Lipid material for nucleic acid delivery and application thereof

Abstract

The invention relates to a drug delivery material and an application thereof. In particular, provided are nanoparticle compositions comprising a lipid component comprising a compound of Formula I, or a salt or isomer thereof. The nanoparticle composition provided by the invention can selectively regulate the liver or spleen, and can efficiently transfect T cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to a lipid material for nucleic acid delivery and its application. Background Technology

[0002] Gene and cell therapy has become a new paradigm for disease treatment and a new focus of drug development. Among these, the safe and efficient delivery of nucleic acid drugs, including pDNA, mRNA, siRNA, and microRNA, is crucial to their clinical efficacy. Lipid nanoparticles (LNPs), as the fastest-growing nucleic acid delivery vector, have seen four drugs (Onpattro®, Comirnaty®, Spikevax®, and mRESVIA®) approved for marketing as of September 2025, fully validating the clinical application value of this technology.

[0003] Despite significant progress in LNP technology, its clinical translation still faces two major bottlenecks: first, its targeting ability in extrahepatic tissues is limited, with the vast majority of LNPs being non-specifically captured by the liver after intravenous administration, making it difficult to effectively reach other target tissues; second, its endosome escape efficiency is low, with studies showing that only about 2%-5% of the administered RNA successfully enters the cytoplasm to exert its effect (Gilleron et al., Nat. Biotechnol. 31:638-646, 2013), which severely limits efficacy and may lead to side effects. Modifying LNPs with tissue- or cell-type specific targeting ligands (such as antibodies or antibody-derived antigen-binding domains) can direct them to specific target tissues or cells. However, even with such strategies, it is still difficult to completely avoid non-specific hepatic uptake, and off-target problems remain prominent. Furthermore, the low cytoplasmic delivery efficiency of existing LNP delivery systems and the potential toxicity caused by long-term lipid accumulation constitute fundamental obstacles to the wider application of LNP technology. Therefore, developing a new generation of safe and efficient ionizable lipids to fundamentally optimize the in vivo delivery behavior of LNPs is of great significance for overcoming current technological bottlenecks. Summary of the Invention

[0004] The purpose of this invention is to provide a compound of general formula (I), its pharmaceutically acceptable salt, or an isomer thereof, to address the problems of insufficient extrahepatic targeting capability, off-target delivery, and low cytoplasmic delivery efficiency in existing LNP technologies.

[0005] The present invention provides a compound of formula I, or a salt or isomer thereof.

[0006]

[0007] I

[0008] In the formula:

[0009] X1, X2, Y1 and Y2 are each independently NR7, O or S, and R7 is selected from H and C1-C10 alkyl groups;

[0010] X3, X4, X5, X6, Y3, Y4, Y5, and Y6 are each independently NR8 or O, or are not present. R8 is selected from H and C1-C10 alkyl groups. X3 and X4 are not present at the same time; X5 and X6 are not present at the same time; Y3 and Y4 are not present at the same time; Y5 and Y6 are not present at the same time.

[0011] L1 is -C(O)-, or it does not exist;

[0012] L2 is NR6 or O, or is absent; R6 is selected from H and optionally substituted C1-C10 alkyl groups;

[0013] L3 is a substituted or unsubstituted C1-C5 alkylene group, or it is absent;

[0014] R3 represents substituted or unsubstituted -NR a R b or hydroxyl group; R a and R b Each is independently selected from H and substituted or unsubstituted C1-C6 alkyl groups, or R a R b Together with the nitrogen atoms they are attached to, they form substituted or unsubstituted 4-9 membered heterocyclic groups;

[0015] R1, R2, R4 and R5 are each independently selected from substituted or unsubstituted C4-C14 alkyl, substituted or unsubstituted C4-C14 alkenyl and substituted or unsubstituted C4-C14 alkynyl groups;

[0016] m1 and m2 can be 2, 3, 4, 5 or 6 independently;

[0017] n1, n2, t1, t2, s1, and s2 are each independently 0, 1, 2, 3, 4, 5, or 6.

[0018] The present invention also provides nanoparticle compositions comprising lipid components, said lipid components comprising a compound of formula I of the present invention or a salt or isomer thereof.

[0019] In one or more embodiments, the lipid component further includes phospholipids, PEG lipids, and structural lipids.

[0020] In one or more embodiments, the PEG lipid is an unfunctionalized PEG lipid and / or a functionalized PEG lipid; preferably, the PEG lipid includes an unfunctionalized PEG lipid and a functionalized PEG lipid.

[0021] In one or more embodiments, the nanoparticle composition specifically targets an organ; preferably, the organ is the liver or spleen.

[0022] In one or more embodiments, the nanoparticle composition comprises a compound of Formula I or a salt or isomer thereof, phospholipids, structural lipids, and PEG lipids; the molar ratio of the compound of Formula I or a salt or isomer thereof, phospholipids, structural lipids, and PEG lipids is 50-65:15-25:8-34.5:0.5-2; preferably, the nanoparticle composition is used to deliver therapeutic and / or preventive agents to the spleen;

[0023] Preferably, the molar ratio of the compound of formula I or its salt or isomer to the PEG lipid is (25-130):1, for example (25-100):1, (25-80):1, (30-60):1, (35-50):1, (35-40):1 or (38-39):1;

[0024] Preferably, the molar ratio of phospholipid to PEG lipid is (7.5-50):1, for example (7.5-30):1, (7.5-20):1, (10-15):1, (12-14):1, (13-14):1 or (13-13.5):1;

[0025] Preferably, the molar ratio of structural lipid to PEG lipid is (4-69):1, for example (5-50):1, (6-30):1, (10-20):1, (12-14):1, (13-14):1 or (13.5-14):1.

[0026] In one or more embodiments, the nanoparticle composition comprises a compound of Formula I or a salt or isomer thereof, phospholipids, structural lipids, and PEG lipids; the molar ratio of the compound of Formula I or a salt or isomer thereof, phospholipids, structural lipids, and PEG lipids is 50:10:38.5:1.5; preferably, the nanoparticle composition is used to deliver therapeutic and / or preventive agents to the liver.

[0027] In one or more embodiments, the nanoparticle composition further includes therapeutic and / or preventative agents.

[0028] In some or more embodiments, the lipid component is conjugated with a ligand that specifically targets an organ, tissue, or cell;

[0029] Preferably, the ligand is selected from peptides, antibodies, and antibody-derived antigen-binding domains;

[0030] Preferably, a portion of the PEG lipid is conjugated with the ligand; preferably, the molar ratio of the PEG lipid without ligand to the PEG lipid with ligand is 14-2:1;

[0031] Preferably, the ligand is an antibody, and more preferably one or more of CD3, CD4, CD5, CD7 and CD8 antibodies that target T cells.

[0032] The present invention also provides a pharmaceutical composition comprising (1) the nanoparticle composition of the present invention; and (2) pharmaceutically acceptable excipients.

[0033] The present invention also provides a non-therapeutic method for producing polypeptides in cells, the method comprising the step of contacting cells with a nanoparticle composition comprising: (1) a lipid component, including phospholipids, PEG lipids, structural lipids, and a compound of formula I of the present invention or a salt or isomer thereof, and (2) a nucleic acid encoding the polypeptide, wherein the nucleic acid is capable of being translated in cells to produce a protein.

[0034] Preferably, the cell is a mammalian cell;

[0035] Preferably, the nucleic acid is selected from one or more of siRNA, microRNA, shRNA, mRNA and pDNA, and is preferably mRNA.

[0036] The present invention also provides a method for delivering therapeutic and / or preventive agents to cells, the method comprising the steps of administering (e.g., injecting) the nanoparticle composition and pharmaceutical composition of the present invention to a subject, the nanoparticle composition comprising: (1) a lipid component, including phospholipids (e.g., polyunsaturated lipids), PEG lipids, structural lipids, and compounds of formula I of the present invention, or salts and isomers thereof, and (2) a therapeutic and / or preventive agent.

[0037] In one or more embodiments, the therapeutic and / or preventive agent is a nucleic acid.

[0038] In one or more embodiments, the therapeutic and / or preventive agent is mRNA or siRNA.

[0039] In one or more embodiments, the cell is a mammalian cell.

[0040] In one or more embodiments, the delivery is specific (rather than whole-body) delivery.

[0041] The present invention also provides a treatment for a disease or symptom, the method comprising the step of administering a therapeutically effective amount of the nanoparticle composition of the present invention to a subject in need, the nanoparticle composition comprising (1) a lipid component, including phospholipids, PEG lipids, structural lipids, and compounds of formula I of the present invention, or salts and isomers thereof, and (2) a therapeutic agent and / or a preventive agent.

[0042] The present invention also provides a method for preparing compounds of formula I of the present invention or their salts or isomers.

[0043] The present invention also provides a method for preparing the nanoparticle composition of the present invention. The method includes the step of mixing (e.g., microfluidizing) the components of the nanoparticle composition, the nanoparticle composition being defined as described in any embodiment herein.

[0044] In one or more embodiments, the components are mixed according to the content of each component in the nanoparticle composition.

[0045] In one or more embodiments, the method includes mixing (e.g., microfluidizing) the components of the nanoparticle composition described herein (e.g., lipids, phospholipids, PEG lipids, structural lipids, and RNA of Formula I) at a weight ratio of lipid components to therapeutic and / or preventative agents of about 5:1 to about 60:1.

[0046] The present invention also provides a method for preparing a nanoparticle composition that can be conjugated with an antibody, the method comprising the step of mixing (e.g., microfluidizing) the components of the nanoparticle composition, wherein the PEG lipids include unfunctionalized PEG lipids and functionalized PEG lipids.

[0047] In one or more embodiments, the method includes the steps of:

[0048] (1) Provide lipid components and dissolve them in anhydrous ethanol to obtain an ethanol phase lipid solution;

[0049] (2) Dissolve the nucleic acid in a buffer solution of appropriate pH to obtain an aqueous nucleic acid solution;

[0050] (3) The ethanol phase lipid solution is mixed with the aqueous phase nucleic acid solution, and then the ethanol is removed to obtain a lipid nanoparticle solution loaded with nucleic acid.

[0051] In one or more embodiments, the PEG lipids include functionalized PEG lipids; preferably, the method further includes (4) co-incubating the nucleic acid-loaded lipid nanoparticle solution with a ligand specifically targeting an organ, tissue or cell to obtain a tissue or cell-specific targeting lipid nanoparticle solution loaded with nucleic acid.

[0052] In one or more embodiments, in step (1), the lipid component is provided according to the content of the lipid component in the obtained nanoparticle composition.

[0053] In one or more embodiments, in step (2), the pH is 3-6; and / or, the buffer solution is selected from citrate buffer.

[0054] In one or more embodiments, in step (3), a microfluidic device is used for mixing; and / or, an ethanol phase lipid solution and an aqueous phase nucleic acid solution are mixed according to the mass ratio of the lipid component and nucleic acid in the prepared nanoparticle composition.

[0055] In one or more embodiments, in step (3), ethanol is removed using methods conventional in the art, including but not limited to ultrafiltration or dialysis.

[0056] The present invention also provides the use of compounds of Formula I of the present invention or their salts or isomers, and / or the nanoparticle compositions of the present invention in the preparation of medicaments for treating or preventing diseases or conditions, or in the preparation of medicaments containing immune cells.

[0057] In one or more embodiments, the nanoparticle compositions described herein or nanoparticle compositions formed from compounds of Formula I herein are used for the specific delivery of therapeutic and / or preventative agents.

[0058] In one or more embodiments, the specific delivery is delivery to an organ and / or cell selected from the heart, liver, spleen, lung, and kidney, and the cell selected from hepatocytes, immune cells, and epithelial cells.

[0059] In one or more embodiments, the specific delivery is delivery to the liver, spleen, T cells, or spleen and T cells.

[0060] In one or more embodiments, the disease or condition is one that benefits from the specific delivery of therapeutic and / or preventative agents.

[0061] In one or more embodiments, the disease is selected from tumors, autoimmune diseases, and infectious diseases.

[0062] In one or more embodiments, the disease or condition is one that benefits from immune cells; preferably, the disease or condition is one that benefits from T cells; preferably, the disease or condition is one that benefits from T cell proliferation and / or activation.

[0063] In one or more embodiments, the immune cells include T cells, NK cells, NKT cells, or TILs.

[0064] The present invention also provides a method for transfecting cells, the method comprising the step of contacting the cells with a nanoparticle composition of the present invention, the composition comprising: a lipid component, including phospholipids, PEG lipids, structural lipids, and a compound of formula I as described in any embodiment herein, or a salt or isomer thereof, the nanoparticle composition optionally further comprising a therapeutic agent and / or a preventive agent.

[0065] In one or more embodiments, the cells are immune cells.

[0066] In one or more embodiments, the immune cells include T cells, NK cells, NKT cells, or TILs.

[0067] In one or more embodiments, the therapeutic and / or preventive agent is a nucleic acid, preferably mRNA.

[0068] The present invention also provides a method for preparing engineered T cells, the method comprising the step of contacting the T cells with the nanoparticle composition of the present invention.

[0069] The present invention has the following beneficial effects:

[0070] 1. Liver-targeted LNPs showed comparable liver delivery efficiency to the positive control SM-102 LNPs;

[0071] 2. The spleen delivery efficiency of spleen-targeted LNPs was significantly better than that of the positive control SM-102 LNPs;

[0072] 3. Spleen-targeted LNPs modified with anti-CD3 antibody showed high CD3+ T cell transfection efficiency, providing a technical platform for in vivo T cell reprogramming. Attached Figure Description

[0073] Figure 1 It refers to the in vivo delivery efficiency of liver-targeted LNPs.

[0074] Figure 2 It is the in vivo delivery efficiency of spleen-targeted LNPs.

[0075] Figure 3 This refers to the transfection efficiency of CD3 antibody-modified LNPs on mouse spleen CD3+ T cells.

[0076] Figure 4 This refers to the transfection efficiency of CD3 antibody-modified LNP on mouse liver CD45- and CD45+ cells. Detailed Implementation

[0077] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0078] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0079] In this document, the terms “contains,” “includes,” “containing,” and similar terms encompass the meanings of “basically composed of” and “composed of.” For example, when this document discloses “A contains B and C,” “A is basically composed of B and C” and “A is composed of B and C” should be considered as having been disclosed in this document.

[0080] In this document, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0081] Unless otherwise specified, percentages refer to mass percentages and proportions refer to mass ratios in this article.

[0082] In this document, when describing embodiments or examples, it should be understood that it is not intended to limit the invention to those embodiments or examples. Rather, all alternatives, modifications, and equivalents of the methods and materials described herein are covered within the scope defined by the claims.

[0083] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0084] This invention provides a novel ionizable cationic lipid and an LNP delivery system containing it. The system is characterized by its tunable delivery targeting: it exhibits highly efficient liver delivery under one formulation; with a different formulation, it can be directed to spleen delivery; further, after antibody functionalization modification, it can achieve specific transfection of CD3+ T cells while significantly reducing off-target expression in the liver, effectively avoiding the risk of liver off-target effects. The LNP delivery system provided by this invention successfully overcomes the technical shortcomings of existing LNPs in terms of strong liver targeting, providing an ideal delivery carrier for achieving cell-specific mRNA therapeutic expression.

[0085] Ionizable cationic lipids

[0086] This invention provides an ionizable cationic lipid having the structure shown in Formula I:

[0087]

[0088] I

[0089] In the formula:

[0090] X1, X2, Y1 and Y2 are each independently NR7, O or S, and R7 is selected from H and C1-C10 alkyl groups;

[0091] X3, X4, X5, X6, Y3, Y4, Y5, and Y6 are each independently NR8 or O, or are not present. R8 is selected from H and C1-C10 alkyl groups. X3 and X4 are not present at the same time; X5 and X6 are not present at the same time; Y3 and Y4 are not present at the same time; Y5 and Y6 are not present at the same time.

[0092] L1 is -C(O)-, or it does not exist;

[0093] L2 is NR6 or O, or is absent; R6 is selected from H and optionally substituted C1-C10 alkyl groups;

[0094] L3 is a substituted or unsubstituted C1-C5 alkylene group, or it is absent;

[0095] R3 represents substituted or unsubstituted -NR a R b or hydroxyl group; R a and R b Each is independently selected from H and substituted or unsubstituted C1-C6 alkyl groups, or R a R b Together with the nitrogen atoms they are attached to, they form substituted or unsubstituted 4-9 membered heterocyclic groups;

[0096] R1, R2, R4 and R5 are each independently selected from substituted or unsubstituted C4-C14 alkyl, substituted or unsubstituted C4-C14 alkenyl and substituted or unsubstituted C4-C14 alkynyl groups;

[0097] m1 and m2 can be 2, 3, 4, 5 or 6 independently;

[0098] n1, n2, t1, t2, s1, and s2 are each independently 0, 1, 2, 3, 4, 5, or 6.

[0099] In some implementations, both X1 and X2 are 0.

[0100] In some implementations, X3 is 0 and X4 is non-existent.

