Lipid nano-composition based on quinoline and capable of ionizing lipid and application of lipid nano-composition

Abstract

The invention relates to the technical field of biology, in particular to a lipid nano-composition based on quinoline and capable of ionizing lipid and application of the lipid nano-composition. The invention discloses a nano composition which comprises ionizable lipid, neutral phospholipid, cholesterol and PEG (polyethylene glycol) lipid, and the structure of the ionizable lipid is as shown in formula I in the specification. Experiments prove that the nano composition disclosed by the invention has good safety and nucleic acid delivery effect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a lipid nanocomposition based on quinoline ionizable lipids and its applications. Background Technology

[0002] mRNA is a single-stranded ribonucleic acid transcribed from a single DNA strand. It carries the coding information for protein synthesis and can be further transcribed and processed in the cytoplasm to express pathogens or tumor-associated antigens, thereby inducing an immune response and achieving disease treatment.

[0003] However, nucleic acid drugs still face several challenges in clinical application. First, nucleic acid drugs are easily degraded by nucleases in vivo, exhibiting poor stability, and their negative charge makes them difficult to effectively penetrate cell membranes, limiting their entry into cells to exert their effects. Second, nucleic acid drugs may trigger off-target effects and immune responses, leading to potential toxic side effects. Therefore, there is an urgent need to develop effective delivery vectors to protect nucleic acid drugs from degradation, improve their intracellular delivery efficiency, and reduce toxic side effects.

[0004] Existing delivery vectors for nucleic acid drugs are mainly divided into viral vectors and non-viral vectors. Although viral vectors have high delivery efficiency, their inherent immunogenicity poses safety risks. In contrast, lipid nanoparticles (LNPs) in non-viral vectors are widely used for nucleic acid drug delivery. LNPs typically consist of four components: ionizable lipids, phospholipids, cholesterol, and polyethylene glycol lipids.

[0005] Liposomes (LNPs) are key components in nucleic acid drug delivery. The structure of an ionizable lipid typically consists of three parts: a positively charged head, a hydrophobic tail, and an intermediate linker. The positively charged head binds to negatively charged nucleic acid molecules, facilitating efficient encapsulation and intracellular delivery. The intermediate linker, which connects the head and tail, typically influences the lipid's stability and degradability. The hydrophobic tail, usually composed of one or more fatty acid chains, determines the lipid's physicochemical properties.

[0006] Currently, several ionizable lipids have been approved by the FDA for the preparation of LNPs, including DLin-MC3-DMA (for...). ), SM-102 (for ) and ALC-0315 (for However, there is still a lack of ionizable cationic lipids that are effective for the efficient delivery of nucleic acid drugs such as mRNA. Further efforts are needed to expand the selection of ionizable lipid compounds to improve their delivery efficiency and safety. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a lipid nanocomposition based on quinoline ionizable lipids and its application.

[0008] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0009] Technical Topic 1

[0010] A lipid nanocomposition comprising an ionizable lipid component, neutral phospholipids, cholesterol, and PEG lipids, wherein the structure of the ionizable lipid component is shown in Formula I:

[0011]

[0012] R1 is H, -C(O)-C1-C40 alkyl, -C(O)-C2-C40 alkenyl, -C(O)-C2-C40 alkynyl, C1-C40 alkyl, C2-C40 alkenyl or C2-C40 alkynyl;

[0013] R2 is H, C1-C40 alkyl, -C(O)-C1-C40 alkyl, C2-C40 alkenyl, C2-C40 ynyl, -C(O)-C2-C40 alkenyl or -C(O)-C2-C40 ynyl;

[0014] Ra1 and Ra2 are H, F, Cl, Br, I;

[0015] Rb1 and Rb2 are H or C1-C8 alkyl groups;

[0016] m1 and m2 can be 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0017] As a further improvement of the present invention, the structure of the ionizable lipid component is shown in Formula II:

[0018]

[0019] R1 is a C1-C30 straight-chain alkyl, a C2-C30 straight-chain alkenyl, or a C2-C30 straight-chain alkynyl.

[0020] m1 and m2 can be 1, 2, 3 or 4.

[0021] As a further improvement of the present invention, R1 is n is an integer between 6 and 24.

[0022] As a further improvement of the present invention, n is an integer from 11 to 17.

[0023] As a further improvement of the present invention, the cationic lipid structure is specifically as follows:

[0024]

[0025]

[0026] As a further improvement of the present invention, the neutral phospholipid is selected from one or more of the following compounds: distearylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin, phosphatidic acid, and sphingomyelin;

[0027] The PEG lipid is selected from one or more of the following compounds: PEG-modified distearylphosphatidylethanolamine, PEG-modified dimyristoylglycerol, PEG-modified dipalmitoylphosphatidylethanolamine, PEG-modified dimyristoylphosphatidylethanolamine, PEG-modified dilauroylphosphatidylethanolamine, PEG-modified ceramide, and mPEG-modified bis(tetradecyl)acetamide; more preferably, the molecular weight of the PEG or mPEG is 1000-5000 Da, and most preferably, DMG-PEG2000.

[0028] As a further improvement of the present invention, the nanocomposition further comprises a therapeutic agent and / or a preventive agent, preferably, the therapeutic agent and / or preventive agent is a nucleic acid;

[0029] Preferably, the nucleic acid is selected from one or more of siRNA, aiRNA, miRNA, dsRNA, shRNA, snRNA, tRNA, rRNA, circRNA, saRNA, antisense oligonucleotide, lncRNA, ssDNA, mRNA, nucleic acid aptamers or ribozymes, with mRNA being the most preferred.

[0030] Technical Theme Two

[0031] A pharmaceutical composition comprising the lipid nanocomposition described in any one of the technical subjects and a pharmaceutically acceptable carrier.

[0032] Technical Theme 3

[0033] The application of the lipid nanocomposition described in any one of Technical Subject One in nucleic acid drug delivery.

[0034] The beneficial effects of adopting the above technical solution are as follows:

[0035] The nanocomposition disclosed in this application has excellent delivery performance, small average particle size, high encapsulation efficiency, and high expression, and has broad application prospects in the field of drug delivery. Attached Figure Description

[0036] Figure 1This is a diagram showing the safety evaluation of DC 2.4 cells for formulations 3, 4, 5, and 9.

[0037] Figure 2 These are fluorescence micrographs of DC 2.4 cell uptake results for formulations 1 and 8.

[0038] Figure 3 The graph shows the DC 2.4 cell uptake results for formulations 5 and 9.

