Lipid nanoparticles for targeting secondary lymphatic organ as well as preparation method and application of lipid nanoparticles

Abstract

The invention discloses lipid nanoparticles targeting a secondary lymphatic organ as well as a preparation method and application of the lipid nanoparticles. According to the invention, PG phospholipids are adopted as auxiliary phospholipids to prepare the lipid nanoparticles, so that an mRNA nano-drug delivery system of a targeted secondary lymphatic organ (spleen) is obtained, and the spleen targeting property of the lipid nanoparticles is enhanced. The lipid nanoparticles prepared by the invention show good stability, excellent physical and chemical data and efficient cell expression effect, are suitable for a drug targeted drug delivery system, and have important practical significance in scientific research and medical value.

Description

Technical Field

[0001] The present invention relates to the field of nano drug preparations, and in particular to lipid nanoparticles targeting secondary lymphoid organs, and a preparation method and application thereof. Background Art

[0002] Lipid nanoparticles (LNP) are one of the important technologies in lipid delivery drug delivery systems and have become the most popular delivery technology for nucleic acid therapeutic drugs. LNP is a multi-component system, usually composed of four components: ionizable cationic lipids, auxiliary phospholipids, cholesterol, and polyethylene glycol-modified phospholipids. Ionizable cationic lipids affect the delivery efficiency and biological properties of LNPs, auxiliary phospholipids help to encapsulate nucleic acids and stabilize LNPs, cholesterol mainly mediates LNP endocytosis and ensures the bilayer structure and lipid fluidity of LNPs, and polyethylene glycol-modified phospholipids can improve the stability of lipid nanoparticles, reduce the binding of nanoparticles to plasma proteins in the body, and prolong the circulation time in the body.

[0003] In 2018, the FDA approved the LNP-based drug patisiran (trade name: ), used to treat hereditary transthyretin-mediated amyloidosis (hATTR), becoming the first gene therapy drug with a non-viral delivery system. In 2020, the FDA authorized the emergency use of Moderna and BioNTech's new crown vaccine. The successful use of LNP in the COVID-19 mRNA vaccine has once again attracted attention to it as a gene delivery vehicle. Cancer immunotherapy has great prospects, but its main challenge is the lack of a delivery system that can target therapeutic drugs to target organs, and mRNA vaccines are an innovative approach to cancer immunotherapy that can produce a powerful anti-tumor immune response. Therefore, the development of an mRNA delivery system that can target immune organs is crucial to the development of safe and efficient mRNA cancer vaccines.

[0004] Secondary lymphoid organs are the sites where immune responses are induced, such as the spleen and lymph nodes. The spleen is the largest secondary lymphoid organ in the human body and has the highest density of antigen presenting cells, B lymphocytes, and T lymphocytes. Tumor-associated antigens (TAA) or new antigens produced by antigen presenting cells (APC) are presented to T lymphocytes with the major histocompatibility complex (MHC) on the surface, activating CD4 + and CD8 +T lymphocytes, ultimately killing tumor cells. Lymph nodes, as peripheral immune organs, also produce immune responses to foreign antigens. The APC cells in the lymph nodes quickly present antigens to B lymphocytes and T lymphocytes to induce immunity, and are used in the field of tumor immunotherapy. Therefore, targeting secondary lymphoid organs can stimulate effective systemic immune responses and exert efficient anti-tumor effects. The development of delivery systems targeting secondary lymphoid organs is particularly important for immunotherapy, especially tumor immunotherapy.

[0005] SORT technology predictably targets lipid nanoparticles to specific organs, becoming a strategy for achieving organ-targeted delivery. Compared with the traditional four-component LNP, the technical feature of SORT-LNP is the introduction of the "fifth element" into LNP, forming its unique lipid formula design, which targets the delivery of mRNA or gene editing systems to specific organ tissues. Cheng et al. precisely changed the in vivo mRNA delivery performance by adding new lipid SORT molecules, achieving specific targeting of organs such as mouse liver, lungs, and spleen.

[0006] Xu Qiaobing and others are committed to developing organ-specific LNP delivery vectors that have the ability to target specific tissues and organs. By adjusting the structure of LNP by chemical methods, they found that imidazole-based synthetic lipid compounds preferentially target mRNA to the spleen, achieving specific targeting of LNP to organs other than the liver (such as the lungs and spleen). At the same time, the research team screened out the 113-O12B lipid molecule, which can highly specifically target lymph nodes.

[0007] Li Siwen's team at China Pharmaceutical University has developed a new type of tumor vaccine: a multifunctional antigen carrier targeting the spleen. The modified lipid material is used to expand the red blood cell membrane to make a nanocarrier, which not only retains the biological properties of the red blood cell membrane, but also makes up for the defect of insufficient drug loading. In mice, it shows obvious spleen accumulation, stimulates systemic specific anti-tumor immunity, and effectively inhibits tumor expansion and metastasis.

[0008] Han Xuexiang et al. developed a new LNP component, adjuvant lipid (C12-TLRa), which significantly enhanced the activation of dendritic cells in lymph nodes and the production of proinflammatory cytokines. + T cells, CD8 + T cells and B cells work together to exert a powerful adaptive immune response, thereby enhancing the immunogenicity of the vaccine.

[0009] However, there are still problems with the targeting of lipid nanoparticles to secondary lymphoid organs. For example, Cheng et al. added negatively charged 1,2-di-(9Z-octadecenoyl)-sn-glycerol-3-phosphate as a SORT molecule into LNP. Although SORT-LNP completely and selectively targeted spleen tissue, it had poor solubility and needed to be dissolved in a highly polar organic solvent. Generally, highly polar organic solvents are highly toxic and are not suitable for biological products. Secondly, in the past development process, a large number of systematic studies have been devoted to the design of ideal ionizable lipids, but the design and screening process of the compound molecular library is complicated, especially the ionizable lipids for the target organs need to be redesigned. The introduction of the "fifth component" lipid complicates the development process of LNP and increases the difficulty of transformation of results.

