Nano-delivery carrier and preparation method therefor

Abstract

The present disclosure relates to a nano-delivery carrier and a preparation method therefor. Specifically, the present disclosure relates to an improved nanoscale lipid carrier for nucleic acid delivery or a delivery system thereof. The carrier comprises an ionizable lipid, a helper lipid, a PEGylated lipid, cholesterol, and a cationic lipid. The nano-delivery carrier of the present disclosure solves the problems of low encapsulation efficiency, batch-to-batch inconsistency of artificially mixed particles, high cationic lipid ratio, high lipid-to-drug ratio, and weak immunogenicity in the prior art.

Description

A nanodelivery carrier and a preparation method thereof

[0001] The present disclosure claims priority to Chinese patent application 2024112975078, filed on September 14, 2024, the entire contents of which are incorporated herein. TECHNICAL FIELD

[0002] The present disclosure belongs to the field of immunotherapy. In particular, the present disclosure relates to lipid nanocarriers for delivering nucleic acids to cells or tissues. BACKGROUND

[0003] Traditional nucleic acid drug (such as mRNA) delivery systems are prepared by dissolving nucleic acids in an acidic aqueous solution, dissolving lipids in an ethanol solution, mixing the two-phase solutions, and then purifying the target product. This preparation process is tedious, especially when applied to personalized tumor vaccine research of nucleic acids, as the nucleic acid sequences of each patient are different, resulting in separate production for each production to avoid cross-contamination between samples, which makes the production cost of each patient's vaccine high and the preparation period long. Therefore, it is necessary to develop a new nucleic acid delivery system and its preparation method to shorten the preparation period and reduce the production cost.

[0004] Currently, researchers have attempted to use non-LNP delivery systems to prepare mRNA vaccines. For example, BioNTech has adopted a lipoplex (LPX) technology, which mixes cationic lipids and auxiliary phospholipids to prepare blank liposomes, and then mixes the blank liposomes and mRNA by vortexing or blowing to prepare mRNA vaccines.

[0005] LPX uses DOTMA / DOPE (molar ratio = 2 / 1) and DOTMA / cholesterol (molar ratio = 2 / 1) as the composition to prepare blank liposomes, and uses the thin film hydration method to prepare blank liposomes, and then uses the extrusion method to control the particle size of the nanocarriers. In use, mRNA is diluted in physiological saline, and mRNA is wrapped to prepare LPX by manually mixing mRNA and blank liposomes, with an average particle size of about 300 nm. Since the cationic lipids are positively charged in a physiological environment, they easily and irreversibly bind to negatively charged cell membranes, causing cell damage and toxicity, making the cytotoxicity of LPX large. Since LPX does not contain PEG-lipids in its composition, it is difficult to filter bacteria and has high production cost and difficulty. In addition, due to the difference in mixing methods of each operator, the physicochemical properties of each vaccine product are often not uniform.

[0006] For example, Jiachen West Sea uses a Ready to use (RTU) delivery system and preparation method to prepare mRNA vaccine, which uses ionizable lipids, auxiliary phospholipids, cholesterol, PEG lipids, and lipid materials to prepare blank liposomes in an acidic solution by microfluidic method; then by increasing the mass ratio of blank liposomes and mRNA, mRNA is wrapped by manually mixing mRNA and blank liposomes in a low pH solution to prepare mRNA vaccine. Because ionizable lipids are not charged under neutral conditions, the RTU particles have low charge, which cannot effectively and completely wrap mRNA, resulting in a high lipid-drug ratio, high cost, high toxicity, and incomplete mRNA encapsulation. In addition, due to the difference in the operation of personnel each time, the physicochemical properties of the vaccine product are often not uniform each time.

[0007] In view of the above, there is still a need in the art for an improved nucleic acid drug delivery system to solve the problems of low encapsulation efficiency, batch inconsistency of manually mixed particles, high cationic lipid ratio, high lipid-drug ratio, and weak immunogenicity in the prior art. SUMMARY

[0008] Delivery carrier

[0009] In view of the above needs in the art, the present disclosure provides a delivery carrier comprising or consisting of:

[0010] • a first lipid composition; and

[0011] • optionally, a second lipid composition consisting of a cationic lipid;

[0012] wherein,

[0013] The first lipid composition comprises or consists of:

[0014] - an ionizable lipid,

[0015] - an auxiliary lipid,

[0016] - a PEGylated lipid, and

[0017] - a sterol, (e.g., cholesterol, coprostanol, sitosterol, ergosterol, elaidosterol, brassicasterol, stigmasterol, calendula sterol, tomatine, ursolic acid, alpha-tocopherol).

