Ionizable lipid compound and application thereof

Abstract

The invention relates to the technical field of drug delivery, in particular to an ionizable lipid compound and application thereof. The invention provides an ionizable lipid compound simultaneously containing # imgabs0 # fragments. The chemical structure of the ionizable lipid compound is shown as a formula (I). The invention also provides an application of the ionizable lipid compound. The mRNA-LNP containing the ionizable lipid compound provided by the invention can integrally improve the biocompatibility of the LNP and the mRNA transfection efficiency, can efficiently deliver a chimeric antigen receptor (CAR) to cells which are extremely difficult to transfect, such as T cells, NK cells, macrophages and the like, especially when being applied to the field of cell therapy, and has an unexpected technical effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to an ionizable lipid compound and its application. Background Art

[0002] Nucleic acid drugs, compared with traditional small molecule chemical drugs and antibody drugs, are theoretically not restricted by the druggability of target proteins. With the advancement of clinical trials and the maturity of related technologies, they have a wider range of application scenarios.

[0003] As an exogenous drug, nucleic acid drugs need to overcome multiple obstacles to exert their effects in the body: instability, immunogenicity, low cell uptake efficiency, difficulty in endosomal escape, etc. An efficient and safe delivery system is an important guarantee for nucleic acid drugs to overcome the above defects and target to exert stable drug effects.

[0004] Currently, lipid nanoparticles (LNPs) have shown high delivery efficiency and good safety in vivo due to their unique structure and physicochemical properties, and have become one of the mainstream delivery systems for nucleic acid drugs.

[0005] Chimeric antigen receptor (CAR)-modified immune cell therapy is a hot research field in current cancer treatment. It is necessary to prepare modified cells in vitro using transfection technology. The mainstream transfection technologies are lentiviral vector transfection or electroporation. However, although lentiviral vector transfection technology can mediate permanent CAR expression, it also has many disadvantages, such as high cost, inability to repeat dosing, complex operation process, and high technical threshold, which limit the wide application of lentiviral vector transfection in the field of cell transfection. Electroporation technology can cause high cytotoxicity, resulting in low yields and activities of cell products. Cells need time to recover from the damage caused by electric current stimulation, resulting in a longer production cycle of cell products. Compared with viral vectors and electroporation technology, mRNA-LNP has the advantages of low cost, high transfection efficiency, good safety, repeatable dosing, and mature scale-up process. It is a key technology for efficient transfection of immune cells including T cells and plays an irreplaceable important role in the field of cell therapy.

[0006] Ionizable lipid compounds are very crucial core components in the LNP delivery system, and have functions such as binding to negatively charged nucleic acids, promoting cell uptake and endosomal escape, and enhancing in vivo transfection of nucleic acid drugs.

[0007] Although various ionizable lipid compounds have been disclosed in the prior art, most of these ionizable lipid compounds have problems such as low transfection efficiency and poor stability. In particular, for cells that are extremely difficult to transfect (such as immune cells, primary cells, and stem cells), there are no commercially available products. Therefore, there is still a need in the art to find ionizable lipid compounds with higher biocompatibility and transfection efficiency and better stability to improve the effect of cell therapy. Summary of the Invention

[0008] In view of the above-mentioned disadvantages of the prior art, the object of the present invention is to provide a novel ionizable lipid compound, which simultaneously contains a fragment, and this specific structural feature can overall improve the biocompatibility of LNP and the mRNA transfection efficiency.

[0009] To achieve the above object and other related objects, the first aspect of the present invention provides an ionizable lipid compound, the chemical structure of which is shown in the following formula (Ⅰ):

[0010]

[0011] Wherein, a and b independently selected from natural numbers from 0 to 25; G1 and G2 independently selected from linear C2-C 10 alkylene;

[0012] G3 is selected from linear or branched C1-C 10 alkylene; or G3 is selected from (CH2) d -O-(CH2) e , wherein, d and e independently selected from 1, 2, 3, 4, 5, 6, 7, 8 or 9, and d + e is an integer from 2 to 10; or G3 is selected from Wherein, g is selected from 2, 3, 4;

[0013] L1 and L2 independently selected from -(C=O)O-, -O(C=O)-, -(C=O)S-, -S(C=O)-, -(C=O)NR-, -NR(C=O)-, -O(C=O)O-, -NR(C=O)O-, -O(C=O)NR-, and R independently selected from H, C1-C 12 hydrocarbon group.

[0014] The second aspect of the present invention provides a lipid nanoparticle, which comprises one or more combinations of the above-mentioned ionizable lipid compound, its stereoisomer, its tautomer or its pharmaceutically acceptable salt.

[0015] In some embodiments of the present invention, the lipid nanoparticle further comprises any one or more combinations of a structural lipid, a helper lipid, a PEG-lipid, and a polymer.

[0016] In some embodiments of the present invention, the lipid nanoparticles further comprise the loaded drug and / or pharmaceutically acceptable excipients.

[0017] The third aspect of the present invention provides the use of the lipid nanoparticles in the preparation of a pharmaceutical composition, and the pharmaceutical composition further comprises the loaded drug and / or pharmaceutically acceptable excipients.

[0018] In some embodiments of the present invention, 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.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The ionizable lipid compound provided by the present invention has an N atom as the ionizable center, a hydrophilic group as the head, and two hydrophobic groups as the tail, and also contains a fragment. Among them, introducing two hydroxyl groups into the head of the ionizable lipid compound can increase the hydrophilicity of the head of the lipid compound, and introducing a methyl group into its tail can appropriately increase the space occupied by the tail structure. The nano-carrier self-assembled by the ionizable lipid compound with such structural characteristics can improve the intracellular delivery efficiency and transfection efficiency.

[0021] 2. The mRNA-LNP prepared by using the ionizable lipid compound provided by the present invention has a size of 60-120 nm, a nucleic acid encapsulation rate greater than 94%, and good low-temperature storage stability. The cell transfection efficiency experiment shows that compared with the commercialized products at the cell levels of T cells, NK cells, macrophages, etc., the transfection efficiency can be increased by 2 orders of magnitude, and it has higher biocompatibility.

[0022] 3. The ionizable lipid compound provided by the present invention can efficiently deliver CAR to cells that are extremely difficult to transfect, such as T cells, NK cells, macrophages, etc., and is particularly suitable for application in the field of cell therapy. Moreover, the synthesis steps are simple, the cost is controllable, it is easy to scale up, which is conducive to the industrialization of biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the 1H NMR spectrum of the ionizable lipid compound H-10 provided by the present invention.

