Lipid compounds and compositions for tissue-specific delivery of active substances

Abstract

The present invention relates to a novel lipid compound for tissue-specific delivery, and a lipid nano-particle (LNP) composition comprising the same, the lipid nano-particle comprising a modified lipid compound as a component, according to the present invention, internal active substances are selectively delivered into cells of specific tissues such as lymph nodes, spleen, retina, cancer, brain, liver and the like in vivo, thereby preventing side effects and safely exhibiting a desired level of effect. The tissue-specific non-viral LNP delivery vectors can be effectively used for prevention of infectious diseases and treatment of rare and refractory (hereditary) diseases (diseases which are effectively and selectively delivered to in-vivo targeted sites, such as macular degeneration, diabetic retinal degeneration, hereditary retinal degeneration, cancer, cerebral diseases, liver diseases and the like).

Description

Technical Field

[0001] This invention relates to a lipid compound that selectively delivers an active substance into the cells of a specific tissue in vivo, and a lipid nanoparticle composition comprising said lipid compound. Background Technology

[0002] In the recent crisis of the large-scale COVID-19 pandemic and the emergence of new variants, only mRNA vaccines, which offer rapid response and high efficacy, have proven to be effective. This can be achieved by using lipid nanoparticles (LNPs) as delivery carriers. These nanoparticles can be mass-produced at a relatively low cost, are versatile, and relatively safe; only the mRNA sequence needs to be replaced. Beyond vaccines, LNP-based therapeutic technologies are also rapidly developing. LNP delivery carriers can stabilize the structure of mRNA and enable efficient intracellular delivery and smooth endosomal escape, thereby improving the efficiency of mRNA delivery to the cytoplasm and thus enhancing the efficacy of mRNA vaccines or therapeutics. The demand for LNPs is expected to increase significantly.

[0003] Lipid nanoparticles (LNPs) were used as a delivery vector for Onpattro, the world's first approved siRNA-based therapeutic agent, in 2018, thus enabling its commercial application. LNPs can circumvent the limitations of gene therapy agents that use existing viral vectors such as adeno-associated virus (AAV) and lentivirus as delivery vectors, namely the problems of immune response and long-term toxicity. At the same time, they can also overcome the limitation of the deliverable gene size (<4.7kb) of the representative viral vector AAV. Therefore, it is expected to be able to deliver various genes (RNA, siRNA, shRNA, rRNA, tRNA, mRNA, miRNA, saRNA, circRNA, DNA, cDNA, plasmid, DNAzyme, ribozyme, PNA, aptamer, antisense oligonucleotide, CRISPR, etc.) for the treatment of cancer, various rare and refractory genetic diseases (brain diseases, retinal diseases, etc.) more safely and effectively.

[0004] Furthermore, in order to maximize the efficacy of gene therapy agents using LNPs as delivery vectors and to avoid side effects caused by delivery to non-target sites (off-target) to ensure safety, it is necessary to achieve specific delivery (targeting) to the disease site (tissue). Experts regard this precise tissue targeting characteristic as one of the core directions for the development of next-generation gene therapy agents.

[0005] As a specific example, inherited retinal dystrophy (IRD) is a progressive disease caused by congenital abnormalities in retinal cell genes, leading to visual impairments such as decreased vision, night blindness, and narrowed visual field, eventually resulting in blindness. Because the eyeball is a particularly immune-exempt region, the development of gene therapy agents utilizing delivery vectors such as viral vectors has been actively pursued. In 2017, Spark Therapeutics' Luxterna, the world's first gene therapy for Leber congenital amaurosis (LCA), one type of IRD, received FDA approval. Luxterna is a therapeutic agent that uses AAV as a delivery vector and is administered via subretinal injection. AAV is a viral vector commonly used in recently commercialized gene therapy agents. Subretinal administration can effectively deliver gene therapies to the retinal pigment epithelium (RPE) or photoreceptor cells, which are often involved in pathological conditions. However, it has drawbacks such as requiring highly delicate surgery, the potential for complications, and limitations on repeated administration. Furthermore, AAV can only carry a subset of smaller genes; therefore, pathogenic genes such as EYS, USH2A, and ABCA4, which are more prevalent in IRD patients, are limited by their larger size and cannot be delivered using AAV. In addition, a significant proportion of individuals possess AAV antibodies. Although the eye is an immune-immune region, the possibility of serious side effects due to immune (inflammatory) responses triggered by the route of administration has been mentioned. In other words, after years of administering gene therapies using AAV as a delivery vehicle to the retina of primates or humans, there have been reports of perifoveal retinal pigment epithelial cells atrophying due to an immune response to AAV, potentially exacerbating concerns about the long-term safety of gene therapies administered to the retina using AAV as a delivery vehicle.

[0006] Therefore, there is a growing demand for the development of novel gene delivery vectors that can overcome the limitations of existing AAV delivery vectors and can be administered via safer and easier methods such as intravitreal injection, which, like subretinal injection, requires advanced technology and limits repeated administration. Although there have been reports of gene therapy agents using non-viral LNPs as delivery vectors via subretinal injection, there are currently no cases of effective delivery to the RPE and photoreceptor cells in the pathological outer retina via intravitreal injection.

[0007] These tissue-specific nonviral LNP delivery vectors can selectively deliver to cells in specific tissues such as lymph nodes, spleen, retina, cancer, brain, and liver, thus effectively enabling their use in the prevention of infectious diseases and the treatment of rare and refractory (hereditary) diseases (macular degeneration, diabetic retinopathy, hereditary retinal degeneration, cancer, brain diseases, liver diseases, etc.).

[0008] Therefore, in order to solve the above problems, the inventors developed a lipid compound that selectively delivers active substances such as genes into specific tissue cells in the body and a lipid nanoparticle composition containing the lipid compound, and confirmed its efficacy, thereby completing the present invention. Summary of the Invention

[0009] Technical issues The object of this invention is to provide a lipid compound, its isomer, or a pharmaceutically acceptable salt thereof for tissue-specific delivery of an active substance.

[0010] Another object of the present invention is to provide a lipid nanoparticle composition for tissue-specific delivery of active substances.

[0011] Another object of the present invention is to provide a method for preparing active substance-lipid nanoparticles.

[0012] Another object of the present invention is to provide a composition for delivering nucleic acids.

[0013] Another object of the present invention is to provide a tissue-specific drug delivery carrier.

[0014] Another object of the present invention is to provide a drug delivery carrier for treating retinal diseases.

[0015] Another object of the present invention is to provide a drug delivery carrier for the prevention of infectious diseases.

[0016] Another object of the present invention is to provide a drug delivery carrier for treating cancer.

[0017] Another object of the present invention is to provide a drug delivery carrier for treating brain diseases.

[0018] Another object of the present invention is to provide a drug delivery carrier for treating liver diseases.

[0019] Technical solution To achieve the above objectives, one aspect of the present invention provides a lipid compound represented by the following chemical formula 1, an isomer thereof, or a pharmaceutically acceptable salt thereof.

[0020] [Chemical Formula 1]

[0021] In chemical formula 1, T represents the targeting ligand, which is selected from monosaccharides, polysaccharides, folic acid, transferrin, RGD peptide, cyclic RGD peptide, TAT peptide, R9 peptide, CATY peptide, HA2 peptide, monoclonal antibody, antigen-binding fragment or antibody fragment, single-chain variable fragment (scFv) and aptamer, or a branched-chain targeting ligand represented by the following chemical formula 2; X 1 X 2 X 3 and X 4 Each independently is selected from one or a combination of the following groups: single bond, C 1-14 Alkylene, C 1-14 Heteroalkylene, C 2-14 imidene group, C 1-6 Alkyl-C(=O)-, C 2-6 Alkenyl-C(=O)-, -(OCH2CH2) m -、-(CH2CH2O) m-, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR)-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(= O)O-, -C(=O)C(=O)-, -C(=N-OR)-, -ON=CR-, -OC=NO-, -C(=O)NR-, -NRC(=O)-, -OC(=O) NR-, -NRC(=O)O-, -C(=O)NRC(=O)-, -S(=O)2NR-, -NRS(=O)2-, -NRC(=O)NR-, -NRC(=S) NR-, -C(=O)NH-N=CR-, -RC=N-NHC(=O)-, -NH-N=CR-, -RC=N-NH-, -NR-, -N(OR)-, -SS-, And functional groups that bond with each other through click reactions. Where m is an integer from 0 to 10, Z is -NH-, -O-, or -S-. d and e are each integers from 1 to 10; L 1 and L 2 Each is independently a single bond, C 1-14 Alkylene, C 2-14 sub-alkenyl, MR a M 1 Or R a MR b ; P is a single bond, -(CH2CH2O) q -、-(OCH2CH2) q -、-CH2O(CH2CH2O) q CH2- or -CH2CH2O(CH2CH2O) q CH2-, where q is an integer from 0 to 120; A is CR d Or N; R 1 and R 2 Each independently is C 1-30 Alkyl, C 2-30 alkenyl, or R c MR d , wherein, the C 1-30 Alkyl and C 2-30 The alkenyl groups are either independently unsubstituted or occupied by 1-3 carbons. 1-16 Alkyl or C 2-16 Alkenyl substitution; The M and M 1 Each independently is C 1-14 Alkylene, C2-14 alkenyl, -NHC(=O)-, -C(=O)NH-, -C(=O)O-, -OC(=O)-, -C(=O)-, -NH-, -N + R2-, -S-, -SS-, -O-, -S(O)2-, -C(=O)S-, -SC(=O)-, -NHC(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, -OP(=O)(OR)O- or -OP(=O)(OR)O(CH2) g - where g is an integer from 1 to 10; R is hydrogen, C 1-20 Alkyl, C 2-20 alkenyl, C 2-20 alkynyl or C 1-20 Heteroalkyl, The R a and R b Each is independently a single bond or C 1-10 Alkylene; The R c For single bond, C 1-14 Alkylene or C 2-14 alkenyl; The R d For hydrogen, C 1-18 Alkyl or C 2-18 alkenyl, wherein the C 1-18 Alkyl and C 2-18 The alkenyl groups are either independently unsubstituted or occupied by 1-3 carbons. 1-12 Alkyl or C 2-12 Alkenyl substitution; Each of the heteroalkyl, heteroalkylene, heterocyclic and heteroaryl groups independently comprises 1-6 heteroatoms selected from N, O and S; The branched-chain targeting ligand is represented by the following chemical formula 2: [Chemical Formula 2]

[0022] In the chemical formula 2, a, b, and c are each independently 0 or 1, of which at least one of a, b, and c must be 1, and h and j are 1-3; T 1 T 2 and T 3 The targeting ligands are each independently selected from the group consisting of: monosaccharides, polysaccharides, folic acid, transferrin, RGD peptides, cyclic RGD peptides, TAT peptides, R9 peptides, CATY peptides, HA2 peptides, monoclonal antibodies, antigen-binding fragments or antibody fragments, single-chain variable fragments (scFv), and aptamers. X 5 X 6 X 7 X 8 X 9 X 10 X 11 and X 12 Each independently is selected from one or a combination of the following groups: single bond, C 1-14 Alkylene, C 1-14 Heteroalkylene, C 2-14 imidene group, C 1-6 Alkyl-C(=O)-, C 2-6 Alkenyl-C(=O)-, -(OCH2CH2) m -、-(CH2CH2O) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR)-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(= O)O-, -C(=O)C(=O)-, -C(=N-OR)-, -ON=CR-, -OC=NO-, -C(=O)NR-, -NRC(=O)-, -OC(=O) NR-, -NRC(=O)O-, -C(=O)NRC(=O)-, -S(=O)2NR-, -NRS(=O)2-, -NRC(=O)NR-, -NRC(=S) NR-, -C(=O)NH-N=CR-, -RC=N-NHC(=O)-, -NH-N=CR-, -RC=N-NH-, -NR-, -N(OR)-, -SS-, And functional groups that bond with each other through click reactions. Where m is an integer from 0 to 10, Z is -NH-, -O-, or -S-. d and e are each integers from 1 to 10; L 3 L 4 L 5 L 6 and L 7 Each is independently a single bond, C 1-14 Alkylene, C 2-14 sub-alkenyl, MR a M 1 Or R a MR b ; A 1 A 2 and A 3 Each is independently a single bond, CR d Or N; Each of the heteroalkyl and heteroalkylene groups independently comprises 1 to 6 heteroatoms selected from N, O, and S.

[0023] Another aspect of the present invention provides a lipid nanoparticle composition comprising a lipid compound represented by the chemical formula 1, an isomer thereof, or a pharmaceutically acceptable salt thereof.

[0024] Another aspect of the present invention provides a method for preparing active substance-lipid nanoparticles, comprising the step of mixing an organic phase and an aqueous phase, wherein the organic phase comprises a lipid compound represented by the chemical formula 1, its isomers or pharmaceutically acceptable salts thereof, and the aqueous phase contains the active substance dissolved therein.

[0025] Another aspect of the present invention provides a tissue-specific drug delivery carrier comprising a lipid compound represented by the chemical formula 1, an isomer thereof, or a pharmaceutically acceptable salt thereof.

[0026] Furthermore, the present invention can provide a drug delivery carrier for treating retinal diseases, comprising a lipid compound represented by the chemical formula 1, an isomer thereof, or a pharmaceutically acceptable salt thereof.

[0027] Furthermore, the present invention can provide a drug delivery carrier for the prevention of infectious diseases, comprising a lipid compound represented by the chemical formula 1, an isomer thereof, or a pharmaceutically acceptable salt thereof.

[0028] Furthermore, the present invention can provide a drug delivery carrier for treating cancer, brain diseases, and liver diseases, comprising a lipid compound represented by the chemical formula 1, its isomers, or a pharmaceutically acceptable salt thereof.

[0029] Furthermore, the present invention can provide a method for delivering a drug, comprising the step of administering to a desired individual a lipid compound represented by said chemical formula 1, an isomer thereof, or a pharmaceutically acceptable salt thereof.

[0030] Furthermore, the present invention can provide the use of a lipid compound represented by the chemical formula 1, its isomers, or pharmaceutically acceptable salts thereof as a drug delivery carrier.

[0031] Furthermore, the present invention can provide a pharmaceutical composition or pharmaceutical dosage form comprising lipid nanoparticles and a payload, wherein the lipid nanoparticles comprise a lipid compound represented by the chemical formula 1, an isomer thereof, or a pharmaceutically acceptable salt thereof.

[0032] Furthermore, the present invention can provide a method for treating a disease, which includes the step of administering the pharmaceutical composition or pharmaceutical dosage form to an individual.

[0033] Technical effect The lipid nanoparticle compositions containing lipid compounds according to the present invention have excellent tissue-specific delivery efficiency and can therefore be used as targeted drug delivery carriers. Attached Figure Description

[0034] Figure 1 This is a schematic diagram showing the structure of mRNA-lipid nanoparticles (LNPs) for tissue-specific delivery of active substances according to an embodiment of the present invention. A is an LNP without tissue-specific ligands, B is an LNP with only part of its surface modified by polyethylene glycol-modified lipids linked to tissue-specific ligands, C is an LNP with most of its surface modified by polyethylene glycol-modified lipids linked to tissue-specific ligands, D is an LNP with only part of its surface modified by phospholipids linked to tissue-specific ligands, and E is an LNP with most of its surface modified by phospholipids linked to tissue-specific ligands.

[0035] Figure 2 IVIS images were taken at 24 and 48 hours after fLuc mRNA-LNP containing the modified PEG-lipid compound 5 synthesized according to Example 5 of the present invention was prepared according to Example 14 and administered to the eye of mice by subretinal injection according to Example 5.

[0036] Figure 3 The images are IVIS images taken at 24 and 48 hours after fLuc mRNA-LNP containing the modified PEG-lipid compound 5 synthesized according to Example 5 of the present invention was prepared according to Example 14 and then injected into the eye of mice via intravitreal injection according to Example 6.

[0037] Figure 4 The images are IVIS images taken at 24 hours after fLuc mRNA-LNP containing modified PEG-lipid compounds 1-5 synthesized according to Examples 1-5 of the present invention was prepared according to Example 14 and administered to the eye of mice via subretinal injection according to Example 5.

[0038] Figure 5 Following Example 14, fLuc mRNA-LNP containing modified PEG-lipid compounds 1-5 synthesized according to Examples 1-5 of the present invention was prepared and then injected into the eye of mice via intravitreal injection as described in Example 6. IVIS images were taken at 24 hours later.

[0039] Figure 6IVIS images were taken at 1 hour, 3 hours, 6 hours and 24 hours after fLuc mRNA-LNP containing modified PEG-lipid compounds 1-5 synthesized according to Examples 1-5 of the present invention was prepared according to Example 14 and administered to the eye of mice via tail vein injection according to Example 7.

[0040] Figure 7 Images were captured using a fluorescence microscope after EGFP mRNA-LNP was applied to ARPE-19 retinal cells according to an embodiment of the present invention, in an attempt to confirm the GFP expression pattern over time. Red fluorescence corresponds to sulfocyanine 5.5-DSPE, which is included as a phospholipid component (1 mol%) for tracing LNP. Detailed Implementation

[0041] The present invention will now be described in detail.

[0042] the term Unless otherwise stated, the term "halogen" refers to F, Cl, Br, or I.

[0043] The term "hydroxyl group" refers to the -OH group.

[0044] In this invention, the term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group (functional group). As an example, "C..." 1-6 Alkyl groups have 1-6 carbon atoms. Specifically, C 1-6 Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, 1-methylbutyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 4-methyl-2-pentyl, 3,3-dimethylbutyl, 2-ethylbutyl, etc. The alkyl group can be independently substituted by one or more substituents; for example, the methyl group can be substituted by 1-3 halogens, hydroxyl groups, thiols, selenol, etc., or C... 1-6 Alkyl groups and other hydrocarbon groups are substituted.

[0045] The term "alkylene" refers to a straight-chain or branched saturated hydrocarbon chain that does not contain hydrogen at either end, such as ethylene (-CH2CH2-) and propylene (-CH2CH2CH2-). The alkylene may be independently substituted by one or more substituents.

[0046] The term "alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon group containing one or more double bonds, wherein the alkenyl group can be independently substituted by one or more substituents. As an example, "C..." 2-6 "Alkenyl" has 2-6 carbon atoms. Specifically, C1-6 Alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, n-pentenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, n-hexenyl, etc.

[0047] The term "alkenylene" refers to a straight-chain or branched unsaturated hydrocarbon chain having at least one double bond and being free of hydrogen atoms at both ends, such as allylene (-CH=CHCH2-) or 2-pentenylene (-CH2CH=CHCH2CH2-). The alkenylene group may be independently substituted by one or more substituents.

[0048] The term "alkoxy" refers to a chemical formula '-O-alkyl', where the alkoxy group can be independently substituted by one or more substituents. For example, C 1-6 Alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentoxy, isopentoxy, tert-pentoxy, secondary-pentoxy, neopentoxy, and hexoxy. In one instance, the alkoxy group may be substituted with more than one substituent, for example, it may be substituted with 1-3 halogens, hydroxyl groups, or C- groups. 1-6 Alkyl groups and other hydrocarbon groups are substituted.

[0049] The term "alkenyloxy" refers to a chemical formula '-O-alkenyl', which can be independently substituted by one or more substituents. For example, C 2-6 Alkenyloxy groups include, but are not limited to, vinyloxy, n-propenyloxy, isopropenyloxy, n-butenyloxy, 1-methyl-2-propenyloxy, 2-methyl-2-propenyloxy, n-pentenyloxy, 1-methyl-2-butenyloxy, 2-methyl-2-butenyloxy, n-hexenyloxy, etc. In one example, the alkenyloxy group may be substituted with more than one substituent, for example, it may be substituted with 1-3 halogens, hydroxyl groups, or C- groups. 1-6 Alkyl groups and other hydrocarbon groups are substituted.

[0050] The term "cycloalkyl" refers to one or more saturated rings or one or more non-aromatic cyclic hydrocarbons, wherein the non-aromatic rings may have a certain degree of unsaturation. Unless otherwise defined, cycloalkyl can be monocyclic or polycyclic. Polycyclic refers to all fused, bridged, or spirocyclic rings. As an example, "C3-C..." 14 "Cycloalkyl" refers to a cycloalkyl group containing 3-14 cyclic carbon atoms. Specifically, C 3-14Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cyclooctyl, bicyclo[1.1.0]butyl, bicyclo[2.2.1]heptyl, adamantyl, etc. In one example, the cycloalkyl group may optionally be substituted with more than one substituent. In one example, 0, 1, 2, 3, or 4 atoms of each ring of the cycloalkyl group may be substituted with a substituent.

