 mg of anionic liposome raw materials DMG-PEG, 1.55 mg of CHO-HP, 4.02 mg of DOPE, and 0.41 mg of B16-Asp were weighed, and 0.12 mg of the fluorescent dye DiR was dissolved in 2 mL of a methanol-chloroform mixed solution (methanol:chloroform = 3:1). This solution was added to a round-bottom flask and ultrasonically dispersed in a water bath for 2 minutes. Liposomes were prepared using a thin-film dispersion method. The uniformly dispersed solution in the round-bottom flask was rotary evaporated at 300 mbar for 10 minutes to remove the organic solvent, then the temperature was lowered to 0 mbar and rotary evaporated for 10 minutes to keep the round-bottom flask dry, thus preparing anionic liposome films. 2 mL of ultrapure water was added to the round-bottom flask for hydration at 60°C, and stirred for 2 hours until complete hydration. The product was then transferred to a 5 mL EP tube. EP tubes were placed on ice, and liposomes were homogenized using ultrasound. The ultrasound power of the ice bath probe was 300W, and the ultrasound was performed for 30 seconds with a 30-second interval, for a total of 10 times, to obtain anionic liposomes (denoted as Asp5-Lipo). Anionic liposome raw materials were weighed according to the formulas 1-1, 2-1, 2-3, 3-1, 3-3, 4-1, and 4-3 in Table 1. At the same time, 0.12 mg of fluorescent dye DiR was weighed for each formula. The liposome raw materials and DiR were dissolved in mL of methanol-chloroform mixed solution (methanol:chloroform = 3:1). The remaining preparation methods were the same as for Asp5-Lipo, to obtain Asp50-Lipo, Glu50-Lipo, Glu5-Lipo, Ser50-Lipo, Ser5-Lipo, Tyr50-Lipo, and Tyr5-Lipo.

[0115] Twenty-four healthy male C57BL / 6 mice (6-8 weeks old) were used, with three mice in each group. 100 μL of liposomes was injected subcutaneously into the base of the tail. Six hours after administration, axillary and abdominal lymph nodes of the mice were collected. The DiR fluorescence signal intensity of the lungs, liver, spleen, kidneys, axillary lymph nodes (Brachial LNs), and inguinal lymph nodes (Inguinal LNs) of the mice after administration was detected using a small animal in vivo imaging system.

[0116] The results are as follows Figure 8 As shown, Ser5-Lipo, Asp5-Lipo, Tyr5-Lipo, and Glu5-Lipo were mainly enriched in the liver, with weak fluorescence signals in the lymph nodes; while the liver uptake of Ser50-Lipo, Asp50-Lipo, Tyr50-Lipo, and Glu50-Lipo was significantly reduced in all groups; all liposomes showed no significant retention in the lungs and spleen. The fluorescence intensity of the mouse inguinal lymph nodes was also statistically analyzed, and the results are as follows: Figure 9The results show that liposomes with a potential concentrated between -5mV and -30mV exhibit good lymphatic drainage effects.

[0117] Example 3: Flow cytometry determination of the effect of BMDC on delivery of peptides by different LNPs Liposomes loaded with the OVA antigen peptide SIINFEKL were prepared using a method similar to the preparation example. 0.25 mg of DMG-PEG, 1.55 mg of CHO-HP, 4.02 mg of DOPE, and 0.41 mg of B16-Asp anionic liposome raw materials were weighed and dissolved in 2 mL of a methanol-chloroform mixed solution (methanol:chloroform = 3:1), and added to a round-bottom flask. The solution was ultrasonically dispersed in a water bath for 2 minutes. Liposomes were prepared using a thin-film dispersion method. The uniformly dispersed solution in the round-bottom flask was rotary evaporated at 300 mbar for 10 min to remove the organic solvent, and then the temperature was lowered to 0 mbar and rotary evaporated for 10 min to keep the round-bottom flask dry, thus preparing anionic liposome films. 2 mL of a 1 mg / mL SIINFEKL aqueous solution was added to the round-bottom flask for hydration at 60 °C. After stirring for 2 h to ensure complete hydration, the product was transferred to a 5 mL EP tube. The 5 mL... The EP tube was placed on ice, and the liposomes were homogenized using ultrasound. The ultrasound power of the ice bath probe was 300W, and the ultrasound was performed for 30 seconds with a 30-second interval, for a total of 10 times, to obtain anionic liposomes (denoted as SIINFEKL@Asp5-Lipo). SIINFEKL@Asp50-Lipo and SIINFEKL@Dotap5-Lipo were prepared using the same method.

[0118] Healthy male C57BL / 6 mice (6-8 weeks old) were euthanized by cervical dislocation and disinfected with 75% ethanol for 5 min. The tibia and femur were removed in a laminar flow hood and then immersed in 75% ethanol for 2-5 min for disinfection. The bones were cut at the joints with scissors. Pre-cooled RPMI-1640 complete culture medium (RPMI-1640 + antibiotics + 10% FBS + 2-ME + HEPES) was drawn into a syringe, and the needle was inserted into the bone cavity to flush the bone marrow into a sterile culture dish containing RPMI-1640 complete culture medium. The flushing was repeated 2-3 times. The cell suspension was collected and filtered through a 70 μm cell filter to remove small debris and muscle tissue. The filtrate was centrifuged at 1000 rpm, 4°C for 5 min, and the supernatant was discarded. The mice were washed once with PBS, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded.

[0119] The collected cells were resuspended in RPMI-1640 complete medium and the cell density was adjusted to 1×10⁻⁶. 6Cells / ml. Seed the cells into 6-well plates, 2 mL of cell suspension per well. Add recombinant Mouse GM-CSF to a final concentration of 200 U / ml. Incubate at 37°C in a 5% CO2 incubator. Every two days, perform a half-volume medium change, discarding 1 mL of medium and adding 1 mL of fresh RPMI-1640 complete medium containing 200 U / ml Mouse GM-CSF. On day 7, harvest suspension cells and loosely adherent cells, which are the BMDCs.