[0024] Figure 2 is the particle size distribution diagram of the mRNA-LNP prepared from the ionizable lipid compound H-10 provided by the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0025] To solve the deficiencies of the prior art, the object of the present invention is to provide a kind of containing Ionizable lipid compounds, pharmaceutical compositions and uses thereof. The pharmaceutical compositions prepared from the ionizable lipid compounds with novel structures of the present invention have high drug transfection efficiency, good biocompatibility and high stability. Therefore, when applied to the field of cell therapy, they can efficiently deliver chimeric antigen receptors (CARs) to cells that are extremely difficult to transfect, such as T cells, NK cells, macrophages, etc.

[0026] To achieve the above objectives, the present invention adopts the following technical solutions:

[0027] In the first aspect of the present invention, an ionizable lipid compound is provided, and its chemical structure is shown in the following formula (I):

[0028]

[0029] Wherein, a and b each independently selected from natural numbers from 0 to 25; G1 and G2 each independently selected from linear C2-C 10 alkylene;

[0030] G3 is selected from linear or branched C1-C 10 alkylene; or G3 is selected from (CH2) d -O-(CH2) e , wherein d and e each independently selected from 1, 2, 3, 4, 5, 6, 7, 8 or 9, and d + e is an integer from 2 to 10; or G3 is selected from Wherein, g is selected from 2, 3, 4;

[0031] L1 and L2 each independently selected from -(C=O)O-, -O(C=O)-, -(C=O)S-, -S(C=O)-, -(C=O)NR-, -NR(C=O)-, -O(C=O)O-, -NR(C=O)O-, -O(C=O)NR-, and R is independently selected from H, C1-C 12 hydrocarbon group.

[0032] In the present invention, the ionizable lipid compound has an N atom as the ionizable center, a hydrophilic group as the head, and two hydrophobic groups as the tails, and also contains fragments. Among them, introducing two hydroxyl groups into the head of the ionizable lipid compound can increase the hydrophilicity of the head of the lipid compound, and introducing a methyl group into its tail can appropriately increase the space occupied by the tail structure. The nanocarriers self-assembled from the ionizable lipid compounds with such structural characteristics can improve the intracellular delivery efficiency and transfection efficiency.

[0033] In some embodiments of the present invention, a and b each independently selected from natural numbers from 10 to 20; G1 and G2 each independently selected from linear C3-C9 alkylene;

[0034] G3 is selected from a linear C2-C6 alkylene group; or G3 is selected from (CH2) d -O-(CH2) e , where d and e are each independently selected from 1, 2, 3, 4, 5, and d + e is an integer from 2 to 6; or G3 is selected from where g is selected from 2, 3;

[0035] L1 and L2 are each independently selected from -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, O(C=O)O-, -(C=O)S-, -S(C=O)-, -(C=O)NH-, -NH(C=O)-.

[0036] In some embodiments of the present invention, a and b are each independently selected from natural numbers from 5 to 17; G1 and G2 are each independently selected from 1,3-propylene, 1,4-butylene, 1,5-pentylene, 1,6-hexylene, 1,7-heptylene, 1,8-octylene;

[0037] G3 is selected from 1,2-ethylene, 1,3-propylene, 1,4-propylene; or G3 is selected from (CH2) d -O-(CH2) e , where d and e are each independently selected from 1, 2, 3, and d + e is an integer from 2 to 4;

[0038] L1 and L2 are each independently selected from -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, -O(C=O)O-, -(C=O)S-, -S(C=O)-, -(C=O)NH-, -NH(C=O)-.

[0039] In some embodiments of the present invention, the ionizable lipid compound is selected from the following group:

[0040]

[0041] In some embodiments of the present invention, the ionizable lipid compound is selected from the following group:

[0042] In some embodiments of the present invention, the ionizable lipid compound is selected from the following group:

[0043] In the present application, the compound represented by formula (I) can be prepared and obtained by referring to the following method:

[0044] The preparation method of the ionizable lipid compounds shown in the above H-1 to H-19 includes the following steps:

[0045] (1) Esterify an isoalkyl carboxylic acid (or alcohol) with a terminal alkenyl alkyl alcohol (or carboxylic acid) to provide a first intermediate product;

[0046] (2) Oxidize the double bond of the first intermediate product to form an epoxy group to provide a second intermediate product;

[0047] (3) Subject an amine to a ring-opening reaction with the epoxy group of the second intermediate product to provide an ionizable lipid compound.

[0048] The method for preparing the ionizable lipid compound shown in H-20 to H-21 above comprises the following steps:

[0049] (1) Esterify an isoalkyl carboxylic acid with a terminal alkenyl alkyl mercaptan to provide a first intermediate product;

[0050] (2) Oxidize the double bond of the first intermediate product to form an epoxy group to provide a second intermediate product;

[0051] (3) Subject an amine to a ring-opening reaction with the epoxy group of the second intermediate product in H-1 to H-19 to provide a third intermediate product (secondary amine intermediate);

[0052] (4) Subject an epoxy compound containing an ester bond to a ring-opening reaction with the epoxy group of the second intermediate product to provide an ionizable lipid compound.

[0053] The method for preparing the ionizable lipid compound shown in H-22 to H-23 above comprises the following steps:

[0054] (1) Condense an isoalkyl carboxylic acid (or amine) with a terminal alkenyl alkyl amine (or carboxylic acid) to provide a first intermediate product;

[0055] (2) Oxidize the double bond of the first intermediate product to form an epoxy group to provide a second intermediate product;

[0056] (3) Subject an epoxy compound containing an ester bond (the third intermediate in H-20 to H-21) to a ring-opening reaction with the epoxy group of the second intermediate product to provide an ionizable lipid compound.

[0057] The compound shown in formula (I) is a lipid whose protonation degree is affected by pH, thereby affecting its charge property. Preferably, it is a lipid that is almost uncharged under normal neutral physiological pH conditions, but can carry a positive charge at acidic pH and thus bind to negatively charged nucleic acids.

[0058] The second aspect of the present invention provides a lipid nanoparticle, which comprises one or a combination of the above ionizable lipid compounds, their stereoisomers, their tautomers or their pharmaceutically acceptable salts.

[0059] The term "stereoisomer" refers to isomers that have the same atomic connection order but different spatial arrangements of atoms.

[0060] The term "tautomer" refers to the phenomenon where the structure of a compound undergoes a balanced interconversion between two functional group isomers, and the corresponding isomers are called tautomers.

[0061] The term "pharmaceutically acceptable salt" refers to acid addition salts or base addition salts. All compounds of the present invention in free base or free acid form can be converted into their pharmaceutically acceptable salts by treatment with appropriate inorganic or organic bases or acids according to methods known to those skilled in the art. The salts of the compounds of the present invention can be converted into their free base or acid form by standard techniques.

