A cationic lipid molecule and its use for the preparation of lipid nanoparticle systems

Abstract

The application discloses a cationic lipid molecule and a preparation method of the cationic lipid molecule for a lipid nanoparticle system, and provides a compound of formula I or a pharmaceutically acceptable salt thereof. In the application, a PEG lipid component is not contained in a three-component LNP composition, so that biological toxicity caused by the PEG lipid component is avoided, and safety is improved. Four-component LNP is simplified, and transfection efficiency is improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a cationic lipid molecule and its use in the preparation of lipid nanoparticle systems. Background Technology

[0002] mRNA drugs have been applied in many fields, such as the treatment of infectious diseases and cancer. However, due to the presence of a large number of RNases in the in vivo and in vitro environments, naked mRNA faces the problem of degradation. Therefore, mRNA drugs need effective delivery vectors.

[0003] Currently, the most commonly used delivery system for mRNA drugs is lipid nanoparticles (LNPs). Commercially available LNP systems, including those used in coronavirus vaccines, consist of four components: cationic lipid compounds, cholesterol, polyethylene glycol lipids, and auxiliary lipids. The cationic lipid compound, with its positively charged surface, binds to the negatively charged mRNA during encapsulation through a positive-negative charge interaction, playing a crucial role among the various components of the LNP. Some LNP systems include SM-102, cholesterol, DMG-PEG-2000, and DSPC; others contain ALC-0315, cholesterol, ALC-0159, and DSPC. Summary of the Invention

[0004] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a cationic lipid molecule and its application in the preparation of lipid nanoparticle systems.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A first aspect of the invention provides a compound of formula I or a pharmaceutically acceptable salt thereof:

[0007]

[0008] Among them, R1 is selected from deuterium, H, R2 is selected from L is selected from C1 to C2. 10 Alkylene, substituted C1-C 10 Alkylene; G1 is selected from C1-C6 straight-chain or branched alkylene groups substituted with 1 to 8 hydroxyl groups; G2 is selected from C1-C6 straight-chain or branched alkylene groups substituted with -C(O)O-, -OC(O)-, or -OC(O)O-, or C1-C6 straight-chain or branched alkylene groups substituted with -OC(O)-; X1 and X2 are each independently selected from C1-C6 straight-chain or branched alkylene groups. 60Straight-chain or branched alkyl; n is selected from natural numbers 1 to 6; m and p are each independently selected from natural numbers 1 to 20.

[0009] In some embodiments of the present invention, m and p are each independently selected from natural numbers from 1 to 10.

[0010] In some embodiments of the present invention, L is selected from C1-C4 alkylene groups and substituted C1-C4 alkylene groups; G1 is selected from C1-C3 straight-chain or branched alkylene groups substituted with 1 to 3 hydroxyl groups; G2 is selected from C1-C3 straight-chain or branched alkylene groups substituted with -C(O)O-, -OC(O)-, and -C(O)O-, and C1-C3 straight-chain or branched alkylene groups substituted with -OC(O)-; X1 and X2 are each independently selected from C1-C4 alkylene groups. 40 Straight-chain or branched alkyl; n is selected from natural numbers 1 to 4; m and p are each independently selected from natural numbers 1 to 8.

[0011] In some embodiments of the present invention, L is selected from methylene or substituted methylene; G1 is selected from hydroxylated methylene or ethylene; G2 is selected from -C(O)O-, -OC(O)-, -C(O)O- substituted methylene or ethylene, -OC(O)- substituted methylene or ethylene; X1 and X2 are each independently selected from C1 to C1. 40 Straight-chain or branched alkyl; n is 2; m and p are independently selected from 1 or 2.

[0012] In some embodiments of the present invention, in the compound of formula I, R1 is selected from deuterium, H,

[0013]

[0014] R2 is selected from

[0015]

[0016] L is selected from C1 to C2. 10 Alkylene, substituted C1-C 10 Alkylene; n is a natural number selected from 1 to 6; t1 to t6 are each independently selected from natural numbers from 1 to 20.

[0017] In some embodiments of the present invention, R1 is selected from deuterium, H, ...

[0018] In some embodiments of the present invention, R2 is selected from...

[0019] In some embodiments of the present invention, R1 is selected from deuterium, H, ...

[0020] In some embodiments of the present invention, R2 is selected from...

[0021] In some embodiments of the present invention, C1 to C are replaced. 10 Alkyl groups include C1-C6 groups substituted with deuterium, hydroxyl, or ester groups. 10 Alkylene.

[0022] In some embodiments of the present invention, n is selected from natural numbers from 1 to 4; t1 to t6 are each independently selected from natural numbers from 1 to 10.

[0023] In some embodiments of the present invention, the compound of formula I is... Furthermore, the substituents on the benzene ring are positioned ortho, meta, or para.

[0024] In some embodiments of the present invention, the compound of formula I is selected from...

[0025]

[0026] In compounds of formula IA to formula ID, the definitions of L, t1 to t4 are as described above.

[0027] In some embodiments of the present invention, the compound of formula I is selected from the following compounds:

[0028]

[0029]

[0030]

[0031] A second aspect of the present invention provides a method for preparing a compound, comprising the following steps:

[0032] i) Compound of Formula II With Formula III compounds The reaction yields a compound of formula IA or formula IB; or

[0033] ii) Compound of formula II With Formula IV compounds The reaction yields compounds of formula IC or formula ID.

[0034] In some embodiments of the present invention, the molar ratio of compound II to compound III is 1:(2-5).

[0035] In some embodiments of the present invention, the molar ratio of compound II to compound IV is 1:(2-5).

[0036] In some embodiments of the present invention, the compound of formula III includes at least one of 1,2-epoxydecane, 1,2-epoxydodecane, 1,2-epoxytetradecane, 1,2-epoxyhexadecane, and 1,2-epoxyoctadecane.

[0037] In some embodiments of the present invention, the compound of formula IV includes at least one of decyl acrylate, dodecyl acrylate, tetradecyl acrylate, hexadecyl acrylate, octadecyl acrylate, and dodecyl acrylate.

