Lipid nanoparticles, methods of making and using the same

Abstract

The present application relates to the technical field of nucleic acid gene therapy, and particularly relates to a lipid nanoparticle and a preparation method and application thereof.The lipid nanoparticle provided by the present application has a molar percentage of (33-50)%: (10-13.33)%: (1.5-2)%: (38.5-51.33)% of cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or derivatives thereof.Targeted LNP synthesis is carried out in the molar percentage range, has a higher encapsulation rate, and can significantly improve the transduction efficiency of the targeted LNP.Not only can the encapsulation rate and the loading capacity of the particles be improved, but also the targeting function of T cells is improved, and the efficient transfection of T cells is realized.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid gene therapy technology, and in particular to lipid nanoparticles, their preparation methods, and applications. Background Technology

[0002] LNP, or Lipid Nanoparticle, also known as lipid nanoparticle, consists of lipid vesicles composed of cationic lipids, cholesterol, polyethylene glycol lipids, and auxiliary lipids (or neutral lipids), which can be used for nucleic acid delivery.

[0003] The encapsulation efficiency of LNPs is a core quality indicator for LNP formulations. Encapsulation efficiency determines the quality and efficacy of the drug and is incorporated into the core quality control standards for LNP-related products. Traditional LNPs are non-targeted applications. With technological advancements, targeted delivery applications are increasingly combining the delivery characteristics of LNPs with the targeting principles of target proteins to cells in vivo. This necessitates the insertion of exogenous target proteins into the LNP structure to form targeted LNPs.

[0004] Existing technologies can utilize targeted LNP particles to encapsulate CAR nucleic acids and then deliver them directly into the body, enabling T cells to express CAR. This approach avoids complex in vitro cell editing and culture processes, potentially significantly shortening the treatment cycle and reducing costs. However, in vivo CAR-T preparation methods typically use CD3 ScFv as the target protein, which has limited targeted transduction efficiency. This means that LNP-encapsulated CAR nucleic acids...

[0005] It may not be able to be efficiently delivered to the target immune cells, thus affecting the final effect of CAR cell therapy.

[0006] Therefore, it is crucial to develop new LNPs to improve the in vivo editing efficiency of CAR nucleic acids. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide lipid nanoparticles, their preparation methods and applications.

[0008] The lipid nanoparticles provided by this invention have packaging materials comprising: cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or its derivatives; the molar percentages of the cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or its derivatives are (33%–50%): (10%–13.33%): (1.5%–2%): (38.5%–51.33%).

[0009] To improve the transfection ability of LNP on T cells, the PEG content in the coating was optimized. However, the results showed that simply changing the PEG content did not improve the transfection ability of the targeting particles on T cells. Therefore, this invention optimized the ratio of each component in the coating material. The results showed that, at a specific ratio, the transfection ability of LNP on cells was significantly improved. Specifically, the molar percentages of cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or its derivatives were (33%–35%): (10%–13.33%): (1.5%–2%): (50%–51.33%).

[0010] For example, the molar percentages of cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or their derivatives are (33%–34%): (13%–13.33%): (1.8%–2%): (51%–51.33%). More specifically, the molar percentages of cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or their derivatives are 33.33%: 13.33%: 2%: 51.33%.

[0011] In a specific embodiment, using an optimized lipid molar percentage range for targeted LNP synthesis results in higher encapsulation efficiency and significantly improved targeted LNP transduction efficiency. This not only enhances particle encapsulation efficiency and loading capacity but also improves T cell targeting function, achieving highly efficient T cell transfection.

[0012] In this invention, the cationic lipids are selected from at least one of SM102, DLin-MC3-DMA, ALC-0315, or ATX-126;

[0013] In this invention, the neutral lipids are selected from at least one of DSPC, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), distearate phosphatidylethanolamine (DSPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), or phosphatidylserine (PS).

[0014] In this invention, the cholesterol derivative includes: 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride (DC-CHOL).

[0015] In this invention, the PEG is a PEG with an average molecular weight of 500 to 5000; for example, at least one of PEG500, PEG1000, PEG1500, PEG2000, PEG3000, PEG4000 or PEG5000.

[0016] In this invention, the PEGylated lipid is at least one of ALC-0159, DMG-PEG, PEG2000-C-DMG, DSPE-PEG, DPPE-MPEG(2000), DSPE-PEG-Amine, DSPE-PEG-NHS,MW 2000, DSPE-PEG-Maleimide (also known as DSPE-PEG-MAL or DSPE-PEG-maleimide), DMG-PEG-MAL (DMG-PEG-maleimide), aminated-PEG-DMG, and hydroxylated-PEG-DMG, wherein the molecular weight of PEG can be selected from 500-5000, preferably PEG2000.

[0017] In some embodiments, the packaging material for the lipid nanoparticles is composed of SM102, DSPC, PEGylated lipids, and cholesterol. In this embodiment, the molar percentages of SM102, DSPC, PEGylated lipids, and cholesterol in the packaging material are 33.33%: 13.33%: 2%: 51.33%.

[0018] In the embodiments described above, the PEGylated lipids are PEG2000-DMG, PEG2000-DSPC, amino-PEG-DMG, and hydroxylated-PEG-DMG;

[0019] In this invention, the packaging material further includes PEGylated proteins; the PEGylated proteins include the protein shown in SEQ ID NO:1 and the PEGylated lipids.

[0020] In this invention, the method for preparing the PEGylated protein formed by the targeting protein and PEGylated lipids includes mixing the targeting protein with a TCEP solution for reaction, adding a solution of PEGylated lipids, and obtaining the PEGylated protein after the reaction.

[0021] This invention uses the TCEP method to modify the fusion protein to form a targeted protein-PEG2000-DSPE complex. The fusion protein-PEG2000-DSPE complex and other lipid components of LNP are directly synthesized into target nanoparticles through microfluidics. The synthesis process is a one-step process, which is simpler and solves the problem of dimer formation in SATS synthesis. It also improves the encapsulation efficiency, coupling efficiency and transduction efficiency of targeted LNP synthesis.

[0022] In this embodiment of the invention, the molar ratio of the fusion protein, TCEP and DSPE-PEG-MAL as described above is 1:25:10.

[0023] Specifically, the preparation of the complex includes:

[0024] 1) Preparation of targeted protein solutions.

[0025] 2) Add an appropriate amount of 0.5M TCEP solution to the targeted protein solution and react at room temperature in the dark.

[0026] 3) Weigh an appropriate amount of DSPE-PEG-MAL and dissolve it in DMSO to prepare a 5mM (14.7mg / ml) DSPE-PEG-MAL working solution.

[0027] 4) Add an appropriate amount of 5mM DSPE-PEG-MAL working solution to the solution in step 2), with the target protein:TCEP:DSPE-PEG-MAL ratio being 1:25:10. React at room temperature in the dark.