[0062] The pharmaceutically acceptable salts of the compounds of the present invention include those salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or salts formed with amino groups by using other methods such as ion exchange used in the art. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, fumarates, glucoheptanoates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxyethanesulfonates, lactates, lactobionates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, etc. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, and ammonium salts. Representative alkali metal salts or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, etc. In appropriate cases, additional pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and arylsulfonates. Additional pharmaceutically acceptable salts include salts formed by quaternization of amines, which quaternization is carried out using appropriate electrophiles (e.g., alkyl halides) to form quaternized alkylated ammonium salts.

[0063] The lipid nanoparticles provided by the present invention further comprise a combination of one or more of a structural lipid, a co-lipid, a PEG-lipid, and a polymer.

[0064] In some specific embodiments of the present invention, in the lipid nanoparticles, the molar percentages of the ionizable lipid compound: co-lipid: structural lipid: PEG-lipid are (20 - 65):(0 - 60):(0 - 60):(0 - 10), and at least any one of the co-lipid, structural lipid, and PEG-lipid is not 0. Preferably, the molar percentages of the ionizable lipid compound: co-lipid: structural lipid: PEG-lipid are (20 - 65):(3 - 50):(15 - 60):(0.1 - 10). In some other specific embodiments of the present invention, in the lipid nanoparticles, the molar ratio of the ionizable lipid compound to the polymer is 0.5:1 - 100:1, preferably 10:1 - 80:1, and more preferably 40:1 - 80:1.

[0065] In some specific embodiments of the present invention, the lipid nanoparticles comprise 20% - 65% ionizable lipid compound, 3% - 40% co-lipid, 20% - 60% structural lipid, and 0.1% - 10% PEG-lipid, where % refers to molar percentage.

[0066] In some specific embodiments of the present invention, the lipid nanoparticles comprise 35% - 49% ionizable lipid compound, 5% - 20% co-lipid, 35% - 50% structural lipid, and 1% - 2% PEG-lipid, where % refers to molar percentage.

[0067] The structural lipid mentioned refers to a structure that can stabilize the composition, including but not limited to a combination of one or more of sterols and their derivatives and non-sterols and their derivatives.

[0068] In some specific embodiments, the structural lipid includes but is not limited to: a combination of one or more of sterols and their derivatives, non-sterols, sitosterol, ergosterol, cholestanone, cholestenone, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, coprosterol, α-tocopherol, or corticosteroids. Sterols are preferably cholesterol and its derivatives; non-limiting examples of cholesterol derivatives include: polar analogs such as 5α-cholestanol, 5α-coprosterol, cholesterolyl-(2'-hydroxy)ethyl ether, cholesterolyl-(4'-hydroxy)butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestane, cholestenone, 5α-cholestenone, and cholesteryl caprate; and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analog such as cholesterolyl-(4'-hydroxy)butyl ether. This is not an exhaustive list, and the selection of the structural lipid is not limited, and any structural lipid can be applied to the present invention.

[0069] In some specific embodiments, the structured lipid is one or a combination of cholesterol, sitosterol, ergosterol, corticosteroids and their derivatives.

[0070] In some specific embodiments, the structured lipid is cholesterol.

[0071] There is no limitation on the type of the "auxiliary lipid", and phospholipid lipids are preferred, including but not limited to: one or a combination of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dimyristoyl phosphatidylglycerol.

[0072] In some specific embodiments, the auxiliary lipid can be selected from one or a combination of more than one of: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-docosanoyl-sn-glycerophosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenoyl-sn-glycero-3-phosphocholine (18:0 diether PC), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), dipalmitoylphosphatidylethanolamine (DPPE), 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-O-hexadecyl-sn-glycerophosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-docosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), diacetyl-phosphatidylethanolamine (DEPE), stearoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, sphingomyelin.

[0073] In some specific embodiments, the phosphatidylcholine is a combination of one or more of DSPC, DPPC, DMPC, DOPC, POPC.

[0074] In some specific embodiments, the auxiliary lipid is phosphatidylcholine, specifically DSPC.

[0075] In some specific embodiments, the auxiliary lipid is phosphatidylethanolamine, specifically DOPE.

[0076] In some specific embodiments, the auxiliary lipid is selected from one or more combinations of DOTAP ((1,2-dioleoyloxypropyl) trimethylammonium chloride), DODAP (1,2-dioleoyl-3-dimethylammonium-propane), 18:1PA (1,2-DI(cis-9-octadecenoyl)-sn-glycero-3-phosphate sodium salt), HS15 (polyethylene glycol (15)-hydroxystearate), GL67 (N4-arginyl-cholesteryl carbamate).

[0077] The PEG-lipid described in the present invention generally refers to a conjugate formed by chemically linking PEG (polyethylene glycol) with a lipid molecule. It includes but is not limited to PEG-modified phospholipids and derived lipids, such as, by way of example, 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 methoxypolyethylene glycol ditetradecylacetamide.

[0078] In some specific embodiments, the PEG-lipid includes but is not limited to PEG-C-DMG, PEG-C-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, PEG-DPPC, PEG-distearoyl phosphatidylethanolamine (PEG-DSPE), PEG-DS, Chol (cholesterol)-PEG, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (PEG-DMG), PEG-S-DMG, polyethylene glycol phosphatidylethanolamine, polyethylene glycol ceramide, polyethylene glycol dimethacrylate (PEG-DMA), PEG distearyl glycerol, PEG dipalmitoleyl, PEG dioleyl, PEG-based distearyl, PEG-based diacylglycamide, PEG dipalmitoyl phosphatidylethanolamine, PEG-based phosphatidylethanol, PEG-based phosphatidylethylene myristyloxypropyl-3-amine, PEG-based oxypropylolamine, 1,2-distearoyloxypropyl-3-amine-N[methoxy(polyethylene glycol)] (PEG-DSA), methoxypolyethylene glycol laurate, and methoxypolyethylene glycol ditetradecylacetamide (ALC0159).

[0079] In some specific embodiments, the PEG-lipid is PEG-DMG.

[0080] There is no limitation on the type of the "polymer", and the polymer may include but is not limited to amphiphilic block copolymers. The amphiphilic block copolymer is a block copolymer composed of a hydrophobic polymer and a hydrophilic compound, including but not limited to polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(lactide-co-caprolactone) (PLCG), polycaprolactone (PCL), polyorthoester, polyanhydride (PAH), polyphosphazene, poly(β - amino ester) (PBAE), poly(α - hydroxy acid), lactide / glycolide copolymer (PLGA or PLG) (including lactide / glycolide copolymer, D - lactide / glycolide copolymer, L - lactide / glycolide copolymer and D,L - lactide / glycolide copolymer), polyglycolide (PGA), polyorthoester (POE), linear or branched polyethylene glycol (PEG), conjugate of poly(α - hydroxy acid), polyaspirins, polyphosphazenes, D - lactide, D,L - lactide - caprolactone, D,L - lactide - glycolide - caprolactone, dextran, vinylpyrrolidone, polyvinyl alcohol (PVA), methacrylate, poly(N - isopropylacrylamide), SAIB (sucrose acetate isobutyrate), hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose, carboxymethyl cellulose or its salt, carbopol, poly(2 - hydroxyethyl methacrylate), poly(2 - methoxyethyl methacrylate), poly(2 - methoxyethoxy - ethyl methacrylate), polymethyl methacrylate (PMMA), methyl methacrylate (MMA), PVA - g - PLGA, PEGT - PBT copolymer, PEO - PPO - PEO (pluronics), PEO - PPO - PAA copolymer, PLGA - PEO - PLGA, PEG - PLGA, PLA - PLGA, PEG - PLA, PEG - PCL, poloxamer 407, PEG - PLGA - PEG triblock copolymer, PEG - PLA - PEG triblock copolymer, PEG - PCL - PEG triblock copolymer or their block copolymers with polyethylene glycol (PEG), or a combination of one or more of the above polymers or copolymers.