[0038] In some embodiments of the present invention, the solvent for the reaction is a lower alcohol solution; preferably ethanol, propanol, or isopropanol; more preferably a 90% to 99% ethanol solution.

[0039] In some embodiments of the present invention, the reaction temperature is 70°C to 100°C and the time is 12h to 72h, preferably 80°C to 90°C and the time is 24h to 60h.

[0040] In some embodiments of the present invention, the solvent for the reaction described in i) is ethanol; preferably a 90% to 99% ethanol solution.

[0041] In some embodiments of the present invention, the solvent for the reaction described in ii) is propanol; preferably isopropanol.

[0042] A third aspect of the invention provides a pharmaceutical composition comprising a carrier comprising a cationic lipid comprising a compound of formula I or a pharmaceutically acceptable salt thereof.

[0043] In some embodiments of the present invention, the composition is a nanoparticle formulation, wherein the average size of the nanoparticle formulation is 10 nm to 300 nm, preferably 90 nm to 280 nm; and the polydispersity index of the nanoparticle formulation is ≤50%, preferably ≤40%, and more preferably ≤30%.

[0044] In some embodiments of the present invention, the molar ratio of the cationic lipid to the carrier is 10% to 75%, for example 10%, 20%, 30%, 40%, 49%, 55%, 60%, 65%, 70%, and 75%.

[0045] This carrier can be used to deliver active ingredients such as therapeutic and / or preventative agents. The active ingredient can be encapsulated within the carrier or bound to the carrier.

[0046] For example, the therapeutic or preventative agent comprises one or more of nucleic acid molecules, small molecule compounds, peptides, or proteins. The nucleic acid includes, but is not limited to, single-stranded DNA, double-stranded DNA, and RNA. Suitable RNAs include, but are not limited to, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

[0047] This carrier can be used to deliver photosensitizers and / or photothermal agents for photothermal therapy. The photosensitizers and / or photothermal agents can be encapsulated within the carrier or combined with the carrier, such as in phototherapy for gliomas.

[0048] In some embodiments of the present invention, the photosensitizer and / or photothermal agent includes at least one such as Fs, Fc, Fo, Ce6, ICG, etc.

[0049] neutral lipids

[0050] The carrier may contain neutral lipids. In this invention, neutral lipids refer to lipids that are uncharged at a selected pH value or exist in a zwitterionic form and play an auxiliary role. These neutral lipids may modulate the flowability of nanoparticles to form a lipid bilayer structure and improve efficiency by promoting lipid phase transitions, and may also affect the specificity of target organs.

[0051] In some embodiments of the invention, the molar ratio of the cationic lipid to the neutral lipid is about 1:1 to 15:1, for example, about 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, and 2:1. In a preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 1.5:1. In another preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 4.9:1.

[0052] For example, neutral lipids may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and their derivatives.

[0053] The carrier component of a composition comprising cationic lipids may include one or more neutral lipid-phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. Generally, phospholipids may include a phospholipid moiety and one or more fatty acid moieties.

[0054] The neutral lipid moiety may be selected from the non-restricted group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-restricted group consisting of lauric acid, myristic acid, myristenoic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, benzanoic acid, docosapentaenoic acid, and docosahexaenoic acid. It also encompasses non-natural species including natural species with modifications and substitutions, such modifications and substitutions include branching, oxidation, cyclization, and alkynes. For example, phospholipids may be functionalized with or crosslinked with one or more alkynes (e.g., alkenyl groups with one or more double bonds replaced by triple bonds). Under appropriate reaction conditions, the alkyne group may undergo a copper-catalyzed cycloaddition reaction upon exposure to azides. These reactions can be used to functionalize the lipid bilayer of a composition to facilitate membrane permeation or cell recognition, or to conjugate the composition with useful components such as targeting or imaging components (e.g., dyes).

[0055] The neutral lipids that can be used in these compositions may be selected from the non-limiting group of the following: 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 Diether 1,2-Dilinoleoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate choline), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME) 16.0PE), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl 1-Steayl-2-oleoyl-stearoyl-ethanolamine (POPE), 1-stearoyl-2-oleoyl-stearoyl-ethanolamine (DSPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl-phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0056] In some embodiments of the present invention, neutral lipids include DSPC. In some embodiments of the present invention, neutral lipids include DOPE. In some embodiments of the present invention, neutral lipids include both DSPC and DOPE.

[0057] structural lipids

[0058] The carrier of the composition comprising cationic lipids may also include one or more structural lipids. In this invention, structural lipids refer to lipids that enhance the stability of nanoparticles by filling the gaps between lipids.

[0059] In some embodiments of the present invention, the molar ratio of the cationic lipid to the structural lipid is about 0.6:1 to 3:1, for example, about 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2.0:1.

[0060] Structural lipids may be selected from, but are not limited to, the group consisting of: cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, α-tocopherol, corticosteroids, and mixtures thereof. In some embodiments of the invention, the structural lipid is cholesterol. In some embodiments of the invention, the structural lipid includes cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or combinations thereof.

[0061] In one embodiment of the composition / carrier of the present invention, the carrier comprises cationic lipids, neutral lipids, and structural lipids; the molar ratio of the cationic lipids, the neutral lipids, and the structural lipids is (10-70):(5-60):(5-50); for example, (20-60):(20-50):(10-30), (30-60):(25-40):(15-28), (40-55):(25-35):(18-25), 47.4:31.6:21.1, 49.4:30.6:20.1, 51.4:29.6:19.1.

[0062] Polymer conjugated lipids

[0063] The carrier of the composition containing cationic lipids may also include one or more polymer-conjugated lipids. Polymer-conjugated lipids primarily refer to polyethylene glycol (PEG)-modified lipids. Hydrophilic PEG stabilizes LNPs, modulates nanoparticle size by restricting lipid fusion, and increases the half-life of nanoparticles by reducing non-specific interactions with macrophages.

[0064] In one embodiment, the polymeric conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. The molecular weight of the PEG-modified PEG is typically 350-5000 Da.

[0065] For example, the polymeric conjugated lipid is selected from one or more of the following: distearate phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and methoxy polyethylene glycol bis(tetradecyl)acetamide (ALC-0159).