[0028] 5) Add the solution obtained in step 4) to an equilibrated desalting column to remove impurities. This yields the PEGylated protein CD3 ScFv-CD86-PEG-DSPE linked to the target protein.

[0029] In a specific embodiment, a step can be added whereby solutions obtained from different steps are added to an equilibrated desalting column for solution displacement or impurity removal. In another specific embodiment, the TCEP method is used to modify the fusion protein, which can also be CD3 ScFv-CD86-PEG-DMG. The preparation of the complex differs from the above preparation scheme only in that DSPE-PEG-MAL is replaced with DMG-PEG-MAL.

[0030] In this invention, the lipid nanoparticles also include nucleic acids coated within the packaging material.

[0031] The nucleic acid includes, but is not limited to, at least one of mRNA, siRNA, miRRNA, DNA, dsRNA, and sDNA. The mRNA includes mRNA encoding CAR, mRNA encoding fluorescent protein, siRNA, miRRNA, DNA, dsRNA, sDNA, small interfering RNA, miRNA, aiRNA, shRNA, tRNA, ssDNA, dsDNA, plasmids, etc.

[0032] In some embodiments, the nucleic acid is a chimeric antigen receptor (CAR) nucleic acid sequence that recognizes CD19-positive tumor cells; in other embodiments, the chimeric antigen receptor (CAR) nucleic acid sequence that recognizes CD19-positive tumor cells can be replaced with a target molecule nucleic acid sequence that recognizes at least one surface-expressed target molecule, including solid tumors, hematologic malignancies, and / or tissues, wherein the target molecule includes any one or more of the following combinations: CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEA, CEACAM5, CEACAM6, CE ACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), and TAG-72. It can be used to treat tumors such as colon cancer, rectal cancer, small intestine cancer, anal cancer, bile duct cancer, stomach cancer, esophageal cancer, gallbladder cancer, lung cancer, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, kidney cancer, bladder cancer, cancers of the central nervous system, glioblastoma, skin cancer, melanoma, lymphoma, head and neck cancer, multiple myeloma, and leukemia.

[0033] In some embodiments, the chimeric antigen receptor (CAR) nucleic acid sequence that recognizes CD19-positive tumor cells can be replaced with an antigenic peptide nucleic acid sequence that can elicit an immune response in the organism, such as a combination of at least one or more nucleic acid fragments of HPV genome E1-E7, L1-L2, or antigenic peptide nucleic acid sequences or sequences of viruses or bacteria related to diseases such as hepatitis A, hepatitis B, poliomyelitis, influenza, and pneumonia, thereby obtaining a vaccine drug for treating the above-mentioned diseases or as a component of a drug combination.

[0034] In some embodiments, the chimeric antigen receptor (CAR) nucleic acid sequence that recognizes CD19-positive tumor cells can be replaced with active agents or therapeutic agents related to diseases such as ophthalmic diseases, metabolic diseases, hyperlipidemia, hepatitis B, pseudohypertrophic muscular dystrophy, non-alcoholic fatty liver disease, heart disease, amyotrophic lateral sclerosis, and cystic fibrosis, such as siRNA, miRNA, ASO, small activating RNA (saRNA), aptamers, and transfer RNA (tRNA) fragments, as drugs or drug combinations for the treatment of ophthalmic diseases, metabolic diseases, hyperlipidemia, hepatitis B, pseudohypertrophic muscular dystrophy, non-alcoholic fatty liver disease, heart disease, amyotrophic lateral sclerosis, and cystic fibrosis.

[0035] Furthermore, the present invention also provides a method for preparing the lipid nanoparticles, comprising:

[0036] The lipid nanoparticle packaging material described above is dissolved in ethanol to obtain the alcohol phase;

[0037] The PEG-conjugated fusion protein and the nucleic acid as described above were dissolved in citrate buffer to obtain the citrate phase; the alcohol phase and the citrate phase were then prepared into lipid nanoparticles via microfluidic control.

[0038] In a specific embodiment, the citrate buffer solution has a pH value of 3 to 5 and a concentration of 0.01 M to 0.03 M. For example, the citrate buffer solution has a pH value of 3.0, 3.5, 4.0, 4.5 or 5.0; and a concentration of 0.01 M, 0.015 M, 0.02 M, 0.025 M or 0.03 M.

[0039] In the microfluidic step, the ratio of alcohol phase flow rate to citric acid phase flow rate is 1:(1-5). For example, the ratio of alcohol phase flow rate to citric acid phase flow rate is 1:1, 1:2, 1:3, 1:4 or 1:5.

[0040] Furthermore, the present invention also provides the use of the lipid nanoparticles in the preparation of products for in vivo delivery of nucleic acids to mammalian subjects.

[0041] Furthermore, the present invention also provides a nucleic acid delivery reagent comprising the aforementioned lipid nanoparticles and buffer solution.

[0042] Furthermore, the present invention also provides a pharmaceutical composition comprising the lipid nanoparticles as described above.

[0043] Furthermore, the present invention also provides the use of the lipid nanoparticles as described above in the preparation of products that introduce nucleic acids into cells.

[0044] Furthermore, the present invention also provides the use of the lipid nanoparticles as described above in the preparation of medicaments for treating diseases or functional disorders in mammalian subjects requiring treatment.

[0045] In this invention, the disease includes at least one of the following: disease caused by infection, metabolic disease, hyperlipidemia, hepatitis B, pseudohypertrophic muscular dystrophy, non-alcoholic fatty liver disease, heart disease, amyotrophic lateral sclerosis, cystic fibrosis, genetic defect disease, or tumor.

[0046] Furthermore, the present invention also provides a cell transfection method, which includes incubating cells after contacting them with the cell transfection reagent as described above. The cell transfection reagent of the present invention can be used for cell transfection, such as animal cells, human cells, plant cells, or microorganisms. When the PEGylated protein therein is CD3ScFv-CD86-PEG-DSPE or CD3ScFv-CD86-PEG-DMG, it targets T cells, and the cell transfection reagent is a T cell-targeting transfection reagent.

[0047] In some embodiments, the cells are in vivo cells, such as those that recognize targeting molecules that target B cells, such as CD19, CD56, CD141, CD1c, CD11b, and CD14, or APC cells that recognize macrophages and dendritic cells. In a specific embodiment, the cells are T cells.

[0048] The lipid nanoparticles provided by this invention have a packaging material in which the molar percentages of cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or its derivatives are (33%–50%): (10%–13.33%): (1.5%–2%): (38.5%–51.33%). Targeted LNP synthesis within this molar percentage range results in higher encapsulation efficiency and significantly improved targeted LNP transduction efficiency. This not only enhances the encapsulation efficiency and loading capacity of the particles but also improves the targeting function of T cells, achieving highly efficient T cell transfection. Attached Figure Description

[0049] Figure 1 shows the validation of the ability of PEG-DMG synthesized in different proportions of targeted LNP to transduce GFP in T cells. In Figure 1, A represents the GFP positivity rate of PEG-DMG synthesized in different proportions of targeted LNP to transduce T cells; and B represents the GFP expression intensity (MFI) of PEG-DMG synthesized in different proportions of targeted LNP to transduce T cells.