[0018] "Delivery carrier" refers to a lipid nanoparticle for loading one or more nucleic acids (such as mRNA).

[0019] It should be understood that the ordinal terms "first lipid composition", "second lipid composition", and the like in the present disclosure are merely used to distinguish different technical features, elements, components, steps, and are not intended to constitute a limitation on the level, order, or number.

[0020] "Ionizable lipid" refers to a lipid molecule that can change its charge state under certain conditions. They are usually designed to carry a positive charge in an acidic environment (such as endosomes or lysosomes), so as to bind to negatively charged nucleic acids (such as mRNA or siRNA) and form stable complexes for the delivery of nucleic acids. However, in a neutral or alkaline environment, the charge state of these lipid molecules will change, usually becoming neutral or close to neutral, thereby improving their biocompatibility and stability.

[0021] In some embodiments, the ionizable lipid is selected from the group consisting of: DODAP, DLin-KC2-DMA, Lipid-29, C12-200, ALC-0315, SM-102, Lipid-5.

[0022] In some specific embodiments, the ionizable lipid is selected from the group consisting of: ALC-0315, SM-102, Lipid-5.

[0023] "Helper lipid" is a class of non-primary functional lipids that work together with the primary active lipids (such as ionizable lipids) in the lipid nanoparticle delivery system. They are designed to enhance the stability, fluidity, and delivery efficiency of the lipid nanoparticle, but are not directly responsible for the binding or endosomal escape of nucleic acids. Common helper lipids include phospholipids, cholesterol and its derivatives, etc., which adjust the physicochemical properties of the lipid nanoparticle to optimize its performance in vitro and in vivo.

[0024] In some embodiments, the helper lipid is selected from 1,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearyl-sn- glycero-3-phosphocholine (DSPC), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DUPC), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn- glycero-3-phosphoethanolamine, palmitoyloleoyl phosphatidyl ethanolamine (POPE), distearoyl- phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1- stearoyl-2-oleoyl-phosphatidylcholine (SOPC), or a combination thereof.

[0025] In some specific embodiments, the helper lipid is DOPE, DSPC, DMPC, or DSPE.

[0026] The term "PEG-modified lipid" or "PEG-modified lipid" or "PEGylated lipid" refers to a molecule comprising a polyethylene glycol moiety and a lipid moiety, which is a lipid modified with polyethylene glycol. The "PEGylated lipid" can increase the hydrophilicity of the surface of the delivery vehicle of the present disclosure, the aggregation between nanoparticles, and can improve the stability of the particles.

[0027] In some embodiments, the PEGylated lipid is selected from ALC-0159, mPEG-DMG, mPEG- DSPE, PEG-DSG, mPEG-DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol), mPEG-DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol), mPEG- DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol).

[0028] In some specific embodiments, the PEGylated lipid is mPEG-DMG, ALC-0159, mPEG-DPPE.

[0029] The "cationic lipid" enables the delivery vehicle of the present disclosure to condense negatively charged nucleic acids (such as mRNA molecules) through electrostatic interactions. A cationic lipid is a lipid with a positively charged functional group (e.g. containing primary, secondary, tertiary, quaternary amines) in its molecular structure, which is positively charged in physiological environment or neutral pH conditions. This positive charge confers unique chemical properties to cationic lipids, enabling them to tightly bind to negatively charged biological macromolecules (such as DNA and RNA) through electrostatic interactions, forming stable complexes.

[0030] In some specific embodiments, the cationic lipid is selected from the group consisting of: EPC, DC-Cholesterol, DOTAP, DOSPA.

[0031] In some specific embodiments, the cationic lipid is EPC; DOTAP, DC-Cholesterol next.

[0032] In some specific embodiments, in the delivery vehicle: the first lipid composition comprises 45% to 99% w / w of the delivery vehicle (for example but not limited to 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, and any number between any two of the numbers is considered to be sufficiently specified); the second lipid composition comprises 1% to 55% w / w of the delivery vehicle (for example but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55%, and any number between any two of the numbers is considered to be sufficiently specified).