[0101] In some implementations, X5 is 0 and X6 is non-existent.

[0102] In some implementations, both Y1 and Y2 are 0.

[0103] In some implementations, Y3 is 0 and Y4 is non-existent.

[0104] In some implementations, Y5 is 0 and Y6 is non-existent.

[0105] In some implementations, X1 and X2 are both 0, X3 is 0, X4 is non-existent, X5 is 0, and X6 is non-existent; and / or, Y1 and Y2 are both 0, Y3 is 0, Y4 is non-existent, Y5 is 0, and Y6 is non-existent.

[0106] In some implementations, m1 and m2 are 2, 3, or 4.

[0107] In some implementations, n1 and n2 are 0, 1, 2, 3, or 4.

[0108] In some implementations, t1, t2, s1 and s2 are each independently 0, 1, 2, 3 or 4, preferably 0 or 1.

[0109] In some implementations, m1 and m2 are each independently 2 or 3; n1 and n2 are each independently 0, 1, 2 or 3; t1, t2, s1 and s2 are each independently 1, 2 or 3. Preferably, m1 and m2 are both 2; n1 and n2 are each independently 0, 1 or 2; t1, t2, s1 and s2 are each independently 0 or 1.

[0110] When substituted, the number of substituents on R1, R2, R4, and R5 can each be independently 1, 2, 3, 4, or 5. The substituents on R1, R2, R4, and R5 are each independently selected from halogens and C1-C10 alkoxy groups. In some embodiments, R1, R2, R4, and R5 are each independently a substituted or unsubstituted C5-C12 alkyl group, such as a substituted or unsubstituted C6-C11 alkyl group, a substituted or unsubstituted C7-C10 alkyl group, a substituted or unsubstituted C7-C9 alkyl group, a substituted or unsubstituted C7 alkyl group, or a substituted or unsubstituted C8 alkyl group.

[0111] -NR in R3 a R b When substituted, the number of substituents can be independently 1, 2, 3, 4, or 5. When substituted, the -NR... a R b The substituents are each independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, hydroxyl, 3-8 heterocyclic groups having 1, 2, or 3 heteroatoms selected from N, O, and S, and amino groups optionally substituted with 1 or 2 C1-C6 alkyl groups. In some embodiments, R a and R b Each is independently selected from H and substituted or unsubstituted C1-C6 alkyl groups; when substituted, the substituents on the C1-C6 alkyl groups are C1-C6 alkyl, C1-C6 alkoxy, halogen, or hydroxyl groups; preferably, R a and R b Each is independently H or an unsubstituted C1-C6 alkyl group. In some other embodiments, R a R b Together with the nitrogen atoms connected to them, they form substituted or unsubstituted 4-9 membered heterocyclic groups; when substituted, the substituents on the 4-9 membered heterocyclic groups are selected from C1-C6 alkyl, hydroxyl and amino groups optionally substituted by 1 or 2 C1-C6 alkyl groups; preferably, when substituted, the substituents on the 4-9 membered heterocyclic groups are amino groups optionally substituted by 1 or 2 C1-C6 alkyl groups.

[0112] In some implementations, R3 is selected from the group of substituents:

[0113] ;

[0114] The wavy line indicates the position where R3 connects to other parts of the compound of formula I;

[0115] Preferably, R3 is selected from the group consisting of:

[0116] .

[0117] When the C1-C10 alkyl group in R6 is substituted, the number of substituents can be 1, 2, 3, 4, or 5, each independently. When substituted, the substituents of the C1-C10 alkyl group are each independently selected from C1-C6 alkyl groups, C1-C6 alkoxy groups, halogens, hydroxyl groups, 3-8 heterocyclic groups having 1, 2, or 3 heteroatoms selected from N, O, and S, and amino groups optionally substituted with 1 or 2 C1-C6 alkyl groups. Preferably, R6 is H or an unsubstituted C1-C10 alkyl group, preferably H.

[0118] Preferably, L2 is O.

[0119] When the C1-C5 alkylene group in L3 is substituted, the number of substituents can be 1, 2, 3, 4, or 5. When substituted, the substituents of the C1-C5 alkylene group are each independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, hydroxyl, 3-8 heterocyclic groups having 1, 2, or 3 heteroatoms selected from N, O, and S, and amino groups optionally substituted by 1 or 2 C1-C6 alkyl groups. Preferably, L3 is an unsubstituted C1-C5 alkylene group; more preferably, L3 is an unsubstituted C2-C4 alkylene group.

[0120] In some embodiments, L1 is -C(O)-, or absent; L2 is NR6 or O, or absent; L3 is a substituted or unsubstituted C1-C4 alkylene group; R3 is a substituted or unsubstituted -NR group. a R b Or hydroxyl group.

[0121] In some implementations, L1 is -C(O)-; L2 is absent; L3 is absent; and R3 is substituted or unsubstituted -NR. a R b Preferably, R a R b Together with the nitrogen atoms connected to them, they form 4-7 membered heterocyclic groups; preferably, when substituted, the substituents on R3 are amino groups optionally substituted by one or two C1-C6 alkyl groups.

[0122] In some embodiments, L1 is absent; L2 is absent; L3 is a C1-C5 alkylene group optionally substituted with 1, 2, 3, or 4 substituents selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, and hydroxyl groups; R3 is a substituted or unsubstituted -NR group. a R b or hydroxyl, substituted or unsubstituted -NR a R b The definition is as described in any of the embodiments herein.

[0123] In some embodiments, L1 is -C(O)-; L2 is absent; L3 is a C1-C5 alkylene group optionally substituted with 1, 2, 3, or 4 substituents selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, and hydroxyl groups; R3 is substituted or unsubstituted -NR. a R b or hydroxyl, substituted or unsubstituted -NR a R b The definition is as described in any of the embodiments herein.

[0124] In some embodiments, L1 is -C(O)-; L2 is NR6 or O; L3 is a substituted or unsubstituted C2-C5 alkylene group; and R3 is a substituted or unsubstituted -NR group. a R b L3, R6, substituted or unsubstituted -NR a R b The definition is as described in any of the embodiments herein.

[0125] In some embodiments, L1 is -C(O)-; L2 is O; L3 is a substituted or unsubstituted C2-C5 alkylene group; and R3 is a substituted or unsubstituted -NR group. a R b L3, substituted or unsubstituted -NR a R b The definition is as described in any of the embodiments herein.

[0126] In some embodiments, L1 is -C(O)-; L2 is O; L3 is a substituted or unsubstituted C2-C5 alkylene group; and R3 is a substituted or unsubstituted -NR group. a R b R a and R b Each is independently selected from H and substituted or unsubstituted C1-C6 alkyl groups, or R a R b The nitrogen atoms connected to them together form substituted or unsubstituted 4, 5, 6 or 7-membered heterocyclic groups; m1 and m2 are each independently 2 or 3; X1 and X2 are both O; Y1 and Y2 are both O; n1 and n2 are each independently 1 or 2; t1, t2, s1 and s2 are each independently 1 or 2; X3, X5, Y3 and Y5 are all O; X4, X6, Y4 and Y6 are not present; R1, R2, R4 and R5 are each independently C4-C14 alkyl (or C6-C9 alkyl).

[0127] In some embodiments, the compound of formula I is selected from the group consisting of:

[0128]

[0129]

[0130] .

[0131] This invention relates to novel ionizable cationic lipids (compounds of formula I, or salts or isomers thereof) and lipid nanoparticle compositions comprising such novel ionizable cationic lipids. The invention also provides methods for delivering therapeutic and / or preventative agents to mammalian cells, particularly methods for specifically delivering therapeutic and / or preventative agents to mammalian cells, tissues, and / or organs, producing an objective effect in mammalian cells, tissues, and / or organs, and methods for treating diseases or conditions in mammals.

[0132] Methods for delivering contrast agents, therapeutic agents, and / or preventive agents to mammals specifically involve administering a nanoparticle composition comprising a contrast agent, a therapeutic agent, and / or a preventive agent to a subject, wherein administration involves contacting the subject's cells, tissues, and / or organs with the composition, thereby delivering the contrast agent, therapeutic agent, and / or preventive agent to the cells, tissues, and / or organs.

[0133] The inventors have discovered that pharmaceuticals prepared using compounds of Formula I or their salts or isomers (such as the nanoparticle compositions or pharmaceutical compositions described herein) can achieve localized effects in organs, tissues, and / or cells after systemic administration, thereby achieving specific delivery of therapeutic and / or prophylactic agents. In some embodiments, the nanoparticle compositions of the present invention are suitable for preparing systemically administered, locally effective pharmaceuticals. Hereinafter, “locally effective pharmaceutical” or “locally effective pharmaceutical composition” refers to a pharmaceutical that can be applied to exert its effect at the site of application after systemic administration; such pharmaceuticals that produce systemic effects are considered unintended pharmaceutical effects. Targets of specific delivery may include cells only, organs only (spleen, liver, etc.) and / or tissues only. In some embodiments, when administered to a subject, the drug is delivered only to the liver or spleen.

[0134] In some embodiments, specific delivery may refer to the fact that, within 4-8 hours (e.g., 6 hours) after administration (e.g., intravenous administration) of the LNP composition described herein, the therapeutic agent and / or the prophylactic agent (mRNA) is maintained primarily in the liver or spleen; in particular, within 4-8 hours (e.g., 6 hours) after administration, the concentration of the LNP composition, the therapeutic agent and / or the prophylactic agent (mRNA) in the liver or spleen is significantly higher than its concentration in non-target tissues.

[0135] In this article, "systemic administration" in "systemically administered drugs with local effects" includes conventional administration methods that allow for systemic exposure, such as intravenous injection, intramuscular injection, and subcutaneous injection, with intravenous injection being preferred.

[0136] In this article, the immune cells are selected from one or more of T cells, NK cells, NKT cells, TILDC cells, monocytes, and macrophages. For example, the immune cells include any one or more selected from CAR-T, TCR-T, TIL, DC-CIK, NK, CAR-NK, LAK, DNT, CTL, and CAR-M.

[0137] In some respects, the ionizable cationic lipids described herein are compounds of formula I or their salts or isomers. Since lipids of formula I may possess a positive or partially positive charge at physiological pH, such lipids may be referred to as cationic lipids.

[0138] As used herein, the term "alkyl" or "alkyl group" refers to a straight-chain or branched saturated hydrocarbon containing one or more carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms), optionally substituted. The symbol "C1-C10 alkyl" refers to a straight-chain or branched saturated hydrocarbon containing 1-10 carbon atoms, optionally substituted. Exemplary alkyl groups also include C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, and C1-C2 alkyl. Unless otherwise stated, alkyl as used herein refers to both unsubstituted and substituted alkyl groups. An alkylene group is the same as an alkyl group but has a divalent charge.

[0139] As used herein, the term "alkenyl" or "alkenyl group" refers to a straight-chain or branched hydrocarbon comprising two or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms) and at least one carbon-carbon double bond, optionally substituted. The symbol "C2-C18 alkenyl" refers to an optionally substituted straight-chain or branched hydrocarbon comprising 2-18 carbon atoms and at least one carbon-carbon double bond. Alkenyl groups may include 1, 2, 3, 4 or more carbon-carbon double bonds. Unless otherwise stated, alkenyl groups referred to herein are both unsubstituted and substituted alkenyl groups.

[0140] As used herein, the term "alkynyl" or "alkynyl group" refers to a straight-chain or branched hydrocarbon comprising two or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms) and at least one carbon-carbon triple bond, optionally substituted. The symbol "C2-C18 alkynyl" refers to a straight-chain or branched hydrocarbon comprising 2-18 carbon atoms and at least one carbon-carbon triple bond, optionally substituted. An alkynyl group may include 1, 2, 3, 5 or more carbon-carbon triple bonds. Unless otherwise stated, the alkynyl group referred to herein refers to both unsubstituted and substituted alkynyl groups.

[0141] As used herein, the term "alkoxy" refers to -O-alkyl, and the definition of alkyl is as described herein.

[0142] As used herein, the term "carbocyclic" or "carbocyclic group" refers to a monocyclic or polycyclic system comprising one or more optionally substituted carbon atom rings. The ring can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more membered rings. The symbol "C3-C6 carbocyclic" refers to a carbocyclic ring comprising a monocyclic ring having 3-6 carbon atoms. The carbocyclic ring may include one or more carbon-carbon double or triple bonds and may be non-aromatic or aromatic (e.g., cycloalkyl or aryl). Examples of carbocyclic rings include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, and naphthyl. Unless otherwise stated, the carbocyclic ring referred to herein means unsubstituted and substituted carbocyclic groups, i.e., optionally substituted carbocyclic rings.

[0143] As used herein, “cycloalkyl” refers to a non-aromatic carbon ring and may or may not include any double or triple bonds, such as C3-C8 cycloalkyl, C4-C7 cycloalkyl, and C5-C6 cycloalkyl.

[0144] As used herein, the term "heterocycle" or "heterocyclic group" refers to a optionally substituted monocyclic or polycyclic system comprising one or more rings, wherein at least one ring comprises at least one heteroatom. The heteroatom may be, for example, a nitrogen, oxygen, or sulfur atom. The ring may be a 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more membered ring. A heterocycle may include one or more double or triple bonds and may be non-aromatic or aromatic (e.g., heterocyclic alkyl or heteroaryl). Examples of heterocycles include imidazolyl, imidazoalkyl, oxazolyl, oxazolyl, thiazolyl, thiazoalkyl, pyrazolyl, pyrazolyl, isoxazolyl, isoxazolyl, isothiazolyl, isothiazolyl, morpholinyl, pyrroleyl, pyrrolealkyl, furanyl, tetrahydrofuranyl, thiophenyl, pyridinyl, piperidinylynyl, quinolinyl, and isoquinolinyl. The heterocyclic group can be a 3-14 member heterocyclic group, a 4-12 member heterocyclic group, a 4-9 member heterocyclic group, or a 5-7 member heterocyclic group.

[0145] As used herein, the term "heterocyclic alkyl" refers to a non-aromatic heterocycle and may or may not contain any double or triple bonds. Unless otherwise stated, the heterocycle referred to herein means unsubstituted and substituted heterocyclic groups, i.e., optionally substituted heterocycles.

[0146] Unless otherwise stated, alkyl, alkenyl, and cyclic groups (e.g., carbocyclic and heterocyclic groups) may optionally be substituted. Optional substituents may be selected from, but are not limited to, halogen atoms (e.g., chlorine, bromine, fluorine, or iodine groups), carboxylic acids (e.g., -C(O)OH), and alcohols (e.g., hydroxyl, -OH), esters (e.g., -C(O)OR or -OC(O)R), aldehydes (e.g., -C(O)H), carbonyl groups (e.g., -C(O)R, or represented by C=O), acyl halides (e.g., -C(O)X, where X is a halide selected from bromides, fluorides, chlorides, and iodides), carbonates (e.g., -OC(O)OR), alkoxy groups (e.g., -OR), acetals (e.g., -C(OR)2R', where each OR is an alkoxy group that may be the same or different, and R' is an alkyl or alkenyl group), phosphates (e.g., P(O)4), and phosphates (e.g., P(O)4). 3+ ), thiols (e.g., -SH), sulfoxides (e.g., -S(O)R), sulfinic acids (e.g., -S(O)OH), sulfonic acids (e.g., -S(O)2OH), thiols (e.g., -C(S)H), sulfates (e.g., S(O)4) 2+ ), sulfonyl (e.g., -S(O)2-), amide (e.g., -C(O)NR or -N(R)C(O)R), azide (e.g., -N3), nitro (e.g., -NO2), cyano (e.g., -CN), isocyano (e.g., -NC), acyloxy (e.g., -OC(O)R), amino (e.g., -NRH or -NH2), carbamoyl (e.g., -OC(O)NR, -OC(O)NRH or -OC(O)NH2), sulfonamides (e.g., -S(O)2NR, -S(O)2NRH, -S(O)2NH2, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)S(O)2H or -N(H)S(O)2H), alkyl, alkenyl and cyclic (e.g., carbocyclic or heterocyclic). In any of the foregoing, R is an alkyl or alkenyl group as defined herein.

[0147] In some embodiments, the substituent itself may be further substituted with, for example, 1, 2, 3, 4, 5, or 6 substituents as defined herein. For example, C1-C6 alkyl groups may be further substituted with 1, 2, 3, 4, 5, or 6 substituents as described herein.

[0148] As used herein, the terms “about” and “approximately” when applied to one or more values ​​of interest refer to values ​​similar to specified reference values. In some embodiments, the terms “about” or “approximately” mean falling within any of the following directions (greater than or less than) the reference value: 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less, unless otherwise stated or apparent from the context (unless the number exceeds 100% of the possible value). For example, when used in the context of a given amount of compound in the lipid component of a nanoparticle composition, “about” can represent + / - 10% of the value. For example, a nanoparticle composition containing a lipid component having about 40% of a given compound may contain 30-50% of the compound.

