Organ-specific delivery compositions for nucleic acids

Abstract

The invention provides an organ-specific delivery composition for nucleic acid. The organ-specific delivery composition comprises a targeted lipid delivery system lipid; further, the composition may comprise a helper lipid; still further, the composition may comprise a cationic lipid. Wherein the lipid of the targeted lipid delivery system can be selected from one or more of ionizable anionic steroids and / or ionizable anionic polymer conjugated lipids; the auxiliary lipid is optionally one or more of phospholipid, steroid, polymer conjugated lipid and modifiable lipid; the cationic lipid may be selected from one or more of permanent cationic lipids and / or ionizable cationic lipids. The delivery composition is capable of specifically delivering a prophylactic / therapeutic agent, particularly a nucleic acid component, to a target organ.

Description

Organ-specific delivery compositions for nucleic acids

[0001] Cross-references

[0002] This application claims priority to Chinese patent application No. 202210809436.X, filed on July 11, 2022, entitled “Organ-specific delivery composition for nucleic acids,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates generally to the field of molecular biology and more particularly to the use of lipid nanoparticle compositions in the organ-specific delivery of substances such as nucleic acids. Background Art

[0004] Treatment or preventive nucleic acid has the potential to completely change vaccination, gene therapy, protein replacement therapy and other genetic disease therapies. Since the first clinical study of therapeutic nucleic acid began in the 2000s, the design of nucleic acid molecules and the research of their delivery method have made significant progress. However, nucleic acid drugs (comprising therapeutic drugs and preventive drugs) are still faced with several challenges, such as most liposome preparations accumulate by the biological process in the liver, thereby reducing the effectiveness of the composition delivered into the target organ. Similarly, other therapeutic agents such as proteins and small molecule drugs can also benefit from organ-specific delivery. Many different types of compounds such as chemicals show significant cytotoxicity. If these compounds can be better directed to be delivered to the desired organ, less off-target effects and side effects will be seen.

[0005] Currently, most lipid nanoparticle delivery systems passively target the liver. To change the target organ of lipid nanoparticles, the common strategy is to adjust the composition of the lipid nanoparticles. According to some reports, lipid nanoparticles composed of one or two lipids can achieve spleen targeting by adjusting the mixing ratio of nucleic acids and nanoparticles (Stephan Grabbe et al., Translating Nanoparticulate-personalized Cancer Vaccines into Clinical Applications: Case Study with RNA-Lipoplexes for the Treatment of Melanoma, 2016). However, this strategy needs to be improved in terms of formulation stability and encapsulation efficiency. In addition, according to some reports (Cheng Qiang et al., Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing, 2020), the addition of permanent anionic lipids to LNPs composed of four components: cationizable lipids, steroids, phospholipids, and PEG lipids can also specifically target the spleen, but at the cost of increased complexity in components and formulation processes. Therefore, lipid nanoparticle delivery systems with high delivery capacity, high stability and low complexity still need to be developed.

[0006] Summary of the Invention

[0007] The present application provides a lipid nanoparticle that can target different tissues and organs for drug delivery. The lipid nanoparticle provided by the present application comprises a targeted lipid delivery system (GOLD) lipid of the present application, wherein the targeted lipid delivery system (GOLD) lipid is selected from one or more combinations of ionizable anionic steroids and / or ionizable anionic polymer-conjugated lipids; further, the lipid nanoparticle provided by the present application further comprises an auxiliary lipid; further, the lipid nanoparticle provided by the present application further comprises a cationic lipid.

[0008] The auxiliary lipid is optionally one or more of a phospholipid, a steroid, a polymer-conjugated lipid and a modifiable lipid.

[0009] Preferably, the phospholipid is selected from any one of DOPE, DSPC, DPPC, DMPC, DOPC, POPC, SM, or a combination thereof.

[0010] Preferably, the steroid is selected from one or more of cholesterol, sitosterol, stigmasterol and ergosterol.

[0011] Preferably, the polymer-conjugated lipid, wherein the polymer refers to a high molecular weight compound formed by covalently bonding one or more small molecule repeating units; the polymer can be selected from polyethylene glycol, polylactic acid, polyamide, cationic polymer, polysarcosine (pSar), polylactic acid-glycolic acid copolymer (PLGA), polyamino acid, polypeptide, polypeptide, etc.; preferably, the polymer-conjugated lipid is selected from polyethylene glycol-conjugated lipid; further, the polyethylene glycol-conjugated lipid is selected from one or more of PEG1000-DMG, PEG5000-DMG, PEG2000-DMG and PEG2000-DSPE.

[0012] Preferably, the modifiable lipids include lipids modified with small molecule compounds, vitamins, carbohydrates, peptides, proteins, nucleic acids, lipopolysaccharides, inorganic molecules or particles, metal ions or particles, and combinations thereof.

[0013] The cationic lipid is selected from one or more combinations of permanent cationic lipids and / or ionizable cationic lipids. The permanent cationic lipid is selected from one or more combinations of DOTAP, DODMA, DSTAP, DMTAP, DDA, and DOBAQ; the ionizable cationic lipid is selected from one or more combinations of SM-102, Lipid 5, A6, DC-chol, C12-200, CKK-E12, 5A2-SC8, GO-C14, OF-2, 306Oi10, OF-Deg-Lin, 92-O17S, OF-C4-Deg-Lin, A18-iso5-2DC18, TT3, FTT5, BAMEA-O16B, Vc-Lipid, C14-4, Lipid 14, 4A3-Cit, and ssPalmO-Phe.

[0014] Tissue and organ specificity is achieved by the targeted lipid delivery system (GOLD) lipids in lipid nanoparticles; further achieved by the combination of the targeted lipid delivery system (GOLD) lipids and auxiliary lipids; and still further achieved by the combination of the targeted lipid delivery system (GOLD) lipids, auxiliary lipids and cationic lipids.

[0015] The lipid nanoparticles preferentially deliver the therapeutic / prophylactic agent to the following target organs: lung, heart, brain, spleen, lymph node, bone, skeletal muscle, stomach, small intestine, large intestine / colorectum, kidney, bladder, breast, testicle, ovary, uterus, thymus, brainstem, cerebellum, cerebrum, spinal cord, eye, ear, tongue or skin. Preferably, the target organ is the spleen.

[0016] The GOLD lipid in the lipid nanoparticles is optionally one or more of an ionizable anionic steroid and / or an ionizable anionic polymer-conjugated lipid.

[0017] Preferably, the ionizable anionic steroid is selected from the following compounds of formula I or pharmaceutically acceptable salts, prodrugs, stereoisomers or deuterated derivatives thereof:

[0018] Among them L 1 is absent, a carbon-carbon single bond, a carbon-carbon double bond, a carbon-carbon triple bond, or -O(C=O)-, -(C=O)O-, -C(=O)-, -O(C=O)O-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、-NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-;Q 1 is absent or is a C1-C8 linear or branched hydrocarbon group, specifically a C1, C2, C3, C4, C5, C6, C7 or C8 linear or branched hydrocarbon group;

[0019] L 2 is absent, a carbon-carbon single bond, a carbon-carbon double bond, a carbon-carbon triple bond, or -O(C=O)-, -(C=O)O-, -C(=O)-, -O(C=O)O-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、-NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-;Q 2 is absent or is a C1-C8 linear or branched hydrocarbon group, specifically a C1, C2, C3, C4, C5, C6, C7 or C8 linear or branched hydrocarbon group;

[0020] L 3 is absent, a carbon-carbon single bond, a carbon-carbon double bond, a carbon-carbon triple bond, or -O(C=O)-, -(C=O)O-, -C(=O)-, -O(C=O)O-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NRa C(=O)-, -C(=O)NR a -、-NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-;Q 3 is absent or is a C1-C8 linear or branched hydrocarbon group, specifically a C1, C2, C3, C4, C5, C6, C7 or C8 linear or branched hydrocarbon group;

[0021] R a is H or deuterium or a C1-C8 straight or branched hydrocarbon group or a C1-C8 straight or branched hydrocarbon group substituted with a carboxyl group, specifically a C1, C2, C3, C4, C5, C6, C7 or C8 straight or branched hydrocarbon group; or -Q 4 -M; where Q 4 is absent or is a C1-C8 straight or branched hydrocarbon group, specifically a C1, C2, C3, C4, C5, C6, C7 or C8 straight or branched hydrocarbon group, and M is

[0022] x is 0, 1, or 2;

[0023] R is X is C, O, or NR b or S; R b It is H or deuterium or a C1-C8 straight or branched hydrocarbon group, specifically a C1, C2, C3, C4, C5, C6, C7 or C8 straight or branched hydrocarbon group.

[0024] According to the above general formula, Table 1 lists some of the ionizable anionic steroid compounds without limitation.

[0025] Table 1. Representative compounds of ionizable anionic steroids

[0026] Preferably, the ionizable anionic polymer-conjugated lipid is selected from the following compound of formula II or a pharmaceutically acceptable salt, prodrug, stereoisomer or deuterated derivative thereof:

[0027] L 1is absent, a carbon-carbon single bond, a carbon-carbon double bond, a carbon-carbon triple bond, or -O(C=O)-, -(C=O)O-, -C(=O)-, -O(C=O)O-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、 -NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-; Q is absent or a C1-C8 linear or branched hydrocarbon group, specifically a C1, C2, C3, C4, C5, C6, C7 or C8 linear or branched hydrocarbon group;

[0028] R a is H or deuterium or a C1-C8 straight or branched hydrocarbon group or a C1-C8 straight or branched hydrocarbon group substituted with a carboxyl group, specifically a C1, C2, C3, C4, C5, C6, C7 or C8 straight or branched hydrocarbon group or a C1, C2, C3, C4, C5, C6, C7 or C8 straight or branched hydrocarbon group substituted with a carboxyl group;

[0029] X is C or N;

[0030] R is

[0031] Z is C, O, or NR b or S;

[0032] Y 1 or Y 2 One of the is absent, a carbon-carbon single bond, a carbon-carbon double bond, a carbon-carbon triple bond, or -O(C=O)-, -C(OH)-, -(C=O)O-, -C(=O)-, -O(C=O)O-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、-NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-, and Y 1 or Y 2The other is absent, a carbon-carbon single bond, a carbon-carbon double bond, a carbon-carbon triple bond, or -O(C=O)-, -C(OH)-, -(C=O)O-, -C(=O)-, -O(C=O)O-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、-NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-;R a is H or deuterium or a C1-C8 straight or branched hydrocarbon group or a C1-C8 straight or branched hydrocarbon group substituted with a carboxyl group, specifically a C1, C2, C3, C4, C5, C6, C7 or C8 straight or branched hydrocarbon group or a C1, C2, C3, C4, C5, C6, C7 or C8 straight or branched hydrocarbon group substituted with a carboxyl group;

[0033] G 1 and G 2 Each is independently absent or substituted C1-C12 linear or branched hydrocarbon group, specifically C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11 or C12 linear or branched hydrocarbon group;

[0034] R 1 and R 2 Each is independently a substituted C6-C24 straight or branched hydrocarbon group, specifically a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23 or C24 straight or branched hydrocarbon group;

[0035] x is 0, 1, or 2.

[0036] According to the above general formula, Table 2 lists some of the ionizable anionic polymer-conjugated lipid compounds in a non-limiting manner.

[0037] Table 2. Representative compounds of ionizable anionic polymer-conjugated lipids

[0038] Furthermore, the lipid nanoparticles may further include auxiliary lipids. The auxiliary lipids are optionally one or more of phospholipids, steroids, polymer-conjugated lipids, and modifiable lipids.

[0039] Preferably, the phospholipid is selected from any one of DOPE, DSPC, DPPC, DMPC, DOPC, POPC, SM, or a combination thereof.

[0040] Preferably, the steroid is selected from one or more of cholesterol, sitosterol, stigmasterol and ergosterol.

[0041] Preferably, in the polymer-conjugated lipid, the polymer refers to a high molecular weight compound formed by covalently bonding one or more small molecule repeating units; the polymer can be selected from polyethylene glycol, polylactic acid, polyamide, cationic polymer, polysarcosine (pSar), polylactic acid-glycolic acid copolymer (PLGA), polyamino acid, polypeptide, polymeric peptoid, etc.; preferably, the polymer-conjugated lipid is selected from polyethylene glycol-conjugated lipid; further, the polyethylene glycol-conjugated lipid is selected from one or more of PEG1000-DMG, PEG5000-DMG, PEG2000-DMG and PEG2000-DSPE.

[0042] Preferably, the modifiable lipids include lipids modified with small molecule compounds, vitamins, carbohydrates, peptides, proteins, nucleic acids, lipopolysaccharides, inorganic molecules or particles, metal ions or particles, and combinations thereof.

[0043] Furthermore, the lipid nanoparticles contain cationic lipids.

[0044] The cationic lipid in the lipid nanoparticles comprises an ammonium group that is positively charged at a given pH and contains at least two hydrophobic groups. The cationic lipid is a dendrimer or dendron. The cationic lipid comprises at least two C6-C24 hydrocarbon groups. The cationic lipid can be one or a combination of permanent cationic lipids and / or ionizable cationic lipids. The permanent cationic lipid can be selected from one or more of DOTAP, DODMA, DSTAP, DMTAP, DDA, and DOBAQ, and the ionizable cationic lipid can be selected from one or more of SM-102, Lipid 5, A6, DC-chol, C12-200, CKK-E12, 5A2-SC8, G0-C14, OF-2, 306Oi10, OF-Deg-Lin, 92-O17S, OF-C4-Deg-Lin, A18-iso5-2DC18, TT3, FTT5, BAMEA-O16B, Vc-Lipid, C14-4, Lipid 14, 4A3-Cit, and ssPalmO-Phe.

[0045] In the lipid nanoparticles, the molar ratio of the cationic lipid to the targeted delivery system lipid is 1:1 to 11:1.

[0046] In the lipid nanoparticles, the molar ratio of the cationic lipid, the targeted delivery system lipid, and the auxiliary lipid is 1:(0.1-1):(0.5-2).

[0047] In other aspects, the present application provides a composition comprising a therapeutic agent or a prophylactic agent and the above-mentioned lipid nanoparticles.

[0048] In a preferred aspect, the therapeutic / prophylactic agent is a nucleic acid.

[0049] Preferably, the nucleic acid includes any and all forms of nucleic acid molecules, including but not limited to single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, short isomers, plasmid DNA, complementary DNA / cDNA, antisense nucleic acid molecules / ASO, small interfering nucleic acid / siRNA, small activating nucleic acid / saRNA, asymmetric interfering nucleic acid / aiRNA, micronucleic acid / miRNA, miRNA inhibitors (agomir, antagomir), Dicer enzyme substrate nucleic acid, small hairpin nucleic acid (shRNA), transfer RNA (tRNA), messenger RNA / mRNA, circular RNA / circRNA, self-replicating mRNA / samRNA, aptamer / aptamer and other forms of nucleic acid molecules known in the art.

[0050] The nucleic acid molecules may include natural nucleotides, nucleotide mimetics or functional analogs, and chemically modified forms of nucleotides.

[0051] Functional nucleotide analogs include, but are not limited to, locked nucleic acid (LNA), peptide nucleic acid (PNA), or morpholino oligonucleotide nucleic acid mimetics or functional analogs, or a combination of more than one thereof.

[0052] Chemical modifications of nucleotides can be located on the backbone bonds of the nucleic acid molecule. The backbone bonds can be modified by replacing one or more oxygen atoms. Modifications to the backbone bonds can include replacing at least one phosphodiester bond with a phosphorothioate bond.

[0053] The chemical modification of the nucleotide can be located on the nucleoside. The modification on the nucleoside can be located on the sugar and base of the nucleoside. The sugar on the nucleoside can be selected from one or more of the following: 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, hexose.

[0054] The chemically modified form of the nucleotide can be selected from one or more of the following: 5-methylcytosine, pseudouridine, 1-methylpseudouridine, pyridine-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinemethyluridine, 1-taurinemethyl-pseudouridine, 5-taurinemethyl-2-thio-uridine, 1-taurinemethyl-4-thio-uridine, 5-methyl- Uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine Cytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2- Aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylaminoformyladenosine, N6-threonylaminoformyladenosine, 2-methylthio-N6-threonylaminoformyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wyobutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine and N2,N2-dimethyl-6-thio-guanosine.

[0055] In a preferred embodiment, the nucleic acid is mRNA, which can encode at least one antigen, fragment, or epitope thereof, or a therapeutic protein, wherein the antigen is selected from pathogenic antigens, such as tumor-associated antigens or pathogenic microorganism antigens. The mRNA can be monocistronic or polycistronic.

[0056] The present application also provides the use of the above composition in preparing medicines.

[0057] The present application also provides a drug comprising the above-mentioned composition and pharmaceutically acceptable excipients (also referred to as pharmaceutical excipients). Pharmaceutical excipients refer to excipients and additives used in the production of drugs and the preparation of prescriptions; they are substances other than active ingredients that have been reasonably evaluated in terms of safety and are included in pharmaceutical preparations. In addition to excipients, acting as carriers, and improving stability, pharmaceutical excipients also have important functions such as solubilization, solubilization, and sustained-release. They are important ingredients that may affect the quality, safety, and effectiveness of drugs. According to their functions and uses, pharmaceutical excipients can be divided into solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc.

[0058] Beneficial effects of this application:

[0059] The present application provides a new lipid nanoparticle that can deliver therapeutic / prophylactic agents, especially nucleic acid components, to specific organs, especially preferentially to organs other than the liver, providing more options for the delivery of nucleic acid drugs, gene drugs, vaccines, etc., and is of great significance for the development and application of nucleic acid preventive and therapeutic agents.

[0060] Most traditional lipid nanoparticles are targeted for liver delivery. By adding targeted lipid delivery system (GOLD) lipids to lipid nanoparticles, organ targeting is greatly changed. In lipid nanoparticles such as TMF1, TMF2, TMF7 and TMF11-TMF38 with the addition of GOLD lipids, the distribution (mean fluorescence intensity) ratio of nucleic acid drugs in the spleen and liver is 1.6 to 80 times (Tables 7 and 8). In traditional lipid nanoparticles without the addition of GOLD lipids, higher delivery efficiency is usually achieved in the liver (Ansell, SM; Du, X. Novel Lipids and Lipid Nanoparticle Formulations for Delivery of Nucleic Acids. WO2017075531 A1).

[0061] Therefore, targeted lipid delivery system (GOLD) lipids play an important role in the organ targeting of lipid nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] One or more embodiments are exemplarily illustrated by the accompanying figures, and these exemplary illustrations do not limit the embodiments. The word "exemplary" is used herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0063] Figure 1 shows the NMR of compound 1-1.

[0064] Figure 2 shows the NMR of compound 1-2.

[0065] Figure 3 shows the NMR of compound 1-3.

[0066] Figure 4 shows the NMR of compound 1-4.

[0067] Figure 5 shows the NMR of compound 1-5.

[0068] Figure 6 shows the NMR of compound 1-6.

[0069] Figure 7 shows the NMR of compound 1-7.

[0070] Figure 8 shows the NMR of compound 1-8.

[0071] Figure 9 shows the NMR of compound 1-9.

[0072] Figure 10 shows the NMR of compound 1-25.

[0073] Figure 11 shows the NMR of compound 1-39.

[0074] Figure 12 shows the NMR of compound 1-42.

[0075] Figure 13 shows the NMR of compound 1-44.

[0076] Figure 14 shows the NMR of compound 1-45.

[0077] Figure 15 shows the NMR of compound 1-47.