[0050] Figure 2 shows the ability of targeted LNP particles with different formulations to transduce GFP in T cells, where A in Figure 2 represents the GFP positivity rate and B in Figure 2 represents the GFP expression intensity.

[0051] Figure 3 The encapsulation efficiency of targeted LNP particles with different formulations is shown.

[0052] Figure 4 The encapsulation efficiency of targeted LNP particles for different formulations with further optimization is shown.

[0053] Figure 5 shows the results of the transduction efficiency and intensity of the targeted LNP synthesized by the molar percentage described in this application for CD19 CAR, where A in Figure 5 is the transduction efficiency graph and B in Figure 5 is the transduction intensity (MFI) graph.

[0054] Figure 6 The molar percentage of targeted LNP encapsulation efficiency of CD19 CAR nucleic acid synthesized according to this application is shown.

[0055] Figure 7 This demonstrates the encapsulation efficiency of a target protein synthesized using the TCPE method in targeted LNP synthesis.

[0056] Figure 8 : Validation of the in vivo efficacy of targeting the LNP fusion protein particles. Detailed Implementation

[0057] This invention provides lipid nanoparticles, their preparation methods, and applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0058] LNP (Lipid Nanoparticle) is a lipid vesicle composed of cationic lipids, accessory lipids, PEG / PEG derivatives (also known as PEGylated lipids), and cholesterol, which can be used for nucleic acid delivery. LNPs have a well-defined composition and can be mass-produced on a large scale using ethanol hydration in specific proportions. Their main mechanism of action involves intracellular drug delivery via endocytosis or pinocytosis and binding to cellular LDLRs. LNPs dissociate based on the pH of the intracellular lysosomes, releasing the drug and allowing it to exert its function.

[0059] Antibody: Commonly referred to as "immunoglobulin," it encompasses antibodies with the structural features of natural antibodies and antibody-like molecules with structural features different from natural antibodies but exhibiting specificity for binding to antigen molecules. In this application, the term "antibody" has its broadest meaning, encompassing immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites or domains, and can be used to refer to antigen-binding structural fragments (e.g., antigen-binding fragments) or complexes of one or more antigen-binding fragments (e.g., scFv). Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), type (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0060] ScFv: Single-chain antibody, a synthetic antibody formed by linking the variable regions of the antibody heavy chain and light chain through a short peptide of 15-20 amino acids (sometimes referred to as a linker). It retains the antibody's activity against the antigen, has a small molecular weight, strong penetrability, and weak immunogenicity. The anti-CD3 antibody described in this application is an anti-CD3 ScFv, represented as CD3ScFv in the specific structure. Anti-CD3 ScFv can be derived from publicly disclosed antibodies targeting CD3, such as OKT3 antibody, UCHT1, and SP34.

[0061] Fusion Protein: The fusion protein described in this application refers to the expression product of two recombined genes obtained through DNA recombination technology. In this application, the fusion protein can be a protein complex formed by the above-mentioned antibody binding to one or more other proteins, peptides, or protein functional domains in any manner. In some embodiments, the binding is a direct and / or indirect connection between the antibody and the other one or more proteins, peptides, or protein functional domains. In some embodiments, the carbon (C) terminus of the antibody in the fusion protein is connected to the nitrogen (N) terminus of the other protein, peptide, or protein functional domain. In some embodiments, the nitrogen (N) terminus of the antibody in the fusion protein is connected to the carbon (C) terminus of the other protein, peptide, or protein functional domain. In some embodiments, the nitrogen (N) terminus of the antibody in the fusion protein is connected to the nitrogen (N) terminus of the other protein, peptide, or protein functional domain. In some embodiments, the carbon (C) terminus of the antibody in the fusion protein is connected to the carbon (C) terminus of the other protein, peptide, or protein functional domain. In some embodiments, the antibody in the fusion protein is tandem with one or more other proteins, peptides, or protein functional domains. In some embodiments, the C-terminus and / or N-terminus of the antibody in the fusion protein is linked to at least two or more other proteins, peptides, or protein functional domains, and these other proteins, peptides, or protein functional domains are not linked in tandem. In some embodiments, the other one or more proteins, peptides, or protein functional domains bind the same or different antigens or different antigenic epitopes to the antibody.

[0062] Cationic lipids are a class of biomacromolecules containing cationic hydrophilic groups. Common cationic polymers include polyethyleneimine (PEI), polylysine (PLL), dendritic macromolecules, and cationic lipids. Cationic lipids are widely used due to their good biocompatibility, simple and well-defined structure, reproducibility, and large-scale production capabilities. Cationic lipid components mainly consist of amphiphilic organic small molecules linked by nonpolar hydrophobic tails and polar hydrophilic heads via linkages. The hydrophilic head is generally composed of a single or multiple protonated amino groups, ensuring electrostatic interaction with negatively charged nucleic acids. The hydrophobic structure is usually composed of steroidal compounds such as cholesterol and alkyl chains. Based on the different linkage methods of the hydrophilic head and hydrophobic tail, cationic lipids can be structurally classified into three types: head-tail, geminal, and dumbbell-shaped, with the head-tail structure being the most common. Cationic lipids constitute the most important part of LNP formulations; they are key components of LNPs, facilitating binding to negatively charged cell membranes and serving as a core component for nucleic acid drug delivery. Common cationic lipids include: DLin-MC3-DMA, ALC-0315, SM102, ATX-126, etc.

[0063] Neutral lipids are structural components of LNPs, also known as accessory lipids. Common examples include DOPE (dioleoylphosphatidylethanolamine), DSPC (dispalmitoylphosphatidylcholine), and DOPC (dioleoyllecithin). Accessory lipids often serve as structural lipids in LNP formulations, spontaneously forming a bilayer membrane structure. This enhances the membrane stability of LNPs, preventing their rapid metabolism and excretion in the bloodstream. Simultaneously, they can disrupt endosome stability, improving nucleic acid delivery efficiency.