[0081] In some embodiments of the present invention, the weight - average molecular weight of PEG in the PEG - lipid is 1000 - 10000, such as 1000 - 2000, 2000 - 4000, 4000 - 6000, 6000 - 8000, 8000 - 10000, and preferably 2000.

[0082] The third aspect of the present invention provides the application of the lipid nanoparticles in the preparation of a pharmaceutical composition, and the pharmaceutical composition further comprises the loaded drug and / or pharmaceutically acceptable excipients.

[0083] The "loaded drug" described in the present invention includes, but is not limited to, any one or a combination of nucleic acids, small molecules, and proteins.

[0084] The "nucleic acid" described in the present invention can be a nucleotide polymer of any length. It includes, but is not limited to, single-stranded DNA, double-stranded DNA, plasmid DNA, short isoforms, mRNA, tRNA, rRNA, long non-coding RNA (lncRNA), micro non-coding RNA (miRNA and siRNA), telomerase RNA, small nuclear RNA (snRNA and scRNA), circular RNA (circRNA), synthetic miRNA (miRNA mimics, miRNA agomir, miRNA antagomir), antisense oligonucleotides (ASO), ribozymes, asymmetric interfering RNA (aiRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), guide RNA (gRNA), small guide RNA (sgRNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), morpholino antisense oligonucleotides, morpholino oligonucleotides, or a combination of one or more of biocustom oligonucleotides.

[0085] In certain embodiments of the present invention, the nucleic acid is mRNA. The mRNA is a type of single-stranded ribonucleic acid transcribed from one strand of DNA as a template and carrying genetic information to guide protein synthesis. The mRNA can be monocistronic mRNA or polycistronic mRNA.

[0086] The "small molecule" described in the present invention refers to a compound that is not a protein or nucleic acid molecule. Small molecules can be small molecule therapeutic agents and / or prophylactic agents, such as antibiotics, anti-inflammatory drugs, anti-cancer drugs, anti-viral drugs, immunosuppressants, analgesics, anti-fungal drugs, anti-parasitic drugs, anti-convulsants, anti-depressants, anti-anxiety drugs, anti-psychotic drugs, etc.

[0087] The "protein" described in the present invention refers to a molecule or complex containing one or more polypeptides with secondary, tertiary, and / or quaternary structures. The secondary, tertiary, and / or quaternary structures of proteins are usually stabilized by non-covalent bonds such as ionic bonds, hydrogen bonds, hydrophobic interactions, and / or van der Waals force interactions. Additionally, or alternatively, proteins can include disulfide bonds, for example, between the 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, etc.

[0088] The pharmaceutical composition of the present invention further comprises pharmaceutically acceptable excipients. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 4 - 8, preferably about 5 - 7, although the pH value can vary depending on the nature of the substances being formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (such as intraperitoneal), intracranial injection, intracavitary injection, inhalation administration, implant administration, etc.

[0089] "Pharmaceutically acceptable" as used in the present invention means that when the drugs are properly administered to animals or humans, they do not produce adverse, allergic or other adverse reactions.

[0090] "Pharmaceutically acceptable excipients" should be compatible with the active ingredient, that is, they can be blended with it without significantly reducing the efficacy of the drug under normal circumstances. Specific examples of some substances that can be used as pharmaceutically acceptable excipients can be sugars, such as glucose, mannitol, sucrose, lactose, trehalose, maltose, etc.; starches, such as corn starch and potato starch, etc.; cellulose and its derivatives, such as sodium methylcellulose, ethyl cellulose and methyl cellulose, etc.; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate, etc.; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter, etc.; alcohols, such as ethanol, propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol, etc.; alginic acid; emulsifiers, such as Tween, etc.; wetting agents, such as sodium lauryl sulfate, etc.; surfactants; lyoprotectants; coloring agents; flavoring agents; tabletting agents; stabilizers; diluents; excipients; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; buffer solutions, etc., and their combinations. These substances are used as needed to improve the stability of the formulation or to help improve the activity or its bioavailability or to produce an acceptable taste or odor in the case of oral administration.

[0091] The pharmaceutical composition of the present invention can be made into inhalation aerosol preparations (such as dry powder preparations, aerosol preparations, inhaled droplet preparations, etc.), implantable gel preparations, microneedle preparations, and can also be made into injection forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day.

[0092] In some embodiments of the present invention, the pharmaceutical composition comprises lipid nanoparticles (Lipid Nanoparticle, LNP), and the size of the lipid nanoparticles is 60-120 nm, which can be 60-70 nm, 70-80 nm, 80-90 nm, 90-100 nm, 100-110 nm, 110-120 nm, or can also be 60-80 nm, 80-100 nm, 100-120 nm.

[0093] In some embodiments of the present invention, the pharmaceutical composition comprises nucleic acid lipid nanoparticles, and the encapsulation efficiency of the nucleic acid in the nano lipid particles is greater than 80%, which can be 80%-85%, 85%-90%, 90%-95%, 95%-97%, 97%-99% or more than 99%.

[0094] In some embodiments of the present invention, the pharmaceutical composition is delivered to target cells; preferably, the target cells are eukaryotic cells, and further preferably, the target cells are one or more of immune cells, stem cells or nerve cells.

[0095] In some embodiments of the present invention, the immune cells include, but are not limited to, one or more of T cells, macrophages, dendritic cells, natural killer cells, monocytes, myeloid cells, B cells, lymphocytes, tumor infiltrating lymphocytes; and / or, the stem cells include, but are not limited to, one or more of induced pluripotent stem cells (iPSC), human embryonic stem cells, adult stem cells, mesenchymal stem cells, hematopoietic stem cells; and / or, the nerve cells include, but are not limited to, one or more of neuron cells, astrocytes, oligodendrocytes, microglia, Schwann cells, neuroblasts.