[0078] Figure 16 shows the NMR of compound 1-48.

[0079] Figure 17 shows the NMR of compound 1-49.

[0080] Figure 18 shows the NMR of compound 1-50.

[0081] Figure 19 shows the NMR of compound 1-51.

[0082] Figure 20 shows the NMR of compound 1-52.

[0083] Figure 21 shows the NMR of compound 1-55.

[0084] Figure 22 shows the NMR of compound 1-56.

[0085] Figure 23 shows fluorescence signals in different organs of mice 4 hours after intravenous administration of a luciferase mRNA-encapsulated lipid nanoparticle TMF1 sample. As shown, the fluorescence signal is strongest in the spleen, while it is very weak in the liver. This indicates that luciferase mRNA expression in the spleen is higher than that in the liver.

[0086] Figure 24 summarizes the fluorescence intensity ratio of spleen to liver after luciferase mRNA was delivered by different lipid nanoparticles. DETAILED DESCRIPTION

[0087] A. Chemical and Formulation Definitions

[0088] When used in the context of chemical groups: "hydrogen" refers to -H; "deuterium" refers to 2H or D; "hydroxy" means -OH; "oxo" means =O; "carbonyl" means -C(=O)-; "carboxy" means -C(=O)OH (also written as -COOH or -CO2H); "halo" independently means -F, -Cl, -Br or -I; "amino" means -NH2; "hydroxyamino" means -NHOH; "nitro" means -NO2; imino means =NH; "cyano" means -CN; "isocyanate" means -N=C=O; "azido" is refers to -N3; ​​in the context of a monovalent group, "phosphate" refers to -OP(O)(OH)2 or a deprotonated form thereof; in the context of a divalent group, "phosphate" refers to -OP(O)(OH)O- or a deprotonated form thereof; "thiol" refers to -SH; and "thio" refers to =S; "sulfonyl" refers to -S(O)2-; "hydroxysulfonyl" refers to -S(O)2OH; "sulfonamide" refers to -S(O)2NH2; and "sulfinyl" refers to -S(O)-. In the context of a chemical formula, the symbol "-" refers to a single bond, "=" refers to a double bond, and "≡" refers to a triple bond. The symbol "----" represents an optional bond, which, if present, is a single bond or a double bond. When drawn vertically through a bond, the symbol Indicates the point of attachment for this group. It should be noted that attachment points are usually only identified in this way for larger groups to help the reader clearly identify the point of attachment. refers to a single bond where the group attached to the thick end of the wedge "comes out of the page". refers to a single bond where the group attached to the thick end of the wedge "enters the page." The symbol =Referring to a single bond, wherein the geometry (e.g., E or Z) around the double bond is undefined. Therefore, both options and their combinations are contemplated. Any undefined valence on an atom of the structure shown in this application implicitly represents a hydrogen atom bonded to that atom. Bold dots on carbon atoms indicate that the hydrogen attached to that carbon is out of the plane of the paper.

[0089] The term "alkyl" used without the "substituted" modifier means a monovalent saturated aliphatic radical having a carbon atom as the point of attachment, having a straight or branched acyclic structure, and having no atoms other than carbon and hydrogen. The groups -CH3(Me), -CH2CH3(Et), -CH2CH2CH3(n-Pr or propyl), -CH(CH3)2(i-Pr, iPr or isopropyl), -CH2CH2CH2CH3(n-Bu), -CH(CH3)CH2CH3(sec-butyl), -CH2CH(CH3)2(isobutyl), -C(CH3)3(tert-butyl, t-Bu or tBu), and -CH2C(CH3)3(neopentyl) are non-limiting examples of alkyl radicals. The term "alkanediyl," used without the "substituted" modifier, means a divalent saturated aliphatic radical having one or two saturated carbon atoms as points of attachment, having a straight or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH2-(methylene), -CH2CH2-, -CH2C(CH3)2CH2-, and -CH2CH2CH2- are non-limiting examples of alkanediyl radicals. "Alkanes" refers to the class of compounds having the formula HR, in which R is alkyl, as that term is defined above. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have independently been replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -C02H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The following groups are non-limiting examples of substituted alkyl groups: -CH2OH, -CH2Cl, -CF3, -CH2CN, -CH2C(O)OH, -CH2C(O)OCH3, -CH2C(O)NH2, -CH2C(O)CH3, -CH2OCH3, -CH2OC(O)CH3, -CH2NH2, -CH2N(CH3)2, and -CH2CH2Cl. The term "haloalkyl" is a subset of substituted alkyl groups in which the hydrogen atom replacement is limited to halo (i.e., -F, -Cl, -Br, or -I) such that no atoms other than carbon, hydrogen, and halogen are present. The group -CH2Cl is a non-limiting example of a haloalkyl group. The term "fluoroalkyl" is a subset of substituted alkyl groups in which the hydrogen atom replacement is limited to fluoro such that no atoms other than carbon, hydrogen, and fluorine are present. The groups -CH2F, -CF3, and -CH2CF3 are non-limiting examples of fluoroalkyl groups.

[0090] The term "alkenyl" used without the "substituted" modifier means a monovalent unsaturated aliphatic radical having a carbon atom as the point of attachment, a straight or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CH=CH2 (vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2 (allyl), -CH2CH=CHCH3, and -CH=CHCH=CH2. The term "alkenediyl" used without the "substituted" modifier means a divalent unsaturated aliphatic radical having two carbon atoms as the point of attachment, a straight or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH3)CH2-, -CH=CHCH2-, and -CH2CH=CHCH2- are non-limiting examples of alkene diyl groups. It should be noted that although alkene diyl groups are aliphatic, this does not preclude the group from forming part of an aromatic structure once attached at both ends. The terms "alkene" and "alkene" are synonymous and refer to the class of compounds having the formula HR, where R is an alkenyl group, as that term is defined above. Similarly, the terms "terminal alkene" and "α-olefin" are synonymous and refer to an alkene having exactly one carbon-carbon double bond, where the bond is part of a vinyl group at the end of the molecule. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH, -NO, -C0H, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH. The groups -CH=CHF, -CH=CHCl, and -CH=CHBr are non-limiting examples of substituted alkenyl groups.

[0091] The term "alkynyl" as used without the "substituted" modifier refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, a straight or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. The term alkynyl as used herein does not exclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C≡CH, -C≡CCH3, and -CH2C≡CCH3 are non-limiting examples of alkynyl groups. "Alkynes" refers to the class of compounds having the formula HR, where R is an alkynyl group. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have independently been replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0092] "Prodrug" refers to a compound, such as a therapeutic agent, that can be converted into a biologically active compound under physiological conditions or by dissolution. Prodrugs are generally rapidly converted in vivo to produce the parent compound, for example by hydrolysis in the blood. Prodrug compounds generally have advantages in solubility, tissue compatibility, or delayed release in mammalian organisms. The term "prodrug" is also intended to include any covalently bonded carrier that releases the active compound in vivo when the prodrug is administered to a mammalian subject. Prodrugs include compounds that have hydroxyl, amino, or sulfhydryl groups bound to any group that, when the prodrug is administered to a mammalian subject, cleaves to form free hydroxyl, free amino, or free sulfhydryl groups, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives or amide derivatives of amine functional groups.

[0093] "Pharmaceutically acceptable salts" refers to salts of the compounds of the present disclosure that are pharmaceutically acceptable as defined above and that possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, Pharmaceutically acceptable salts include hydroxybenzoic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, enanthic acid, caproic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl) benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acid, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tert-butylacetic acid, trimethylacetic acid, etc. Pharmaceutically acceptable salts also include base addition salts that can form when the acidic protons present can react with inorganic bases or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc. It will be recognized that the particular anion or cation forming part of any salt of the present disclosure is not critical so long as the salt as a whole is pharmacologically acceptable.

[0094] "Stereoisomers" or "optical isomers" are isomers of a given compound in which the same atoms are bonded to the same other atoms, but in which the three-dimensional configurations of those atoms are different. "Enantiomers" are stereoisomers of a given compound that are mirror images of each other, like the left and right hands. "Diastereomers" are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain chiral centers (also called stereocenters or stereocenters), which are any points (although not necessarily atoms) in a molecule that carry multiple groups such that the interchange of any two groups produces stereoisomers. In organic compounds, chiral centers are usually carbon, phosphorus, or sulfur atoms, although other atoms may also serve as stereocenters in organic and inorganic compounds. A molecule can have multiple stereocenters, resulting in its many stereoisomers. In compounds whose stereoisomerism is attributed to tetrahedral stereocenters (e.g., tetrahedral carbon), it is assumed that the total number of possible stereoisomers will not exceed 2n, where n is the number of tetrahedral stereocenters. Molecules with symmetry often have a number smaller than the maximum possible number of stereoisomers. A 50:50 mixture of enantiomers is called a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically enriched so that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and / or diastereomers can be split or separated using techniques known in the art. It is expected that for any stereochemically undefined stereocenter or chiral axis, the stereocenter or chiral axis can exist in its R form, S form, or as a mixture of the R form and S form (including racemic mixtures and non-racemic mixtures). The phrase "substantially free of other stereoisomers" as used herein means that the composition contains ≤15%, more preferably ≤10%, even more preferably ≤5%, or most preferably ≤1% of one or more other stereoisomers.

[0095] deuterium( 2 H (or D) is a stable and non-radioactive isotope of hydrogen, having approximately twice the mass of hydrogen (H) and is the most common isotope of hydrogen.

[0096] "Prevention" or "preventing" includes: (1) inhibiting the onset of a disease in a subject or patient who may be at risk for and / or susceptible to the disease but who does not yet experience or display any or all symptoms or signs of the disease; and / or (2) slowing the onset of symptoms or signs of a disease in a subject or patient who may be at risk for and / or susceptible to the disease but who does not yet experience or display any or all symptoms or signs of the disease.

[0097] "Treatment" or "treating" includes (1) inhibiting a disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease (e.g., arresting further development of the symptoms and / or signs), (2) ameliorating a disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease (e.g., reversing the symptoms and / or signs), and / or (3) achieving any measurable reduction in a disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease.

[0098] "Protein", "polypeptide" or "peptide" refers to a polymer of amino acid residues and includes a wide range of protein molecules such as cytokines, chemokines, interleukins, interferons, growth factors, coagulation factors, anticoagulants, blood factors, bone morphogenic proteins, immunoglobulins and enzymes. Some non-limiting examples of therapeutic proteins include the following therapeutic proteins or fragments, variants or derivatives thereof: therapeutic proteins for the treatment of metabolic or endocrine disorders, including acid sphingomyelinase, Adipotide, Agalsidase-β, Alglucosidase, α-galactosidase A, α-glucosidase, α-L-iduronidase, α-N-acetylglucosidase, amphiregulin, angiopoietin (Ang1, Ang2, Ang3, Ang4, ANGPTL2, ANGPTL3, ANGPTL4, ANGPTL5, ANGPTL6, ANGPTL7), beta-cellulose , β-glucuronidase, bone morphogenetic proteins BMPs (BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP15), CLN6 protein, epidermal growth factor (EGF), Epigen, epidermal regulin, fibroblast growth factors (FGF, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23), Galsulphase, Glucocerebrosidase, GM-CSF, Heparin-binding EGF-like growth factor (HB-EGF), Hepatocyte growth factor (HGF), Hepcidin, Human albumin, Increased albumin loss, Iduronate sulfatase (iduronate-2-sulfatase), Integrins αVβ3, αVβ5, and α5β1, Iduronate sulfatase, Laronease, N-acetylgalactosamine-4-sulfatase (rhASB; galsulf ase, arylsulfatase A (ARSA), arylsulfatase B (ARSB)), N-acetylglucosamine-6-sulfatase, nerve growth factor (NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin 4 / 5 (NT-4 / 5), neuregulin (NRG1, NRG2, NRG3, NRG4), neuropilin (NRP-1, NRP-2), obestatin, platelet-derived growth factor (PDGF (PDFF-A, PDGF-B, PDGF-C, PDGF-D), TGFβ receptors (endothelin, TGF-β1 receptor,TGF-β2 receptor, TGF-β3 receptor), thrombopoietin (THPO) (megakaryocyte growth and development factor (MGDF)), transforming growth factor (TGF (TGF-a, TGF-β (TGFβ1, TGFβ2, and TGFβ3))), VEGF (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PIGF), nesiritide, trypsin, adrenocorticotropic hormone (ACTH), atrial natriuretic peptide (ANP), cholecystokinin, gastrin, leptin, oxytocin, somatostatin, vasopressin (antidiuretic hormone), calcitonin, exenatide, growth hormone (GH), somatropin, insulin, insulin-like growth factor 1 IGF-1, mecasermin rinfabate, an IGF-1 analog, mecasermin, an IGF-1 analog, pegvisomant, pramlintide, teriparatide (human parathyroid hormone residues 1-34), becaplermin, dibotermin-α (bone morphogenetic protein 2), histrelin acetate (gonadotropin-releasing hormone; GnRH), octreotide, and palifermin (keratinocyte growth factor; KGF); for the treatment of blood disorders, circulatory system diseases, respiratory system diseases, cancer or tumor diseases, infectious diseases, or immunodeficiency Therapeutic proteins, including alteplase (tissue plasminogen activator; tPA), anistreplase, antithrombin III (AT-III), bivalirudin, darbepoietin alfa, drotrexone alfa (activated protein C, erythropoietin, epoetin alfa, erythropoietin, erythropoietin, factor IX, factor VIIa, factor VIII, lepirudin, protein C concentrate, reteplase (a deletion mutant of tPA), streptokinase, tenecteplase, urokinase, angiostatin, anti-CD22 immunotoxin , denileukin-toxin conjugate, Immunocyanin, MPS (Metallopanstimulin), Aflibercept, Endostatin, Collagenase, Human deoxyribonuclease I, Dornase, Hyaluronidase, Papain, L-asparaginase, Peg-asparaginase, Rasburicase, Human chronic gonadotropin (HCG), Human follicle-stimulating hormone (FSH), Luteinizing hormone-alpha, Prolactin, Alpha-1 proteinase inhibitor, Lactase, Pancreatic enzymes (lipase, amylase) enzyme, protease), adenosine deaminase (bovine pegastatin, PEG-ADA), abatacept, alefacept, anakinra, etanercept, interleukin-1 (IL-1) receptor antagonist, thymosin, TNF-α antagonist, enfuvirtide, and thymosin α1; therapeutic proteins selected from adjuvants or immunostimulatory proteins, including: human adjuvant proteins, in particular pattern recognition receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10,TLR11; NOD1, NOD2, NOD3, NOD4, NOD5, NALP1, NALP2, NALP3, NALP4, NALP5, NALP6, NALP7, NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14, 1IPAF, NAIP, CIITA, RIG-I, MDA5 and LGP2, signal transducers of TLR signaling, including adaptor proteins, including, for example, Trif and Cardif; components of small-GTPase signaling (RhoA, Ras, Rac1, Cdc42, Rab, etc.), components of PIP signaling (PI3K, Src-kinase, etc.), components of MyD88-dependent signaling (MyD88, IRAK1, IRAK2, IRAK4, TIRAP, TRAF6, etc.), Components of MyD88-independent signaling (TICAM1, TICAM2, TRAF6, TBK1, IRF3, TAK1, IRAK1, etc.); activated kinases, including, for example, Akt, MEKK1, MKK1, MKK3, MKK4, MKK6, MKK7, ERK1, ERK2, GSK3, PKC kinase, PKD kinase, GSK3 kinase, JNK, p38 MAPK, TAK1, IKK, and TAK1; activated Transcription factors, including, for example, NF-κB, c-Fos, c-Jun, c-Myc, CREB, AP-1, Elk-1, ATF2, IRF-3, IRF-7, heat shock proteins, such as HSP10, HSP60, HSP65, HSP70, HSP75 and HSP90, gp96, fibrinogen, TypIII repeat extra domain A of fibronectin; or components of the complement system, including C1q, MBL, C1r, C1s, C 2b, Bb, D, MASP-1, MASP-2, C4b, C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9, CR1, CR2, CR3, CR4, C1qR, C1INH, C4bp, MCP, DAF, H, I, P and CD59, or induced target genes, including, for example, beta-defensins, cell surface proteins; or human adjuvant proteins, including trif, flt-3 ligand, Gp96 or fibronectin, to induce or Cytokines that enhance the innate immune response, including IL-1α, IL1β, IL-2, IL-6, IL-7, IL-8, IL-9, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, TNFα, IFNα, IFNβ, IFNγ, GM-CSF, G-CSF, M-CSF; chemokines, including IL-8, IP-10, MCP-1, MIP-1α,RANTES, eotaxin, CCL21; cytokines released by macrophages, including IL-1, IL-6, IL-8, IL-12, and TNF-α; as well as IL-1R1 and IL-1α; bacterial (adjuvant) proteins, particularly bacterial heat shock proteins or chaperones, including Hsp60, Hsp70, Hsp90, Hsp100; OmpA (outer membrane protein) from Gram-negative bacteria; bacterial porins, including OmpF; bacterial toxins, including those from Bordetella pertussis pertussis toxin (PT), pertussis adenylate cyclase toxins CyaA and CyaC from Bordetella pertussis, PT-9K / 129G mutant from pertussis toxin, tetanus toxin, cholera toxin (CT), cholera toxin B-subunit, CTK63 mutant from cholera toxin, CTE112K mutant from CT, Escherichia coli heat-labile enterotoxin (LT), B subunit (LTB) from heat-labile enterotoxin, Escherichia coli heat-labile enterotoxin mutants with reduced toxicity, including LTK63, LTR72; phenol-soluble regulatory protein; neutrophil-activating protein (HP-NAP) from Helicobacter pylori; surfactant protein D; outer surface protein A lipoprotein from Borrelia burgdorferi, lipoprotein B from Mycobacterium tuberculosis. tuberculosis; proteins from bacterial fimbriae; enterotoxin CT from Vibrio cholerae, fimbriae proteins from fimbriae of Gram-negative bacteria, and surfactant protein A and bacterial flagellin, protozoan (adjuvant) proteins, in particular Tc52 from Trypanosoma cruzi, PFTG from Trypanosoma gondii, protozoan heat shock proteins, LeIF from Leishmania spp., LeIF from Toxoplasma gondii ... gondii), viral (adjuvant) proteins, in particular respiratory syncytial virus fusion glycoprotein (F-protein), envelope proteins from MMT virus, murine leukemia virus proteins, hemagglutinin protein of wild-type measles virus, fungal (adjuvant) proteins, in particular fungal immunomodulatory protein (FIP; LZ-8); and keyhole limpet hemocyanin (KLH), OspA; therapeutic proteins for hormone replacement therapy, in particular estrogens, progesterone or progesterone, and testosterone; therapeutic proteins for reprogramming somatic cells into pluripotent or totipotent stem cells, in particular Oct-3 / 4,Sox gene family (Sox1, Sox2, Sox3 and Sox15), Klf family (Klf1, Klf2, Klf4 and Klf5), Myc family (c-myc, L-myc and N-myc), Nanog and LIN28; and therapeutic antibodies selected from antibodies for the treatment of cancer or tumor diseases, in particular 131T-Tositumomab, 3F8, 8H9, Abafuzumab, Adelimumab, Afutuzumab, Alacizumab pegol, Alemtuzumab, Amatuximab, AME-133v, AMG102, Maanimumab (Maanatumomab), Apolizumab, Batumuximab, Betumumab, Belimumab, Bevacizumab, Bivalve-DM1, Blinatumomab, Brentuximab vedotin, Cantuzumab, Mecanizumab, Cantuzumab ravtansine, Carlosomab pendetide, Carlumab, Catuzumab, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clivatuzumab tetraxetan, CNTO328, CNTO 95, Conatumumab, Dacetuzumab, Dalotuzumab, Desutuzumab, Desutuzumab, Drozitumab, Ecomeximab, Edelecomab, Elotuzumab, Elsilimomab, Enavatuzumab, Ensituximab, Epratuzumab, Ertumaxomab, Etaracizumab, Farletuzumab, FBTA05, Ficlatuzumab, Figitumumab, Flanvotumab, Galiximab, Galiximab, Ganitumab, GC1008, Gemtuzumab, Gemtuzumab Ozogacin, Girentuximab, Glemnbatumumab vedotin, GS6624, HuC242-DM4, HuHMFG1, HuN901-DM1, Ibritumomab Tiuxetan, Icrucumab, ID09C3, Indatuximab ravtansine, Inotuzumab ozogamicin, Intetumumab, Ipilimumab, Iratumumab, Labetuzumab, Lexitumab, Lintuzumab, Lorvotuzumab mertansine, Lucatumumab, Ruximab, Mapatumumab, Matuzumab, MDX-060, MEDI 522, Mitumomab, Mogamulizumab, MORab-003,MORab-009, Moxetumomab pasudotox, MT103, Tarceva, Naptumomab estafenatox, Narnatumab, Necitumumab, Nimotuzumab, Olaratumab, Onartuzumab, Oportuzumab monatox, Oregovomab, PAM4, Panitumab, Patritumab, Pemtumomab, Pertuzumab, Pliximab, Racotumomab, Radretumab, Ramucirumab, Rilotumumab, Rituximab, Robatumumab, Samalizumab, SGN-30, SGN-40, Silobuzumab, Siltuximab, Tabalumab, Tacatuzumab Tetraxetan, Palatatumomab, Tenatumomab, Teprotumumab, TGN1412, Ticilimumab (= tremelimumab), Tigatuzumab, TNX-650, Tositumomab, Trastuzumab, TRBS07, Tremelimumab, Tucotuzumab celmoleukin, Ublituximab, Urelumab, Veltuzumab (IMMU-106), Volociximab, Futumomab, WX-G250, zalutumumab and Talizumab; antibodies for the treatment of immune disorders, particularly Efalizumab, Epratuzumab, Etrolizumab, Fontolizumab, Ixekizumab, Mepolizumab, Milatuzumab, pooled immunoglobulins, Priliximab, Rituximab, Rontalizumab, Rulizumab, Sarilumab, Vedolizumab, Visilizumab mab, Reslizumab, Adalimumab, Aselizumab, Atinumab, Atlizumab, Belimumab, Besilesomab, BMS-945429, Briakinumab, Brodalumab, Canakinumab, Becilizumab, Erlizumab, Fezakinumab, Golimumab, Gomiliximab, Infliximab, Mavrilimumab, Natalizumab, Ocrelizumab, Odomumab, Ofatumumab, Ozoralizumab, Pexelizumab, Rovizumab, SBI-087, Secukinumab, Sirukumab, Talizumab, Tocilizumab, Toralizumab, TRU-015, TRU-016,Ustekinumab, Vepramumab, Azotumomab, Sifalimumab, Ruximab, and Rho(D) immune globulin; antibodies used to treat infectious diseases, particularly Afelimomab, CR6261, Ebalizumab, Efungumab, Avelumab, Felvelumab, Foravirumab, Ibalizumab, Libivirumab, Motavizumab, Nebacumab, Tuvirumab, Ubivirumab, and Batuximab , Pagibaximab, Palivizumab, Panobacumab, PRO140, Refaviru, Raxibacumab, Rigavir, Sevir, Suvizumab, and Tilatuzumab; Antibodies for the treatment of blood disorders, particularly Abciximab, Adalimumab, Eculizumab, Mepolizumab, and Milatuzumab; Antibodies for immunomodulation, particularly Antithymocyte Globulin, Basiliximab, Ciliizumab, Daclizumab, Ga vilimomab, inokizumab, moromonab-CD3, odulumab and siplizumab; antibodies for the treatment of diabetes, in particular gevokizumab, otelixizumab and teplizumab; antibodies for the treatment of Alzheimer's disease, in particular bapineuzumab, crenezumab, gantenerumab, ponezumab, R1450 and solanezumab; antibodies for the treatment of asthma, in particular benralizumab, enokizumab, keliximab, lebrikizumab, omalizumab, oxelumab, pascolizumab and tralokinumab; antibodies for the treatment of various conditions, in particular blosozumab, caroRx, fresolimumab, fulranumab, romosozumab, setamol,Tanezumab and ranibizumab; erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), α-galactosidase A, α-L-iduronidase, thyrotropin α, N-acetylgalactosamine-4-sulfatase (rhASB), dornase alfa, tissue-type plasminogen activator (TPA) activase, glucocerebrosidase, interferon (IF) β-1α, interferon β-1b, interferon γ, interferon α, TNF-α, IL-1 to IL-36, human growth hormone (rHGH), human insulin (BHI), human chorionic gonadotropin α, darbepoetin α, follicle-stimulating hormone (FSH), and factor VIII, antibodies and antibody derivatives such as bispecific antibodies, multispecific antibodies, and ADCs, all used to treat various conditions. Peptides are compounds formed by α-amino acids linked together by peptide bonds and are also intermediate products of protein hydrolysis. Generally, the number of amino acids contained in a peptide is from two to nine. Depending on the number of amino acids in the peptide, peptides are called different names: dipeptides, tripeptides, tetrapeptides, pentapeptides, etc. Peptides composed of three or more amino acid molecules are called polypeptides. Their molecular weight is less than 10,000 Da, they can pass through semipermeable membranes, and are not precipitated by trichloroacetic acid and ammonium sulfate. There are also literatures that call peptides composed of 2 to 10 amino acids oligopeptides (small molecule peptides); peptides composed of 10 to 50 amino acids are called polypeptides; peptides composed of more than 50 amino acids are called proteins. In other words, proteins are sometimes also called polypeptides.