[0064] PEG (polyethylene glycol) lipids are polymers in which PEG is chemically bonded to the ends of lipids. They are crucial components in lipid peroxides (LNPs), regulating half-life and cellular uptake, influencing LNP population size and dispersibility, preventing LNP aggregation, and ensuring particle stability during preparation and storage. The length and structure of the PEG chain significantly impact its role in LNPs. Longer PEG chains provide better protection but may result in slower PEG removal from the LNP surface, affecting cellular uptake. Shorter PEG chains are easier to remove but may be less stable. In LNP applications, PEG with an average molecular weight of 500-5000 is commonly chosen, with PEG2000 (average molecular weight 2000) being preferred, to ensure LNP stability and effective PEG removal from the LNP. PEGylated proteins: These are proteins that link polyethylene glycol (PEG) polymers to protein molecules. They are single-chain antibodies, peptides, proteins, or fusion proteins expressed through gene recombination that are chemically modified to react with PEG or PEG derivatives (such as DSPE-PEG-MAL, DMG-PEG-MAL, etc.) to form polymers, which are called PEGylated (fusion) proteins. In some embodiments, these proteins can be proteins or fusion proteins that target biological tissues, organs, or specific cells, and can also be called targeted PEGylated proteins.

[0065] Cholesterol is an abundant cell membrane component and is often used as a structural lipid in LNP formulations. Cholesterol is primarily found in the outer shell of LNPs, and modifications to its sterol structure can cause tissue changes on the LNP surface. When bound to high Tm (low gel-liquid crystal phase transition) lipids, cholesterol increases membrane fluidity and narrows the bilayer. In both cases, cholesterol pulls the lipids towards a liquid state.

[0066] Molar ratio: Under specific preparation conditions, cationic lipids and nucleic acids bind effectively via electrostatics. The molar concentration of protonated nitrogen ions in the cationic lipids and the molar concentration of phosphorus atoms in the nucleic acids are in a specific ratio, such as an N / P ratio ranging from 2:1 to 10:1. LNPs are synthesized simultaneously through ethanol hydration in specific molar ratios. Different lipid proportions result in different LNP assemblies, such as a bilayer membrane structure formed on the surface of PEG polymerization, or, when the cationic lipid proportion is high, a bilayer membrane encapsulating cationic lipids and nucleic acid polymer particles. Molar percentage refers to the percentage of an element in a compound. Within a certain range of lipid molar percentages, lipid nanoparticles can be formed and effectively packaged into LNPs.

[0067] SATA protein modification method: also known as SATA-maleimide chemical coupling method, its working principle is as follows: SATA (N-succinimide-S-acetylthioacetate) is used to modify the primary amine group of the target protein to introduce an active group. Then, the reactant reacts with hydroxylamine to give the antibody free thiol groups. Finally, a thioether coupling chemical reaction is used to link the active thiol groups on the target protein to the MAL groups on the MAL-PEG-DSPE lipid to form a PEGylated protein complex.

[0068] TCEP protein modification method: also known as antibody site-directed modification of thiol-maleimide coupling method, its working principle is as follows: disulfide bonds are designed at specific sites of the target protein to be modified, and the disulfide bonds are reduced by TCEP reducing agent, so that the modified protein has free thiol groups. Then, the free thiol groups on the antibody are linked to the MAL groups on the MAL-PEG-DSPE lipid through a thioether coupling chemical reaction to form a PEGylated protein complex.

[0069] Nucleic acids: Biological macromolecules with nucleotides as their basic building blocks, capable of carrying and transmitting genetic information. They are a general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and can be mRNA, small interfering RNA, tRNA, miRNA, aiRNA, ssRNA, short hairpin RNA (shRNA), dsRNA, gene sequences, ssDNA, dsDNA, or plasmids.

[0070] mRNA, also known as messenger RNA, is a type of single-stranded ribonucleic acid transcribed from one strand of DNA as a template. It carries genetic information and can guide protein synthesis.

[0071] siRNA: Interfering RNA, also known as small interfering RNA, short interfering RNA, or silent RNA, is a double-stranded RNA of 20 to 25 nucleotides in length. It can be used to regulate gene expression and participate in some RNAi-related response pathways, such as antiviral mechanisms or changes in chromatin structure.

[0072] Delivery: The delivery described in this application refers to the delivery of the lipid nanoparticles described in this application, which encapsulate nucleic acids such as mRNA or interfering RNA, or other active agents or therapeutic agents, to the whole body or target sites within an organism. This is achieved through the fusion of the lipid nanoparticles with the cell membrane at the delivery site, endocytosis by relevant cells, or degradation by the environment at the delivery site, thereby releasing the nucleic acid molecule at the delivery site, cell, or tissue. In some embodiments, the target mRNA sequence is encapsulated in the lipid nanoparticles described in this application, which can be widely distributed within the organism, thereby delivering the target RNA throughout the body. In some embodiments, the target mRNA sequence is encapsulated in the targeted lipid nanoparticles described in this application, which can be distributed at target sites within the organism depending on the target molecules they carry, thereby delivering the target RNA to the target site or target cells. In some embodiments, the target mRNA can be a nucleic acid of a marker molecule such as GFP described in this application, or a chimeric antigen receptor (CAR) molecule mRNA, or an mRNA sequence of an antigenic polypeptide that can elicit an immune response. In some embodiments, the nucleic acid can also be small interfering RNA. The target molecules may include tRNA, miRNA, aiRNA, ssRNA, short hairpin RNA (shRNA), dsRNA, gene sequences, ssDNA, dsDNA, or plasmids. In some embodiments, the target molecules may be T-cell targeting molecules that recognize CD3, CD4, CD8, CD5, CD7, etc., or B-cell targeting molecules that recognize CD19, etc., or APC cells such as macrophages and DC cells. In some embodiments, the organism refers to mammals, such as mice, monkeys, dogs, cats, and humans. In some embodiments, lipid nanoparticles encapsulating nucleic acids such as mRNA or interfering RNA, or other active agents or therapeutic agents, are provided to the organism via any of the following methods: intravenous, subcutaneous, or intraperitoneal administration.

[0073] Preparation of 0.02M citric acid reagent:

[0074] 1) Weigh 19.213g of citric acid (manufacturer SPECTRUM Lot No.1IE0871 FW192.13) and dissolve it in a certain amount of ultrapure water. After it is completely dissolved, dilute it to 1L in a 1L volumetric flask to obtain a 0.1M / L mother liquor.

[0075] 2) Weigh 25.807g of sodium citrate (manufacturer SPECTRUM Lot No.1HK0873 FW258.07) and dissolve it in a certain amount of ultrapure water. After it is completely dissolved, dilute it to 1L in a 1L volumetric flask to obtain a 0.1M / L mother liquor.

[0076] 3) Prepare 50ml of 0.02M citrate buffer: 40ml ultrapure water + 6.55ml 0.1M / L citrate stock solution + 3.45ml 0.1M / L sodium citrate stock solution.

[0077] The fusion protein used in this application is CD3 ScFv-CD86-G4H12, with the amino acid sequence shown in SEQ ID NO.1. The PEGylated protein ultimately used as the LNP component is CD3 ScFv-CD86-PEG-DSPE or CD3ScFv-CD86-PEG-DMG, where PEG is PEG2000. The PEGylated protein linked to the targeting protein can be synthesized using the SATA protein modification method or the TCEP protein modification method.