[0039] Figure 4 The results of B16F10-OVA administration to tumor-bearing mice are shown in Figure A, which shows the tumor growth inhibition in the intravenous injection group; Figure C shows the tumor growth inhibition in the intramuscular injection group; Figure B shows the weight change in the intravenous injection group; Figure D shows the weight change in the intramuscular injection group; Figure E shows the survival analysis in the intravenous injection group; and Figure F shows the survival analysis in the intramuscular injection group (n=6).

[0040] Figure 5 Figure A shows the concentration of OVA-specific IgG antibodies in B16F10-OVA-bearing mice administered the drug in the examples. Figure B shows the intravenous injection group and the intramuscular injection group (*P<0.05, **P<0.01, ***P<0.001).

[0041] Figure 6 The figures show the T cell composition in the spleen and inguinal lymph nodes of B16F10-OVA tumor-bearing mice administered intravenously in the examples. A shows the CD8 and CD4 cell content in the spleen; B shows the CD8 and CD4 cell content in the inguinal lymph nodes (*P<0.05, **P<0.01).

[0042] Figure 7 The figures show the T cell composition in the spleen and inguinal lymph nodes of B16F10-OVA tumor-bearing mice administered via intramuscular injection in the examples. Figure A shows the CD8 and CD4 cell content in the spleen; Figure B shows the CD8 and CD4 cell content in the inguinal lymph nodes (*P<0.05, **P<0.01, ***P<0.001).

[0043] Figure 8 The CD8+ in the tumors of B16F10-OVA-bearing mice in the examples is... + IFNγ + The percentage of M2 / M1 macrophages, and the ratio of M2 to M1 macrophages, where A represents the CD8+ of the intravenous injection group. + IFNγ + Percentage, B represents CD8 in the intramuscular injection group + IFNγ +Percentage, C is the ratio of M2 to M1 macrophages in the intravenous injection group, and D is the ratio of M2 to M1 macrophages in the intramuscular injection group (*P<0.05, **P<0.01, ***P<0.001);

[0044] Figure 9 The images show the H&E staining of major organs in B16F10-OVA tumor-bearing mice injected intravenously. From left to right, these are the heart, liver, spleen, lung, and kidney.

[0045] Figure 10 The images show the H&E staining of major organs in B16F10-OVA tumor-bearing mice injected intramuscularly. From left to right, these are the heart, liver, spleen, lung, and kidney.

[0046] Figure 11 The image shows the TUNEL staining results of tumor tissue from B16F10-OVA tumor-bearing mice, where IV represents the intravenous injection group and IM represents the intramuscular injection group. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of the present invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0048] Terms and Definitions

[0049] It should be noted that the structural and chemical formula descriptions in the embodiments or implementations of this invention are intended to cover all alternative, modified, and equivalent technical solutions, all of which should be considered within the scope of this invention. Those skilled in the art should understand that many similar or equivalent methods and materials can be used to implement this invention; therefore, this invention is not limited to the specific methods and materials described. In the event of any discrepancies or contradictions between the cited documents, patents, or similar materials and this application (including but not limited to terminology definitions, terminology application, and described techniques), the content of this invention shall prevail.

[0050] In this document, the minimum and maximum carbon atom content in hydrocarbon groups are indicated by prefixes, for example, the prefix Ca-Cb indicates the presence of "a" to "b" carbon atoms. Exemplarily, "C1-Cn" refers to a straight or branched saturated / unsaturated carbon chain containing 1, 2, 3, 4, 5, ..., or n carbon atoms; further understood, "C1-Cn" should be interpreted as any subrange included, such as C1-C40, C2-C40, C6-C30, C1-C10, C1-C8, C1-C6, C1-C5, C1-C3, etc.

[0051] As used herein, “alkyl” means a fully saturated (without double or triple bonds) straight-chain or branched hydrocarbon chain group. An alkyl group can have 1 to 40 carbon atoms (wherever it appears herein, a numerical range of “1 to 40” refers to each integer within the given range; for example, “1 to 40 carbon atoms” means that an alkyl group can consist of 1, 2, 3, etc., up to and including 40 carbon atoms, although this definition also covers the occurrence of the term “alkyl” without specifying a numerical range). An alkyl group can also be a medium-sized alkyl group having 1 to 10 carbon atoms, such as “C1-6”. An alkyl group can also be a lower alkyl group having 1 to 4 carbon atoms. The alkyl group of a compound can be specified as “C1-C4 alkyl”, “C1-4 alkyl”, or similar names. By way of example only, "C1-C4 alkyl" or "C1-4 alkyl" indicates that there are one to four carbon atoms in the alkyl chain, meaning the alkyl chain is selected from the group consisting of: methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, and hexyl.

[0052] As used herein, “alkenyl” refers to an alkyl group containing one or more double bonds in a straight-chain or branched hydrocarbon chain. If more than one double bond is present, the double bonds may be concatenated or unconcatenated. Alkenyl groups can have 2 to 40 carbon atoms (wherever it appears herein, a numerical range of “2 to 40” refers to each integer within the given range; for example, “2 to 40 carbon atoms” means that an alkenyl group can consist of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, etc., up to and including 40 carbon atoms). In alkenyl groups, stereochemically unspecified C=C double bonds (e.g., -CH=CHCH3) can be (E)- or (Z)- double bonds.

[0053] As used herein, “alkynyl” refers to an alkyl group containing one or more triple bonds in a straight or branched hydrocarbon chain. An alkynyl group can have 2 to 40 carbon atoms (whenever it appears herein, for example, “2 to 40” means every integer within a given range; for example, “2 to 40 carbon atoms” means that an alkynyl group can consist of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, etc., up to and including 40 carbon atoms).

[0054] The term "treatment" generally refers to the use of medication to improve, alleviate, or cure a disease state, meaning partial or complete relief, improvement, delay of its onset, inhibition of its progression, reduction of its severity, and / or reduction of the incidence of one or more of its symptoms or features. It can also refer to a specific infection, disease, symptom, and / or condition. For example, "treatment" of cancer can refer to inhibiting tumor survival, growth, and / or spread. To reduce risk, treatment can be administered to subjects who do not exhibit disease, symptom, and / or condition and / or to subjects who only exhibit early signs of disease, symptom, and / or condition. It can also refer to the pathological development of a disease, symptom, and / or condition.

[0055] The terms "therapeutic agent" or "preventive agent" refer to any pharmaceutical agent that, when administered to a subject, has a therapeutic, diagnostic, and / or preventive effect and / or induces the desired biological and / or pharmacological action. Therapeutic agents are also referred to as "active agents" or "active components." In this invention, therapeutic agents can be nucleic acids, small molecule compounds, macromolecule compounds, peptides, immunomodulators, antigens or fragments thereof, vaccines, antitumor drugs, antibiotics, or mixtures thereof.