[0051] The term "aryl" refers to a monocyclic or bicyclic aromatic hydrocarbon. That is, in this specification, unless otherwise defined, aryl can include biaryl groups such as phenyl and naphthyl. As an example, C 6-10 An aryl group is an aromatic ring with 6-10 carbon atoms. In one instance, 0, 1, 2, 3, 4, 5, or 6 atoms in each aryl ring can be substituted by substituents.

[0052] The terms “heteroalkyl” and “heteroalkylene” each independently contain 1-6 heteroatoms selected from N, O and S.

[0053] The term "heteroaryl" refers to an aromatic 5- to 10-membered monocyclic or bicyclic heterocycle containing 1 to 6 heteroatoms selected from N, O, and S. Unless otherwise defined, a heteroaryl can be monocyclic or polycyclic. As an example, a "5- to 10-membered heteroaryl" can be a 5- or 6-membered aromatic heterocycle containing 1 to 4 heteroatoms selected from N, O, and S, or a bicyclic ring fused with a benzene ring or another heteroaryl ring. In one instance, 0, 1, 2, 3, or 4 atoms of each ring of the heteroaryl may be substituted with substituents. Examples of monocyclic heteroaryl groups include pyrroleyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, 1,2,3-triazolyl, 1,3,4-triazolyl, 1-oxa-2,3-diazolyl, 1-oxa-2,4-diazolyl, 1-oxa-2,5-diazolyl, 1-oxa-3,4-diazolyl, 1-thia-2,3-diazolyl, 1-thia-2,4-diazolyl, 1-thia-2,5-diazolyl, 1-thia-3,4-diazolyl, tetrazolyl, pyridinyl, pyridinyl, pyrazinyl, and similar groups, but are not limited thereto. Examples of bicyclic heteroaryl groups include indolyl, isoyindolyl, benzofuranyl, benzothiophenyl, indazoleyl, benzimidazolyl, benzotriazolyl, pyrrolo[2,3-b]pyridyl, pyrrolo[2,3-c]pyridyl, pyrrolo[3,2-c]pyridyl, pyrrolo[3,2-b]pyridyl, imidazo[4,5-b]pyridyl, imidazo[4,5-c]pyridyl, pyrazolo[4,3-d]pyridyl, pyrazolo[4,3-c]pyridyl, pyrazolo[3,4-c]pyridyl, pyrazolo[3,4-b]pyridyl, purineyl, indolazinyl, imidazo[1,2-a]pyridyl, imidazo[1,5-a]pyridyl, pyrazolo[1,5-a]pyridyl, pyrrolo[1,5-a]pyridyl, pyrrolo[2,3-b]pyridyl, pyrrolo[2,3-c]pyridyl, pyrrolo[3,2-c]pyridyl, pyrrolo[3,4-b]pyridyl, purineyl, indolazinyl, imidazo[1,2-a]pyridyl, imidazo[1,5-a]pyridyl, pyrazolo[1,5-a]pyridyl, pyrrolo[3,3-b]pyridyl, pyrrolo[3,3-c ...purineyl, indolazinyl, imidazo[1,2-a]pyrid [1,2-b]pyridazinyl, imidazo[1,2-c]pyrimidinyl, quinolinyl, isoquinolinyl, cenolinyl, azirquinazolinyl, quinoxalinyl, phthalazinyl, 1,6-naphridinyl, 1,7-naphridinyl, 1,8-naphridinyl, 1,5-naphridinyl, 2,6-naphridinyl, 2,7-naphridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[4,3-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrido[2,3-b]pyrimidinyl, pyrido[2,3-b]pyrazinyl, pyrido[3,4-b]pyrazinyl, pyrimido[5,4-d]pyrimidinyl, pyrazino[2,3-b]pyrazinyl, pyrimido[4,5-d]pyrimidinyl and similar groups, but not limited thereto.

[0054] The term "heterocyclyl" refers to a saturated or partially unsaturated ring containing 1 to 6 heteroatoms selected from N, O, and S, excluding a carbon atom. Unless otherwise defined, a heterocyclyl group can be monocyclic or polycyclic. As an example, "3-14 membered heterocyclyl group" refers to an aliphatic heterocycle containing 3-14 cyclic atoms, which may include 3-6 membered aliphatic heterocycles, or the heterocyclyl group may be fused with a benzene ring or another heterocyclyl group to form a bicyclic ring. In one instance, 0, 1, 2, 3, or 4 atoms of each ring of the heterocyclyl group may be substituted with substituents. For example, heterocyclic groups may include, but are not limited to, aziridine, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrazolyl, imidazoyl, oxazolyl, isoxazolyl, thiazoyl, dioxacyclopentenyl, piperidinyl, tetrahydropyranyl, tetrahydrothiaranyl, piperazinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholin-4-yl, aziridine heptyl, diaziridine heptyl, homopiperazinyl, oxazine heptyl, indolyl, isoindolyl, dihydroindolyl, dioxoisoindolinyl, dihydrofuranyl, dihydroimidazolinyl, dihydrooxazolyl, dihydrobenzodioxacyclohexenyl, tetrahydropyridinyl, dihydropyranyl, dihydrobenzofuranyl, benzodioxazolyl or benzodioxylyl and similar groups.

[0055] The term "substitution" refers to replacing a hydrogen atom in a molecule with a substituent, such that, without exceeding the valence of the specified atom, this substitution produces a chemically stable compound. For example, "group A is substituted by substituent B" can mean that the hydrogen atom bonded to the carbon atoms or other atoms forming the skeleton of group A is replaced by substituent B, causing group A to form a covalent bond with substituent B.

[0056] The term "substituent" refers to another group bonded to the parent nucleus, and there can be more than one substituent. When multiple substituents are present, they can be the same or different. When both the parent nucleus and the substituent are hydrocarbon groups, the number of carbon atoms in the parent nucleus does not include the number of carbon atoms in the substituent. For example, butyl (-C4H9) with a methoxy (-O-CH3) substituent is classified as both a C1 alkoxy and a C4 alkyl group.

[0057] The term “N / P ratio” refers, for example, in a nanoparticle composition containing lipid components and RNA, the molar ratio of ionizable (e.g., within a physiological pH range) nitrogen atoms in lipids (or multiple lipids) to phosphate groups in nucleic acid molecular entities (or multiple nucleic acid molecular entities).

[0058] The term "isomer" refers to a variety of stereoisomers. Examples of the present invention can generate a variety of stereoisomers during the preparation process. If no specific stereoisomer is specified, all stereoisomers that may be formed during the reaction process may be included.

[0059] In the embodiments of this invention, the following abbreviations are used throughout: "Ac" refers to acetyl, "AcO" or "OAc" refers to acetoxy, "ACN" refers to acetonitrile, "aq" refers to aqueous, "BOC", "Boc" or "boc" refers to N-tert-butyloxycarbonyl, "Bn" refers to benzyl, "Bu" refers to butyl, "nBu" refers to n-butyl, "tBu" refers to tert-butyl, "Cbz" refers to benzyloxycarbonyl, "DCC" refers to N,N′-dicyclohexylcarbodiimide, "DCM" refers to dichloromethane (CH2Cl2), "DEA" refers to diethylamine, "DIPEA" refers to N,N-diisopropylethylamine, "DMF" refers to N,N-dimethylformamide, "DMSO" refers to dimethyl sulfoxide, "EDC" refers to 1-ethyl-3-(3-dimethylaminopropyl)carboxydiimide, and "EDTA" refers to ethylenediaminetetraethylamine. Acids, "Et" refers to ethyl, "EtOAc" refers to ethyl acetate, "EtOH" refers to ethanol, "HATU" refers to (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, "HOAc" or "AcOH" refers to acetic acid, "IPA" refers to isopropanol, "LAH" refers to lithium aluminum hydride, "mCPBA" refers to m-chloroperoxybenzoic acid, "Me" refers to methyl, "MeOH" refers to methanol, "MS" refers to mass spectrometry, "MTBE" refers to methyl tert-butyl ether, "NHS" refers to N-hydroxysuccinimide, "Ph" refers to phenyl, "PyBOP" refers to benzotriazol-1-yloxytripyrrolidinylphosphine hexafluorophosphate, "TFA" refers to trifluoroacetic acid, "THF" refers to tetrahydrofuran, "TLC" refers to thin-layer chromatography, "R" refers to... f "" refers to the retention fraction, "rt" refers to the retention time, "rt" refers to room temperature, "h" refers to the hour, "min" refers to the minute, "s" refers to the second, "equiv." refers to the equivalent, and "sat." refers to saturation.

[0060] The terms "active substance," "drug," or "payload" refer to a bioactive substance, meaning any substance that, when administered to a subject, has therapeutic, diagnostic, and / or preventative effects and / or induces desired biological and / or pharmacological effects. These bioactive substances include, but are not limited to, cytotoxic substances, radioactive ions, chemotherapeutic drugs, small molecule drugs, proteins, nucleic acids, etc. When delivered to cells or organs, the bioactive substance can induce preferred changes in cells, organs, or other body tissues or systems and may be useful for treating more than one disease, disorder, or condition. As a specific example, the bioactive substance may refer to RNA, siRNA, shRNA, rRNA, tRNA, mRNA, miRNA, saRNA, circRNA, DNA, cDNA, plasmids, DNAzymes, ribozymes, PNA, aptamers, antisense oligonucleotides, CRISPR, proteins, carbohydrates, or drugs, etc.

[0061] The term "prevention" as used in this invention refers to all actions that inhibit or delay the occurrence, spread, and recurrence of the disease by applying compounds or pharmaceutical compositions according to the invention, and the term "treatment" refers to all actions that improve or cause beneficial changes in the symptoms of the disease by applying compounds or pharmaceutical compositions according to the invention.

[0062] The terms “poly(ethylene glycol)”, “PEG”, “PEG moiety”, and “PEG derivative” as used herein refer to functional groups corresponding to polyethylene glycol and are used interchangeably. All possible combinations of elements described herein are included unless otherwise stated herein or the context clearly contradicts it.

[0063] "Drug delivery unit" refers to a form of delivery of a pharmaceutical preparation, such as a pill, tablet, or other drug delivery unit known in the art. In some embodiments, the drug delivery unit is a vial containing lyophilized antisense oligonucleotides. In some embodiments, the drug delivery unit is a vial containing reconstituted antisense oligonucleotides.

[0064] "Dosage" refers to a specific amount of a pharmaceutical preparation administered in a single dose or over a specific time period. In some embodiments, the dosage may be administered by one, two, or more boluses, tablets, or injections. For example, in some embodiments requiring subcutaneous administration, the required dosage may require a volume that cannot be easily contained in a single injection, thus requiring more than two injections to achieve the desired amount. In some embodiments, the pharmaceutical preparation is administered over an extended period of time or via continuous infusion. Dosage may be expressed as the amount of pharmaceutical preparation administered hourly, daily, weekly, or monthly. Dosage may be expressed as mg / kg or g / kg.

[0065] This invention provides compounds of Formula 1, their isomers, or pharmaceutically acceptable salts thereof: [Chemical Formula 1]

[0066] In chemical formula 1, T is the targeting ligand, which is selected from monosaccharides, polysaccharides, folic acid, transferrin, RGD peptide, cyclic RGD peptide, TAT peptide, R9 peptide, CATY peptide, HA2 peptide, monoclonal antibody, antigen-binding fragment or antibody fragment, single-chain variable fragment (scFv) and aptamer, or is a branched-chain targeting ligand represented by the following chemical formula 2; X 1 X 2 X 3 and X 4 Each independently is selected from one or a combination of the following groups: single bond, C 1-14 Alkylene, C 1-14 Heteroalkylene, C 2-14 imidene group, C 1-6 Alkyl-C(=O)-, C 2-6 Alkenyl-C(=O)-, -(OCH2CH2) m -、-(CH2CH2O) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR)-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(= O)O-, -C(=O)C(=O)-, -C(=N-OR)-, -ON=CR-, -OC=NO-, -C(=O)NR-, -NRC(=O)-, -OC(=O) NR-, -NRC(=O)O-, -C(=O)NRC(=O)-, -S(=O)2NR-, -NRS(=O)2-, -NRC(=O)NR-, -NRC(=S) NR-, -C(=O)NH-N=CR-, -RC=N-NHC(=O)-, -NH-N=CR-, -RC=N-NH-, -NR-, -N(OR)-, -SS-, And functional groups that bond with each other through click reactions. Where m is an integer from 0 to 10, Z is -NH-, -O-, or -S-. d and e are each integers from 1 to 10; L 1 and L 2 Each is independently a single bond, C 1-14 Alkylene, C 2-14 sub-alkenyl, MR a M1 Or R a MR b ; P is a single bond, -(CH2CH2O) q -、-(OCH2CH2) q -、-CH2O(CH2CH2O) q CH2-、-CH2CH2O(CH2CH2O) q CH2-, where q is 0 or an integer from 1 to 120; A is CR d Or N; R 1 and R 2 Each independently is C 1-30 Alkyl, C 2-30 alkenyl, or R c MR d , wherein, the C 1-30 Alkyl and C 2-30 The alkenyl groups are either independently unsubstituted or occupied by 1-3 carbons. 1-16 Alkyl or C 2-16 Alkenyl substitution; The M and M 1 Each independently is C 1-14 Alkylene, C 2-14 alkenyl, -NHC(=O)-, -C(=O)NH-, -C(=O)O-, -OC(=O)-, -C(=O)-, -NH-, -N + R2-, -S-, -SS-, -O-, -S(O)2-, -C(=O)S-, -SC(=O)-, -NHC(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, -OP(=O)(OR)O- or -OP(=O)(OR)O(CH2) g - where g is an integer from 1 to 10; R is hydrogen, C 1-20 Alkyl, C 2-20 alkenyl, C 2-20 alkynyl or C 1-20 Heteroalkyl, The R a and R b Each is independently a single bond or C 1-10 Alkylene; The R c For single bond, C 1-14 Alkylene or C 2-14 alkenyl; The R d For hydrogen, C 1-18 Alkyl or C 2-18alkenyl, wherein the C 1-18 Alkyl and C 2-18 The alkenyl groups are either independently unsubstituted or occupied by 1-3 carbons. 1-12 Alkyl or C 2-12 Alkenyl substitution; Each of the heteroalkyl, heteroalkylene, heterocyclic and heteroaryl groups independently comprises 1-6 heteroatoms selected from N, O and S; The branched-chain targeting ligand is represented by the following chemical formula 2: [Chemical Formula 2]

[0067] In the chemical formula 2, a, b, and c are each independently 0 or 1, of which at least one of a, b, and c must be 1, and h and j are 1-3; T 1 T 2 and T 3 Each is independently selected from one of the following groups: monosaccharides, polysaccharides, folic acid, transferrin, RGD peptide, cyclic RGD peptide, TAT peptide, R9 peptide, CATY peptide, HA2 peptide, monoclonal antibody, antigen-binding fragment or antibody fragment, single-chain variable fragment (scFv), and aptamer; X 5 X 6 X 7 X 8 X 9 X 10 X 11 and X 12 Each independently is selected from one or a combination of the following groups: single bond, C 1-14 Alkylene, C 1-14 Heteroalkylene, C 2-14 imidene group, C 1-6 Alkyl-C(=O)-, C 2-6 Alkenyl-C(=O)-, -(OCH2CH2) m -、-(CH2CH2O) m-, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR)-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(= O)O-, -C(=O)C(=O)-, -C(=N-OR)-, -ON=CR-, -OC=NO-, -C(=O)NR-, -NRC(=O)-, -OC(=O) NR-, -NRC(=O)O-, -C(=O)NRC(=O)-, -S(=O)2NR-, -NRS(=O)2-, -NRC(=O)NR-, -NRC(=S) NR-, -C(=O)NH-N=CR-, -RC=N-NHC(=O)-, -NH-N=CR-, -RC=N-NH-, -NR-, -N(OR)-, -SS-, And functional groups that bond with each other through click reactions. Where m is an integer from 0 to 10, Z is -NH-, -O-, or -S-. d and e are each integers from 1 to 10; L 3 L 4 L 5 L 6 and L 7 Each is independently a single bond, C 1-14 Alkylene, C 2-14 sub-alkenyl, MR a M 1 Or R a MR b ; A 1 A 2 and A 3 Each is independently a single bond, CR d Or N; Each of the heteroalkyl and heteroalkylene groups independently comprises 1 to 6 heteroatoms selected from N, O, and S.

[0068] As an example, T represents a targeting ligand, which can be a monosaccharide, polysaccharide, folic acid, transferrin, RGD peptide, cyclic RGD peptide, TAT peptide, R9 peptide, CATY peptide, HA2 peptide, monoclonal antibody, antigen-binding fragment or antibody fragment, single-chain variable fragment (scFv), or aptamer. The antigen-binding fragment or antibody fragment refers to a fragment with antigen-binding function and may include Fab, F(ab`), F(ab`)2, and Fv, etc. As specific examples, antibodies that can be used as the target ligand include, but are not limited to, anti-CD3, anti-CD19, anti-CD20, anti-CD22, anti-CD33, anti-CD38, anti-CD54, anti-CD74, anti-CD138, anti-CD166, anti-CD209, anti-cMET, anti-EGFR, anti-HER2, anti-HIV-gp120, anti-HLA-DR, and anti-transferrin receptor (TfRscFv).

[0069] The monosaccharides may be selected from the following group: glucose, N-acetyl-glucose, galactose, N-acetyl-galactose, mannose, allose, azulose, arabinose, cladinose, erythrose, erythritol, fructose, D-fucoitol, L-fucoitol, fucose, fucose, fucosyl saccharide, galactosamine, D-galactosamine, N-acetyl-galactosamine, glucosamine, N-acetyl-glucosamine, glucosamine alcohol, glucose-6- Phosphoric acid, glyceraldehyde, L-glycerol-D-mannose-heptanose, glycerol, glycerone, gulose, idose, lysose, mannosamine, mannosamine-6-phosphate, allulose, quinose, quinoside, rhamnitol, rhamnosamine, rhamnose, ribose, ribulose, sedoheptulose, sorbose, tagatose, tartaric acid, threose, xylose, xylulose, N,N,N-trimethyl-glucosamine or N,N,N-trimethyl-galactosamine.

[0070] Furthermore, the monosaccharides can be D- or L-stereotypes. The monosaccharides can include those substituted with at least one or more substituents. For example, the monosaccharides can be deoxyglycans (hydroxyl groups replaced by hydrogen), aminoglycans (hydroxyl groups replaced by amino groups), thioglycans (hydroxyl groups replaced by thiols, or C=O replaced by C=S, or cyclic epoxides replaced by sulfur), selenoglycans, telluriumglycans, azo-heterosaccharides (cyclic carbons replaced by nitrogen), iminoglycans (epoxides replaced by nitrogen), phosphatoses (epoxides replaced by phosphorus), phosphatoses (cyclic carbons replaced by phosphorus), C-substituted monosaccharides (hydrogen atoms on non-terminal carbon atoms replaced by carbon), unsaturated monosaccharides, sugar alcohols (carbonyl groups replaced by CHOH groups), aldonic acids (aldehyde groups replaced by carboxyl groups), ketaluronic acids, uronic acids, glycosidic acids, etc. Amino sugars may include amino monosaccharides, preferably galactosamine, glucosamine, mannosamine, fucosesamine, quinoside, neuraminic acid, muramic acid, lactosidine, acosamine, bacitrasamine, daunosamine, deoxysamine, phosphatase, galoside, carnosamine, carnosamine, carbamosamine, inosamine, perosesamine, pneumosamine, crimson glycosamine, and erythromycin.