[0010] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0011] In view of the problems existing in the prior art, the present invention provides a nano drug delivery system solution targeting secondary lymphoid organs, which improves the distribution and expression activity of active ingredients, especially mRNA, in tissues and organs by formula design, screening and adjustment of lipid nanoparticles, overcomes the disadvantage of excessive liver homing caused by intravenous injection of LNP, improves the delivery efficiency in extrahepatic tissues, and achieves secondary lymphoid organ targeting of drugs such as mRNA. Specifically, the present invention includes the following contents.

[0012] In a first aspect of the present invention, a lipid nanoparticle targeting a secondary lymphoid organ is provided, comprising an ionizable cationic lipid, an auxiliary phospholipid and an active ingredient, wherein the auxiliary phospholipid is a compound having a structure represented by formula (I) or a salt thereof:

[0013]

[0014] wherein R1 and R2 each independently represent a substituted or unsubstituted straight chain or branched chain alkyl group, including a saturated or unsaturated alkyl group, or a substituted or unsubstituted straight chain or branched chain alkenyl group.

[0015] In certain embodiments, according to the lipid nanoparticles targeting secondary lymphoid organs as described in the first aspect of the present invention, the auxiliary phospholipid is selected from at least one of 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), L-α-phosphatidyl-DL-glycero-dioleoyl (DOPG) and 1,2-palmitoylphosphatidylglycerol (DPPG).

[0016] In certain embodiments, according to the lipid nanoparticles targeting secondary lymphoid organs according to the first aspect of the present invention, the ionizable cationic lipids include but are not limited to the structure of formula (II):

[0017]

[0018] wherein Q is a substituted or unsubstituted straight-chain C2-20 alkylene, wherein one or more C atoms of the alkylene are optionally replaced by heteroatoms independently selected from O, S and N; or Q is a substituted or unsubstituted, saturated or unsaturated 4-6-membered ring, wherein the ring atoms of the 4-6-membered ring optionally contain one or more heteroatoms independently selected from O, S and N; wherein the substituted substituent is selected from halogen, -OH, straight-chain or branched C1-20 alkyl, straight-chain or branched C1-20 alkoxy, straight-chain or branched C2-20 alkenyl, straight-chain or branched C2-20 alkynyl, -CH2CH(OH)R5,

[0019]

[0020] R1, R2, R3, and R4 may be the same or different, and are independently selected from hydrogen, substituted or unsubstituted straight or branched C1-30 alkyl, substituted or unsubstituted straight or branched C2-30 alkenyl, substituted or unsubstituted straight or branched C2-30 alkynyl, wherein one or more C atoms of the alkyl, alkenyl or alkynyl are optionally replaced by heteroatoms independently selected from O, S and N, or -CH2CH(OH)R5; the substituted substituents are selected from halogen, -OH, straight or branched C1-10 alkyl, straight or branched C1-10 alkoxy.

[0021] In certain embodiments, according to the lipid nanoparticles targeting secondary lymphoid organs according to the first aspect of the present invention, the ionizable cationic lipid is present in a molar percentage of 25.0-60.0% of the lipid nanoparticle composition.

[0022] In certain embodiments, according to the lipid nanoparticles targeting secondary lymphoid organs according to the first aspect of the present invention, the auxiliary phospholipid is present in a molar percentage of 10.0-40.0% of the lipid nanoparticle composition.

[0023] In certain embodiments, the lipid nanoparticles targeting secondary lymphoid organs according to the first aspect of the present invention further comprise cholesterol.

[0024] In certain embodiments, according to the lipid nanoparticles targeting secondary lymphoid organs according to the first aspect of the present invention, the cholesterol is present in a molar percentage of 20.0-50.0% of the lipid nanoparticle composition.

[0025] In certain embodiments, the lipid nanoparticles targeting secondary lymphoid organs according to the first aspect of the present invention further comprise a phospholipid modified with polyethylene glycol.

[0026] In certain embodiments, according to the lipid nanoparticles targeting secondary lymphoid organs according to the first aspect of the present invention, the polyethylene glycol-modified phospholipid is present in a molar percentage of 0.5-10.0% of the lipid nanoparticle composition.

[0027] In certain embodiments, according to the lipid nanoparticles targeting secondary lymphoid organs as described in the first aspect of the present invention, the active ingredient is selected from at least one of small molecule compounds, nucleic acids, proteins and polypeptides.

[0028] In certain embodiments, according to the lipid nanoparticles targeting secondary lymphoid organs as described in the first aspect of the present invention, the ratio of the total mass of lipid molecules in the lipid nanoparticles to the mass of the active ingredients is 1:1-60:1.

[0029] The second aspect of the present invention provides a pharmaceutical composition comprising the lipid nanoparticles according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

[0030] The third aspect of the present invention provides a method for preparing lipid nanoparticles targeting secondary lymphoid organs according to the first aspect, comprising:

[0031] The step of preparing an organic phase by mixing an ionizable cationic lipid and an auxiliary phospholipid and optionally a phospholipid modified with cholesterol and polyethylene glycol;

[0032] The step of dissolving the active ingredient in an aqueous solvent to prepare an aqueous phase; and

[0033] The step of mixing the organic phase and the aqueous phase.

[0034] In certain embodiments, according to the method for preparing lipid nanoparticles targeting secondary lymphoid organs as described in the third aspect of the present invention, the organic phase and the aqueous phase are mixed using microfluidics or collision jet technology.

[0035] The fourth aspect of the present invention provides use of the lipid nanoparticles described in the first aspect in the preparation of a drug targeting secondary lymphoid organs.

[0036] The lipid nanoparticles prepared by the present invention exhibit good stability, excellent physicochemical data, and efficient cell expression effect, and are suitable for drug targeted drug delivery systems, and have important practical significance in both scientific research and medical value.

[0037] The lipid nanoparticles of the present invention can induce immunity of an individual to active ingredients such as mRNA. Taking advantage of this advantage, the lipid nanoparticles can be used as autologous cancer vaccines and ultimately can be used to prevent or treat cancer.