[0033] In some specific embodiments, in the delivery vehicle: the first lipid composition comprises 90% w / w of the delivery vehicle; the second lipid composition comprises 10% w / w of the delivery vehicle.

[0034] In the present disclosure, when referring to a specific numerical value, a certain error range should be considered, which is mainly based on the fact that it is difficult to achieve absolute accuracy, either by experimental measurement to obtain data, or by theoretical calculation to obtain results, due to the inherent inaccuracy of measurement and experiment. There are precision limitations in the measurement instruments themselves, for example, even if the precision of a common electronic balance is very high, there will be a certain measurement error on the last decimal place. During the experimental operation process, environmental factors (such as slight fluctuations in temperature and humidity), differences in the operation of the operator, etc. will all cause deviations in the data. Taking the measurement of the pH value of a solution as an example, even if a high-precision pH meter is used, multiple measurements of the same solution may result in slightly different values.

[0035] As some examples, in different scenarios:

[0036] - If referring to "content 90%", the reasonable error interval can be set as "90% ± 1%". Because of the conventional accuracy of weighing, and trace impurities are also normal.

[0037] - If referring to "temperature 30°C", the error interval can be set as "30°C ± 3°C", which is mainly considering the temperature control accuracy of the constant temperature equipment.

[0038] - If referring to the concentration "50 mg / ml", the error interval is usually "50 mg / ml ± 5%", which is consistent with the pharmacopoeia standard of content uniformity in drug production.

[0039] If not specifically mentioned, the error range is in accordance with the conventional error range in the art. Values within the error range should be considered to fall within the scope of the present disclosure. In some embodiments, in the first lipid composition:

[0040] The mole fraction of the ionizable lipid is 10 to 90;

[0041] The mole fraction of the helper lipid is 0 to 30;

[0042] The mole fraction of the PEGylated lipid is 0 to 10;

[0043] The mole fraction of the sterol is 9 to 70.

[0044] In some specific embodiments, in the first lipid composition:

[0045] The mole fraction of the ionizable lipid is 30 to 60 (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or any integer or decimal within this range); and

[0046] the mole fraction of the ionizable lipid is 40, the mole fraction of the helper lipid is 10, the mole fraction of the PEGylated lipid is 2, and the mole fraction of the sterol is 48.

[0047] the mole fraction of the ionizable lipid is 40, the mole fraction of the helper lipid is 10, the mole fraction of the PEGylated lipid is 2, and the mole fraction of the sterol is 48.

[0048] the mole fraction of the ionizable lipid is 40, the mole fraction of the helper lipid is 10, the mole fraction of the PEGylated lipid is 2, and the mole fraction of the sterol is 48.

[0049] It should be noted that "mole fraction" does not represent a number of moles. "Mole fraction" requires comparison between two or more components. For example, "the mole fraction of the ionizable lipid is 40, the mole fraction of the helper lipid is 10" means the ratio of the two is 40:10, or 4:1, or equivalent ratios, regardless of the actual number of moles.

[0050] In some embodiments, the mole ratio of the ionizable lipid:the helper lipid:the PEGylated lipid:the sterol is 40:15:1.5:43.5.

[0051] In some embodiments, the mole ratio of the ionizable lipid:the helper lipid:the PEGylated lipid:the sterol is 40:10:2:48.

[0052] In some embodiments, the mole ratio of the ionizable lipid:the helper lipid:the PEGylated lipid:the sterol is 40:10:2.5:47.5.

[0053] In some embodiments, the mole ratio of the ionizable lipid:the helper lipid:the PEGylated lipid:the sterol is 40:15:2:43.

[0054] In some embodiments, the mole ratio of the ionizable lipid:the helper lipid:the PEGylated lipid:the sterol is 40:20:1.5:38.5.

[0055] In some specific embodiments, the molar ratio of said ionizable lipid: said helper lipid: said PEG lipid: said sterol is 45:5:2:48.

[0056] In some specific embodiments, the molar ratio of said ionizable lipid: said helper lipid: said PEG lipid: said sterol is 45:15:1.5:38.5.

[0057] In some specific embodiments, the molar ratio of said ionizable lipid: said helper lipid: said PEG lipid: said sterol is 50:10:1.5:38.5.