[0071] The polysaccharides may be selected from the group consisting of disaccharides, trisaccharides, and oligosaccharides, specifically one of the following: abicose, acarbose, amylose, amylopectin, amylose, celery sugar, acanose, artemisia sugar, ascorbic acid, boyrenose, cellobiose, cellotriose, cellulose, potato trisaccharide, charose, chitin, colistin, cyclodextrin, stigmacanose, dextrin, 2-deoxyribose, 2-deoxyglucose, deoxydigitoxin, digitoxin, digitalis toxin, iwasose, iouimose, fructooligosaccharides, galactooligosaccharides, gentiotriose, gentiobiose, glucan, glycogen, glycogen, witch hazel, heparin, inulin, and iso-L-glucan. Sugar ketones, isomaltose, isomalttriose, isoglucosylmaltose, maltodextrose, lactose, lactosamine, lactosidamine, laminarin, L-glucose, L-glucose-enolone, β-maltose, maltodextrose, mannan-oligosaccharides, mannotriose, melitriose, melibiose, muramic acid, carbamoylose, deoxyaloose, neuraminic acid, aspergillus niger, nojirimycin, novose, oleanolic acid, panose, porase, plantain, primrose, raffinose, rose syrup, rutinose, sarcopene, sedoheptulose, solanine, sophorose, stachyose, streptose, sucrose, α,α-trehalose, trehalosamine, melitriose, tivasugar, xylobiose, umbelliferose.

[0072] Furthermore, the polysaccharide may comprise polysaccharides substituted with at least one or more substituents. For example, the disaccharide may comprise amino sugars and their derivatives, particularly carbamoglycosamines derived at the C-4' position or 4-deoxy-3-aminoglycans derived at the C-6' position.

[0073] As an example, the functional groups that are mutually bonded through the click reaction can be used , , , , , , Any functional groups known in the art to be mutually bonded through click reactions can be used without any restrictions.

[0074] The T, T 1 T 2 and T 3 Each of the following can be independently selected from glucose (Glu), N-acetyl-glucosamine (GlcNAc), galactose (Gal), N-acetylgalactosamine (GalNAc), and mannose (Man). Preferably, T can be selected from N-acetylgalactosamine (GalNAc) or mannose (Man).

[0075] Specific examples of compounds of chemical formula 1 according to the present invention are as follows, but are not limited thereto: (1) Glu-DBCO-PEG2000-DSPE; (2)GlcNAc-DBCO-PEG2000-DSPE; (3) Gal-DBCO-PEG2000-DSPE; (4)GalNAc-DBCO-PEG2000-DSPE; (5) Man-DBCO-PEG2000-DSPE; (6) Glu-succinamide-PEG2000-DMG; (7) GlcNAc-succinamide-PEG2000-DMG; (8) Gal-succinamide-PEG2000-DMG; (9) GalNAc-succinamide-PEG2000-DMG; (10) Man-succinamide-PEG2000-DMG; (11)(R)-2,3-bis(stearoyloxy)propyl(2-(dimethyl(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)ammonyl)ethyl)phosphate; (12) 2-((2-(((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)dimethylammonium)ethyl((R)-2,3-bis(stearoyloxy)propyl)phosphate; (13)(R)-2,3-bis(stearoyloxy)propyl(2-(dimethyl(2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)ammonyl)ethyl)phosphate; (14) 2-((2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)dimethylammonium)ethyl((R)-2,3-bis(stearoyloxy)propyl)phosphate; (15)(R)-2,3-bis(stearoyloxy)propyl(2-(dimethyl(2-(((2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)ammonyl)ethyl)phosphate; (16)Man-1,2,3-triazole-PEG2000-DSPE; (17) Man-DIFO-PEG2000-DSPE; (18)Man-BCN-PEG2000-DSPE; (19) Man-triarylphosphine-PEG2000-DSPE; (20)Man-TCO-PEG2000-DSPE; (21) Man-succinamide-PEG2000-carbamate-DSPE; (22) Man-amide-PEG2000-amide-DSPE; (23) Man-valerate-PEG2000-amide-DSPE; (24) Man-succinamide-PEG2000-DMG; (25) Man-amide-PEG2000-DMG; (26) Man-valerate-PEG2000-DMG; (27)(R)-2,3-bis(stearoyloxy)propyl(2-(dimethyl(2-(4-oxo-4-((2-(((2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)amino)butamido)ethyl)ammono)ethyl)phosphate; (28)(R)-2,3-bis(stearoyloxy)propyl(3,3-dimethyl-10,13-dioxo-16-(((2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6-oxa-3,9,14-triazahexadecane-3-onthium-1-yl)phosphate; (29) Man-succinamide-PEG2000-DSG; (30) Man-succinamide-PEG2000-DOG; (31) Man-succinamide-PEG2000-DLG; (32)Man-PEG2000-(10Z,29Z)-tetradec-10,29-diene-20-yl; (33)Man-PEG2000-(7Z, 10Z, 29Z, 32Z)-tetracarbazide-7,10, 29,32-tetraene-20-yl; (34)Tri-Man-DBCO-PEG2000-DMG; (35)Tri-Man-PEG-DBCO-PEG2000-DMG; (36)Tri-(PEG-Man)-PEG-DBCO-PEG2000-DMG; (37) Tri-Man-PEG2000-DMG; (38) Tri-Man-PEG2000-DSPE; (39)(R)-2,3-bis(stearoyloxy)propyl(2-(4-((2-(((2R,3R,4R,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-(trimethylammonium)tetrahydro-2H-pyran-2-yl)oxy)ethyl)amino)-4-oxobutamido)ethyl)phosphate; (40)(R)-2,3-bis(stearoyloxy)propyl(2-(4-((2-(((2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-(trimethylammonium)tetrahydro-2H-pyran-2-yl)oxy)ethyl)amino)-4-oxobutamido)ethyl)phosphate; (41)(R)-2,3-bis(stearoyloxy)propyl(2-(4-((2-(((2R,3R,4R,5S,6R)-3-(dimethylammonium)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)amino)-4-oxobutamido)ethyl)phosphate; (42)(R)-2,3-bis(stearoyloxy)propyl(2-(4-((2-(((2R,3R,4R,5R,6R)-3-(dimethylammonium)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)amino)-4-oxobutamido)ethyl)phosphate; (43) Glu-carbamate-ethylethoxy-PEG2000-DMG; (44)GlcNAc-carbamate-ethylethoxy-PEG2000-DMG; (45) Gal-carbamate-ethylethoxy-PEG2000-DMG; (46) GalNAc-carbamate-ethylethoxy-PEG2000-DMG; (47) Man-carbamate-ethylethoxy-PEG2000-DMG; (48) Di(tris(man)methylamide)amidoamine-PEG2000-DMG; (49) Tris(2-(tris-man-amidoamine)ethoxy)methylamine-PEG2000-DMG; (50) Di(tri(Man)methylamine)thiourea ethylamine-PEG2000-DMG; (51)G1-bis-MPA-(Man-succinamide)-PEG2000-DMG; (52) Di((Di(Man-ethylamidoamine))Ethylamidoamine)Ethylamine-PEG2000-DMG; (53) Di(Di((Di(Man-Ethylamidoamine))Ethylamidoamine)Ethylamidoamine)Ethylamine-PEG2000-DMG; (54)G1-polylysine-(Man-succinamide)-ethylamine-PEG2000-DMG; (55)G2-polylysine-(Man-succinamide)-ethylamine-PEG2000-DMG.

[0076] The lipid compounds according to the invention can be advantageously used in lipid nanoparticle compositions to deliver active substances to vertebrate cells or organs. For example, lipid nanoparticle compositions containing the lipid compounds described herein exhibit excellent safety when administered in vivo and excellent long-term stability at refrigeration and room temperature.

[0077] Lipid nanoparticle composition In another aspect, the present invention provides a lipid nanoparticle composition comprising a lipid compound of Formula 1, an isomer thereof, or a salt thereof. Specifically, the lipid nanoparticle composition may comprise an ionizable lipid; a first phospholipid; a second phospholipid; a structural lipid (e.g., cholesterol); a first PEG-lipid; and a second PEG-lipid.

[0078] In this invention, the term "phospholipid" refers to a lipid that may contain phosphate residues and more than one carbon chain (e.g., an unsaturated fatty acid chain). Phospholipids may contain more than one multiple (e.g., double or triple) bond (e.g., more than one unsaturated group). Some phospholipids can promote membrane fusion.

[0079] The lipid nanoparticle compositions according to the present invention may contain more than one phospholipid, for example, more than one (poly)unsaturated lipid. The phospholipids may be assembled into more than one lipid bilayer. Typically, the phospholipids may contain phospholipid residues and more than one fatty acid residue. The phospholipid residues may be selected from, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid residues may be selected from, but are not limited to, 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, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0080] The present invention provides a lipid nanoparticle composition comprising: an ionizable lipid; a lipid compound represented by the first chemical formula 1, its isomers or pharmaceutically acceptable salts thereof as a first phospholipid; a second phospholipid; a structural lipid; a lipid compound represented by the first chemical formula 1, its isomers or pharmaceutically acceptable salts thereof as a first polyethylene glycol-modified lipid; and a second polyethylene glycol-modified lipid.

[0081] The ionizable lipids promote the formation of the core structure of the active ingredient, enhance intracellular delivery, and facilitate endosome escape after endocytosis. As an example, two or more of the following groups can be used alone or in combination: (6Z, 9Z, 28Z, 31Z)-hexadecano-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid (SM-102), 1-linoleyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleylcarbamoyloxy-3-di Methylaminopropane (DLin-C-DAP), 1,2-dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleoyloxy)propylamine (DODMA), bis(octadecylamide)glycyl carboxyl ester Amines (DOGS), spermine cholesterol carbamate (GL-67); bis-guanidinium-spermine cholesterol (BGTC), 3β-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydecyl)amino)ethyl)(2-hydroxydecyl)amino)ethyl)piperazin-1-yl)ethylazadiyl)bisdodecane-2-ol (C12-200), N-tert-butyl-N'-tetradecylamino-propanediamine (bisC14-amanediamine); dimethyl bis(octadecyl)ammonium bromide (DDAB), N-(1,2-dimyristyloxypropyl-3-yl)-N, N-Dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-diolenoyl-N,N-dimethylammonium chloride (DODAC), dioleoyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), N-(1-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarbamoyl)ethyl)-N,N-dimethyltrifluoroacetate ammonium (DOSPA), 1,2-dioleoyltrimethylpropane ammonium chloride (DOTAP), N-(1-(2,3-diolenoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), and aminopropyl-dimethyl-bis(dodecyloxy)propylammonium bromide (GAP-DLRIE), etc.

[0082] The structural lipid may be selected from the group consisting of, for example, cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, tomatine, ursolic acid, α-tocopherol, and mixtures thereof, but is not limited thereto. In some instances, the structural lipid is cholesterol. In some embodiments, the structural lipid includes, but is not limited to, cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisolone, and hydrocortisone) or combinations thereof.

[0083] The first phospholipid may comprise a lipid compound represented by Formula 1, its isomers, or a pharmaceutically acceptable salt thereof. The first phospholipid functions as a tissue-specific delivery agent by introducing a targeting ligand to the terminal portion.

[0084] The second phospholipid can be any phospholipid known in the art. For example, DOPE (1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine), DSPC (1,2-distearyl-sn-glycerol-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine), EPC (egg yolk phosphatidylcholine), DOPC (1,2-dioleoyl-sn-glycerol-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycerol-3-phosphocholine), DOPG (1,2-dioleoyl-sn-glycerol-3-phosphocholine), DPPG (1,2-dipalmitoyl-sn-glycerol-3-phosphocholine), and DPPG (1,2-dipalmitoyl-sn-glycerol-3-phosphocholine) can be used. 1,2-Distearate-sn-glycerol-3-phosphatidylethanolamine), PE (phosphatidylethanolamine), DPPE (1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine), DOPE (1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine), POPE (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylethanolamine), DOPS (1,2-dioleoyl-sn-glycerol-3-[phospho-L-serine]), ceramide, sphingomyelin, and other known phospholipids. For phospholipids, modified phospholipids such as compounds 11-15, 27, 28, 39-42 in Table 1 can be used alone, or two or more known phospholipids can be used in combination.

[0085] The first polyethylene glycol-modified lipid may comprise a lipid compound represented by Formula 1, its isomers, or a pharmaceutically acceptable salt thereof. The first polyethylene glycol-modified lipid functions as a tissue-specific delivery agent by introducing a targeting ligand to the terminal portion.

[0086] The second polyethylene glycol-modified lipid can be any polyethylene glycol-modified lipid known in the art. The second polyethylene glycol-modified lipid protects the nanoparticles by reducing recognition by the immune system and improving their distribution in vivo. Examples include PEG-dilauroyl glycerol, PEG-dimyristoyl glycerol (PEG-DMG), PEG-dipalmitoyl glycerol, PEG-distearylphosphatidylethanolamine (PEG-DSPE), PEG-dipalmitoyl glycerol, PEG-distearyl glycerol (PEG-DSG), PEG-dilauroyl glycine, PEG-dimyristoyl glycine, PEG-dipalmitoyl glycine, PEG-distearyl glycine, PEG-cholesterol, and PEG-DMB (3,4-dimyristoxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), as well as 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-200]. For polyethylene glycol-modified lipids, compounds 1-10, 16-26, 29-38, and 43-55 listed in Table 1 can be used alone to modify polyethylene glycol-modified lipids, or two or more known polyethylene glycol-modified lipids can be used in combination.

[0087] Additionally, stealth lipids, as is known, may be included. Stealth lipids can refer to lipids that alter the duration for which nanoparticles can remain in vivo (e.g., in the blood). Stealth lipids, for example, can reduce particle aggregation and control particle size, thereby facilitating formulation processes. Stealth lipids used herein can modulate the pharmacokinetic properties of LNPs. Stealth lipids suitable for lipid compositions of this disclosure may include, but are not limited to, stealth lipids having a hydrophilic head group attached to the lipid moiety.

[0088] The lipid nanoparticles of the present invention can encapsulate active substances such as RNA, siRNA, shRNA, rRNA, tRNA, mRNA, miRNA, saRNA, circRNA, DNA, cDNA, plasmids, DNAzymes, ribozymes, PNA, aptamers, ASO (antisense oligonucleotides), and CRISPR in their core.

[0089] In applications as drug delivery carriers for treating retinal diseases, when using intravitreal injection or subretinal injection methods, the T, T 1 T 2 and T 3 Mannose (Man) or N-acetylglucosamine (GlcNAc) can be preferred.

[0090] In drug delivery carrier applications for the prevention of infectious diseases, when using intramuscular or intranasal injection methods, the T, T 1 T 2 and T3 Mannose can be preferred.

[0091] In drug delivery carrier applications for the treatment of cancer and brain diseases, intravenous injection methods can be used.

[0092] In the use of a drug delivery carrier for treating liver diseases, when using an intravenous injection method, the T, T 1 T 2 and T 3 N-acetylgalactosamine (GalNAc) can be preferred.

[0093] Methods for preparing active substances-lipid nanoparticles In another aspect, the present invention provides a method for preparing active material-lipid nanoparticles comprising a lipid compound of the stated chemical formula 1. Specifically, a method for preparing active material-lipid nanoparticles is provided, comprising the step of mixing an organic phase and an aqueous phase, wherein the organic phase comprises a lipid compound represented by the stated chemical formula 1, its isomers, or a pharmaceutically acceptable salt thereof, and the aqueous phase contains the active material dissolved therein.

[0094] This invention provides a method for preparing active substance-lipid nanoparticles, comprising: an organic phase in which ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids are mixed in a molar ratio of (20-60):(0-25):(30-60):(0-10); and an aqueous phase in which the active substance is dissolved; and a mixing step thereof; wherein the phospholipids are a mixture of a first phospholipid and a second phospholipid in a molar ratio of (0.005-100):(0-99.995), the polyethylene glycol-modified lipids are a mixture of a first polyethylene glycol-modified lipid and a second polyethylene glycol-modified lipid in a molar ratio of (0.005-100):(0-99.995), and the ionizable lipids and the active substance are mixed in a weight ratio of (2-80):1.

[0095] Specific examples of the ionizable lipids, phospholipids, cholesterol, PEGylated lipids, and active substances are as described above.

[0096] Composition for delivering nucleic acids In another aspect, the present invention provides a composition for delivering nucleic acids, comprising a lipid compound represented by said chemical formula 1, an isomer thereof, or a pharmaceutically acceptable salt thereof.

[0097] Drug delivery carrier In another aspect, the present invention provides a method for delivering an active substance (e.g., mRNA) to cells (e.g., vertebrate cells). The invention includes the step of administering a lipid nanoparticle composition to a subject, said administration involving contacting cells in the subject's body with the lipid nanoparticle composition, and enabling delivery of the active substance to target tissue cells.

[0098] According to one embodiment, the present invention can provide a tissue-specific drug delivery carrier comprising the lipid nanoparticle composition. The tissue-specific drug delivery carrier according to the present invention can select a target tissue based on the type of targeting ligand in chemical formulas 1 and 2.

[0099] Furthermore, the present invention can provide a drug delivery carrier for treating retinal diseases comprising the lipid nanoparticle composition. The drug delivery carrier for treating retinal diseases can be administered via subretinal injection or intravitreal injection.

[0100] Furthermore, the present invention can provide a drug delivery carrier for the prevention of infectious diseases comprising the lipid nanoparticle composition. The drug delivery carrier for the prevention of infectious diseases can be administered via intramuscular injection or intranasal injection.

[0101] Furthermore, the present invention can provide a drug delivery carrier comprising the lipid nanoparticle composition for treating cancer, brain diseases, and liver diseases. The drug delivery carrier for treating cancer, brain diseases, or liver diseases may contain different types of targeting ligands depending on the type of cancer, brain disease, or liver disease.

[0102] According to one embodiment, the drug delivery carrier for treating cancer, brain diseases, and liver diseases can be administered via intravenous injection, direct injection to the cancerous lesion site, or direct injection into the brain. Most preferably, the drug delivery carrier for treating cancer, brain diseases, and liver diseases can be administered via intravenous injection.

[0103] Pharmaceutically acceptable salts The active ingredient of this invention can be used in the form of a pharmaceutically acceptable salt, which is an acid addition salt formed from a pharmaceutically acceptable free acid. A pharmaceutically acceptable salt refers to any organic or inorganic addition salt of the basic compound of the active ingredient, the concentration of which has a relatively non-toxic and harmless effect on the patient, and the side effects caused by the salt do not reduce the beneficial efficacy of the basic compound of the active ingredient. These salts can use inorganic and organic acids as free acids. As inorganic salts, hydrochloric acid, bromic acid, nitric acid, sulfuric acid, perchloric acid, phosphoric acid, etc., can be used; as organic acids, citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, mesylic acid, glycolic acid, succinic acid, tartaric acid, galacturonic acid, M acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, mesylic acid, ethanesulfonic acid, 4-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid, or malonic acid, etc., can be used. In addition, these salts include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.). For example, acid addition salts can include acetates, aspartates, benzoates, benzenesulfonates, bicarbonates / carbonates, bisulfates / sulfates, borates, camphorsulfonates, citrates, edetates, ethanesulfonates, formates, fumarates, glucohepanoates, gluconates, glucuronates, hexafluorophosphates, benzoates, hydrochlorides / chlorides, hydrobromates / bromines, hydroiodates / iodides, hydroxyethanesulfonates, lactates, malates, maleates, and malonates. Methanesulfonates, methyl sulfates, naphthates, 2-naphthalenesulfonates, nicotinates, nitrates, orotates, oxalates, palmitates, dihydroxynaphthalates, phosphates / hydrogen phosphates / dihydrogen phosphates, glycosides, stearates, succinates, tartrates, toluenesulfonates, trifluoroacetates, aluminum salts, arginine salts, benzyl benzoate, calcium salts, choline salts, diethylamine salts, diethanolamine salts, glycine salts, lysine salts, magnesium salts, meglumine salts, ethanolamine salts, potassium salts, sodium salts, tromethamine salts, zinc salts, etc., with hydrochloride salts or trifluoroacetates being preferred.

[0104] The acid addition salts according to the present invention can be prepared by conventional methods, for example, by dissolving the active substance in an organic solvent, such as methanol, ethanol, acetone, dichloromethane, acetonitrile, etc., adding an organic or inorganic acid, and filtering and drying the resulting precipitate; or by drying after vacuum distillation of the solvent and excess acid, or by crystallizing in an organic solvent.

[0105] Furthermore, pharmaceutically acceptable metal salts can be prepared using bases. Alkali metal or alkaline earth metal salts, for example, can be obtained by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering out the insoluble compound salt, and evaporating and drying the filtrate. In this case, sodium, potassium, or calcium salts are suitable for pharmaceutical preparation. Additionally, the corresponding silver salts can be obtained by reacting the alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0106] Furthermore, the present invention includes not only the active substance and its pharmaceutically acceptable salt, but also all possible solvates, hydrates, isomers, optical isomers, etc. that can be prepared therefrom.