[0038] In a specific embodiment, the lipid nanoparticles of the present invention can induce immunity to the active ingredient by effectively delivering the active ingredient to secondary lymphoid organs.

[0039] In a specific embodiment, the present invention uses DSPG as an auxiliary phospholipid to prepare lipid nanoparticles, thereby obtaining an mRNA nano drug delivery system targeting a secondary lymphoid organ (spleen), thereby enhancing the spleen targeting of lipid nanoparticles. The DSPG material used in the present invention is artificially synthesized, low in price, and is conducive to industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 In vitro imaging of different types of phospholipid-assisted (PG-based) nanodrug delivery systems.

[0041] Figure 2 Cell results for different types of phospholipid-assisted (PG-type) nanodrug delivery systems.

[0042] Figure 3 This is a data diagram of the organ distribution of different types of auxiliary phospholipids (PG-type) nano drug delivery systems. From this, we can directly see the distribution and expression of the nano drug delivery system in various tissues and organs.

[0043] Figure 4 In vitro data of nanodrug delivery systems with different formulations.

[0044] Figure 5 Nanodrug delivery systems composed of different formulations are distributed in various tissues and organs.

[0045] Figure 6 The in vitro data of different auxiliary phospholipid nanodrug delivery systems show that DSPG-based nanodrug delivery has a significant effect in targeting secondary lymphoid organs (spleen), and DSPC-based nanodrug delivery partially targets secondary lymphoid organs (lymph nodes).

[0046] Figure 7 In vivo and in vitro data of SM102 nanodrug delivery system. DETAILED DESCRIPTION

[0047] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0048] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0049] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0050] Herein, the term "lipid nanoparticle" is abbreviated as "LNP" in English and refers to any vesicle formed by one or more lipid components. Unlike liposomes, it generally includes a solid core and a lipid layer. The solid core contains ionizable cationic lipids and active ingredients, particularly electronegative nucleic acid-like substances, while the lipid layer includes auxiliary phospholipids.

[0051] [Lipid Nanoparticles]

[0052] The first aspect of the present invention provides a lipid nanoparticle, which is a lipid nanoparticle that targets active ingredients to secondary lymphoid organs. Generally speaking, it includes ionizable cationic lipids, auxiliary phospholipids and active ingredients, and optionally cholesterol and / or polyethylene glycol-modified phospholipids.

[0053] Ionizable cationic lipids

[0054] The ionizable cationic lipid of the present invention is not particularly limited, as long as it can be combined with the active ingredient to form a core, any ionizable cationic lipid material known in the art can be used. Exemplarily, examples of ionizable cationic lipids include, but are not limited to, 3β-[N-(N', N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Chol); 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); 1,2-dioleoyl-3-dimethylammonium propane (DODAP); dimethyldioctadecyl ammonium bromide (DDAB); 1,2-dilauroyl-sn-glycero-3-chloroethylphosphocholine salt (DL-EPC); N-[1-(2,3-dioleyl)propyl]-NNN-trimethylammonium chloride (DOTMA); N-[1-(2,3-dioleyl)propyl]-NNN-dimethylammonium chloride (DODMA); N,N-dioctadecane-N,N-dimethylammonium chloride (DODAC); N-(1-(2,3-dioleyl)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA); 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE); dioctadecylaminoglycyl spermine (DOGS); neutral lipids conjugated to cationic modifying groups; and combinations thereof. In addition, cationic lipids in many available formulations may be used, such as (from GIBCO / BRL), (from GIBCO / MRL), siPORT (from Applied Biosystems), (from Promega) and (from Bio-Rad Laboratories, Inc.).

[0055] In the present invention, the amount of ionizable cationic lipid is generally present in a molar percentage of 25.0-60.0% of the lipid nanoparticle composition. Preferably, the amount of ionizable cationic lipid is 27%, 30%, 35%, 40%, 45%, 50%, 55% of the lipid nanoparticle composition.

[0056] In certain embodiments, the ionizable cationic lipids of the present invention include, but are not limited to, structures of formula (II):

[0057]

[0058] wherein Q is a substituted or unsubstituted straight-chain C2-20 alkylene, wherein one or more C atoms of the alkylene are optionally replaced by heteroatoms independently selected from O, S and N; or Q is a substituted or unsubstituted, saturated or unsaturated 4-6-membered ring, wherein the ring atoms of the 4-6-membered ring optionally contain one or more heteroatoms independently selected from O, S and N; wherein the substituted substituent is selected from halogen, -OH, straight-chain or branched C1-20 alkyl, straight-chain or branched C1-20 alkoxy, straight-chain or branched C2-20 alkenyl, straight-chain or branched C2-20 alkynyl, -CH2CH(OH)R5,

[0059]

[0060] R1, R2, R3, and R4 may be the same or different, and are independently selected from hydrogen, substituted or unsubstituted straight or branched C1-30 alkyl, substituted or unsubstituted straight or branched C2-30 alkenyl, substituted or unsubstituted straight or branched C2-30 alkynyl, wherein one or more C atoms of the alkyl, alkenyl or alkynyl are optionally replaced by heteroatoms independently selected from O, S and N, or -CH2CH(OH)R5; the substituted substituents are selected from halogen, -OH, straight or branched C1-10 alkyl, straight or branched C1-10 alkoxy.

[0061] The condition is that at least one of R1, R2, R3, and R4 is

[0062] R5 is selected from hydrogen, substituted or unsubstituted straight or branched C1-30 alkyl, substituted or unsubstituted straight or branched C2-30 alkenyl, substituted or unsubstituted straight or branched C2-30 alkynyl, wherein one or more C atoms of the alkyl, alkenyl or alkynyl are optionally replaced by heteroatoms independently selected from O, S and N; the substituted substituents are selected from halogen, -OH, straight or branched C1-10 alkyl, straight or branched C1-10 alkoxy;

[0063] R6 is selected from hydrogen, C1-3 alkyl, C1-3 alkoxy, -OH;

[0064] n is an integer selected from 1-8, m is an integer selected from 0-8, n and m are independent of each other and may be the same or different;

[0065] When at least two of R1, R2, R3, and R4 are When n and m in each of the groups are independent of each other and may be the same or different.