[0056] In this invention, the term "pharmaceutical acceptable" means that a substance or composition, when exposed to mammals, will not produce unreasonable toxicity, irritation, allergic reactions, or other adverse side effects.

[0057] In this invention, the term "pharmaceutically acceptable salt" refers to the organic and inorganic salts of the compounds of this invention. These salts not only retain the pharmacological activity of the compounds but also possess favorable pharmaceutical properties, such as enhanced solubility, stability, or bioavailability. These salts include, but are not limited to, organic acid salts such as acetates, citrates, fumarates, maleates, oxalates, malates, citrates, succinates, tartrates, lactates, camphor sulfonates, benzene sulfonates, p-toluene sulfonates, methanesulfonates, trifluoroacetates, trifluoromethanesulfonates, etc.; and inorganic acid salts such as hydrohalides, sulfates, phosphates, nitrates, etc. Furthermore, the active pharmaceutical ingredient can also form salts with amino acids such as glutamic acid or aspartic acid, i.e., glutamate or aspartate.

[0058] In this invention, the compounds and their stereoisomers described are all within the scope of protection, including but not limited to enantiomers, diastereomers, and cis-trans isomers. Therefore, the compounds protected by this invention include not only all possible single stereoisomers, but also their optically active mixtures and racemates. Furthermore, those skilled in the art will understand that different stereoisomers may exhibit significant differences in biological activity, pharmacodynamics, and toxicity; therefore, selecting specific stereoisomers or combinations thereof may optimize therapeutic effects. Stereoisomers can be obtained by known chemical or physical methods, such as chiral catalysis, chiral synthesis, or chiral resolution by chromatographic or chemical methods. This invention also includes stereoisomers obtained by these methods and their pharmaceutically acceptable salts.

[0059] The term "ionizable lipid" as used in this invention refers to lipids that exhibit a positive charge under specific pH conditions and can bind to negatively charged nucleic acid molecules through electrostatic interactions.

[0060] As used herein, a “lipid nanocomposition” is a composition comprising one or more lipids. Lipid nanocompositions typically have particle sizes on the order of micrometers or smaller and may comprise a lipid bilayer. Lipid nanocompositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipid complexes. For example, a lipid nanocomposition may be a liposome having a lipid bilayer with a diameter of 500 nm or smaller.

[0061] As used herein, “PEG lipid” or “PEGylated lipid” refers to lipids containing polyethylene glycol.

[0062] As used herein, "phospholipid" is a lipid comprising a phosphate ester moiety and one or more carbon chains, such as unsaturated fatty acid chains. Phospholipids may contain one or more (e.g., double or triple) bonds (e.g., one or more unsaturated bonds). Certain phospholipids can facilitate fusion with membranes. For example, cationic phospholipids can interact with one or more negatively charged phospholipids in a membrane (e.g., a cell membrane or intracellular membrane). Fusion of phospholipids with membranes can allow one or more elements of a lipid-containing composition to cross the membrane, thereby allowing, for example, the delivery of one or more elements to the cell.

[0063] As used herein, “encapsulation ratio” refers to the amount of therapeutic and / or preventive agents that are part of the lipid nanocomposition, relative to the total amount of therapeutic or preventive agents used in the preparation of the lipid nanocomposition. For example, if 97 mg of therapeutic and / or preventive agents are encapsulated in the lipid nanocomposition out of a total of 100 mg initially provided to the composition, the encapsulation ratio can be 97%. As used herein, “encapsulation” can mean complete, substantial, or partial encapsulation, closure, enclosure, or sealing.

[0064] As used herein, in the context of lipid nanocomposites, “particle size” or “average particle size” refers to the average diameter of the lipid nanocomposites.

[0065] The term "nucleic acid" refers to biological macromolecules composed of nucleotide units, primarily of two types: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). They play crucial roles in the storage, transmission, and expression of genetic information. Nucleic acids include: messenger RNA (mRNA), small interfering RNA (siRNA), double-stranded RNA (dsRNA), small nuclear RNA (snRNA), transfer RNA (tRNA), ribosomes (rRNA), circular RNA (circRNA), self-amplifying RNA (saRNA), antisense oligonucleotides (ASO), microRNA (miRNA), long non-coding RNA (lncRNA), small hairpin RNA (shRNA), single-stranded DNA (ssDNA), nucleic acid aptamers, and ribozymes. In some therapeutic regimens, the RNA used is mRNA.

[0066] In some implementations, the therapeutic or preventative agent is mRNA, and OVA (ovalbumin) is used as a model antigen to stimulate a specific immune response against melanoma.

[0067] In one embodiment, the lipid nanoparticles are administered via intravenous injection.

[0068] In one embodiment, the lipid nanoparticles are administered via intramuscular injection.

[0069] The term "targeting" generally refers to the ability of a delivery system to deliver drugs to a predetermined target area in order to enhance the therapeutic or diagnostic effect.

[0070] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., rather than events that occur within a living organism (e.g., an animal, plant, or microorganism).

[0071] As used herein, the term "in vivo" refers to events that occur within an organism (such as an animal, plant, or microorganism, or its cells or tissues).

[0072] As used herein, the term "ex vivo" refers to an event that occurs outside an organism (e.g., an animal, plant, or microorganism, or its cells or tissues). Ex vivo events can occur in environments with minimal alteration from the natural (e.g., internal) environment.

[0073] In the embodiments of this patent, all reagents and instruments used in the experiments are conventional items in the art, and their functions and uses are known. The technical means involved are conventional operating methods commonly mastered by those skilled in the art, and experiments without special instructions are all conducted under normal temperature and pressure conditions. For operating steps without specified special requirements, the standard procedures in the product instructions of the reagents or instruments are followed to ensure the repeatability and reliability of the results.

[0074] The reagents used in this application are from the following sources:

[0075] Bright-Glo reagent was purchased from Prometheus, Inc., USA, catalog number E2610;

[0076] The TUNEL apoptosis detection kit was purchased from Wuhan Saiweier Biotechnology Co., Ltd., product number G1507;

[0077] All the following formulations were purchased from Thermo Fisher Scientific (China) Co., Ltd. (eBioscience);

[0078] Ribogreen reagent kit, catalog number R11491;

[0079] Anti-Mouse CD4-FITC, part number 11-0042;

[0080] Anti-Mouse CD3-PerCP-eFluor TM 710, item number 46-0032;

[0081] Anti-Mouse CD45-PE-Cyanine7, item number 25-0451;

[0082] Anti-Mouse CD8-APC, part number MCD0805;

[0083] Anti-Mouse IFNγ-PE, part number 12-7311;

[0084] Anti-Mouse CD11b-FITC, part number 11-0112;

[0085] Anti-Mouse F4\80-PE, part number 12-4801;

[0086] Anti-Mouse CD163-APC, part number 17-1631;

[0087] Anti-Mouse CD86-PerCP-eFluor TM 710, item number 46-0862.