[0149] As used herein, the term "compound" is intended to include all isomers and isotopes of the structures shown. "Isotope" refers to atoms having the same atomic number but different mass numbers due to different numbers of neutrons in their nuclei; for example, isotopes of hydrogen include tritium and deuterium. Furthermore, the compounds, salts, or complexes of this disclosure can be prepared by conventional methods by combining with solvents or water molecules to form solvates and hydrates.

[0150] As used herein, the term "contact" means establishing a physical connection between two or more entities. For example, contacting mammalian cells with a nanoparticle composition means establishing a physical connection between the mammalian cells and the nanoparticles. Methods for contacting cells with external entities in vivo and in vitro are well known in the field of biology. For example, the nanoparticle composition can be contacted with mammalian cells via different routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous), and can involve varying amounts of the nanoparticle composition. Furthermore, the nanoparticle composition can contact more than one type of mammalian cell. In some embodiments, the cells are immune cells (such as T cells). In other embodiments, the nanoparticle composition can also contact more than one mammalian organ (e.g., the liver or spleen).

[0151] As used herein, the term “delivery” means providing an entity to a destination. For example, delivering a therapeutic and / or preventative agent to a subject may involve administering a nanoparticle composition containing the therapeutic and / or preventative agent to the subject (e.g., via intravenous, intramuscular, intradermal, or subcutaneous routes). Applying a nanoparticle composition to a mammal or mammalian cells may involve contacting one or more cells with the nanoparticle composition. “Specific delivery” means delivering the drug primarily to a specific location in the subject, such as an organ (e.g., the liver or spleen), a tissue, or a cell, with no or minimal delivery to organs, tissues, or cells other than said organ, tissue, or cell. For example, when a drug is administered to a subject, it is specifically delivered to the liver or spleen.

[0152] In some embodiments, nanoparticle compositions comprising compounds according to Formula I have substantially the same level of specific delivery effect, regardless of the route of administration. For example, some of the compounds disclosed herein exhibit similar specific delivery when used for intravenous or intramuscular delivery of therapeutic and / or prophylactic drugs.

[0153] As used herein, the term "specific delivery" refers to the delivery of more (e.g., at least 1.5 times, at least 2 times, at least 3 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times) therapeutic and / or prophylactic agents to target cells, tissues, or organs of interest (e.g., liver or spleen) than to non-target tissues via nanoparticles. The level of nanoparticle delivery to a specific tissue can be identified by comparing the amount of protein produced in the tissue to the weight of the tissue, comparing the amount of therapeutic and / or prophylactic agents in the tissue to the weight of the tissue, comparing the amount of protein produced in the tissue to the total amount of protein in the tissue, or comparing the amount of therapeutic and / or prophylactic agents in the tissue to the total amount of therapeutic and / or prophylactic agents in the tissue. It should be understood that the ability of nanoparticles to specifically deliver to target cells, target tissues, and target organs does not need to be determined in treated subjects; it can be determined in surrogate models such as animal models (e.g., mouse models).

[0154] As used herein, “encapsulation efficiency” refers to the amount of therapeutic and / or preventive agents that become part of the nanoparticle composition relative to the initial total amount of therapeutic and / or preventive agents used to prepare the nanoparticle composition. For example, if 97 mg of a total of 100 mg of therapeutic and / or preventive agents initially provided to the composition are encapsulated in the nanoparticle composition, the encapsulation efficiency can be 97%.

[0155] As used herein, “expression” of a nucleic acid sequence includes the translation of mRNA into a polypeptide or protein and / or post-translational modifications of the polypeptide or protein.

[0156] As used herein, the term "in vitro" refers to an event that occurs in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, or in a Petri dish, rather than in a living organism (e.g., an animal, plant, or microorganism). As used herein, the term "in vivo" refers to an event that occurs within an organism (e.g., an animal, plant, or microorganism, or its cells or tissues). As used herein, the term "ex vivo" refers to an event that occurs outside of an organism (e.g., an animal, plant, or microorganism, or its cells or tissues). Ex vivo events may occur in environments with minimal variation compared to natural (e.g., in vivo) environments.

[0157] As used herein, the term "isomer" refers to any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound. A compound may include one or more chiral centers and / or double bonds, and thus may exist as a stereoisomer. This disclosure covers any and all isomers of the compounds described herein, including pure stereoisomeric forms and mixtures of enantiomers and stereoisomers, such as racemates. Methods for determining enantiomers and stereoisomer mixtures of compounds and for resolving them into their enantiomeric or stereoisomeric components are well known.

[0158] As used herein, a "lipid component" is a component of a nanoparticle composition comprising one or more lipids. For example, a lipid component may comprise one or more cationic / ionizable, polyethylene glycol-modified, structured, or other lipids, such as phospholipids. In some embodiments, the lipid component comprises a compound of Formula I of the present invention or a salt or isomer thereof. Further, the lipid component further comprises phospholipids. Further, the lipid component further comprises PEG lipids. Further, the lipid component further comprises structured lipids. Further, the lipid component further comprises a solvent, such as an organic solvent or a buffer solution; commonly used organic solvents include alcohols, such as ethanol, preferably anhydrous ethanol, and commonly used buffer solutions include PBS buffer. Preferably, the lipid component further comprises phospholipids, PEG lipids, and structured lipids. Those skilled in the art can self-assemble the lipid components of the present invention using conventional methods, or can self-assemble the lipid components of the present invention with a loaded component (e.g., a therapeutic agent and / or a preventative agent, such as mRNA) using conventional methods.

[0159] As used herein, “administration” may include intravenous, intramuscular, intradermal, subcutaneous or other methods of delivering the composition to a subject; preferably intravenous, such as intravenous injection.

[0160] As used in this article, "modified" means non-natural. For example, RNA can be modified RNA. That is, RNA can include one or more non-naturally occurring nucleobases, nucleosides, nucleotides, or linkers. RNA also includes any sequence optimizations performed on the RNA.

[0161] As used herein, a “nanoparticle composition” is a composition comprising one or more lipids. Nanoparticle compositions are typically on the order of micrometers or smaller and may include a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipid complexes. For example, a nanoparticle composition may be a liposome having a lipid bilayer with a diameter of 500 nm or smaller.

[0162] As used in this article, "patient" means a subject who may seek or need treatment, needs treatment, is receiving treatment, will receive treatment, or is receiving care from a trained professional due to a specific disease or condition.

[0163] As used herein, "PEG lipid" or "PEGylated lipid" refers to lipids containing polyethylene glycol components.

[0164] The phrase “pharmaceutically acceptable” as used in this article means compounds, materials, compositions, and / or dosage forms that, within reasonable medical judgment, are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0165] As used herein, the phrase "pharmaceutically acceptable excipient" means any component other than the compounds described herein (e.g., a carrier capable of suspending, complexing, or dissolving an active compound) and having substantially non-toxic and non-toxic properties. Excipients may include, for example: anti-adhesives, antioxidants, adhesives, coating agents, compression aids, disintegrants, dyes (pigments), emollients, emulsifiers, fillers (diluents), film-forming agents or coating agents, fragrances, flavorings, flow enhancers (flow improvers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydrates. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (binary), calcium stearate, croscarmellose, croscarmellose, citric acid, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl cellulose methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silica, sodium carboxymethyl cellulose, sodium citrate, sodium glycolate starch, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, xylitol, and other substances disclosed herein.

[0166] In this specification, the structural formulas of compounds are sometimes used for convenience to represent a certain isomer, but this disclosure includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, etc. It should be understood that not all isomers may have the same level of activity.

[0167] For compounds represented by the chemical formulas herein, polymorphism can exist. It should be noted that any crystal form, mixture of crystal forms, or its anhydrides or hydrates are included within the scope of this disclosure. The terms "polymorph," "polymorph," or "crystal form" refer to crystalline structures in which a compound (or its salts or solvates) can crystallize in different crystalline arrangements, all having the same elemental composition. Different crystal forms typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optical and electrical properties, stability, and solubility. Recrystallization solvents, crystallization rates, storage temperatures, and other factors can cause one crystal form to dominate. Polymorphs of compounds can be prepared by crystallization under different conditions.

[0168] The composition may also include salts of one or more compounds. The salt may be a pharmaceutically acceptable salt. As used herein, a "pharmaceutically acceptable salt" refers to a derivative of the disclosed compound in which the parent compound is modified by converting an existing acid or base moiety into its salt form (e.g., by reacting the free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amino groups; alkali metal or organic salts of acidic residues such as carboxylic acids; and the like.

[0169] Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, diglucuronate, dodecyl sulfate, ethanesulfonate, fumarate, glucohepanoate, glyceryl phosphate, hemisulfate, heptaate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, dodecyl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, etc.

[0170] Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc.

[0171] Pharmaceutically acceptable salts disclosed herein include, for example, conventional non-toxic salts of parent compounds formed from non-toxic inorganic or organic acids.

[0172] The pharmaceutically acceptable salts disclosed herein can be synthesized from parent compounds containing a basic or acidic moiety using conventional chemical methods. Typically, such salts are prepared by reacting the free acidic or basic form of these compounds with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture thereof; typically, non-aqueous media such as diethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. A list of suitable salts is available in any edition of Remington's Pharmaceutical Sciences, which is incorporated herein by reference in its entirety.

[0173] As used herein, "phospholipid" is a lipid comprising a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. Phospholipids may include one or more multiple (e.g., double or triple) bonds (e.g., one or more degrees of unsaturation). Certain phospholipids can facilitate fusion with membranes. For example, cationic phospholipids can interact with one or more negatively charged phospholipids in a membrane (e.g., a cell membrane or intracellular membrane). Fusion of phospholipids with membranes can allow one or more components of a lipid-containing composition to cross the membrane, thereby allowing, for example, the delivery of one or more components to cells.

[0174] As used herein, the term “polypeptide” or “polypeptide of interest” refers to a polymer of amino acid residues typically linked by peptide bonds, which may be produced naturally (e.g., isolated or purified) or synthetically.

[0175] As used herein, “RNA” refers to ribonucleic acid, which may be naturally occurring or non-natural. For example, RNA may include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. RNA may include cap structures, chain-terminating nucleosides, stem-loops, polyA sequences, and / or polyadenylation signals. RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, RNA may be messenger RNA (mRNA). Translation of mRNA encoding a specific polypeptide, such as in vivo translation of mRNA within mammalian cells, can produce the encoded polypeptide. RNA may be selected from small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), and mRNA.

[0176] As used herein, “single unit dose” refers to the dose of any therapeutic agent administered in a single dose / single route / single point of contact, i.e., a single dosing event. As used herein, “fractionated dose” refers to dividing a single unit dose or total daily dose into two or more doses. As used herein, “total daily dose” refers to the amount administered or prescribed over a 24-hour period. It can be administered as a single unit dose.

[0177] As used herein, “size” or “average size” in the context of nanoparticle compositions refers to the average diameter of the nanoparticle composition.

[0178] As used herein, the terms “subject,” “object,” or “patient” mean any organism to which the compositions according to this disclosure may be administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. Preferably, the subject described herein suffers from a disease that benefits from specific drug delivery, such as a lesion in an organ or tissue to which it is desired to deliver the drug only to that organ or tissue.

[0179] As used herein, "target cell" refers to any one or more cells of interest. Cells can be found in vitro, in vivo, in situ, or in the tissues or organs of an organism. The organism can be an animal, preferably a mammal, more preferably a human, and most preferably a patient. In some embodiments, the target cell is a T cell.

[0180] As used herein, “target tissue” refers to any one or more tissue types of interest, wherein delivery of treatment and / or prevention will result in the desired biological and / or pharmacological effects. Examples of target tissues of interest include specific tissues, organs, and systems or groups thereof. In some embodiments, the target organ is the spleen or liver.

[0181] The terms "therapeutic agent" or "prophylactic agent" refer to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or preventative effect and / or induces the desired biological and / or pharmacological action. Therapeutic agents are also referred to as "active substances" or "active agents" and include, but are not limited to, cytotoxic substances, radioactive ions, chemotherapeutic drugs, small molecule drugs, proteins, and nucleic acids, such as RNA. In some embodiments, the nucleic acid is selected from one or more of pDNA, mRNA, siRNA, saRNA, microRNA, and shRNA.

[0182] As used herein, the term "therapeutic effective dose" means, when administered to a subject who has or is susceptible to an infection, disease, condition and / or symptom, to treat, improve the symptoms of the infection, disease, condition and / or symptom, to diagnose, prevent and / or delay the onset of the infection, disease, condition and / or symptom.

[0183] As used in this article, "transfection" refers to the introduction of a substance (such as RNA) into cells. Transfection can occur, for example, in vitro, outside the body, or in vivo.

[0184] As used herein, the term “treatment” refers to the partial or complete reduction, improvement, alleviation, delay of the onset, inhibition of progression, reduction of severity, and / or decrease of incidence of a particular disease, disorder, and / or condition. For example, “treatment” for cancer may refer to the inhibition of tumor survival, growth, and / or spread. To reduce risk, treatment may be administered to subjects who do not exhibit signs of disease, condition, and / or symptoms, and / or subjects who only exhibit early signs of disease, condition, and / or symptoms, to develop pathology associated with the disease, condition, and / or symptoms.

[0185] Another aspect of this disclosure is a nanoparticle composition comprising a lipid component, said lipid component comprising a compound of formula I described herein, or a salt or isomer thereof.

[0186] Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, lipid vesicles, and lipid complexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In some embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by aqueous compartments. The lipid bilayers may be functionalized and / or cross-linked with each other. The lipid bilayer may include one or more ligands, proteins, or channels.

[0187] The nanoparticle composition comprises a lipid component, including at least one compound of formula I, or a salt or isomer thereof. The nanoparticle composition may also include a variety of other components. For example, in addition to lipids of formula I compounds, the nanoparticle composition may include one or more cationic lipids.

[0188] The lipid component of the nanoparticle composition may include one or more PEG lipids. PEG lipids are lipids modified with polyethylene glycol, and may be alternatively referred to as PEGylated lipids. PEG lipids may be selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, PEG lipids may be PEG-c-DOMG, PEG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids, preferably PEG-DMG or PEG-DSPE lipids.

[0189] In some embodiments, the tail chain of the PEGylated lipid includes, but is not limited to, a saturated or unsaturated alkane chain of C14-C18 length, comprising a polyethylene glycol moiety having a molecular weight of 1000-5000 Da, preferably 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (PEG). 2000 -DMG), distearate phosphatidylethanolamine-polyethylene glycol 2000 (PEG2) 000 -DSPE) or cholesterol-polyethylene glycol 2000 (PEG) 2000 One or more of -Cho1), preferably PEG 2000 -DMG or PEG2 000 -DSPE, more preferably PEG 2000 -DMG.

[0190] In some embodiments, the PEG lipid may optionally be functionalized. When functionalized, the PEG lipid is a functionalized PEG lipid. The functional group is a conventional functional group in the art that can be used to covalently couple with an antibody, such as maleimide, NHS ester, DBCO, TCO hydrazide, etc. An exemplary functionalized PEG lipid may be a maleimide-modified polyethylene glycol lipid.

[0191] The lipid component of the nanoparticle composition may include one or more structural lipids. The structural lipids may be selected from, but are not limited to, cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisolone, and hydrocortisone) or combinations thereof.

[0192] The lipid component of the nanoparticle composition may include one or more phospholipids, such as one or more unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. Typically, phospholipids may include a phospholipid moiety and one or more fatty acid moieties. For example, phospholipids may be lipids according to Formula II:

[0193]

[0194] (II)

[0195] In Equation II, R p The symbol R1 and R2 represent the fatty acid moieties, which may or may not have unsaturated fatty acids and can be the same or different. The phospholipid moieties can be selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moieties can be selected from lauric acid, myristic acid, myristone acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, benzolic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0196] Phospholipids that can be used in the compositions and methods described herein may be selected from soybean phospholipids (SPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate choline (SOPC), 1-palmitoyl-2-oleoyl lecithin (POPC), disorhoyl lecithin (DEPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), dipalmitoyl lecithin (DPPC), 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), DLPC, myristoylphosphatidylcholine (DMPC), DUPC, OChemsPC, DOPG, and sphingomyelin. In some embodiments, the nanoparticle composition comprises DSPC. In some embodiments, the nanoparticle composition comprises DOPE. In some embodiments, the nanoparticle composition comprises DSPC and / or DOPE.

[0197] In some embodiments, the nanoparticle composition described herein comprises a compound of formula I, a PEG lipid, DSPC, and cholesterol. In some embodiments, the nanoparticle composition described herein comprises a compound of formula I, an unfunctionalized PEG lipid, DSPC, and cholesterol. In other embodiments, the nanoparticle composition described herein comprises a compound of formula I, a functionalized PEG lipid, an unfunctionalized PEG lipid, DSPC, and cholesterol; preferably, the molar ratio of the unfunctionalized PEG lipid to the functionalized PEG lipid is (14-2):1, for example (10-2):1, (5-2):1.

[0198] In some embodiments, the nanoparticle composition comprising one or more lipids described herein may also comprise one or more adjuvants, such as aluminum hydroxide.