[0107] route and method of administration The drug formulations described herein can be administered to subjects in various ways via multiple routes of administration, including but not limited to oral, non-oral (e.g., intravenous, subcutaneous, intramuscular, intraspinal, spinal, intrathecal, direct intracardiac, intraperitoneal, intralymphatic, intranasal), intranasal, buccal, local, or transdermal routes of administration. The drug formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid delivery forms, powders, immediate-release formulations, controlled-release formulations, instantaneous solvents, tablets, capsules, pills, delayed-release formulations, extended-release formulations, pulsatile-release formulations, multi-microparticle formulations, and mixed immediate-release and controlled-release formulations.

[0108] On one hand, this document discloses a method for treating diseases or conditions in vertebrates, comprising administering a therapeutically effective dose of the pharmaceutical composition described herein. In one embodiment, the disease or condition can be treated by administering a payload.

[0109] As those skilled in the art will recognize, the appropriate dosage or effective dose depends on individual subject parameters, duration of treatment, nature of concurrent therapies (if any), route of administration, and similar factors including the knowledge and competence of the medical personnel, such as the specific condition being treated, severity of the condition, age, physical condition, body type, sex, and weight. It is known to those skilled in the art that factors relating to dosage determination do not require additional experiments beyond routine testing. Generally, the maximum dose of a single ingredient or combination thereof is preferred, i.e., the safest dose based on reasonable medical judgment. Empirical considerations such as half-life often help in determining the dosage used.

[0110] Dosing frequency can be determined and adjusted during treatment, typically but not necessarily based on the treatment and / or suppression and / or improvement and / or delay of the disease. In some embodiments, the dosage is used daily, every other day, every 3 days, every 4 days, every 5 days, or every 6 days. In some embodiments, the dosing frequency is once every 1 week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every 1 month, every 2 months, every 3 months, or for longer periods. Progression of these therapies can be easily monitored using existing technologies and analyses.

[0111] The dosing regimen may vary over time. In some embodiments, for normal-weight adult subjects, a dose ranging from approximately 0.01 to 1000 mg / kg may be administered. In some embodiments, the dose is 1 to 200 mg. In some implementations, the dosage may be about 0.01-0.05 mg / kg, about 0.01-0.1 mg / kg, about 0.01-1 mg / kg, about 0.01-10 mg / kg, about 0.01-100 mg / kg, 0.01-500 mg / kg, about 0.1-1 mg / kg, about 0.1-5 mg / kg, about 0.1-10 mg / kg, about 0.1-100 mg / kg, about 0.1-500 mg / kg, about 0.1-1000 mg / kg, about 1-5 mg / kg, about 1-100 mg / kg, about 1-500 mg / kg, about 1-1000 mg / kg, or about 10-1000 mg / kg. The range is 100 mg / kg, approximately 10-500 mg / kg, approximately 10-1000 mg / kg, or approximately 100-1000 mg / kg.

[0112] As will be clearly understood by those skilled in the art, the appropriate dosage of the therapeutic agents described herein will depend on the specific formulation (or combination thereof) used, the dosage form and route of administration, the type and severity of the disease, whether it is for preventive or therapeutic purposes, prior therapy, the subject's clinical history and response to the antagonist, and the judgment of the attending physician.

[0113] This invention provides a method for preparing the compound.

[0114] Specifically, the compounds of Formula 1 can be prepared by, but are not limited to, the methods described in the following examples. In particular, those skilled in the art will fully understand that the compounds of Formula 1 of the present invention can be prepared using various methods with techniques well known in the art. The following examples illustrate step-by-step methods for preparing representative compounds according to the present invention. Various compounds of the present invention can be prepared by changing the reagents and solvents used in the following preparation steps or by changing the reaction order.

[0115] The present invention will now be described in more detail through the following embodiments. However, the following embodiments are for illustrative purposes only, and the scope of the present invention is not limited to the following embodiments.

[0116] Example 1. Preparation of Glu-DBCO-PEG2000-DSPE (Compound 1)

[0117] Step 1: Synthesis of 2-aminoethyl-β-D-glucopyranoside (compound 1b) 2-Azideethyl-β-D-glucopyranoside (purchased from Synthose, 46.5 mg, 0.177 mmol) was added to a 10 mL sealable flask, dissolved in methanol (1.0 mL), and then Pd / C was added. The reaction solution was saturated with hydrogen and stirred at room temperature under hydrogen atmosphere for 2 hours. The flask containing the mixed solution was passed through a diatomaceous earth filter to remove Pd / C. The reaction product was extracted with methanol and dried under vacuum to obtain 2-aminoethyl-β-D-glucopyranoside (compound 1b, 45.5 mg, 0.203 mmol, 100%) as a transparent solid. Compound 1b was used directly in the next step of the reaction without further purification.

[0118] 1 H NMR (600 MHz, D2O): δ4.88 (d, J = 8.0 Hz, 1H), 3.98 (ddd, J = 10.9,5.9, 4.3 Hz, 1H), 3.92 (dd, J = 12.3, 2.3 Hz, 1H), 3.76 (ddd, J = 10.7, 6.4, 4.5Hz 1H), 3.72 (dd, J = 12.3, 6.0 Hz, 1H), 3.50 (dd, J = 10.3, 9.3 Hz, 1H), 3.46(dt, J = 5.7, 2.3 Hz, 1H), 3.38 (dd, J = 10.4, 9.5 Hz, 1H), 3.30 (dd, J = 9.4, 8.0Hz, 1H), 2.93 (td, J = 5.7, 4.3 Hz, 2H); 13C NMR (151 MHz, D2O): δ102.28, 75.90,75.62, 73.09, 69.59, 60.66, 39.88.; HRMS (ESI) m / z: [M+H] + The calculated value is C8H 18 NO6224.1134; Measured value is 224.1132.

[0119] Step 2: Synthesis of Glu-PEG5-DBCO (Compound 1d) 2-Aminoethyl-β-D-glucopyranoside (compound 1b, 11.0 mg, 46.9 μmol) and DBCO-PEG5-N-hydroxysuccinimide (NHS) ester (purchased from Click Chemistry Tools, compound 1c, 29.9 mg, 42.3 μmol) were added to a 10 mL flask and dissolved in DMSO. d 6. After adding 0.5 mL of the solution, inject 0.06 mL of DIPEA. Then, stir the flask at room temperature for 5 hours and concentrate. The crude reaction product was purified by reversed-phase C18 silica gel chromatography (eluting with MeOH containing 35% H2O) to obtain Glu-PEG5-DBCO (compound 1d, 11.3 mg, 14.0 μmol, 33%), which is a pale orange liquid.

[0120] 1 H NMR (600 MHz, DMSO- d 6): δ7.84 (t, J = 5.6 Hz, 1H), 7.68 (t, J = 5.7 Hz,1H), 7.63 (d, 1H) 7.56 (m, 1H), 7.54 - 7.43 (m, 3H), 7.54 - 7.43 (m, 3H),5.04 (d, J = 14.1 Hz, 1H), 4.99 (d, J = 4.3 Hz, 1H), 4.96 (d, J = 4.9 Hz, 1H), 4.92(d, J = 5.3 Hz, 1H), 4.54 (t, J = 5.8 Hz, 1H), 4.12 (d, J = 7.8 Hz, 1H), 3.77 -3.65 (m, 2H), 3.63 (d, J = 14.1 Hz, 1H), 3.58 (t,J = 6.5 Hz, 1H), 3.54 - 3.37(m, 19H), 3.30 - 3.18 (m, 1H), 3.18 - 3.06, (m, 2H), 3.06 - 3.00 (m, 1H),3.00 - 2.86 (m, 2H), 2.44 (ddd, J = 15.8, 8.5, 5.8 Hz, 1H), 2.32 (t, J = 6.5 Hz, 2H), 2.17 (t, J = 6.5 Hz, 2H), 1.81 (ddd, J = 15.1, 8.5, 5.8 Hz, 1H). 13 C NMR (151MHz, DMSO- d 6): δ170.62, 170.35, 151.87, 148.84, 132.86, 130.03, 129.44,128.70, 128.54, 128.21, 127.28, 125.69, 122.94, 121.88, 114.76, 108.55,103.62, 77.36, 77.00, 73.92, 70.51, 70.22, 70.13, 70.09, 69.99, 69.92, 68.39,67.25, 67.14, 61.54, 55.30, 39.56, 39.20, 36.52, 36.41, 35.41, 34.64.; HRMS(ESI) m / z: [M+H] + The calculated value is C 40 H 56 N3O 14 802.3762; measured value is 802.3752.

[0121] Step 3: Synthesis of Glu-DBCO-PEG2000-DSPE (Compound 1) DSPE-PEG2000-azide (purchased from Avanti Polar Lipids, compound 1e, 5.32 mg, 1.85 μmol) was added to a 10 mL flask and dissolved in DMSO- d 6 (0.15 mL). Then, Glu-PEG5-DBCO (compound 1d, 6.28 mg, 7.44 μmol) was dissolved alone in DMSO- d6 (0.1 mL) was then injected into the reaction flask solution, and after stirring at room temperature for 5 hours, Glu-PEG5-DBCO (compound 1d, 2.36 mg, 2.80 μmol) was dissolved in DMSO- d 6 (0.1 mL) was injected into the reaction solution, and then stirred at room temperature for 18 hours and concentrated. The crude reaction product was purified in methanol using an SEC column (Sephadex LH-20, H 26 cm × OD 1 cm, manufacturer: GE Healthcare) to obtain Glu-DBCO-PEG2000-DSPE (compound 1, 5.71 mg, 1.57 μmol, 85%), as a white solid.

[0122] 1 H NMR (600 MHz, DMSO- d 6): δ7.91 - 7.80 (m, 2H), 7.72 - 7.19 (m, 10H), 5.87 (dd, J = 38.4, 16.9 Hz, 1H), 5.05 (m, 1H), 4.85 (d, J = 3.9 Hz, 1H), 4.72(d, J = 5.3 Hz, 1H), 4.60 (q, J = 7.0 Hz, 1H), 4.64 - 4.39 (m, 4H), 4.16 - 3.96 (m, 5H), 3.91 - 2.77 (m, 318H), 2.37 - 2.13 (m, 6H), 1.97 - 1.79 (m, 1H), 1.49 (d, J = 7.1 Hz, 5H), 1.23 (s, 56H), 0.85 (t, J = 6.9 Hz, 6H). MS (MALDI-TOF, DHB matrix) m / z: [M] - The calculated value is C 172 H 315 N7O 68 P - 3598.1; the measured value is 3598.1.

[0123] Example 2. Preparation of GlcNAc-DBCO-PEG2000-DSPE (Compound 2)

[0124] Step 1: Synthesis of 2-aminoethyl-2-acetamido-2-deoxy-β-D-glucopyranoside (compound 2b) 2-Azideethyl-2-acetamido-2-deoxy-β-D-glucopyranoside (purchased from Synthose, 39.2 mg, 0.128 mmol) was added to a 10 mL sealable flask, dissolved in methanol (1.0 mL), and then Pd / C was added. The reaction solution was saturated with hydrogen and stirred at room temperature under hydrogen atmosphere for 2 hours. The flask containing the mixed solution was passed through a diatomaceous earth filter to remove Pd / C. The reaction product was extracted with methanol and dried under vacuum to give 2-aminoethyl-2-acetamido-2-deoxy-β-D-glucopyranoside (compound 2b, 37.4 mg, 0.141 mmol, 100%), a clear solid. Compound 2b was used directly in the next reaction without further purification.

[0125] 1 H NMR (600 MHz, D2O): δ4.32 (d, J = 8.0 Hz, 1H), 3.82 - 3.77 (m, 2H,H), 3.63 - 3.56 (m, 2H), 3.50 (dd, J = 10.3, 9.3 Hz), 3.38 (dd, J = 9.4, 8.0 Hz,1H), 3.26 - 3.18 (m, 2H), 2.7 (td, J = 5.7, 4.3 Hz, 2H), 1.91 (s, 3H); 13 C NMR (151 MHz, D2O): δ174.76, 101.29, 75.82, 74.74, 73.69, 69.85, 60.66, 55.54,39.88, 22.10. HRMS (ESI) m / z: [M+H] + The calculated value is C 10 H 21 N2O6 265.1400; measured value 265.1398.

[0126] Step 2: Synthesis of GlcNAc-PEG5-DBCO (compound 2c) 2-Aminoethyl-2-acetamido-2-deoxy-β-D-glucopyranoside (compound 2b, 14.6 mg, 52.5 μmol) and DBCO-PEG5-N-hydroxysuccinimide (NHS) ester (purchased from Click Chemistry Tools, compound 1c, 30.2 mg, 42.7 μmol) were added to a 10 mL flask and dissolved in DMSO. d 6. After adding 0.5 mL of the solution, inject 0.06 mL of DIPEA. Then, stir the flask at room temperature for 5 hours and concentrate. The crude reaction product was purified by reversed-phase C18 silica gel chromatography (eluting with MeOH containing 35% H2O) to obtain GlcNAc-PEG5-DBCO (compound 2c, 14.6 mg, 17.5 μmol, 41%), which is a pale orange liquid.

[0127] 1 H NMR (600 MHz, DMSO- d 6): δ7.73 (t, J = 5.6 Hz, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 7.5 Hz, 1H), 7.61 - 7.56 (m, 1H), 7.53 - 7.28 (m, 7H), 5.13 -4.97 (m, 2H), 4.94 (d, J = 5.2 Hz, 1H), 4.58 (t, J = 5.7 Hz, 1H), 4.29 (d, J = 8.4Hz, 1H), 4.09 (q, J = 5.2 Hz, 1H), 3.75 - 3.53 (m, 6H), 3.52 - 3.01 (m, 81H), 2.93 (td, J = 13.9, 5.9 Hz, 1H), 2.43 (dt, J = 15.2, 7.2 Hz, 1H), 2.31 (t, J = 6.5Hz, 2H), 2.16 (t, J = 6.5 Hz, 2H), 1.81 (dt, J = 15.2, 7.2 Hz, 1H), 1.81 (s, 3H). 13 C NMR (151 MHz, DMSO- d6): δ170.61, 170.34, 169.76, 151.87, 148.84, 132.85,130.02, 129.43, 128.69, 128.53, 128.20, 127.27, 125.68, 122.93, 121.88,114.75, 108.54, 101.55, 77.52, 74.86, 71.08, 70.21, 70.11, 70.08, 69.97,69.91, 67.58, 67.21, 67.14, 61.55, 55.85, 55.30, 40.54, 39.63, 39.15, 36.47,36.41, 35.41, 34.63, 23.54.; HRMS (ESI) m / z: [M+H] + The calculated value is C 42 H 59 N4O 14 843.4028; the measured value is 843.4005.

[0128] Step 3: Synthesis of GlcNAc-DBCO-PEG2000-DSPE (Compound 2) DSPE-PEG2000-azide (purchased from Avanti Polar Lipids, compound 1e, 6.72 mg, 2.34 μmol) was added to a 10 mL flask and dissolved in DMSO- d 6 (0.15 mL). Then, GlcNAc-PEG5-DBCO (compound 2c, 9.36 mg, 10.5 μmol) was dissolved separately in DMSO- d 6 (0.1 mL) was then injected into the reaction flask solution, and after stirring at room temperature for 5 hours, GlcNAc-PEG5-DBCO (compound 2c, 1.76 mg, 2.03 μmol) was dissolved in DMSO- d 6 (0.1 mL) was injected into the reaction solution, and then stirred at room temperature for 18 hours and concentrated. The crude reaction product was purified in methanol using a SEC column (model: Sephadex LH-20, H 26cm × OD 1cm, manufacturer: GE Healthcare) to obtain GlcNAc-DBCO-PEG2000-DSPE (compound 2, 6.78 mg, 1.85 μmol, 79%), as a white solid.

[0129] 1 H NMR (600 MHz, DMSO- d6): δ7.98 - 7.11 (m, 12H), 5.87 (dd, J = 38.2, 16.9 Hz, 1H), 5.00 (d, J = 39.2 Hz, 3H), 4.58 (s, 1H), 4.48 (td, J = 16.2, 7.2Hz, 2H), 4.41 - 4.22 (m, 3H), 4.14 - 3.96 (m, 4H), 3.87 - 2.82 (m, 253H), 2.26 (ddt, J = 31.3, 26.3, 6.5 Hz, 8H), 1.81 (s, 4H), 1.62 - 1.40 (m, 5H), 1.23(s, 55H), 0.85 (t, J = 6.9 Hz, 6H). MS (MALDI-TOF, DHB matrix) m / z: [M] - The calculated value is C 174 H 318 N8O 68 P - 3639.1; the measured value is 3639.0.

[0130] Example 3. Preparation of Gal-DBCO-PEG2000-DSPE (Compound 3)

[0131] Step 1: Synthesis of 2-aminoethyl-β-D-galactopyranoside (compound 3b) 2-Azideethyl-β-D-galactopyranoside (purchased from Synthose, 22.3 mg 85.1 μmol) was added to a 10 mL sealable flask, dissolved in methanol (1.0 mL), and then Pd / C was added. The reaction solution was saturated with hydrogen and stirred at room temperature under hydrogen atmosphere for 2 hours. The flask containing the mixed solution was passed through a diatomaceous earth filter to remove Pd / C. The reaction product was extracted with methanol and dried under vacuum to obtain 2-aminoethyl-β-D-galactopyranoside (compound 3b, 24.5 mg, 17.1 μmol, 100%) as a transparent solid. Compound 3b was used directly in the next reaction without further purification.

[0132] 1 H NMR (600 MHz, D2O): δ4.32 (d, J = 7.8 Hz, 1H), 3.89 (ddd, J= 10.9,5.9, 4.3 Hz, 1H), 3.84 (d, J = 3.4 Hz, 1H), 3.74 - 3.63 (m, 3H) 3.61 (dt, J =7.4, 4.3 Hz, 1H), 3.57 (dd, J = 10.0, 3.4 Hz, 1H), 3.45 (dd, J = 9.9, 7.8 Hz,1H), 2.84 (m, 2H).; 13 C NMR (151 MHz, D2O): δδ102.89, 75.15, 72.66, 70.79,70.28, 68.61, 60.97, 40.01.; HRMS (ESI) m / z: [M+H] + The calculated value is C8H 18 NO6224.1134; Measured value is 224.1131.

[0133] Step 2: Synthesis of Gal-PEG5-DBCO (compound 3c) 2-Aminoethyl-β-D-galactopyranoside (compound 3b, 11.8 mg, 50.1 μmol) and DBCO-PEG5-N-hydroxysuccinimide (NHS) ester (purchased from Click Chemistry Tools, compound 1c, 29.2 mg, 41.2 μmol) were added to a 10 mL flask and dissolved in DMSO. d 6. After adding 0.5 mL of the solution, inject 0.06 mL of DIPEA. Then, stir the flask at room temperature for 5 hours and concentrate. The crude reaction product was purified by reversed-phase C18 silica gel chromatography (eluting with MeOH containing 35% H2O) to obtain Gal-PEG5-DBCO (compound 3c, 13.0 mg, 16.1 μmol, 39%), which is a pale orange liquid.

[0134] 1 H NMR (600 MHz, DMSO- d 6): δ7.84 (t, J = 5.6 Hz, 1H), 7.68 (t, J = 5.7 Hz,1H), 7.63 (d, 1H), 7.56 (m, 1H), 7.54 - 7.43 (m, 3H), 7.54 - 7.43 (m, 3H),5.04 (d, J= 14.1 Hz, 1H), 4.85 (d, J = 4.3 Hz, 1H), 4.73 (d, J = 4.9 Hz, 1H), 4.60(t, J = 5.8 Hz, 1H), 4.37 (d, J = 5.3 Hz, 1H), 4.07 (d, J = 7.8 Hz, 1H), 3.70 (dt,J = 10.1, 5.9 Hz, 2H), 3.66 - 3.55 (m, 4H), 3.55 - 3.37 (m, 21H), 3.37 - 3.23(m, 3H), 3.19 (m, 1H), 3.10 (m, 1H), 2.93 (m, 1H), 2.43 (m, 1H), 2.32 (t, J =6.5 Hz, 2H), 2.17 (t, J = 6.5 Hz, 2H), 1.81 (m, 1H). 13 C NMR (151 MHz, DMSO- d 6): δ170.62, 170.57, 170.34, 151.87, 148.84, 132.85, 130.02, 129.43, 128.69,128.53, 128.20, 127.27, 125.68, 122.93, 121.88, 114.75, 108.54, 104.21,75.72, 73.73, 71.04, 70.22, 70.12, 70.08, 69.99, 69.91, 68.59, 68.22, 67.24,67.14, 60.94, 55.30, 40.54, 39.64, 39.20, 36.53, 36.41, 35.41, 34.63.; HRMS(ESI) m / z: [M+H] + The calculated value is C 40 H 56 N3O 14 802.3762; measured value is 802.3749.