[0058] In some specific embodiments, the molar ratio of said ionizable lipid: said helper lipid: said PEG lipid: said sterol is 50:10:2:38.

[0059] In some specific embodiments, the molar ratio of said ionizable lipid: said helper lipid: said PEG lipid: said sterol is 50:15:2.5:32.5.

[0060] In some specific embodiments, the molar ratio of said ionizable lipid: said helper lipid: said PEG lipid: said sterol is 50:15:1.5:33.5.

[0061] In some specific embodiments, the molar ratio of said ionizable lipid: said helper lipid: said PEG lipid: said sterol is 50:15:2:33.

[0062] In some specific embodiments, the molar ratio of said ionizable lipid: said helper lipid: said PEG lipid: said sterol is 50:20:1.5:28.5.

[0063] In some specific embodiments, the molar ratio of said ionizable lipid: said helper lipid: said PEG lipid: said sterol is 30:10:2:58.

[0064] In some embodiments, the delivery vehicle has an average particle size ranging from 40 to 1000 nm.

[0065] In some specific embodiments, the delivery vehicle has an average particle size ranging from 40 to 200 nm; for example, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nm, or any integer or decimal within this range.

[0066] It should be noted that the nominal particle size is generally a general description of the particle population size, which can correspond to a certain characteristic particle size (such as the average particle size, the number average particle size, the median diameter D50, etc.). The average particle size described herein does not mean that each specific lipid particle in the population is the same particle size. For example, the nominal particle size of "50 nm" refers to the average particle size of the batch of particles, but there can be a certain distribution range (such as 40 to 60 nm) in actual detection.

[0067] It is a consensus in the art that the average particle size of the delivery carrier is a statistical parameter reflecting the central tendency of the particle population size, which is calculated by statistical analysis of the particle size of a certain number of particles, and is used to summarize the overall size characteristics of the particle system. Since the particle size distribution can present different morphologies (such as normal distribution, skew distribution, etc.), there are many methods for calculating the average particle size, including number average particle size, volume average particle size, Z-average particle size, etc. As an example, when the dynamic light scattering method (instrument model not limited) is used to measure the particle population, the average particle size obtained falls within the specified range, then it is considered to fall within the scope of the present disclosure. In some embodiments, the zeta potential of the delivery carrier is in the range of -10 to +60 mV.

[0068] In some specific embodiments, the zeta potential of the delivery carrier is in the range of +15 to +35 mV; 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 mV, or any integer or decimal within this range.

[0069] The zeta potential (ζ potential) of the delivery carrier is a key electrochemical parameter for describing the stability of the dispersion system, which refers to the potential difference between the sliding surface of the particle and the bulk of the dispersion medium. It reflects the charged state of the particle surface and the ability of the particles to repel or attract each other. Common measurement methods include: laser Doppler electrophoresis, microelectrophoresis, and electroacoustic method. As an example, when the electrophoretic light scattering method (instrument model not limited) is used to measure the particle population, the zeta potential obtained falls within the specified range, then it is considered to fall within the scope of the present disclosure.

[0070] In some specific embodiments, the delivery carrier is selected from the group consisting of:

[0071] 1) 90% first lipid composition, 10% cationic lipid;

[0072] The first lipid composition comprises: Lipid-5: DOPE: mPEG-DMG: cholesterol in a molar ratio of 40:15:1.5:43.5;

[0073] 2) 90% first lipid composition, 10% cationic lipid;

[0074] the first lipid composition comprises: ALC-0315 : DSPC : ALC-0159 : cholesterol at a molar ratio of 50 : 10 : 1.5 : 38.5;

[0075] 3) 90% first lipid composition, 10% cationic lipid;

[0076] the first lipid composition comprises: SM102 : DSPC : mPEG-DMG : cholesterol at a molar ratio of 50 : 10 : 1.5 : 38.5;

[0077] 4) 90% first lipid composition, 10% cationic lipid;

[0078] the first lipid composition comprises: SM-102 : DSPC : mPEG-DMG : sitosterol at a molar ratio of 40 : 10 : 2 : 48;

[0079] 5) 90% first lipid composition, 10% cationic lipid;

[0080] the first lipid composition comprises: SM-102 : DSPC : mPEG-DMG : sitosterol at a molar ratio of 45 : 5 : 2 : 48;