[0199] Therapeutic agents and / or preventative agents

[0200] The nanoparticle composition may include one or more therapeutic and / or preventive agents. The present invention relates to methods for delivering therapeutic and / or preventive agents to mammalian cells or organs, generating polypeptides of interest in mammalian cells, and treating diseases or conditions in mammals in need, including administering and / or contacting mammalian cells having a nanoparticle composition comprising therapeutic and / or preventive agents.

[0201] Therapeutic agents and / or preventative agents can be substances that, once delivered to cells or organs, bring about desired changes in those cells, organs, or other body tissues or systems. Such species can be used to treat one or more diseases or conditions. In some embodiments, therapeutic agents and / or preventative agents are small-molecule pharmaceuticals that can be used to treat specific diseases or conditions.

[0202] Examples of pharmaceuticals that can be used in nanoparticle compositions include, but are not limited to, antitumor agents (e.g., doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, streptozotocin, actinomycin D, vincristine, vinblastine, cystine arabinoside, anthracyclines, alkylating agents, platinum compounds, antimetabolites and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs), anti-infective agents, local anesthetics (e.g., debucaine and chlorpromazine), β-adrenergic blockers (e.g., propranolol, timolol, and labetalol), and antihypertensive drugs (e.g., chlorpromazine). Drugs containing: Loratadine and hydralazine, antidepressants (e.g., imipramine, amitriptyline, and doxepin), antidepressants (e.g., phenytoin sodium), antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterial agents (e.g., gentamicin, ciprofloxacin, and cefoxitin), antifungals (e.g., miconazole, terconazole, econazole, isconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, anesthetics, and imaging agents.

[0203] In some implementations, the therapeutic and / or prophylactic agents are cytotoxins, radioactive ions, chemotherapeutic agents, vaccines, compounds that elicit an immune response, and / or another therapeutic and / or prophylactic agent. Cytotoxins or cytotoxic agents include any reagent that may be harmful to cells. Examples include, but are not limited to, paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, imidin, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, photomycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansine, e.g., maytanol, receramide, and their analogues or homologues. Radioactive ions include, but are not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium.

[0204] Other treatment and / or preventive medications include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., nitrogen mustard, thiotepiperazine, resveratrol, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatin(II)(DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly known as doxorubicin) and doxorubicin), antibiotics (e.g., dermatomycin (formerly known as actinomycin), bleomycin, photomycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine, vinblastine, paclitaxel, and maytansine alkaloids).

[0205] In other embodiments, the therapeutic and / or preventative agents are proteins. Therapeutic proteins useful in the nanoparticles of this disclosure include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), factor VIR, luteinizing hormone-releasing hormone (LHRH) analogs, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0206] In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term "polynucleotide," in its broadest sense, includes any compound and / or substance that is or may be incorporated into an oligonucleotide chain. Exemplary polynucleotides used according to this disclosure include, but are not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), their hybrids, RNAi inducers, one or more of RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, vectors, etc.

[0207] In some embodiments, the therapeutic and / or preventative agent is RNA. The RNA that can be used in the compositions and methods described herein may be selected from, but is not limited to, short chains, antagonists, antisense strands, ribozymes, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA, small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some embodiments, the RNA is mRNA or siRNA.

[0208] In some embodiments, the therapeutic and / or preventative agent is mRNA. The mRNA may encode any polypeptide of interest, including any naturally occurring or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can be of any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA may have a therapeutic effect when expressed in cells. In some embodiments, the mRNA is TPA mRNA.

[0209] In other embodiments, the therapeutic and / or preventative agent is siRNA. The siRNA may be capable of selectively knocking down or downregulating the expression of genes of interest. For example, the siRNA may be selected to silence genes associated with a specific disease or condition when administered to a subject in need of a nanoparticle composition containing siRNA. The siRNA may contain a sequence complementary to the mRNA sequence encoding a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.

[0210] In some implementations, the therapeutic and / or prophylactic agent is shRNA or a vector or plasmid encoding it. shRNA can be generated within target cells after delivery of a suitable construct to the cell nucleus. Constructs and mechanisms associated with shRNA are well known in the relevant fields.

[0211] Nucleic acids and polynucleotides used in this disclosure typically include a first region (e.g., a coding region) encoding a linker nucleoside of a polypeptide of interest, a first flanking region (e.g., a 5-UTR) located at the 5' end of the first region, a second flanking region (e.g., a 3-UTR) located at the 3' end of the first region, at least one 5'-cap region, and a 3'-stabilizing region. In some embodiments, the nucleic acid or polynucleotide also includes a poly-A region or a Kozak sequence (e.g., in the 5'-UTR). In some cases, the polynucleotide may contain one or more intronic nucleotide sequences that can be cleaved from the polynucleotide. In some embodiments, the polynucleotide or nucleic acid (e.g., mRNA) may include a 5' cap structure, a chain-terminating nucleotide, a stem-loop, a polyadenylated sequence, and / or a polyadenylation signal. Any region of the nucleic acid may include one or more alternative components (e.g., alternative nucleosides).

[0212] Typically, the shortest length of a polynucleotide can be a polynucleotide sequence long enough to encode a dipeptide. In another embodiment, the length of the polynucleotide sequence is sufficient to encode a tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. In some cases, the length of the polynucleotide is at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 80, at least 90, at least 100, at least 120, at least 150, at least 180, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, or at least 5000 nucleotides.

[0213] Nucleic acids and polynucleotides may include one or more naturally occurring components, including any typical nucleotide A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine). In one embodiment, all or substantially all of the nucleotides comprise (a) a 5'-UTR, (b) an open reading frame (ORF), (c) a 3'-UTR, (d) a polyadenylated tail, and any combination of a, b, c, or d above.

[0214] Polynucleotides and nucleic acids may include one or more modified (e.g., altered or substituted) nucleobases, nucleosides, nucleotides, or combinations thereof. Nucleic acids and polynucleotides used in nanoparticle compositions may include any useful modifications or alterations, such as to the inter-nucleobase, sugar, or nucleoside bonds (e.g., linking phosphate / phosphodiester bonds / phosphodiester backbones).

[0215] In some cases, nucleic acids do not induce an innate immune response in cells into which polynucleotides (such as mRNA) are introduced.

[0216] Other components

[0217] In addition to those described in the foregoing sections, nanoparticle compositions may include one or more components. For example, nanoparticle compositions may include one or more hydrophobic small molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0218] The nanoparticle composition may also include one or more permeability-enhancing molecules, carbohydrates, polymers, surface modifiers, or other components. Permeability-enhancing molecules may be, for example, those described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs). Polymers may be included in and / or used to encapsulate or partially encapsulate the nanoparticle composition. Polymers may be biodegradable and / or biocompatible. Polymers may be selected from, but are not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, polystyrene, polyimides, polysulfones, polyurethanes, polyacetylene, polyethylene, polyethyleneimine, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitrile, and polyarylates. Surface modifiers may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants, such as dimethyl dioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytics (e.g., acetylcysteine, nettinib, and erdosteine), and DNases (e.g., rhDNase). Surface modifiers may be disposed within nanoparticles and / or on the surface of nanoparticle compositions (e.g., through coating, adsorption, covalent bonding, or other processes).

[0219] The nanoparticle composition may also contain one or more functionalized lipids. For example, lipids can be functionalized with alkynyl groups, which can undergo cycloaddition reactions when exposed to azides under appropriate reaction conditions. In particular, lipid bilayers can be functionalized in this way with one or more groups that can be used to promote membrane permeation, cell recognition, or imaging.

[0220] The surface of the nanoparticle composition can also be conjugated with one or more useful antibodies. Functional groups and conjugates for targeted cell delivery, imaging, and membrane permeation are well known in the art. In some embodiments, the antibody is an antibody capable of targeting immune cells. In some embodiments, the immune cells are T cells, and the antibody is a CD3 antibody. Therefore, the present invention also provides a nanoparticle composition of conjugated antibodies in which the lipid component contains fragments conventional in the art capable of conjugating antibodies, such as maleimide structures. In some embodiments, the lipid component of the nanoparticle composition of conjugated antibodies includes a compound of formula I herein, or a salt or isomer thereof, unfunctionalized PEG lipids, antibody-conjugated functionalized PEG lipids, structural lipids (such as cholesterol), and other lipids (such as phospholipids). Antibody-conjugated functionalized PEG lipids refer to lipids obtained by conjugating functionalized PEG lipids with antibodies.

[0221] In addition to these components, nanoparticle compositions may include any substance that can be used in pharmaceutical compositions. For example, nanoparticle compositions may include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersants, suspending agents, granulation aids, disintegrants, fillers, flow aids, liquid carriers, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, and other substances. Excipients may also be included, such as waxes, butters, colorants, coating agents, flavoring agents, and fragrances. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's *The Science and Practice of Pharmacy*, 21st edition).

[0222] The lipid component of the nanoparticle composition may include, for example, lipids according to Formula I, phospholipids (e.g., unsaturated lipids, such as DOPE or DSPC), PEG lipids, and structural lipids. Each lipid component may be provided in a specific fraction. In some embodiments, the lipid component of the nanoparticle composition comprises about 50 mol% to about 65 mol% of a compound of Formula I, about 15 mol% to about 25 mol% of phospholipids, about 8 mol% to about 34.5 mol% of structural lipids, and about 0.5 mol% to about 2 mol% of PEG lipids. In some embodiments, the lipid component of the nanoparticle composition comprises about 55 mol% to about 60 mol% of a compound of Formula I, about 20 mol% to about 25 mol% of phospholipids, about 20 mol% to about 30 mol% of structural lipids, and about 1.5 mol% to about 2 mol% of PEG lipids. In a particular embodiment, the lipid component comprises about 58 mol% of a compound of Formula I, about 20 mol% of phospholipids, about 20.5 mol% of structural lipids, and about 1.5 mol% of PEG lipids. In another specific embodiment, the lipid component comprises about 50 mol% of a compound of formula I, about 10 mol% of a phospholipid, about 38.5 mol% of a structural lipid, and about 1.5 mol% of a PEG lipid. In some embodiments, the phospholipid may be DOPE or DSPC. In other embodiments, the PEG lipid may be PEG-DMG (e.g., PEG). 2000 -DMG) or PEG-DSPE (e.g., PEG-DMG) or PEG-DSPE 2000 -DSPE). In some implementations, the structural lipid can be cholesterol.

[0223] The amount of therapeutic and / or prophylactic agents in a nanoparticle composition can depend on the size, composition, desired target and / or application, or other properties of the nanoparticle composition, as well as the nature of the therapeutic and / or prophylactic agents. For example, the amount of RNA that can be used in a nanoparticle composition may depend on the size, sequence, and other characteristics of the RNA. The relative amounts of therapeutic and / or prophylactic agents and other components (e.g., lipids) in a nanoparticle composition can also vary. In some embodiments, the weight / weight ratio of the lipid component to the therapeutic agent and / or preventive agent in the nanoparticle composition may be from about 3:1 to about 60:1, for example, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 46:1, 47:1, 48:1, 50:1, 60:1. For example, the weight / weight ratio of the lipid component to the therapeutic agent and / or preventive agent may be from about 10:1 to about 40:1, about 20:1 to about 50:1, about 30:1 to about 50:1, about 40:1 to about 50:1, or about 45:1 to about 50:1. In some embodiments, the weight / weight ratio is about 20:1, about 46:1, about 47:1, or about 48:1. The amount of therapeutic and / or preventative agents in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., UV-Vis spectroscopy).

[0224] The average size of the nanoparticle composition can be between 30 nanometers and 200 nanometers, for example, measured by methods well known in the art. For example, the average size can be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm or 150 nm. In some embodiments, the average size of the nanoparticle composition may be about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, from about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In some embodiments, the average size of the nanoparticle composition may be about 70 nm to about 120 nm, or about 80 nm to about 110 nm. In specific embodiments, the average size may be about 58 nm, about 59 nm, about 60 nm, about 63 nm, about 64 nm, about 65 nm, about 66 nm, about 67 nm, or about 68 nm.

[0225] The nanoparticle composition can be relatively homogeneous. A polydispersity index can be used to indicate the uniformity of the nanoparticle composition, such as the particle size distribution. Small polydispersity indices (e.g., less than 0.3) typically indicate a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition can be 0–0.2, 0–0.15, 0–0.13, or 0–0.12.

[0226] Encapsulation efficiency of therapeutic and / or preventative agents describes the amount of therapeutic and / or preventative agents that are encapsulated or otherwise bound to the nanoparticle composition after preparation, relative to the initial amount provided. Encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or preventative agents in a solution containing the nanoparticle composition before and after the nanoparticle composition is broken down with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or preventative agents (e.g., RNA) in solution. For the nanoparticle compositions described herein, the encapsulation efficiency of the therapeutic and / or preventative agents can be at least 50%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In some embodiments, the encapsulation efficiency can be at least 90%.

[0227] The nanoparticle composition may optionally include one or more coatings. For example, the nanoparticle composition may be formulated in coated capsules, films, or tablets. The capsules, films, or tablets of the compositions described herein may have any useful dimensions, tensile strength, hardness, or density.

[0228] The nanoparticle compositions of the present invention can be prepared in various forms suitable for a variety of routes of administration, such as liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable dosage forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical (including buccal and sublingual), transdermal and / or transdermal administration (e.g., creams, ointments, pastes, lotions, gels, powders, solutions, sprays, inhalers, and patches), dosage forms for vaginal application (e.g., vaginal suppositories, tampons, creams, gels, pastes, foams, and sprays), dosage forms for implant application (e.g., solids, semi-solids, gels), suspensions, powders, and other dosage forms.

[0229] Pharmaceutical Composition

[0230] Nanoparticle compositions can be formulated wholly or partially as pharmaceutical compositions. Pharmaceutical compositions may include one or more nanoparticle compositions.

[0231] For example, a pharmaceutical composition may include one or more nanoparticle compositions, comprising one or more different therapeutic and / or preventative agents. The pharmaceutical composition may also include one or more pharmaceutically acceptable excipients, such as those described herein. General guidance on formulating and manufacturing pharmaceutical compositions and agents can be found in Remington's *The Science and Practice of Pharmacy*, 21st edition. Conventional excipients and adjuvants may be used in any pharmaceutical composition unless any conventional excipient or adjuvant may be incompatible with one or more components of the nanoparticle composition. The amount of excipients in a pharmaceutical composition may be determined by someone skilled in the art as needed.

[0232] The relative amounts of one or more nanoparticle compositions, one or more pharmaceutically acceptable excipients, and / or any additional ingredients in the pharmaceutical compositions according to this disclosure will vary depending on the characteristics and size of the nanoparticles and / or the condition of the treated subject, and further depend on the route of administration of the composition. For example, the pharmaceutical composition may comprise 0.1% to 100% (wt / wt) of one or more nanoparticle compositions.

[0233] In some embodiments, the nanoparticle compositions and / or pharmaceutical compositions of this disclosure are refrigerated or frozen for storage and / or transport (e.g., stored at temperatures of 4°C or lower, such as temperatures between -150°C) and between about 0°C or about -80°C and about -20°C (e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C).

[0234] For example, the nanoparticle compositions and / or pharmaceutical compositions disclosed herein are stable for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months, for example, at a temperature of 4°C or lower (e.g., between about 4°C and -20°C). In one embodiment, the formulation is stable at 4°C for at least 4 weeks. In some embodiments, the pharmaceutical compositions disclosed herein comprise the nanoparticle compositions disclosed herein and pharmaceutically acceptable excipients selected from one or more of Tris, acetates (e.g., sodium acetate), citrates (e.g., sodium citrate), saline, PBS, and sucrose. In some embodiments, the pharmaceutical compositions of this disclosure have a pH value between about 7 and 8 (e.g., between 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or between 7.5).

[0235] In the context of this disclosure, "stability" and "stable" mean that the nanoparticle compositions and / or pharmaceutical compositions disclosed herein remain unchanged or nearly unchanged in response to chemical or physical changes (e.g., degradation, particle size change, aggregation, altered encapsulation, etc.) under given conditions of manufacture, preparation, transport, storage and / or use, such as when stresses such as shear force, freeze / thaw stress are applied.

[0236] Nanoparticle compositions and / or pharmaceutical compositions comprising one or more nanoparticle compositions may be administered to any patient or subject, including those patient or subject cells, tissues, organs, or systems or groups thereof who may benefit from therapeutic effects provided by delivering therapeutic agents and / or preventative agents to one or more specific pharmaceutical agents.

[0237] Although the term "composition" primarily refers to compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to any other mammal.

[0238] It is well known that compositions suitable for human administration are modified to make them suitable for administration to a variety of animals, and ordinary skilled veterinary pharmacologists can design or perform such modifications simply through routine (if any) experiments. Intended subjects for administration of the compositions include, but are not limited to, humans, other primates and other mammals, including commercially relevant mammals such as cattle, pigs, tube animals, sheep, cats, dogs, mice and / or rats.