[0099] "Small molecule compounds" include 7-methoxypteridine, 7-methylpteridine, abacavir, abafungin, abarelix, acebutolol, acenaphthene, acetaminophen, acetanilide, acetazolamide, acetohexamide, acitretin, acrivastine, adenine, adenosine, alafloxacin, albendazole, salbutamol, alclofenac, aldesleukin, alemtuzumab, alfuzosin, alitretinoin, alobarbital, allopurinol, all-trans retinoic acid (ATRA), aloprin, alprazolam, alprenolol, hexamethylenetetramine, amifostine, amiloride, aminoglutethimide, aminopyrine, amiodarone hydrochloride, amitriptyline, amlodipine, amobarbital, amodiaquine, amoxapine, amphetamine, amphetamine, amphetamine, amphetamine B, ampicillin, and amprenavir , amsacrine, amyl nitrate, amobarbital, anastrozole, amrinone, anthracene, anthracycline antibiotics, aprobital, arsenic trioxide, asparaginase, aspirin, astemizole, atenolol, atorvastatin, atovaquone, atrazine, atropine, azathioprine atropine, auranofin, azacitidine, azapropazone, azathioprine, azintamide, azithromycin, aztreonam, baclofen, barbiturates, live BCG, beclomethasone, bendroflumethiazide, benezepril, benidipine, benoyl ester, benzyl alcohol, benzazepam, benzamide, benzanthracene, benzathine penicillin, benzhexyphenidyl hydrochloride, benznidazole, benzodiazepines, benzoic acid, hydroxynaphthyphenidin, benzyl alcohol Tamethasone, bevacizumab (atorvastatin), bexarotene, bezafibrate, bicalutamide, bifonazole, biperiden, bisacodyl, bisantrene, bleomycin, bleomycin, bortezomib, brinzolamide, bromazepam, bromocriptine mesylate, bromperidol, brotizolam, budesonide, bumetanide, bupropion, busulfan, butalbital, butamben, butenafine hydrochloride, butalbital, butalbital (n-butalbital), butoconazole, butoconazole nitrate, butylparaben, caffeine, calcifediol, calciprotriene, calcitriol, captestosterone, cambendazole, camphor, camptothecin, camptothecin analogs, candesartan, capecitabine, capsaicin, captopril, carbamazepine, carboplatin Bimazole, chloramphenicol, carboplatin, carbimazole, carimazole, carmustine, cefamandole, cefazolin, cefixime, ceftazidime, cefuroxime axetil, celecoxib, cephradine, cerivastatin, cetrizine, cetuximab, chlorambucil, chloramphenicol, chlordiazepoxide, clomethiazole, chloroquine, chlorothiazide, chlorpheniramine, chlorproguanil hydrochloride, chlorpromazine, chlorsulfuronamide, chlorprothixene, chlorpyrifos, chlortetracycline, chlorthalidone, chlorzoxazone, cholecalciferol, cilostazol, cimetidine, cinnarizine, cinoxacin, ciprofibrate, ciprofloxacin hydrochloride, cisapride, cisplatin, citalopram, cladribine, clarithromycin, clemastine fumarate, clioquinol, clobazam, clofarabine, clofazimine, clofibrate,Clomiphene citrate, clomipramine, clonazepam, clopidogrel, clotiazepam, clotrimazole, clotrimazole, cloxacillin, clozapine, cocaine, codeine, colchicine, colistin, conjugated estrogens, corticosterone, cortisone, cortisone acetate, cyclizine, cyclohexanebarbital, cyclobenzaprine, cyclobutane-spirobarbiturate, cyclohexane-spirobarbiturate, cycloheptane-spirobarbiturate, cyclohexane- Spirobarbiturate, cyclopentane-spirobarbiturate, cyclophosphamide, cyclopropane-spirobarbiturate, cycloserine, cyclosporine, cyproheptadine, cyproheptadine hydrochloride, cytarabine, cytosine, dacarbazine, dactinomycin, danazol, dananthrone, dantrolene sodium, dapsone, erythropoietin alfa, darodipine, daunorubicin, decoquinate, dehydroepiandrosterone, delavirdine, demeclocycline, denileukin, deoxycholic acid Corticosterone, desoximetasone, dexamethasone, dextroamphetamine, dexchlorpheniramine, dexfenfluramine, dexrazoxane, dextropropoxyphene, heroin, diatrizoate, diazepam, diazoxide, dichlorophen, 2,4-dichlorprop, diclofenac, dicoumarol, didanosine, diflunisal, digitoxin, digoxin, dihydrocodeine, dihydroequilinone, dihydroergotamine mesylate, diiodohydroxyquinoline, diltiazem hydrochloride, Diloxanide furoate, dimenhydrinate, desmopramine, dinitolamide, diosgenin, diphenoxylate hydrochloride, biphenyl, dipyridamole, erythromycin, disopyramide, disulfiram, diuron, docetaxel, domperidone, donepezil, doxazosin, doxazosin hydrochloride, doxorubicin (neutral), doxorubicin hydrochloride, doxycycline, drostanolone propionate, droperidol, diprophylline, echinocandin, econazole, Econazole nitrate, efavirenz, elitizine, enalapril, emtamol, enoximone, epinephrine, epipodophyllotoxin derivatives, epirubicin, epoetin alfa, iprosartan (eposartan), dehydroequilin, equilin, ergocalciferol, ergotamine tartrate, erlotinib, erythromycin, estradiol, estramustine, estriol, estrone, ethacrynic acid, ethambutol, ethinyl tert-butyl amine, ethionamide, profenamide hydrochloride, ethyl 4-aminobenzoate (benzocaine), ethyl parahydroxybenzoate, ethinyl estradiol, etodolac, etomidate, etoposide, acitretin, exemestane, felbamate, felodipine, fenbendazole azole, fenbuconazole, fenbufen, fenfos, fenclorac, fenfluramine, fenofibrate, fenoldepam, fenoprofen calcium, fenoxycarb, fenpicron, fentanyl, fenticonazole, fexofenadine, filgrastim, finasteride, flecainide acetate, floxuridine, fludarabine, fluconazole, fluconazole, flucytosine, fludioxonil, fludrocortisone, fludrocortisone acetate, flufenamic acid, fluanisone, flunarizine hydrochloride, flunisolide, flunitrazepam, fluocortolone, flumethasone, fluorene, fluorouracil, fluoxetine hydrochloride, fluoxymesterone, flupentixol decanoate,Fluphenthixol decanoate, flurazepam, flurbiprofen, fluticasone propionate, fluvastatin, folic acid, fosenopril, fosphenytoin sodium, frovatriptan, furosemide, fulvestrant, furazolidone, gabapentin, G-BHC (lindane), gefitinib, gemcitabine, gemfibrozil, gemtuzumab, glafenine, glyburide, gliclazide, glimepiride, glipizide, glutethimide, glyburide, glyceryl trinitrate (nitroglycerin), goserelin acetate, grepafloxacin, griseofulvin, guaifenesin, guanabenzyl acetate, guanine, halofantrine hydrochloride, haloperidol, hydrochlorothiazide, helbarbital, heroin, hesperetin, hexachlorobenzene, hexamethylenetetracycline tal, histrelin acetate, hydrocortisone, hydroflumethiazide, hydroxyurea, scopolamine, hypoxanthine, ibritumomab tiuxetan, ibuprofen, idarubicin, allyl butalbital, ifosfamide, ihydroequilenin, imatinib mesylate, imipenem, indapamide, indinavir, indomethacin, indoprofen, interferon alfa-2a, interferon alfa-2b, iodamide, iopanoic acid, iprodione, irbesartan, irinotecan, isavuconazole, isocarboxazid, isoconazole, isoguanidine, isoniazid, isopropyl barbiturate, isoproturon, isosorbide dinitrate, isosorbide mononitrate, isradipine, itraconazole, itraconazole, itraconazole Itra, ivermectin, ketoconazole, ketoprofen, ketorolac, kalin, labetalol, lamivudine, lamotrigine, lansoprazole, L-DOPA, leflunomide, lenalidomide, letrozole, folinic acid, leuprolide acetate, levamisole, levofloxacin, lidocaine, linalool, lisinopril, lomefloxacin, lomustine, loperamide, loratadine, lorazepam, lorefloxacin, clomethazepam, losartan mesylate, lovastatin, lisuride maleate, maprotiline hydrochloride, mazindol, mebendazole, meclizine hydrochloride, Meclofenamic acid, medazepam, digoxin, medroxyprogesterone acetate, mefenamic acid, mefloquine hydrochloride, megestrol acetate, melphalan, mepentate bromide, meprobamate, meptazol, mercaptopurine, mesalamine, mesna, mesoridazine, mestranol, methadone, methaqualone, methocarbamol, mephenytoin, methotrexate, methoxsalen, methsuximide, methylclothiazide, methylphenidate, methylphenobarbital, methylparaben, methylprednisolone, methyltestosterone, methysergide maleate, metoclopramide, metolazone, metoprolol, metronidazole, mianserin hydrochloride, miconazole, midazolam, mifepristone, miglitol, minocycline, minoxidil, Mitomycin C, mitotane, mitoxantrone, mycophenolate mofetil, molindone, montelukast, morphine, moxifloxacin hydrochloride, nabumetone, nadolol, nalbuphine, nalidixic acid, nandrolone, tetracene, naphthalene, naproxen, naratriptan hydrochloride, natamycin, nelarabine, nelfinavir, nevirapine, nicardipine hydrochloride,Nicotinamide, niacin, acenocoumarol, nifedipine, nilutamide, nimodipine, nimozole, nisoldipine, nitrazepam, nitrofurantoin, nitrofurazone, nizatidine, novofloxacin, norethindrone, norfloxacin, norgestrel, nortriptyline hydrochloride, nystatin, estradiol, ofloxacin, olanzapine, omeprazole, omoconazole, ondansetron hydrochloride, oprelleukin, Ornidazole, oxaliplatin, oxaliquine, octadyl pamoate, oxaprozin, oxamol, oxazepam, oxcarbazepine, oxfendazole, oxiconazole, oxprenolol, hydroxybutazone, hydroxyphenoxylamine hydrochloride, paclitaxel, palifermin, pamidronate, para-aminosalicylic acid, pantoprazole, mefenamic acid, paroxetine hydrochloride, pegaspargase, pegfilgrastim, pemetrexed Sodium succinate, penicillamine, pentaerythritol tetranitrate, pentazocine, pentazocine, pentobarbital, pentobarbitone, pentostatin, pentoxifylline, perphenazine, perphenazine pimozide, perylene, phenylacetylurea, phenacetin, phenanthren, phenindione, phenobarbital, phenolbarbital, phenolphthalein, phenoxybenzamine, phenoxybenzamine hydrochloride, phenoxymethylpenicillin, phensuximide, phenylbutazone, phenytoin, pindolol, pioglitazone, pipobroman, piroxicam, pizotifen maleate, platinum compounds, plicamycin, polyene, polymyxin B, porfibril sodium, posaconazole (Posa), pramipexole, prasterone, pravastatin, praziquantel, Prazosin, prazosin hydrochloride, prednisolone, prednisone, primidone, probarbital, probenecid, probucol, procarbazine, prochlorperazine, progesterone, proguanil hydrochloride, promethazine, propofol, propoxur, propranolol, propylparaben, propylthiouracil, prostaglandin, pseudoephedrine, pteridine-2-methyl-thiol, pteridine-2-thiol, pteridine-4-methyl-thiol, pteridine-4-thiol, pteridine-7-methyl-thiol, pteridine-7-thiol, pyrantel pamoate, pyrazinamide, pyrene, pyridostigmine, pyrimethamine, quetiapine, imipenem, quinapril, quinidine, quinidine sulfate, quinine, quinine sulfate, rabeprazole sodium, ranitidine hydrochloride, rasburicase, ravuconazole, repaglinide, dicyclopentyl, reserpine , retinoids, rifabutin, rifampicin, rifapentine, rimexolone, risperidone, ritonavir, rituximab, rizatriptan benzoate, rofecoxib, ropinirole hydrochloride, rosiglitazone, saccharin, salbutamol, salicylamide, salicylic acid, saquinavir, sargramostim, butabarbital, secobarbital, sertaconazole, sertindole, sertraline hydrochloride, simvastatin, sirolimus, sorafenib, sparfloxacin, spiramycin, spironolactone, dihydrotestosterone, stanozolol, stavudine, diethylstilbestrol, streptozocin, strychnine, sulconazole, sulconazole nitrate, sulfacetamide, sulfadiazine, sulfamethazine, sulfamethazine, sulfamethoxazole, sulfa, sulfathiazole, sulindac, sulphabenzamide,sulfacetamide, sulphadiazine, sulphadoxine, sulfisoxazole, sulphamerazine, sulpha-methoxazole, sulphapyridine, sulfasalazine, sulfinpyridine, sulphapyridine, sulfathiazide, sulpiride, thiothiazide, sumatriptan succinate, Sunitinib maleate, tacrine, tacrolimus, talbutal, tamoxifen citrate, tamulosin, targretin, taxanes, tazarotene, telmisartan, temazepam, temozolomide, teniposide, tenoxicam, terazosin, terazosin hydrochloride, terbinafine hydrochloride, terbutaline sulfate, terconazole, terfenadine, testosterone, tetracycline, tetrahydrocannabinol, tetraoxaprine, thalidomide, thebaine, theobromine, Theophylline, thiabendazole, thiamphenicol, thioguanine, thioridazine, thiotepa, ethotoin, thymine, tiagabine hydrochloride, tibolone, ticlopidine, tinidazole, tioconazole, tirofiban, tizanidine hydrochloride, tolazamide, tolbutamide, tolcapone, topiramate, topotecan, toremifene, tositumomab, tramadol, trastuzumab, trazodone hydrochloride, tretinoin, triamcinolone, triamterene, triazolam, triazoles, triflupromazine, trimethoprim, horse Trimipramine acetate, triphenylene amine, troglitazone, tromethamine, tropicamide, trovafloxacin, tadalafil, ubidecarenone (coenzyme Q10), undecylenic acid, uracil, uracil mustard, uric acid, valproic acid, valrubicin, valsartan, vancomycin, venlafaxine hydrochloride, vigabatrin, pentobarbital, vinblastine, vincristine, vinorelbine, voriconazole, xanthine, zafirlukast, zidovudine, zileuton, zoledronic acid, zolmitriptan, zolpidem, and zopiclone.