[0066] In a preferred embodiment of the present invention, Q is a substituted or unsubstituted straight-chain C2-20 alkylene group, wherein one or more C atoms of the alkylene group are optionally replaced by heteroatoms independently selected from O, S and N;

[0067] Preferably, Q is wherein R8 and R9 are independently selected from substituted or unsubstituted straight-chain C1-10 alkylene, wherein one or more C atoms of the alkylene are optionally replaced by heteroatoms independently selected from O, S and N; R7 is hydrogen, halogen, -OH, straight-chain or branched C1-20 alkyl, straight-chain or branched C2-20 alkenyl, straight-chain or branched C2-20 alkynyl, or -CH2CH(OH)R5, or the substituted substituent is halogen, -OH, straight-chain or branched C1-10 alkyl, straight-chain or branched C1-10 alkoxy;

[0068] Preferably, Q is Wherein: x and y may be the same or different and independently selected from integers of 1-8; R7 is defined as above; preferably, x or y are the same or different and selected from integers of 1-3, for example, 1, 2 or 3; preferably, R7 is a straight chain or branched C1-4 alkyl, for example, methyl, ethyl, n-propyl, n-butyl, etc.

[0069] In some embodiments of the present invention, the saturated or unsaturated 4-6 membered ring is piperazinyl or cyclohexyl. In a preferred embodiment of the present invention, R6 is -OH. In a preferred embodiment of the present invention, n is selected from an integer of 4-8, and m is selected from an integer of 4-8.

[0070] In a preferred embodiment of the present invention, the ionizable cationic compound of the present invention has the following formula A, B, C or D:

[0071]

[0072] Wherein each n1 is independent of each other, and may be the same or different, each n1 is selected from an integer of 1-8, each m1 is independent of each other, and may be the same or different, each m1 is selected from an integer of 0-8; preferably, each n1 is selected from an integer of 4-8, and each m1 is selected from an integer of 4-8; preferably, each n1 is the same as each other, and each m1 is the same as each other.

[0073]

[0074] Wherein each n2 is independent of each other, and may be the same or different, each n2 is selected from an integer of 1-8, each m2 is independent of each other, and may be the same or different, each m2 is selected from an integer of 0-8; preferably, each n2 is selected from an integer of 4-8, and each m2 is selected from an integer of 4-8; preferably, each n2 is the same as each other, and each m2 is the same as each other.

[0075]

[0076] Wherein each n3 is independent of each other, and may be the same or different, each n3 is selected from an integer of 1-8, each m3 is independent of each other, and may be the same or different, each m3 is selected from an integer of 0-8; preferably, each n3 is selected from an integer of 4-8, and each m3 is selected from an integer of 4-8; preferably, each n3 is the same as each other, and each m3 is the same as each other.

[0077]

[0078] Wherein each n4 is independent of each other, and may be the same or different, each n4 is selected from an integer of 1-8, each m4 is independent of each other, and may be the same or different, each m4 is selected from an integer of 0-8; preferably, each n4 is selected from an integer of 4-8, and each m4 is selected from an integer of 4-8; preferably, each n4 is the same as each other, and each m4 is the same as each other.

[0079] In a preferred embodiment, the ionizable cationic lipid of the present invention has a structure shown in the following formula (i.e., C2 compound):

[0080]

[0081] Auxiliary phospholipids

[0082] In the present invention, the auxiliary phospholipid is an electronegative compound, which is a compound having a structure represented by formula (I) or a salt thereof:

[0083]

[0084] Wherein R1 and R2 each independently represent a substituted or unsubstituted straight or branched alkyl or unsaturated alkyl, or a substituted or unsubstituted straight or branched alkenyl. Here, the alkyl is preferably a saturated hydrocarbon group or an unsaturated hydrocarbon group obtained by removing a single hydrogen atom from a hydrocarbon portion containing 1 to 23 carbon atoms. Here, the carbon number of the alkyl is preferably 1-17, such as 1-10, and another example is 2-8. Examples of alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, n-dodecyl and n-tridecyl. Here, the alkenyl is preferably a monovalent group obtained by removing a single hydrogen atom from a hydrocarbon portion having at least one carbon-carbon double bond. The carbon number of the alkenyl is preferably 1-17, such as 4-8, such as 6, etc. Examples of alkenyl include, but are not limited to, for example, vinyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, 1-methyl-2-butene-1-yl, etc. In addition, substituted substituents include, but are not limited to, halogen, -OH, straight or branched C1-20 alkyl, straight or branched C1-20 alkoxy, straight or branched C2-20 alkenyl, straight or branched C2-20 alkynyl, etc. Preferably, the number of carbon atoms of the straight or branched C1-20 alkyl is preferably 1-17 or 2-10, such as 4, 6, 8, etc. The number of carbon atoms of the straight or branched C1-20 alkoxy is preferably 1-17 or 2-10, such as 4, 6, 8, etc. The number of carbon atoms of the straight or branched C2-20 alkenyl is preferably 2-17 or 2-10, such as 4, 6, 8, etc. The number of carbon atoms in the straight or branched C2-20 alkynyl group is preferably 2-15 or 2-10, such as 4, 6, 8, etc. In addition, the length of the branch chain is generally 1-8 carbon atoms, preferably 1-5 carbon atoms.

[0085] In certain embodiments, the auxiliary phospholipids of the present invention are PG phospholipids, examples of which include but are not limited to 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), L-α-phosphatidyl-DL-glycero-dioleoyl (DOPG), 1,2-palmitoylphosphatidylglycerol (DPPG). The present invention may use a combination of one or more of the above phospholipids.