[0239] Pharmaceutical compositions comprising one or more nanoparticles can be prepared by any method known or subsequently developed in the field of pharmacology. Generally, such preparation methods involve combining the active ingredient with excipients and / or one or more other auxiliary ingredients, and then, if desired or necessary, segmenting, shaping, and / or packaging the product into the desired single or multiple forms. Multiple dosage units are also possible.

[0240] The pharmaceutical compositions according to this disclosure can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as multiple single unit doses. As used herein, a “unit dose” is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient (e.g., a nanoparticle composition). The amount of active ingredient is generally equal to the dose of active ingredient to be given to a subject and / or a convenient fraction of that dose, such as one-half or one-third of the dose.

[0241] Pharmaceutical compositions can be formulated in various forms suitable for a variety of routes and methods of administration. For example, pharmaceutical compositions can be formulated as liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable dosage forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical (including buccal and sublingual), transdermal and / or transdermal administration (e.g., creams, ointments, pastes, lotions, gels, powders, solutions, sprays, inhalers, and patches), dosage forms for vaginal administration (e.g., vaginal suppositories, tampons, creams, gels, pastes, foams, and sprays), dosage forms for implant administration (e.g., solids, semi-solids, gels), suspensions, powders, and other dosage forms.

[0242] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitol fatty acid esters, and mixtures thereof. Besides inert diluents, oral compositions may include additional therapeutic and / or prophylactic agents, additional agents such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and / or aromatizers. In some embodiments of parenteral administration, the composition is mixed with a solubilizer, such as alcohol, oil, modified oil, ethylene glycol, polysorbate, cyclodextrin, polymer and / or combinations thereof.

[0243] Injectable formulations, such as sterile injectable aqueous or oily suspensions, can be formulated using suitable dispersants, wetting agents, and / or suspending agents according to known techniques. Sterile injectable formulations can be sterile injectable solutions, suspensions, and / or emulsions in non-toxic, parenteral diluents and / or solvents. Acceptable carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. Sterile oils can be used as solvents or suspension media. For this purpose, any mild, non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used to prepare injections. Injectable formulations can be sterilized, for example, by filtration through a bacterial trap filter, and / or by incorporating a sterilizing agent in the form of a sterile solid composition, which can be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

[0244] Compositions for rectal or vaginal administration are typically suppositories, which can be prepared by mixing the composition with a suitable non-irritating excipient that is solid at ambient temperature but liquid at body temperature and thus melts in the rectum or vaginal cavity and releases the active ingredient.

[0245] This disclosure also contemplates the use of transdermal patches, such dosage forms which can be prepared, for example, by dissolving and / or dispersing the compound in a suitable medium. Alternatively or additionally, the rate can be controlled by providing a rate-controlled membrane and / or by dispersing the compound in a polymer matrix and / or gel.

[0246] Suitable devices for delivering the intradermal pharmaceutical compositions described herein include any type of liquid or solid injection device, such as conventional syringes, fine syringes, short needle syringes, liquid jet syringes, compressed gas accelerated powder syringes, ballistic powder delivery devices, etc.

[0247] Pharmaceutical compositions can be prepared, packaged, and / or sold in formulations suitable for administration via the buccal cavity or lung. Such formulations may comprise dry granules containing the active ingredient. These compositions are conveniently available in dry powder form for administration using devices comprising dry powder reservoirs and / or self-propelled solvent / powder dispensing containers. Such formulations can also be prepared, packaged, and / or sold as aqueous solutions and / or dilute alcoholic solutions and / or suspensions (optionally sterile, containing the active ingredient) and can be conveniently administered using any nebulizer and / or atomizing device. Such formulations may also contain one or more additional ingredients, including but not limited to flavoring agents such as sodium saccharin, volatile oils, buffers, surfactants, and / or preservatives such as methylparaben.

[0248] The formulations described herein for pulmonary delivery can also be used for intranasal delivery of pharmaceutical compositions. Such formulations are administered by inhalation, i.e., rapid inhalation through the nasal passages from a powder container near the nose. Pharmaceutical compositions can be prepared, packaged, and / or marketed as formulations suitable for oral administration. Such formulations can be, for example, in the form of tablets and / or lozenges prepared using conventional methods.

[0249] Methods and uses

[0250] This disclosure provides a method for generating a target polypeptide in mammalian cells. The method involves contacting cells with a nanoparticle composition containing mRNA encoding the target polypeptide. When cells are contacted with the nanoparticle composition, the mRNA can be absorbed and translated within the cells to generate the target polypeptide.

[0251] Generally, the process of contacting mammalian cells with a nanoparticle composition containing mRNA encoding a target polypeptide can be performed in vivo, in vitro, in culture, or in vitro. The amount of nanoparticle composition contacted with the cells and / or the amount of mRNA therein can depend on the type of cells or tissues contacted, the method of administration, the physiological and chemical characteristics of the nanoparticle composition and mRNA (e.g., size, charge, and chemical composition), and other factors. Efficiency metrics may include polypeptide translation (represented by polypeptide expression), mRNA degradation levels, and immune response indicators.

[0252] The step of contacting a nanoparticle composition containing mRNA with cells can involve or cause transfection. Phospholipids contained in the lipid component of the nanoparticle composition can, for example, promote transfection and / or enhance transfection efficiency by interacting with and / or fusing with the cell membrane or intracellular membrane. Transfection can allow the mRNA to be translated within the cell. In some embodiments, the cells are selected from immune cells, such as T cells.

[0253] In some embodiments, the nanoparticle compositions described herein can be used for therapeutic purposes. For example, the mRNA contained in the nanoparticle composition may encode a therapeutic peptide (e.g., in a translatable region) and produce the therapeutic peptide upon contact with and / or entry into (e.g., transfection) cells. In other embodiments, the mRNA contained in the nanoparticle composition may encode a peptide that improves or increases the immunity of a subject.

[0254] In some embodiments, the mRNA contained in the nanoparticle composition may encode a recombinant polypeptide that may replace one or more polypeptides that are substantially absent in the cells in contact with the nanoparticle composition. Alternatively, the recombinant polypeptide generated by mRNA translation may antagonize the activity of endogenous proteins in, on, or secreted by cells. In another alternative, the recombinant polypeptide generated by mRNA translation may indirectly or directly antagonize the activity of biological moieties in, on, or secreted by cells. The antagonized biological moieties may include, but are not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., low-density lipoprotein), nucleic acids, carbohydrates, and small molecule toxins.

[0255] In some implementations, mRNA is as described in any of the implementations herein.

[0256] Methods for delivering therapeutic agents to cells and organs

[0257] This disclosure provides methods for delivering therapeutic and / or prophylactic agents to mammalian cells or organs. Delivery of therapeutic and / or prophylactic agents to cells involves administering a nanoparticle composition comprising the therapeutic and / or prophylactic agent to a subject, wherein administration of the composition involves contacting cells with the composition. For example, proteins, cytotoxic agents, radioactive ions, chemotherapeutic agents, or nucleic acids (e.g., RNA, such as mRNA) can be delivered to cells or organs. In the case where the therapeutic and / or prophylactic agent is mRNA, when cells are contacted with the nanoparticle composition, the translatable mRNA can be translated within the cells to produce a polypeptide of interest. However, substantially untranslatable mRNA can also be delivered to cells. In one or more embodiments, the therapeutic and / or prophylactic agent comprises a coding sequence (DNA and / or RNA) of a chimeric antigen receptor.

[0258] In some embodiments, the nanoparticle composition can target specific types of cells (e.g., cells of a specific organ or system thereof). For example, a nanoparticle composition containing a therapeutic and / or preventative agent of interest can be specifically delivered to the liver or spleen of a mammal. Specific delivery to a specific type of cell, organ, or system or tissue thereof means that a higher proportion of the nanoparticle composition containing the therapeutic and / or preventative agent is delivered to the destination of interest (e.g., cells, tissues, organs) relative to other destinations.

[0259] As another example of targeted or specific delivery, mRNA encoding a protein-binding ligand (e.g., an antibody or a functional fragment thereof, a scaffold protein, or a peptide) or receptor on the cell surface can be contained in the nanoparticle composition. In some embodiments, the ligand may be a surface-bound antibody, which can allow modulation of cell-targeting specificity. These approaches can improve the affinity and specificity of targeting interactions. The ligand can be selected by those skilled in the art, for example, biology, based on the desired localization or function of the cell. Targeted cells may include, but are not limited to, hepatocytes, spleen cells, epithelial cells, hematopoietic cells, endothelial cells (vascular endothelial cells), lung cells, osteocytes, stem cells, mesenchymal cells, nerve cells, cardiomyocytes, adipocytes, vascular smooth muscle cells, skeletal muscle cells, β cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells, preferably T cells.

[0260] Methods of treating diseases and symptoms

[0261] Nanoparticle compositions can be used to treat diseases or conditions. In particular, such compositions can be used to treat diseases or conditions characterized by the absence or abnormality of protein or peptide activity. For example, a nanoparticle composition containing mRNA encoding a missing or abnormal peptide can be administered or delivered to cells. Subsequent translation of the mRNA can produce a peptide, thereby reducing or eliminating the problems caused by the absence or abnormal activity of the peptide. The methods and compositions can be used to treat acute diseases or conditions such as sepsis, stroke, and myocardial infarction. Therapeutic agents and / or preventative agents contained in the nanoparticle compositions may also be able to alter the transcription rate of a given species, thereby affecting gene expression. Diseases and / or conditions characterized by dysfunctional or abnormal protein or peptide activity that can be administered to the compositions include, but are not limited to, rare diseases, infectious diseases (as vaccines and therapeutics), cancers and proliferative disorders, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0262] In this document, the disease or condition may also be selected from diseases or conditions that benefit immune cells, preferably, the immune cells are T cells. In one or more embodiments, the disease or condition is one that benefits T cell proliferation and / or activation. In one or more embodiments, the disease or condition is selected from viral infectious diseases (e.g., HIV, EB virus-related diseases, COVID-19, hepatitis B, hepatitis C, etc.), bacterial / parasitic infectious diseases (e.g., tuberculosis, leprosy), cancer (solid tumors and hematologic malignancies), autoimmune diseases (e.g., type 1 diabetes, multiple sclerosis, rheumatoid arthritis, etc.), and immunodeficiency diseases (allergic diseases, etc.).

[0263] This disclosure provides methods relating to the administration of nanoparticle compositions comprising one or more therapeutic and / or preventive agents, and pharmaceutical compositions comprising the same. The terms therapeutic and preventive are used interchangeably herein. The therapeutic composition or its imaging, diagnostic, or preventive composition may be administered to a subject in any reasonable amount and via any route of administration for the effective prevention, treatment, diagnosis, or imaging of a disease, condition, and / or illness. The specific amount administered to the subject may vary depending on the subject's species, age, and general condition, the specific ingredients, and the route of administration. Compositions according to this disclosure may be formulated in unit dosage form to facilitate administration and uniformity of dosage. However, it should be understood that the specific dosage of the compositions of this disclosure will be determined by the attending physician within the bounds of reasonable medical judgment.

[0264] Nanoparticle compositions comprising one or more therapeutic and / or prophylactic agents can be administered via any route. In some embodiments, compositions, including prophylactic, diagnostic, or imaging compositions comprising one or more nanoparticle compositions described herein, are administered via one or more of a variety of routes, including oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, percutaneous or intradermal, intradermal, rectal, intravaginal, intraocular, subretinal, vitreous, mucosal, nasal, oral, intestinal, intratumoral, sublingual, intranasal; intratracheal instillation, bronchial instillation and / or inhalation, as oral sprays and / or powders, nasal sprays and / or aerosols, and / or via portal vein catheters. Intravenous injection is a preferred route of administration. A suitable route of administration will depend on a variety of factors, including the nature of the nanoparticle composition, the therapeutic and / or prophylactic agents, the patient's condition, etc.

[0265] In some embodiments, the compositions according to this disclosure can be administered at levels sufficient to deliver the following doses: about 0.0001 mg / kg to about 10 mg / kg, about 0.001 mg / kg to about 10 mg / kg, about 0.005 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg. kg, about 0.0001 mg / kg to about 5 mg / kg, about 0.001 mg / kg to about 5 mg / kg, about 0.005 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 5 mg / kg, about 2 mg / kg to about 5 mg / kg, about 0.0001 mg / kg to about 2.5 mg / kg, about 0.0 ... mg / kg to about 2.5 mg / kg, about 0.01 mg / kg to about 2.5 mg / kg, about 0.05 mg / kg to about 0.05 mg / kg to about 2.5 mg / kg, about 2.5 mg / kg, about 0.1 mg / kg to about 2.5 mg / kg, about 1 mg / kg to about 2.5 mg / kg, about 2 mg / kg to about 2.5 mg / kg, about 0.0001 mg / kg to about 1 mg / kg, about 0.001 mg / kg to about 1 mg / kg, about 0.005 mg / kg to about 1 mg / kg, about 0.01 mg / kg to about 1 mg / kg, about 0.05 mg / kg to about 1 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 0.0001 mg / kg to about 0.25 mg / kg, about 0.001 mg / kg to about 0.25 mg / kg, about 0.005 mg / kg to about 0.25 mg / kg, about 0.01 mg / kg to about 0.25 mg / kg, about 0.05 mg / kg to about 0.25 mg / kg, or about 0.1 mg / kg to about 0.25 mg / kg of therapeutic agents and / or prophylactic agents (e.g., mRNA).

[0266] The dosage can be administered once or multiple times daily in the same or different amounts to achieve the desired level of mRNA expression and / or therapeutic, diagnostic, preventative, or imaging effects. The desired dosage can be delivered, for example, three times daily, twice daily, once daily, every other day, every three days, weekly, every two weeks, every three weeks, or every four weeks. In some embodiments, the desired dosage can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).

[0267] Nanoparticle compositions comprising one or more therapeutic and / or preventative agents may be used in combination with one or more other therapeutic, preventative, diagnostic, or imaging agents. Each agent will be administered at a dosage and / or schedule determined for that agent. Therapeutic, preventative, diagnostic, or imaging agents used in combination may be administered together in a single composition or individually in different compositions.

[0268] Those skilled in the art will recognize or be able to determine many equivalents of specific embodiments of the disclosure described herein using only conventional experiments. The scope of this disclosure is not intended to be limited to the foregoing description, but rather as set forth in the appended claims.

[0269] In the claims, articles such as “a,” “an,” and “the” may refer to one or more, unless there is a contrary indication or it is obvious from the context.

[0270] The term "comprising" is intended to be open and allows, but does not require, the inclusion of additional elements or steps. When the term "comprising" is used herein, the terms "substantially consisting of" and "consisting of" are therefore also covered and disclosed. Furthermore, it should be understood that the order of steps or the order in which certain actions are performed is not important, as long as the invention remains operable. Two or more steps or actions may be performed simultaneously.

[0271] The compounds disclosed herein can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or readily prepared intermediates, employing standard synthetic methods and procedures known or to be understood by those skilled in the art. The synthesis of the compounds disclosed herein will be apparent to those skilled in the art based on the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules, as well as for the transformation and manipulation of functional groups, are available from relevant scientific literature or standard textbooks in the field, such as Smith, MB, March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5 thJohn Wiley & Sons, New York, 2001. The synthetic methods described in the embodiments herein are intended to illustrate, but not limit, the general procedures for preparing the compounds disclosed herein. Those skilled in the art will notice that the order of certain steps may be changed during the reaction sequence and synthetic schemes described herein, such as the introduction and removal of protecting groups. Multiple stereoisomers can be generated in the reaction schemes described herein. Those skilled in the art will recognize that the reaction can be optimized to preferentially generate one isomer, or new schemes can be designed to generate a single isomer. If mixtures are generated, techniques such as preparative thin-layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC can be used to separate the isomers.

[0272] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0273] Experimental instruments

[0274]

[0275] Experimental reagents

[0276]

[0277] Example 1

[0278] The chemical structure of compound XH-1 is as follows:

[0279]

[0280] Step 1. Synthesis of intermediate A-1

[0281]

[0282] To a dry round-bottom flask equipped with a stir bar, octanoic acid (69.3 mmol, 10.00 g), 1,3-dihydroxyacetone (34.7 mmol, 3.12 g), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (86.7 mmol, 16.44 g), 4-dimethylaminopyridine (6.9 mmol, 847 mg), and dichloromethane (150 mL) were added sequentially, and the mixture was stirred at room temperature for 24 hours. After the reaction was detected by thin-layer chromatography (TLC), the reaction solution was washed once with an appropriate amount of saturated NaHCO3 solution, twice with 10% citric acid aqueous solution, and twice with saturated brine. After drying with anhydrous sodium sulfate, the solution was filtered and concentrated to obtain a pale yellow oily crude product, which was then purified by column chromatography (petroleum ether / ethyl acetate = 10:1) to obtain intermediate A-1 (10.36 g, yield 81%). 1 H NMR (400 MHz, Chloroform-d) δ 4.75 (s, 4H), 2.42 (t, J = 7.6 Hz, 4H), 1.66 (t, J = 7.4 Hz, 4H), 1.34 – 1.26 (m, 16H), 0.90 – 0.86 (m, 6H).