[0081] 6) 90% first lipid composition, 10% cationic lipid;

[0082] the first lipid composition comprises: SM-102 : DSPC : mPEG-DMG : sitosterol at a molar ratio of 45 : 15 : 1.5 : 38.5;

[0083] 7) 90% first lipid composition, 10% cationic lipid;

[0084] the first lipid composition comprises: SM-102 : DSPC : mPEG-DMG : sitosterol at a molar ratio of 50 : 10 : 1.5 : 38.5;

[0085] 8) 90% first lipid composition, 10% cationic lipid;

[0086] the first lipid composition comprises: SM-102 : DSPC : mPEG-DMG : sitosterol at a molar ratio of 50 : 15 : 2 : 33;

[0087] 9) 90% first lipid composition, 10% cationic lipid;

[0088] the first lipid composition comprises: SM-102 : DOPE : mPEG-DMG : sitosterol at a molar ratio of 50 : 20 : 1.5 : 28.5;

[0089] 10) 90% first lipid composition, 10% cationic lipid;

[0090] The first lipid composition comprises: SM-102: DOPE: mPEG-DMG: phytosteryl in a molar ratio of 50: 15: 1.5: 33.5;

[0091] 11) 90% first lipid composition, 10% cationic lipid;

[0092] The first lipid composition comprises: SM-102: DSPC: mPEG-DMG: (phytosteryl and cholesterol) in a molar ratio of 45: 15: 1.5: 38.5;

[0093] 12) 90% first lipid composition, 10% cationic lipid;

[0094] The first lipid composition comprises: Lipid-5: DSPC: mPEG-DPPE: cholesterol in a molar ratio of 50: 10: 1.5: 38.5;

[0095] 13) 90% first lipid composition, 10% cationic lipid;

[0096] The first lipid composition comprises: Lipid-5: DSPC: mPEG-DMG: brassicasterol in a molar ratio of 50: 10: 1.5: 38.5;

[0097] 14) 90% first lipid composition, 10% cationic lipid;

[0098] The first lipid composition comprises: ALC-0315: DSPC: ALC-0159: cholesterol in a molar ratio of 50: 10: 1.5: 38.5;

[0099] 15) 90% first lipid composition, 10% cationic lipid;

[0100] The first lipid composition comprises: ALC-0315: DSPE: ALC-0159: cholesterol in a molar ratio of 50: 10: 1.5: 38.5;

[0101] 16) 90% first lipid composition, 10% cationic lipid;

[0102] The first lipid composition comprises: ALC-0315: DMPC: ALC-0159: cholesterol in a molar ratio of 50: 10: 1.5: 38.5;

[0103] 17) 90% first lipid composition, 10% cationic lipid;

[0104] The first lipid composition comprises ALC-0315 : DSPC : mPEG-DMG : cholesterol in a molar ratio of 50:10:1.5:38.5;

[0105] 18) 90% first lipid composition, 10% cationic lipid;

[0106] The first lipid composition comprises ALC-0315 : DSPE : mPEG-DMG : cholesterol in a molar ratio of 50:10:1.5:38.5;

[0107] 19) 90% first lipid composition, 10% cationic lipid;

[0108] The first lipid composition comprises ALC-0315 : DSPC : mPEG-DMG : cholesterol in a molar ratio of 50:10:1.5:38.5;

[0109] 20) 90% first lipid composition, 10% cationic lipid;

[0110] The first lipid composition comprises ALC-0315 : DSPC : mPEG-DMG : cholesterol in a molar ratio of 40:10:2.5:47.5;

[0111] 21) 90% first lipid composition, 10% cationic lipid;

[0112] The first lipid composition comprises ALC-0315 : DSPC : mPEG-DMG : cholesterol in a molar ratio of 40:15:2:43;

[0113] 22) 90% first lipid composition, 10% cationic lipid;

[0114] The first lipid composition comprises ALC-0315 : DSPC : mPEG-DMG : cholesterol in a molar ratio of 40:20:1.5:38.5;

[0115] 23) 90% first lipid composition, 10% cationic lipid;

[0116] The first lipid composition comprises ALC-0315 : DSPC : mPEG-DMG : cholesterol in a molar ratio of 50:10:2:38;

[0117] 24) 90% first lipid composition, 10% cationic lipid;

[0118] The first lipid composition comprises ALC-0315 : DSPC : mPEG-DMG : cholesterol in a molar ratio of 50:15:2.5:32.5;

[0119] 25) 90% first lipid composition, 10% cationic lipid;

[0120] The first lipid composition comprises ALC-0315 : DSPC : mPEG-DMG : cholesterol in a molar ratio of 30 : 10 : 2 : 58.