[0088] The synthetic routes of the compounds in this application are shown below:

[0089]

[0090] Synthetic route for compound BAQ (Example 1):

[0091]

[0092] Compound 1 (1.2 g, 6 mmol) was added to a 10 mL reaction flask at room temperature and kept at 80 °C for 2 hours without stirring. Then, diethylenetriamine (0.22 mL, 2 mmol) was added. The reaction mixture was stirred at 130 °C for 6 hours. TLC analysis showed that the reaction was complete. The reaction mixture was cooled to room temperature, filtered, and dried under vacuum to obtain a white solid BAQ compound (470 mg, 55%) that could be used directly in the next reaction. 1 H NMR(600MHz,Chloroform-d)δ8.43(d,J=4.4Hz,1H),7.85–7.78(m,2H),7.30(dd,J=8.7,1.9Hz,1H ), 6.57 (d, J = 4.4Hz, 1H), 6.33 (t, J = 4.9Hz, 1H), 3.47 (dt, J = 4.9, 4.1Hz, 2H), 2.88 (q, J = 4.3Hz, 2H). 13 C NMR (151MHz, CDCl3) δ150.01,149.26,149.05,134.65,127.64,126.78,125.56,119.42,101.58,48.88,43.64.ESI-HRMS:

[0093] C 22 H 22 Cl2N5 + m / z[M+H] + The calculated value is 426.1247, and the measured value is 426.1243.

[0094] Preparation Example 2: Preparation of CQN-A

[0095] Compound BAQ (426 mg, 1.0 mmol) was added to a 25 mL reaction flask, followed by 3 mL of anhydrous methanol and 1 mL of anhydrous dichloromethane. Dodecane (2 mmol) and acetic acid (20 μL) were added at 0 °C, and the mixture was stirred for 20 minutes. Then, sodium cyanoborohydride (126 mg, 2 mmol) was added. The reaction mixture was brought to room temperature and stirred for 12 hours. TLC analysis showed the reaction was complete. The reaction solution was concentrated under reduced pressure, and then extracted with deionized water (20 mL) and dichloromethane (20 mL × 2). The organic layer was separated, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and the crude product obtained was concentrated by vacuum distillation. The crude product was purified by silica gel column chromatography using an eluent containing 0.1% triethylamine (dichloromethane:methanol = 30:1 V / V) to give a pale yellow oily compound CQN-A (320 mg, 54%). 1 H NMR (600MHz, Methanol-d4) δ8.17(d,J=5.6Hz,2H),7.57(d,J=2.3Hz,2H),7.47(d,J=8.9Hz,2H),6.87(dd,J=8.8,2.1Hz,2H),6.34(d,J=5 .6Hz,2H),3.30(t,J=5.9Hz,4H),2.79(t,J=5.9Hz,4H),2.55(t,J=7.0Hz,2H),1.44(q,J=7.3Hz,2H),1.18(s,20H),0.82(t,J=7.1Hz,3H). 13 C NMR(151MHz,MeOD)δ152.52,151.67,148.68,136.42,127.19,125.96,123.34,118.25,99.78,55.58,5 3.18,41.73,33.07,30.95,30.88,30.81,30.80,30.77,30.52,28.81,28.61,23.76,14.50ESI-HRMS:C 34 H 46 Cl2N5 + m / z[M+H] + The calculated value is 594.3125, found 594.3127.

[0096] Preparation Example 3: Preparation of CQN-B

[0097] Compound BAQ (426 mg, 1.0 mmol) was added to a 25 mL reaction flask, followed by 3 mL of anhydrous methanol and 1 mL of anhydrous dichloromethane. Tetradecanoal (2 mmol) and acetic acid (20 μL) were added at 0 °C, and the mixture was stirred for 20 minutes. Then, sodium cyanoborohydride (126 mg, 2 mmol) was added. The reaction mixture was brought to room temperature and stirred for 12 hours. TLC analysis showed the reaction was complete. The reaction solution was concentrated under reduced pressure, and then extracted with deionized water (20 mL) and dichloromethane (20 mL × 2). The organic layer was separated, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and the crude product was concentrated by vacuum distillation. The crude product was purified by silica gel column chromatography using an eluent containing 0.1% triethylamine (dichloromethane:methanol = 30:1 V / V) to give a pale yellow oily compound CQN-B (295 mg, 47%). 1 H NMR(600MHz, Methanol-d4)δ8.24(t,J=5.3Hz,2H),7.56(dd,J=8.9,3.4Hz,2H),7.00–6.94(m,2H),6.46(dd,J=8.8,5.4Hz,2H),3 .41(q,J=5.7Hz,4H),2.89(d,J=5.9Hz,2H),2.66(q,J=6.7Hz,4H),1.54(q,J=7.4Hz,2H),1.29–1.22(m,40H),0.92–0.86(m,3H). 13 C NMR(151MHz,MeOD)δ152.88,151.15,148.08,136.75,126.77,126.73,126.14,123.42,118.13,99.81,60.15,55.65, 53.15,41.83,33.08,30.94,30.88,30.84,30.79,30.76,30.49,28.83,28.58,23.74,14.47,14.45,8.47.ESI-HRMS:C 36 H 50 Cl2N5 + m / z[M+H] + The calculated value is 622.3438, and the found value is 622.3440.