[0283] Step 2. Synthesis of intermediate A-2

[0284]

[0285] To a dry round-bottom flask equipped with a stir bar, intermediate A-1 (30.3 mmol, 10.36 g), sodium triacetoxyborohydride (45.4 mmol, 9.62 g), acetic acid (30.1 mmol, 1.82 g), and tetrahydrofuran (100 mL) were added sequentially. The mixture was stirred at room temperature for 24 hours under nitrogen protection. After the reaction was completed, the reaction solution was washed once with an appropriate amount of saturated NaHCO3 solution, twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a pale yellow oily crude product. This crude product was then purified by column chromatography (petroleum ether / ethyl acetate = 5:1) to obtain intermediate A-2 (9.68 g, yield 93%).

[0286] Step 3. Synthesis of intermediate A-3

[0287]

[0288] Intermediate A-2 (28.1 mmol, 9.68 g) was dissolved in 50 mL of DCM. 4-Nitrophenylchloroformate (42.2 mmol, 8.50 g) and triethylamine (84.3 mmol, 8.53 g) were slowly added at 0 °C, and the mixture was stirred at room temperature for 24 hours. After the reaction was completed by TLC, the reaction solution was washed twice with 10% citric acid aqueous solution and twice with saturated brine. After drying with anhydrous sodium sulfate, the solution was filtered and concentrated to obtain a pale yellow oily crude product. This crude product was then purified by column chromatography (petroleum ether:ethyl acetate = 10:1) to give intermediate A-3 (10.45 g, yield 73%). 1 H NMR (400 MHz, Chloroform-d) δ 8.36 –8.24 (m, 2H), 7.48 – 7.35 (m, 2H), 5.18 (tt, J = 5.9, 3.9 Hz, 1H), 4.50 (dd,J = 12.3, 3.9 Hz, 2H), 4.25 (dd, J = 12.3, 5.9 Hz, 2H), 2.36 (t, J = 7.5 Hz, 4H), 1.68 – 1.59 (m, 4H), 1.33 – 1.22 (m, 16H), 0.90 – 0.85 (m, 6H).

[0289] Step 4. Synthesis of intermediate A-4

[0290]

[0291] Intermediate A-3 (20.5 mmol, 10.45 g) was dissolved in 50 mL of DMF, and tert-butyl di(2-hydroxyethyl)carbamate (10.3 mmol, 2.10 g), 4-dimethylaminopyridine (20.5 mmol, 2.51 g), and triethylamine (20.5 mmol, 2.08 g) were added sequentially. The mixture was stirred at room temperature for 24 hours. After the reaction was completed by TLC, the reaction solution was washed three times with an appropriate amount of saturated NaHCO3 solution, once with 10% citric acid aqueous solution, and twice with saturated brine. After drying with anhydrous sodium sulfate, the solution was filtered and concentrated to obtain a pale yellow oily crude product, which was then purified by column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain intermediate A-4 (5.68 g, yield 59%). 1H NMR (400 MHz, Chloroform-d) δ 5.01 (t, J = 5.1 Hz, 2H), 4.27 (dd, J = 12.1, 4.3 Hz, 4H), 4.23 – 4.15 (m, 4H), 4.12 (dd, J = 12.1, 5.8Hz, 4H), 3.47 (dt, J = 16.0, 5.6 Hz, 4H), 2.25 (t, J = 7.6 Hz, 8H), 1.88 (s,2H), 1.54 (t, J = 7.4 Hz, 8H), 1.39 (s, 9H), 1.25 – 1.16 (m, 32H), 0.83 –0.78 (m, 12H).

[0292] Step 5. Synthesis of intermediate A-5

[0293]

[0294] Intermediate A-4 (6.0 mmol, 5.68 g) was dissolved in 30 mL of DCM, and 20 mL of HCl / EA solution was added. The mixture was stirred at room temperature for 2 hours. After the reaction was completed by TLC, the organic solvent was removed by rotary evaporation to obtain the crude product. The crude product was redissolved in DCM, washed once with an appropriate amount of saturated NaHCO3 solution, washed twice with saturated brine, dried over anhydrous sodium sulfate, and concentrated by filtration to obtain a pale yellow oily substance, which was intermediate A-5 (5.10 g). This substance did not require purification and was used directly in the next reaction.

[0295] Step 6. Synthesis of compound XH-1

[0296]

[0297] Intermediate A-5 (1.0 mmol, 1.00 g) was dissolved in 50 mL of DCM. Triphosgene (0.5 mmol, 153 mg) and triethylamine (3.0 mmol, 314 mg) were added sequentially under ice bath conditions. The mixture was then stirred at room temperature for 4 hours. N,N-dimethylethanolamine (3.0 mmol, 277 mg) was then added, and the mixture was stirred overnight. After the reaction was detected by TLC, the reaction solution was washed twice with 10% citric acid aqueous solution, once with saturated NaHCO3 solution, and twice with saturated brine. After drying with anhydrous sodium sulfate, the solution was filtered and concentrated to obtain a yellow oily crude product. This crude product was then purified by column chromatography (dichloromethane:methanol = 30:1) to give 538 mg of compound XH-1 (pale yellow oily liquid). 1H NMR (400 MHz, Chloroform-d) δ 5.07 (p, J =4.5 Hz, 2H), 4.38 – 4.16 (m, 14H), 3.67 – 3.55 (m, 4H), 2.57 (t, J = 5.9 Hz,2H), 2.39 – 2.25 (m, 14H), 1.61 (t, J = 7.4 Hz, 8H), 1.29 (qd, J = 8.3, 7.9,4.0 Hz, 32H), 0.90 – 0.86 (m, 12H). MS, ESI + m / z: calcd C 49 H 88 N2O 16 [M+H] + ,961.6; found, 961.7.

[0298] Example 2

[0299] The chemical structure of compound XH-2 is as follows:

[0300]

[0301] Referring to the synthesis method in step 6 of Example 1, N,N-dimethylethanolamine was replaced with 4-(dimethylamino)butyl-1-ol to obtain 545 mg of compound XH-2 (pale yellow oily liquid). 1 H NMR (400 MHz, Chloroform-d) δ5.07 (dt, J = 10.0, 5.0 Hz, 2H), 4.36 – 4.12 (m, 14H), 3.59 (q, J = 6.3 Hz,4H), 2.68 (s, 2H), 2.53 (s, 6H), MS, ESI + m / z: calcd C 51 H 92 N2O 16 [M+H] + , 989.6; found, 989.7.

[0302] Example 3

[0303] The chemical structure of compound XH-3 is as follows:

[0304]

[0305] Referring to the synthesis method in step 6 of Example 1, N,N-dimethylethanolamine was replaced with 2-dimethylaminoethylamine to obtain 525 mg of compound XH-3 (pale yellow oily liquid). 1 H NMR (400 MHz, Chloroform-d) δ 5.54(t, J = 4.9 Hz, 1H), 5.01 (tt, J = 5.8, 4.2 Hz, 2H), 4.27 (dd, J = 12.1, 4.3Hz, 4H), 4.21 (t, J = 5.7 Hz, 4H), 4.12 (dd, J = 12.1, 5.8 Hz, 4H), 3.51 (t,J = 5.8 Hz, 4H), 3.26 (q, J = 5.6 Hz, 2H), 2.42 (t, J = 6.0 Hz, 2H), 2.26 (t,J = 7.6 Hz, 8H), 2.22 (s, 6H), 1.54 (t, J = 7.3 Hz, 8H), 1.26 – 1.18 (m,32H), 0.84 – 0.78 (m, 12H). MS, ESI + m / z: calcd C 49 H 89 N3O 15 [M+H] + , 960.6;found, 960.7.

[0306] Example 4

[0307] The chemical structure of compound XH-4 is as follows:

[0308]

[0309] Referring to the synthesis method in step 6 of Example 1, N,N-dimethylethanolamine was replaced with 3-(dimethylamino)propylamine to obtain 561 mg of compound XH-4 (pale yellow oily liquid). 1H NMR (400 MHz, Chloroform-d) δ 6.70(t, J = 4.7 Hz, 1H), 5.07 (tt, J = 5.8, 4.2 Hz, 2H), 4.34 (dd, J = 12.1, 4.3Hz, 4H), 4.27 (t, J = 5.8 Hz, 4H), 4.18 (dd, J = 12.1, 5.8 Hz, 4H), 3.56 (t,J = 5.9 Hz, 4H), 3.32 (q, J = 5.7 Hz, 2H), 2.55 (s, 2H), 2.40 – 2.26 (m,14H), 1.75 (d, J = 13.0 Hz, 2H), 1.65 – 1.58 (m, 8H), 1.32 – 1.25 (m, 32H), 0.91 – 0.85 (m, 12H). MS, ESI + m / z: calcd C 50 H 91 N3O 15 [M+H] + , 974.6; found, 974.7.

[0310] Example 5

[0311] The chemical structure of compound XH-5 is as follows:

[0312]

[0313] Step 1. Synthesis of intermediate B-1

[0314]

[0315] To a dry round-bottom flask equipped with a stir bar, nonanoic acid (69.2 mmol, 10.00 g), 2-hydroxymethyl-1,3-propanediol (31.6 mmol, 3.35 g), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (80.0 mmol, 14.98 g), 4-dimethylaminopyridine (6.3 mmol, 772 mg), and dichloromethane (150 mL) were added sequentially, and the mixture was stirred at room temperature for 24 hours. After the reaction was detected by thin-layer chromatography (TLC), the reaction solution was washed once with an appropriate amount of saturated NaHCO3 solution, twice with 10% citric acid aqueous solution, and twice with saturated brine. After drying with anhydrous sodium sulfate, the solution was filtered and concentrated to obtain a pale yellow oily crude product, which was then purified by column chromatography (petroleum ether / ethyl acetate = 10:1) to obtain intermediate B-1 (8.24 g, yield 67%).

[0316] Step 2. Synthesis of intermediate B-2

[0317]

[0318] Intermediate B-1 (23.0 mmol, 8.24 g) was dissolved in 50 mL of DCM. 4-Nitrophenylchloroformate (34.5 mmol, 6.95 g) and triethylamine (69 mmol, 6.98 g) were slowly added at 0 °C, and the mixture was stirred at room temperature for 24 hours. After the reaction was completed by TLC, the reaction solution was washed twice with 10% citric acid aqueous solution and twice with saturated brine. After drying with anhydrous sodium sulfate, the solution was filtered and concentrated to obtain a pale yellow oily crude product. This crude product was then purified by column chromatography (petroleum ether:ethyl acetate = 10:1) to give intermediate B-2 (8.26 g, yield 69%).

[0319] Step 4. Synthesis of intermediate B-3

[0320]

[0321] Intermediate B-2 (15.0 mmol, 8.26 g) was dissolved in 50 mL of DMF, and tert-butyl di(2-hydroxyethyl)carbamate (7.5 mmol, 1.54 g), 4-dimethylaminopyridine (15.0 mmol, 1.83 g), and triethylamine (15.0 mmol, 1.52 g) were added sequentially. The mixture was stirred at room temperature for 24 hours. After the reaction was completed by TLC, the reaction solution was washed three times with an appropriate amount of saturated NaHCO3 solution, once with 10% citric acid aqueous solution, and twice with saturated brine. After drying with anhydrous sodium sulfate, the solution was filtered and concentrated to obtain a pale yellow oily crude product, which was then purified by column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain intermediate B-3 (5.35 g, yield 69%).

[0322] Step 5. Synthesis of intermediate B-4

[0323]

[0324] Intermediate B-3 (5.2 mmol, 5.35 g) was dissolved in 30 mL of DCM, and 20 mL of HCl / EA solution was added. The mixture was stirred at room temperature for 2 hours. After the reaction was completed by TLC, the organic solvent was removed by rotary evaporation to obtain the crude product. The crude product was redissolved in DCM, washed once with an appropriate amount of saturated NaHCO3 solution, washed twice with saturated brine, dried over anhydrous sodium sulfate, and concentrated by filtration to obtain a pale yellow oily substance, which was intermediate B-4 (4.92 g). This substance did not require purification and was used directly in the next reaction.

[0325] Step 6. Synthesis of compound XH-5

[0326]

[0327] Intermediate B-4 (1.0 mmol, 1.00 g) was dissolved in 50 mL of ACN. Potassium carbonate (3 mmol, 429 mg) and 2-bromoethanol (1.5 mmol, 194 mg) were added sequentially under ice bath conditions, and the mixture was stirred at 70 °C for 12 hours. After the reaction was completed by TLC, the reaction solution was washed twice with 10% citric acid aqueous solution, once with saturated NaHCO3 solution, and twice with saturated brine. After drying with anhydrous sodium sulfate, the solution was filtered and concentrated to obtain a yellow oily crude product. This crude product was then purified by column chromatography (dichloromethane:methanol = 30:1) to give 429 mg of compound XH-5 (pale yellow oily liquid). 1H NMR (400 MHz, Chloroform-d) δ 4.21 (dt, J = 5.8, 2.8 Hz, 8H), 4.14 (d, J = 5.9 Hz, 8H), 3.57 (dt, J =10.6, 4.3 Hz, 2H), 2.88 (t, J = 5.8 Hz, 4H), 2.75 (t, J = 5.2 Hz, 2H), 2.68(s, 1H), 2.43 (p, J = 6.0 Hz, 2H), 2.31 (t, J = 7.6 Hz, 8H), 1.59 (s, 8H),1.32 – 1.25 (m, 40H), 0.92 – 0.85 (m, 12H). MS, ESI + m / z: calcd C 52 H 95 NO 15 [M+H] + , 974.7; found, 974.7.

[0328] Example 6

[0329] The chemical structure of compound XH-6 is as follows:

[0330]

[0331] Referring to the synthesis method in step 6 of Example 5, 2-bromoethanol was replaced with 2-bromopropanol to obtain 398 mg of compound XH-6 (pale yellow oily liquid). 1 H NMR (400 MHz, Chloroform-d) δ 4.27 – 4.18 (m, 8H), 4.14 (d, J = 6.0 Hz, 8H), 3.73 (t, J = 5.5 Hz, 2H), 3.59 (t, J = 5.4 Hz, 2H), 3.51 – 3.44 (m, 2H), 2.84 (t, J = 5.9 Hz, 2H), 2.75 (t, J = 6.0 Hz, 1H), 2.43(p, J = 6.0 Hz, 2H), 2.31 (t, J = 7.5 Hz, 8H), 1.75 – 1.54 (m, 10H), 1.29 (m,40H), 0.88 (t, J = 6.6 Hz, 12H). MS, ESI + m / z: calcd C 53 H 97 NO15 [M+H] + , 988.7;found, 988.7

[0332] Example 7

[0333] The chemical structure of compound XH-7 is as follows:

[0334]

[0335] Intermediate B-4 (1.0 mmol, 1.00 g) was dissolved in 50 mL of DCM. Triphosgene (0.5 mmol, 153 mg) and triethylamine (3.0 mmol, 314 mg) were added sequentially under ice bath conditions. The mixture was then stirred at room temperature for 4 hours. N,N-dimethylethanolamine (3.0 mmol, 277 mg) was added, and the mixture was stirred overnight. After the reaction was detected by TLC, the reaction solution was washed twice with 10% citric acid aqueous solution, once with saturated NaHCO3 solution, and twice with saturated brine. After drying with anhydrous sodium sulfate, the solution was filtered and concentrated to obtain a yellow oily crude product. This crude product was then purified by column chromatography (dichloromethane:methanol = 30:1) to give 594 mg of compound XH-7 (pale yellow oily liquid). 1 H NMR (400 MHz, Chloroform-d) δ 4.30 – 4.11(m, 18H), 3.58 (d, J = 6.0 Hz, 4H), 2.58 (t, J = 5.9 Hz, 2H), 2.43 (q, J =5.9 Hz, 2H), 2.36 – 2.24 (m, 14H), 1.61 (s, 8H), 1.32 – 1.23 (m, 40H), 0.91 –0.85 (m, 12H). MS, ESI + m / z: calcd C 55 H 100 N2O 16 [M+H] + , 1045.7; found, 1045.8.

[0336] Example 8

[0337] The chemical structure of compound XH-8 is as follows:

[0338]

[0339] Following the synthesis method of Example 7, N,N-dimethylethanolamine was replaced with 3-dimethylamino-1-propanol to obtain 564 mg of compound XH-8 (pale yellow oily liquid). 1 H NMR (400 MHz, Chloroform-d) δ 4.29 – 4.12(m, 18H), 3.58 (dd, J = 9.5, 5.1 Hz, 4H), 2.42 (td, J = 11.9, 6.3 Hz, 4H), 2.34 – 2.23 (m, 14H), 1.86 – 1.80 (m, 2H), 1.61 (t, J = 7.4 Hz, 8H), 1.32 –1.23 (m, 40H), 0.88 (t, J = 6.6 Hz, 12H). MS, ESI + m / z: calcd C 56 H 102 N2O 16 [M+H] + , 1059.7; found, 1059.8.