[0078] Synthesize PEGylated proteins linked to target proteins using the SATA protein modification method:

[0079] 1) After removing the desalting column preservative solution from the Thermofisher 89889 desalting column, equilibrate the desalting column with 1 ml of 0.1M PB, 0.15M NaCl pH7.4 solution.

[0080] 2) 512 μl of aqueous solution of CD3 ScFv-CD86-G4H12 fusion protein, with a concentration of 3.90 mg / ml.

[0081] 3) Weigh 6.3 mg SATA (sigmaaldrich A9043) and dissolve it in 1364 μl DMSO (Origen CP-70) to prepare a 20 mM (4.62 mg / ml) SATA working solution.

[0082] 4) Add 4.5 μl of 20 mM SATA working solution to 256 μl of fusion protein solution (the molar ratio of fusion protein to SATA is 1:5), and react at room temperature in the dark for 0.5 h.

[0083] 5) Add the solutions obtained in step 4) to the desalting column after equilibration in step 1), centrifuge at 1000g for 2 min, and collect the flow-through liquid. Collect 340 μl of each solution.

[0084] 6) Add 34 μl of 0.5 M hydroxylamine, 25 mM EDTA, 0.1 M PB, and 0.15 M NaCl pH 7.4 solution (1 / 10 of the liquid volume) to the above solution and react at room temperature in the dark for 2 h.

[0085] 7) Weigh 6.0 mg of DSPE-PEG-MAL (WH-0010205 from Huasheng Biotechnology) and dissolve it in 408 μl of DMSO (OrigenCP-70) to prepare a 5 mM (14.7 mg / ml) DSPE-PEG-MAL working solution.

[0086] 8) Add 17.8 μl of 5 mM DSPE-PEG-MAL working solution to each of the solutions in 6), wherein the fusion protein:SATA:DSPE-PEG-MAL = 1:5:5. Incubate at room temperature in the dark for 0.5 h.

[0087] 9) Add 370 μl of the solution obtained in step 8) to the equilibrated desalting column, centrifuge at 1000 g for 2 min, and collect the flow-through. This yields the PEGylated protein CD3 ScFv-CD86-PEG-DSPE.

[0088] In some embodiments, step 4 is: take 1.8 μl of 20 mM SATA working solution and add it to 256 μl of fusion protein solution (the molar ratio of fusion protein to SATA is 1:2), react at room temperature in the dark for 0.5 h, and the fusion protein:SATA:DSPE-PEG-MAL in step 8 is 1:2:5.

[0089] The PEGylated protein CD3ScFv-CD86-PEG-DSPE, linked to a target protein, was synthesized using the TCEP protein modification method.

[0090] 1) After removing the desalting column preservative solution from the Thermofisher 89889 desalting column, equilibrate the desalting column with 1 ml of 0.1M PB, 0.15M NaCl pH7.4 solution.

[0091] 2) 660 μl of aqueous solution of CD3 ScFv-CD86-G4H12 fusion protein, with a concentration of 5.05 mg / ml.

[0092] 3) Add 1.6 μl of 0.5 M TCEP (Aladdin T107252-25g) aqueous solution to 350 μl of fusion protein solution, and react at room temperature in the dark for 1 h. (The molar ratio of fusion protein to TCEP is 1:25).

[0093] 4) Add the solution obtained in step 3) to the equilibrated desalting column, centrifuge at 1000g for 2 min, and collect the flow-through liquid.

[0094] 5) Weigh 13.3 mg of DSPE-PEG-MAL (WH-0010205 from Huasheng Biotechnology) and dissolve it in 905 μl of DMSO (Origen CP-70) to prepare a 5 mM (14.7 mg / ml) DSPE-PEG-MAL working solution.

[0095] 6) Add 63.2 μl of 5 mM DSPE-PEG-MAL working solution to the flow-through solution obtained in step 4), with the fusion protein:TCEP:DSPE-PEG-MAL ratio being 1:25:10. Incubate at room temperature in the dark for 0.5 h.

[0096] 7) Add 400 μl of the solution obtained in step 6) to the equilibrated desalting column, centrifuge at 1000g for 2 min, and collect the flow-through. This yields the PEGylated protein CD3 ScFv-CD86-PEG-DSPE.

[0097] CD3 ScFv-CD86-PEG-DMG was synthesized according to the above scheme, except that DSPE-PEG-MAL was replaced with DMG-PEG-MAL (manufacturer: Aivito, product number 160743-62-4).

[0098] Encapsulation rate detection principle:

[0099] Assessing the encapsulation effect of LNP on mRNA by measuring total mRNA and free mRNA in the formulation is a key indicator of the biological activity of nucleic acid drugs. Encapsulation efficiency is the core indicator of this. Encapsulation efficiency is measured using the RiboGreen fluorescent dye quantitative detection kit (manufacturer: Invitrogen, catalog number R11490). After the fluorescent dye binds to nucleic acid, it excites fluorescence at a specific wavelength, and the signal values ​​are analyzed and compared. First, the concentration of free RNA in the LNP-RNA solution is measured. Then, the LNP structure is destroyed using Triton-100, and the concentration of all RNA in the solution is measured. The fluorescence value of the test sample is assigned according to the concentration of the standard. The encapsulation efficiency is calculated as: (Total RNA amount - Free RNA amount) / Total RNA amount * 100%.

[0100] The specific steps are as follows:

[0101] Preparation of 1X TF Buffer: Stock solution: 1M Tris-HCl (pH 7.4), 0.5M EDTA (pH 8.0), prepare 100mL.

[0102] The 50ul sample to be tested was gradually diluted 100-fold using 1X TF Buffer;

[0103] 0.5% Triton 100 preparation: Add 5 ml of Triton 100 to 995 ml of TE buffer (V / V) and shake gently.

[0104] Total LNP-RNA assay: Add 100 μL of a 100-fold diluted sample to 900 μL of 0.5% Triton 100 (named Sample 1). Cell-free LNP-RNA assay: Add 100 μL of a 100-fold diluted sample to 900 μL of 1*TE buffer (named Sample 2).

[0105] Preparation of standard: Add 10 μL of standard to 990 μL of 1*TE buffer (the original concentration of the standard is 1 mg / mL), and then dilute the above sample with TE buffer to 200, 100, 20, 4, 0 ng / mL;

[0106] Add 100 μL of standard, sample 1, or sample 2 to each well, and repeat for three replicates.

[0107] Fluorescent dye preparation (2000-fold dilution): Add 5 μL of fluorescent dye to 10 mL of TE buffer;

[0108] After adding 100 μL of dye to each well, the OD value was measured in the dark under excitation light of 485 nm and emission light of 520 nm.