[0121] Alternatively, as an alternative, when each of the above groups does not contain a cationic lipid, the first lipid composition accounts for 100%.

[0122] Use of the delivery vehicle

[0123] The present disclosure also provides use of the aforementioned delivery vehicle in the manufacture of a medicament.

[0124] In some embodiments, the delivery vehicle of the present disclosure is used in the manufacture of a nucleic acid cell transfection reagent.

[0125] In some embodiments, the delivery vehicle of the present disclosure is used in the manufacture of a nucleic acid protein replacement drug.

[0126] In some embodiments, the delivery vehicle of the present disclosure is used in the manufacture of a nucleic acid vaccine.

[0127] In some embodiments, the delivery vehicle of the present disclosure is used in the manufacture of a nucleic acid vaccine; the nucleic acid vaccine is used to treat a tumor or prevent recurrence of a tumor.

[0128] In some embodiments, the delivery vehicle of the present disclosure is used in the manufacture of a nucleic acid vaccine; the nucleic acid is selected from the group consisting of siRNA, mRNA, sgRNA, DNA, or a combination thereof.

[0129] The present disclosure also provides the aforementioned delivery vehicle for use in delivering a nucleic acid to a cell or tissue. The nucleic acid is selected from the group consisting of siRNA, mRNA, sgRNA, DNA, or a combination thereof; preferably mRNA. The cell or tissue is located in the liver or spleen.

[0130] The delivery vehicle of the present disclosure is capable of packaging, carrying, and delivering a negatively charged nucleic acid.

[0131] In some embodiments, the nucleic acid (exemplarily, mRNA) suitable for delivery with the delivery vehicle of the present disclosure is linear or circular.

[0132] In some embodiments, the nucleic acid (exemplarily, mRNA) suitable for delivery with the delivery vehicle of the present disclosure is 20 nt to 20000 nt in length; for example, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000 nt, or any integer or decimal number within this range. In exemplary embodiments, the nucleic acid is 1000 to 5000 nt in length.

[0133] In some embodiments, the mass ratio of the delivery vehicle of the present disclosure and the nucleic acid to be delivered is 1 : 1 to 80: 1; preferably 10: 1 to 80: 1; for example, 1 : 1, 2: 1, 2.5: 1, 3: 1, 4: 1, 5: 1, 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, 55: 1, 60: 1, 65: 1, 70: 1, 75: 1, 80: 1.

[0134] In some specific embodiments, the mass ratio of the delivery vehicle of the present disclosure and the nucleic acid to be delivered is 15: 1 to 20: 1; for example, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, or any integer or decimal number within this range.

[0135] The present disclosure also provides the aforementioned delivery vehicle for use in delivering a cargo to a cell or tissue. The cargo is selected from the group consisting of: a nucleic acid, a protein, a complex of a nucleic acid and a protein, a mixture of a nucleic acid and a protein. The cell or tissue is located in the liver or the spleen.

[0136] The delivery vehicle of the present disclosure is capable of packaging, carrying, delivering the aforementioned cargo molecules.

[0137] In some embodiments, the protein suitable for delivery with the delivery vehicle of the present disclosure.

[0138] In some embodiments, the mass ratio of the delivery vehicle of the present disclosure and the cargo to be delivered is 1 : 1 to 80: 1; preferably 10: 1 to 80: 1; for example, 1 : 1, 2: 1, 2.5: 1, 3: 1, 4: 1, 5: 1, 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, 55: 1, 60: 1, 65: 1, 70: 1, 75: 1, 80: 1.

[0139] In some specific embodiments, the mass ratio of the delivery carrier of the present disclosure and the cargo to be delivered is 15:1 to 20:1; for example, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, or any integer or decimal number within this range.