[0086] In certain embodiments, the auxiliary phospholipid of the present invention is a compound salt, preferably a monovalent salt, more preferably an alkali metal salt, examples of which include but are not limited to sodium salt and potassium salt. The present invention can use two or more compound salts.

[0087] In the present invention, the amount of auxiliary phospholipids is generally 10.0-40.0% of the molar percentage of the lipid nanoparticle composition. Preferably, the amount of auxiliary phospholipids is 12.0-33.0%, more preferably 15.0-30.0%, such as 16.0%, 18%, 20%, 22%, 25%, 26%, 28% or 30%.

[0088] Active ingredients

[0089] The active ingredient of the present invention is not particularly limited, preferably an electronegative substance. Exemplary active ingredients include, but are not limited to, small molecule compounds, nucleic acids, proteins, polypeptides, etc. Among them, small molecule compounds are preferably amphipathic compounds. Examples of small molecule compounds include, but are not limited to, antitumor drugs, such as small molecule weak base antitumor drugs in salts: anthracyclines, vinca alkaloids, methotrexate, chlorambucil, doxorubicin, etc. Among them, examples of nucleic acids include, but are not limited to, mRNA, antisense nucleic acids (ASOs), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), aptamers, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA). In addition, mRNA is RNA encoding proteins, antigens or enzymes.

[0090] In certain embodiments, the antigen of the present invention is a pathogen antigen, such as an immunogenic peptide fragment of bacteria, virus, fungus, or other pathogens.

[0091] In certain embodiments, the antigen of the present invention is a tumor antigen. Tumor antigens can be expressed on the surface, cytoplasm or nucleus of tumor cells. Tumor antigens can also be selected from proteins that are overexpressed in tumor cells compared to normal cells. Tumor antigens can be further divided into tumor-associated antigens and tumor-specific antigens.

[0092] In the present invention, the ratio of the total mass of lipid molecules to the mass of active ingredients in the lipid nanoparticles is 1:1-60:1, such as 2:1-55:1, 5:1-50:1, 10:1-40:1, 15:1-45:1, 20:1-30:1.

[0093] cholesterol

[0094] The lipid nanoparticles of the present invention may optionally contain cholesterol. Cholesterol is present in a molar percentage of about 20.0-50.0% of the lipid nanoparticle composition. In some embodiments, the molar percentage of cholesterol is about 25.0-45.0% of the total lipid molecules thereof. In some embodiments, the molar percentage of cholesterol is about 25.0-40.0%.

[0095] PEGylated phospholipids

[0096] The lipid nanoparticles of the present invention optionally include phospholipids modified by polyethylene glycol. In some embodiments, the phospholipids modified by polyethylene glycol are present in a lipid nanoparticle composition molar percentage of about 0.5-10.0%. In some embodiments, the phospholipids modified by polyethylene glycol account for about 1.0-5.0% of its overall lipid molecules. In some embodiments, the molar percentage of the phospholipids modified by polyethylene glycol is about 1.5-2.5%. In some embodiments, the phospholipids modified by polyethylene glycol include a PEG component of about 1000 to about 10000 daltons, for example, the molecular weight of PEG is 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000 daltons. In one embodiment, the phospholipids modified by polyethylene glycol are DMG-PEG2000.

[0097] In certain embodiments, examples of polyethylene glycol-modified phospholipids of the present invention include, but are not limited to, polysorbate 80, TPGS, mPEG-DSPE, PEG-DMG, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol. The present invention can use one of the above ingredients or a combination of multiple. Polyethylene glycol-modified phospholipids can also use known products, and specific examples thereof include C14-PEG2000 (1,2-dimyristoyl-rac-glycerol, methoxy polyethylene glycol-2000 (DMG-PEG2000)) and C18-PEG5000 (1,2-distearoyl-rac-glycerol, methoxy polyethylene glycol-5000 (DSG-PEG5000)).

[0098] [Drug composition]

[0099] The second aspect of the present invention provides a pharmaceutical composition comprising the lipid nanoparticles described in the first aspect of the present invention and a pharmaceutically acceptable carrier. The lipid nanoparticles have been described above. The pharmaceutically acceptable carrier is described in detail below.

[0100] In the present invention, pharmaceutically acceptable carriers refer to molecular entities and compositions that do not produce harmful, allergic or other adverse reactions when administered to animals such as humans. According to the disclosure of the present invention, the preparation of pharmaceutical compositions containing at least one compound or additional active ingredients is known to those skilled in the art, as illustrated by Remington's Pharmaceutical Sciences, 2003. In addition, for animal (e.g., human) administration, it should be understood that LNP preparations should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards.

[0101] The pharmaceutical composition of the present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, transmucosally, intrauterinely, orally, topically, locally, by inhalation (e.g., spray inhalation), by injection, by infusion, by continuous infusion, by local perfusion of direct bathing target cells, by catheter, by lavage, in emulsion, or by other methods or any combination of the above methods, which is known to those skilled in the art. In a certain embodiment, the pharmaceutical composition of the present invention is a lyophilized powder.

[0102] The actual dosage of the pharmaceutical composition administered to an animal or human patient can be determined by physical and physiological factors such as body weight or surface area, severity of the condition, type of disease to be treated, previous or concurrent therapeutic interventions, idiopathic conditions of the patient, and route of administration. Depending on the dosage and route of administration, the preferred dosage and / or number of administrations of the effective amount may vary according to the subject's response. In any case, the physician responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for an individual subject.

[0103] In certain embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may contain, for example, about 2% to about 75% of the unit weight, or about 25% to about 60%, and any range therein.

[0104] A solution of a composition as a free alkali or a pharmaceutically acceptable salt can be prepared in water appropriately mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof and in oil. Under conventional storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms. The pharmaceutical form suitable for injection includes sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injections or dispersions. In all cases, the form must be sterile and must have a fluidity to the extent that easy injection capability exists. It must be stable under manufacturing and storage conditions, and must prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (i.e., glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and / or vegetable oils. It can be, for example, by using a coating, such as lecithin, in the case of a dispersion, by maintaining the desired particle size and by using a surfactant to maintain appropriate fluidity. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate or gelatin.