[0100] An antigen (Ag) is any substance that triggers the production of antibodies and is considered to be any substance capable of inducing an immune response. Foreign molecules can be recognized by immunoglobulins on B cells or processed by antigen-presenting cells, where they bind to the major histocompatibility complex (MHC), forming complexes that reactivate T cells and trigger a continuous immune response.

[0101] Epitopes, also known as antigenic determinants, can be composed of a continuous sequence (protein primary structure) or a discontinuous three-dimensional protein structure, and are specific chemical groups that determine antigenicity. Most epitopes are found on the surface of antigenic substances, while some are found internally and are exposed only after enzymatic or other treatment. A natural antigen can have multiple determinants. The larger the antigen molecule, the greater the number of epitopes.

[0102] Tumor-associated antigens (TAAs) are antigenic molecules present on both tumor cells and normal cells. These include embryonic proteins, glycoprotein antigens, and squamous cell antigens, and are commonly used in clinical tumor diagnosis. TAAs are not unique to tumor cells; they are synthesized in trace amounts by normal cells and highly expressed during tumor cell proliferation, hence the term "associated antigens." Tumors originating from the same tissue type may harbor the same TAAs in different individuals.

[0103] Pathogens are microorganisms that can invade the human body and cause infection or even infectious diseases. Among pathogens, bacteria and viruses are the most harmful. Pathogens include prions, fungi, bacteria, spirochetes, mycoplasmas, rickettsiae, chlamydiae, and viruses. Pathogen antigens are substances derived from pathogens that have the ability to trigger an immune response.

[0104] "Lipid encapsulation" refers to lipid nanoparticles that provide active or therapeutic agents (e.g., nucleic acids (e.g., mRNA)), with full encapsulation, partial encapsulation, or intermediate between the two. In some embodiments, the nucleic acid (e.g., mRNA) is completely encapsulated in the lipid nanoparticles.

[0105] In various embodiments, the lipid nanoparticles have an average diameter of about 90 nm to about 600 nm, about 100 nm to about 550 nm, about 150 nm to about 500 nm, about 200 nm to about 400 nm, about 250 nm to about 300 nm, about 200 nm to about 300 nm, and are substantially non-toxic.

[0106] B. Targeted Lipid Delivery System (GOLD) Lipids

[0107] In some aspects, present disclosure includes one or more targeted delivery systems (GOLD) lipids, which cause the selective delivery of lipid nanoparticles to specific organs. The targeted delivery system (GOLD) lipid can be one or more of ionizable anion steroids and / or ionizable anion polymer conjugated lipids. In some embodiments, the targeted delivery system (GOLD) lipid is present in the compositions in a mol ratio of about 5%, 10%, 15%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 45%, to about 50% or any range that can be derived therefrom. In some embodiments, the targeted delivery system (GOLD) lipid can exist in a mol ratio of about 5% to about 50%, about 5% to about 45%, about 10% to about 40%, or about 20% to about 35%, or about 20% to about 30%.

[0108] In some embodiments, the GOLD compound can be an ionizable anionic steroid. In some embodiments, the present disclosure provides one or more lipids with one or more steroids and ionizable anionic groups. Ionizable anionic steroids can contain a group with a negative charge (regardless of pH). The ionizable anionic groups that can be used in ionizable anionic steroids are carboxylate groups, sulfonate groups, or phosphate groups. The ionizable anionic groups can be carboxylate groups. The carboxylate groups can be compounds that have a negative charge at a pH lower than 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. The steroids and steroid derivatives can be compounds with a tetracyclic 17-carbon ring structure that can further include one or more substitutions, including alkyl, alkoxy, hydroxyl, oxo, acyl, or a double bond between two or more carbon atoms. In some aspects, the ring structure of the steroid includes three fused cyclohexyl rings and a fused cyclopentyl ring.

[0109] In some embodiments, the GOLD compound can be an ionizable anionic polymer conjugated lipid. A lipid is a small molecule having two or more C6-C24 alkyl or alkenyl or alkynyl chains. In some aspects, the present disclosure provides one or more lipids having one or more polymer conjugated components and an ionizable anionic group. The ionizable anionic group that can be used in the ionizable anionic polymer conjugated lipid is a carboxylate group, a sulfonate group, or a phosphate group. The ionizable anionic group can be a carboxylate group. The carboxylate group can be a compound having a negative charge at a pH lower than 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. The polymer conjugated component can be a compound comprising a PEG chain connected to a glycerol group or containing one or more C6-C24 long-chain alkyl or alkenyl or C6-C24 fatty acid groups connected to a linker group using a PEG chain.

[0110] C. Helper lipids

[0111] In some aspects of the present disclosure, the composition containing one or more auxiliary lipids is mixed with cationic lipids and targeted delivery system lipids to produce lipid nanoparticles. In some embodiments, the cationic lipids and targeted delivery system lipids are mixed with 1, 2, 3, 4 or 5 different types of auxiliary lipids. It is contemplated that cationic lipids and targeted delivery system lipids can be mixed with a variety of different auxiliary lipids of a single type. In some embodiments, the auxiliary lipids include but are not limited to one or more of phospholipids, steroids or steroid derivatives, polymer-conjugated lipids and modified lipids.

[0112] "Phospholipid" refers to any lipid that contains a phosphate group. In some embodiments, the phospholipid is a structure containing one or two long-chain C6-C24 alkyl or alkenyl groups, glycerol or sphingosine, one or two phosphate groups, and an optional small organic molecule. In some embodiments, the small organic molecule is an amino acid, a sugar, or an amino-substituted alkoxy group, such as choline or ethanolamine. In some embodiments, the phospholipid is phosphatidylcholine. In some embodiments, the phospholipid is DOPE, DSPC, DPPC, DMPC, DOPC, POPC, or SM. In some embodiments, the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine.

[0113] "Steroids and steroid derivatives" include any steroid or steroid derivative. As used herein, in some embodiments, the term "steroid" is a class of compounds with a tetracyclic 17-carbon ring structure, which may further include one or more substitutions, including alkyl, alkoxy, hydroxyl, oxo, acyl, or a double bond between two or more carbon atoms. In one aspect, the ring structure of a steroid includes three fused cyclohexyl rings and a fused cyclopentyl ring. In some embodiments, a steroid derivative includes the above-mentioned ring structure substituted with one or more non-alkyl groups. In some embodiments, a steroid or a steroid derivative is a sterol. In some embodiments of the present disclosure, the steroid or steroid derivative is cholestane or a cholestane derivative. As mentioned above, a cholestane derivative includes the non-alkyl substitution of one or more of the above-mentioned ring systems. In some embodiments, the cholestane or cholestane derivative is cholestene or a cholestene derivative or a sterol or a sterol derivative. In other embodiments, the cholestane or cholestane derivative is cholestene and a sterol or a derivative thereof.

[0114] "Polymer-conjugated lipids" refer to lipids that inhibit aggregation of lipid nanoparticles or improve lipid nanoparticle stability or change immune responses or change circulation time in the body. The polymer-conjugated lipids include, but are not limited to, polyethylene glycol-conjugated lipids, polylactic acid-conjugated lipids, polyamide-conjugated lipids, cationic polymer-conjugated lipids, polysarcosine (pSar)-conjugated lipids, polylactic acid-co-glycolic acid (PLGA)-conjugated lipids, polyamino acids-conjugated lipids, polypeptides-conjugated lipids, poly-peptide-conjugated lipids or mixtures thereof. In some embodiments, the "polyethylene glycol-conjugated lipids" refer to any lipid to which a PEG group has been attached. In some embodiments, the PEG lipid is a diglyceride, which also includes a PEG chain connected to a glycerol group. In other embodiments, the PEG lipid is a compound containing one or more C6-C24 long-chain alkyl or alkenyl groups or C6-C24 fatty acid groups connected to a linker group with a PEG chain. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-conjugated ceramides, PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropane-3-amines, PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, PEG-modified distearoyl phosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol. In some embodiments, PEG modification is measured by the molecular weight of the PEG component of the lipid. In some embodiments, the molecular weight of the PEG used for modification is about 100 to about 15,000. In some embodiments, the molecular weight is about 200 to about 500, about 400 to about 5000, about 500 to about 3000, or about 1200 to about 3000. The molecular weight of PEG used for modification is about 100, 200, 400, 500, 600, 800, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 12500 to about 15000. "Modified lipids" include lipids modified with small molecule compounds, vitamins, carbohydrates, peptides, proteins, nucleic acids, lipopolysaccharides, inorganic molecules or particles, metal ions or particles, and combinations thereof.

[0115] List of English abbreviations

[0116] Example

[0117] In order to more clearly describe the purpose, features and advantages of the present application, the present application will be described in detail below with reference to the accompanying drawings and specific implementation methods. However, the implementation methods of the present application described in detail below are only for the purpose of illustrating the contents of the present application and do not constitute any limitation to the present application.

[0118] Example 1. Compound Synthesis

[0119] The following reagents used for compound synthesis were all from Shanghai Titan Technology Co., Ltd., AR grade.

[0120] The synthesis method of compound 1-1 is as follows:

[0121] Procedure: Succinic anhydride (259 mg, 2.59 mmol) was dissolved in DCM (5 mL) in a 25 mL flask, followed by DMAP (32 mg, 0.259 mmol). After stirring at room temperature for 30 min, cholesterol (500 mg, 1.29 mmol) was added to the solution and stirred overnight at room temperature. The reaction progress was monitored by TLC (10% MeOH / DCM). Upon completion, the solution was diluted with 10 mL of DCM. The solution was then washed with 10 mL of water and 10 mL of saturated sodium chloride solution, dried over anhydrous Na2SO4, filtered, and the organic phase concentrated to yield approximately 610 mg of crude product. Purification was performed using a Flash column (10 g silica gel, loaded with 5 mL of DCM; mobile phases: 50 mL of DCM; 50 mL of 1% methanol + 99% DCM; 50 mL of 2% methanol + 98% DCM; 50 mL of 5% methanol + 95% DCM) to afford 360 mg of the pure product (Compound 1-1) in a 52% yield.

[0122] The NMR results of compound 1-1 are shown in Figure 1. 1 H NMR (400MHz, CDCl3): δ5.37 (m, 1H), 4.50 (m, 1H), 2.65 (t, J = 6.0Hz, 2H), 2.60 (t, J = 6.0Hz, 2H), 2.35 (d, J = 7.0Hz, 2H), 0.5-2.1 (m, 41H).

[0123] The synthesis method of compound 1-2 is as follows:

[0124] Procedure: Malonic acid (1.1 g, 10.4 mmol) was dissolved in DCM (20 mL) in a 50 mL flask. DMAP (127 mg, 1.04 mmol) and DCC (536 mg, 2.6 mmol) were then added. After stirring at room temperature for 30 min, cholesterol (1 g, 2.6 mmol) was added to the solution and stirred overnight at room temperature. The reaction progress was monitored by TLC (10% MeOH / DCM). After completion of the reaction, the solution was diluted with 20 mL of DCM. The mixture was then washed with 20 mL of water and 20 mL of saturated sodium chloride solution, dried over anhydrous Na2SO4, filtered, and the organic phase concentrated to yield approximately 1.1 g of crude product. The pure product (compound 1-2) was separated and purified using a flash column (15 g silica gel, loaded with 10 mL DCM; the mobile phases were: 50 mL DCM; 50 mL 1% methanol + 99% DCM; 50 mL 2% methanol + 98% DCM; 50 mL 5% methanol + 95% DCM) to obtain 840 mg of the pure product (compound 1-2) in a yield of 68%.

[0125] The NMR results of compound 1-2 are shown in Figure 2. 1 H NMR (400MHz, CDCl3): δ5.42 (m, 1H), 4.71-4.79 (m, 1H), 3.44 (s, 2H), 2.39 (d, J = 8.0Hz, 2H), 0.5-2.1 (m, 41H).

[0126] The synthesis method of compound 1-3 is as follows:

[0127] A 100 mL flask was charged with adipic acid (4.38 g, 30 mmol), DMAP (1.22 g, 10 mmol), DCC (6.2 g, 30 mmol), and DCM (40 mL). The mixture was stirred at room temperature for 30 min, and cholesterol (3.86 g, 10 mmol) was added to the reaction mixture. The reaction progress was monitored by TLC (10% MeOH / DCM). After the reaction was complete, the reaction mixture was filtered to remove solids. The filtrate was washed with 15 mL of water and 15 mL of saturated sodium chloride solution, dried over anhydrous Na2SO4, filtered, and the organic phase was concentrated to yield approximately 4.5 g of crude product. The pure product, compound 1-3, 2.2 g, was isolated and obtained using a flash column purification (120 g silica gel, mobile phases: 500 mL of n-heptane; 300 mL of 1% EtOAc + 99% of n-heptane; 300 mL of 2% EtOAc + 98% of n-heptane; 200 mL of 5% EtOAc + 95% of n-heptane; 1000 mL of 8% EtOAc + 92% of n-heptane).

[0128] The NMR results of compounds 1-3 are shown in Figure 3. 1H NMR (400MHz, CDCl3): δ5.37-5.36(m,1H),4.65-4.58(m,1H),2.39-2.29(m,6H),2.03-1.78(m,6H),1.69-0.85(m,40H),0.67(s,3H).

[0129] The synthesis method of compound 1-4 is as follows:

[0130] To a 50 mL flask, add glutaric anhydride (2.0 g, 20 mmol), DMAP (245 mg, 2 mmol), cholesterol (4.0 g, 10 mmol), and DCM (40 mL). Stir at room temperature overnight. TLC (30% EtOAc / n-heptane) is used to monitor the reaction progress. Upon completion, the mixture is washed with 15 mL of water and 15 mL of saturated brine, dried over anhydrous NaSO, filtered, and the organic phase concentrated to yield approximately 4.5 g of crude product. The pure product, compound 1-4, 1.6 g, was isolated and obtained using a flash column purification (20 g silica gel, mobile phases: 500 mL of n-heptane; 300 mL of 1% EtOAc + 99% of n-heptane; 300 mL of 2% EtOAc + 98% of n-heptane; 200 mL of 5% EtOAc + 95% of n-heptane; 1000 mL of 8% EtOAc + 92% of n-heptane).

[0131] The NMR results of compounds 1-4 are shown in Figure 4. 1 H NMR (400MHz, CDCl3): δ5.38-5.36(m,1H),4.66-4.58(m,1H),2.45-2.30(m,6H),2.03-1.80(m,8H),1.64-0.83(m,38H),0.67(s,3H).

[0132] The synthesis method of compound 1-5 is as follows:

[0133] Step 1: Synthesis of compound 1-5-1

[0134] The reaction product, cholesterol (5 g, 12.9 mmol), and Boc-glycine (2.26 g, 12.9 mmol) were added to a 250 mL flask and dissolved in DCM (50 mL). DMAP (157 mg, 1.29 mmol) and DCC (4 g, 19.4 mmol) were then added. The mixture was stirred at room temperature overnight. The progress of the reaction was monitored by TLC (20% EtOAc / n-heptane). After the reaction was complete, the reaction solution was filtered to remove the solids. The filtrate was washed with 25 mL of water and 25 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and the organic phase was concentrated to obtain approximately 7.1 g of crude product. The product was purified using a flash column (60 g silica gel, loaded with 50 mL of n-heptane; the mobile phases were: 300 mL of n-heptane; 300 mL of 1% EtOAc + 99% n-heptane; 300 mL of ethanol). 2% EtOAc + 98% n-heptane) to separate and obtain 5.3 g of pure product (1-5-1) with a yield of 76%.

[0135] Step 2: Synthesis of compound 1-5-2

[0136] 1-5-1 (5.3 g, 9.7 mmol) was added to a 100 mL flask and dissolved in DCM (30 mL), followed by TFA (6 mL). The mixture was stirred at room temperature for 2 h, and the reaction was detected by TLC (20% EtOAc / normal heptane). After complete reaction, the reaction solution was concentrated, normal heptane (50 mL) was added for pulping, and solid was obtained by filtration. The solid was then dissolved in DCM (25 mL), washed with 25 mL 10% Na2CO3 solution, 25 mL water, and 25 mL saturated aqueous common salt water, and dried over anhydrous Na2SO4, filtered, and the concentrated organic phase gave 3.3 g of pure product (1-5-2) in a yield of 77%.

[0137] Step 3: Synthesis of Compounds 1-5

[0138] To a 25 mL flask, succinic anhydride (225 mg, 2.25 mmol) was dissolved in DCM (5 mL), and DMAP (27 mg, 0.225 mmol) was added. After stirring at room temperature for 30 min, 1-5-2 (500 mg, 1.13 mmol) was added to the solution and stirred at room temperature overnight. The reaction progress was monitored by TLC (10% MeOH / DCM, 0.1% acetic acid). After completion of the reaction, the solution was diluted with 10 mL of DCM. The product was then washed with 10 mL of water and 10 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and the organic phase concentrated to yield approximately 590 mg of crude product. The pure product (compound 1-5) of 320 mg was isolated and obtained using a flash column (10 g silica gel, loaded with 5 mL DCM; the mobile phases were: 50 mL 1% methanol + 99% DCM; 50 mL 2% methanol + 98% DCM; 50 mL 5% methanol + 95% DCM; 50 mL 10% methanol + 90% DCM). The yield was 52%.

[0139] The NMR results of compounds 1-5 are shown in Figure 5. 1 H NMR (400MHz, CDCl3): δ5.38 (m, 1H), 4.65-4.71 (m, 1H), 4.02 (d, J = 4.0Hz, 2H) ,2.70-2.74(m,2H),2.57-2.60(m,2H),2.32-2.35(m,2H),0.5-2.1(m,44H).

[0140] The synthesis method of compound 1-6 is as follows:

[0141] A 50 mL flask was charged with malonic acid (500 mg, 4.8 mmol), DMAP (58.6 mg, 0.48 mmol), DCC (248 mg, 1.2 mmol), and DCM (10 mL). The mixture was stirred at room temperature for 30 min, and 1-5-2 (700 mg, 1.2 mmol) was added to the reaction mixture. The reaction progress was monitored by TLC (30% EtOAc / n-heptane). After completion of the reaction, the reaction mixture was filtered to remove solids. The filtrate was washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and the organic phase concentrated to yield approximately 1.1 g of crude product. Flash column purification (20 g silica gel, mobile phases: 200 mL of n-heptane; 150 mL of 1% EtOAc + 99% of n-heptane; 150 mL of 2% EtOAc + 98% of n-heptane; 200 mL of 5% EtOAc + 95% of n-heptane; 500 mL of 8% EtOAc + 92% of n-heptane) was used to separate the pure product, compound 1-6, 290 mg, with a yield of 77%.