[0066] In one embodiment of the composition / carrier of the present invention, the polymer conjugated lipid is DMG-PEG2000 and / or ALC-0159.

[0067] In one embodiment of the composition / carrier of the present invention, the carrier comprises cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids, wherein the molar ratio of the cationic lipids, the neutral lipids, the structural lipids, and the polymer-conjugated lipids is (25–75):(5–25):(15–65):(0.5–10), for example (45–55):(7.5–15):(35–55):(1–5), 48:10:50.5:1.5, or 55:10:43.5:1.5, or 50:10:38.5:1.5.

[0068] Therapeutic agents and / or preventative agents

[0069] The composition may include one or more therapeutic and / or preventive agents. In one embodiment, the mass ratio of the carrier to the therapeutic or preventive agent is 1:10 to 30:1, for example 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, or 25:1.

[0070] In one embodiment, the mass ratio of the carrier to the therapeutic or preventative agent is 12.5:1 to 20:1, preferably 15:1.

[0071] The nitrogen-to-phosphorus ratio (N:P) refers to the molar ratio of nitrogen in cationic lipids to phosphorus in RNA in therapeutic or preventative agents.

[0072] In one embodiment of the composition / carrier of the present invention, the nitrogen-to-phosphorus ratio (N:P) in the composition is 0.375-36.

[0073] In one embodiment, the method for preparing a composition comprising a therapeutic or preventative agent includes the following steps:

[0074] Ethanol phase preparation: The support is dissolved in the ethanol phase;

[0075] Aqueous phase preparation: Dissolve the therapeutic or preventative agent in a buffer solution;

[0076] The ethanol phase is added to the aqueous phase and mixed to obtain the composition.

[0077] The therapeutic or preventive agent includes, but is not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides, or proteins.

[0078] For example, the therapeutic or preventative agent is a vaccine or compound that can elicit an immune response.

[0079] The carriers of the present invention can deliver therapeutic and / or preventive agents to mammalian cells or organs, and thus the present invention also provides methods for treating diseases or conditions in mammals in need, including administering a composition comprising therapeutic and / or preventive agents to mammals and / or contacting mammalian cells with the composition.

[0080] Therapeutic agents and / or preventive agents include biologically active substances and are alternatively referred to as "active agents." Therapeutic agents and / or preventive agents can be substances that, upon delivery to a cell or organ, induce a desired change in that cell or organ or other body tissue or system. Such species can be used to treat one or more diseases, conditions, or illnesses. In some embodiments, therapeutic agents and / or preventive agents are small molecule drug substances that can be used to treat a specific disease, condition, or illness.Examples of pharmaceuticals that can be used in a composition include, but are not limited to, anti-hypertrophic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), and antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside). Arabinoside, anthracycline, alkylating agents, platinum compounds, antimetabolites and nucleoside analogs such as methotrexate and purine and pyrimidine analogs, anti-infectives, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blockers (e.g., propranolol, timolol, and labetalol), antihypertensives (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anticonvulsants (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterial agents (e.g., gentamicin, ciprofloxacin, and cefoxitin), antifungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma medications, vitamins, sedatives, and imaging agents.

[0081] In some implementations, the therapeutic and / or prophylactic agents are cytotoxins, radioactive ions, chemotherapeutic agents, vaccines, compounds that elicit an immune response, and / or another therapeutic and / or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, and dihydroxyanthraquinone. Anthracindione, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol, rachelmycin (CC-1065), and their analogues or homologues. Radioactive ions include, but are not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium. Vaccines include compounds and formulations capable of providing immunity against one or more conditions associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis, and tuberculosis, and may include mRNA encoding infectious disease-derived antigens and / or epitopes. Vaccines may also include compounds and formulations that direct an immune response against cancer cells and may include mRNA encoding tumor cell-derived antigens, epitopes, and / or novel epitopes. Compounds that elicit an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species. In some embodiments, vaccines and / or compounds capable of eliciting an immune response are administered intramuscularly by a composition comprising compounds according to formulas (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), or (III) (e.g., compounds 3, 18, 20, 25, 26, 29, 30, 60, 108-112, or 122).Other therapeutic and / or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., nitrogen mustard, thiotepa, chlorambucil, lactamase (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), and cyclophosphamides). Phosphoramide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine cycloplatin (II) (DDP, cisplatin), anthracyclines (e.g. daunomycin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g. dactinomycin (formerly known as actinomycin), bleomycin, mithramycin and antramycin (AMC)), and antimitotic agents (e.g. vincristine, vinblastine, paclitaxel and levothyroxine).

[0082] In other embodiments, the therapeutic and / or preventative agents are proteins. Therapeutic proteins that can be used in the nanoparticles of this invention include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), factor VIR, luteinizing hormone-releasing hormone (LHRH) analogs, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0083] In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The broadest meaning of the term "polynucleotide" includes any compound and / or substance that is an oligonucleotide chain or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides used according to the invention include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA); ribonucleic acid (RNA), including messenger mRNA (mRNA), hybrids thereof; RNAi inducible factors; RNAi factors; siRNA; shRNA; miRNA; antisense RNA; ribonuclease; catalytic DNA; RNA that induces triple helix formation; aptamers, etc. In some embodiments, the therapeutic and / or preventive agent is RNA. RNA that can be used in the compositions and methods described herein can be selected from, but is not limited to, the group consisting of: shortmer, antagomir, antisense RNA, ribonuclease, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some embodiments, the RNA is mRNA.

[0084] In some embodiments, the therapeutic and / or preventative agent is mRNA. The mRNA may encode any polypeptide of interest, including any polypeptide that is naturally occurring or non-naturally present or otherwise modified. The polypeptide encoded by the mRNA may have any size and may possess any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA may have a therapeutic effect when expressed in cells.

[0085] In some embodiments, the mRNA includes at least one tumor antigen mRNA, such as breast cancer vaccine antigen mRNA, colorectal cancer breast cancer vaccine antigen mRNA, glioma vaccine antigen mRNA, melanoma vaccine antigen mRNA, prostate cancer vaccine antigen mRNA, lymphoma breast cancer vaccine antigen mRNA, and hematologic mRNA.