[0096] In some specific embodiments of the present invention, the pharmaceutical composition is delivered to target cells for cell therapy to treat cancer. The cell therapy includes, but is not limited to, one or a combination of more of chimeric antigen receptor T cell (CAR-T cell) therapy, T-cell receptor engineered T cells (TCR-T) therapy, chimeric antigen receptor natural killer cell (CAR-NK cell) therapy, chimeric antigen receptor natural killer T cell (CAR-NKT cell) therapy, chimeric antigen receptor macrophage cell (CAR-M cell) therapy, and tumor infiltrating lymphocytes cell (TIL cell) therapy. The cancer includes, but is not limited to, one or more of leukemia, lymphoma, melanoma, cervical cancer, and sarcoma. The leukemia includes, but is not limited to, one or more of childhood acute lymphoblastic leukemia and chronic lymphocytic leukemia. The lymphoma includes, but is not limited to, one or more of advanced adult lymphoma and recurrent or refractory non-Hodgkin lymphoma.

[0097] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments, rather than limiting the protection scope of the present invention.

[0098] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. Except for the specific methods, devices, and materials used in the examples, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0099] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt the conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in this technology field.

[0100] Example 1: Synthesis of Ionizable Lipid Compounds

[0101] 1. Synthesis of Ionizable Lipid Compound H-07

[0102] The synthesis route is as follows:

[0103]

[0104] (1) Synthesis of Compound C

[0105] Dissolve isostearic acid (Compound B, 14.2 g, 49.9 mmol) in 150 mL of dichloromethane (DCM), add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 6.8 g, 59.9 mmol), 4-dimethylaminopyridine (DMAP, 0.6 g, 4.99 mmol), and N,N-diisopropylethylamine (DIPEA, 8.39 g, 64.9 mmol). After stirring for 10 min, add 5-hexen-1-ol (Compound A, 5.00 g, 49.9 mmol), and stir at room temperature overnight. After monitoring the reaction to completion by thin-layer chromatography (TLC), remove dichloromethane under reduced pressure using a rotary evaporator. Add 300 mL of ethyl acetate (EA), wash with an equal volume of saturated sodium chloride solution three times, dry the organic phase over anhydrous sodium sulfate for 30 min, remove ethyl acetate under reduced pressure using a rotary evaporator, and purify by column chromatography (silica gel column, eluent: petroleum ether (PE):EA = 3:1 (volume ratio)) to obtain 17.3 g of a colorless liquid with a yield of 94.5%.

[0106] (2) Synthesis of Compound D

[0107] Dissolve Compound C (15.0 g, 40.91 mmol) in 200 mL of DCM, add meta-chloroperoxybenzoic acid (m-CPBA, 10.8 g, 53.19 mmol, 85% mass fraction) under ice bath conditions, stir for 15 min, then remove the ice bath and stir overnight. After monitoring the reaction to completion by TLC, add an excess of saturated sodium bisulfite solution (15 mL) to consume the unreacted m-CPBA, and remove DCM under reduced pressure using a rotary evaporator. Add 300 mL of ethyl acetate, wash with 300 mL of saturated sodium bicarbonate solution three times and 300 mL of saturated sodium chloride solution once, dry the organic phase over anhydrous sodium sulfate for 30 min, remove ethyl acetate under reduced pressure using a rotary evaporator, and purify by column chromatography (silica gel column, eluent: PE:EA = 1:1 (volume ratio)) to obtain 13.1 g of a colorless liquid with a yield of 83.7%.

[0108] (3) Synthesis of Ionizable Lipid Compound H-07

[0109] Dissolve diethanolamine (Compound E, 0.55 g, 5.23 mmol) in 10 mL of anhydrous dimethylformamide (DMF), add Compound D (5.0 g, 13.07 mmol), stir at room temperature for 10 min, and then react at 100 °C for 12 h. After monitoring the reaction to completion by TLC, remove the solvent by rotary evaporation under reduced pressure, add 300 mL of EA, wash with 300 mL of saturated sodium bicarbonate solution three times and 300 mL of saturated sodium chloride solution once, dry the organic phase over anhydrous sodium sulfate for 30 min, and remove EA by rotary evaporation under reduced pressure. Purify by column chromatography (silica gel column, eluent: DCM:methanol (MeOH) = 200:1 (volume ratio)) to obtain 3.75 g of a colorless liquid with a yield of 82.4%.

[0110] Using the above method and replacing the corresponding starting materials, Compounds H-01 to H-19 and comparative sample h-2 can be prepared. For example, replacing diethanolamine with 4-amino-1-butanol can prepare H-08; replacing isostearic acid with 8-methylnonanoic acid can prepare H-01.

[0111] 2. Synthesis of ionizable lipid Compound H-20

[0112] The synthesis route is as follows:

[0113]

[0114] (1) Synthesis of Compound D-1

[0115] Referring to the synthesis method of Compound D, Compound D-1 can be synthesized.

[0116] (2) Synthesis of Compound F

[0117] Dissolve diethanolamine (Compound E, 4.12 g, 39.20 mmol) in 20 mL of anhydrous dimethylformamide (DMF), add Compound D (3.0 g, 7.84 mmol), stir at room temperature for 10 min, and then react at 100 °C for 12 h. After monitoring the reaction to completion by TLC, remove the solvent by rotary evaporation under reduced pressure, add 200 mL of EA, wash with 200 mL of saturated sodium bicarbonate solution three times and 200 mL of saturated sodium chloride solution once, dry the organic phase over anhydrous sodium sulfate for 30 min, and remove EA by rotary evaporation under reduced pressure. Purify by column chromatography (silica gel column, eluent: DCM:methanol (MeOH) = 50:1 (volume ratio)) to obtain 2.26 g of a colorless liquid with a yield of 59.1%.

[0118] (3) Synthesis of Compound H-20

[0119] Dissolve compound F (0.30 g, 0.62 mmol) in 2 mL of anhydrous dimethylformamide (DMF), add compound D-1 (0.25 g, 0.62 mmol), stir at room temperature for 10 min, and then react at 100 °C for 12 h. After monitoring the reaction to completion by TLC, remove the solvent under reduced pressure using a rotary evaporator. Add 30 mL of EA, wash with 30 mL of saturated sodium bicarbonate solution three times and 30 mL of saturated sodium chloride solution once. Dry the organic phase over anhydrous sodium sulfate for 30 min, and then remove EA under reduced pressure using a rotary evaporator. Purify by column chromatography (silica gel column, eluent: DCM: methanol (MeOH) = 100:1 (v / v)) to obtain 0.36 g of a colorless liquid with a yield of 66.0%.

[0120] Using the above method and replacing the corresponding starting materials, compounds H-20 to H-21 can be prepared. For example: replacing isostearyl mercaptan with isostearic acid and replacing 5-hexenoic acid with 5-hexenethiol can prepare H-21.