[0142] The NMR results of compounds 1-6 are shown in Figure 6. 1 H NMR (400MHz, CDCl3): δ5.96(s,1H),5.39-5.38(m,1H),4.70-4.64(m,1H),4.01(d, J=4Hz,2H),2.39-2.33(m,2H),2.08-1.78(m,5H),1.68-0.85(m,43H),0.68(s,3H).

[0143] The synthesis method of compound 1-7 is as follows:

[0144] A 100 mL flask was charged with adipic acid (701.6 mg, 4.8 mmol), DMAP (58.6 mg, 0.48 mmol), DCC (248 mg, 1.2 mmol), and DCM (20 mL). The mixture was stirred at room temperature for 30 min, and 1-5-2 (700 mg, 1.2 mmol) was added to the reaction mixture. The reaction progress was monitored by TLC (30% EtOAc / n-heptane). After completion of the reaction, the reaction mixture was filtered to remove solids. The filtrate was washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and the organic phase concentrated to yield approximately 1.3 g of crude product. The pure product, compound 1-7, 360 mg, was isolated and obtained by purification using a flash column (20 g silica gel, mobile phases: 200 mL of n-heptane; 150 mL of 1% EtOAc + 99% of n-heptane; 150 mL of 2% EtOAc + 98% of n-heptane; 200 mL of 5% EtOAc + 95% of n-heptane; 500 mL of 8% EtOAc + 92% of n-heptane). The yield was 44%.

[0145] The NMR results of compound 1-7 are shown in Figure 7. 1 H NMR (400MHz, d6-DMSO): δ11.99 (s, 1H), 8.22 (t, J = 8Hz, 1H), 5.35-5.33 (m, 1H), 4.51-4.43 (m, 1H), 3.7 7(d,J=4Hz,2H),3.33(s,2H),2.27-2.10(m,6H),1.98-1.75(m,5H),1.59-0.83(m,38H),0.65(s,3H).

[0146] The synthesis method of compound 1-8 is as follows:

[0147] To a 50 mL flask, glutaric anhydride (194 mg, 1.7 mmol), DMAP (21 mg, 0.17 mmol), and DCM (5 mL) were added. The mixture was stirred at room temperature for 30 min, and 1-5-2 (500 mg, 0.85 mmol) was added. The reaction progress was monitored by TLC (30% EtOAc / n-heptane). After completion of the reaction, the reaction mixture was filtered to remove solids. The filtrate was washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and the organic phase concentrated to yield approximately 720 mg of crude product. The pure product, compound 1-8, 320 mg, was isolated and obtained using a flash column purification (20 g silica gel, mobile phases: 200 mL of n-heptane; 150 mL of 1% EtOAc + 99% of n-heptane; 150 mL of 2% EtOAc + 98% of n-heptane; 200 mL of 5% EtOAc + 95% of n-heptane; 500 mL of 8% EtOAc + 92% of n-heptane).

[0148] The NMR results of compound 1-8 are shown in Figure 8. 1 H NMR (400MHz, CDCl3): δ6.42-6.39(m,1H),5.38-5.36(m,1H),4.71-4.63(m,1H),4.02 -4.01(m,2H),2.45-2.32(m,6H),2.09-1.78(m,8H),1.63-0.85(m,36H),0.67(s,3H).

[0149] The synthesis method of compound 1-9 is as follows:

[0150] Step 1: Synthesis of compound 1-9-1

[0151] To a 100 mL flask, add cholesterol (5.0 g, 12.9 mmol), triethylamine (2.6 g, 25.8 mmol), and DCM (50 mL). Place in an ice bath at 0-5°C. Add methanesulfonic anhydride (3.4 g, 19.4 mmol) dropwise. After complete addition, allow to react at 0-5°C for 2 h. Slowly warm to room temperature and stir overnight. Monitor the reaction progress by TLC (20% EtOAc / n-heptane). After completion, wash with 5 mL of water and 5 mL of saturated brine, dry over anhydrous Na2SO4, filter, and concentrate the organic phase to yield approximately 7 g of crude product. The pure product 1-9-1 was separated and purified using a flash column (200 g silica gel, loaded with 500 mL of n-heptane; the mobile phases were: 500 mL of n-heptane; 500 mL of 0.3% EtOAc + 99.7% of n-heptane; 500 mL of 1% EtOAc + 99% of n-heptane; 2000 mL of 2% EtOAc + 98% of n-heptane); 3.6 g of the product was obtained with a yield of 60%.

[0152] Step 2: Synthesis of compound 1-9-2

[0153] To a 100 mL flask, add 1-9-1 (4.0 g, 8.6 mmol), potassium phthalimide (1.75 g, 9.5 mmol), and DMF (40 mL). Heat and stir at 90°C for 16 h. Monitor the reaction progress by TLC (30% EtOAc / n-heptane). After the reaction is complete, add 15 mL of water. The aqueous layer is extracted twice with 20 mL of ethyl acetate. The combined organic phases are washed with 15 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated to yield approximately 3 g of crude product. The pure product, 1-9-2, 1 g, was isolated and obtained by flash column purification (200 g silica gel, loaded with 500 mL n-heptane; the mobile phases were: 300 mL n-heptane; 300 mL 1% EtOAc + 99% n-heptane; 300 mL 3% EtOAc + 97% n-heptane; 300 mL 5% EtOAc + 95% n-heptane; 2000 mL 10% EtOAc + 90% n-heptane). The yield was 22%.

[0154] Step 3: Synthesis of compound 1-9-3

[0155] To a 100 mL flask, add 1-9-2 (200 mg, 38 mmol), hydrazine hydrate (133 mg, 0.95 mmol), and anhydrous ethanol (2 mL). Heat at 80-85°C and stir for 3 h. Monitor the reaction progress by TLC (30% EtOAc / n-heptane). After completion, filter the mixture, concentrate the filtrate to dryness, add 2 mL of DCM, dry over anhydrous NaSO, filter, and use the filtrate directly in the next step.

[0156] Step 4: Synthesis of Compound 1-9

[0157] To a 50 mL flask, add succinic anhydride (76.1 mg, 0.76 mmol), DMAP (9.3 mg, 0.076 mmol), and DCM (1 mL). Stir at room temperature for 30 min, then add the solution of 1-9-3 obtained in the previous step. Monitor the reaction progress by TLC (30% EtOAc / n-heptane). Upon completion, dilute with 10 mL of DCM, wash with 5 mL of water and 5 mL of saturated brine, dry over anhydrous NaSO, filter, and concentrate the organic phase to yield approximately 180 mg of crude product. The pure product, compound 1-9, 80 mg, was isolated and obtained by purification using a Flash column (20 g silica gel, mobile phases: 100 mL of n-heptane; 100 mL of 1% EtOAc + 99% n-heptane; 100 mL of 2% EtOAc + 98% n-heptane; 100 mL of 5% EtOAc + 95% n-heptane; 300 mL of 8% EtOAc + 92% n-heptane).

[0158] The NMR results of compound 1-9 are shown in Figure 9. 1 H NMR (400MHz, CDCl3): δ6.03-6.02(m,1H),3.47-3.43(m,1H),2,71-2.68(m,2H),2.55 -2.52(m,2H),2.07-1.98(m,2H),1.87-1.71(m,4H),1.59-0.82(m,48H),0.71(s,3H).

[0159] The synthesis method of compound 1-25 is as follows:

[0160] Step 1: Synthesis of compound 1-25-1

[0161] Cholesterol (11.6 g, 30 mmol), triethylamine (9.2 g, 90 mmol), DSC (11.5 g, 45 mmol), and acetonitrile (110 mL) were added to a 250 mL three-necked flask. The mixture was heated to 45°C and stirred overnight. The reaction progress was monitored by TLC (20% EtOAc / n-heptane). After completion, the organic phase was concentrated to yield approximately 10 g of crude product. Purification was performed using a flash column (100 g silica gel, mobile phases: 300 mL of n-heptane; 200 mL of 1% EtOAc + 99% n-heptane; 200 mL of 5% EtOAc + 95% n-heptane; 300 mL of 8% EtOAc + 92% n-heptane; 1000 mL of 15% EtOAc + 85% n-heptane) to isolate the pure product, 1-25-1, 6.1 g, in a 38% yield.

[0162] Step 2: Synthesis of compound 1-25-2

[0163] To a 25 mL three-necked flask was added 1-25-1 (541.8 mg, 1.03 mmol), triethylamine (114.3 mg, 1.13 mmol), glycine tert-butyl ester (131.2 mg, 1.1 mmol), and DCM (5 mL). The mixture was stirred at room temperature overnight. The reaction progress was monitored by TLC (20% EtOAc / n-heptane). After completion of the reaction, 10 mL of DCM was added, and the mixture was washed with 5 mL of aqueous sodium bicarbonate solution and 5 mL of aqueous sodium chloride solution. The mixture was dried over anhydrous Na2SO4, filtered, and the organic phase was concentrated to yield approximately 800 mg of crude product. The pure product, 1-25-2, 350 mg, was isolated and obtained using a flash column (10 g silica gel, mobile phases: 200 mL of n-heptane; 200 mL of 1% EtOAc + 99% n-heptane; 200 mL of 5% EtOAc + 95% n-heptane; 300 mL of 8% EtOAc + 92% n-heptane). The yield was 38%.

[0164] Step 3: Synthesis of compound 1-25

[0165] 1-25-2 (350 mg, 0.64 mmol) was added to a 50 mL flask and dissolved in DCM (9 mL). TFA (3 mL) was then added. After stirring at room temperature for 2 h, the reaction was monitored by TLC (20% EtOAc / n-heptane). After completion of the reaction, the reaction solution was concentrated and dissolved in MTBE (20 mL). The solution was then adjusted to pH 8-9 with 10% NaHCO₃ solution and pH 1-2 with 1 M dilute hydrochloric acid. The mixture was stirred for 20 min, separated, and the aqueous phase was extracted with 20 mL MTBE. The organic phases were combined, dried over anhydrous Na₂SO₄, filtered, and concentrated to yield 300 mg of a crude product. The pure product, compound 1-25, 100 mg, was isolated and obtained by purification using a flash column (8 g silica gel, mobile phases: 200 mL of n-heptane; 100 mL of 5% EtOAc + 95% of n-heptane; 100 mL of 10% EtOAc + 90% of n-heptane; 100 mL of 20% EtOAc + 80% of n-heptane; 200 mL of 30% EtOAc + 70% of n-heptane). The yield was 32%.

[0166] The NMR results of compound 1-25 are shown in Figure 10. 1H NMR (400MHz, d6-DMSO): δ7.33 (t, J = 8Hz, 1H), 5.35-5.32 (m, 1H), 4.36-4.29 (m, 1H), 3.6 3-3.61(m,2H),2.32-2.20(m,2H),2.03-1.75(m,6H),1.56-0.84(m,41H),0.66(s,3H).

[0167] The synthesis method of compound 1-32 is as follows:

[0168] To a 50 mL flask, 1-5-2 (500 mg, 0.9 mmol), EDTA anhydride (236 mg, 0.9 mmol), and toluene (5 mL) were added. The mixture was heated at 90°C and stirred for 16 h. The reaction progress was monitored by TLC (10% MeOH / DCM). After the reaction was complete, 5 mL of water and 15 mL of THF were added and stirred overnight. The mixture was separated and the aqueous layer was extracted with 10 mL of THF. The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to yield the product, 1-32, 40 mg, in a 6.2% yield.

[0169] The synthesis method of compound 1-36 is as follows:

[0170] Step 1: Synthesis of compound 1-36-1

[0171] Bromoacetyl chloride (13.03 g, 82.8 mmol) was dissolved in DCM (50 mL) in a 500 mL flask and cooled to 0-5°C in an ice-salt bath. Cholesterol (16 g, 41.4 mmol) in DCM (110 mL) was slowly added dropwise to the solution. After addition, the mixture was stirred overnight at room temperature. The reaction progress was monitored by TLC (20% EtOAc / n-heptane). After completion of the reaction, 50 mL of water was added to quench the reaction. The layers were separated, and the aqueous phase was extracted with DCM (50 mL x 2). The combined organic phases were washed with 50 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated to yield approximately 22 g of crude product. The pure product, 1-36-1, was isolated and obtained by purification using a flash column (200 g silica gel, loaded with 500 mL of n-heptane; the mobile phases were: 500 mL of n-heptane; 500 mL of 0.1% EtOAc + 99.9% n-heptane; 500 mL of 0.3% EtOAc + 99.7% n-heptane; 500 mL of 0.5% EtOAc + 99.5% n-heptane; 2000 mL of 1% EtOAc + 99% n-heptane). The yield was 76%.

[0172] Step 2: Synthesis of compound 1-36-2

[0173] To a 50 mL flask, add 1-36-1 (8.0 g, 15.8 mmol) and dissolve DIPEA (4.1 g, 31.6 mmol) in DCM (40 mL). Add tert-butyl glycine (4.1 g, 31.6 mmol). Stir overnight at room temperature. Monitor the reaction progress by TLC (20% EtOAc / n-heptane). After completion, wash with 10 mL of water and 10 mL of saturated brine, dry over anhydrous NaSO, filter, and concentrate the organic phase to yield approximately 12.1 g of crude product. The pure product 1-36-2 was separated and purified using a flash column (100 g silica gel, loaded with 300 mL of n-heptane; the mobile phases were: 500 mL of n-heptane; 500 mL of 0.1% EtOAc + 99.9% of n-heptane; 500 mL of 0.3% EtOAc + 99.7% of n-heptane; 2000 mL of 0.5% EtOAc + 99.5% of n-heptane) to obtain 7.5 g of the product with a yield of 85%.

[0174] Step 3: Synthesis of compound 1-36-3

[0175] Succinic anhydride (174.9 mg, 1.75 mmol), DMAP (106.8 mg, 0.87 mmol), and DCM (10 mL) were added to a 50 mL flask. Stirring was continued at room temperature for 30 min, and 1-36-2 (1.0 g, 1.75 mmol) was added to the reaction mixture. The reaction progress was monitored by TLC (5% MeOH / DCM). After completion of the reaction, the mixture was washed with 5 mL of water and 5 mL of saturated sodium chloride solution, dried over anhydrous Na2SO4, filtered, and the organic phase concentrated to yield approximately 1.8 g of crude product. Purification was performed using a flash column (40 g silica gel, mobile phases: 200 mL of DCM; 200 mL of 1% MeOH + 99% DCM; 500 mL of 1.5% MeOH + 98.5% DCM) to obtain the pure product, 1-36-3, 1.1 g, in a 92% yield.

[0176] Step 4: Synthesis of compound 1-36

[0177] 1-36-3 (300 mg, 0.46 mmol) was added to a 50 mL flask and dissolved in DCM (5 mL). TFA (2 mL) was then added. After stirring at room temperature for 2 h, the reaction was monitored by TLC (5% MeOH / DCM). After completion of the reaction, the reaction solution was concentrated and dissolved in MTBE (20 mL). The solution was then adjusted to pH 8-9 with 10% NaHCO₃ solution and pH 1-2 with 1 M dilute hydrochloric acid. The mixture was stirred for 20 min, separated, and the aqueous phase was extracted with 20 mL MTBE. The organic phases were combined, dried over anhydrous Na₂SO₄, filtered, and concentrated to yield 300 mg of a crude product. The pure product, 1-36, 140 mg, was isolated and obtained using a flash column (8 g silica gel, mobile phases: 100 mL of DCM; 100 mL of 1% MeOH + 99% of DCM; 100 mL of 1.5% MeOH + 98.5% of DCM; 100 mL of 2% MeOH + 98% of DCM; 100 mL of 3% MeOH + 97% of DCM; 300 mL of 8% MeOH + 92% of DCM). The yield was 52%.

[0178] The synthesis method of compound 1-38 is as follows:

[0179] Step 1: Synthesis of compound 1-38-1

[0180] Succinic anhydride (4.08 g, 40.8 mmol), DMAP (498 mg, 4.08 mmol), and DCM (50 mL) were added to a 100 mL flask. Stir at room temperature for 10 min, then the starting material (5.0 g, 5.4 mmol) was added to the reaction mixture. The reaction progress was monitored by TLC (5% MeOH / DCM). After completion of the reaction, the reaction mixture was filtered to remove solids. The filtrate was washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous NaSO, filtered, and the organic phase concentrated to yield the crude product, 1-38-1, approximately 8 g. Yield: 100%.

[0181] Step 2: Synthesis of compound 1-38-2

[0182] To a 50 mL flask, 1-38-1 (1.0 g, 2.9 mmol) was dissolved in DCM (10 mL). DMAP (176.9 mg, 1.45 mmol) and DCC (597.4 mg, 2.9 mmol) were added. After stirring at room temperature for 30 min, cholesterol (1.12 g, 2.9 mmol) was added to the solution, and the mixture was stirred overnight at room temperature. The reaction progress was monitored by TLC (20% EtOAc / n-heptane). After completion of the reaction, the reaction mixture was filtered to remove solids. The filtrate was washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and the organic phase concentrated to yield approximately 1.9 g of crude product. The pure product, 1-38-2, 1.1 g, was isolated and obtained by purification using a Flash column (20 g silica gel, mobile phases: 300 mL of n-heptane; 200 mL of 1% EtOAc + 99% n-heptane; 200 mL of 5% EtOAc + 95% n-heptane; 300 mL of 8% EtOAc + 92% n-heptane). The yield was 53%.

[0183] Step 3: Synthesis of compound 1-38

[0184] 1-38-2 (400 mg, 0.56 mmol) was added to a 50 mL flask and dissolved in DCM (2 mL). A hydrochloric acid ethyl acetate solution (4 mL) was added. After stirring at room temperature for 2 h, the reaction was monitored by TLC (20% EtOAc / n-heptane). After completion of the reaction, the reaction solution was concentrated and then dissolved in MTBE (20 mL). The solution was then adjusted to pH 8-9 with 10% NaHCO₃ solution and pH 1-2 with 1 M dilute hydrochloric acid. The mixture was stirred for 20 min, separated, and the aqueous phase was extracted with 20 mL MTBE. The organic phases were combined, dried over anhydrous Na₂SO₄, filtered, and concentrated to yield 300 mg of a crude product. The pure product, 1-38, 120 mg, was isolated and obtained using a flash column (8 g silica gel, mobile phases: 200 mL of DCM; 100 mL of 1% MeOH + 99% of DCM; 100 mL of 1.5% MeOH + 98.5% of DCM; 100 ml of 2% MeOH + 98% of DCM; 200 mL of 3% MeOH + 97% of DCM).

[0185] The synthesis method of compound 1-39 is as follows:

[0186] Cholesterol (1.16 g, 3 mmol, 1.0 eq) and 6 mL of ethyl acetate were added to a 100 mL flask. Cool to -5°C in an ice-salt bath, and chlorosulfonic acid (384.5 mg, 3.3 mmol, 1.1 eq) was slowly added dropwise. Stir at this temperature for 3 h. The reaction progress was monitored by TLC (20% EtOAc / n-heptane). After completion of the reaction, the mixture was filtered, and the filtrate was washed with 5 mL of water and 5 mL of saturated brine. The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to yield approximately 3 g of crude product. Flash column purification (80 g silica gel, loaded with 500 mL n-heptane; mobile phases: 200 mL n-heptane; 100 mL 0.5% EtOAc + 99.5% n-heptane; 100 mL 0.8% EtOAc + 99.2% n-heptane; 100 mL 1% EtOAc + 99% n-heptane; 100 mL 1.5% EtOAc + 98.5% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 100 mL 3% EtOAc + 97% n-heptane; 600 mL 10% EtOAc + 90% n-heptane) was used to separate the pure product, compound 1-39, 150 mg, with a yield of 28%.