[0086] In other embodiments, the therapeutic and / or preventative agent is siRNA. siRNA is capable of selectively reducing or downregulating the expression of a gene of interest. For example, the siRNA may be chosen such that, upon administration of a composition comprising the siRNA to a subject in need, a gene associated with a specific disease, symptom, or condition is silenced. The siRNA may contain a sequence complementary to the mRNA sequence encoding the gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.

[0087] In some implementations, the therapeutic and / or preventative agents are sgRNA and / or cas9 mRNA. sgRNA and / or cas9 mRNA can be used as gene editing tools. For example, the sgRNA-cas9 complex can affect the mRNA translation of cellular genes.

[0088] In some implementations, the therapeutic and / or prophylactic agent is shRNA or its encoding vector or plasmid. shRNA can be generated within the target cell after delivery of an appropriate construct into the nucleus. Constructs and mechanisms associated with shRNA are well known in the relevant field.

[0089] Disease or ailment

[0090] The compositions / carriers of the present invention can deliver therapeutic or preventative agents to subjects or patients. These therapeutic or preventative agents include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, peptides, or proteins. Therefore, the compositions of the present invention can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, peptide or protein drugs. Due to the wide variety of such therapeutic or preventative agents, the compositions of the present invention can be used to treat or prevent a variety of diseases or conditions.

[0091] In one embodiment, the disease or condition is characterized by dysfunctional or abnormal protein or polypeptide activity.

[0092] For example, the disease or condition is selected from the group consisting of: infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0093] In one embodiment, the infectious disease is selected from diseases caused by coronavirus, influenza virus, or HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies, and various herpes diseases.

[0094] Other components

[0095] The composition may include one or more components other than those described in the foregoing sections. For example, the composition may include one or more hydrophobic small molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0096] The composition may also include one or more permeability-enhancing molecules, carbohydrates, polymers, surface modifiers, or other components. Permeability-enhancing molecules may be, for example, those described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogues).

[0097] Surface modifiers may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyl dioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), and mucolytics (e.g., acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocisteine, and eprazinone). The composition may contain mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, streptococcal DNase α (dornasealfa), neltenexine, and erdosteine), and DNases (e.g., rhDNase). Surface modifiers may be placed within and / or on the surface of the nanoparticles of the composition (e.g., by coating, adsorption, covalent bonding, or other methods).

[0098] The composition may also contain one or more functionalized lipids. For example, the lipids may be functionalized with an alkynyl group, which may undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. Specifically, the lipid bilayer can be functionalized in this way with one or more groups that can effectively promote membrane permeation, cell recognition, or imaging. The surface of the composition may also be conjugated to one or more useful antibodies. Functional groups and conjugates that can be used for targeted cell delivery, imaging, and membrane permeation are well known in the art.

[0099] In addition to these components, the composition may include any substance that can be used in a pharmaceutical composition. For example, the composition may include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersants, suspending agents, granulation agents, disintegrants, fillers, flow aids, liquid media, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, etc. Excipients include, for example, starch, lactose, or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's *The Science and Practice of Pharmacy*, 21st edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0100] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar and / or combinations thereof.

[0101] In some embodiments, compositions comprising one or more lipids described herein may further comprise one or more adjuvants, such as glucopyranosyl lipid adjuvants (GLA), CpG oligodeoxyribonucleotides (e.g., class A or class B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

[0102] The compositions of the present invention can be formulated into solid, semi-solid, liquid, or gaseous preparations, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, and aerosols. The compositions of the present invention can be prepared by methods well known in the pharmaceutical industry. For example, a sterile injectable solution can be prepared by incorporating the desired amount of the therapeutic or preventative agent with the various other desired ingredients described above into a suitable solvent, such as sterile distilled water, followed by filtration and sterilization. Surfactants may also be added to promote the formation of a homogeneous solution or suspension.

[0103] For example, the compositions of the present invention can be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In one embodiment, the composition is administered subcutaneously.

[0104] The compositions of the present invention are administered in therapeutically effective amounts, which can vary not only with the specific reagent chosen, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and can ultimately be determined by the attending physician or clinician. For example, the therapeutic or preventative agent can be administered to mammals (e.g., humans) at doses of about 0.001 mg / kg to about 10 mg / kg.

[0105] The term "pharmaceutically acceptable salt" refers to a relatively non-toxic inorganic or organic acid addition salt of the compounds of this invention. For example, see SMBerge et al., "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19. Inorganic acids include, for example, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, or nitric acid; organic acids include, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, bamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, etc. Pteropenic acid, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid, dodecyl sulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheponic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, etc. For example, HCl (or hydrochloric acid), HBr (or hydrobromic acid solution), methanesulfonic acid, sulfuric acid, tartaric acid, or fumaric acid can be used to form pharmaceutically acceptable salts with the compounds shown in formula (I).

[0106] The term "alkyl" in this invention refers to a branched or straight-chain saturated aliphatic monovalent hydrocarbon group having a specified number of carbon atoms. The term "alkylene" in this invention refers to a branched or straight-chain saturated aliphatic divalent hydrocarbon group having a specified number of carbon atoms. C n ~C m This refers to groups that include a number of carbon atoms from n to m. For example, C2 to C5 alkylene groups include C2 alkylene, C3 alkylene, C4 alkylene, and C5 alkylene.

[0107] "Therapeutic effective amount" is the amount of a therapeutic agent that, when administered to a patient, improves the disease or symptoms. "Prophylactic effective amount" is the amount of a preventive agent that, when administered to a subject, prevents the disease or symptoms. The amount of a therapeutic agent constituting a "therapeutic effective amount" or a preventive agent constituting a "prophylactic effective amount" varies depending on the therapeutic / preventive agent, the disease state and its severity, the age and weight of the patient / subject to be treated / prevented, etc. Those skilled in the art can conventionally determine the therapeutic and preventive effective amounts based on their knowledge and the present invention.