[0120] Collect BMDCs, according to 3×10 5 BMDCs were seeded in 24-well plates at a density of 1 / 2 well and administered the following drugs according to the following groups: PBS, LPS, SIINFEKL@Asp5-Lipo (120 μg / mL), SIINFEKL@Asp50-Lipo (120 μg / mL), SIINFEKL@Dotap5-Lipo (120 μg / mL), SIINFEKL@Asp5-Lipo (180 μg / mL), SIINFEKL@Asp50-Lipo (180 μg / mL), and SIINFEKL@Dotap5-Lipo (180 μg / mL). The plates were incubated for 36 h, and BMDCs were collected, washed, and incubated with antibodies. The positive percentage of SIINFEKL on the surface of the DCs was determined by flow cytometry to evaluate the delivery efficiency of different anionic liposomes for peptides.

[0121] Figure 10 The results showed that, compared with the PBS and LPS control groups, SIINFEKL@Asp5-Lipo, SIINFEKL@Asp50-Lipo, and SIINFEKL@Dotap5-Lipo all significantly increased SIINFEKL-H-2Kb in BMDC. + The positive rate achieves efficient antigen cross-presentation, and the delivery efficiency increases with increasing liposome concentration.

[0122] This invention simultaneously measures the positive ratios of co-stimulatory molecules CD80 and CD86 to evaluate the stimulatory effects of different anionic liposomes on the maturation of BMDCs.

[0123] Figure 11 The results showed that, compared with the PBS control group, the CD80 levels in each liposome treatment group were significantly higher. + CD86 + The proportion of mature dendritic cells (DCs) was significantly increased, and there was no significant difference compared with the positive stimulant LPS group. This series of anionic liposomes can effectively induce the maturation and activation of dendritic cells and has the dual functions of excellent antigen delivery and immune adjuvant.

[0124] As can be seen from the above embodiments, the present invention provides anionic liposomes, their preparation method, and applications, which can improve the immune response of therapeutic tumor vaccines. The anionic liposomes of the present invention are made from readily available, non-toxic, environmentally friendly, and biodegradable materials, and have excellent safety and biocompatibility. Furthermore, they can be transported to lymph nodes via skin lymphatic endothelial cells, where they are phagocytosed by immune cells and enhance their function, thereby effectively improving drug efficacy.

[0125] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An anionic lipid compound of formula (I), its stereoisomers, tautomers, or pharmaceutically acceptable salts thereof. (I) In the formula, R1 and R2 are each independently C 10-20 alkyl; R3 is an aspartic acid residue, a glutamic acid residue, a tyrosine residue, or a serine residue.

2. The anionic lipid compound, its stereoisomers, tautomers, or pharmaceutically acceptable salts as described in claim 1, characterized in that, R1 and R2 are each independently C 14-18 alkyl.

3. The anionic lipid compound, its stereoisomers, tautomers, or pharmaceutically acceptable salts as described in claim 1, characterized in that, The anionic lipid compounds are selected from the following group: 、 、 、 。 4. A method for preparing the anionic lipid compound as described in claim 1, characterized in that, Including the following steps: (s1) In the presence of an organic solvent and a basic catalyst, 3,4-dihydroxybenzaldehyde reacts with R1Br / R2Br to generate intermediate compound A; (s2) In the presence of a catalyst, intermediate compound A reacts with dimethyl phosphite and an amino acid selected from the group consisting of aspartic acid, glutamic acid, tyrosine, or serine to obtain the anionic lipid compound as described in claim 1. The definitions of R1 and R2 are as described in claim 1.

5. The method as described in claim 4, characterized in that, In step (s1), the molar ratio of 3,4-dihydroxybenzaldehyde to R1Br / R2Br is 1-3:1-5, preferably 1:1-3, and more preferably 1:2; In step (s2), the molar ratio of the intermediate compound A to the amino acid is 1-3:1-5, preferably 1-3:1-3, more preferably 1:1-3, for example about 1:1; The molar ratio of the intermediate compound A to the dimethyl phosphite is 1-3:1-5, preferably 1-3:1-3, more preferably 1:1-3, for example about 1:

1.

6. A lipid nanoparticle, characterized in that, The lipid nanoparticles include the anionic lipid compound as described in claim 1.

7. The lipid nanoparticles as described in claim 6, characterized in that, The lipid nanoparticles also include other lipids; The other lipids are selected from the group consisting of phospholipids, sterol lipids, PEG lipids, or combinations thereof.

8. Use of an anionic lipid compound as claimed in claim 1, its stereoisomers, tautomers, or pharmaceutically acceptable salts, or the lipid nanoparticles as claimed in claim 6, characterized in that, Used for: (1) Encapsulated therapeutic and / or preventative agents; (2) Preparation of in vivo delivery systems for therapeutic agents and / or preventative agents; and / or (3) Therapeutic tumor vaccines.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the anionic lipid compound of claim 1, its stereoisomers, tautomers, or pharmaceutically acceptable salts, or the lipid nanoparticles of claim 6, a bioactive agent encapsulated in the lipid nanoparticles, and pharmaceutically acceptable carriers, excipients, and excipients.

10. Use of an anionic lipid compound as claimed in claim 1, its stereoisomers, tautomers, or pharmaceutically acceptable salts, lipid nanoparticles as claimed in claim 6, or pharmaceutical compositions as claimed in claim 9, characterized in that, Drugs used to prepare for the prevention and / or treatment of inflammatory, infectious, cancer, proliferative, genetic, autoimmune, or metabolic diseases.

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