[0098] Preparation Example 4: Preparation of CQN-C

[0099] Compound BAQ (426 mg, 1.0 mmol) was added to a 25 mL reaction flask, followed by 3 mL of anhydrous methanol and 1 mL of anhydrous dichloromethane. Hexadecane (2 mmol) and acetic acid (20 μL) were added at 0 °C, and the mixture was stirred for 20 minutes. Then, sodium cyanoborohydride (126 mg, 2 mmol) was added. The reaction mixture was brought to room temperature and stirred for 12 hours. TLC analysis showed the reaction was complete. The reaction solution was concentrated under reduced pressure, and then extracted with deionized water (20 mL) and dichloromethane (20 mL × 2). The organic layer was separated, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and the crude product was concentrated by vacuum distillation. The crude product was purified by silica gel column chromatography using an eluent containing 0.1% triethylamine (dichloromethane:methanol = 30:1 V / V) to give a pale yellow oily compound CQN-C (280 mg, 43%). 1 H NMR (600MHz, Methanol-d4) δ8.24(d,J=6.0Hz,2H),7.62(d,J=2.2Hz,2H),7.04(dd,J=8.9,2.2Hz,2H),6.53(d,J=6. 0Hz, 2H), 3.46 (t, J=5.7Hz, 4H), 2.92 (dd, J=6.6, 4.9Hz, 4H), 2.71 (t, J=7.2Hz, 2H), 1.29 (s, 26H), 0.95–0.86 (m, 3H). 13 CNMR(151MHz,MeOD)δ152.88,151.15,148.08,136.75,126.77,126.73,126.14,123.42,118.13,99.81,60.15,55.65, 53.15,41.83,33.08,30.94,30.88,30.84,30.79,30.76,30.49,28.83,28.58,23.74,14.47,14.45,8.47.ESI-HRMS:C 38 H 54 Cl2N5 + m / z[M+H] + The calculated value is 650.3751, found 650.3763.

[0100] Preparation Example 5: Preparation of CQN-D

[0101] Compound BAQ (426 mg, 1.0 mmol) was added to a 25 mL reaction flask, followed by 3 mL of anhydrous methanol and 1 mL of anhydrous dichloromethane. Hexadecane (2 mmol) and acetic acid (20 μL) were added at 0 °C, and the mixture was stirred for 20 minutes. Then, sodium cyanoborohydride (126 mg, 2 mmol) was added. The reaction mixture was brought to room temperature and stirred for 12 hours. TLC analysis showed the reaction was complete. The reaction solution was concentrated under reduced pressure, and then extracted with deionized water (20 mL) and dichloromethane (20 mL × 2). The organic layer was separated, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and the crude product was concentrated by vacuum distillation. The crude product was purified by silica gel column chromatography using an eluent containing 0.1% triethylamine (dichloromethane:methanol = 30:1 V / V) to give a pale yellow oily compound CQN-D (290 mg, 43%). 1 H NMR (600MHz, Methanol-d4) δ8.27(d,J=5.5Hz,2H),7.66(d,J=2.2Hz,2H),7.55(d,J=9.0Hz,2H),6.94(dd,J=8.9,2.2Hz,2H),6.46(d,J=5 .6Hz,2H),3.41(t,J=5.8Hz,4H),2.90(t,J=5.8Hz,4H),2.65(t,J=6.9Hz,2H),1.53(p,J=7.2Hz,2H),1.29(s,32H),0.90(d,J=7.0Hz,3H). 13 C NMR(151MHz,MeOD)δ152.37,152.24,149.34,136.23,127.64,125.87,123.28,118.45,99.84,55.55,53.2 9,41.69,33.07,30.90,30.84,30.81,30.78,30.74,30.68,30.47,28.80,28.72,23.73,14.44.ESI-HRMS:C 40 H 58 Cl2N5 + m / z[M+H] + The calculated value is 678.4064, found 678.4060.

[0102] Example 1: Preparation of lipid nanoparticles by microfluidic injection

[0103] In this embodiment, the neutral lipid in the lipid nanoparticles is distearate phosphatidylcholine (DSPC), and the PEG lipid is DMG-PEG2000.

[0104] Add 6 μL of EGFP mRNA (1 mg / mL), Luc mRNA (1 mg / mL), or OVA mRNA (1 mg / mL) to 1072 μL of citrate buffer solution at pH 4.0 to prepare an aqueous phase solution. Prepare a 3.24 mL organic phase mixture solution using ethanol as the solvent according to the molar ratio of MC3:CHOL:DSPC:DMG-PEG2000 = 50:38.5:10:1.5. The amount of organic phase added is calculated according to the N / P ratio in Table 1.

[0105] Add 3 μL of EGFP mRNA (1 mg / mL), Luc mRNA (1 mg / mL), or OVA mRNA (1 mg / mL) to 1077 μL of citrate buffer solution at pH 4.0. Prepare a 3.24 mL organic phase mixture solution using ethanol as solvent according to the molar ratio of CQN series ionizable lipids:CHOL:DSPC:DMG-PEG2000 of 30:58.5:10:1.5. The amounts of the four components added (ionizable lipids, CHOL, DSPC, and DMG-PEG2000) were calculated according to the N / P ratio in Table 1.

[0106] EGFP mRNA LNPs, Luc mRNA LNPs, and OVA mRNA LNPs of the CQN series and MC3 were prepared using a microfluidic mixing method. The aqueous and organic phases were mixed at a flow rate of 1:3 using a microfluidic device. The mixture was then replaced with 1×PBS using tangential flow filtration and filtered through a 0.22 μm filter membrane for sterilization to obtain the LNPs.

[0107] The LNP formulations prepared by the above methods are shown in Table 1.

[0108] Table 1

[0109]

[0110] The particle size and PDI of LNP were determined using dynamic light scattering.

[0111] The total and free mRNA content in LNPs were detected using the Ribogreen kit, and the encapsulation efficiency of LNPs was calculated.

[0112] The particle size, PDI, and encapsulation efficiency results for some formulations are shown in Table 2.

[0113] preparation Particle size (nm) PDI Encapsulation rate Formulation 1 104.80±2.69 0.25±0.01 95.08±0.01％ Formulation 2 120.07±1.16 0.26±0.01 97.04±0.01％ Formulation 5 96.50±1.39 0.25±0.02 93.35±0.01％ Formulation 6 112.53±1.60 0.25±0.01 93.96±0.01％ Formulation 7 114.53±1.17 0.24±0.01 96.84±0.01％ Formulation 8 92.69±2.05 0.13±0.03 92.91±0.01％ Formulation 9 96.81±1.29 0.24±0.02 94.29±0.89％ Formulation 10 103.30±3.21 0.12±0.02 94.92±0.01％

[0114] Example 2 LNP Security

[0115] DC2.4 cells were loaded at 5 × 10⁻⁶ 3Cells were evenly seeded at a density of 100 μL of RPMI-1640 complete medium in 96-well plates and incubated at 37°C in a 5% CO2 cell culture incubator for 24 h before the medium was discarded.

[0116] The concentration of Luc mRNA in formulations 3, 4, 5, and 9 was determined using the RiboGreen kit. LNP was diluted in RPMI-1640 complete medium to a concentration of 0.5 mg / mL and then added to DC2.4 cells.