[0105] [Preparation method]

[0106] The third aspect of the present invention provides a method for preparing lipid nanoparticles targeting secondary lymphoid organs. Exemplarily, the preparation method at least comprises:

[0107] The step of mixing an ionizable cationic lipid, an auxiliary phospholipid, and optionally a phospholipid modified with cholesterol and polyethylene glycol to prepare an organic phase comprises: dissolving the ionizable cationic lipid, the auxiliary phospholipid, cholesterol, and the phospholipid modified with polyethylene glycol in an organic solvent at a molar ratio to prepare a mixed lipid solution, and using the mixed lipid solution as the organic phase;

[0108] The step of dissolving the active ingredient in an aqueous solvent to prepare an aqueous phase is as follows: according to the volume of aqueous phase required in the formulation, adding the object to be delivered (e.g., nucleic acid) into the citric acid buffer system and mixing well to form the aqueous phase;

[0109] The step of mixing the organic phase and the aqueous phase is as follows: the prepared organic phase and aqueous phase are rapidly mixed by using a microfluidic chip or collision jet technology.

[0110] In the present invention, the organic phase can be used to dissolve ionized cationic lipids and auxiliary phospholipids and optional cholesterol and polyethylene glycol-modified phospholipids by alcohol solvents. Examples of alcohol solvents include, but are not limited to, ethanol. In the present invention, the aqueous solvent is generally an aqueous buffer of salt, such as a citric acid buffer. The manner in which the organic phase is mixed with the aqueous phase is not limited, for example, rapid mixing is performed by microfluidics or collision jet technology. The mixing volume ratio of the organic phase and the aqueous phase during mixing is not limited, and generally the volume of the organic phase is required to be less than the volume of the aqueous phase. For example, in the range of 1:2 to 1:10, preferably in the range of 1:3 to 1:7, etc.

[0111] Example 1

[0112] The specific method for preparing the nano drug delivery system targeting secondary lymphoid organs in this embodiment is as follows: respectively measure the corresponding volume of C2 (manufacturer: Qichensheng Biotechnology Co., Ltd.), DSPG, POPG, DOPG, DPPG, CHOL and DMG-PEG2000 in the formula, mix them and set aside (organic phase). Dissolve the corresponding volume of mRNA in the formula in a certain volume of citric acid buffer for use (aqueous phase), and quickly mix the organic phase for dissolving lipids and the aqueous phase for dissolving mRNA in a volume ratio of 1:3 using microfluidics technology, and then remove the ethanol component by ultrafiltration and concentration.

[0113] Nano drug delivery systems with different types of auxiliary phospholipids were designed, and the dosage of each component in the nano delivery system is shown in Table 1 below.

[0114] Table 1 Composition of nano drug delivery systems with different types of auxiliary phospholipids

[0115]

[0116] Table 2 Physicochemical data of nano drug delivery systems with different types of auxiliary phospholipids

[0117]

[0118] The in vivo distribution study was conducted using normal female C57BL / 6N mice aged 6-8 weeks, with 5 mice in each group. Luc-mRNA was injected intravenously into the mice at a dosage of 10 μg / mouse. The pre-configured D-luciferin potassium salt substrate (100 μl / mouse) was injected 6h and 24h later. After a single dose, the expression of isolated tissues and organs was observed after dissection 6h later (from top to bottom, heart, liver, spleen, lung, kidney, lymph node). The expression of different types of auxiliary phospholipid nanodrug delivery systems in the liver, spleen, lymph nodes and other parts after intravenous administration was investigated.

[0119] Figure 1In vitro imaging of nano-drug delivery systems with different types of auxiliary phospholipids is shown, with representative images of the expression of drug delivery systems in organs such as heart, liver, spleen, lung, kidney and lymph nodes from top to bottom. Figure 2 The firefly luciferase activity expression of nano drug delivery systems with different types of auxiliary phospholipids in RAW264.7 cell line was shown. Figure 3 The quantitative statistical results of the expression of nano drug delivery systems with different types of auxiliary phospholipids in organs such as heart, liver, spleen, lung, kidney, lymph node, etc. Among them, the spleen expression in LNP-1, LNP-2, LNP-3 and LNP-4 was 71.6%, 81.1%, 73.6% and 88.6%, respectively.

[0120] Depend on Figure 1 It can be seen that the results of in vivo targeting studies of nano drug delivery systems with different types of auxiliary phospholipids show that PG-type auxiliary phospholipids such as DSPG, DOPG, POPG and DPPG drug delivery systems all have mRNA expression in secondary lymphoid organs (spleen). Figure 3 The targeting efficiency of the DSPG delivery system to the secondary lymphoid organ (spleen) reached 88.6%, which increased the relative enrichment of mRNA in the spleen to a certain extent. The spleen fluorescence intensity of LNP-4 was the highest, indicating that DSPG has the potential to enhance the enrichment of mRNA in the spleen. Figure 2 The fireny luciferase activity expression results in the culture medium showed that the cellular RLU expression values ​​of PG-type auxiliary phospholipids were all higher, so DSPG can be used for subsequent research.

[0121] Example 2

[0122] The specific method for preparing the secondary lymphoid organ targeted nano-delivery carrier in this embodiment is as follows: the corresponding volumes of C2, DSPG, CHOL and DMG-PEG2000 in the formula are measured respectively, mixed and set aside (organic phase). The corresponding volume of mRNA in the formula is dissolved in a certain volume of citric acid buffer for use (aqueous phase), and the organic phase for dissolving lipids and the aqueous phase for dissolving mRNA are quickly mixed in a volume ratio of 1:3 using microfluidics technology, and then the ethanol component is removed by ultrafiltration and concentration.

[0123] Nano drug delivery systems with different formulation ratios were designed, and the amounts of each component in the nano drug delivery system are shown in Table 3 below.