[0121] 3. Synthesis of ionizable lipid compound H-23

[0122] The synthesis route is as follows:

[0123]

[0124] (1) Synthesis of compound D-2

[0125] Referring to the synthesis method of compound D, compound D-2 can be synthesized

[0126] (2) Synthesis of compound H-23

[0127] Dissolve compound F (0.50 g, 1.03 mmol) in 3 mL of anhydrous dimethylformamide (DMF), add compound D-2 (0.39 g, 1.03 mmol), stir at room temperature for 10 min, and then react at 100 °C for 12 h. After monitoring the reaction to completion by TLC, remove the solvent under reduced pressure using a rotary evaporator. Add 30 mL of EA, wash with 30 mL of saturated sodium bicarbonate solution three times and 30 mL of saturated sodium chloride solution once. Dry the organic phase over anhydrous sodium sulfate for 30 min, and then remove EA under reduced pressure using a rotary evaporator. Purify by column chromatography (silica gel column, eluent: DCM: methanol (MeOH) = 80:1 (v / v)) to obtain 0.32 g of a colorless liquid with a yield of 35.9%.

[0128] 4. Synthesis of ionizable lipid compound H-24

[0129]

[0130] (1) Synthesis of compound C-3

[0131] Dissolve A (5-hexen-1-ol, 2.00 g, 19.97 mmol) in 50 mL of DCM, add Et3N (4.45 g, 43.93 mmol) and CDI (4.24 g, 19.97 mmol), and react at 50 °C for 1.0 h. Then add B-2 (5.38 g, 19.97 mmol) and react at 50 °C for 16 h. Cool to room temperature, wash successively with 5% citric acid (50 mL × 2) and saturated brine. The organic phase is dried over anhydrous sodium sulfate and concentrated to obtain the crude product. The crude product is purified by column chromatography (PE:EA = 10:1) to obtain 6.31 g of the product, with a yield of 79.9%.

[0132] (2) Synthesis of compound D-3

[0133] Referring to the synthesis method of compound D, compound D-3 can be synthesized.

[0134] (3) Synthesis of compound H-24

[0135] Dissolve compound F (0.50 g, 1.03 mmol) in 3 mL of anhydrous dimethylformamide (DMF), add compound D-3 (0.42 g, 1.03 mmol), stir at room temperature for 10 min, and then react at 100 °C for 12 h. After monitoring the reaction to completion by TLC, remove the solvent under reduced pressure using a rotary evaporator, add 30 mL of EA, wash 3 times with 30 mL of saturated sodium bicarbonate solution and 1 time with 30 mL of saturated sodium chloride solution. The organic phase is dried over anhydrous sodium sulfate for 30 min, and the EA is removed under reduced pressure using a rotary evaporator. Purify by column separation (silica gel column, eluent is DCM:MeOH = 50:1 (volume ratio)) to obtain 0.51 g of a colorless liquid, with a yield of 55.3%.

[0136] Using the above method and replacing the corresponding starting materials, compound H-24 can be prepared. For example: replacing isostearylamine with isostearyl alcohol and 5-hexen-1-ol with 5-hexenamine can prepare H-24. In addition, referring to the third step in the synthesis method of compound H-07 and replacing compound D in its synthesis process with compound D-3, compound H-25 can be synthesized.

[0137] 5. Synthesis of ionizable lipid compound H-27

[0138]

[0139] (1) Synthesis of compound C-4

[0140] Synthesize compound C-4 by referring to the synthesis method of compound C-3.

[0141] (2) Synthesis of compound D-4

[0142] Synthesize compound D-4 by referring to the synthesis method of reference compound D.

[0143] (3) Synthesis of compound H-27

[0144] Dissolve compound F (0.50 g, 1.03 mmol) in 3 mL of anhydrous dimethylformamide (DMF), add compound D-4 (0.42 g, 1.03 mmol), stir at room temperature for 10 min, and then react at 100 °C for 12 h. After monitoring the reaction to completion by TLC, remove the solvent under reduced pressure using a rotary evaporator, add 50 mL of EA, wash once with 50 mL of saturated sodium chloride solution, dry the organic phase over anhydrous sodium sulfate for 30 min, and remove EA under reduced pressure using a rotary evaporator. Purify by column chromatography (silica gel column, eluent is DCM:MeOH = 100:1 (volume ratio)) to obtain 0.26 g of a colorless liquid with a yield of 28.2%.

[0145] In addition, referring to the third step in the synthesis method of reference compound H-07, replacing compound D in its synthesis process with compound D-4, compound H-28 can be synthesized.

[0146] 6. Synthesis of ionizable lipid compound h-3

[0147]

[0148] (1) Synthesis of compound C0

[0149] Synthesize compound C0 by referring to the synthesis of reference compound C.

[0150] (2) Synthesis of compound h-3

[0151] Dissolve compound C0 (1.00 g, 2.23 mmol) in 10 mL of anhydrous dimethylformamide (DMF), add compound E (93.97 mg, 0.89 mmol) and N,N-diisopropylethylamine (0.29 mg, 2.23 mmol), and react at 100 °C for 12 h. After monitoring the reaction to completion by TLC, add 50 mL of EA, wash three times with 50 mL of saturated sodium chloride solution, dry the organic phase over anhydrous sodium sulfate for 30 min, and remove EA under reduced pressure using a rotary evaporator. Purify by column chromatography (silica gel column, eluent is DCM:MeOH = 200:1 (volume ratio)) to obtain 0.56 g of a colorless liquid with a yield of 72.0%.

[0152] Table 1 Comparison table of 1H NMR data of exemplary ionizable lipid compounds H-1 to H-18 in Example 1

[0153]

[0154]

[0155]

[0156] Example 2: Preparation and screening of nucleic acid lipid nanoparticles (mRNA-LNP)

[0157] 1. Preparation of Nucleic Acid-Lipid Nanoparticles

[0158] (1) The ionizable lipid compounds (Lipid) H-1 to H-28, DSPC, cholesterol, and PEG-lipid (PEG2000-DMG) prepared in Example 1 were dissolved in ethanol according to the molar ratio of 46.3 / 9.4 / 42.7 / 1.6 to prepare different lipid ethanol solutions (Lipid concentration 20 mg / mL).

[0159] (2) Prepare mRNA at a lipid nanoparticle (LNP) to mRNA mass ratio of 10:1 to 30:1 (the ratio used in this example was 15:1), and dilute the mRNA to 0.2 mg / mL using citrate or sodium acetate buffer (pH = 3 or 5) to obtain an mRNA solution.