[0108] The term cationic lipid, as used in this article, refers to lipids that carry a positive charge at a selected pH value. Cationic liposomes readily bind to negatively charged nucleic acids, that is, through electrostatic interactions with the negatively charged phosphate groups present in nucleic acids, forming lipid nanoparticles (LNPs). LNPs are currently one of the mainstream delivery carriers.

[0109] The beneficial effects of this invention are:

[0110] The three-component LNP in this invention does not contain PEG lipid components, thus avoiding the biotoxicity caused by PEG lipid components and improving safety; the four-component LNP is simplified, thereby improving transfection efficiency. Attached Figure Description

[0111] Figure 1 This is the NMR spectrum of AO12 in this invention.

[0112] Figure 2 The NMR spectrum of AM12 in this invention is shown.

[0113] Figure 3 This is the NMR spectrum of AP12 of the present invention.

[0114] Figure 4 This is the NMR spectrum of CO14 in this invention.

[0115] Figure 5 This is the NMR spectrum of CM14 of the present invention.

[0116] Figure 6 This is the NMR spectrum of CP14 of the present invention.

[0117] Figure 7 This is the result of the cationic lipid molecule delivery performance test in Experiment Example 2 of the present invention. Detailed Implementation

[0118] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0119] Example 1: Synthesis of cationic lipid compounds

[0120] The synthesis route diagram for AO10, AO12, AO14, AO16, and AO18 is shown below:

[0121]

[0122] AO10, AO12, AO14, AO16, and AO18 were prepared by reacting 1,2-phenylenediamine with 1,2-epoxydecane, 1,2-epoxydodecane, 1,2-epoxytetradecane, 1,2-epoxyhexadecane, or 1,2-epoxyoctadecane at a molar ratio of 1:2.2 in 95% ethanol at 85°C for 48 hours.

[0123] Among them, the NMR spectrum of AO12 is as follows Figure 1 As shown.

[0124] Example 2: Synthesis of cationic lipid compounds

[0125] The synthesis route diagram for AM10, AM12, AM14, AM16, and AM18 is shown below:

[0126]

[0127]

[0128] 1,3-phenylenediamine was reacted with 1,2-epoxydecane, 1,2-epoxydodecane, 1,2-epoxytetradecane, 1,2-epoxyhexadecane, or 1,2-epoxyoctadecane in 95% ethanol at a molar ratio of 1:2.2 at 85°C for 48 hours to prepare AM10, AM12, AM14, AM16, and AM18 respectively.

[0129] Among them, the NMR spectrum of AM12 is as follows Figure 2 As shown.

[0130] Example 3: Synthesis of cationic lipid compounds

[0131] The synthesis routes of AP10, AP12, AP14, AP16, and AP18 are shown in the following diagram:

[0132]

[0133] AP10, AP12, AP14, AP16, and AP18 were prepared by reacting 1,4-phenylenediamine with 1,2-epoxydecane, 1,2-epoxydodecane, 1,2-epoxytetradecane, 1,2-epoxyhexadecane, or 1,2-epoxyoctadecane at a molar ratio of 1:2.2 in 95% ethanol at 85°C for 48 hours.

[0134] Among them, the NMR spectrum of AP12 is as follows Figure 3 As shown.

[0135] Example 4: Synthesis of cationic lipid compounds

[0136] The synthesis routes for BO10, BO12, BO14, BO16, and BO18 are shown in the following diagram:

[0137]

[0138] 1,2-phenylenediamine was reacted with 1,2-epoxydecane, 1,2-epoxydodecane, 1,2-epoxytetradecane, 1,2-epoxyhexadecane, or 1,2-epoxyoctadecane in 95% ethanol at a molar ratio of 1:4.4 at 85°C for 48 hours to prepare BO10, BO12, BO14, BO16, and BO18 respectively.

[0139] Example 5: Synthesis of cationic lipid compounds

[0140] The synthesis routes of BM10, BM12, BM14, BM16, and BM18 are shown in the following diagram:

[0141]

[0142] BM10, BM12, BM14, BM16, and BM18 were prepared by reacting 1,3-phenylenediamine with 1,2-epoxydecane, 1,2-epoxydodecane, 1,2-epoxytetradecane, 1,2-epoxyhexadecane, or 1,2-epoxyoctadecane in 95% ethanol at a molar ratio of 1:4.4 at 85°C for 48 hours.

[0143] Example 6: Synthesis of cationic lipid compounds

[0144] The synthesis routes of BP10, BP12, BP14, BP16, and BP18 are shown in the following diagram:

[0145]

[0146] BP10, BP12, BP14, BP16, and BP18 were prepared by reacting 1,4-phenylenediamine with 1,2-epoxydecane, 1,2-epoxydodecane, 1,2-epoxytetradecane, 1,2-epoxyhexadecane, or 1,2-epoxyoctadecane in 95% ethanol at a molar ratio of 1:4.4 at 85°C for 48 hours.

[0147] Example 7: Synthesis of cationic lipid compounds

[0148] The synthesis routes for CO10, CO12, CO14, CO16, CO18, and CO22 are shown in the following diagram:

[0149]

[0150]

[0151] 1,2-phenylenediamine was reacted with decyl acrylate, dodecyl acrylate, tetradecyl acrylate, hexadecyl acrylate, octadecyl acrylate, or dodecyl acrylate in isopropanol at a molar ratio of 1:2.2 for 48 hours at 85°C to prepare CO10, CO12, CO14, CO16, CO18, and CO20 respectively.

[0152] The NMR spectrum of CO14 is as follows: Figure 4 As shown.

[0153] Example 8: Synthesis of cationic lipid compounds

[0154] The synthesis route diagram for CM10, CM12, CM14, CM16, CM18, and CM22 is shown below:

[0155]

[0156]

[0157] CM10, CM12, CM14, CM16, CM18, and CM20 were prepared by reacting 1,3-phenylenediamine with decyl acrylate, dodecyl acrylate, tetradecyl acrylate, hexadecyl acrylate, octadecyl acrylate, or dodecyl acrylate in isopropanol at a molar ratio of 1:2.2 at 85°C for 48 hours.

[0158] The NMR spectrum of CM14 is as follows: Figure 5 As shown.