[0109] A standard curve was constructed based on the standard curve. Sample 1, representing the OD value of total RNA, was substituted into the standard curve. The value was multiplied by 1000 (the original solution was diluted 1000 times) to obtain the total RNA OD value. Similarly, Sample 2 was recorded as the free RNA OD value. Encapsulation efficiency = (Total RNA OD value - Free RNA OD value) / Total RNA OD value * 100%

[0110] The sequences involved in this invention:

[0111]

[0112]

[0113] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0114] The present invention will be further illustrated below with reference to the embodiments:

[0115] Example 1: Optimizing PEG content alone cannot improve the transduction ability of T cells to LNP particles.

[0116] Ionizable cationic lipids such as SM102, neutral lipids, PEGylated lipids, and cholesterol or other modified cholesterol were dissolved in ethanol at a molar percentage ratio of 50%:10%:1.5%:38.5%. mRNA (target mRNA was GFP RNA, RNA sequence as shown in SEQ ID NO.3) was dissolved in 0.02M citrate buffer at pH 4.0 at a P:N ratio of 1:3 with the ionizable cationic lipids. The targeted PEGylated protein CD3ScFv-CD86-PEG-DSPE was also dissolved in the same citrate buffer. Using a microfluidic system (Aitesen), the flow rate of the alcohol phase was set to a 1:3 ratio of the citrate phase flow rate. LNP solutions were synthesized via microfluidic equipment and then dialyzed using PBS buffer or TFF tangential flow (Replekin). The solution was replaced using the Research2i [KR2i] TFF system, and the LNP was finally stored in 0.01M PBS buffer. mRNA quantification was performed using an RNA nucleic acid quantification kit (probe method, manufacturer: Thermo, catalog number: R11490). The molar percentage of PEGylated lipids was determined, and the specific molar percentage is as follows:

[0117] The current formulation is PEG-DMG 1.5%: SM102 (manufacturer: Senobeng, item number 2089251-47-6) molar percentage 50%, DSPC (manufacturer: Aveta, item number 816-94-4) molar percentage 10%, DMG-PEG-MAL molar percentage 1.5% (manufacturer: Aveta, item number 160743-62-4), and CHOL (manufacturer: Aveta, item number 57-88-5) molar percentage 38.85%.

[0118] PEG-DMG 1.8%: SM102 (manufacturer: Senobeng, item number 2089251-47-6) molar percentage 49.85%, DSPC (manufacturer: Aveta, item number 816-94-4) molar percentage 9.97%, DMG-PEG-MAL molar percentage 1.8% (manufacturer: Aveta, item number 160743-62-4), CHOL (manufacturer: Aveta, item number 57-88-5) molar percentage 38.38%.

[0119] PEG-DMG 2%: SM102 (manufacturer: Senobeng, item number 2089251-47-6) molar percentage 49.75%, DSPC (manufacturer: Aveta, item number 816-94-4) molar percentage 9.94%, DMG-PEG-MAL molar percentage 2% (manufacturer: Aveta, item number 160743-62-4), CHOL (manufacturer: Aveta, item number 57-88-5) molar percentage 38.31%.

[0120] In the targeted LNP synthesis process, the targeted PEGylated protein is prepared by modifying the PEGylated lipid DSPE-PEG-MAL and the target protein (CD3ScFv-CD86-G4H12 in this example) using different protein modification methods, such as the SATA method or the TCEP method.

[0121] The transduction efficiency of the particles was verified. Three batches of T cells were randomly selected, and the positivity rate of GFP transfection in T cells and the average fluorescence intensity were analyzed using a paired t-test. The results are shown in Figure 1. Figure 1A Results of GFP positivity rate in T cells transduced with targeted LNP synthesized in different proportions of PEG-DMG; Figure 1B The results show the molecular weight index (MFI) of GFP expression in T cells transduced with targeted LNPs synthesized at different ratios of PEG-DMG. The results indicate that the transduction efficiency is superior with a PEG-DMG content of 1.5% in the current protocol. The results also demonstrate that simply increasing the molar percentage of PEG-DMG did not improve the transduction efficiency or expression intensity of the targeting particles in T cells.

[0122] Example 2: Multi-component optimization of LNP components to enhance the transduction ability of targeted LNP particles to T cells.

[0123] Table 1: LNP components and proportions

[0124]

[0125] The components were proportioned according to the ratios in Table 1, and the targeted LNP was synthesized according to the scheme in Example 1. The PEGylated protein linked to the target protein was either CD3 ScFv-CD86-PEG-DMG or CD3 ScFv-CD86-PEG-DSPE (synthesized using the SATA protein modification method).

[0126] Simultaneously, different batches of T cells were transfected to verify the transfection efficiency and encapsulation rate of the targeting particles. The results are shown in Figure 2 and... Figure 3 As shown in Figure 2. Figure 2 shows the ability of targeted LNP particles with different formulations to transduce GFP in T cells, where A in Figure 2 represents the GFP positivity rate and B in Figure 2 represents the GFP expression intensity.

[0127] Analysis using paired t-tests showed that the 1.3x and 2x optimized formulations were significantly superior to the current process and the 1.6x formulation. Specifically, the formulations containing SM102 (33.33%), DSPC (13.33%), PEG-DMG (2%), and CHOL (51.33%) exhibited better targeting of LNPs to enhance T cell transduction and also demonstrated superior encapsulation efficiency.

[0128] At the same time, the ratio is increased further on top of the 2x ratio, and the result is as follows: Figure 4As shown, the results indicate that the encapsulation efficiency of the targeted particles did not increase. At the same time, increasing the ratio of cationic lipids in the overall formulation is not conducive to particle stability and therefore detrimental to product development.

[0129] SM102 is a cationic lipid. Other cationic lipids such as DLin-MC3-DMA, ALC-0315, and ATX-126 can replace SM102 to achieve the same effect. DSPC is a neutral lipid. Other neutral lipids such as dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), distearate phosphatidylethanolamine (DSPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylserine (PS) can all achieve the same effect. PEG-DMG is a structural component. Other PEG-DMG derivatives include, but are not limited to, aminated-PEG-DMG, hydroxylated-PEG-DMG, or other active groups. Others include PEG with different chain lengths, such as PEG3000-DMG. Cholesterol is the main membrane component of LNP, and other cholesterol derivatives such as 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride (DC-CHOL) can achieve the same effect.

[0130] In summary, targeted LNPs synthesized from ionizable cationic lipids such as SM102, neutral lipids, PEGylated lipids, and cholesterol or other modified cholesterol in a molar percentage range of 33.33%: 13.33%: 2%: 51.33% exhibit superior transduction efficiency and encapsulation rate.

[0131] Example 3: The LNP component of this application exhibits superior transduction efficiency and encapsulation efficiency in transducing CAR nucleic acids.

[0132] The targeted LNP was prepared according to the steps in Example 1, wherein the target nucleic acid molecule was a CARRNA sequence targeting CD19, and the RNA sequence is shown in SEQ ID NO.2. The transduction ability of the targeted LNP encapsulating the target nucleic acid to target cells was verified by in vitro transduction.