[0187] The NMR results of compound 1-39 are shown in Figure 11. 1 H NMR (400MHz, CDCl3): δ5.38-5.36(m,1H),4.64-4.58(m,1H),2.33-2.30(m,2H),2.02-1.83(m,6H),1.61-0.86(m,36H),0.68(s,3H).

[0188] The synthesis method of compound 1-41 is as follows:

[0189] A 50 mL flask was charged with 1-5-2 (543.8 mg, 1.0 mmol), pyridine (237.3 mg, 3.0 mmol), and THF (10 mL). The mixture was cooled to -40°C with dry ice, and phosphorus oxychloride (536.6 mg, 3.5 mmol) was added dropwise rapidly. The mixture was stirred at -10°C for 3 h. The reaction progress was monitored by TLC (10% MeOH / DCM). After the reaction was complete, 2 mL of dilute hydrochloric acid was added and stirred for 1 h. The layers were separated, and the aqueous phase was extracted with THF (5 mL x 2). The combined organic phases were washed with 5 mL of aqueous sodium chloride solution, dried over anhydrous Na2SO4, filtered, and the organic phases were concentrated to obtain the pure product, 1-41, approximately 260 mg, in a 50% yield.

[0190] The synthesis method of compound 1-42 is as follows:

[0191] Step 1: Synthesis of compound 1-42-1

[0192] To a 25 mL three-necked flask was added 1-25-1 (500 mg, 0.95 mmol), ethanolamine (58 mg, 0.95 mmol), triethylamine (105 mg, 1.04 mmol), and DCM (5 mL). The mixture was stirred at room temperature for 3 h. The reaction progress was monitored by TLC (30% EtOAc / n-heptane). After completion of the reaction, DCM (10 mL) was added, and the mixture was washed with 5 mL of aqueous sodium bicarbonate solution and 5 mL of aqueous sodium chloride solution. The mixture was dried over anhydrous NaSO, filtered, and the organic phase was concentrated to yield approximately 800 mg of crude product. The pure product, 1-42-1, 300 mg, was isolated and obtained using a flash column (10 g silica gel, mobile phases: 200 mL of n-heptane; 200 mL of 1% EtOAc + 99% n-heptane; 200 mL of 5% EtOAc + 95% n-heptane; 300 mL of 8% EtOAc + 92% n-heptane). The yield was 64%.

[0193] Step 2: Synthesis of compound 1-42

[0194] Succinic anhydride (122.6 mg, 1.23 mmol), DMAP (14.97 mg, 0.12 mmol), and DCM (3 mL) were added to a 50 mL flask. The mixture was stirred at room temperature for 30 min, and 1-42-1 (300 mg, 0.61 mmol) was added to the reaction mixture. The reaction progress was monitored by TLC (30% EtOAc / n-heptane). After the reaction was complete, the reaction mixture was filtered to remove solids. The filtrate was washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and the organic phase concentrated to yield approximately 300 mg of crude product. The pure product, compound 1-42, 100 mg, was isolated and obtained in a yield of 28% by using a flash column purification method (20 g silica gel, mobile phases: 500 mL of n-heptane; 300 mL of 1% EtOAc + 99% of n-heptane; 300 mL of 2% EtOAc + 98% of n-heptane; 200 mL of 5% EtOAc + 95% of n-heptane; 300 mL of 8% EtOAc + 92% of n-heptane).

[0195] The NMR results of compound 1-42 are shown in Figure 12. 1 H NMR (400MHz, d6-DMSO): δ7.18(t,J=8Hz,1H),5.35-5.33(m,1H),4.35-4.29(m,1H),4.00(t,J=8H z,2H),3.21-3.17(m,2H),2.32-2.18(m,2H),2.01-1.77(m,6H),1.56-0.84(m,37H),0.66(s,3H).

[0196] The synthesis method of compound 1-44 is as follows:

[0197] Step 1: Synthesis of compound 1-44-1

[0198] To a 50 mL flask, glutaric anhydride (286.3 mg, 2.5 mmol), DMAP (30.7 mg, 0.25 mmol), and DCM (7 mL) were added. The mixture was stirred at room temperature for 30 min, and 1-36-2 (700 mg, 1.25 mmol) was added. The reaction progress was monitored by TLC (30% EtOAc / n-heptane). After completion of the reaction, the reaction mixture was filtered to remove solids. The filtrate was washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and the organic phase concentrated to yield approximately 1.5 g of crude product. The pure product, 1-44-1, 700 mg, was isolated and obtained using a Flash column (20 g silica gel, mobile phases: 200 mL of n-heptane; 150 mL of 1% EtOAc + 99% of n-heptane; 150 mL of 2% EtOAc + 98% of n-heptane; 200 mL of 5% EtOAc + 95% of n-heptane; 500 mL of 8% EtOAc + 92% of n-heptane). 1 H NMR (400MHz, CDCl3): δ5.38-5.36(m,1H),4.70-4.63(m,1H),4.16-4.05(m, 4H),2.72-2.64(m,4H),2.35-2.31(m,2H),2.02-0.85(m,49H),0.67(s,3H).

[0199] Step 2: Synthesis of compound 1-44-2

[0200] To a 50 mL flask, 1-44-1 (500 mg, 0.74 mmol) was dissolved in DCM (5 mL). DMAP (18.2 mg, 0.15 mmol) and DCC (230.3 mg, 1.12 mmol) were added. After stirring at room temperature for 30 min, cholesterol (287.7 mg, 0.74 mmol) was added to the solution and stirred at room temperature overnight. The reaction progress was monitored by TLC (10% MeOH / DCM). After the reaction was complete, the reaction solution was filtered to remove the solids. The filtrate was washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and the organic phase concentrated to yield approximately 700 mg of crude product. The pure product, 1-44-2, 400 mg, was isolated and obtained by purification using a Flash column (12 g silica gel, mobile phases: 300 mL of n-heptane; 200 mL of 1% EtOAc + 99% n-heptane; 200 mL of 5% EtOAc + 95% n-heptane; 300 mL of 8% EtOAc + 92% n-heptane). The yield was 52%. 1 H NMR (400MHz, CDCl3): δ5.36(m,2H),4.60(m,2H),4.09-4.07(m,4H),2.36-2.29(m,9H),2.03-1.83(m,14H),1.62-0.86(m,72H),0.68(s,6H).

[0201] Step 3: Synthesis of compound 1-44

[0202] 1-44-2 (400 mg, 0.38 mmol) was added to a 50 mL flask and dissolved in DCM (12 mL). TFA (4 mL) was then added. After stirring at room temperature for 2 h, the reaction was monitored by TLC (20% EtOAc / n-heptane). After completion of the reaction, the reaction solution was concentrated and dissolved in MTBE (20 mL). The solution was then adjusted to pH 8-9 with 10% NaHCO₃ solution and pH 1-2 with 1 M dilute hydrochloric acid. The mixture was stirred for 20 min, separated, and the aqueous phase was extracted with MTBE (20 mL). The organic phases were combined, dried over anhydrous Na₂SO₄, filtered, and concentrated to yield 520 mg of the crude product. The pure product, compound 1-44, 250 mg, was isolated and obtained by purification using a Flash column (8 g silica gel, mobile phases: 200 mL of n-heptane; 100 mL of 1% EtOAc + 99% of n-heptane; 100 mL of 5% EtOAc + 95% of n-heptane; 100 mL of 10% EtOAc + 90% of n-heptane; 200 mL of 20% EtOAc + 80% of n-heptane). The yield was 67%.

[0203] The NMR results of compound 1-44 are shown in Figure 13. 1H NMR (400MHz, CDCl3): δ5.39-5.35(m,2H),4.71-4.68(m,1H),4.61-4.56(m,1H),4.19 -4,14(m,4H),2.40-2.20(m,8H),2.03-1.83(m,6H),1.65-0.85(m,72H),0.68(s,6H).

[0204] The synthesis method of compound 1-45 is as follows:

[0205] Step 1: Synthesis of compound 1-45-1

[0206] To a 100 mL flask, ethanolamine (341.6 mg, 5.59 mmol) was dissolved in DCM (30 mL). DMAP (136.6 mg, 1.12 mmol) and DCC (1.15 g, 5.59 mmol) were added. After stirring at room temperature for 30 min, 1-4 (2.8 g, 5.59 mmol) was added to the solution, and the mixture was stirred at room temperature overnight. The reaction progress was monitored by TLC (10% MeOH / EtOAc). After completion of the reaction, the reaction mixture was filtered to remove solids. The filtrate was washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and the organic phase concentrated to yield approximately 3.4 g of crude product. Flash column purification (120 g silica gel, mobile phases: 300 mL of DCM; 200 mL of 1% MeOH + 99% of DCM; 200 ml of 2% MeOH + 98% of DCM; 300 mL of 3% MeOH + 97% of DCM; 800 mL of 5% MeOH + 95% of DCM) was used to separate the pure product, 1-45-1, 2.2 g, with a yield of 73%. 1 H NMR (400MHz, CDCl3): δ6.18(s,1H),5.37-5.36(m,1H),4.62-4.58(m,1H),3.73-3.70(m,2H),3.48(s,1H),3.43-3.39(m,2H),3.06(s,1H), 2.37-2.25(m,6H),2.11-1.79(m,13H),1.61-0.85(m,43H),0.68-0.68(m,3H).

[0207] Step 2: Synthesis of compound 1-45

[0208] To a 100 mL flask, glutaric anhydride (209.81 mg, 1.84 mmol), DMAP (44.5 mg, 0.37 mmol), and DCM (10 mL) were added. The mixture was stirred at room temperature for 30 min, and 1-46-1 (1.0 g, 1.84 mmol) was added to the reaction mixture. The reaction progress was monitored by TLC (5% MeOH / DCM). After the reaction was complete, the reaction mixture was filtered to remove solids. The filtrate was washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and the organic phase concentrated to yield approximately 1.7 g of crude product. The pure product, compound 1-45, 180 mg, was isolated and obtained by purification using a flash column (120 g silica gel, mobile phases: 300 mL of DCM; 200 mL of 1% MeOH + 99% DCM; 200 ml of 2% MeOH + 98% DCM; 300 mL of 3% MeOH + 97% DCM; 300 mL of 5% MeOH + 95% DCM). The yield was 15%.

[0209] The NMR results of compound 1-45 are shown in Figure 14. 1 H NMR (400MHz, CDCl3): δ6.31(s,1H),5.37-5.36(m,1H),4.65-4.61(m,1H),4.20-4.18(m,2H), 3.56-3.52(m,2H),2.46-2.24(m,12H),2.04-1.82(m,13H),1.61-0.85(m,43H),0.68(m,3H).

[0210] The synthesis method of compound 1-47 is as follows:

[0211] To a 100 mL flask, add DCC (1.52 g, 7.37 mmol, 1.5 eq), DMAP (300.16 mg, 2.46 mmol, 0.5 eq), and sebacic acid (1.49 g, 7.37 mmol, 1.5 eq) dissolved in DCM (40 mL). Stir for 30 min. Cholesterol (1.9 g, 4.91 mmol, 1.0 eq) is added dropwise to the solution and stirred overnight at room temperature. The reaction progress is monitored by TLC (35% EtOAc / n-heptane). After completion of the reaction, filter, wash the filtrate with 5 mL of water and 5 mL of saturated brine, and dry the organic phase over anhydrous NaSO, filter, and concentrate to yield approximately 3 g of crude product. Flash column purification (80 g silica gel, loaded with 500 mL n-heptane; mobile phases: 200 mL n-heptane; 100 mL 0.5% EtOAc + 99.5% n-heptane; 100 mL 0.8% EtOAc + 99.2% n-heptane; 100 mL 1% EtOAc + 99% n-heptane; 100 mL 1.5% EtOAc + 98.5% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 100 mL 3% EtOAc + 97% n-heptane; 1000 mL 10% EtOAc + 90% n-heptane) was used to separate the pure product, compound 1-47, 1 g, with a yield of 36%.

[0212] The NMR results of compound 1-47 are shown in Figure 15. 1 H NMR (400MHz, CDCl3): δ5.38-5.36(m,1H),4.65-4.57(m,1H),2.36-2.24(m, 6H),2.03-1.95(m,2H),1.87-1.78(m,3H),1.65-0.85(m,48H),0.68(s,3H).

[0213] The synthesis method of compound 1-48 is as follows:

[0214] To a 100 mL flask, add DCC (1.04 g, 5.04 mmol, 1.3 eq), DMAP (236.9 mg, 1.94 mmol, 0.5 eq), and dissolve 1,6-diacid (1.45 g, 5.04 mmol, 1.3 eq) in DCM (30 mL). Stir for 30 min. Cholesterol (1.5 g, 3.88 mmol, 1.0 eq) is added dropwise to the solution, followed by stirring at room temperature overnight. The reaction progress is monitored by TLC (20% EtOAc / n-heptane). Upon completion, the reaction is filtered, and the filtrate is washed with 5 mL of water and 5 mL of saturated brine. The organic phase is dried over anhydrous NaSO, filtered, and concentrated to yield approximately 3 g of crude product. Flash column purification (80 g silica gel, loaded with 500 mL n-heptane; mobile phases: 200 mL n-heptane; 100 mL 0.5% EtOAc + 99.5% n-heptane; 100 mL 0.8% EtOAc + 99.2% n-heptane; 100 mL 1% EtOAc + 99% n-heptane; 100 mL 1.5% EtOAc + 98.5% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 100 mL 3% EtOAc + 97% n-heptane; 600 mL 10% EtOAc + 90% n-heptane) was used to separate and obtain the pure product, compound 1-48, 700 mg, with a yield of 28%.

[0215] The NMR results of compound 1-48 are shown in Figure 16. 1 H NMR (400MHz, CDCl3): δ5.38-5.36(m,1H),4.65-4.57(m,1H),2.36-2.24(m, 6H),2.03-1.93(m,2H),1.87-1.80(m,3H),1.65-0.85(m,60H),0.67(s,3H).

[0216] The synthesis method of compound 1-49 is as follows:

[0217] To a 100 mL flask, add DCC (1.04 g, 5.04 mmol, 1.3 eq), DMAP (236.9 mg, 1.94 mmol, 0.5 eq), and dissolve 18-diacid (1.59 g, 5.04 mmol, 1.3 eq) in DCM (30 mL). Stir for 30 min. Cholesterol (1.5 g, 3.88 mmol, 1.0 eq) is then added dropwise and stirred overnight at room temperature. The reaction progress is monitored by TLC (20% EtOAc / n-heptane). After completion, the reaction is filtered, and the filtrate is washed with 5 mL of water and 5 mL of saturated brine. The organic phase is dried over anhydrous NaSO, filtered, and concentrated to yield approximately 3 g of crude product. Flash column purification (80 g silica gel, loaded with 500 mL n-heptane; mobile phases: 200 mL n-heptane; 100 mL 0.5% EtOAc + 99.5% n-heptane; 100 mL 0.8% EtOAc + 99.2% n-heptane; 100 mL 1% EtOAc + 99% n-heptane; 100 mL 1.5% EtOAc + 98.5% n-heptane; 100 mL 2% EtOAc + 98% n-heptane; 600 mL 3% EtOAc + 97% n-heptane) was used to separate the pure product, compound 1-49, 500 mg, with a yield of 19%.

[0218] The NMR results of compound 1-49 are shown in Figure 17. 1 H NMR (400MHz, CDCl3): δ5.38-5.36(m,1H),4.62-4.58(m,1H),2.37-2.21(m, 6H),2.02-1.95(m,2H),1.87-1.82(m,3H),1.67-0.85(m,63H),0.68(s,3H).

[0219] The synthesis method of compound 1-50 is as follows:

[0220] Succinic anhydride (699.6 mg, 7.0 mmol), DMAP (328.5 mg, 2.7 mmol), and DCM (30 mL) were added to a 50 mL flask. The mixture was stirred at room temperature for 30 min, and 1-36-2 (3.0 g, 5.4 mmol) was added to the reaction mixture. The reaction progress was monitored by TLC (30% EtOAc / n-heptane). After the reaction was complete, the reaction mixture was filtered to remove solids. The filtrate was washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and the organic phase was concentrated to yield approximately 4.5 g of crude product. The pure product, compound 1-50, 2.5 g, was isolated and obtained using a flash column purification (20 g silica gel, mobile phases: 500 mL of n-heptane; 300 mL of 1% EtOAc + 99% of n-heptane; 300 mL of 2% EtOAc + 98% of n-heptane; 200 mL of 5% EtOAc + 95% of n-heptane; 1000 mL of 8% EtOAc + 92% of n-heptane).

[0221] The NMR results of compound 1-50 are shown in Figure 18. 1 H NMR (400MHz, CDCl3): δ5.39-5.36(m,1H),4.70-4.63(m,1H),4.15-4.05(m,4H),2.74-2. 63(m,4H),2.33(t,J=8Hz,2H),2.03-1.80(m,5H),1.64-0.85(m,41H),0.68-0.67(m,3H).

[0222] The synthesis method of compound 1-51 is as follows:

[0223] Step 1: Synthesis of compound 1-51-1

[0224] To a 50 mL flask, add 1-36-1 (4.0 g, 7.9 mmol) and dissolve DIPEA (2.3 g, 15.8 mmol) in DCM (40 mL). Add tert-butyl 3-aminopropionate (4.1 g, 31.6 mmol). Stir at room temperature overnight. TLC (30% EtOAc / n-heptane) monitors the reaction progress. After completion, wash with 10 mL of water and 10 mL of saturated brine, dry over anhydrous NaSO, filter, and concentrate the organic phase to yield approximately 12.1 g of crude product. The pure product, 1-51-1, 2.3 g, was isolated and obtained using a flash column (100 g silica gel, loaded with 300 mL of n-heptane; the mobile phases were: 300 mL of n-heptane; 300 mL of 0.1% EtOAc + 99.9% of n-heptane; 300 mL of 0.3% EtOAc + 99.7% of n-heptane; 1000 mL of 0.5% EtOAc + 99.5% of n-heptane).

[0225] Step 2: Synthesis of compound 1-51

[0226] Succinic anhydride (546 mg, 5.5 mmol), DMAP (256.4 mg, 2.1 mmol), and DCM (30 mL) were added to a 50 mL flask. The mixture was stirred at room temperature for 30 min, and 1-51-1 (2.4 g, 4.2 mmol) was added to the reaction mixture. The reaction progress was monitored by TLC (30% EtOAc / n-heptane). After completion of the reaction, the reaction mixture was filtered to remove solids. The filtrate was washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and the organic phase concentrated to yield approximately 2.0 g of crude product. The pure product, compound 1-51, 1.3 g, was isolated and obtained by purification using a Flash column (20 g silica gel, mobile phases: 300 mL 1% EtOAc + 99% n-heptane; 300 mL 2% EtOAc + 98% n-heptane; 200 mL 5% EtOAc + 95% n-heptane; 300 mL 8% EtOAc + 92% n-heptane; 500 mL 20% EtOAc + 80% n-heptane). The yield was 46%.