[0135] Step 3: Synthesis of Gal-DBCO-PEG2000-DSPE (Compound 3) DSPE-PEG2000-azide (purchased from Avanti Polar Lipids, compound 1e, 6.51 mg, 2.65 μmol) was added to a 10 mL flask and dissolved in DMSO- d 6 (0.15 mL). Then, Gal-PEG5-DBCO (compound 3c, 8.60 mg, 10.2 μmol) was dissolved alone in DMSO- d 6 (0.1 mL) was then injected into the reaction flask solution, and after stirring at room temperature for 5 hours, Gal-PEG5-DBCO (compound 3c, 2.70 mg, 3.19 μmol) was dissolved in DMSO- d 6 (0.1 mL) was injected into the reaction solution, and then stirred at room temperature for 18 hours and concentrated. The crude reaction product was purified in methanol using a SEC column (model: Sephadex LH-20, H 26cm × OD 1cm, manufacturer: GE Healthcare) to obtain Gal-DBCO-PEG2000-DSPE (compound 3, 7.01 mg, 2.25 μmol, 85%), as a white solid.

[0136] 1 H NMR (600 MHz, DMSO- d 6): δ8.10 - 7.75 (m, 1H), 7.76 - 7.03 (m, 10H), 5.91 (d, 16.9 Hz, 1H), 5.84 (d, 16.9 Hz, 1H), 5.03 (m, 1H), 4.85 (d, J = 3.9Hz, 1H), 4.72 (d, J = 5.3 Hz, 1H), 4.60 (q, J = 7.0 Hz, 1H), 4.48 (td, J = 16.5, 7.3 Hz, 1H), 4.37 (d, J = 4.5 Hz, 1H), 4.29 (td, J = 12.7, 3.5 Hz, 1H), 4.20 -3.97 (m, 5H), 3.96 - 2.86 (m, 298H), 2.38 - 2.09 (m, 6H), 1.89 (m, 1H), 1.49(d, J = 7.1 Hz, 5H), 1.23 (s, 56H), 0.85 (t, J= 6.9 Hz, 6H). MS (MALDI-TOF, DHB matrix) m / z: [M] - The calculated value is C 172 H 315 N7O 68 P - 3598.1; the measured value is 3598.0.

[0137] Example 4. Preparation of GalNAc-DBCO-PEG2000-DSPE (Compound 4)

[0138] Step 1: Synthesis of 2-aminoethyl-2-acetamido-2-deoxy-β-D-galactopyranoside (compound 4b) 2-Azideethyl-2-acetamido-2-deoxy-β-D-galactopyranoside (purchased from Synthose, 21.2 mg, 69.1 μmol) was added to a 10 mL sealable flask, dissolved in methanol (1.0 mL), and then Pd / C was added. The reaction solution was saturated with hydrogen and stirred at room temperature under hydrogen atmosphere for 2 hours. The flask containing the mixture was passed through a diatomaceous earth filter to remove Pd / C. The reaction product was extracted with methanol and dried under vacuum to obtain 2-aminoethyl-2-acetamido-2-deoxy-β-D-galactopyranoside (compound 4b, 20.5 mg, 77.6 μmol, 100%), a transparent solid. Compound 4b was used directly in the next reaction without further purification.

[0139] 1 H NMR (600 MHz, D2O): δ4.46 (d, J = 8.0 Hz, 1H), 3.94 - 3.90 (m, 3H), 3.84 -3.65 (m, 5H), 2.91 - 2.7 (td, J = 5.7, 4.3 Hz, 2H), 2.04 (s, 3H); 13 C NMR (151 MHz, D2O): δ174.91, 101.83, 75.08, 70.91, 70.62, 67.76, 60.99, 52.47,40.00, 22.16. HRMS (ESI) m / z: [MH] - The calculated value is C 10 H 19 N2O6263.1243; measured value 263.1242.

[0140] Step 2: Synthesis of GalNAc-PEG5-DBCO (compound 4c) 2-Aminoethyl-2-acetamido-2-deoxy-β-D-galactopyranoside (compound 4b, 11.5 mg, 47.8 μmol) and DBCO-PEG5-N-hydroxysuccinimide (NHS) ester (purchased from Click Chemistry Tools, compound 1c, 26.2 mg, 37.0 μmol) were added to a 10 mL flask and dissolved in DMSO. d 6. After adding 0.5 mL of the solution, inject 0.06 mL of DIPEA. Then, stir the flask at room temperature for 5 hours and concentrate. The crude reaction product was purified by reversed-phase C18 silica gel chromatography (eluting with MeOH containing 35% H2O) to obtain GalNAc-PEG5-DBCO (compound 4c, 8.32 mg, 7.77 μmol, 21%), which is a pale orange liquid.

[0141] 1 H NMR (600 MHz, DMSO- d 6): δ7.75 (t, J = 5.6 Hz, 1H), 7.72 - 7.57 (m,4H), 7.55 - 7.43 (m, 3H), 7.43 - 7.28 (m, 3H), 5.05 (d, J = 14.1 Hz, 1H), 4.63(d, J = 47.3 Hz, 3H), 4.27 (d, J = 8.4 Hz, 1H), 3.71 (dt, J = 10.5, 8.6 Hz, 1H), 3.68 - 3.27 (m, 35H), 3.27 - 3.03 (m, 3H), 2.94 (ddt, J = 13.9, 8.6, 5.8 Hz,1H), 2.44 (ddd, J = 15.4, 8.5, 6.3 Hz, 1H), 2.32 (t, J = 6.5 Hz, 2H), 2.17 (t, J =6.5 Hz, 2H), 1.89 - 1.74 (m, 4H). 13 C NMR (151 MHz, DMSO- d6): δ170.61, 170.60,170.34, 170.26, 151.87, 148.84, 132.85, 130.02, 129.43, 128.69, 128.53,128.20, 127.27, 125.68, 122.93, 121.88, 114.75, 108.54, 101.78, 75.86, 72.22,70.22, 70.11, 70.08, 69.97, 69.91, 67.91, 67.22, 67.14, 60.96, 55.30, 52.47,40.53, 39.80, 39.16, 36.48, 36.41, 35.41, 34.63, 23.53.; HRMS (ESI) m / z: [M+H] + The calculated value is C 42 H 59 N4O 14 843.4028; the measured value is 843.3998.

[0142] Step 3: Synthesis of GalNAc-DBCO-PEG2000-DSPE (Compound 4) DSPE-PEG2000-azide (purchased from Avanti Polar Lipids, compound 1e, 6.43 mg, 2.24 μmol) was added to a 10 mL flask and dissolved in DMSO- d 6 (0.15 mL). Then, GalNAc-PEG5-DBCO (compound 4c, 6.94 mg, 7.91 μmol) was dissolved alone in DMSO- d 6 (0.1 mL) was then injected into the reaction flask solution, and after stirring at room temperature for 5 hours, GalNAc-PEG5-DBCO (compound 4c, 1.28 mg, 1.44 μmol) was dissolved in DMSO- d 6 (0.1 mL) was injected into the reaction solution, and then stirred at room temperature for 18 hours and concentrated. The crude reaction product was purified in methanol using a SEC column (model: Sephadex LH-20, H 26cm × OD 1cm, manufacturer: GE Healthcare) to obtain GalNAc-DBCO-PEG2000-DSPE (compound 4, 7.56 mg, 2.06 μmol, 92%), as a white solid.

[0143] 1 H NMR (600 MHz, DMSO- d6): δ7.98 - 7.11 (m, 12H), 5.87 (dd, J = 38.2, 16.9 Hz, 1H), 5.00 (d, J = 39.2 Hz, 3H), 4.58 (s, 1H), 4.48 (td, J = 16.2, 7.2Hz, 2H), 4.41 - 4.22 (m, 3H), 4.14 - 3.96 (m, 4H), 3.87 - 2.82 (m, 253H), 2.26 (ddt, J = 31.3, 26.3, 6.5 Hz, 8H), 1.81 (s, 4H), 1.62 - 1.40 (m, 5H), 1.23(s, 55H), 0.85 (t, J = 6.9 Hz, 6H). MS (MALDI-TOF, DHB matrix) m / z: [M] - The calculated value is C 174 H 318 N8O 68 P - 3639.1; the measured value is 3639.1.

[0144] Example 5. Preparation of Man-DBCO-PEG2000-DSPE (Compound 5)

[0145] Step 1: Synthesis of 2-aminoethyl-α-D-mannopyranoside (compound 5b) 2-Azideethyl-α-D-mannopyranoside (purchased from Synthose, 39.8 mg, 0.152 mmol) was added to a 10 mL sealable flask, dissolved in methanol (1.0 mL), and then Pd / C was added. The reaction solution was saturated with hydrogen and stirred at room temperature under hydrogen atmosphere for 2 hours. The flask containing the mixed solution was passed through a diatomaceous earth filter to remove Pd / C. The reaction product was extracted with methanol and dried under vacuum to obtain 2-aminoethyl-α-D-mannopyranoside (compound 5b, 37.4 mg, 0.167 mmol, 100%) as a transparent solid. Compound 5b was used directly in the next step of the reaction without further purification.

[0146] 1 H NMR (600 MHz, D2O): δ4.88 (d, J= 8.0 Hz, 1H), 3.97 (m, 1H), 3.92 -3.74 (m, 5H), 3.65 (m, 1H), 3.63 (m, 1H), 2.83 - 2.92 (m, 2H); 13 C NMR (151MHz, D2O): δ99.92, 72.80, 70.56, 69.97, 68.35, 66.77, 60.90, 39.83.; HRMS(ESI) m / z: [M+H] + The calculated value is C8H 18 NO6224.1134; Measured value is 224.1136.

[0147] Step 2: Synthesis of Man-PEG5-DBCO (compound 5c) 2-Aminoethyl-α-D-mannopyranoside (compound 5b, 11.8 mg, 50.1 μmol) and DBCO-PEG5-N-hydroxysuccinimide (NHS) ester (purchased from Click Chemistry Tools, compound 1c, 29.2 mg, 41.2 μmol) were added to a 10 mL flask and dissolved in DMSO. d 6. After adding 0.5 mL of the solution, inject 0.06 mL of DIPEA. Then, stir the flask at room temperature for 5 hours and concentrate. The crude reaction product was purified by reversed-phase C18 silica gel chromatography (eluting with MeOH containing 35% H2O) to obtain Man-PEG5-DBCO (compound 5c, 13.0 mg, 16.1 μmol, 39%), which is a pale orange liquid.

[0148] 1 H NMR (600 MHz, DMSO- d 6): δ7.89 (t, J = 5.6 Hz, 1H), 7.68 (t, J = 5.7 Hz,1H), 7.65 - 7.56 (m, 2H), 7.55 - 7.27 (m, 6H), 5.04 (d, J = 14.0 Hz, 1H), 4.71(dd, J = 14.3, 4.7 Hz, 2H), 4.64 - 4.51 (m, 2H), 4.46 (t, J = 5.9 Hz, 1H), 4.10(q, J= 5.2 Hz, 1H), 3.79 - 3.52 (m, 6H), 3.52 - 3.27 (m, 26H), 3.23 (ddt, J =18.8, 12.5, 6.0 Hz, 2H), 3.17 (d, J = 4.6 Hz, 3H), 3.10 (ddt, J = 14.0, 8.5, 6.1Hz, 1H), 2.93 (ddt, J = 14.0, 8.5, 5.8 Hz, 1H), 2.43 (ddd, J = 15.3, 8.5, 6.3 Hz,1H), 2.31 (t, J = 6.6 Hz, 2H), 2.17 (t, J = 6.5 Hz, 2H), 1.81 (ddd, J = 16.0, 8.5, 5.8 Hz, 1H). 13 C NMR (151 MHz, DMSO- d 6): δ170.62, 170.59, 170.35, 151.87,148.84, 132.86, 130.03, 129.44, 128.70, 128.54, 128.21, 127.28, 125.69,122.94, 121.88, 114.76, 108.54, 100.42, 74.47, 71.37, 70.71, 70.22, 70.12,70.09, 69.97, 69.92, 67.45, 67.27, 67.14, 65.86, 61.71, 55.30, 49.07, 38.87,36.51, 36.41, 35.41, 34.64.; HRMS (ESI) m / z: [M+H] + The calculated value is C 40 H 56 N3O 14 802.3762; measured value is 802.3747.

[0149] Step 3: Synthesis of Man-DBCO-PEG2000-DSPE (Compound 5) DSPE-PEG2000-azide (purchased from Avanti Polar Lipids, compound 1e, 6.31 mg, 2.20 μmol) was added to a 10 mL flask and dissolved in DMSO-d 6 (0.15 mL). Then, Man-PEG5-DBCO (compound 5c, 8.33 mg, 9.87 μmol) was dissolved alone in DMSO- d 6 (0.1 mL) was then injected into the reaction flask solution, and after stirring at room temperature for 5 hours, Man-PEG5-DBCO (compound 5c, 2.58 mg, 3.05 μmol) was dissolved in DMSO- d 6 (0.1 mL) was injected into the reaction solution, and then stirred at room temperature for 18 hours and concentrated. The crude reaction product was purified in methanol using a SEC column (model: Sephadex LH-20, H 26cm × OD 1cm, manufacturer: GE Healthcare) to obtain Man-DBCO-PEG2000-DSPE (compound 5, 7.08 mg, 1.96 μmol, 89%), as a white solid.

[0150] 1 H NMR (600 MHz, DMSO- d 6): δ7.92 - 7.84 (m, 1H), 7.71 - 7.20 (m, 10H), 5.87 (dd, J = 38.3, 16.9 Hz, 1H), 5.04 (td, J = 8.3, 5.2 Hz, 1H), 4.71 (dd, J =16.4, 4.8 Hz, 2H), 4.65 - 4.54 (m, 2H), 4.47 (dd, J = 17.2, 8.2 Hz, 3H), 4.28(dd, J = 12.0, 3.0 Hz, 1H), 4.13 - 3.98 (m, 4H), 2.32 (t, J = 6.6 Hz, 2H), 2.25(td, J = 7.4, 4.4 Hz, 4H), 2.21 (t, J = 6.5 Hz, 2H), 1.98 - 1.80 (m, 1H), 1.49(d, J = 7.1 Hz, 5H), 1.23 (s, 57H), 0.85 (t, J = 7.0 Hz, 6H). MS (MALDI-TOF, DHB matrix) m / z: [M] - The calculated value is C 172 H 315N7O 68 P - 3598.1; the measured value is 3598.0.

[0151] Example 6. Preparation of 2-bromoethyl-β-D-glucopyranoside (compound 11b)

[0152] Step 1: Synthesis of 2-bromoethyl-β-D-glucopyranoside (compound 11b) 2-Bromoethyl-2,3,4,6-tetra-O-acetate-β-D-glucopyranoside (Angene, 0.0824 g, 0.172 mmol) was added to a 10 mL sealable flask and dissolved in anhydrous methanol (1.6 mL). After adding 0.2 mL of 0.5 N sodium methoxide (NaOMe) solution (0.100 mmol), the reaction solution was stirred under argon for 20 minutes. The mixture was then passed through Amberlite IR-120 ion exchange resin (Sigma-Aldrich, H...). + After concentration at the resin chromatography column, 2-bromoethyl-β-D-glucopyranoside (compound 11b, 0.0482 g, 0.168 mmol, 98%) was obtained as a transparent solid.

[0153] 1 H NMR (400 MHz, MeOD): δ 4.33 (d, J = 7.7 Hz, 1H), 4.12 (ddd, J = 11.2,7.0, 6.1 Hz, 1H), 3.91 (ddd, J = 11.2, 7.0, 6.1 Hz, 1H), 3.87 (d, J = 11.9 Hz, 1H), 3.66 (dd, J = 12.1, 3.9 Hz, 1H), 3.56 (m, 2H), 3.40 - 3.25 (m, 3H), 3.19(dd, J = 9.0, 7.8 Hz, 1H). 13 C NMR (101 MHz, MeOD): δ103.12, 76.64, 76.57, 73.59,70.14, 69.51, 61.31, 29.56.; HRMS (ESI) m / z: [M+Na] + The calculated value is C8H 16BrO6Na3 08.9950; measured value 308.9944.

[0154] Example 7. Preparation of Glu-DSPC (Compound 11)

[0155] Step 1: Synthesis of N,N-dimethylamine-DSPE (compound 11d) In a 10 mL sealable flask, DSPE (0.0491 g, 0.0625 mmol, purchased from Avanti Polar Lipids) was dissolved in acetonitrile (0.5 mL), followed by the addition of formaldehyde (0.45 mL, purchased from Sigma-Aldrich). The reaction solution was stirred in an oil bath at 50 °C for 10 minutes under argon atmosphere. Sodium cyanoborohydride (NaCNBH3, purchased from Sigma-Aldrich) solid (0.0312 g, 0.472 mmol) was added. The reaction solution was stirred in an oil bath at 50 °C for 1 hour under argon atmosphere. The mixture was filtered through diatomaceous earth, washed with chloroform, and concentrated. The residue was recrystallized from acetone to give N,N-dimethylamine-DSPE (compound 11d, 0.03404 g, 0.0439 mmol, 70%) as a white solid.

[0156] 1 H NMR (400 MHz, CDCl3): δ 5.21 (m, 1H), 4.37 (dd, J = 12.1, 3.0 Hz,1H), 4.28 - 4.02 (m, 3H), 3.94 (t, J = 6.6 Hz, 2H), 3.01 (t, J = 6.6 Hz, 2H),2.66 (s, 6H), 2.27 (t, J = 7.8 Hz, 4H), 1.57 (qu, 4H), 1.24 (m, 56H), 0.87 (t, J = 6.8 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ173.47, 173.17, 70.50, 63.67, 62.80,58.45, 44.21, 34.31, 34.11, 31.93, 29.75, 29.73, 29.68, 29.61, 29.59, 29.42,29.38, 29.23, 29.20, 24.97, 24.89, 22.69, 14.12.; HRMS (ESI) m / z: [M] - The calculated value is C 43 H 85 NO8P - 774.6013; the measured value is 774.6013.

[0157] Step 2: Synthesis of Glu-DSPC (Compound 11) In a 10 mL sealable flask, compounds 11b (0.0213 g, 0.0706 mmol) and 11d (0.0185 g, 0.0226 mmol) were dissolved in acetonitrile (0.5 mL), followed by the addition of DIPEA (0.02 mL, purchased from Sigma-Aldrich). The reaction solution was stirred in an oil bath at 65 °C for 42 hours under argon atmosphere. The reaction mixture was recrystallized in acetone to give Glu-DSPC (compound 11, 4.21 mg, 4.29 μmol, 20%), a white solid.

[0158] 1 H NMR (500 MHz, CDCl3 / MeOD): δ5.20 (m, 1H), 4.37 (dd, J = 12.1, 3.0 Hz,1H), 4.32 - 3.79 (m, 10H), 3.68 (m, 2H), 3.43-3.01 (m, 12H), 2.29 (td, J = 9.2,5.1 Hz, 4H), 1.57 (q, 4H), 1.24 (m, 56H), 0.85 (t, J = 6.8 Hz, 6H).; HRMS (ESI)m / z: [M+H] + The calculated value is C 51 H 101 NO 14 P + 982.6960; the measured value is 982.6962.