[0133] In vitro transduction was validated according to the above-described in vitro transduction efficiency verification method. T cells from different donors were repeatedly transfected and statistically analyzed. 1 μg of CD19 CAR antibody was added, labeled at 4°C for 30 min, washed with 1 mL of PBS, centrifuged at 350g for 5 min, and resuspended in 100 μL of PBS. CAR positivity and MFI were then detected. The results are shown in Figure 5. Figure 5 shows the transduction efficiency and intensity of the targeted LNP synthesized according to the molar percentage described in this application for CD19 CAR, where A in Figure 5 is the transduction efficiency graph and B is the transduction intensity (MFI) graph.

[0134] The prepared targeted LNPs were encapsulated according to the method in Example 2, such as... Figure 6 The figure shows the encapsulation efficiency of the targeted LNPs containing CD19 CAR nucleic acid synthesized using the molar percentages described in this application. The results indicate that the targeted LNPs containing CD19 CAR nucleic acid synthesized using different molar percentages of the LNP synthesis components described in this application have a better encapsulation efficiency compared to LNPs with conventional ratios.

[0135] In summary, the targeted LNP synthesized from the components described in this application can better transduce target cells after encapsulating the target mRNA, thus achieving targeted delivery capability.

[0136] Example 4: PEGylated lipids synthesized using the TCEP method resulted in better encapsulation of targeted LNPs.

[0137] The formulation components SM102 (33.33%), DSPC (13.33%), PEG-DMG (2%), and CHOL (51.33%) were dissolved in proportion. CD3 ScFv-CD86-PEG-DSPE, synthesized using either the SATA or TCEP protein modification method, was used as the PEGylated protein, and CD19 CAR mRNA was dissolved in 0.02 M citrate buffer at pH 4.0. The remaining components were dissolved in ethanol. CD19 CAR mRNA was dissolved in ethanol at a P:N ratio of 1:3 with ionizable cationic lipids. LNP solutions were synthesized using a microfluidic system (Aitesen) with the ethanol-to-citric acid phase flow rate ratio at 1:3. The solutions were then dialyzed using PBS buffer or TFF tangential flow (Replekin). The solution was replaced using the Research2i [KR2i]TFF system, and the LNP was finally stored in 0.01M PBS buffer. mRNA quantification was performed using an RNA nucleic acid quantification kit (probe method).

[0138] Encapsulation efficiency was determined using an RNA quantification kit (Thermo, catalog number R11490), and the detection method was the same as in Example 3. The results are as follows: Figure 7 As shown, the results indicate that, through t-test and unpaired test, the combination of the LNP formulation described in this application and the targeted PEGylated protein modified by TCEP significantly improves the encapsulation efficiency of the particles, meeting the particle encapsulation rate requirements for industrial-scale production.

[0139] In summary, the targeted LNPs prepared using the ionizable cationic lipids described in this application, such as SM102, neutral lipids, PEGylated lipids, and cholesterol or other modified cholesterol in a molar percentage ratio of 33.33%:13.33%:2%:51.33% in the presence of target nucleic acid molecules can improve the encapsulation efficiency of LNP particles and enhance the targeted transduction efficiency; preferably, the targeting protein is modified using TCEP.

[0140] Example 5: Application of LNP component molar ratio in non-targeted LNP synthesis

[0141] LNP synthesis: Ionizable cationic lipids SM102, DPSC, PEG-DMG (manufacturer: Avitol, catalog number 160743-62-4), and cholesterol (CHOL) were dissolved in ethanol at a molar percentage ratio of 33.33%:13.33%:2%:51.33%. mRNA was dissolved in 0.02M citrate buffer (pH 4.0) at a P:N ratio of 1:3 with the ionizable cationic lipids. The LNP solution was synthesized using a microfluidic device with the alcohol-to-citric acid phase flow rate ratio of 1:3. Solution replacement was performed using PBS buffer dialyze or TFF tangential flow, and the LNP was finally stored in 0.01M PBS buffer. In this embodiment, the PEG-DMG is a PEGylated lipid without any protein components. The PEG-DMG in this embodiment is a pure PEG-DMG compound without modification of the target protein.

[0142] The synthesized LNPs were subjected to encapsulation efficiency and loading capacity assays. The encapsulation efficiency procedure was described above. The loading capacity was determined by the amount of mRNA contained in the LNP. mRNA quantification was performed using an RNA nucleic acid quantification kit (probe method, manufacturer: Thermo, catalog number: R11490). The results are shown in Table 2 below.

[0143] Table 2: LNP Encapsulation Efficiency, Loading Capacity, and Transduction Efficiency Detection

[0144]

[0145] As can be seen from Table 2 above, the LNP synthesis formulation ratio described in this application is not only applicable to the one-step synthesis of lipid components of targeted LNPs, but also applicable to the synthesis of non-targeted conventional LNPs.

[0146] In some embodiments, the LNPs described in this application can also be synthesized first using ionizable cationic lipids such as SM102, neutral lipids, PEG or PEGylated lipids, cholesterol or other modified cholesterol in a molar percentage ratio of 33.33%:13.33%:2%:51.33%, and then a targeting protein or targeting protein-PEG or targeting protein-PEG derivative is added to it, and a targeted LNP is formed by shaking or sonication.

[0147] Example 6: In vivo preparation and functional verification of CAR-T using targeted LNP particles

[0148] Lipid nanoparticles (LNPs) encapsulated with nucleic acids can be administered to organisms via intravenous, subcutaneous, or intraperitoneal administration. This allows for the delivery of targeted nucleic acids with active or therapeutic effects to target sites throughout the body, serving as drugs or drug combinations for treating diseases. To verify the ability of nucleic acid-encapsulated lipid nanoparticles (LNPs) as drugs or drug combinations, a targeted LNP was synthesized using a chimeric antigen receptor (CAR) nucleic acid sequence capable of recognizing CD19-positive tumor cells such as B-cell lymphoma and B-cell leukemia, combined with the targeted LNP component described in this application. This targeted LNP was then intravenously infused into mice bearing B-cell tumors, and the effectiveness of the nucleic acid-encapsulated targeted LNP as a drug or drug combination component was tested. The preparation of the targeted LNP is described in Example 4.

[0149] Human PBMCs (1e7 cells) were injected via tail vein into severely immunodeficient mice, such as NCG mice, to construct a humanized mouse environment that mimics the human immune environment. Twenty-four hours later, 2e6 cells (Nalm6-Luc-GFP cells) were injected via tail vein, and mice were intraperitoneally injected with 15 mg / kg of Luceferase substrate. In vivo imaging was performed using a live imaging system. After in vivo imaging, 40 μg / mouse of a targeted LNP (lnitrogenous protein) encapsulated with nucleic acid, prepared according to the protocol in Example 4, was injected via tail vein. Subsequent in vivo imaging and particle injection were performed approximately once a week, and the efficacy of the particles was evaluated based on changes in fluorescence values. Results are as follows: Figure 8 As shown, the experiment was divided into three groups: the Nalm6 group, the LNP group, and the targeted particle group. Compared with the LNP group, the targeted particle group showed a significant inhibitory effect on tumor proliferation. The results indicate that targeted LNPs encapsulated with nucleic acids can be used as components of drugs or drug combinations for disease treatment, and in this embodiment, they can achieve the goal of treating tumors.