[0159] Example 9: Synthesis of cationic lipid compounds

[0160] The synthesis routes for CP10, CP12, CP14, CP16, CP18, and CP22 are shown in the diagram below:

[0161]

[0162]

[0163] 1,4-phenylenediamine was reacted with decyl acrylate, dodecyl acrylate, tetradecyl acrylate, hexadecyl acrylate, octadecyl acrylate, or dodecyl acrylate in isopropanol at a molar ratio of 1:2.2 for 48 hours at 85°C to prepare CP10, CP12, CP14, CP16, CP18, and CP20 respectively.

[0164] Among them, the NMR spectrum of CP14 is as follows Figure 6 As shown.

[0165] In the following examples or comparative examples, Luci mRNA:EZ Cap TMFirefly Luciferase mRNA reagent; Lipo2000: Lipofectamine2000 liposome 2000; N:P ratio refers to the molar ratio of nitrogen in cationic lipids to phosphorus in RNA.

[0166] Example 10: Preparation of lipid nanoparticles

[0167] Preparation of three-component LNP@Luci mRNA with AO10, AO12, AO14, AO16, AO18, AM10, AM12, AM14, AM16, AM18, AP10, AP12, AP14, AP16, or AP18 as cationic lipids:

[0168] Ethanol phase preparation: Cationic lipids, DOPE, and cholesterol were dissolved in ethanol at a molar ratio of 47.4:31.6:21.1.

[0169] Aqueous phase preparation: Luci-mRNA was dissolved in PBS to a concentration of 22.22 ng / uL.

[0170] Take 1 μL of ethanol phase and 9 μL of aqueous phase. The amounts of cationic lipids in 1 μL of ethanol phase are 0.22 nmol, 0.44 nmol, 0.88 nmol, 1.76 nmol, 3.53 nmol, and 5.29 nmol, respectively. The amount of Luci-mRNA in 9 μL of aqueous phase is 200 ng. Add 1 μL of ethanol phase to 9 μL of aqueous phase with a pipette and mix well. Let stand for 10 minutes to obtain LNP@Luci mRNA with N:P = 0.75, 1.5, 3, 6, 12, and 18, respectively.

[0171] Example 11: Preparation of lipid nanoparticles

[0172] Preparation of four-component LNP@Luci mRNA with AO10, AO12, AO14, AO16, AO18, AM10, AM12, AM14, AM16, AM18, AP10, AP12, AP14, AP16, or AP18 as cationic lipids:

[0173] Ethanol phase preparation: Cationic lipids, DSPC, cholesterol, and ALC-0159 were dissolved in ethanol at a molar ratio of 46.3:9.4:42.7:1.6, respectively.

[0174] Aqueous phase preparation: Luci-mRNA was dissolved in PBS to a concentration of 22.22 ng / uL.

[0175] Take 1 μL of ethanol phase and 9 μL of aqueous phase. The amounts of cationic lipids in 1 μL of ethanol phase are 0.88 nmol, 1.76 nmol, 3.53 nmol, 5.29 nmol, and 7.06 nmol, respectively. The amount of Luci-mRNA in 9 μL of aqueous phase is 200 ng. Add 1 μL of ethanol phase to 9 μL of aqueous phase with a pipette and mix well. Let stand for 10 minutes to obtain LNP@LucimRNA with N:P = 3, 6, 12, 18, and 24, respectively.

[0176] Example 12: Preparation of lipid nanoparticles

[0177] Preparation of three-component LNP@Luci mRNA with BO10, BO12, BO14, BO16, BO18, BM10, BM12, BM14, BM16, BM18, BP10, BP12, BP14, BP16, or BP18 as cationic lipids:

[0178] Ethanol phase preparation: Cationic lipids, DOPE, and cholesterol were dissolved in ethanol at a molar ratio of 47.4:31.6:21.1.

[0179] Aqueous phase preparation: Luci-mRNA was dissolved in citrate-sodium citrate buffer at pH 4 to a concentration of 22.22 ng / μL.

[0180] Take 1 μL of ethanol phase and 9 μL of aqueous phase. The amounts of cationic lipids in the 1 μL ethanol phase are 0.22 nmol, 0.44 nmol, 0.88 nmol, 1.76 nmol, 3.53 nmol, and 5.29 nmol, respectively. The 9 μL aqueous phase contains 200 ng of Luci-mRNA. Add 1 μL of ethanol phase to the 9 μL aqueous phase with a pipette and mix well. Let stand for 10 minutes to obtain LNP@Luci mRNA with N:P = 0.75, 1.5, 3, 6, 12, and 18, respectively.

[0181] Example 13: Preparation of lipid nanoparticles

[0182] Preparation of three-component LNP@Luci mRNA with CO10, CO12, CO14, CO16, CO18, CO22, CM10, CM12, CM14, CM16, CM18, CM22, CP10, CP12, CP14, CP16, CP18, or CP22 as cationic lipids:

[0183] Ethanol phase preparation: Cationic lipids, DOPE, and cholesterol were dissolved in ethanol at a molar ratio of 47.4:31.6:21.1.

[0184] Aqueous phase preparation: Luci-mRNA was dissolved in citrate-sodium citrate buffer at pH 4 to a concentration of 22.22 ng / μL.

[0185] Take 1 μL of ethanol phase and 9 μL of aqueous phase. The amounts of cationic lipids in the 1 μL ethanol phase are 0.44 nmol, 0.88 nmol, 1.76 nmol, 3.53 nmol, 5.29 nmol, and 7.06 nmol, respectively. The 9 μL aqueous phase contains 200 ng of Luci-mRNA. Add 1 μL of ethanol phase to the 9 μL aqueous phase with a pipette and mix well. Let stand for 10 minutes to obtain LNP@Luci mRNA with N:P = 1.5, 3, 6, 12, 18, and 24, respectively.

[0186] Example 14: Preparation of lipid nanoparticles

[0187] Preparation of four-component LNP@Luci mRNA with CO10, CO12, CO14, CO16, CO18, CO22, CM10, CM12, CM14, CM16, CM18, CM22, CP10, CP12, CP14, CP16, CP18, or CP22 as cationic lipids:

[0188] Ethanol phase preparation: Cationic lipids, DSPC, cholesterol, and ALC-0159 were dissolved in ethanol at a molar ratio of 46.3:9.4:42.7:1.6, respectively.