[0159] Example 8.4 Preparation of 4-Nitrophenyl Carbonate-Ethylethoxy-PEG2000-DMG (Compound 43g)

[0160] Step 1: Synthesis of N-Boc-PEG2000-methoxyepoxide (compound 43b) N-Boc-PEG2000 (purchased from BroadPharm, 0.418 g, 0.188 mmol) was added to a 10 mL sealable flask and dissolved in toluene (4.0 mL). Sodium hydroxide (NaOH) solid (0.230 g, 5.64 mmol) was ground in a mortar and added, followed by epichlorohydrin (purchased from Sigma-Aldrich, 0.30 mL). The reaction solution was stirred in an oil bath at 50 °C for 14 hours under argon atmosphere. The mixture was diluted with DCM, the organic layer was washed with brine, dried over sodium sulfate, and concentrated. The residue was purified by silica gel chromatography (DCM / MeOH = 13:1) to give N-Boc-PEG2000-methoxyepoxide (compound 43b, 0.310 g, 0.140 mmol, 75%) as a white solid.

[0161] 1 H NMR (600 MHz, CDCl3): δ5.08 (s, 1H), 3.91 - 3.04 (m, 193H), 2.82(dd, J = 5.0, 4.1 Hz, 1H), 2.63 (dd, J = 5.1 (2.7 Hz, 1H), 1.47 (s, 9H).; MS (MALDI-TOF, DHB matrix) m / z: [M+H] + The calculated value is C 98 H 195 NO 48 2154.3; the measured value is 2154.3.

[0162] Step 2: Synthesis of N-Boc-PEG2000-dihydroxypropoxy (compound 43c) In a 10 mL sealable flask, N-Boc-PEG2000-methoxyepoxide (compound 43b, 0.286 g, 0.127 mmol) was dissolved in H2O (2.0 mL), followed by the addition of 1 N NaOH (4.0 mL). The reaction solution was stirred at room temperature for 14 hours. The mixture was concentrated, dissolved in a small amount of DCM, precipitated in excess diethyl ether, and filtered. The resulting solid was washed several times with diethyl ether, the filtrate was removed from the flask, and the supernatant solid was dissolved in DCM and transferred to a new flask to obtain N-Boc-PEG2000-dihydroxypropoxy (compound 43c, 0.249 g, 0.112 mmol), a white solid. Compound 43c was used in the next step without further purification. MS (MALDI-TOF, DHB matrix) m / z: [M+H] + The calculated value is C 98 H 197 NO 49 2172.3; the measured value is 2172.2.

[0163] Step 3: Synthesis of N-Boc-PEG2000-DMG (compound 43d) N-Boc-PEG2000-dihydroxypropoxy (compound 43c, 0.248 g, 0.108 mmol), myristic acid (purchased from TCI, 0.202 g, 0.865 mmol), and 4-dimethylaminopyridine (0.0215 g, 0.161 mmol) were added to a 10 mL sealable flask and dissolved in DCM (1.0 mL). The mixture was cooled to 0 °C in an ice bath, and EDC (0.169 g, 0.865 mmol) was added. The reaction solution was slowly heated under argon atmosphere and stirred at room temperature for 14 hours. The mixture was concentrated and purified by silica gel chromatography (DCM / MeOH = 13:1), and then purified in DMF using an SEC column (model: Bio Beads S-X1, H 42cm × OD 3.0cm, manufacturer: BioRad) to obtain N-Boc-PEG2000-DMG (compound 43d, 0.133g, 0.0497mmol, 41%), as a white solid.

[0164] 1 H NMR (600 MHz, CDCl3): δ5.19 (m, 1H), 4.35 (dd, J = 11.9, 3.4 Hz, 1H), 4.15 (dd, J= 11.9, 6.8 Hz, 1H), 3.92 - 3.09 (m, 204H), 2.31 (td, J = 7.4, 3.9Hz, 4H), 1.59 (m, 4H), 1.41 (s, 9H), 1.31 (m, 40H), 0.88 (m, 6H).; MS (MALDI-TOF, DHB matrix) m / z: [M+H] + The calculated value is C 126 H 249 NO 51 2592.7; the measured value is 2592.6.

[0165] Step 4: Synthesis of amino-PEG2000-DMG (compound 43e) In a 10 mL sealable flask, N-Boc-PEG2000-DMG (compound 43d, 0.132 g, 0.0510 mmol) was dissolved in DCM (0.2 mL), followed by the addition of TFA (0.8 mL). The reaction solution was stirred at room temperature under argon atmosphere for 1 hour. The mixture was concentrated and purified by silica gel chromatography (DCM / MeOH / NH4OH = 7:1:0.5), and further purified in DMF using an SEC column (model: BioBeads S-X1, H 42 cm × OD 3.0 cm, manufacturer: BioRad) to obtain amine-PEG2000-DMG (compound 43e, 0.0850 g, 0.0347 mmol, 69%) as a white solid.

[0166] 1 H NMR (600 MHz, CDCl3): δ7.94 (s, 1H), 5.23 (m, 1H), 4.36 (dd, J =11.9, 3.4 Hz, 1H), 4.17 (dd, J = 11.9, 6.8 Hz, 1H), 3.92 - 3.09 (m, 183fH), 2.32 (td, J = 7.4, 3.9 Hz, 4H), 1.63 (m, 4H), 1.28 (m, 40H), 0.90 (m, 6H).; MS (MALDI-TOF, DHB matrix) m / z: [M+H] + The calculated value is C 123 H 242 F3NO 51 2606.6; the measured value is 2606.5.

[0167] Step 5: Synthesis of hydroxy-ethylethoxy-PEG2000-DMG (compound 43f) In a 10 mL sealable flask, amine-PEG2000-DMG (compound 43e, 36.1 mg, 0.0141 mmol) and hydroxy-PEG1-acid (purchased from Broadpharm, 4.42 mg, 0.0320 mmol) were dissolved in DMF (1.0 mL), followed by the addition of DIPEA (0.04 mL) and PyBOP (11.1 mg, 0.0208 mmol). The reaction solution was stirred at room temperature under argon atmosphere for 15 hours. The mixture was purified in DMF using an SEC column (model: Bio Beads S-X1, H 42 cm × OD 3.0 cm, manufacturer: BioRad) to obtain hydroxy-ethylethoxy-PEG2000-DMG (compound 43f, 33.4 mg, 0.0130 mmol, 92%) as a white solid.

[0168] 1 H NMR (600 MHz, DMSO- d 6): δ7.89 (s, 1H), 5.10 (m, 1H), 4.56 (t, J = 5.5Hz, 1H), 4.26 (dd, J = 12.0, 3.1 Hz, 1H), 4.08 (dd, J = 12.0, 7.0 Hz, 1H), 3.78 -3.05 (m, 206H), 2.31 (t, J = 6.2 Hz, 2H), 2.25 (td, J = 7.4, 3.9 Hz, 4H), 1.50(m, 4H), 1.23 (m, 40H), 0.85 (m, 6H).; MS (MALDI-TOF, DHB matrix) m / z: [M+H] + The calculated value is C 126 H 249 NO 52 2608.7; the measured value is 2608.7.

[0169] Step 6: Synthesis of 4-nitrophenyl carbonate-ethylethoxy-PEG2000-DMG (compound 43g) In a 10 mL sealable flask, hydroxy-ethylethoxy-PEG2000-DMG (compound 43f, 32.5 mg, 0.0120 mmol) and p-nitrophenyl chloroformate (Sigma-Aldrich, 34.5 mg, 0.164 mmol) were dissolved in THF (1.0 mL), followed by the addition of DIPEA (0.03 mL). The reaction solution was stirred at room temperature under argon atmosphere for 15 hours. The mixture was purified in DMF using an SEC column (Bio Beads S-X1, H 42 cm × OD 3.0 cm, BioRad) to give 4-nitrophenyl carbonate-ethylethoxy-PEG2000-DMG (compound 43 g, 29.6 mg, 0.0108 mmol, 68%) as a white solid. Compound 43 g was used in the next step without further purification. MS (MALDI-TOF, DHB matrix) m / z: [M+H] + The calculated value is C 133 H 252 N2O 56 2773.7; the measured value is 2773.6.

[0170] Example 9. Preparation of Glu-carbamate-ethylethoxy-PEG2000-DMG (compound 43)

[0171] Step 1: Synthesis of Glu-carbamate-ethylethoxy-PEG2000-DMG (Compound 43) In a 10 mL sealable flask, 4-nitrophenyl carbonate-ethylethoxy-PEG2000-DMG (compound 43 g, 15.0 mg, 4.11 μmol) and compound 1b (4.06 mg, 17.3 μmol) were dissolved in DMSO. d Add 0.3 mL of DIPEA (0.01 mL) after 6. Stir the reaction solution at room temperature under argon for 15 hours. After concentrating the mixture, purify it by silica gel chromatography (DCM / MeOH / H2O=6:1:0.1) and then purify it in DMF using an SEC column (model: Bio Beads S-X1, H 42cm×OD3.0cm, manufacturer: BioRad) to obtain Glu-carbamate-ethylethoxy-PEG2000-DMG (compound 43, 6.28 mg, 2.23 μmol, 54%) as a white solid.

[0172] 1 H NMR (600 MHz, DMSO- d 6): δ 7.90 (t,J = 5.6 Hz, 1H), 7.20 (t, J = 6.0Hz, 1H), 5.10 (m, 1H), 4.99 (d, J = 4.1 Hz, 1H), 4.92 (d, J = 4.8 Hz, 1H), 4.90(d, J = 5.1 Hz, 1H), 4.48 (t, J = 5.9 Hz, 1H), 4.26 (dd, J = 12.0, 3.2 Hz, 1H), 4.11 (d, J = 7.8 Hz, 1H), 4.08 (dd, J = 12.0, 7.0 Hz, 1H), 4.05 - 4.00 (m, 2H), 3.89-2.76 (m, 216H), 2.36 - 2.22 (m, 6H), 1.50 (m, 4H), 1.23 (m, 40H), 0.85 (m, 6H). MS (MALDI-TOF, DHB matrix) m / z: [M+H] + The calculated value is C 135 H 264 N2O 59 2857.8; the measured value is 2857.7.

[0173] Example 10. Preparation of GlcNAc-carbamate-ethylethoxy-PEG2000-DMG (compound 44)

[0174] Step 1: Synthesis of GlcNAc-carbamate-ethylethoxy-PEG2000-DMG (Compound 44) In a 10 mL sealable flask, 4-nitrophenyl carbonate-ethylethoxy-PEG2000-DMG (compound 43 g, 14.1 mg, 3.88 μmol) and compound 2b (4.59 mg, 16.5 μmol) were dissolved in DMSO- dAdd 0.3 mL of DIPEA (0.01 mL) after 6. Stir the reaction solution at room temperature under argon for 15 hours. After concentrating the mixture, purify it by silica gel chromatography (DCM / MeOH / H2O=6:1:0.1) and then purify it in DMF using an SEC column (model: Bio Beads S-X1, H 42cm×OD3.0cm, manufacturer: BioRad) to obtain GlcNAc-carbamate-ethylethoxy-PEG2000-DMG (compound 44, 4.11 mg, 1.44 μmol, 35%) as a white solid.

[0175] 1 H NMR (600 MHz, DMSO- d 6): δ 7.91 (t, J = 5.6 Hz, 1H), 7.67 (d, J = 8.9Hz, 1H), 6.99 (t, J = 6.0 Hz, 1H), 5.10 (m, 1H), 4.99 (d, J = 4.6 Hz, 1H), 4.92(d, J = 5.3 Hz, 1H), 4.53 (t, J = 5.9 Hz, 1H), 4.30 (d, J = 8.4 Hz, 1H), 4.26 (dd, J = 12.0, 3.1 Hz, 1H), 4.08 (dd, J = 12.0, 7.0 Hz, 1H), 4.05 - 4.00 (m, 2H), 3.89-2.76 (m, 216H), 2.36 - 2.22 (m, 6H), 1.81 (s, 3H), 1.50 (m, 4H), 1.23 (m, 40H), 0.85 (m, 6H). MS (MALDI-TOF, DHB matrix) m / z: [M+H] + The calculated value is C 137 H 267 N3O 59 2898.8; the measured value is 2898.7.

[0176] Example 11. Preparation of Gal-carbamate-ethylethoxy-PEG2000-DMG (Compound 45)

[0177] Step 1: Synthesis of Gal-carbamate-ethylethoxy-PEG2000-DMG (Compound 45) In a 10 mL sealable flask, 4-nitrophenyl carbonate-ethylethoxy-PEG2000-DMG (compound 43 g, 14.3 mg, 3.92 μmol) and compound 3b (4.41 mg, 18.7 μmol) were dissolved in DMSO- d Add 0.3 mL of DIPEA (0.01 mL) after 6. Stir the reaction solution at room temperature under argon for 15 hours. After concentrating the mixture, purify it by silica gel chromatography (DCM / MeOH / H2O=6:1:0.1) and then purify it in DMF using an SEC column (model: Bio Beads S-X1, H 42cm×OD3.0cm, manufacturer: BioRad) to obtain Gal-carbamate-ethylethoxy-PEG2000-DMG (compound 45, 6.15 mg, 2.19 μmol, 52%) as a white solid.

[0178] 1 H NMR (600 MHz, DMSO- d 6): δ 7.90 (t, J = 5.6 Hz, 1H), 7.19 (t, J = 6.0Hz, 1H), 5.10 (m, 1H), 4.86 (d, J = 4.1 Hz, 1H), 4.71 (d, J = 4.8 Hz, 1H), 4.57(d, J = 5.1 Hz, 1H), 4.37 (t, J = 5.9 Hz, 1H), 4.26 (dd, J = 12.0, 3.2 Hz, 1H), 4.08 (dd, J = 12.0, 7.0 Hz, 1H), 4.06 (d, J = 7.8 Hz, 1H), 4.05 - 4.00 (m, 2H), 3.89-2.76 (m, 224H), 2.36 - 2.22 (m, 6H), 1.50 (m, 4H), 1.23 (m, 40H), 0.85(m, 6H).; MS (MALDI-TOF, DHB matrix) m / z: [M+H] + The calculated value is C 135 H 264 N2O 592857.8; the measured value is 2857.7.

[0179] Example 12. Preparation of GalNAc-carbamate-ethylethoxy-PEG2000-DMG (compound 46)

[0180] Step 1: Synthesis of GalNAc-carbamate-ethylethoxy-PEG2000-DMG (Compound 46) In a 10 mL sealable flask, 4-nitrophenyl carbonate-ethylethoxy-PEG2000-DMG (compound 43 g, 13.13 mg, 3.61 μmol) and compound 4b (4.40 mg, 15.8 μmol) were dissolved in DMSO- d Add 0.3 mL of DIPEA (0.01 mL) after 6. Stir the reaction solution at room temperature under argon for 15 hours. After concentrating the mixture, purify it by silica gel chromatography (DCM / MeOH / H2O=6:1:0.1) and then purify it in DMF using an SEC column (model: Bio Beads S-X1, H 42cm×OD3.0cm, manufacturer: BioRad) to obtain GalNAc-carbamate-ethylethoxy-PEG2000-DMG (compound 46, 5.39 mg, 1.89 μmol, 50%) as a white solid.

[0181] 1 H NMR (600 MHz, DMSO- d 6): δ 7.90 (t, J = 5.6 Hz, 1H), 7.63 (d, J = 8.8Hz, 1H), 6.98 (t, J = 6.0 Hz, 1H), 5.10 (m, 1H), 4.59 (s, 2H), 4.50 (s, 1H), 4.25 (d, J = 8.6 Hz, 1H), 4.25 (dd, J = 12.0, 3.1 Hz, 1H), 4.08 (dd, J= 12.0, 7.0Hz, 1H), 4.05 - 4.00 (m, 2H), 3.89-2.76 (m, 210H), 2.36 - 2.22 (m, 6H), 1.81(s, 3H), 1.50 (m, 4H), 1.23 (m, 40H), 0.85 (m, 6H).; MS (MALDI-TOF, DHB matrix) m / z: [M+H] + The calculated value is C 137 H 267 N3O 59 2898.8; the measured value is 2898.8.

[0182] Example 13. Preparation of Man-carbamate-ethylethoxy-PEG2000-DMG (Compound 47)

[0183] Step 1: Synthesis of Man-carbamate-ethylethoxy-PEG2000-DMG (Compound 47) In a 10 mL sealable flask, 4-nitrophenyl carbonate-ethylethoxy-PEG2000-DMG (compound 43 g, 14.0 mg, 3.85 μmol) and compound 5b (4.65 mg, 19.7 μmol) were dissolved in DMSO. d Add 0.3 mL of DIPEA (0.01 mL) after 6. Stir the reaction solution at room temperature under argon for 15 hours. After concentrating the mixture, purify it by silica gel chromatography (DCM / MeOH / H2O=6:1:0.1) and then purify it in DMF using an SEC column (model: Bio Beads S-X1, H 42cm×OD3.0cm, manufacturer: BioRad) to obtain Man-carbamate-ethylethoxy-PEG2000-DMG (compound 47, 6.43 mg, 2.28 μmol, 56%) as a white solid.

[0184] 1 H NMR (600 MHz, DMSO- d 6): δ 7.90 (t, J = 5.6 Hz, 1H), 7.20 (t, J = 6.0Hz, 1H), 5.10 (m, 1H), 4.99 (d, J = 4.1 Hz, 1H), 4.92 (d, J = 4.8 Hz, 1H), 4.90(d, J= 5.1 Hz, 1H), 4.48 (t, J = 5.9 Hz, 1H), 4.26 (dd, J = 12.0, 3.2 Hz, 1H), 4.11 (d, J = 7.8 Hz, 1H), 4.08 (dd, J = 12.0, 7.0 Hz, 1H), 4.05 - 4.00 (m, 2H), 3.89-2.76 (m, 216H), 2.36 - 2.22 (m, 6H), 1.50 (m, 4H), 1.23 (m, 40H), 0.85 (m, 6H). MS (MALDI-TOF, DHB matrix) m / z: [M+H] + The calculated value is C 135 H 264 N2O 59 2857.8; the measured value is 2857.8.

[0185] Compounds 11-15, 27, and 28, as shown in Table 1 below, were synthesized using the same method as in Example 1, but with phospholipid derivatives instead of compounds 1c and 1e of Example 1.

[0186] Compounds 16-20, as shown in Table 1 below, were synthesized using the same method as in Example 1, but with various click reactions.

[0187] The same method as in Examples 6-7 was followed, but in step 1 of Example 6, other monosaccharides were used instead of compound 11a to synthesize compounds 12-15 as shown in Table 1 below.

[0188] Compounds 27-28 and 39-42, as shown in Table 1, were synthesized by following the same method as in Examples 6-7, but by linking various monosaccharides with simple chemical bonds.

[0189] Compounds 6-10, 21-26, and 29-33, as shown in Table 1 below, were synthesized by means of simple chemical bonds with compounds 1b, 2b, 3b, 4b to 5b used in Examples 1-5.

[0190] Compounds 34-38 and 48-55, as shown in Table 1 below, were synthesized using the same method as in Examples 1 or 8-13, but with branched-chain targeting ligands linked by click reaction and simple chemical bonds.

[0191] In compounds 1-55, n is an integer from 37 to 53, x is from 1 to 3, y is from 1 to 5, and z can be an integer from 1 to 12. Specifically, n is 45, x is 1, y is 2, and z is 3.

[0192] In addition, compounds 11-15, 27, 28 and 39-42, as lipid compounds represented by Formula 1, relate to modified phospholipid compounds, while compounds 1-10, 16-26, 29-38 and 43-55, as lipid compounds represented by Formula 1, relate to modified polyethylene glycol-lipids.

[0193] Table 1

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202] In Table 1, n is 45, x is 1, y is 2, and z is 3.

[0203] Example 14. Preparation of mRNA-lipid nanoparticles (LNP) The ionizable lipids synthesized or commercially available according to Examples 1-13 (SM-102 (Broadpharm, USA; Patent Document 3 [PCT / US2016 / 052352 (BENENARO, KE; KUMARASINGHE, ES; CORNEBISE, M.), 2016.09.16]), ALC-0315 (Broadpharm, USA; Patent Document 4 [PCT / US2016 / 029572 (TAM, Y.; HOPE, MJ; WEISSMAN, D.; PARDI, N.), 2016.04.27]), DLin-MC3-DMA (MC3; Broadpharm, USA; Patent Document 4 [PCT / US2016 / 029572 (TAM, Y.; HOPE, MJ; WEISSMAN, D.; PARDI, N.), 2016.04.27]) were used. The compounds (such as dpharm, USA), phospholipids (compounds 11-15, 27-28, 39-42, DSPC (Sigma-Aldrich, USA), cholesterol (Sigma-Aldrich, USA), and polyethylene glycol-modified lipids (compounds 1-10, 16-26, 29-38, 43-55, ALC-0159 (Broadpharm, USA; Patent Document 5 [PCT / US2015 / 034496 (ANSELL, SM; DU, X) 2015.06.05]), DMG-PEG (DMG-PEG2000; Sigma-Aldrich, USA)) are dissolved in an organic phase (ethanol). Prepare an aqueous phase by diluting 5-100 μg of mRNA (firefly luciferase mRNA, fLuc mRNA, enhanced green fluorescent protein mRNA, EGFP mRNA, etc.; TriLink Biotechnologies, USA) in 0.2-2.0 mL of 5-50 mM sodium acetate or 5-50 mM sodium citrate aqueous solution.