[0140] The delivery carrier of the present disclosure is prepared into a dosage form selected from intravenous administration, local administration, intratumoral administration, intramuscular administration, subcutaneous administration. The present disclosure unexpectedly notes that intravenous injection is more advantageous for spleen targeting; local administration (intratumoral injection, intramuscular injection, subcutaneous injection) has the characteristic of expressing at the injection site and will not be off-target to the liver and other organs.

[0141] Nucleic acid delivery system

[0142] The present disclosure also provides a nucleic acid delivery system comprising the delivery carrier of the present disclosure; and a nucleic acid.

[0143] The nucleic acid delivery system of the present disclosure is a nanoscale delivery system, which refers to a drug delivery system with a particle diameter in the nanometer range (1 to 1000 nm), which mainly concentrates and loads drugs through various forms such as embedding, adsorption, encapsulation or covalent bond combination, and targets the delivery of nucleic acid drugs to specific tissues or cells to play a role. The delivery system can effectively overcome the difficulties faced by nucleic acid drugs (such as mRNA) in clinical application, such as easy degradation, difficulty in entering target organs, tissues or target cells, and low late endosome escape efficiency.

[0144] In some embodiments, the nucleic acid contained in the nucleic acid delivery system of the present disclosure is selected from siRNA, mRNA, sgRNA, DNA, or a combination thereof.

[0145] In some embodiments, the mass ratio of the delivery carrier and the nucleic acid to be delivered in the nucleic acid delivery system of the present disclosure is 1:1 to 80:1; for example, 2.5:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1.

[0146] In some specific embodiments, the mass ratio of the delivery carrier and the nucleic acid to be delivered in the nucleic acid delivery system of the present disclosure is 15:1 to 20:1; for example, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, or any integer or decimal number within this range.

[0147] Cargo delivery system

[0148] The present disclosure also provides a cargo delivery system comprising the delivery carrier of the present disclosure and the cargo.

[0149] In some embodiments, the cargo is selected from the group consisting of: nucleic acid, protein, complex of nucleic acid and protein, mixture of nucleic acid and protein.

[0150] In some embodiments, the mass ratio of the delivery vehicle and the cargo to be delivered in the cargo delivery system of the present disclosure is 1 : 1 to 80: 1; for example, 2.5: 1, 5: 1, 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, 55: 1, 60: 1, 65: 1, 70: 1, 75: 1, 80: 1.

[0151] In some specific embodiments, the mass ratio of the delivery vehicle and the cargo to be delivered in the cargo delivery system of the present disclosure is 15: 1 to 20: 1; for example, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, or any integer or decimal within this range.

[0152] Preparation method

[0153] The present disclosure also provides a method for preparing a nucleic acid delivery system, comprising the steps of:

[0154] 1) providing a first lipid composition and a second lipid composition (the second lipid composition is optional) to form a delivery vehicle, so that the total concentration of lipids in the delivery vehicle is 1 mg / ml to 80 mg / ml, preferably 10 mg / ml; contacting (preferably mixing) the delivery vehicle and a buffer with pH < 9 (preferably less than 7, for example 10 mM pH 4.0 citric acid buffer) by microfluidics, the volume ratio of the delivery vehicle and the buffer being 1:3;

[0155] The parameters of the microfluidics include: flow rate 1 ml / min to 200 ml / min, temperature 4°C to 70°C;

[0156] Preferably, the flow rate is 12 ml / min to 20 ml / min and the temperature is 20°C to 30°C;

[0157] 2) diluting the solution obtained in step 1) with a buffer with pH < 9 (preferably less than 7, for example 10 mM pH 4.0 citric acid buffer) by 1 to 20 times, preferably 7 times, in volume;

[0158] 3) subjecting the solution obtained in step 2) to ultrafiltration,

[0159] The parameters of the ultrafiltration include: ultrafiltration rotation speed 0 rpm to 5000 rpm, centrifugation time 0 min to 180 min, centrifugation temperature 2°C to 30°C;

[0160] Preferably, the ultrafiltration rotation speed is 2000 rpm to 3000 rpm, the centrifugation time is 15 min to 30 min, the centrifugation temperature is 2 °C to 8 °C; the sample is ultrafiltrated to about 1 ml, 5 ml of 10 mM pH 4.0 citric acid buffer is added, and the ultrafiltration is performed again, and the ultrafiltration is stopped again when the volume is about 1 ml;