[0189] Aqueous phase preparation: Luci-mRNA was dissolved in citrate-sodium citrate buffer at pH 4 to a concentration of 22.22 ng / μL.

[0190] Take 1 μL of ethanol phase and 9 μL of aqueous phase. The amounts of cationic lipids in the 1 μL ethanol phase are 0.88 nmol, 1.76 nmol, 3.53 nmol, 5.29 nmol, and 7.06 nmol, respectively. The 9 μL aqueous phase contains 200 ng of Luci-mRNA. Add 1 μL of ethanol phase to the 9 μL aqueous phase with a pipette and mix well. Let stand for 10 minutes to obtain LNP@Luci mRNA with N:P = 1.5, 3, 6, 12, 18, and 24, respectively.

[0191] Comparative Example 1: Preparation of lipo2000@Luci mRNA

[0192] Dissolve lipo2000 to 0.1 mg / ml in PBS, dissolve Luci-mRNA to 25 ng / μL in PBS, take 2 μL of lipo2000 (0.1 mg / ml) and 8 μL of Luci mRNA, mix well and let stand for 10 minutes to obtain lipo2000@Luci mRNA.

[0193] Comparative Example 2: Preparation of Pfizer / BioNTech LNP@Luci-mRNA with ALC-0315 as the cationic lipid:

[0194] Ethanol phase preparation: ALC-0315, DSPC, cholesterol, and ALC-0159 were dissolved in ethanol at a molar ratio of 46.3:9.4:42.7:1.6, respectively.

[0195] Aqueous phase preparation: Luci-mRNA was dissolved in citrate-sodium citrate buffer at pH 4 to a concentration of 22.22 ng / μL.

[0196] Take 1 μL of ethanol phase and 9 μL of water phase. The 1 μL ethanol phase contains 1.76 nmol of ALC-0315 and the 9 μL water phase contains 200 ng of Luci-mRNA. Add the 1 μL ethanol phase to the 9 μL water phase with a pipette and mix well. Let stand for 10 minutes to obtain LNP@Luci mRNA with N:P = 3.

[0197] Experiment 1: Screening the transfection effects of different LNPs on 293T cells

[0198] 293T cells were seeded per well in a 96-well white-based cell culture plate, with a cell culture volume of 90 μL per well. After 24 hours of culture, 10 μL of LNP prepared in the examples or comparative examples was added to the wells. After co-incubation for 24 hours, One-umi was added. TM Firefly luciferase reporter gene assay reagent (100 μL / well), after standing for 10 minutes, its bioluminescence value was measured using an ELISA reader.

[0199] RLU, Raletive luciferase activity, is related to luciferase activity.

[0200] The test results are as follows:

[0201] Table 1. Screening results of three-component LNP@Luci mRNAs of different cationic lipids

[0202]

[0203]

[0204]

[0205] Table 2. Screening results of four-component LNP@Luci mRNAs of different cationic lipids.

[0206]

[0207]

[0208]

[0209] As can be seen from Tables 1 and 2:

[0210] AM12, a three-component LNP@Luci-mRNA with N:P=3 for cationic lipids, showed 1.6 times the transfection efficiency of lipo2000 and 1.4 times the transfection efficiency of ALC-0315 four-component LNP@Luci-mRNA (N:P=3).

[0211] The transfection efficiency of the three-component LNP@Luci mRNA (N:P=6) with CO10 as the cationic lipid was 1.3 times that of lipo2000 and 1.2 times that of the four-component LNP@Luci-mRNA (N:P=3) transfection of ALC-0315.

[0212] The transfection efficiency of the three-component LNP@Luci mRNA (N:P=1.5) with CP10 as the cationic lipid was twice that of lipo2000 and 1.8 times that of the four-component LNP@Luci-mRNA (N:P=3) with ALC-0315.

[0213] The transfection efficiency of the three-component LNP@Luci mRNA (N:P=18) with CM12 as the cationic lipid was 1.6 times that of lipo2000 and 1.4 times that of the four-component LNP@Luci-mRNA (N:P=3) of ALC-0315.

[0214] The transfection efficiency of the three-component LNP@Luci mRNA (N:P=24) with CP12 as the cationic lipid was 1.2 times that of lipo2000 and 1.06 times that of the four-component LNP@Luci-mRNA (N:P=3) of ALC-0315.

[0215] CO14 (N:P=12) is a three-component LNP@Luci mRNA (N:P=24) of cationic lipids, which is 2.25 times that of lipo2000 and 2.02 times that of ALC-0315 four-component LNP@Luci-mRNA (N:P=3).

[0216] CM14 (N:P=3) is 4 times that of lipo2000 and 3.6 times that of ALC-0315 four-component LNP@Luci-mRNA (N:P=3) transfection.

[0217] The transfection efficiency of the three-component LNP@Luci mRNA (N:P=3) with CP12 as the cationic lipid was 1.6 times that of lipo2000 and 1.4 times that of the four-component LNP@Luci-mRNA (N:P=3) transfection of ALC-0315.

[0218] Experimental Example 2: Test of Cationic Lipid Molecular Delivery Performance

[0219] I. Preparation of a three-component LNP@IDH1(R132H) with AM12 as the cationic lipid

[0220] Ethanol phase preparation: Cationic lipids AM12, DOPE, and cholesterol were dissolved in ethanol at a molar ratio of 47.4:31.6:21.1.

[0221] Aqueous phase preparation: IDH1 (R132H) antigen mRNA was dissolved in PBS to a concentration of 22.22 ng / uL. The IDH1 (R132H) antigen sequence is referenced in: Platten, M., Bunse, L., Wick, A. et al. A vaccine targeting mutant IDH1 in newly diagnosed glioma. Nature 592, 463–468 (2021).