[0204] Through microfluidic mixing device (NanoAssemblr) ®Spark Precision Nanosystems (Canada) prepared mRNA-lipid nanoparticles (LNPs) by mixing an organic phase (ethanol) containing dissolved ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids with an aqueous phase (sodium acetate or sodium citrate aqueous solution) containing dissolved mRNA at an average flow rate of 25-250 μL / 10 s. Specifically, the component lipid compounds were dissolved in ethanol at a composition ratio (20-60):(0-25):(30-60):(0-10) (ionizable lipids: phospholipids: cholesterol: polyethylene glycol-modified lipids) (in molar amounts), and the first phospholipid and the first polyethylene glycol-modified lipid, which were linked to the targeting ligand, were mixed at 0.05-100 mol% of total phospholipids and total polyethylene glycol-modified lipids, respectively (see Table 2), where the sum of the composition ratios was 100. The weight ratio of ionizable lipids to mRNA was (4-30):1 by mixing the organic and aqueous phases, thereby preparing lipid nanoparticles. To remove ethanol from the prepared lipid nanoparticles encapsulated with mRNA and to adjust the pH of the lipid nanoparticle aqueous solution to a level similar to the in vivo pH (~7.4), a dialysis kit (Slide-A-Lyzer) was used. TM The dialysis kit (10K MWCO, Thermo Fisher Scientific, USA) was used, and the cells were dialyzed several times with PBS (phosphate-buffered saline, pH 7.4, Invitrogen, USA) for 12-18 hours. The expected structure of the prepared mRNA-lipid nanoparticles is as follows: Figure 1 As shown.

[0205] Figure 1 This is a schematic diagram of the structure of mRNA-lipid nanoparticles (LNPs) for tissue-specific delivery of active substances according to an embodiment of the present invention. A is an LNP without tissue-specific ligands, B is an LNP with only part of its surface modified by polyethylene glycol-modified lipids linked to tissue-specific ligands, C is an LNP with most of its surface modified by polyethylene glycol-modified lipids linked to tissue-specific ligands, D is an LNP with only part of its surface modified by phospholipids linked to tissue-specific ligands, and E is an LNP with most of its surface modified by phospholipids linked to tissue-specific ligands.

[0206] Experimental Example Experimental Example 1. Measurement of pKa of mRNA-LNP The apparent p-value of the lipid nanoparticles prepared in Example 14 was measured using analysis with sodium 6-(p-toluidine)-2-naphthalenesulfonate (TNS; Sigma-Aldrich, USA). K aAnionic TNS typically does not fluoresce in aqueous solution. However, as the pH decreases, the content of positively charged ionizable lipids increases, migrating to the hydrophobic (lipophilic) region via electrostatic interactions, thus gradually increasing the fluorescence intensity. Conversely, as the pH increases, the ionizable lipids are neutralized and cease electrostatic interactions with TNS. Consequently, TNS loses its lipophilicity and leaves the hydrophobic region, resulting in TNS fluorescence quenching. Based on these principles, the change in TNS fluorescence intensity with ambient pH can be plotted as an S-shaped curve, and the logarithm of the inflection point can be calculated to measure the pH of the lipid nanoparticles. K a The p-value of lipid nanoparticles K a The pH value is typically between 6.0 and 7.0. After intravenous injection, the surface charge in plasma (pH ~ 7.4) is close to 0, reducing the likelihood of interaction with in vivo tissues and thus increasing the rate at which it reaches hepatocytes. Once inside the cells, it carries a high charge in endosomal regions (pH 5.5), facilitating the endosomal escape of contained genes, proteins, and synthetic drugs awaiting delivery, resulting in excellent in vivo drug delivery efficiency. (Non-patent literature 1 [Sabnis, S. et al.]) Mol. Ther. 2018, 26

[1509] , Non-Patent Literature 2 [Jayaraman, M. et al.]. Angew. Chem. Int. Ed. 2012, 51 ,8529]).

[0207] Specifically, a master buffer containing 10 mM sodium phosphate, 10 mM sodium borate, 10 mM sodium citrate, and 150 mM sodium chloride (NaCl) was prepared, and the pH was adjusted between 2.5 and 12 using 1 M sodium hydroxide (NaOH) and 1 M hydrochloric acid (HCl) to prepare aqueous solutions with various pH values. Then, TNS was dissolved in DMSO to prepare 150–300 μM TNS standard stock solutions. LNPs were prepared at concentrations of 0.02–0.08 mg / mL mRNA in 1X PBS buffer or 20 mM Tris buffer containing 8% sucrose. Solutions of each pH value were added in 90–94 μL to three wells (n=3) of a 96-well plate with a black background. Add 3.26–4.00 μL of lipid nanoparticles and 2 μL of TNS solution to each well. After carefully mixing each well, measure the fluorescence intensity (λ) of each well using a multi-functional microplate reader (Cary Eclipse, Agilent Technologies, USA). ex 330nm, λ em 435nm).

[0208] Experimental Example 2. Measurement of size, polydispersity index, and surface charge of mRNA-LNP The mRNA contained in each LNP prepared in Example 14 was diluted with PBS to a concentration of 1 μg / mL. The size, polydispersity index (PDI), and surface charge of the LNPs were measured using a Malvern nanoparticle potentiometer ZS90 (Malvern Instruments, UK). The results are shown in Table 2. For particles administered to the systemic circulatory system, the size must be less than 300 nm to pass through the liver endothelium capillaries, and the PDI must be less than 0.3 to be considered as having a uniform particle size distribution, thus expecting consistent efficacy (Non-Patent Literature 3 [Danaei, M.; Dehghankhold, M.; Ataei, S.; Davarani, FH; Javanmard, R.; Dokhani, A.; Khorasani, S.; Mozafari, MR. Pharmaceutics 2018, 10 ,57]).

[0209] Experimental Example 3. Measurement of mRNA encapsulation efficiency Use RiboGreen ® RNA Detection Kit (Quant-iT™ RiboGreen) ® The encapsulation efficiency (EE), %) of LNPs containing mRNA was determined by Invitrogen, and the results are shown in Table 2. The lipid nanoparticles prepared in Example 14 were diluted to 50 μL with 1X TE (Tris-EDTA) buffer or 1X TE buffer containing 2% Triton X-100 to achieve a final mRNA concentration of 20-30 ng / mL, and then added to each well of a 96-well plate. Group A (without Triton X-100) received an additional 50 μL of 1X TE buffer, and Group B (with Triton X-100) received an additional 50 μL of 1X TE buffer containing 2% Triton X-100. The lipid nanoparticles were lysed with Triton X-100 and held at 37°C for 10 minutes to release the contained mRNA. Then, 100 μL of Ribogreen reagent was added to each well. The fluorescence intensity (λ) of groups A and B was measured using a multi-functional microplate reader. ex 480nm, λ em The mRNA encapsulation efficiency (%) was calculated using Formula 1 below (520 nm). The mRNA encapsulation efficiency (%) for each lipid nanoparticle was the average of two repeated measurements.

[0210] Formula 1 mRNA encapsulation efficiency (%) = [(Fluorescence intensity of group B - Fluorescence intensity of group A) / (Fluorescence intensity of group B)] × 100 Table 2

[0211] *The accessory lipids (phospholipids; accounting for 10 mol% of total lipids) consist of 9.9 mol% DSPC and 0.1 mol% sulfonated anthocyanin 5.5-DSPE.

[0212] Experimental Example 4. Evaluation of protein expression efficiency of EGFP mRNA-lipid nanoparticles applied to cells In this experimental example, several EGFP mRNAs encapsulated with LNPs prepared according to the method described in Example 14 were applied to three types of retinal cells (ARPE-19 human retinal pigment epithelial cells (ATCC, USA), 661W mouse retinal photoreceptor cells (provided by Professor Jinwoo Kim's laboratory at KAIST), and rMC-1 rat retinal Müllerian cells (Kerafast, USA)) to compare time-dependent in vitro ( in vitro Protein expression efficiency (related to transfection, endosomal escape, mRNA stability, etc.).

[0213] The lipid compounds constituting the LNP were cholesterol and ionizable lipids (MC3 or SM-102 were used). DSPC was used as the phospholipid, and 1 mol% of the total phospholipids consisted of sulfo-cyanine 5.5-DSPE, used for fluorescence microscopy tracking of the LNP. PEG-lipids, as shown in Table 1, were used in the control group (neutral DMG-PEG) and the experimental group (constituted with terminally modified lipid compounds 1-5 of a portion of DMG-PEG and a portion of anionic DSPE-PEG).

[0214] Specifically, 18 hours before administering mRNA-LNP, ARPE-19, 661W, and rMC-1 cells were inoculated at a concentration of 1×10⁻⁶ mRNA-LNP. 4 Up to 2×10 5 Cells were seeded at a density of 100 ng / well in 8-well plates (ibidi) and incubated at 37°C with 5% CO2. The mRNA-LNP concentration was adjusted to 100 ng / well based on the mRNA concentration, and cells were treated at 5 min, 10 min, 15 min, 30 min, 1 h, 3 h, 6 h, 24 h, and 48 h. After treatment, the culture medium was removed, and the cells were fixed with 4% PFA for 15 min. Images were then captured using a fluorescence microscope. Figure 7 As shown.

[0215] like Figure 7As shown, it was confirmed that green fluorescence from expressed GFP could be detected as early as 5 minutes after treatment of ARPE-19 cells with EGFP mRNA-LNP, and its intensity gradually increased over time. Furthermore, it was confirmed that the overall GFP expression rate (green fluorescence) was significantly increased when SM-102 was used instead of MC as the ionizable lipid; and this increase reached its maximum at 24 and 48 hours. Simultaneously, it was confirmed that in the results using the stronger signal of SM-102 as the ionizable lipid, when 80% of the PEG-lipids in the LNP component contained terminally modified compound 5 for tissue-specific delivery, the fluorescence intensity of GFP was stronger compared to the case where the PEG-lipids consisted only of unmodified DMG-PEG, especially at 24 and 48 hours.

[0216] Therefore, according to one embodiment of the present invention, when LNPs containing terminally modified lipids are applied to cells to impart tissue specificity, it can be seen that selective delivery into the cell interior is more active, and efficient protein expression in the cytoplasm is observed at a relatively early stage through smooth endosomal escape.

[0217] Experimental Example 5. Evaluation of protein expression efficiency of fLuc mRNA-lipid nanoparticles via subretinal injection. All the following animal experiments were reviewed and approved by the Animal Experimentation Ethics Committee of the Korea Institute of Biotechnology. In this example, a Hamilton injection syringe with a 33-gauge blunt needle was used to inject fLuc mRNA-LNP (100 ng - 1 mg mRNA, 300 ng / μL) containing a modified lipid compound subretinally (via the pars plana approach; Non-Patent Literature 4 [Huang, P. et al.]). Nature Protocols 2022, 17

[1468] In 6-8 week old female or male C57BL / 6J mice (Orient Bio) under respiratory anesthesia (3% isoflurane and 97% medical oxygen), to confirm protein expression rates at 24 and 48 hours, 200 μL (15 mg / mL) of luciferin was injected intraperitoneally 10 minutes before imaging, followed by respiratory anesthesia with 3% isoflurane and 97% medical oxygen. Luminescent images were acquired using a small animal imaging system (in vivo imaging system, Lumina III, PerkinElmer, Waltham, MA). Results are as follows: Figure 2 and Figure 4 As shown.

[0218] Cholesterol was used as the lipid compound constituting LNP, MC3 as the ionizable lipid, and DSPC as the phospholipid. PEG-lipids, as shown in Table 1, were used. The control group used neutral DMG-PEG or unmodified DSPE-PEG, with the same content (20 mol% and 80 mol%) of the unmodified DSPE-PEG as the experimental group. The experimental group was used to confirm the effect of anions in the terminally modified anionic DSPE-PEG. The experimental group consisted of terminally modified lipid compounds 1-5 of a portion of DMG-PEG and a portion of anionic DSPE-PEG.

[0219] Figure 2 These are IVIS images taken at 24 and 48 hours after subretinal injection of fLuc mRNA-LNP containing modified PEG-lipid compound 5 according to an embodiment of the present invention into the eye of mice. Compared with a control group containing the same amount of anion, it was confirmed that LNPs containing 20 mol% or 80 mol% of compound 5 achieved significantly higher levels of protein expression. Furthermore, it was observed that the signal was stronger at 24 hours than at 48 hours, and the protein expression rate was significantly higher when the total PEG-lipid contained more terminally modified PEG-lipid compound 5 (i.e., 80 mol% instead of 20 mol%). Therefore, it can be inferred that compound 5 facilitates more efficient and specific delivery to the retinal tissue at the injection site.

[0220] Figure 4The images are IVIS images taken 24 hours after intraocular injection of fLuc mRNA-LNPs containing 80 mol% (20 mol% DMG-PEG) of modified PEG-lipid compounds 1-5 according to an embodiment of the present invention via subretinal injection in mice. It was confirmed that the tissue-specific delivery efficiency of the retinal compounds varied depending on the type of monosaccharide used for terminal modification. Specifically, it was found that protein expression was relatively high when glucose (Glu) and mannose (Man) were used as monosaccharides, and also high when both mannose (Man) and N-acetylgalactosamine (GalNAc) were present in equal amounts, while protein expression was relatively low when only galactose (Gal) and N-acetylgalactosamine (GalNAc) were present. Therefore, by screening and optimizing ligands exhibiting tissue specificity, it is hoped that novel LNPs and their compositions exhibiting significantly superior active substance delivery effects compared to existing levels can be developed.

[0221] Experimental Example 6. Evaluation of protein expression efficiency of fLuc mRNA-lipid nanoparticles via intravitreal injection In this experimental example, fLuc mRNA-LNP (100 ng - 1 mg mRNA, 300 ng / μL) containing a modified lipid compound was injected intravitreally using a 33-gauge blunt-tipped Hamilton syringe (Non-Patent Literature 5 [You, S. et al.)). Biomaterials 2020, 255 [120188] In 6-8 week old female or male C57BL / 6J mice (Oriental Biotech) under respiratory anesthesia (3% isoflurane and 97% medical oxygen), to confirm protein expression rates at 24 and 48 hours, 200 μL (15 mg / mL) of luciferin was injected intraperitoneally 10 minutes before imaging. Respiratory anesthesia was then administered with 3% isoflurane and 97% medical oxygen. Luminescence images were acquired using a small animal imaging system (IVIS Lumina III, PerkinElmer, Waltham, MA). Results are as follows: Figure 3 and Figure 5 As shown.

[0222] The lipid compounds that constitute LNP are the same as those described in Experimental Example 5.

[0223] Figure 3These are IVIS images taken at 24 and 48 hours after intravitreal injection of fLuc mRNA-LNP containing a modified PEG-lipid compound 5 according to an embodiment of the present invention into the eye of mice. In the case of intravitreal injection, unlike subretinal injection, no signal was detected in the eye in the control group LNP composed of unmodified PEG-lipid compounds. However, in the LNP containing only 20 mol% of the modified PEG-lipid compound 5, the signal was very weak, but protein expression was observed in one of the two experimental animals. A relatively strong signal (protein expression) was observed when the LNP contained 80 mol%. Since protein expression increased with increasing content of tissue-specific modified lipid compound 5 in the LNP component, it is speculated that the ligand mannose (Man) linked to the PEG terminus affects the delivery efficiency. Furthermore, it was confirmed that the signal at 48 hours post-injection was significantly lower than at 24 hours post-injection.

[0224] Figure 5 These are IVIS images taken at 24-hour time points after intravitreal injection of fLuc mRNA-LNPs containing 80 mol% (20 mol% DMG-PEG) of modified PEG-lipid compounds 1-5 according to an embodiment of the present invention into the mouse eye. Unlike subretinal injection, most LNPs showed almost no protein expression, but LNPs containing compound 5 showed relatively significant protein expression at the 24-hour time point, and LNPs containing compound 4, although weaker, also showed protein expression. Therefore, it is speculated that LNPs containing compound 5 exhibit superior retinal tissue-specific delivery during intravitreal injection.

[0225] Compared to subretinal injection in Example 5, intravitreal injection is a very safe and convenient method of drug delivery. However, compared to subretinal injection, which involves direct injection into the outer retinal layer (RPE, photoreceptors) where the main pathological phenomena occur, the delivery efficiency is usually significantly lower. This is because during intravitreal injection, most of the substance cannot penetrate into the retina and is excreted. Even if some penetration occurs, it cannot reach the deeper outer layer and mostly remains in the medial ganglion cell layer. Therefore, it is necessary to develop a low-level intraretinal nucleus (LNP) with excellent retinal penetration and significantly high specificity for the outer retinal layer, so that it can effectively transfect into the surrounding RPE and photoreceptor cells upon reaching the outer layer. Therefore, through the optimization process of the LNP containing 80 mol% PEG-lipid content of Compound 5 according to the present invention, it is expected to achieve reusable administration and easy delivery of the active substance to the outer retinal layer via safe intravitreal injection.

[0226] Example 7. Evaluation of protein expression efficiency of fLuc mRNA-lipid nanoparticles via intramuscular injection (im) and intravenous injection (iv). In this experimental example, fLuc mRNA-LNP containing a modified lipid compound was prepared according to the method of Example 14 and administered via intramuscular injection (im) or intravenous injection (iv) to compare the resulting in vivo effects. in vivo Protein expression efficiency (related to transfection, endosomal escape, mRNA stability, etc.).

[0227] The dose of injected mRNA-LNP was adjusted to 0.1-0.5 mg / kg body weight based on mRNA and administered to female C57BL / 6J mice (Orient Bio) aged 6-10 weeks via intramuscular or intravenous injection. Then, at 1, 3, 6, 24, 48, and 72 hours post-injection, 200 μL of luciferin was injected intraperitoneally, and whole-body fluorescence images were acquired using an IVIS Lumina III (PerkinElmer, USA) device.

[0228] Figure 6The images show luminescence images taken at 1 hour, 3 hours, 6 hours, and 24 hours after mouse tail vein injection of fLuc mRNA-LNP containing 80 mol% (20 mol% DMG-PEG) of modified PEG-lipid compounds 1-5 according to an embodiment of the present invention. The mRNA-LNP containing compound 4 with GalNAc as a terminal modification ligand exhibited particularly strong protein expression efficiency in the liver, with a significantly higher signal intensity at 3 hours post-injection than at 1 hour. Subsequently, protein expression was also observed in the liver in mRNA-LNP containing only Man and in a 1:3 ratio of GalNAc / Man, but the signal was significantly lower than that containing GalNAc alone, and almost imperceptible at 1 hour, but reaching a detectable signal level at 3 hours. Therefore, it can be confirmed that when mRNA-LNP containing tissue-specific terminally modified PEG-lipid compounds is administered intravenously in vivo, it exhibits very strong specificity for liver tissue when GalNAc is used as a ligand. This tissue-specific delivery method of LNP holds promise for selective and effective treatment of liver-specific diseases.

[0229] To date, this invention has been studied focusing on its preferred embodiments. Those skilled in the art will understand that the invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustratively rather than restrictively. The scope of the invention is not limited to the foregoing description, but is particularly embodied in the claims, and all differences within the scope of its equivalents should be interpreted as included within the invention.