[0117] After incubating in a cell culture incubator for 24 hours, add 10 μL of CCK-8 reagent and incubate at 37°C in the dark for 2 hours. OD values ​​at 450 nm are measured using a microplate reader. Each group has 5 replicates. Cell viability is calculated using the following formula:

[0118]

[0119] Among them OD s The absorbance value of the sample group (containing cells, culture medium, and drug), OD c The absorbance value (OD) of the negative control group (containing cells and culture medium, but without drugs) is... b The absorbance value is for the blank control (containing culture medium but no cells).

[0120] The safety evaluation results of CQN Luc mRNA LNP in DC 2.4 cells showed that ( Figure 1 At an mRNA concentration of 0.5 mg / mL, formulations 3, 4, and 5 did not show significant toxicity to DC 2.4 cells, and cell viability was greater than 80% in all cases.

[0121] Example 3 LNP uptake experiment

[0122] 1. EGFP mRNA LNP uptake assay

[0123] DC 2.4 cells were divided into groups of 4 × 10⁻⁶. 4 Cells were seeded at a density of 1 mL in glass dishes containing 1 mL of RPMI-1640 complete medium. After being cultured in a 37°C, 5% CO2 incubator for 24 h, the cells were washed with PBS buffer.

[0124] The mRNA concentrations of formulations 1 and 8 prepared in Example 1 were determined using the RiboGreen kit. Formulations 1 and 8 were diluted with RPMI-1640 complete medium to prepare RPMI-1640 complete medium containing 5 ng / μL of EGFP mRNA.

[0125] Add 500 μL of the culture medium containing the formulation to each well of the previously cultured DC2.4 cells in glass dishes. Incubate at 37°C for 2 h, 6 h, and 12 h in a 5% CO2 cell culture incubator, respectively. After incubation, wash twice with cold PBS buffer containing heparin sodium and once with PBS. Add 4% paraformaldehyde (0.5 mL / well) to the dishes, incubate at room temperature for 10 min, and wash twice with cold PBS buffer. Then permeabilize the cells with PBS (0.5 mL / well) containing 0.1% Triton X-100 for 15 min, and wash twice with cold PBS buffer after permeabilization. After washing, stain the cell nuclei with 1 μg / mL DAPI working solution (0.3 mL / well) for 10 min, and wash twice with cold PBS buffer. Finally, add 100 μL of anti-fluorescence quencher, seal with a coverslip, blot dry the inside of the glass dish, and keep the inside and outside of the dish dry. The expression of green fluorescent protein was observed using laser scanning confocal microscopy. The excitation wavelength for the DAPI fluorescence channel was 405 nm, and the excitation wavelength for the green fluorescent protein fluorescence channel was 488 nm.

[0126] The results are as follows Figure 2 As shown, when formulations 1 and 8 were incubated with DC 2.4 cells for 2 hours, both formulations 1 and 8 showed weak green fluorescence; when incubated for 6 hours, the green fluorescence of both formulations 1 and 8 was higher than that of 2 hours; when incubated for 12 hours, almost no green fluorescent protein expression was observed in formulation 8, while green fluorescence was still visible in formulation 1.

[0127] 2. Luc mRNA LNP uptake assay

[0128] DC 2.4 cells were cultured at a concentration of 1.5 × 10⁻⁶. 4 Cells were seeded at a density of 100 μL of RPMI-1640 complete medium in 96-well plates (black transparent bottom). The cells were cultured in a conventional adherent culture at 37°C and 5% CO2 incubator for 24 h and then washed with PBS buffer.

[0129] The mRNA concentrations of formulations 5 and 9 prepared in Example 1 were determined using the RiboGreen reagent kit. Formulations 5 and 9 were diluted with RPMI-1640 complete medium to prepare RPMI-1640 complete medium containing 3.5 ng / μL of mRNA. 100 μL of the medium was added to each well of a previously cultured 96-well plate containing cells. The plates were incubated at 37°C for 2, 4, 8, 12, 24, and 36 h. After incubation, the plates were removed from the incubator and cooled to room temperature. 100 μL of Bright-Glo reagent was added to each well to completely lyse the cells, and bioluminescence was measured at different time points using a multi-mode microplate reader.

[0130] The results are as follows Figure 3 As shown, Luc mRNA was successfully expressed in DC2.4 cells in formulation group 5, and its effect was better than that in formulation group 9.

[0131] Example 4

[0132] Preparation of tumor-bearing mice: Mouse melanoma B16F10-OVA cells were collected, the culture medium was discarded, and the cells were rinsed twice with 4 mL of PBS buffer. 4 mL of trypsin was added to digest the cells, and RPMI 1640 complete medium was added to terminate the digestion. The cells were centrifuged (1000 rpm, 5 min) and the supernatant was discarded. The cells were resuspended in an appropriate amount of RPMI 1640 complete medium, and cell counts were performed using a cell counter. Finally, the cell concentration was adjusted to 1 × 10⁶ cells / mL. 7 Cells / mL. Take 100 μL (1 × 10⁻⁶) of cell suspension. 6 Cells were subcutaneously injected into the right axillary region of C57BL / 6N mice to establish a mouse subcutaneous melanoma model.

[0133] The size of the tumor was measured using calipers when the subcutaneous tumor volume was approximately 50 mm. 3 At that time, tumor-bearing mice were randomly divided into 10 groups (n=6) for drug administration, 5 groups were administered via tail vein injection and 5 groups were administered via intramuscular injection via the right hind leg of the mice;

[0134] The five groups receiving intravenous / intramuscular injection were further divided into:

[0135] The control group received 0.1 mL of physiological saline.

[0136] The free mRNA group consisted of 0.1 mL of physiological saline containing 5 μg of OVA mRNA;

[0137] The formulation groups were selected from 0.1 ml of physiological saline containing 5 μg OVA mRNA from Formulations 6, 7, and 10 in Example 1.

[0138] The medication was administered every three days for a total of four doses. During the administration period, the tumor size was measured every other day using calipers, and the tumor volume was calculated according to the formula. Body weight changes were also recorded. Tumor growth curves and mouse body weight change curves were plotted.

[0139]

[0140] Where V is the tumor volume (mm²) 3 ), where length is the long diameter of the tumor (mm) and width is the short diameter of the tumor (mm).

[0141] Tumor growth curve, mouse body weight change curve, and mouse survival rate graph are shown below. Figure 4 As shown:

[0142] Results from the intravenous injection group showed ( Figure 4 A) The tumors in the blank group grew rapidly, while the tumors in the intravenous injection groups of formulations 6 and 7 grew more slowly, showing tumor growth inhibition ability, which was superior to the blank group and formulation 10 group.