[0227] The NMR results of compound 1-51 are shown in Figure 19. 1H NMR (400MHz, CDCl3): δ5.38-5.35(m,1H),4.70-4.59(m,1H),4.16-4.07(m,4H),3.67(t,J=8Hz,1H),3.61(t,J=8Hz ,1H),2.80-2.67(m,3H),2.57-2.51(m,3H),2.34-2.31(m,2H),2.04-1.78(m,5H),1.61-0.84(m,41H),0.67(m,3H).

[0228] The synthesis method of compound 1-52 is as follows:

[0229] To a 50 mL flask, glutaric anhydride (204.53 mg, 1.79 mmol), DMAP (109.5 mg, 0.89 mmol), and DCM (10 mL) were added. The mixture was stirred at room temperature for 30 min, and 1-36-2 (1.0 g, 1.79 mmol) was added to the reaction mixture. The reaction progress was monitored by TLC (5% MeOH / DCM). After completion of the reaction, the reaction mixture was filtered to remove solids. The filtrate was washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and the organic phase concentrated to yield approximately 4.5 g of crude product. The pure product, compound 1-52, 1.1 g, was isolated and obtained using a flash column (20 g silica gel, mobile phases: 500 mL of n-heptane; 300 mL of 1% MeOH + 99% DCM; 300 mL of 1.5% MeOH + 98.5% DCM; 600 ml of 2% MeOH + 98% DCM). The yield was 92%.

[0230] The NMR results of compound 1-52 are shown in Figure 20. 1 H NMR (400MHz, CDCl3): δ5.37(m,1H),4.69-4.65(m,1H),4.14-4.02(m,4H),2. 47-2.32(m,6H),2.09-1.85(m,8H),1.58-0.85(m,46H),0.67(t,J=4Hz,3H).

[0231] The synthesis method of compound 1-53 is as follows:

[0232] To a 100 mL flask, cholesterol (1.0 g, 2.59 mmol, 1.0 eq), DMAP (315.9 mg, 2.59 mmol, 1.0 eq), and diglycolic anhydride (360.23 mg, 3.1 mmol, 1.2 eq) were dissolved in DCM (10 mL) and stirred at room temperature overnight. The reaction progress was monitored by TLC (20% EtOAc / n-heptane). Upon completion, the reaction was washed with 5 mL of water and 5 mL of saturated brine. The organic phase was dried over anhydrous NaSO, filtered, and concentrated to yield approximately 2 g of crude product. The pure product, 1-53, 500 mg, was isolated and obtained using a flash column (60 g silica gel, loaded with 500 mL of n-heptane; the mobile phases were: 200 mL of n-heptane; 100 mL of 1% EtOAc + 99% of n-heptane; 100 mL of 1.5% EtOAc + 98.5% of n-heptane; 100 mL of 2% EtOAc + 98% of n-heptane; 600 mL of 3% EtOAc + 97% of n-heptane). 1 H NMR (400MHz, CDCl3): δ5.39-5.37(m,1H),4.73-4.69(m,1H),4.20(s,2H),2.35-2. 33(m,2H),2.03-1.94(m,2H),1.90-1.80(m,3H),1.63-0.85(m,36H),0.67(s,3H).

[0233] The synthesis method of compound 1-55 is as follows:

[0234] Step 1: Synthesis of compound 1-55-1

[0235] To a 50 mL flask, 1-52 (473.1 mg, 0.7 mmol) was dissolved in DCM (5 mL). DMAP (18.2 mg, 0.15 mmol) and DCC (230.3 mg, 1.12 mmol) were added. After stirring at room temperature for 30 min, SM-102 (500 mg, 0.7 mmol) was added to the solution and stirred at room temperature overnight. The reaction progress was monitored by TLC (10% MeOH / DCM). After completion of the reaction, the reaction mixture was filtered to remove solids. The filtrate was washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and the organic phase concentrated to yield approximately 700 mg of crude product. The pure product, 1-55-1, 550 mg, was isolated and obtained using a flash column (12 g silica gel, mobile phases: 300 mL of n-heptane; 200 mL of 1% EtOAc + 99% n-heptane; 200 mL of 5% EtOAc + 95% n-heptane; 300 mL of 8% EtOAc + 92% n-heptane). The yield was 57%.

[0236] Step 2: Synthesis of compound 1-55

[0237] 1-55-1 (550 mg, 0.4 mmol) was added to a 50 mL flask and dissolved in DCM (6 mL). TFA (3 mL) was then added. After stirring at room temperature for 2 h, the reaction was monitored by TLC (20% EtOAc / n-heptane). After completion of the reaction, the reaction solution was concentrated and then dissolved in MTBE (20 mL). The solution was then adjusted to pH 8-9 with 10% NaHCO₃ solution and pH 1-2 with 1 M dilute hydrochloric acid. The mixture was stirred for 20 min, separated, and the aqueous phase was extracted with 20 mL MTBE. The organic phases were combined, dried over anhydrous Na₂SO₄, filtered, and concentrated to yield 520 mg of a crude product. The pure product, compound 1-55, 200 mg, was isolated and obtained by purification using a flash column (8 g silica gel, mobile phases: 200 mL of n-heptane; 100 mL of 1% EtOAc + 99% of n-heptane; 100 mL of 5% EtOAc + 95% of n-heptane; 100 mL of 10% EtOAc + 90% of n-heptane; 200 mL of 20% EtOAc + 80% of n-heptane). The yield was 38%.

[0238] The NMR results of compound 1-55 are shown in Figure 21. 1 H NMR (400MHz, CDCl3): δ5.37-5.30(m,1H),4.88-4.82(m,1H),4.69-4.61(m,1H),4.42-4.39(m,2H),4.18-4.04(m,6H),3.40-3.38(m,2 H),3.25-3.16(m,4H),2.46-2.38(m,4H),2.34-2.27(m,6H),2.04-1.94(m,4H),1.88-1.79(m,3H),1.73-0.85(m,105H),0.67(s,3H).

[0239] The synthesis method of compound 1-56 is as follows:

[0240] Step 1: The synthesis method of compound 1-56-1 is as follows:

[0241] A 500 mL flask was charged with N-methylethanolamine (3.4 g, 45.3 mmol) and DCM (50 mL). The mixture was cooled to 0-5°C in an ice-salt bath. A solution of TBDPSCl (12.44 g, 45.3 mmol) in DCM (50 mL) was slowly added dropwise. After addition, the mixture was stirred at room temperature for 2 h. The reaction progress was monitored by TLC (10% MeOH / DCM). After completion of the reaction, 30 mL of water was added to quench the reaction. The mixture was separated and the aqueous phase was extracted with DCM (50 mL x 2). The combined organic phases were washed with 50 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated to yield approximately 12 g of crude product. The product 1-56-1 was purified by flash column (200 g silica gel, loaded with 300 mL n-heptane; the mobile phases were: 300 mL n-heptane; 300 mL 0.1% EtOAc + 99.9% n-heptane; 300 mL 0.3% EtOAc + 99.7% n-heptane; 300 mL 1% EtOAc + 99% n-heptane; 300 mL 1% EtOAc + 99% n-heptane; 300 mL 0.2% MeOH + 99.8% DCM; 500 mL 0.3% MeOH + 99.7% DCM) to obtain pure product 1-56-1,9 g with a yield of 63%.

[0242] Step 2: Synthesis of compound 1-56-2

[0243] To a 100 mL flask, 1-56-1 (5.0 g, 16 mmol), potassium carbonate (5.5 g, 39.9 mmol), and 3-chloro-1,2-propanediol (2.12 g, 19.1 mmol) were added and dissolved in isopropanol (15 mL). The mixture was heated to 80°C and stirred overnight. The reaction progress was monitored by TLC (10% MeOH / DCM). After completion, the reaction was filtered and the organic phase was concentrated to yield approximately 10.1 g of crude product. The pure product, 1-56-2, 3.6 g, was isolated and obtained by purification using a Flash column (100 g silica gel, 200 mL 0.1% MeOH + 99.9% DCM; 200 mL 0.2% MeOH + 99.8% DCM, 200 mL 0.3% MeOH + 99.7% DCM; 200 mL 0.5% MeOH + 99.5% DCM; 300 mL 1% MeOH + 99% DCM). The yield was 58%.

[0244] Step 3: Synthesis of compound 1-56-4

[0245] A 50 mL flask was charged with 1-56-3 (1.0 g, 2.7 mmol), DCC (556.8 mg, 2.7 mmol), DMAP (82.4 mg, 0.67 mmol), and DCM (3 mL). The mixture was stirred at room temperature for 30 min, and 1-56-2 (523 mg, 1.35 mmol) was added to the reaction mixture. The reaction progress was monitored by TLC (30% EtOAc / n-heptane). After completion of the reaction, the reaction mixture was filtered to remove solids. The filtrate was washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and the organic phase concentrated to yield approximately 1.5 g of crude product. The pure product, 1-56-4, 1.2 g, was isolated and obtained using a Flash column (50 g silica gel, mobile phases: 200 mL of n-heptane; 150 mL of 1% EtOAc + 99% of n-heptane; 150 mL of 2% EtOAc + 98% of n-heptane; 200 mL of 5% EtOAc + 95% of n-heptane; 500 mL of 8% EtOAc + 92% of n-heptane).

[0246] Step 4: Synthesis of compound 1-56-5

[0247] To a 50 mL flask, 1-56-4 (1.0 g, 0.92 mmol) was dissolved in THF (5 mL). The mixture was cooled to -5°C in an ice-salt bath, and TBAF (717.9 mg, 2.75 mmol) was added. The mixture was stirred at room temperature overnight. The reaction progress was monitored by TLC (10% MeOH / DCM). Upon completion, the reaction was quenched by the addition of 5 mL of aqueous ammonium chloride. The layers were separated, and the aqueous phase was extracted with EtOAc (10 mL x 2). The combined organic phases were washed with 5 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and concentrated to yield approximately 700 mg of the crude product. The pure product, 1-56-5, 350 mg, was isolated and obtained using a Flash column (12 g silica gel, mobile phases: 300 mL of n-heptane; 200 mL of 1% EtOAc + 99% of n-heptane; 200 mL of 5% EtOAc + 95% of n-heptane; 200 mL of 8% EtOAc + 92% of n-heptane; 200 mL of 15% EtOAc + 85% of n-heptane; 400 mL of 30% EtOAc + 70% of n-heptane). The yield was 45%.

[0248] Step 5: Synthesis of compound 1-56-7

[0249] To a 50 mL flask were added 1-56-6 (113.9 mg, 0.17 mmol), DCC (35 mg, 0.17 mmol), DMAP (10.4 mg, 0.08 mmol), and DCM (5 mL). The mixture was stirred at room temperature for 30 min, and 1-56-5 (140 mg, 0.17 mmol) was added. The reaction progress was monitored by TLC (10% MeOH / DCM). After completion of the reaction, the reaction mixture was filtered to remove the solids. The filtrate was washed with 5 mL of water and 5 mL of saturated brine, dried over anhydrous Na2SO4, filtered, and the organic phase concentrated to yield approximately 350 mg of crude product. The pure product, 1-56-7, 350 mg, was isolated and obtained using a Flash column (20 g silica gel, mobile phases: 100 mL of n-heptane; 100 mL of 1% EtOAc + 99% n-heptane; 100 mL of 2% EtOAc + 98% n-heptane; 100 mL of 5% EtOAc + 95% n-heptane; 100 mL of 8% EtOAc + 92% n-heptane; 200 mL of 20% EtOAc + 80% n-heptane). The yield was 60%.

[0250] Step 6: Synthesis of compound 1-56

[0251] 1-56-7 (180 mg, 0.12 mmol) was added to a 50 mL flask and dissolved in EtOAc (1 mL). 4 M hydrochloric acid ethyl acetate solution (1 mL) was added. After stirring at room temperature for 2 h, the reaction progress was monitored by TLC (20% MeOH / DCM). The organic phase was concentrated to obtain 210 mg of crude product. The product was purified using a Flash column (8 g silica gel, mobile phases were: 100 mL of n-heptane; 100 mL of 5% EtOAc + 95% n-heptane; 100 mL of 10% EtOAc + 90% n-heptane; 100 mL of 20% EtOAc + 80% n-heptane; 200 mL of 30% EtOAc + 70% n-heptane) to obtain the pure product, compound 1-56, 50 mg, in a yield of 29%.

[0252] The NMR results of compound 1-56 are shown in Figure 22. 1 H NMR (400MHz, CDCl3): δ5.39-5.34(m,1H),5.15(m,1H),4.69-4.62(m,1H),4.38-4.34(m,1H),4.25-4.04(m,15H) ,2.67-2.55(m,5H),2.42-2.26(m,17H),2.05-1.92(m,5H),1.89-1.84(m,3H),1.69-0.80(m,150H),0.67(m,3H).

[0253] DODMA, DSPC, cholesterol, PEG2000-DMG, etc. used in the following examples were all from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd., and Luc-mRNA was from Shanghai Zhaowei Technology Development Co., Ltd.

[0254] Example 2. Preparation of lipid nanoparticles TMF1 (containing 20% ​​compound 1-1)

[0255] 1. Dissolve DODMA (cationic lipid), DSPC (auxiliary lipid), cholesterol (auxiliary lipid), PEG2000-DMG (auxiliary lipid) and compound 1-1 (GOLD lipid) in ethanol to obtain oil phase (ethanol phase) stock solutions with molar ratios of 37.0%, 7.5%, 34.2%, 1.3% and 20.0%, respectively.

[0256] 2. Dilute the luciferase-expressing mRNA stock solution to 0.09 mg / mL using pH 4 citric acid buffer to obtain an aqueous phase.

[0257] 3. The oil phase (ethanol phase) containing the five lipid mixtures and the mRNA-containing aqueous phase were quickly mixed at a volume ratio of 1:3 to prepare TMF1 with a total lipid to mRNA weight ratio of 22.5:1. The sample was then concentrated using an ultrafiltration tube with a molecular weight cutoff of 100 kd, followed by elution with pH 7.4 PBS buffer, and finally replaced with 120 mg / mL sucrose PBS buffer to obtain the final sample.

[0258] Example 3. Preparation of lipid nanoparticles TMF2 (containing 5% compound 1-1):

[0259] 1. Dissolve DODMA (cationic lipid), DSPC (auxiliary lipid), cholesterol (auxiliary lipid), PEG2000-DMG (auxiliary lipid) and compound 1-1 (GOLD lipid) in ethanol to obtain oil phase (ethanol phase) stock solutions with molar ratios of 44.0%, 8.9%, 40.6%, 1.5% and 5.0%, respectively.

[0260] 2. Dilute the luciferase-expressing mRNA stock solution to 0.09 mg / mL using pH 4 citric acid buffer to obtain an aqueous phase.

[0261] 3. The oil phase (ethanol phase) containing the five lipid mixtures and the mRNA-containing aqueous phase were quickly mixed at a volume ratio of 1:3 to prepare TMF2 with a total lipid to mRNA weight ratio of 22.6:1. The sample was then concentrated using an ultrafiltration tube with a molecular weight cutoff of 100 kd, followed by elution with pH 7.4 PBS buffer, and finally replaced with 120 mg / mL sucrose PBS buffer to obtain the final sample.

[0262] Example 4. Preparation of lipid nanoparticles TMF3 (containing 20% ​​compound 1-2):

[0263] 1. Dissolve DODMA (cationic lipid), DSPC (auxiliary lipid), cholesterol (auxiliary lipid), PEG2000-DMG (auxiliary lipid) and compound 1-2 (GOLD lipid) in ethanol to obtain oil phase (ethanol phase) stock solutions with molar ratios of 37.0%, 7.5%, 34.2%, 1.3% and 20.0%, respectively.

[0264] 2. Dilute the luciferase-expressing mRNA stock solution to 0.09 mg / mL using pH 4 citric acid buffer to obtain an aqueous phase.

[0265] 3. The oil phase (ethanol phase) containing the five lipid mixtures and the mRNA-containing aqueous phase were quickly mixed at a volume ratio of 1:3 to prepare TMF3 with a total lipid to mRNA weight ratio of 24.2:1. The sample was then concentrated using an ultrafiltration tube with a molecular weight cutoff of 100 kd, followed by elution with pH 7.4 PBS buffer, and finally replaced with 120 mg / mL sucrose PBS buffer to obtain the final sample.

[0266] Example 5. Preparation of lipid nanoparticles TMF4 (containing 20% ​​compound 1-3):

[0267] 1. Dissolve DODMA (cationic lipid), DSPC (auxiliary lipid), sitosterol (auxiliary lipid), PEG2000-DMG (auxiliary lipid) and compounds 1-3 (GOLD lipid) in ethanol to obtain oil phase (ethanol phase) stock solutions with molar ratios of 37.0%, 7.5%, 34.2%, 1.3% and 20.0%, respectively.

[0268] 2. Dilute the luciferase-expressing mRNA stock solution to 0.09 mg / mL using pH 4 citric acid buffer to obtain an aqueous phase.

[0269] 3. The oil phase (ethanol phase) containing the five lipid mixtures and the mRNA-containing aqueous phase were quickly mixed at a volume ratio of 1:3 to prepare TMF4 with a total lipid to mRNA weight ratio of 24.5:1. The sample was then concentrated using an ultrafiltration tube with a molecular weight cutoff of 100 kd, followed by elution with pH 7.4 PBS buffer, and finally replaced with 120 mg / mL sucrose PBS buffer to obtain the final sample.

[0270] Example 6. Preparation of lipid nanoparticles TMF5 (containing 20% ​​compounds 1-5):

[0271] 1. Dissolve DODMA (cationic lipid), DSPC (auxiliary lipid), cholesterol (auxiliary lipid), PEG2000-DMG (auxiliary lipid) and compounds 1-5 (GOLD lipid) in ethanol to obtain oil phase (ethanol phase) stock solutions with molar ratios of 37.0%, 7.5%, 34.2%, 1.3% and 20.0%, respectively.

[0272] 2. Dilute the luciferase-expressing mRNA stock solution to 0.09 mg / mL using pH 4 citric acid buffer to obtain an aqueous phase.

[0273] 3. The oil phase (ethanol phase) containing the five lipid mixtures and the mRNA-containing aqueous phase were quickly mixed at a volume ratio of 1:3 to prepare TMF5 with a total lipid to mRNA weight ratio of 24.8:1. The sample was then concentrated using an ultrafiltration tube with a molecular weight cutoff of 100 kd, followed by elution with pH 7.4 PBS buffer, and finally replaced with 120 mg / mL sucrose PBS buffer to obtain the final sample.

[0274] Example 7. Preparation of lipid nanoparticles TMF6 (containing 20% ​​compounds 1-6):

[0275] 1. Dissolve DODMA (cationic lipid), DOPE (helper lipid), cholesterol (helper lipid), PEG2000-DMG (helper lipid), and compounds 1-6 (GOLD lipid) in ethanol to obtain oil phase (ethanol phase) stock solutions with molar ratios of 37.0%, 7.5%, 34.2%, 1.3%, and 20.0%, respectively.

[0276] 2. Dilute the luciferase-expressing mRNA stock solution to 0.09 mg / mL using pH 4 citric acid buffer to obtain an aqueous phase.

[0277] 3. The oil phase (ethanol phase) containing the five lipid mixtures and the mRNA-containing aqueous phase were quickly mixed at a volume ratio of 1:3 to prepare TMF6 with a total lipid to mRNA weight ratio of 24.7:1. The sample was then concentrated using an ultrafiltration tube with a molecular weight cutoff of 100 kd, followed by elution with pH 7.4 PBS buffer, and finally replaced with 120 mg / mL sucrose PBS buffer to obtain the final sample.