Claims

1. A lipid compound represented by the following chemical formula 1, its isomer, or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In chemical formula 1, T is the targeting ligand, which is selected from the group consisting of monosaccharides, polysaccharides, folic acid, transferrin, RGD peptide, cyclic RGD peptide, TAT peptide, R9 peptide, CATY peptide, HA2 peptide, monoclonal antibody, antigen-binding fragment or antibody fragment, single-chain variable fragment and aptamer, or is a branched-chain targeting ligand represented by the following chemical formula 2. X 1 X 2 X 3 and X 4 Each independently is selected from one or a combination of the following groups: single bond, C 1-14 Alkylene, C 1-14 Heteroalkylene, C 2-14 imidene group, C 1-6 Alkyl-C(=O)-, C 2-6 Alkenyl-C(=O)-, -(OCH2CH2) m -、-(CH2CH2O) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR)-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(= O)O-, -C(=O)C(=O)-, -C(=N-OR)-, -ON=CR-, -OC=NO-, -C(=O)NR-, -NRC(=O)-, -OC(=O) NR-, -NRC(=O)O-, -C(=O)NRC(=O)-, -S(=O)2NR-, -NRS(=O)2-, -NRC(=O)NR-, -NRC(=S) NR-, -C(=O)NH-N=CR-, -RC=N-NHC(=O)-, -NH-N=CR-, -RC=N-NH-, -NR-, -N(OR)-, -SS-, And functional groups that bond with each other through click reactions. in, m is an integer between 0 and 10. Z is -NH-, -O-, or -S-. d and e are each integers from 1 to 10; L 1 and L 2 Each is independently a single bond, C 1-14 Alkylene, C 2-14 sub-alkenyl, MR a M 1 Or R a MR b ; P is a single bond, -(CH2CH2O) q -、-(OCH2CH2) q -、-CH2O(CH2CH2O) q CH2-、-CH2CH2O(CH2CH2O) q CH2-, where q is 0 or an integer from 1 to 120; A is CR d Or N; R 1 and R 2 Each independently is C 1-30 Alkyl, C 2-30 alkenyl, or R c MR d , wherein, the C 1-30 Alkyl and C 2-30 The alkenyl groups are either independently unsubstituted or occupied by 1-3 carbons. 1-16 Alkyl or C 2-16 Alkenyl substitution; The M and M 1 Each independently is C 1-14 Alkylene, C 2-14 alkenyl, -NHC(=O)-, -C(=O)NH-, -C(=O)O-, -OC(=O)-, -C(=O)-, -NH-, -N + R2-, -S-, -SS-, -O-, -S(O)2-, -C(=O)S-, -SC(=O)-, -NHC(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, -OP(=O)(OR)O- or -OP(=O)(OR)O(CH2) g - where g is an integer from 1 to 10; R is hydrogen, C 1-20 Alkyl, C 2-20 alkenyl, C 2-20 alkynyl or C 1-20 Heteroalkyl, The R a and R b Each is independently a single key or C 1-10 Alkylene; The R c For single bond, C 1-14 Alkylene or C 2-14 alkenyl; The R d For hydrogen, C 1-18 Alkyl or C 2-18 alkenyl, wherein the C 1-18 Alkyl and C 2-18 The alkenyl groups are either independently unsubstituted or occupied by 1-3 carbons. 1-12 Alkyl or C 2-12 Alkenyl substitution; Each of the heteroalkyl, heteroalkylene, heterocyclic and heteroaryl groups independently comprises 1-6 heteroatoms selected from N, O and S; The branched-chain targeting ligand is represented by the following chemical formula 2: [Chemical Formula 2] In the chemical formula 2, a, b, and c are each independently 0 or 1, of which at least one of a, b, and c must be 1, and h and j are 1-3; T 1 T 2 and T 3 Each is independently selected from one of the following groups: monosaccharides, polysaccharides, folic acid, transferrin, RGD peptide, cyclic RGD peptide, TAT peptide, R9 peptide, CATY peptide, HA2 peptide, monoclonal antibody, antigen-binding fragment or antibody fragment, single-chain variable fragment, and aptamer; X 5 X 6 X 7 X 8 X 9 X 10 X 11 and X 12 Each independently is selected from one or a combination of the following groups: single bond, C 1-14 Alkylene, C 1-14 Heteroalkylene, C 2-14 imidene group, C 1-6 Alkyl-C(=O)-, C 2-6 Alkenyl-C(=O)-, -(OCH2CH2) m -、-(CH2CH2O) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR)-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(= O)O-, -C(=O)C(=O)-, -C(=N-OR)-, -ON=CR-, -OC=NO-, -C(=O)NR-, -NRC(=O)-, -OC(=O) NR-, -NRC(=O)O-, -C(=O)NRC(=O)-, -S(=O)2NR-, -NRS(=O)2-, -NRC(=O)NR-, -NRC(=S) NR-, -C(=O)NH-N=CR-, -RC=N-NHC(=O)-, -NH-N=CR-, -RC=N-NH-, -NR-, -N(OR)-, -SS-, And functional groups that bond with each other through click reactions. Where m is an integer from 0 to 10, Z is -NH-, -O-, or -S-. d and e are each integers from 1 to 10; L 3 L 4 L 5 L 6 and L 7 Each is independently a single bond, C 1-14 Alkylene, C 2-14 sub-alkenyl, MR a M 1 Or R a MR b ; A 1 A 2 and A 3 Each is independently a single bond, CR d Or N; Each of the heteroalkyl and heteroalkylene groups independently comprises 1 to 6 heteroatoms selected from N, O, and S.

2. The lipid compound, its isomer, or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The monosaccharide is selected from one of the following groups: glucose, N-acetyl-glucose, galactose, N-acetyl-galactose, mannose, allose, arabinose, cladinose, erythrose, erythritol, fructose, D-fucoitol, L-fucoitol, fucose, fucose, fucose, galactosamine, D-galactosamine, N-acetyl-galactosamine, glucosamine, N-acetyl-glucosamine, glucosamine alcohol, glucose-6 - Phosphate, glyceraldehyde, L-glycerol-D-mannose-heptanose, glycerol, glycerone, gulose, idose, lythose, mannosamine, mannosamine-6-phosphate, allulose, quinose, quinoside, rhamnitol, rhamnosamine, rhamnose, ribose, ribulose, sedoheptulose, sorbose, tagatose, tartaric acid, threose, xylose, xylulose, N,N,N-trimethyl-glucosamine and N,N,N-trimethyl-galactosamine, The polysaccharides include one selected from the group consisting of disaccharides, trisaccharides, and oligosaccharides, specifically one selected from the group consisting of: abicosyl, acarbose, amylose, amylopectin, amylose, celery sugar, acanose, artemisia sugar, ascorbic acid, boyrenose, cellobiose, cellotriose, cellulose, potato trisaccharide, charose, chitin, colistin, cyclodextrin, senna sugar, dextrin, 2-deoxyribose, 2-deoxyglucose, deoxydigitoxin, digitoxin, digitalis toxin sugar, iwasose, ioumose, fructooligosaccharides, galactooligosaccharides, gentiotriose, gentiobiose, glucan, glycogen, liver glycogen, witch hazel, heparin, inulin, and iso-L-glucan. Glucosamine, isomaltose, isomalttriose, isoglucosylmaltose, maltodextrose, lactose, lactosamine, lactosidamine, laminobiose, L-glucose, L-glucose-enolone, β-maltose, maltodextrose, mannan-oligosaccharide, mannotriose, melitriose, melibiose, muramic acid, carbamoylose, deoxyaloose, neuraminic acid, aspergillus niger, nojirimycin, novose, oleanolic acid, panose, porase, plantain, primrose, raffinose, rose syrup, rutinose, sarcopentose, sedoheptulose, solanine, sophorose, stachyose, streptose, sucrose, α,α-trehalose, trehalosamine, melitriose, tivasugar, xylobiose, and umbelliferous sugars.

3. The lipid compound, its isomer, or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The functional groups that are mutually bonded through the click reaction are , , , , , , .

4. The lipid compound, its isomer, or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The T, T 1 T 2 and T 3 Each of the following is independently selected from the group consisting of glucose, N-acetyl-glucosamine, galactose, N-acetylgalactosamine, and mannose.

5. The lipid compound, its isomer, or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that, The T, T 1 T 2 and T 3 It is N-acetylgalactosamine, mannose, or N-acetylglucosamine.

6. The lipid compound, its isomer, or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The lipid compound represented by the chemical formula 1 is any one of compounds 1 to 55 in Table 1.

7. The lipid compound, its isomer, or a pharmaceutically acceptable salt thereof according to claim 1. in, The lipid compounds are selected from the group consisting of: (1) Glu-DBCO-PEG2000-DSPE; (2)GlcNAc-DBCO-PEG2000-DSPE; (3) Gal-DBCO-PEG2000-DSPE; (4)GalNAc-DBCO-PEG2000-DSPE; (5) Man-DBCO-PEG2000-DSPE; (6) Glu-succinamide-PEG2000-DMG; (7) GlcNAc-succinamide-PEG2000-DMG; (8) Gal-succinamide-PEG2000-DMG; (9) GalNAc-succinamide-PEG2000-DMG; (10) Man-succinamide-PEG2000-DMG; (11)(R)-2,3-bis(stearoyloxy)propyl(2-(dimethyl(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)ammonyl)ethyl)phosphate; (12) 2-((2-(((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)dimethylammonium)ethyl((R)-2,3-bis(stearoyloxy)propyl)phosphate; (13)(R)-2,3-bis(stearoyloxy)propyl(2-(dimethyl(2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)ammonyl)ethyl)phosphate; (14) 2-((2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)dimethylammonium)ethyl((R)-2,3-bis(stearoyloxy)propyl)phosphate; (15)(R)-2,3-bis(stearoyloxy)propyl(2-(dimethyl(2-(((2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)ammonyl)ethyl)phosphate; (16)Man-1,2,3-triazole-PEG2000-DSPE; (17) Man-DIFO-PEG2000-DSPE; (18)Man-BCN-PEG2000-DSPE; (19) Man-triarylphosphine-PEG2000-DSPE; (20)Man-TCO-PEG2000-DSPE; (21) Man-succinamide-PEG2000-carbamate-DSPE; (22) Man-amide-PEG2000-amide-DSPE; (23) Man-valerate-PEG2000-amide-DSPE; (24) Man-succinamide-PEG2000-DMG; (25) Man-amide-PEG2000-DMG; (26) Man-valerate-PEG2000-DMG; (27)(R)-2,3-bis(stearoyloxy)propyl(2-(dimethyl(2-(4-oxo-4-((2-(((2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)amino)butamido)ethyl)ammono)ethyl)phosphate; (28)(R)-2,3-bis(stearoyloxy)propyl(3,3-dimethyl-10,13-dioxo-16-(((2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6-oxa-3,9,14-triazahexadecane-3-onthium-1-yl)phosphate; (29) Man-succinamide-PEG2000-DSG; (30) Man-succinamide-PEG2000-DOG; (31) Man-succinamide-PEG2000-DLG; (32)Man-PEG2000-(10Z,29Z)-tetradec-10,29-diene-20-yl; (33)Man-PEG2000-(7Z, 10Z, 29Z, 32Z)-tetracarbazide-7,10, 29,32-tetraene-20-yl; (34)Tri-Man-DBCO-PEG2000-DMG; (35)Tri-Man-PEG-DBCO-PEG2000-DMG; (36)Tri-(PEG-Man)-PEG-DBCO-PEG2000-DMG; (37) Tri-Man-PEG2000-DMG; (38) Tri-Man-PEG2000-DSPE; (39)(R)-2,3-bis(stearoyloxy)propyl(2-(4-((2-(((2R,3R,4R,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-(trimethylammonium)tetrahydro-2H-pyran-2-yl)oxy)ethyl)amino)-4-oxobutamido)ethyl)phosphate; (40)(R)-2,3-bis(stearoyloxy)propyl(2-(4-((2-(((2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-(trimethylammonium)tetrahydro-2H-pyran-2-yl)oxy)ethyl)amino)-4-oxobutamido)ethyl)phosphate; (41)(R)-2,3-bis(stearoyloxy)propyl(2-(4-((2-(((2R,3R,4R,5S,6R)-3-(dimethylammonium)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)amino)-4-oxobutamido)ethyl)phosphate; (42)(R)-2,3-bis(stearoyloxy)propyl(2-(4-((2-(((2R,3R,4R,5R,6R)-3-(dimethylammonium)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)amino)-4-oxobutamido)ethyl)phosphate; (43) Glu-carbamate-ethylethoxy-PEG2000-DMG; (44)GlcNAc-carbamate-ethylethoxy-PEG2000-DMG; (45) Gal-carbamate-ethylethoxy-PEG2000-DMG; (46) GalNAc-carbamate-ethylethoxy-PEG2000-DMG; (47) Man-carbamate-ethylethoxy-PEG2000-DMG; (48) Di(tris(man)methylamide)amidoamine-PEG2000-DMG; (49) Tris(2-(tris-man-amidoamine)ethoxy)methylamine-PEG2000-DMG; (50) Di(tri(Man)methylamine)thiourea ethylamine-PEG2000-DMG; (51)G1-bis-MPA-(Man-succinamide)-PEG2000-DMG; (52) Di((Di(Man-ethylamidoamine))Ethylamidoamine)Ethylamine-PEG2000-DMG; (53) Di(Di((Di(Man-Ethylamidoamine))Ethylamidoamine)Ethylamidoamine)Ethylamine-PEG2000-DMG; (54)G1-polylysine-(Man-succinamide)-ethylamine-PEG2000-DMG; (55)G2-polylysine-(Man-succinamide)-ethylamine-PEG2000-DMG.

8. A lipid nanoparticle composition comprising: Ionizable lipids; The lipid compound of claim 1, its isomers, or pharmaceutically acceptable salts thereof as the first phospholipid; Second phospholipid; cholesterol; The lipid compound of claim 1, its isomers, or pharmaceutically acceptable salts thereof, as the first polyethylene glycol-modified lipid; as well as Second polyethylene glycol-modified lipid.

9. The lipid nanoparticle composition according to claim 8, characterized in that, The ionizable lipid is selected from one or more of the group consisting of: (6Z, 9Z, 28Z, 31Z)-heptadecano-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate, [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid, 1-linoleyl-2-linoleyloxy-3-dimethylaminopropane, 1,2-dilinoleylcarbamoyloxy 1,2-Dilinoleoyl-3-dimethylaminopropane, 1,2-Dilinoleoyloxy-N,N-dimethylaminopropane, 2,2-Dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane, 2,2-Dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane, 1,2-dioleoyl-3-dimethylammonium propane, N,N-dimethyl-(2,3-dioleoyloxy)propylamine, bis(octadecylamide)glycylcarboxylic acid, spermine cholesterol aminomethyl Ester esters; bis-guanidinium-spermine-cholesterol, 3β-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol, 1,1'-(2-(4-(2-((2-(bis(2-hydroxydecyl)amino)ethyl)(2-hydroxydecyl)amino)ethyl)piperazin-1-yl)ethylazadiyl)bisdodecane-2-ol, N-tert-butyl-N'-tetradecylamino-propanediol; dimethylbis(octadecyl)ammonium bromide (DDAB), N-(1,2-dimyristyloxypropyl-3-yl)-N,N- Dimethyl-N-hydroxyethyl ammonium bromide, N,N-diolenoyl-N,N-dimethyl ammonium chloride, dioleoyloxypropyl-3-dimethylhydroxyethyl ammonium bromide, N-(1-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarbamoyl)ethyl)-N,N-dimethyltrifluoroacetate ammonium, 1,2-dioleoyltrimethylpropane ammonium chloride, N-(1-(2,3-diolenoyloxy)propyl)-N,N,N-trimethyl ammonium chloride, and aminopropyl-dimethyl-bis(dodecyloxy)propyl ammonium bromide.

10. The lipid nanoparticle composition according to claim 8, characterized in that, The second phospholipid is selected from one or more of the following groups: 1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-distearyl-sn-glycerol-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine, egg yolk phosphatidylcholine, 1,2-dioleoyl-sn-glycerol-3-phosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycerol-3-phosphocholine, 1 2-Dipalmitoyl-sn-glycerol-3-phosphate, 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine, phosphatidylethanolamine, 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol-3-[phospho-L-serine], ceramide, sphingomyelin.

11. The lipid nanoparticle composition according to claim 8, characterized in that, The second polyethylene glycol-modified lipid is selected from one or more of the following groups: PEG-dilauroyl glycerol, PEG-dimyristoyl glycerol, PEG-dipalmitoyl glycerol, PEG-distearylphosphatidylethanolamine, PEG-dipalmitoyl glycerol, PEG-distearyl glycerol, PEG-dilauroyl glycine, PEG-dimyristoyl glycine, PEG-distearyl glycine, PEG-cholesterol and 3,4-dimyristoxybenzyl-[ω]-methyl-poly(ethylene glycol) ether, and 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-200].

12. The lipid nanoparticle composition according to claim 8, characterized in that, The lipid nanoparticles may encapsulate one or more active substances selected from the group consisting of: RNA, siRNA, shRNA, rRNA, tRNA, mRNA, miRNA, saRNA, circRNA, DNA, cDNA, plasmid, DNAzyme, ribozyme, PNA, aptamer, antisense oligonucleotide, and CRISPR.

13. A method for preparing an active substance—lipid nanoparticles, comprising: An organic phase comprising an organic phase in which ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids are mixed in a molar ratio of (20-60):(0-25):(30-60):(0-10), and an aqueous phase in which the active substance is dissolved, are mixed. The phospholipid is a mixture of a first phospholipid and a second phospholipid in a molar ratio of (0.005-100):(0-99.995), wherein the first phospholipid comprises the lipid compound of Formula 1 as claimed in claim 1, its isomers, or pharmaceutically acceptable salts thereof. The PEGylated lipid is a mixture of a first PEGylated lipid and a second PEGylated lipid in a molar ratio of (0.005-100):(0-99.995), wherein the first PEGylated lipid comprises the lipid compound of Formula 1 as claimed in claim 1, its isomers, or pharmaceutically acceptable salts thereof. The ionizable lipids and the active substance are mixed at a weight ratio of (2-80):

1.

14. The method for preparing active substance-lipid nanoparticles according to claim 13, characterized in that, The phospholipids comprise: The lipid compound of claim 1, its isomers, or pharmaceutically acceptable salts thereof as the first phospholipid; and Second phospholipid; The second phospholipid is selected from one or more of the following groups: 1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-distearyl-sn-glycerol-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine, egg yolk phosphatidylcholine, 1,2-dioleoyl-sn-glycerol-3-phosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycerol-3-phosphocholine, 1 2-Dipalmitoyl-sn-glycerol-3-phosphate, 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine, phosphatidylethanolamine, 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol-3-[phospho-L-serine], ceramide, sphingomyelin.

15. The method for preparing active substance-lipid nanoparticles according to claim 13, characterized in that, The polyethylene glycol-modified lipid comprises: The lipid compound of claim 1, its isomers, or pharmaceutically acceptable salts thereof as the first polyethylene glycol-modified lipid; and Second PEGylated lipid; The second polyethylene glycol-modified lipid is selected from one or more of the following groups: PEG-dilauroyl glycerol, PEG-dimyristoyl glycerol, PEG-dipalmitoyl glycerol, PEG-distearylphosphatidylethanolamine, PEG-dipalmitoyl glycerol, PEG-distearyl glycerol, PEG-dilauroyl glycine, PEG-dimyristoyl glycine, PEG-distearyl glycine, PEG-cholesterol and 3,4-dimyristoxybenzyl-[ω]-methyl-poly(ethylene glycol) ether, and 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-200].

16. A composition for delivering nucleic acids, comprising the lipid compound of claim 1, an isomer thereof, or a pharmaceutically acceptable salt thereof.

17. A tissue-specific drug delivery carrier comprising the lipid nanoparticle composition of claim 8.

18. A drug delivery carrier for treating retinal diseases, comprising the lipid nanoparticle composition of claim 8.

19. The drug delivery carrier for treating retinal diseases according to claim 18, characterized in that, The drug delivery carrier for treating retinal diseases is administered via subretinal injection or intravitreal injection.

20. A drug delivery carrier for the prevention of infectious diseases, comprising the lipid nanoparticle composition of claim 8.

21. The drug delivery carrier for preventing infectious diseases according to claim 20, characterized in that, The drug delivery carrier for the prevention of infectious diseases is administered via intramuscular injection or intranasal injection.

22. A drug delivery carrier for treating cancer, brain diseases, and liver diseases, comprising the lipid nanoparticle composition of claim 8.

23. The drug delivery carrier for treating diseases according to claim 22, characterized in that, The drug delivery carrier used to treat cancer, brain diseases, and liver diseases is administered via intravenous injection.

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