[0143] Results from the intramuscular injection group showed ( Figure 4 C), tumors grew rapidly in the blank group and the intramuscular injection group of formulation 10, while the effect of formulation 7 group was better than that of formulation 6 group and the free mRNA group. As shown in the body weight change curve, mice administered via both routes maintained stable body weight during the administration period, with no significant weight loss observed. Figure 4 (B, D)

[0144] Mice were sacrificed 48 hours after the last administration, and inguinal lymph nodes, spleen, and tumor tissue were collected for later use.

[0145] Example 5: Determination of OVA-specific IgG antibody content in plasma

[0146] Blood was collected from the mice used in Example 4 via the orbital venous plexus 48 hours after the last administration. The blood was collected in heparinized tubes, mixed thoroughly, and placed on ice. The samples were centrifuged at low temperature (3500 rpm, 10 min) within 30 min of collection, and the supernatant was collected. The supernatant was aliquoted and stored at -80°C.

[0147] The frozen plasma was retrieved and tested for OVA-specific IgG antibodies. The results were as follows: Figure 5 As shown in Figure A, the plasma IgG antibody levels in mice injected intravenously with formulations 6, 7, and 10 were significantly different from those in the blank control group and the free OVA mRNA group, with the IgG antibody levels in formulation 6 being higher than those in formulation 10. The plasma IgG antibody levels in the intramuscular injection groups of formulations 6, 7, and 10 were also significantly higher than those in the blank control group. Figure 5 B).

[0148] Example 6: Composition of T cells in the spleen and lymph nodes

[0149] Spleens from mice used in Example 4 were mechanically destroyed in DMEM containing 5% FBS under ice bath conditions, and the homogenate was transferred to centrifuge tubes. After low-temperature centrifugation (500g, 5min), the supernatant was discarded, and 3mL of erythrocyte lysis buffer was added. Erythrocytes were lysed at 4°C for 5min. The lysis process was quenched with 10mL of cold PBS, and single cells were collected using a 40μm cell filter. Cells were resuspended in an appropriate volume of RPMI 1640 complete medium to adjust the cell concentration to 1×10⁶ cells / mL. 7 Cells / mL.

[0150] Add 100 μL of cell suspension to each tube, along with Anti-Mouse CD4-FITC and Anti-Mouse CD3-PerCP-eFluor. TM 710, Anti-Mouse CD45-PE-Cyanine7, and Anti-Mouse CD8-APC were added to the cells, gently vortexed, and incubated at 4°C for 60 min. The cells were washed with 500 μL of flow cytometry staining solution, centrifuged at low temperature (500 g, 5 min), and the supernatant was discarded. This process was repeated twice. The cells were resuspended in 500 μL of flow cytometry staining solution, and the T cell composition was analyzed by flow cytometry.

[0151] Inguinal lymph nodes were collected and mechanically destroyed in DMEM containing 5% FBS. Cells were filtered, and the filter was rinsed with DMEM medium. After centrifugation at low temperature (500g, 5min), the supernatant was discarded, and the cells were resuspended in flow cytometry staining solution (repeat twice). The cells were then resuspended in 500μL of flow cytometry staining solution, centrifuged at low temperature (500g, 5min), and the supernatant was discarded (repeat twice).

[0152] Add 100 μL of inguinal lymph node cell suspension to each tube, along with Anti-Mouse CD4-FITC and Anti-Mouse CD3-PerCP-eFluor. TM 710, Anti-Mouse CD45-PE-Cyanine7, and Anti-Mouse CD8-APC were added to the cells, gently vortexed, and incubated at 4°C for 60 min. The cells were washed with 500 μL of flow cytometry staining solution, centrifuged at low temperature (500 g, 5 min), and the supernatant was discarded. This process was repeated twice. The cells were resuspended in 500 μL of flow cytometry staining solution, and the T cell composition was analyzed by flow cytometry.

[0153] The results of the intravenous injection group showed that ( Figure 6 CD8+ in the spleen of the treatment group + The proportion of T cells was numerically higher in the control group than in the control group, and the proportions in formulations 6 and 7 were also higher than those in formulation 10. Figure 6 A) CD4 levels in the spleen of mice in each treatment group + The proportion of T cells was increased in both groups compared to the control group.

[0154] CD8 in the inguinal lymph nodes of mice in different treatment groups + The percentage of T cells increased in all groups, with formulation 6 showing the most significant effect compared to the blank control group; CD4+ levels were also increased in the inguinal lymph nodes of mice treated with formulations 6, 7, and 10. + The proportion of T cells was increased compared to the control group. Figure 6 B).

[0155] Results of the intramuscular injection group were as follows Figure 7 As shown, CD8+ in the spleen of formulations 6 and 7 + The proportion of T cells was increased compared with the blank group and formulation 10 group; CD4+ was found in the spleen of mice in formulations 6 and 7. + The proportion of T cells was increased compared with both the blank group and the preparation group 10, while there was no significant difference between the preparation group 10 and the blank group. Figure 7 A). CD8+ levels in the inguinal lymph nodes of mice in formulations 6 and 7. + The proportion of T cells was numerically higher than that in the blank group and the preparation group; CD4+ was found in the inguinal lymph nodes of mice in all treatment groups. + The proportion of T cells was increased compared with both the blank group and the preparation group. Figure 7 B).

[0156] Example 7CD8 + IFNγ + Cell assay

[0157] The tumor tissue obtained in Example 4 was cut into 2-4 mm pieces using scissors or a scalpel. 5 mL of RPMI 1640 complete culture medium containing collagenase IV (1 mg / mL) was added, and the tumor tissue was digested at 37°C for 1 hour. After digestion, the cells were dispersed by pipetting and filtered through a cell filter to remove clumps and debris. The cell suspension was collected in a centrifuge tube. After low-temperature centrifugation (500 g, 5 min), the supernatant was discarded, and the cells were resuspended in RPMI 1640 complete culture medium. The cell suspension was centrifuged at 400 g for 5 min, and the supernatant was discarded (repeat twice). Finally, the cells were resuspended in an appropriate volume of RPMI 1640 complete culture medium to adjust the cell concentration to 1 × 10⁻⁶. 7 Single-cell suspension with cells / mL.