[0160] (3) The lipid ethanol solution and the mRNA solution were thoroughly mixed at a volume ratio of 1:5 to 1:1 (the ratio used in this example was 1:3). The obtained lipid nanoparticles were purified by ultrafiltration and dialysis. After filtration and sterilization, the average particle size and PDI (polydispersity index) of the mRNA-LNP (mRNA-encapsulated lipid nanoparticles) were characterized using a Malvern Zetasizer Nano ZS, and the mRNA encapsulation efficiency was determined using a Ribogreen RNA quantification kit. The results are shown in Table 2.

[0161] Table 2 Characterization data of each group of mRNA-LNP

[0162]

[0163]

[0164] Results show that: All the ionizable lipid compounds H-1 to H-28 provided in Example 1 of the present invention can form stable nanostructures. The obtained mRNA-LNPs have a narrow size distribution, and the size varies with the structure of different mRNA-LNPs, ranging from 60 to 120 nm. Under the same other conditions, using the comparative samples shown in Table 3, commercially available MC3, ALC0315, comparative sample h-1 (h-1 is from Patent CN20221034495.1), h-2 (prepared in the aforementioned Example 1), and h-3 (prepared in the aforementioned Example 1), respectively replace the above ionizable lipid compounds H-1 to H-28 to prepare mRNA-LNPs, and the average particle size, PDI, and encapsulation efficiency are tested. The structural formulas of MC-3, ALC0315, h-1, h-2, and h-3 are shown in Table 3. The test results are shown in Table 4.

[0165] Table 3 Structural formulas of control samples

[0166]

[0167] Table 4 Characterization data of each group of mRNA-LNPs

[0168]

[0169] From the data comparison in Table 2 and Table 4, it can be seen that the mRNA-LNPs provided in the examples of the present invention can all form stable nanostructures, have a narrow size distribution, the size varies with the structure of different mRNA-LNPs, ranging from 60 to 120 nm, the encapsulation efficiency is greater than 95%, and they have relatively excellent physical and chemical properties.

[0170] 2. Low-temperature storage stability

[0171] The mRNA-LNP provided by the present Example Group H-10 was stored in a dry environment at 4°C. At different time points (0 day, 6 days, 10 days, 15 days, 30 days, 45 days, 60 days, 90 days), the average particle size, PDI, and encapsulation efficiency of the mRNA-LNP were respectively tested, and the measurement results are shown in Table 5.

[0172] Table 5 Effect comparison at different low-temperature storage times

[0173] Storage time (days) Average particle size (nm) PDI Entrapment efficiency (%) 0 69.3 0.094 97.4 6 69.9 0.098 97.3 10 70.1 0.097 97.3 15 70.5 0.103 97.1 30 70.8 0.098 97.0 45 72.4 0.106 96.8 60 73.9 0.107 96.2 90 74.2 0.109 96.1

[0174] As can be seen from Table 5, the average particle size, PDI, and encapsulation efficiency of the mRNA-LNP provided by the present Example Group H-10 basically did not change within 90 days, and it has good low-temperature storage stability, which is convenient for the storage and transportation of the product.

[0175] According to the aforementioned method, the mRNA-LNPs provided in Example groups H-1 to H-9 and H-11 to H-28 were selected, and the measurement results all showed that the average particle size, PDI, and encapsulation efficiency basically did not change within 90 days.

[0176] Example 3: Transfection efficiency and biocompatibility of nucleic acid lipid nanoparticles (mRNA-LNP)

[0177] 1. Cell transfection efficiency

[0178] Suspend NK-92 cells or T cells (100 μL, cell density of 6×10 4 cells / ml) in the exponential growth phase and add them to a 96-well plate. Incubate in a cell culture incubator for 24 h. Replace the complete medium with 60 μL of DMEM serum-free medium. Add 0.4 μg or 0.2 μg of the prepared Luciferase mRNA LNP preparation to each well, with three replicates for each concentration. Place in a 37°C, 6% CO2 incubator and culture for 4 h. Then supplement with 60 μL of complete medium and continue to culture for 24 h. Remove the medium, add 100 μL of PBS and wash gently once, then add 30 μL / well of cell lysate, place on a micro shaker and shake at room temperature for 16 min to fully lyse the cells. Collect the lysate, centrifuge at 12,000 rpm for 6 min, and collect the supernatant. Take 20 μL of the supernatant and add it to a new black opaque 96-well plate. Use the cell lysate as a blank control. Add 100 μL of firefly luciferase detection solution, incubate in the dark for 6 min, then use a multi-functional microplate reader to detect the chemiluminescence intensity RLU, and subtract the RLU of the blank control, and take the average value. In Table 6, E represents the power of 10 in scientific notation. For example, "6.41E+04" represents 6.41×10 4 .

[0179] Table 6 Transfection efficiency comparison table for NK-92 cells

[0180]

[0181]

[0182] Table 7 Transfection efficiency comparison table for T cells

[0183]

[0184]

[0185] 2. Animal transfection efficiency

[0186] Male ICR mice (6 - 8 weeks old, purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd.) were housed under experimental conditions of 22 ± 2 °C and a relative humidity of 45–75%, with a 12-h light / dark cycle. Messenger RNA encoding luciferase (luciferase mRNA) was used as the reporter gene. Luciferase catalyzes luciferin to produce bioluminescence, and the transfection efficiency of LNP was reflected by detecting the bioluminescence intensity per unit time. Taking luciferase mRNA (purchased from ApexBio Technology) as an example, the mRNA-LNP sample H-10 obtained from Experiment 1, commercially available control sample MC3, commercially available control sample ALC0315, control sample h-1, and control sample h-2 were prepared; the above samples were administered by intramuscular injection at a dose of 150 μg / kg mRNA, with two mice per group of samples, injected into two legs. At specific time points, luciferin (20 μg / mL) was injected intraperitoneally into the mice. After 5 minutes, the mice were placed in a small animal in vivo imaging system to measure the fluorescence intensity, and the final results were expressed as the average fluorescence intensity. The experimental results of the fluorescence intensity after intraperitoneal injection of the samples into the mice are shown in Table 8.

[0187] Table 8 Fluorescence intensity comparison table

[0188]

[0189] Compared with the commercially available products, the mRNA-LNP prepared from the ionizable lipid compound provided by the present invention can improve the transfection effect at various cell levels by 1 - 3 orders of magnitude, far exceeding the expected range, having excellent cell transfection efficiency, and greatly expanding its application in the field of cell therapy. Moreover, the transfection efficiency in animals also has a comparable transfection effect compared with the commercially available products.