[0340] Example 9

[0341] The chemical structure of compound XH-9 is as follows:

[0342]

[0343] Following the synthesis method of Example 7, N,N-dimethylethanolamine was replaced with 4-(dimethylamino)butyl-1-ol to obtain 591 mg of compound XH-9 (pale yellow oily liquid). 1 H NMR (400 MHz, Chloroform-d) δ 4.28 –4.11 (m, 18H), 3.57 (dt, J = 10.8, 5.3 Hz, 4H), 2.42 (d, J = 9.8 Hz, 4H), 2.31 (dd, J = 9.6, 5.5 Hz, 14H), 1.68 (d, J = 2.6 Hz, 2H), 1.63 – 1.59 (m,8H), 1.29 (qd, J = 8.3, 5.4, 3.4 Hz, 42H), 0.91 – 0.85 (m, 12H). MS, ESI + m / z: calcd C 56 H 102 N2O 16 [M+H] +, 1073.7; found, 1073.8.

[0344] Example 10

[0345] The chemical structure of compound XH-10 is as follows:

[0346]

[0347] Following the synthesis method of Example 7, N,N-dimethylethanolamine was replaced with N-(2-hydroxyethyl)pyrrolidine to obtain 566 mg of compound XH-10 (pale yellow oily liquid). 1 H NMR (400 MHz, Chloroform-d) δ 4.31 –4.12 (m, 18H), 3.58 (d, J = 5.4 Hz, 4H), 2.76 (s, 2H), 2.57 (s, 4H), 2.42 (p,J = 5.9 Hz, 2H), 2.31 (t, J = 7.6 Hz, 8H), 1.79 (s, 4H), 1.62 (d, J = 7.4 Hz, 8H), 1.33 – 1.24 (m, 40H), 0.91 – 0.85 (m, 12H). MS, ESI + , m / z: calcdC 57 H 102 N2O 16 [M+H] + , 1071.7; found, 1071.8.

[0348] Example 11

[0349] The chemical structure of compound XH-11 is as follows:

[0350]

[0351] Referring to the synthesis method of Example 7, N,N-dimethylethanolamine was replaced with 2-(azacyclobutane-1-yl)ethanol to prepare 514 mg of compound XH-11 (pale yellow oily liquid). 1H NMR (400 MHz, Chloroform-d) δ4.30 – 4.18 (m, 8H), 4.14 (d, J = 5.9 Hz, 8H), 4.08 (t, J = 5.8 Hz, 2H), 3.57(q, J = 6.0 Hz, 4H), 3.27 (t, J = 7.0 Hz, 4H), 2.67 (t, J = 5.8 Hz, 2H), 2.43(p, J = 6.1 Hz, 2H), 2.31 (t, J = 7.6 Hz, 8H), 2.09 (p, J = 7.0 Hz, 2H), 1.65– 1.58 (m, 8H), 1.29 – 1.25 (m, 40H), 0.88 (t, J = 6.5 Hz, 12H). MS, ESI + m / z: calcd C 56 H 100 N2O 16 [M+H] + , 1057.7; found, 1057.9.

[0352] Example 12

[0353] The chemical structure of compound XH-12 is as follows:

[0354]

[0355] Following the synthesis method of Example 7, N,N-dimethylethanolamine was replaced with N-hydroxyethylpiperidine to obtain 578 mg of compound XH-12 (pale yellow oily liquid). 1 H NMR (400 MHz, Chloroform-d) δ 4.31 – 4.18 (m,10H), 4.14 (d, J = 5.9 Hz, 8H), 3.57 (dt, J = 12.0, 5.5 Hz, 4H), 2.59 (t, J =6.1 Hz, 2H), 2.43 (q, J = 5.9 Hz, 6H), 2.31 (t, J = 7.6 Hz, 8H), 1.64 – 1.56(m, 14H), 1.42 (q, J = 2.5 Hz, 2H), 1.27 (td, J = 6.0, 3.4 Hz, 40H), 0.88 (t,J = 6.6 Hz, 12H). MS, ESI + m / z: calcd C 58H 104 N2O 16 [M+H] + , 1085.7; found,1085.7.

[0356] Example 13

[0357] The chemical structure of compound XH-13 is as follows:

[0358]

[0359] Intermediate B-4 (1.0 mmol, 1.00 g) was dissolved in 50 mL of DCM. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (80.0 mmol, 14.98 g), 1-hydroxybenzotriazole (80.0 mmol, 14.98 g), N,N-dimethyl-β-alanine (3.0 mmol, 314 mg) and triethylamine (3.0 mmol, 314 mg) were added sequentially. The mixture was brought to room temperature and stirred for 4 hours. Then N,N-dimethylethanolamine (3.0 mmol, 277 mg) was added, and the mixture was stirred overnight. After the reaction was detected by TLC, the reaction solution was washed twice with 10% citric acid aqueous solution, once with saturated NaHCO3 solution, and twice with saturated saline solution. After drying with anhydrous sodium sulfate, the solution was filtered and concentrated to obtain a yellow oily crude product, which was then purified by column chromatography (dichloromethane:methanol = 30:1) to obtain 594 mg of compound XH-13 (pale yellow oily liquid). 1 H NMR (400 MHz, Chloroform-d) δ 4.27 (dt, J = 8.1, 5.5 Hz, 4H), 4.20 (dd, J = 6.1, 1.6 Hz, 4H), 4.14 (dd, J = 6.0, 4.6 Hz, 8H), 3.66 (dt, J = 18.7, 5.5 Hz, 4H), 2.71(t, J = 7.2 Hz, 2H), 2.59 (t, J = 7.4 Hz, 2H), 2.42 (h, J = 6.0 Hz, 2H), 2.30(t, J = 3.8 Hz, 14H), 1.64 – 1.57 (m, 8H), 1.29 – 1.25 (m, 40H), 0.90 – 0.86(m, 12H). MS, ESI + m / z: calcd C 55 H 100 N2O 15 [M+H] +, 1029.7; found, 1029.8.

[0360] Example 14

[0361] The chemical structure of compound XH-14 is as follows:

[0362]

[0363] Referring to the synthesis method of Example 13, N,N-dimethyl-β-alanine was replaced with 4-(dimethylamino)butyric acid to obtain 527 mg of compound XH-14 (pale yellow oily liquid). 1 H NMR (400 MHz, Chloroform-d) δ 4.28 –4.13 (m, 16H), 3.55 (t, J = 8.0 Hz, 4H), 2.40 (ddd, J = 17.1, 7.0, 3.2 Hz, 6H), 2.31 (t, J = 7.6 Hz, 14H), 1.84 (d, J = 7.3 Hz, 2H), 1.61 (p, J = 6.0,4.9 Hz, 8H), 1.32 – 1.26 (m, 40H), 0.90 – 0.86 (m, 12H). MS, ESI + , m / z: calcdC 56 H 102 N2O 15 [M+H] + , 1043.7; found, 1043.8.

[0364] Example 15

[0365] The chemical structure of compound XH-15 is as follows:

[0366]

[0367] Referring to the synthesis method in step 6 of Example 5, 2-bromoethanol was replaced with 2-bromo-N,N-dimethylethylamine to obtain 515 mg of compound XH-15 (pale yellow oily liquid). 1H NMR (400 MHz, Chloroform-d) δ 4.20 (dt,J = 6.5, 3.3 Hz, 8H), 4.14 (d, J = 6.0 Hz, 8H), 3.73 – 3.67 (m, 1H), 3.56 (s,1H), 2.95 – 2.80 (m, 6H), 2.62 (s, 2H), 2.44 (ddd, J = 11.9, 8.2, 5.4 Hz,6H), 2.31 (t, J = 7.6 Hz, 8H), 1.61 (d, J = 14.7 Hz, 8H), 1.30 – 1.25 (m,40H), 0.88 (t, J = 6.7 Hz, 12H). MS, ESI + m / z: calcd C 54 H 100 N2O 14 [M+H] + ,1001.7; found, 1001.8.

[0368] Example 16

[0369] The chemical structure of compound XH-16 is as follows:

[0370]

[0371] Referring to the synthesis method in step 6 of Example 5, 2-bromoethanol was replaced with 1-(3-bromopropyl)pyrrolidine to obtain 562 mg of compound XH-16 (pale yellow oily liquid). 1 H NMR (400 MHz, Chloroform-d) δ 4.17 (dt,J = 18.1, 6.1 Hz, 16H), 3.72 (q, J = 7.0 Hz, 2H), 3.25 – 3.18 (m, 2H), 2.81(t, J = 5.6 Hz, 4H), 2.70 (t, J = 6.2 Hz, 2H), 2.49 – 2.39 (m, 2H), 2.31 (t,J = 7.6 Hz, 8H), 2.17 (d, J = 7.1 Hz, 4H), 2.08 (dq, J = 11.9, 6.2 Hz, 2H), 1.61 (t, J = 7.3 Hz, 10H), 1.28 (dq, J = 6.3, 3.5, 2.9 Hz, 40H), 0.88 (t, J =6.5 Hz, 12H). MS, ESI+ m / z: calcd C 57 H 104 N2O 14 [M+H] + , 1041.7; found, 1041.8.

[0372] Example 17

[0373] The chemical structure of compound XH-17 is as follows:

[0374]

[0375] Following the synthesis method of Example 7, N,N-dimethylethanolamine was replaced with (R)-3-dimethylaminopyrrolidine to prepare 571 mg of compound XH-17 (pale yellow oily liquid). 1 H NMR (400 MHz, Chloroform-d) δ 4.27 –4.11 (m, 16H), 3.62 – 3.34 (m, 8H), 3.23 (d, J = 9.4 Hz, 1H), 2.65 (p, J =7.6 Hz, 2H), 2.30 (dd, J = 14.7, MS, ESI + m / z: calcd C 57 H 103 N3O 15 [M+H] + , 1070.7; found, 1011.1.

[0376] Example 18

[0377] The chemical structure of compound XH-18 is as follows:

[0378]

[0379] Following the synthesis method of Example 7, 2-dimethylaminoethylamine was replaced with N,N-dimethylethanolamine to obtain 492 mg of compound XH-18 (pale yellow oily liquid). 1H NMR (400 MHz, Chloroform-d) δ 5.50 (t, J = 4.9Hz, 1H), 4.26 (t, J = 5.7 Hz, 4H), 4.20 (d, J = 6.1 Hz, 4H), 4.14 (d, J = 6.0Hz, 8H), 3.56 (t, J = 5.7 Hz, 4H), 3.31 (q, J = 5.7 Hz, 2H), 2.44 (dt, J =12.2, 6.1 Hz, 4H), 2.30 (dd, J = 16.3, 8.7 Hz, 14H), 1.66 – 1.58 (m, 8H),1.33 – 1.24 (m, 40H), 0.88 (t, J = 6.6 Hz, 12H). MS, ESI + , m / z: calcdC 55 H 101 N3O 15 [M+H] + , 1044.7; found, 1044.9.

[0380] Example 19

[0381] The chemical structure of compound XH-19 is as follows:

[0382]

[0383] Referring to the synthesis method of Example 5, the starting material nonanoic acid was replaced with heptanoic acid to obtain 486 mg of compound XH-19 (pale yellow oily liquid). 1 H NMR (400 MHz, Chloroform-d) δ 4.33 – 4.09 (m, 18H), 3.58(q, J = 5.8 Hz, 4H), 2.57 (t, J = 6.0 Hz, 2H), 2.42 (p, J = 5.9 Hz, 2H), 2.35– 2.25 (m, 14H), 1.63 (d, J = 3.0 Hz, 8H), 1.34 – 1.25 (m, 32H), 0.88 (t, J =6.6 Hz, 12H). MS, ESI + m / z: calcd C 51 H 92 N2O 16 [M+H] + , 989.7; found, 989.8.

[0384] Example 20

[0385] The chemical structure of compound XH-20 is as follows:

[0386]

[0387] Following the synthesis method of Example 18, N,N-dimethylethanolamine was replaced with N-(2-hydroxyethyl)pyrrolidine to obtain 577 mg of compound XH-20 (pale yellow oily liquid). 1 H NMR (400 MHz, Chloroform-d) δ 4.29 –4.11 (m, 18H), 3.58 (q, J = 5.5 Hz, 4H), 2.76 (t, J = 6.1 Hz, 2H), 2.58 (s,4H), 2.42 (p, J = 6.0 Hz, 2H), 2.31 (t, J = 7.6 Hz, 8H), 1.82 – 1.76 (m, 4H), 1.61 (d, J = 14.7 Hz, 8H), 1.28 (ddd, J = 8.5, 6.0, 3.2 Hz, 32H), 0.92 – 0.84(m, 12H). MS,ESI + m / z: calcd C 53 H 94 N2O 16 [M+H] + , 1015.7; found, 1015.8.

[0388] Example 21

[0389] The chemical structure of compound XH-21 is as follows:

[0390]

[0391] Step 1. Synthesis of intermediate C-1

[0392]

[0393] To a dry round-bottom flask equipped with a stir bar, nonanoic acid (63.2 mmol, 10.00 g), 3-(benzyloxy)propane-1,2-diol (31.6 mmol, 5.76 g), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (80.0 mmol, 14.98 g), 4-dimethylaminopyridine (6.3 mmol, 772 mg), and dichloromethane (150 mL) were added sequentially, and the mixture was stirred at room temperature for 24 hours. After the reaction was detected by thin-layer chromatography (TLC), the reaction solution was washed once with an appropriate amount of saturated NaHCO3 solution, twice with 10% citric acid aqueous solution, and twice with saturated brine. After drying with anhydrous sodium sulfate, the solution was filtered and concentrated to obtain a pale yellow oily crude product, which was then purified by column chromatography (petroleum ether / ethyl acetate = 10:1) to obtain intermediate C-1 (10.28 g, yield 70%).

[0394] Step 2. Synthesis of intermediate C-2

[0395]

[0396] Intermediate C-1 (22.2 mmol, 10.28 g), 500 mg palladium on carbon, and 100 mL of ethanol were added sequentially to a dry round-bottom flask equipped with a stir bar. The mixture was stirred at room temperature under hydrogen atmosphere for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was concentrated to obtain a pale yellow oily crude product. This crude product was then purified by column chromatography (petroleum ether / ethyl acetate = 5:1) to obtain intermediate C-2 (7.56 g, 91% yield).

[0397] Step 2. Synthesis of intermediate C-3

[0398]

[0399] Intermediate C-2 (20.3 mmol, 7.56 g) was dissolved in 100 mL of DCM. 4-Nitrophenylchloroformate (30.4 mmol, 6.14 g) and triethylamine (60.9 mmol, 6.16 g) were slowly added at 0 °C, and the mixture was stirred at room temperature for 24 hours. After the reaction was completed by TLC, the reaction solution was washed twice with 10% citric acid aqueous solution and twice with saturated brine. After drying with anhydrous sodium sulfate, the solution was filtered and concentrated to obtain a pale yellow oily crude product. This crude product was then purified by column chromatography (petroleum ether:ethyl acetate = 10:1) to give intermediate C-3 (8.02 g, yield 74%).

[0400] Step 4. Synthesis of intermediate C-4

[0401]

[0402] Intermediate C-3 (14.9 mmol, 8.02 g) was dissolved in 50 mL of DMF, and tert-butyl di(2-hydroxyethyl)carbamate (7.5 mmol, 1.53 g), 4-dimethylaminopyridine (15.0 mmol, 1.83 g), and triethylamine (15.0 mmol, 1.51 g) were added sequentially. The mixture was stirred at room temperature for 24 hours. After the reaction was completed by TLC, the reaction solution was washed three times with an appropriate amount of saturated NaHCO3 solution, once with 10% citric acid aqueous solution, and twice with saturated brine. After drying with anhydrous sodium sulfate, the solution was filtered and concentrated to obtain a pale yellow oily crude product, which was then purified by column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain intermediate C-4 (5.18 g, yield 69%).

[0403] Step 5. Synthesis of intermediate C-5

[0404]

[0405] Intermediate C-4 (5.2 mmol, 5.18 g) was dissolved in 30 mL of DCM, and 20 mL of HCl / EA solution was added. The mixture was stirred at room temperature for 2 hours. After the reaction was completed by TLC, the organic solvent was removed by rotary evaporation to obtain the crude product. The crude product was redissolved in DCM, washed once with an appropriate amount of saturated NaHCO3 solution, washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a pale yellow oily substance, which was intermediate C-5 (4.92 g). This substance did not require purification and was used directly in the next reaction.

[0406] Step 6. Synthesis of compound XH-21

[0407] Referring to the synthesis method of Example 7, intermediate B-4 was replaced with intermediate C-5 to obtain 481 mg of compound XH-21 (pale yellow oily liquid). 1 H NMR (400 MHz, Chloroform-d) δ 5.32 – 5.22 (m, 2H), 4.37– 4.13 (m, 14H), 3.58 (q, J = 5.8 Hz, 4H), 2.57 (t, J = 6.0 Hz, 2H), 2.38 –2.24 (m, 14H), 1.62 (dt, J = 7.5, 3.8 Hz, 8H), 1.33 – 1.24 (m, 40H), 0.88 (t,J = 6.7 Hz, 12H). MS, ESI + m / z: calcd C 53 H96 N2O 16 [M+H] + , 1017.7; found, 1018.0.