[0158] Add ovalbumin and protein transport inhibitors to the above single-cell suspension and continue culturing in a 37°C, 5% CO2 incubator for 24 h. After culturing, add an equal volume of 100 μL of cell suspension to each tube and add Anti-Mouse CD4-FITC and Anti-Mouse CD3-PerCP-eFluor. TM710, Anti-Mouse CD45-PE-Cyanine7, and Anti-Mouse CD8-APC were added to the cells, gently vortexed, and incubated at 4°C for 60 min. The cells were washed with 500 μL of flow cytometry staining solution, centrifuged at low temperature (500 g, 5 min), and the supernatant was discarded. This process was repeated twice. The cells were resuspended in 100 μL of flow cytometry staining solution, and 100 μL of IC50 fixative was added. After incubation at room temperature in the dark for 30 min, 500 μL of 1× permeabilization buffer was added, and the cells were centrifuged at low temperature (500 g, 5 min), and the supernatant was discarded. This process was repeated twice. The cell pellet was resuspended in 100 μL of 1× permeabilization buffer, Anti-Mouse IFNγ-PE was added, and the cells were incubated at room temperature in the dark for 30 min. 500 μL of 1× permeabilization buffer was added, and the cells were centrifuged at 500 g for 5 min at room temperature, and the supernatant was discarded. This process was repeated twice. Cells were resuspended in 500 μL of flow cytometry staining solution and analyzed by flow cytometry to measure CD8. + IFNγ + The percentage.

[0159] Results from the intravenous injection group showed differences in CD8 levels between the blank control group, OVA mRNA, formulation 6, and formulation 7. + IFNγ + There was no difference in the percentage of CD8 in formulation 10. + IFNγ + The percentage increased. Figure 8 A).

[0160] CD8 in the intramuscular injection group and the formulation 6 and formulation 7 groups + IFNγ + The percentages were significantly higher than those in group 10 of the formulation. Figure 8 B).

[0161] Resuspend the cells in 500 μL of flow cytometry staining solution, centrifuge at low temperature (500 g, 5 min), and discard the supernatant (repeat twice). Add an equal volume of 100 μL of cell suspension to each tube, and add Anti-Mouse CD11b-FITC, Anti-Mouse F4\80-PE, Anti-Mouse CD163-APC, and Anti-Mouse CD86-PerCP-eFluor. TM 710 was added to the cells, gently vortexed, and incubated at 4°C in the dark for 60 min. The cells were washed with 500 μL of flow cytometry staining solution, centrifuged at low temperature (500 g, 5 min), and the supernatant was discarded. This process was repeated twice. The cells were resuspended in 500 μL of flow cytometry staining solution, and the M2 / M1 macrophage ratio was analyzed by flow cytometry. The results are as follows: Figure 8 C Figure 8As shown in D, the M2 / M1 ratio in formulations 6 and 7 was significantly reduced, demonstrating a good anti-tumor effect.

[0162] Example 8

[0163] In Example 4, mice were sacrificed 48 hours after the last administration, and tumor tissue and major organs (heart, liver, spleen, lung, and kidney) were collected. The major organs were fixed with 4% paraformaldehyde, prepared into sections, stained with H&E, and the tissue sections were observed and photographed using an optical microscope. The differences in tissue morphology among the treatment groups were analyzed. Results for the intravenous injection group are as follows: Figure 9 As shown, the results of the intramuscular injection group are as follows: Figure 10 As shown;

[0164] The results showed that both the intravenous injection group and the intramuscular injection group exhibited good biocompatibility, and there were no obvious lesions in the major organs, indicating that the preparation had no acute or serious toxicity at the administered dose.

[0165] Tumor tissue sections were stained using the TUNEL apoptosis detection kit, and the sections were observed and photographed under an optical microscope. The results are as follows: Figure 11 As shown, there were almost no apoptotic cells in the blank group, while the proportion of apoptotic cells was relatively high in the intravenously injected preparation group 6 and the intramuscularly injected preparation group 7, suggesting that the preparations in these groups have strong anti-tumor capabilities, which are consistent with the tumor growth curve.

[0166] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A lipid nanocomposition, characterized in that, It comprises an ionizable lipid component, neutral phospholipids, cholesterol, and PEG lipids, wherein the structure of the ionizable lipid component is shown in Formula I: R1 is H, -C(O)-C1-C40 alkyl, -C(O)-C2-C40 alkenyl, -C(O)-C2-C40 alkynyl, C1-C40 alkyl, C2-C40 alkenyl or C2-C40 alkynyl; R2 is H, C1-C40 alkyl, -C(O)-C1-C40 alkyl, C2-C40 alkenyl, C2-C40 ynyl, -C(O)-C2-C40 alkenyl or -C(O)-C2-C40 ynyl; Ra1 and Ra2 are H, F, Cl, Br, I; Rb1 and Rb2 are H or C1-C8 alkyl groups; m1 and m2 can be 0, 1, 2, 3, 4, 5, 6, 7 or 8.

2. The lipid nanocomposition according to claim 1, characterized in that, The structure of the ionizable lipid component is shown in Formula II: R1 is a C1-C30 straight-chain alkyl, C2-C30 straight-chain alkenyl, or C2-C30 straight-chain alkynyl. m1 and m2 can be 1, 2, 3 or 4.

3. The lipid nanocomposition according to claim 1, characterized in that, R1 is n is an integer between 6 and 24.

4. The lipid nanocomposition according to claim 1, characterized in that, Its specific structure is as follows:

5. The lipid nanocomposition according to claim 1, characterized in that, The neutral phospholipids are selected from one or more of the following compounds: distearylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin, phosphatidic acid, and sphingomyelin; The PEG lipid is selected from one or more of the following compounds: PEG-modified distearylphosphatidylethanolamine, PEG-modified dimyristoylglycerol, PEG-modified dipalmitoylphosphatidylethanolamine, PEG-modified dimyristoylphosphatidylethanolamine, PEG-modified dilauroylphosphatidylethanolamine, PEG-modified ceramide, and mPEG-modified bis(tetradecyl)acetamide; more preferably, the molecular weight of the PEG or mPEG is 1000-5000 Da, and most preferably, DMG-PEG2000.

6. The lipid nanocomposition according to claim 5, characterized in that, It further includes therapeutic and / or preventive agents, preferably nucleic acids.

7. The lipid nanocomposition according to claim 6, characterized in that, The nucleic acid is selected from one or more of siRNA, aiRNA, miRNA, dsRNA, shRNA, snRNA, tRNA, rRNA, circRNA, saRNA, antisense oligonucleotide, lncRNA, ssDNA, mRNA, nucleic acid aptamers or ribozymes, with mRNA being the most preferred.

8. A pharmaceutical composition comprising the lipid nanocomposition of any one of claims 1-7 and a pharmaceutically acceptable carrier.

9. The use of the lipid nanocomposition according to any one of claims 1-7 in nucleic acid drug delivery.

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