[0190] 3. Biocompatibility

[0191] The CCK-8 kit was used to measure cell viability. The NK-92 cell suspension in the exponential growth phase (100 μL, cell density of 2×10 4 / mL) were added to a 96-well plate and incubated in a cell culture incubator for 24 hours. The cell culture medium was then removed, and 100 μL of fresh cell culture medium containing 20 μg / mL of mRNA for each mRNA-LNP sample obtained in Example 2 was added and incubated with the cells for 4 hours. Subsequently, the cell supernatant was removed, fresh cell culture medium was added, and the cells were incubated for a further 20 hours. The supernatant was removed, and 100 μL of fresh cell culture medium containing CCK-8 working solution (10 μL / mL) was added and incubated for 2 hours. At the same time, a blank group was set up: an equal amount of CCK-8 working solution was used instead of mRNA-LNP, and the other conditions were exactly the same. The absorbance of each well at 450 nm was measured using a multifunctional microplate reader (no bubbles should appear in the well plate during the detection process), and the viability of cells without any treatment (control group) was set to 100%. The cell viability of each group was calculated as follows: Cell viability (%) = [A1-A0] / [A2-A0]×100. Among them, A1 is the absorbance of the drug-dosed group, A0 is the absorbance of the blank group, and A2 is the absorbance of the control group. The experimental results are shown in Table 9.

[0192] Table 9 Cell viability effect comparison table

[0193]

[0194]

[0195] The experimental results showed that within the limited LNP concentration range, the cell viability was greater than 95% and no obvious cytotoxicity was observed.

[0196] 4. Preparation of CAR-T cells

[0197] According to the reference (Science 375, 91-96 (2022)), the FAP protein sequence was designed and FAP CAR mRNA was synthesized by in vitro transcription. The LNP-FAP CAR was prepared using the method for preparing LNP in Example 1, and lipid H-10 was selected as an example. The 48-well plate was pretreated with 5 μg / mL human or mouse CD3 / CD28, and the sorted human or mouse CD8+ T cells were inoculated into a 48-well plate (4×105 cells / well). After 36 hours of cell activation, the transfection experiment was performed. Before transfection, the complete culture medium was replaced with 100 μL DMEM serum-free medium, and a transfection reagent with an mRNA concentration of 60 μg / mL was added. After 6 hours of transfection, complete culture medium containing 20% FBS was added. After 24 hours of culture, the FAP-CAR-T cell positive rate was detected by flow cytometry, and the cell positive rate was 91%.

[0198] According to the aforementioned method, other lipids synthesized in Example 1 were selected, and the cell positive rate was above 85%, and efficient transfection of CAR into T cells was achieved.

[0199] The above embodiments are only illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. An ionizable lipid compound, the chemical structure of which is shown in the following formula (I): Among them, a and b are each independently selected from natural numbers of 0 to 25; G1 and G2 are each independently selected from linear C2 to C 10 alkylene; G3 is selected from a linear or branched C1-C 10 alkylene; or G3 is selected from (CH2) d -O-(CH2) e , where d and e are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8 or 9, and d + e is an integer from 2 to 10; or G3 is selected from where g is selected from 2, 3, 4; L1 and L2 are each independently selected from -(C=O)O-, -O(C=O)-, -(C=O)S-, -S(C=O)-, -(C=O)NR-, -NR(C=O)-, -O(C=O)O-, -NR(C=O)O-, -O(C=O)NR-, and R is independently selected from H, C1-C 12 hydrocarbyl group.

2. The ionizable lipid compound according to claim 1, wherein a and b each independently represent a natural number from 10 to 20; G1 and G2 each independently represent a linear C3-C9 alkylene group; G3 is selected from a linear C2-C6 alkylene group; or G3 is selected from (CH2) d -O-(CH2) e , wherein d and e are each independently selected from 1, 2, 3, 4, 5, and d+e is an integer from 2 to 6; or G3 is selected from wherein g is selected from 2 and 3; L1 and L2 each independently represent -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, -(C=O)S-, -S(C=O)-, -(C=O)NH-, -NH(C=O)-.

3. The ionizable lipid compound according to claim 2, wherein, a and b each independently represent a natural number from 5 to 17; G1 and G2 each independently represent 1,3-propylene, 1,4-butylene, 1,5-pentylene, 1,6-hexylene, 1,7-heptylene, 1,8-octylene; G3 is selected from 1,2-ethylene, 1,3-propylene, 1,4-butylene; or G3 is selected from (CH2) d -O-(CH2) e , where d and e are each independently selected from 1, 2, 3, and d + e is an integer from 2 to 4; L1 and L2 each independently represent -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, -O(C=O)O-, -(C=O)S-, -S(C=O)-, -(C=O)NH-, -NH(C=O)-.

4. The ionizable lipid compound according to any one of claims 1-3, wherein, The ionizable lipid compound is selected from the following group:

5. A lipid nanoparticle, characterized in that A combination comprising one or more of an ionizable lipid compound, its stereoisomer, its tautomer or its pharmaceutically acceptable salt as described in any one of claims 1-4.

6. The lipid nanoparticle according to claim 5, wherein It further comprises a combination of any one or more of a structural lipid, a helper lipid, a PEG-lipid, and a polymer.

7. The lipid nanoparticle according to claim 6, wherein, The structural lipid is selected from a combination of one or more of a sterol, a non-sterol or their respective derivatives; and / or, the helper lipid is selected from a combination of one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dimyristoyl phosphatidylglycerol, DOTAP, DODAP, 18:1PA, HS15, GL67; and / or, the PEG-lipid is selected from a combination of one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, methoxypolyethylene glycol ditetradecylacetamide.

8. The lipid nanoparticle according to claim 6, wherein The molar percentage of the ionizable lipid compound: helper lipid: structural lipid: PEG-lipid is (20-65):(0-60):(0-60):(0-10), and at least any one of the helper lipid, the structural lipid, and the PEG-lipid is not 0; preferably, the molar percentage of the ionizable lipid compound: helper lipid: structural lipid: PEG-lipid is (20-65):(3-50):(15-60):(0.1-10); or, the molar ratio of the ionizable lipid compound to the polymer is 0.5:1-100:1, preferably 10:1-80:1, more preferably 40:1-80:

1.

9. Use of the lipid nanoparticle according to any one of claims 5-8 in the preparation of a pharmaceutical composition, wherein the pharmaceutical composition further comprises the loaded drug and / or a pharmaceutically acceptable excipient.

10. The application according to claim 9, characterized in that, The drug carried therein includes one or more of nucleic acid molecules, small molecule compounds, and proteins.

11. The application according to claim 10, characterized in that, 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.

12. The application according to claim 11, wherein The immune cells are one or more of T cells, macrophages, dendritic cells, natural killer cells, monocytes, myeloid cells, B cells, lymphocytes, tumor-infiltrating lymphocytes; and / or, the stem cells are one or more of induced pluripotent stem cells, human embryonic stem cells, adult stem cells, mesenchymal stem cells, hematopoietic stem cells, neural stem cells; and / or, the nerve cells are one or more of neuron cells, astrocytes, oligodendrocytes, microglia, Schwann cells, neuroblasts.

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