[0408] Example 22

[0409] The chemical structure of compound XH-22 is as follows:

[0410]

[0411] Following the synthesis method of Example 21, N,N-dimethylethanolamine was replaced with N-(2-hydroxyethyl)pyrrolidine to prepare 516 mg of compound XH-22 (pale yellow oily liquid). 1 H NMR (400 MHz, Chloroform-d) δ 5.33 –5.21 (m, 2H), 4.42 – 4.08 (m, 14H), 3.58 (q, J = 6.0 Hz, 4H), 2.73 (t, J =6.2 Hz, 2H), 2.57-2.54 (m, MS, ESI + m / z: calcd C 55 H 198 N2O 16 [M+H] + , 1043.7; found, 1043.9.

[0412] Example 23

[0413] The chemical structure of compound XH-23 is as follows:

[0414]

[0415] Referring to the synthesis method of Example 21, the starting material 3-(benzyloxy)propane-1,2-diol in step 1 was replaced with 4-(benzyloxy)butane-1,2-diol to obtain 556 mg of compound XH-23 (pale yellow oily liquid). 1H NMR (400MHz, Chloroform-d) δ 5.19 (qd, J = 6.2, 3.4 Hz, 2H), 4.33 – 4.14 (m, 12H), 4.06 (dd, J = 12.0, 5.9 Hz, 2H), 3.65 – 3.50 (m, 4H), 2.57 MS, ESI + m / z: calcd C 55 H 100 N2O 16 [M+H] + ,1044.7; found, 1046.1.

[0416] Example 24

[0417] The chemical structure of compound XH-24 is as follows:

[0418]

[0419] Referring to the synthesis method of Example 23, N,N-dimethylethanolamine was replaced with N-(2-hydroxyethyl)pyrrolidine to obtain 508 mg of compound XH-24 (pale yellow oily liquid). 1 H NMR (400 MHz, Chloroform-d) δ 5.19(qd, J = 6.2, 3.4 Hz, 2H), 4.32 – 4.14 (m, 12H), 4.06 (dd, J = 12.0, 6.0 Hz,2H), 3.57 (q, J = 5.9, 5.1 Hz, 4H), 2.74 (t, J = 6.2 Hz, 2H), 2.56 (d, J =5.9 Hz, 4H), 2.30 (t, J = 7.6 Hz, 8H), 1.98 (q, J = 6.5 Hz, 4H), 1.80 – 1.75(m, 4H), 1.65 – 1.56 (m, 8H), 1.33 – 1.24 (m, 40H), 0.88 (t, J = 6.6 Hz, 12H). MS, ESI + m / z: calcd C 57 H102 N2O 16 [M+H] + , 1071.7; found, 1072.1.

[0420] Example 25: General preparation and characterization methods for lipid nanoparticles

[0421] Preparation method: The required lipids were dissolved in anhydrous ethanol to a concentration of 30 mg / mL. The lipid phase was prepared according to a specific formulation (ionizable lipids: neutral phospholipids: cholesterol: polyethylene glycol-modified lipids) with a nitrogen-to-phosphoprotein ratio of 6:1. The mRNA was dissolved in 25 mM citrate buffer (pH = 4.0) to prepare a 100 μg / mL aqueous solution. Lipid nanoparticles were prepared using microfluidics at a total flow rate of 16 mL / min and a flow rate ratio of lipid phase:aqueous phase = 1:3, with a preparation volume of approximately 12 mL. Ultrafiltration was performed using a 100 kDa ultrafiltration tube. The lipid nanoparticles were diluted 4-fold with 1×PBS (0.01 M) and ultrafiltered at 3000 g for 20 min, repeated three times, to obtain lipid nanoparticles with a 1×PBS buffer system. These were then stored at 4°C for later use.

[0422] Characterization methods: The particle size and polydispersity index of LNPs were determined using a Malven particle size potentiometer. The encapsulation efficiency and mRNA content of LNPs were determined using the ThermoFisher Quant-iTTMRiboGreen™ kit, as detailed below:

[0423] Preparation of standard curve: Dilute the mRNA stock solution with 1×TE to 2 μg / mL. Take 0, 2, 10, 25, 50 and 100 μL of each concentration standard solution into a 96-well black ELISA plate. Then add 1×TE to each well to a total volume of 100 μL. Add 1×Ribo to each well for 100 μL. Detect the ELISA plate with an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

[0424] LNP measurement sample preparation: Ultrafiltered LNP was first diluted with 1×TE to approximately 1 μg / mL of the theoretical concentration; permeabilization: The diluted LNP was permeabilized with 1% Triton X-100 at room temperature for 10 min; no permeabilization: The diluted LNP was added to an equal volume of DEPC water and incubated at room temperature for 10 min. Then, 100 μL of each sample was transferred to a 96-well black microplate, and 100 μL of 1×Ribo was added to each well. Each sample was tested in triplicate. The microplate reader was used for detection at an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

[0425] In this paper, the mRNA used for LNP characterization is the mRNA encoding firefly luciferase (mRNA-Luci).

[0426] In this article, mRNA-Luci was purchased from Kaituo, with the trade number P22J012.

[0427] In this article, mRNA-EGFP was purchased from Kaituo.

[0428] In this paper, when preparing lipid nanoparticles, the proportions of each lipid in the lipid component are all molar ratios.

[0429] Example 26: Preparation and Characterization of Liver-Targeting Lipid Nanoparticles

[0430] Referring to the preparation method in Example 25, the following formulation was used (ionizable lipids: DSPC: cholesterol: PEG) 2000 LNPs were prepared using a mixture of DMG (50:10:38.5:1.5), with XH-10 as the ionizable lipid and SM-102 as the positive control. Their physicochemical properties are shown in Table 1.

[0431] Table 1: Particle size, PDI, and encapsulation efficiency of liver-targeted LNPs

[0432]

[0433] Example 27: Evaluation of the in vivo mRNA delivery capability of liver-targeting lipid nanoparticles

[0434] Lipid nanoparticles SM-102 LNP and XH-10 LNP loaded with mRNA-Luci were prepared according to the method in Example 26. The LNPs were injected intravenously into mice (n = 3, dose: 1.0 mg / kg, injection volume: 200 µL / mouse). Six hours later, Luciferase substrate was injected intraperitoneally (dose: 2 mg / kg, injection volume: 100 µL / mouse). Fifteen minutes later, the mice were euthanized by cervical dislocation. The heart, liver, spleen, lungs, and kidneys were quickly removed, rinsed in pre-cooled PBS in the dark, and observed under in vivo imaging. The fluorescence intensity of the ROI region was recorded. The results are as follows: Figure 1 As shown, the XH-10 LNP exhibited liver delivery capacity comparable to the positive control group SM-102 LNP.

[0435] Example 28: Preparation and characterization of spleen-targeting lipid nanoparticles

[0436] Referring to the preparation method in Example 25, the following formulation was used (ionizable lipids: DSPC: cholesterol: PEG) 2000LNPs were prepared using a DSPE ratio of 58:20:20.5:1.5, wherein the ionizable lipids were XH-1 to XH-20, with the commercially available lipid SM-102 serving as a positive control. Their physicochemical properties are shown in Table 2.

[0437] Table 2: Particle size, PDI and encapsulation efficiency of spleen-targeted LNPs

[0438]

[0439] Example 29: Evaluation of the in vivo mRNA delivery capability of spleen-targeting lipid nanoparticles

[0440] Lipid nanoparticles SM-102 LNP, XH-7 LNP, XH-10 LNP, XH-19 LNP, XH-20 LNP, XH-21 LNP, XH-22 LNP, XH-23 LNP, and XH-24 LNP loaded with mRNA-Luci were prepared according to the method in Example 28. The above LNPs were injected intravenously into mice (n = 3, dose: 1.0 mg / kg, injection volume: 200 µL / mouse). Six hours later, Luciferase substrate was injected intraperitoneally (dose: 2 mg / kg, injection volume: 100 µL / mouse). Fifteen minutes later, the mice were euthanized by cervical dislocation. The heart, liver, spleen, lungs, and kidneys were quickly removed, rinsed in pre-cooled PBS in the dark, and observed under in vivo imaging. The fluorescence intensity of the ROI region was recorded. The results are as follows: Figure 2 As shown, after prescription adjustment, the targeting of LNPs was successfully reprogrammed from the liver to the spleen. Among them, XH-7, XH-19, XH-21, XH-22 and XH-23 LNPs showed particularly outstanding performance, with their spleen delivery efficiency being significantly better than the positive control SM-102 LNP.

[0441] Example 30: Preparation and characterization of CD3 antibody-modified lipid nanoparticles

[0442] Lipids were dissolved in anhydrous ethanol to a concentration of 30 mg / mL. The nitrogen-to-phosphorus ratio of ionizable lipids to mRNA was 6:1, following a formulation (ionizable lipids: DSPC: cholesterol: polyethylene glycol-modified lipids: maleimide-modified polyethylene glycol lipids = 58:20:20.5:1.0:0.5) to obtain the lipid phase. The mRNA was dissolved in 25 mM citrate buffer (pH = 4.0) to prepare a 100 μg / mL mRNA solution, yielding the aqueous phase. Lipid nanoparticles were prepared using microfluidics at a total flow rate of 16 mL / min and a flow rate ratio of lipid phase:aqueous phase = 1:3, with a preparation volume of approximately 12 mL. Ultrafiltration was performed using a 100 kDa ultrafiltration tube. The lipid nanoparticles were diluted 4-fold with 1×PBS (0.01 M) and ultrafiltered at 3000 g for 20 min, repeated three times, to obtain lipid nanoparticles with a 1×PBS buffer system. LNP encapsulation efficiency and mRNA concentration were determined using the Thermo Fisher Quant-iTTMRiboGreen™ kit. Antibody activated with tris(2-carboxyethyl)phosphonic acid hydrochloride was added to ultrafiltered fresh LNP at an antibody-to-mRNA mass ratio of 0.5. The mixture was then shaken at 37°C and 800 rpm for 2 hours. After shaking, an appropriate amount of cysteine ​​was added to bring the cysteine ​​concentration in the LNP to 0.5 mM. Ionizable lipids included XH-7 LNP, XH-10 LNP, XH-19 LNP, and XH-20 LNP, with the commercially available lipid SM-102 used as a positive control. PEGylated lipids were used. 2000 -DMG or PEG 2000 -DSPE. The antibody is a mouse CD3-targeting antibody (Baiying Biotechnology, 00KHYT001 V6 Anti-Mouse CD3E(1E11.D)-scfv-His), and its physicochemical properties are shown in Table 3.

[0443] Table 3: Particle size, PDI and encapsulation efficiency of CD3-modified LNPs

[0444]

[0445] Example 31: Evaluation of the transfection ability of CD3 antibody-modified lipid nanoparticles on mouse spleen T cells

[0446] Lipid nanoparticles modified with CD3 antibody and loaded with mRNA-EGFP were prepared according to the method in Example 30. The above-mentioned LNPs were injected intravenously into mice (n = 3, dose: 1.0 mg / kg, injection volume: 200 µL / mouse). Six hours later, the mice were euthanized by cervical dislocation, and the liver and spleen were quickly removed and washed in pre-cooled PBS in the dark. After grinding and digestion, the mixture was filtered through a 200 µm sieve and lysed with erythrocyte lysis buffer at 4°C in the dark for 5 min. The lysate was immediately washed twice with PBS to remove the erythrocyte lysis buffer. The resulting cell suspensions of liver and spleen were stained with flow cytometry antibody at 4°C in the dark for 40 min, washed twice with PBS, and the proportions of GFP-positive cells in mouse spleen CD3+ T cells, liver CD45- cells, and liver CD45+ cells were examined using flow cytometry. The results are as follows: Figure 3 and 4 As shown, LNPs 4 and 6 exhibited better transfection efficiency in spleen T cells than the positive control group (benchmark LNP). At the same time, both showed lower transfection efficiency in both immune and non-immune cells of the liver, indicating that they can effectively avoid the risk of off-target effects in the liver.

Claims

1. A compound of formula I, or a salt or isomer thereof, I In the formula: X1, X2, Y1 and Y2 are each independently NR7, O or S, and R7 is selected from H and C1-C10 alkyl groups; X3, X4, X5, X6, Y3, Y4, Y5, and Y6 are each independently NR8 or O, or are not present. R8 is selected from H and C1-C10 alkyl groups. X3 and X4 are not present at the same time; X5 and X6 are not present at the same time; Y3 and Y4 are not present at the same time; Y5 and Y6 are not present at the same time. L1 is -C(O)-, or it does not exist; L2 is NR6 or O, or is absent; R6 is selected from H and optionally substituted C1-C10 alkyl groups; L3 is a substituted or unsubstituted C1-C5 alkylene group, or it is absent; R3 represents substituted or unsubstituted -NR a R b or hydroxyl group; R a and R b Each is independently selected from H and substituted or unsubstituted C1-C6 alkyl groups, or R a R b Together with the nitrogen atoms they are attached to, they form substituted or unsubstituted 4-9 membered heterocyclic groups; R1, R2, R4 and R5 are each independently selected from substituted or unsubstituted C4-C14 alkyl, substituted or unsubstituted C4-C14 alkenyl and substituted or unsubstituted C4-C14 alkynyl groups; m1 and m2 can be 2, 3, 4, 5 or 6 independently; n1, n2, t1, t2, s1, and s2 are each independently 0, 1, 2, 3, 4, 5, or 6.

2. A nanoparticle composition comprising a lipid component, said lipid component comprising the compound of formula I according to claim 1, or a salt or isomer thereof. Preferably, the lipid component further includes phospholipids, PEG lipids, and structural lipids.

3. The nanoparticle composition according to claim 2, characterized in that: The nanoparticle composition comprises the compound of formula I or its salt or isomer as claimed in claim 1, phospholipids, structural lipids, and PEG lipids; the molar ratio of the compound of formula I or its salt or isomer, phospholipids, structural lipids, and PEG lipids is 50-65:15-25:8-34.5:0.5-2; preferably, the nanoparticle composition is used to deliver therapeutic and / or preventive agents to the spleen; or The nanoparticle composition comprises the compound of formula I or its salt or isomer as claimed in claim 1, phospholipids, structural lipids, and PEG lipids; the molar ratio of the compound of formula I or its salt or isomers, phospholipids, structural lipids, and PEG lipids is 50:10:38.5:1.5; preferably, the nanoparticle composition is used to deliver therapeutic and / or preventive agents to the liver.

4. The nanoparticle composition according to claim 2 or 3, characterized in that, The nanoparticle composition also includes therapeutic and / or preventative agents.

5. The nanoparticle composition according to claim 4, characterized in that, The lipid component is conjugated with a ligand that specifically targets organs, tissues or cells. Preferably, the ligand is selected from peptides, antibodies, and antibody-derived antigen-binding domains; Preferably, a portion of the PEG lipid is conjugated with the ligand; preferably, the molar ratio of the PEG lipid without ligand to the PEG lipid with ligand is 14-2:1; Preferably, the ligand is an antibody, and more preferably one or more of CD3, CD4, CD5, CD7 and CD8 antibodies that target T cells.

6. A pharmaceutical composition comprising (1) the nanoparticle composition of claim 4 or 5; and (2) a pharmaceutically acceptable excipient.

7. A method for delivering a therapeutic and / or preventative agent to cells, the method comprising the step of administering to a subject the nanoparticle composition of any one of claims 2-5 or the pharmaceutical composition of claim 6, wherein the nanoparticle composition in the nanoparticle composition and the pharmaceutical composition comprises: (1) lipid components, including phospholipids, PEG lipids, structured lipids, and compounds of formula I as claimed in claim 1, or their salts and isomers, and (2) therapeutic agents and / or preventive agents; Preferably, the therapeutic agent and / or preventive agent is a nucleic acid; Preferably, the nucleic acid is selected from one or more of siRNA, microRNA, shRNA, mRNA and pDNA, and is preferably mRNA.

8. A method for preparing the compound of formula I according to claim 1 or its salt or isomer, or a method for preparing the nanoparticle composition according to any one of claims 2-5.

9. Use of the compound of claim 1 or its salt or isomer, and / or the nanoparticle composition of any one of claims 2-5 in the preparation of a medicament for treating or preventing a disease or condition; or in the preparation of a medicament containing immune cells; Preferably, the nanoparticle compositions described herein or nanoparticle compositions formed from compounds of Formula I herein are used for the specific delivery of therapeutic agents and / or preventative agents; Preferably, the disease or condition is one that benefits from the specific delivery of therapeutic and / or preventative agents.

10. A method for preparing engineered T cells, the method comprising the step of contacting the T cells with the nanoparticle composition of claim 5.

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