[0278] Example 8. Preparation of lipid nanoparticles TMF7 (containing 38.5% compound 1-1):

[0279] 1. Dissolve DODMA (cationic lipid), DSPC (auxiliary lipid), compound 1-1 (GOLD lipid) and PEG2000-DMG (auxiliary lipid) in ethanol to obtain oil phase (ethanol phase) stock solutions with molar ratios of 50%, 10%, 38.5% and 1.5%

[0280] 2. Dilute the luciferase-expressing mRNA stock solution to 0.3 mg / mL using pH 4 citric acid buffer to obtain an aqueous phase.

[0281] 3. The oil phase (ethanol phase) containing the four lipid mixtures and the mRNA-containing aqueous phase were quickly mixed at a volume ratio of 1:3 to prepare TMF7 with a total lipid to mRNA weight ratio of 24.9:1. The sample was then concentrated using an ultrafiltration tube with a molecular weight cutoff of 100 kd, followed by elution with pH 7.4 PBS buffer, and finally replaced with 120 mg / mL sucrose PBS buffer to obtain the final sample.

[0282] Example 9. Preparation of lipid nanoparticles TMF8 (containing 25% compound 1-1):

[0283] 1. Dissolve DODMA (cationic lipid), DSPC (auxiliary lipid), and compound 1-1 (GOLD lipid) in ethanol to obtain oil phase (ethanol phase) stock solutions with molar ratios of 50%, 25%, and 25%.

[0284] 2. Dilute the luciferase-expressing mRNA stock solution to 0.09 mg / mL using pH 4 citric acid buffer to obtain an aqueous phase.

[0285] 3. The oil phase (ethanol phase) containing the three lipid mixtures and the aqueous phase containing mRNA were quickly mixed at a volume ratio of 1:3 to prepare TMF8 with a total lipid to mRNA weight ratio of 30.7:1. The sample was then concentrated using an ultrafiltration tube with a molecular weight cutoff of 100 kd, followed by elution with pH 7.4 PBS buffer, and finally replaced with 120 mg / mL sucrose PBS buffer to obtain the final sample.

[0286] Example 10. Preparation of lipid nanoparticles TMF9 (containing 40% compound 1-1):

[0287] 1. Dissolve DODMA (cationic lipid) and compound 1-1 (GOLD lipid) in ethanol to obtain a 60% and 40% oil phase (ethanol phase) stock solution.

[0288] 2. Dilute the luciferase-expressing mRNA stock solution to 0.09 mg / mL using pH 4 citric acid buffer to obtain an aqueous phase.

[0289] 3. The oil phase (ethanol phase) containing the two lipid mixtures and the aqueous phase containing mRNA were quickly mixed at a volume ratio of 1:3 to prepare TMF9 with a total lipid to mRNA weight ratio of 31.4:1. The sample was then concentrated using an ultrafiltration tube with a molecular weight cutoff of 100 kd, followed by elution with pH 7.4 PBS buffer, and finally replaced with 120 mg / mL sucrose PBS buffer to obtain the final sample.

[0290] Example 11. Preparation of lipid nanoparticles TMF10 (containing 38.5% compound 1-1 and 10% compound 1-56):

[0291] 1. Dissolve DODMA (cationic lipid), compound 1-56 (GOLD lipid), compound 1-1 (GOLD lipid) and PEG2000-DMG (auxiliary lipid) in ethanol to obtain oil phase (ethanol phase) stock solutions with molar ratios of 50%, 10%, 38.5% and 1.5%.

[0292] 2. Dilute the luciferase-expressing mRNA stock solution to 0.09 mg / mL using pH 4 citric acid buffer to obtain an aqueous phase.

[0293] 3. The oil phase (ethanol phase) containing the four lipid mixtures and the aqueous phase containing mRNA were quickly mixed at a volume ratio of 1:3 to prepare TMF10 with a total lipid to mRNA weight ratio of 26.2:1. The sample was then concentrated using an ultrafiltration tube with a molecular weight cutoff of 100 kd, followed by elution with pH 7.4 PBS buffer, and finally replaced with 120 mg / mL sucrose PBS buffer to obtain the final sample.

[0294] Example 12. Preparation of lipid nanoparticles TMF38 (containing 100% compound 1-56):

[0295] 1. Dissolve compound 1-56 (GOLD lipid) in ethanol to obtain a 100% oil phase (ethanol phase) stock solution.

[0296] 2. Dilute the luciferase-expressing mRNA stock solution to 0.09 mg / mL using pH 4 citric acid buffer to obtain an aqueous phase.

[0297] 3. The oil phase (ethanol phase) containing the lipid mixture and the aqueous phase containing the mRNA were quickly mixed at a volume ratio of 1:3 to prepare TMF38 with a total lipid to mRNA weight ratio of 24.8:1. The sample was then concentrated using an ultrafiltration tube with a molecular weight cutoff of 100 kd, followed by elution with pH 7.4 PBS buffer, and finally replaced with 120 mg / mL sucrose PBS buffer to obtain the final sample.

[0298] Lipid nanoparticles TMF11-37 were prepared in a similar manner to lipid nanoparticles TMF7 using the corresponding starting materials. TMF38 was prepared in a similar manner to TMF9 using the corresponding starting materials. Detailed information on the lipid nanoparticle components is provided in Table 3.

[0299] Table 3. List of components of lipid nanoparticles prepared from representative GOLD lipid compounds

[0300] Example 13: Detection of lipid nanoparticles

[0301] The size and polydispersity index (PDI) of the lipid nanoparticles were determined using dynamic light scattering (DLS) at a 90° side scattering angle using a NanoBrook 90plus PLAS (Brookhaven Instruments, US). The results are shown in Tables 4, 5, and 6. The content and encapsulation efficiency of the lipid nanoparticles were determined using the Qubit RNA HS Quantification Kit (ThermoFisher Scientific, UK) according to the manufacturer's instructions. The results are shown in Tables 4, 5, and 6.

[0302] Table 4. Characterization data of lipid nanoparticles prepared from representative GOLD lipid compounds

[0303] Table 5. Characterization data of lipid nanoparticles prepared from representative GOLD lipid compounds

[0304] Table 6. Characterization data of different lipid nanoparticles prepared by GOLD lipids 1-56

[0305] Example 14: Delivery system organ distribution detection

[0306] Liposomes prepared in Examples 1, 2, 3, 4, 5, 6, 7, 8, 10, and 12 were injected into 6-8 week old female ICR mice via the epicanthal venous plexus at the fundus at a dose of 0.5 mg / kg. Four hours after administration, 15 mg / mL of D-luciferin potassium salt was injected intraperitoneally at a dose of 150 mg / kg. Ten minutes after injection of the luciferase substrate, the mice were placed in an in vivo imaging system (IVIS Lumina XRMS Series III, PerkinElmer) to observe the fluorescence intensity and distribution in the mice. The mice were then sacrificed, and their organs (heart, liver, spleen, lungs, and kidneys) were isolated and imaged in vitro to observe the fluorescence intensity and distribution in different organs. The organ distribution of Luc-mRNA delivered by representative lipid compounds is shown in Tables 7 and 8 and Figure 23. The selectivity data of spleen and liver are summarized as shown in Figure 24. As long as the ratio of spleen to liver is higher than 1, it is shown that the selectivity of spleen is better, and the effect of spleen targeting can be achieved. The higher the ratio, the better the spleen targeting effect. Among them, lipid nanoparticles TMF36, TMF17, TMF32 and TMF35 have the highest selectivity for spleen and liver. 4 hours after intravenous administration to mice, the ratio of the mean fluorescence intensity of TMF36, TMF17, TMF32 and TMF35 in spleen and liver (spleen / liver) is 57.1, 59.7, 61.7 and 80.3 respectively. The mean fluorescence intensity in different organs represents the delivery efficiency of the corresponding delivery system in different organs. The lipid nanoparticles of the present application can successfully deliver nucleic acid molecules to the spleen and express them, and the delivery efficiency for the spleen is significantly higher than the delivery efficiency for the liver and other tissues / organs.

[0307] Table 7. Expression intensity of Luc-mRNA delivered by lipid nanoparticles prepared with representative GOLD lipids.

[0308] Table 8. Luc-mRNA expression intensity delivered by different lipid nanoparticles prepared with GOLD lipid 1-56

[0309] The above detailed description of the present application is intended to enable persons familiar with the technology in this field to understand the mechanism and content of the present application and implement it. It does not limit the scope of protection of the present application. Any equivalent changes or modifications made according to the spirit of the present application should be included in the scope of protection of the present application. Industrial Applicability

[0310] The organ-specific delivery composition for nucleic acids provided in this application can specifically deliver preventive / therapeutic agents (especially nucleic acid components) to target organs, and in particular can preferentially deliver to organs other than the liver, providing more options for the delivery of nucleic acid drugs, gene drugs, vaccines, etc., and is of great significance for the development and application of nucleic acid preventive and therapeutic agents.

Claims

1. A lipid nanoparticle for organ-targeted delivery, comprising a targeted lipid delivery system lipid, wherein the targeted lipid delivery system lipid is selected from one or more combinations of ionizable anionic steroids and / or ionizable anionic polymer-conjugated lipids.

2. The lipid nanoparticle according to claim 1, wherein the ionizable anionic steroid is selected from the compound of the following general formula I or a pharmaceutically acceptable salt, prodrug, stereoisomer or deuterated derivative thereof: Among them L 1 is absent, a carbon-carbon single bond, a carbon-carbon double bond, a carbon-carbon triple bond, or -O(C=O)-, -(C=O)O-, -C(=O)-, -O(C=O)O-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、-NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-;Q 1 is absent or is a C1-C8 straight or branched hydrocarbon group; L 2 is absent, a carbon-carbon single bond, a carbon-carbon double bond, a carbon-carbon triple bond, or -O(C=O)-, -(C=O)O-, -C(=O)-, -O(C=O)O-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、-NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-;Q 2 is absent or is a C1-C8 straight or branched hydrocarbon group; L 3 is absent, a carbon-carbon single bond, a carbon-carbon double bond, a carbon-carbon triple bond, or -O(C=O)-, -(C=O)O-, -C(=O)-, -O(C=O)O-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、-NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-;Q 3 is absent or is a C1-C8 straight or branched hydrocarbon group; R a is H or deuterium or C1-C8 straight chain or branched chain hydrocarbon group or C1-C8 straight chain or branched chain hydrocarbon group substituted with carboxyl; or -Q 4 -M; where Q 4 is absent or is a C1-C8 straight or branched hydrocarbon group, M is x is 0, 1, or 2; R is X is C, O, or NR b or S; R b It is H or deuterium or a C1-C8 straight or branched hydrocarbon group.

3. The lipid nanoparticle according to claim 2, wherein the ionizable anionic steroid is selected from the compounds in Table 1 or pharmaceutically acceptable salts, prodrugs, stereoisomers or deuterated derivatives thereof.

4. The lipid nanoparticle according to claim 1, wherein the ionizable anionic polymer-conjugated lipid is selected from the compound of the following general formula II or a pharmaceutically acceptable salt, prodrug, stereoisomer or deuterated derivative thereof: L 1 is absent, a carbon-carbon single bond, a carbon-carbon double bond, a carbon-carbon triple bond, or -O(C=O)-, -(C=O)O-, -C(=O)-, -O(C=O)O-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、-NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-; Q is absent or a C1-C8 linear or branched hydrocarbon group; R a is H or deuterium or a C1-C8 straight or branched hydrocarbon group or a C1-C8 straight or branched hydrocarbon group substituted with a carboxyl group; X is C or N; R is Z is C, O, or NR b or S; Y 1 or Y 2 One of the is absent, a carbon-carbon single bond, a carbon-carbon double bond, a carbon-carbon triple bond, or -O(C=O)-, -C(OH)-, -(C=O)O-, -C(=O)-, -O(C=O)O-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、-NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-, and Y 1 or Y 2 The other is absent, a carbon-carbon single bond, a carbon-carbon double bond, a carbon-carbon triple bond, or -O(C=O)-, -C(OH)-, -(C=O)O-, -C(=O)-, -O(C=O)O-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、-NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-;R a is H or deuterium or a C1-C8 straight or branched hydrocarbon group or a C1-C8 straight or branched hydrocarbon group substituted with a carboxyl group; G 1 and G 2 Each is independently absent or substituted C1-C12 straight or branched hydrocarbon group; R 1 and R 2 Each is independently a substituted C6-C24 straight or branched hydrocarbon group; x is 0, 1, or 2.

5. The lipid nanoparticle according to claim 4, characterized in that The ionizable anionic polymer-conjugated lipid is selected from the compounds in Table 2 or pharmaceutically acceptable salts, prodrugs, stereoisomers or deuterated derivatives thereof.

6. The lipid nanoparticle according to claim 1, characterized in that The lipid nanoparticles further include auxiliary lipids, which are selected from one or more combinations of phospholipids, steroids, polymer-conjugated lipids, and / or modified lipids.

7. The lipid nanoparticle according to claim 6, characterized in that The phospholipid is selected from one or a combination of more than one of DOPE, DSPC, DPPC, DMPC, DOPC, POPC and SM.

8. The lipid nanoparticle according to claim 6, characterized in that The steroid is selected from any one or a combination of more than one of cholesterol, sitosterol, stigmasterol and ergosterol.

9. The lipid nanoparticle according to claim 6, characterized in that The polymer-conjugated lipid is selected from one or more of polyethylene glycol-conjugated lipids, polylactic acid-conjugated lipids, polyamide-conjugated lipids, cationic polymer-conjugated lipids, polysarcosine pSar-conjugated lipids, polylactic acid-glycolic acid copolymer PLGA-conjugated lipids, polyamino acid-conjugated lipids, polypeptide-conjugated lipids, and poly-peptide-conjugated lipids.

10. The lipid nanoparticle according to claim 9, characterized in that The polyethylene glycol-conjugated lipid is selected from any one or a combination of more than one of PEG1000-DMG, PEG5000-DMG, PEG2000-DMG and PEG2000-DSPE.

11. The lipid nanoparticle according to claim 6, characterized in that The modified lipid is selected from lipids modified by any one or a combination of small molecule compounds, vitamins, carbohydrates, peptides, proteins, nucleic acid lipopolysaccharides, inorganic molecules or particles, metal ions or particles.

12. The lipid nanoparticle according to claim 6, characterized in that The lipid nanoparticles further include a cationic lipid, wherein the cationic lipid is selected from one or more combinations of permanent cationic lipids and / or ionizable cationic lipids.

13. The lipid nanoparticle according to claim 12, characterized in that The permanent cationic lipid is selected from one or a combination of more than one of DOTAP, DODMA, DSTAP, DMTAP, DDA, and DOBAQ.

14. The lipid nanoparticle according to claim 12, characterized in that The ionizable cationic lipid is selected from one or more of SM-102, Lipid 5, A6, DC-chol, C12-200, CKK-E12, 5A2-SC8, G0-C14, OF-2, 306Oi10, OF-Deg-Lin, 92-O17S, OF-C4-Deg-Lin, A18-iso5-2DC18, TT3, FTT5, BAMEA-O16B, Vc-Lipid, C14-4, Lipid 14, 4A3-Cit, and ssPalmO-Phe.

15. The lipid nanoparticle according to claim 12, characterized in that The molar ratio of the targeted delivery system lipid, auxiliary lipid and cationic lipid is (0.1-1): (0.5-2):

1.

16. The lipid nanoparticle according to any one of claims 1 to 15, characterized in that The lipid nanoparticles are targeted to the following organs: lung, heart, brain, spleen, lymph nodes, bone, skeletal muscle, stomach, small intestine, large intestine / colorectum, kidney, bladder, breast, testicle, ovary, uterus, thymus, brainstem, cerebellum, cerebrum, spinal cord, eye, ear, tongue or skin, preferably to the spleen.

17. A composition comprising a therapeutic or prophylactic agent and the lipid nanoparticle of any one of claims 1 to 16.

18. The composition according to claim 17, characterized in that The preventive or therapeutic agent is selected from any one of nucleic acids, proteins, polypeptides, small molecule compounds, and cells, or a combination of more than one of them.

19. The composition according to claim 17, characterized in that The mass ratio of lipid nanoparticles to the preventive or therapeutic agent is 10:1 to 100:

1.

20. The composition according to claim 17, characterized in that The average particle size of the composition is 20 nm to 600 nm.

21. The composition according to claim 17, characterized in that The polydispersity index PDI of the composition is from 0.001 to 0.

5.

22. The composition according to claim 18, characterized in that The nucleic acid is selected from single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, short isomers, plasmid DNA, complementary DNA / cDNA, antisense nucleic acid molecules / ASO, small interfering nucleic acid / siRNA, small activating nucleic acid / saRNA, asymmetric interfering nucleic acid / aiRNA, micronucleic acid / miRNA, micronucleic acid agonist / miRNA agomir, micronucleic acid inhibitor / miRNA antagomir, Dicer enzyme substrate nucleic acid / dsRNA, small hairpin nucleic acid / shRNA, transfer RNA / tRNA, messenger RNA / mRNA, circular RNA / circRNA, self-replicating mRNA / samRNA, and aptamer / aptamer.

23. The composition of claim 22, wherein the therapeutic or prophylactic agent comprises at least one mRNA encoding an antigen or a fragment thereof or an epitope thereof, or an mRNA encoding a therapeutic protein.

24. The composition of claim 23, wherein the mRNA is selected from a monocistronic mRNA or a polycistronic mRNA.

25. The composition of claim 23, wherein the mRNA comprises one or more functional nucleotide analogs or nucleotide chemical modifications.

26. The composition according to claim 25, wherein the functional nucleotide analogue is selected from one or a combination of locked nucleic acid (LNA), peptide nucleic acid (PNA), or morpholino oligonucleotide nucleic acid mimetics or functional analogues.

27. The composition of claim 25, wherein the nucleotide chemical modifications are selected from one or more of the following modifications: (1) modification of the backbone connecting nucleotides in the nucleotide sequence of the nucleic acid molecule; (2) modification of the ribose sugar in the nucleotide sequence of the nucleic acid molecule; (3) Modification of bases in the nucleotide sequence of the nucleic acid molecule.

28. The composition of claim 27, wherein the backbone modification is a phosphorothioate bond.

29. The composition according to claim 27, wherein the modification of the ribose is selected from one or a combination of 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose.

30. The composition of claim 25, wherein the nucleotide chemical modification is selected from the group consisting of 5-methylcytosine, pseudouridine, 1-methylpseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl -pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2 -aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylaminoformyladenosine, N6-threonylaminoformyladenosine, 2-methylthio-N6-threonylaminoformyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wyosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, or a combination of more than one thereof.

31. The composition of claim 23, wherein the antigen is a pathogenic antigen.

32. The composition according to claim 31, wherein the pathogenic antigen is selected from any one or a combination of more than one of tumor-associated antigens or pathogenic microorganism antigens.

33. The composition according to any one of claims 17 to 32, characterized in that The composition is targeted to the following organs: lung, heart, brain, spleen, lymph node, bone, skeletal muscle, stomach, small intestine, large intestine / colorectum, kidney, bladder, breast, testicle, ovary, uterus, thymus, brainstem, cerebellum, cerebrum, spinal cord, eye, ear, tongue or skin; preferably, it is targeted to the spleen.

34. Use of the composition according to any one of claims 17 to 33 in the preparation of a medicament.

35. A drug characterized in that Comprising the composition according to any one of claims 17 to 33 and pharmaceutically acceptable excipients.

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