[0150] In some embodiments, the chimeric antigen receptor (CAR) nucleic acid sequence recognizing CD19-positive tumor cells can be replaced with a target molecule nucleic acid sequence recognizing at least one surface-expressed molecule, including solid tumors, hematologic malignancies, and / or tissues. The target molecule includes any one or more of the following combinations: CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEA, CEACAM5, CEACAM6, CEACAM7, Mesothelin, and MUC1. CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), and TAG-72. It can be used to treat tumors such as colon cancer, rectal cancer, small intestine cancer, anal cancer, bile duct cancer, stomach cancer, esophageal cancer, gallbladder cancer, lung cancer, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, kidney cancer, bladder cancer, cancers of the central nervous system, glioblastoma, skin cancer, melanoma, lymphoma, head and neck cancer, multiple myeloma, and leukemia.

[0151] In some embodiments, the chimeric antigen receptor (CAR) nucleic acid sequence that recognizes CD19-positive tumor cells can be replaced with an antigenic peptide nucleic acid sequence that can elicit an immune response in the organism, such as a combination of at least one or more nucleic acid fragments of HPV genome E1-E7, L1-L2, or antigenic peptide nucleic acid sequences or sequences of viruses or bacteria related to diseases such as hepatitis A, hepatitis B, poliomyelitis, influenza, and pneumonia, thereby obtaining a vaccine drug for treating the above-mentioned diseases or as a component of a drug combination.

[0152] In some embodiments, the chimeric antigen receptor (CAR) nucleic acid sequence that recognizes CD19-positive tumor cells can be replaced with active agents or therapeutic agents related to ophthalmic diseases, metabolic diseases, hyperlipidemia, hepatitis B, pseudohypertrophic muscular dystrophy, non-alcoholic fatty liver disease, heart disease, amyotrophic lateral sclerosis, cystic fibrosis, etc., such as siRNA, miRNA, ASO, small activating RNA (saRNA), aptamers, transfer RNA (tRNA) fragments, etc., as drugs or drug combinations to treat ophthalmic diseases, metabolic diseases, hyperlipidemia, hepatitis B, pseudohypertrophic muscular dystrophy, non-alcoholic fatty liver disease, heart disease, amyotrophic lateral sclerosis, cystic fibrosis, etc.

[0153] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Lipid nanoparticles, whose packaging materials include: The ingredients include cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or their derivatives; wherein the molar percentages of the cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or their derivatives are (33%–50%): (10%–13.33%): (1.5%–2%): (38.5%–51.33%).

2. The lipid nanoparticles according to claim 1, characterized in that, The cationic lipids are selected from at least one of SM102, DLin-MC3-DMA, ALC-0315 or ATX-126; The neutral lipids are selected from at least one of DSPC, DOPE, DPPC, PC, PE, PG, PI or PS; The PEG has an average molecular weight of 500 to 5000. The PEGylated lipid is at least one of ALC-0159, DMG-PEG, PEG2000-C-DMG, DSPE-PEG, DPPE-MPEG(2000), DSPE-PEG-Amine, DSPE-PEG-NHS,MW 2000, DSPE-PEG-Maleimide, DMG-PEG-MAL, DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-MAL, DMG-PEG2000-MAL, aminated-PEG-DMG, or hydroxylated-PEG-DMG.

3. The lipid nanoparticles according to claim 1 or 2, characterized in that, The packaging material is composed of SM102, DSPC, PEGylated lipids and cholesterol, wherein the molar percentages of SM102, DSPC, PEGylated lipids and cholesterol are 33.33%: 13.33%: 2%: 51.33%.

4. The lipid nanoparticles according to any one of claims 1 to 3, characterized in that, The packaging material also includes PEGylated proteins; the PEGylated proteins include the protein shown in SEQ ID NO:1 and the PEGylated lipids.

5. The lipid nanoparticles according to any one of claims 1 to 4, characterized in that, It also includes nucleic acids encapsulated within packaging materials.

6. The lipid nanoparticles according to claim 5, characterized in that, The nucleic acid includes at least one of mRNA, siRNA, miRRNA, DNA, dsRNA, sDNA, small interfering RNA, miRNA, aiRNA, shRNA, tRNA, ssDNA, dsDNA, and plasmid.

7. A method for preparing lipid nanoparticles, comprising: The lipid nanoparticle packaging material according to any one of claims 1 to 4 is dissolved in ethanol to obtain an alcohol phase; The PEG-conjugated fusion protein and the nucleic acid according to any one of claims 5-6 were dissolved in citrate buffer to obtain the citrate phase; the alcohol phase and the citrate phase were then prepared into lipid nanoparticles by microfluidic control.

8. The preparation method according to claim 7, characterized in that, The citrate buffer solution has a pH of 4.0 and a concentration of 0.02M. In the microfluidic step, the ratio of alcohol phase flow rate to citric acid phase flow rate is 1:

3.

9. The method according to claim 7, characterized in that, The PEG-coupled fusion protein is formed by a targeting protein and PEGylated lipids.

10. Use of the lipid nanoparticles according to any one of claims 1 to 6 in the preparation of a product for in vivo delivery of nucleic acids to be administered to mammalian subjects.

11. A nucleic acid delivery reagent comprising the lipid nanoparticles and buffer solution as described in any one of claims 1 to 6.

12. A pharmaceutical composition comprising the lipid nanoparticles according to claims 1 to 6.

13. Use of the lipid nanoparticles of claim 5 or 6 in the preparation of products that introduce nucleic acids into cells.

14. Use of the lipid nanoparticles of claim 5 or 6 in the preparation of a medicament for treating a disease or functional disorder in a mammalian subject in need of treatment.

15. The application according to claim 14, characterized in that, Diseases include at least one of the following: infectious diseases, metabolic diseases, ophthalmic diseases, hyperlipidemia, hepatitis B, pseudohypertrophic muscular dystrophy, non-alcoholic fatty liver disease, heart disease, amyotrophic lateral sclerosis, cystic fibrosis, genetic defects, or tumors.

16. A method for nucleic acid transduction, comprising lipid nanoparticles as described in any one of claims 1-6, wherein the lipid nanoparticles are incubated after contact with cells to transduce target nucleic acids of the target cells.

17. The transduction method according to claim 16, characterized in that, The target cells are any one or a combination of T lymphocytes, B cells, NK cells, macrophages, and DC cells.

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