[0124] Table 3 Formulation composition of nano drug delivery system with different formulation ratios

[0125]

[0126] Table 4 Physicochemical data of nano drug delivery systems with different formulation ratios

[0127]

[0128] The in vivo distribution study was conducted using normal female C57BL / 6N mice aged 6-8 weeks, with 3 mice in each group. Luc-mRNA was injected intravenously into the mice at a dosage of 10 μg / mouse. The pre-configured D-luciferin potassium salt substrate (100 μl / mouse) was injected 6h and 24h later. After a single dose, the expression of isolated tissues and organs was observed after dissection 6h later (from top to bottom, heart, liver, spleen, lung, kidney, lymph node). The expression of nanodrug delivery systems with different formulation ratios in the liver, spleen, lung and other parts after intravenous administration was investigated.

[0129] Figure 4 The results of in vitro organ fluorescence intensity of nano drug delivery systems with different formulations are shown in Figure 2. The results of in vivo targeting studies of lipid nanoparticle drug delivery systems with different proportions show that the higher the proportion of auxiliary phospholipid DSPG in the formula, the more obvious the targeting to secondary lymphoid organs (spleen). Figure 5 The results showed that the distribution of lipid nanoparticles composed of 5 different formulations in the spleen reached more than 80%, indicating that increasing the proportion of DSPG in the formulation to a certain extent can increase the relative enrichment of mRNA in the spleen. When the proportion of DSPG in the formulation reached 30%, the proportion of the drug delivery system in the secondary lymphoid organ (spleen) reached 93.3%, which was conducive to the specific delivery of mRNA to the spleen for expression. Figure 5 The spleen distribution corresponding to LNP-5, LNP-6, LNP-7, LNP-8 and LNP-9 were 93.6%, 81.6%, 87.8%, 93.3% and 87.4%, respectively.

[0130] Example 3

[0131] The specific method for preparing the nano drug delivery system targeting secondary lymphoid organs (spleen) in this embodiment is as follows: respectively measure the corresponding volumes of C2, DSPG (electronegativity) or DSPC (electroneutrality), CHOL and DMG-PEG2000 in the formula, mix them and set aside (organic phase). The corresponding volume of mRNA in the formula is dissolved in a certain volume of citric acid buffer and set aside (aqueous phase), and the organic phase dissolving lipids and the aqueous phase dissolving mRNA are quickly mixed in a volume ratio of 1:3 using microfluidics technology, and then the ethanol component is removed by ultrafiltration and concentration.

[0132] Two auxiliary phospholipid nano drug delivery systems with different electronegativity were designed, in which the DSPG phospholipid surface was negatively charged compared with the DSPC phospholipid surface, and the distribution of auxiliary phospholipid nano drug delivery systems with different electronegativity in tissues and organs was verified. The dosage of each component in the nano delivery system is shown in Table 5 below.

[0133] Table 5 Composition of the auxiliary phospholipid nanodrug delivery system with different electronegativity

[0134]

[0135] Table 6 Physicochemical data of phospholipid nano-drug delivery systems with different electronegativity

[0136]

[0137] The in vivo distribution study was conducted using normal female C57BL / 6N mice aged 6-8 weeks, with 3 mice in each group. Luc-mRNA was injected intravenously into the mice at a dosage of 10 μg / mouse. The pre-configured D-luciferin potassium salt substrate (100 μl / mouse) was injected 6h and 24h later. After a single dose, the expression of isolated tissues and organs was observed after dissection 6h later (from top to bottom, heart, liver, spleen, lung, kidney, lymph node). The expression of electronegative and electroneutral assisted phospholipid nanodrug delivery systems in the liver, spleen, lymph nodes and other parts after intravenous administration was investigated.

[0138] Figure 6 The results of electronegativity and electroneutrality-assisted phospholipid nanoparticle drug delivery systems in vitro were shown. Figure 6 It can be seen that the results of the in vivo targeting study of DSPG (electronegative) and DSPC (electronegative) lipid nanoparticle drug delivery systems showed that the electronegative auxiliary phospholipid DSPG had a higher spleen fluorescence intensity and a significant spleen organ targeting effect. The neutral auxiliary phospholipid DSPC had a significant liver organ targeting effect, and DSPC partially targeted secondary lymphoid organs (lymph nodes).

[0139] Example 4

[0140] This example is an experiment on the applicability of PG-based auxiliary phospholipids targeting secondary lymphoid organs. Specifically, SM102 is the main component of Moderna's new crown mRNA vaccine and is an ionizable amino lipid approved for marketing. Currently, PG-based auxiliary phospholipids show good secondary lymphoid organ targeting (spleen and lymph nodes) in C2. To verify whether the introduction of PG-based auxiliary phospholipids is universal for secondary lymphatic targeting, we conducted a study using the marketed ionizable lipid SM102 as an example.

[0141] The specific method for preparing the SM102 nano drug delivery system in this embodiment is as follows: the corresponding volumes of SM-102, DSPG, CHOL and DMG-PEG2000 in the formula are measured respectively, mixed and set aside (organic phase). The corresponding volume of mRNA in the formula is dissolved in a certain volume of citric acid buffer for use (aqueous phase), and the organic phase for dissolving lipids and the aqueous phase for dissolving mRNA are quickly mixed in a volume ratio of 1:3 using microfluidics technology, and then the ethanol component is removed by ultrafiltration and concentration.

[0142] The in vivo distribution study was conducted using normal female C57BL / 6N mice aged 6-8 weeks, with 3 mice in each group. Luc-mRNA was injected intravenously into the mice at a dosage of 10 μg / mouse. The pre-configured D-luciferin potassium salt substrate (100 μl / mouse) was injected 6h and 24h later. After a single dose, the expression of isolated tissues and organs was observed after dissection 6h later (from top to bottom, heart, liver, spleen, lung, kidney, lymph node). The expression of the SM102 nano drug delivery system in the liver, spleen, lymph nodes and other parts after intravenous administration was investigated.