[0222] Take 1 μL of ethanol phase and 9 μL of aqueous phase. The 1 μL of ethanol phase contains 0.88 nmol and 1.76 nmol of cationic lipids, respectively. Add 1 μL of ethanol phase to 9 μL of aqueous phase with a pipette and mix by blowing and stirring. Let stand for 10 minutes to obtain LNP@IDH1(R132H) with N:P = 3 and 6, respectively.

[0223] II. Western blot experiment on LNP@IDH1(R132H) expression in 293T cells

[0224] Cell seeding: 500,000 cells / well were seeded into a six-well plate. 2.5 mL of DMEM complete medium was added to each well. After 24 hours of seeding, the medium in the wells was replaced with DMEM medium (1 mL / well) without fetal bovine serum. LNP@IDH1(R132H) with N:P=3, LNP@IDH1(R132H) with N:P=6, and free IDH1(R132H) mRNA were added accordingly and incubated for 24 hours.

[0225] Protein sample preparation

[0226] Add 90 μL of RIPA lysis buffer to each well of a six-well plate, lyse on ice for 30 minutes, collect the lysate in an EP tube, centrifuge at 15,000 rpm for 10 minutes, collect the supernatant, add about 10 μL of 5x loading buffer to each well of the supernatant, mix well, and heat at 100 °C for 10 minutes.

[0227] SDS-PAGE electrophoresis

[0228] Place the SDS-PAGE gel in the electrophoresis tank, add 1X electrophoresis buffer, and then load 5 μL of protein marker, 15 μL of N:P=3 LNP@IDH1(R132H), 15 μL of N:P=6 LNP@IDH1(R132H), and 15 μL of free IDH1(R132H) mRNA onto the SDS-PAGE gel. Electrophoresis is performed at 150V for 45 minutes.

[0229] Transfer membrane

[0230] Prepare a 0.2µm PVDF membrane approximately 5*7cm in size (pre-activated by soaking in methanol for 1 minute). In an enamel tray containing transfer buffer, open the clamp so the black side is facing down. Place a sponge pad, three layers of filter paper, the PVDF membrane, SDS-PAGE gel, three layers of filter paper, and another sponge pad in the following order: clamp, filter paper, and sponge pad. Place the clamp into the transfer tank (black side of the clamp facing the black side of the tank), and transfer the membrane at 300mA for 70 minutes on ice.

[0231] Closed

[0232] The PVDF membrane was immersed in TBST containing 5% skim milk and sealed on a shaker at room temperature for 2 hours.

[0233] Washing film

[0234] After sealing, the PVDF membrane is soaked in TBST on a shaker at room temperature for 10 minutes. Then, the old TBST is poured off and new TBST is added. This process is repeated three times.

[0235] Primary Antibody Incubation

[0236] The primary antibody was diluted 1:20000 with antibody dilution buffer. The PVDF membrane was then immersed in the diluted primary antibody and incubated on a shaker at 4°C for 10 hours.

[0237] Washing film

[0238] After incubating the PVDF membrane with the primary antibody, soak it in TBST on a shaker at room temperature for 10 minutes, then pour off the old TBST and add new TBST. Repeat this process three times.

[0239] Secondary antibody incubation

[0240] The secondary antibody was diluted 1:5000 with TBST containing 2.5% skim milk. The PVDF membrane was then immersed in the diluted secondary antibody and incubated on a shaker at room temperature for 1.5 hours.

[0241] Washing film

[0242] After incubating the PVDF membrane with the secondary antibody, soak it in TBST on a shaker at room temperature for 10 minutes, then pour off the old TBST and add new TBST. Repeat this process three times.

[0243] Chemiluminescence imaging

[0244] The developer solutions A and B were mixed in a 1:1 ratio and dropped onto the surface of the PVDF membrane. After about 30 seconds, the membrane was developed using a chemiluminescence analyzer.

[0245] The results are as follows Figure 7 As shown.

[0246] from Figure 7 It can be seen that the three-component LNP with AM12 as the cationic lipid and N:P=3 can effectively deliver glioma tumor antigens, and this LNP has the potential to be developed into a glioma tumor vaccine vector.

[0247] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof: Compounds of Formula I are selected from: ； In formula IA, L is selected from C1~C 10 Alkylene; t1 is selected from natural numbers from 3 to 10; ； In formula IB, when the substituents on the benzene ring are in the ortho or para position, L is selected from C1 to C2. 10 Alkylene; t1 is selected from natural numbers from 3 to 10; when the positional relationship of the substituents on the benzene ring is meta, L is selected from C1 to C2. 10 Alkylene; t1 is 3; ； In formula IC, when the substituents on the benzene ring are in the ortho or para position, L is selected from C1 to C2. 10 Alkylene; t4 is selected from natural numbers from 3 to 10; When the substituents on the benzene ring are in a meta position, L is selected from C1~C2. 10 Alkylene; t4 is selected from natural numbers from 4 to 10.

2. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: Compounds of Formula I are selected from the following compounds: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 3. A method for preparing the compound of formula I according to claim 1 or 2, characterized in that: Includes the following steps: i) Compound of Formula II With Formula III compounds The reaction yields a compound of formula IA or formula IB; or ii) Compound of formula II With Formula IV compounds The reaction yields a compound of formula IC; Where n=2, and the positional relationships of the substituents on the benzene ring in the compound of formula II are defined as described in claim 1 or 2.

4. A pharmaceutical composition, characterized in that: The carrier comprises a cationic lipid comprising a compound of formula I as claimed in claim 1 or 2 or a pharmaceutically acceptable salt thereof, or at least one of the following compounds: 、 、 、 。 5. The pharmaceutical composition according to claim 4, characterized in that: The cationic lipid accounts for 10% to 75% of the carrier molar ratio.

6. The pharmaceutical composition according to claim 4, characterized in that: The carriers include cationic lipids, neutral lipids, and structural lipids.

7. The pharmaceutical composition according to claim 4, characterized in that: The carriers include cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids.

8. The pharmaceutical composition according to any one of claims 4 to 7, characterized in that: The pharmaceutical composition further includes at least one therapeutic agent, a preventive agent, a photosensitizer, and / or a photothermal agent.

9. The use of the compound of Formula I according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 4 to 8, in the preparation of a medicament for treating a disease or condition selected from the group consisting of: infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

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