[0143] Figure 7 The in vivo and in vitro imaging results of the SM102 nanodrug delivery system are shown. Figure 7 It can be seen that the auxiliary phospholipid DSPG+SM102 nano drug delivery system can target secondary lymphoid organs (spleen and lymph nodes), and the fluorescence intensity of the spleen and lymph nodes is relatively high, and the effect is significant. This shows that the auxiliary phospholipid DSPG is also suitable for the design of the commercially available ionizable lipid-targeted secondary lymphoid organ drug delivery system.

[0144] Example 5

[0145] This example is an example of particle size and potential detection of a nano drug delivery system.

[0146] The "Intensity" refers to the hydrodynamic diameter of the particle distribution measured by dynamic light scattering (DLS). Zeta potential is a scientific term for the electrokinetic potential in a colloidal system.

[0147] To characterize the nano drug delivery system, the size and zeta potential of the delivery system were checked by dynamic light scattering, and the drug delivery system was measured 5 times. The method is as follows: a certain volume of the nano drug delivery system was taken, diluted to a certain concentration with purified water, and the particle size and surface potential of the drug delivery system targeting secondary lymphoid organs were measured in a laser particle size analyzer.

[0148] Example 6

[0149] This example is an in vitro luciferase expression test example of a nano drug delivery system.

[0150] The cell biological activity of the nano drug delivery system was verified, and the cell line used for the test was RAW264.7 (mouse mononuclear macrophage leukemia cells). The experimental group was the nano drug delivery system, and the final transfection concentration was 1ug / ml, with 6 parallel wells in each group. After 24 hours of cell transfection, the intracellular firefly luciferase activity was determined by chemiluminescence.

[0151] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. A lipid nanoparticle targeting secondary lymphoid organs, characterized in that: The invention comprises an ionizable cationic lipid, an auxiliary phospholipid and an active ingredient, wherein the auxiliary phospholipid is a compound having a structure shown in formula (I) or a salt thereof: wherein R1 and R2 each independently represent a substituted or unsubstituted straight chain or branched chain alkyl group, or a substituted or unsubstituted straight chain or branched chain alkenyl group.

2. The lipid nanoparticle targeting secondary lymphoid organs according to claim 1, characterized in that: The auxiliary phospholipid is selected from at least one of 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), L-α-phosphatidyl-DL-glycero-dioleoyl (DOPG) and 1,2-palmitoylphosphatidylglycerol (DPPG).

3. The lipid nanoparticle targeting secondary lymphoid organs according to claim 1, characterized in that: The ionizable cationic lipids include, but are not limited to, structures of formula (II): wherein Q is a substituted or unsubstituted straight-chain C2-20 alkylene, wherein one or more C atoms of the alkylene are optionally replaced by heteroatoms independently selected from O, S and N; or Q is a substituted or unsubstituted, saturated or unsaturated 4-6-membered ring, wherein the ring atoms of the 4-6-membered ring optionally contain one or more heteroatoms independently selected from O, S and N; wherein the substituted substituent is selected from halogen, -OH, straight-chain or branched C1-20 alkyl, straight-chain or branched C1-20 alkoxy, straight-chain or branched C2-20 alkenyl, straight-chain or branched C2-20 alkynyl, -CH2CH(OH)R5, R1, R2, R3, and R4 may be the same or different, and are independently selected from hydrogen, substituted or unsubstituted straight or branched C1-30 alkyl, substituted or unsubstituted straight or branched C2-30 alkenyl, substituted or unsubstituted straight or branched C2-30 alkynyl, wherein one or more C atoms of the alkyl, alkenyl or alkynyl are optionally replaced by heteroatoms independently selected from O, S and N, or -CH2CH(OH)R5; the substituted substituents are selected from halogen, -OH, straight or branched C1-10 alkyl, straight or branched C1-10 alkoxy.

4. The lipid nanoparticle targeting secondary lymphoid organs according to claim 1, characterized in that: The ionizable cationic lipid is present in a molar percentage of 25.0-60.0% of the lipid nanoparticle composition.

5. The lipid nanoparticle targeting secondary lymphoid organs according to claim 1, characterized in that: The helper phospholipid is present in a molar percentage of 10.0-40.0% of the lipid nanoparticle composition.

6. The lipid nanoparticle targeting secondary lymphoid organs according to claim 1, characterized in that: Further included is cholesterol.

7. The lipid nanoparticle targeting secondary lymphoid organs according to claim 6, characterized in that: The cholesterol is present in a molar percentage of 20.0-50.0% of the lipid nanoparticle composition.

8. The lipid nanoparticle targeting secondary lymphoid organs according to claim 1, characterized in that: Phospholipids further modified with polyethylene glycol.

9. The lipid nanoparticle targeting secondary lymphoid organs according to claim 8, characterized in that: The polyethylene glycol-modified phospholipid is present in a molar percentage of 0.5-10.0% of the lipid nanoparticle composition.

10. The lipid nanoparticle targeting secondary lymphoid organs according to claim 1, characterized in that: The active ingredient is selected from at least one of small molecule compounds, nucleic acids, proteins and polypeptides.

11. The lipid nanoparticle targeting secondary lymphoid organs according to claim 1, characterized in that: The ratio of the total mass of lipid molecules to the mass of active ingredients in the lipid nanoparticles is 1:1-60:

1.

12. A pharmaceutical composition, characterized in that Comprising the lipid nanoparticle according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

13. A method for preparing lipid nanoparticles targeting secondary lymphoid organs according to any one of claims 1 to 11, characterized in that: include: The step of preparing an organic phase by mixing an ionizable cationic lipid and an auxiliary phospholipid and optionally a phospholipid modified with cholesterol and polyethylene glycol; The step of dissolving the active ingredient in an aqueous solvent to prepare an aqueous phase; and The step of mixing the organic phase and the aqueous phase.

14. The preparation method according to claim 13, characterized in that: The organic phase and the aqueous phase are mixed using microfluidics or collision jet technology.

15. Use of the lipid nanoparticle according to any one of claims 1 to 11 in the preparation of a medicament targeting secondary lymphoid organs.

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