[0161] 4) providing the nucleic acid and the solution obtained in step 3) in a buffer, so that the concentration of the nucleic acid is 10 pg / ml to 1000 pg / ml (for example, but not limited to, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, 100 pg / ml, 200 pg / ml, 300 pg / ml, 400 pg / ml, 500 pg / ml, 600 pg / ml, 700 pg / ml, 800 pg / ml, 900 pg / ml, and any range between the foregoing values is deemed to have been fully listed in the present disclosure), preferably 50 pg / ml;

[0162] When the nucleic acid and the solution obtained in step 3) are provided, the pH of the buffer used is independently 4 to 9 (preferably 4 to 7.4) for each;

[0163] The buffer is independently selected from the group consisting of: phosphate buffer, citrate buffer, acetic acid buffer, histidine buffer, histidine salt buffer, HEPES, Tris salt, preferably phosphate buffer, citrate buffer; for example, 10 mM pH 4.0 citric acid buffer;

[0164] 5) contacting (preferably mixing) the delivery vehicle and the nucleic acid by microfluidics, the volume ratio of the delivery vehicle and the nucleic acid being 20:1 to 1:20;

[0165] The parameters of the microfluidics include: flow rate 1 ml / min to 200 ml / min, flow rate ratio 0.1:1 to 1:0.1, volume ratio 1:10 to 10:1, temperature 4 °C to 70 °C;

[0166] Preferably, the flow rate is 12 ml / min to 20 ml / min, the flow rate ratio is 0.3:1 to 1:0.3, the volume ratio is 1:5 to 5:1, and the temperature is 20 °C to 30 °C;

[0167] 6) obtaining the nucleic acid delivery system.

[0168] Convenient alternative to clinical scenarios

[0169] The present disclosure also provides a method for preparing a nucleic acid delivery system, comprising the steps of:

[0170] - steps 1) to 4) as defined in the foregoing method of the present disclosure;

[0171] - 5) contacting the delivery vehicle and the nucleic acid in a container by manual mixing and / or vortexing, the volume ratio of the delivery vehicle and the nucleic acid being from 20: 1 to 1 :20;

[0172] - 6) obtaining the nucleic acid delivery system;

[0173] wherein steps 1) to 3) as a whole and step 4) can be interchanged in order or performed in parallel.

[0174] In some specific embodiments of the method of preparation, the order of steps 1) to 3) as a whole and step 4) of the foregoing is interchangeable, or parallel.

[0175] In some embodiments of the method of preparation, the first lipid composition comprises or consists of an ionizable lipid, a helper lipid, a PEGylated lipid, and a sterol (cholesterol or analogs such as coprostanol, sitosterol, ergosterol, elaidosterol, brassicasterol, stigmasterol, campesterol, tomatidine, ursolic acid, a-tocopherol).

[0176] In some embodiments of the method of preparation, the ionizable lipid is selected from the group consisting of DODAP, DLin-KC2-DMA, Lipid-29, C12-200, ALC-0315, SM-102, Lipid-5.

[0177] In some embodiments of the method of preparation, the helper lipid is selected from the group consisting of DMPC, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC), 1,2-didodecanoyl-sn-glycero-phosphocholine (DUPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, palmitoyloleoyl phosphatidyl ethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), or a combination thereof.

[0178] In some embodiments of the method of preparation, the PEGylated lipid is selected from the group consisting of: ALC-0159, mPEG-DMG, mPEG-DSPE, PEG-DSG, mPEG-DPPE, mPEG-DOPE, mPEG-DMPE.

[0179] In some embodiments of the method of preparation, the second lipid composition consists of a cationic lipid.

[0180] In some embodiments of the method of preparation, the cationic lipid is selected from the group consisting of: EPC, DC-Cholesterol, DOTAP, DOSPA, DOTMA.

[0181] In some embodiments of the method of preparation, the first lipid composition comprises 45% to 99% w / w of the delivery vehicle; the second lipid composition comprises 1% to 55% w / w of the delivery vehicle.

[0182] In some specific embodiments of the method of preparation, the first lipid composition comprises 90% w / w of the delivery vehicle; the second lipid composition comprises 10% w / w of the delivery vehicle.

[0183] In some embodiments of the method of preparation, in the first lipid composition:

[0184] the mole fraction of the ionizable lipid is 10 to 90;

[0185] the mole fraction of the helper lipid is 0 to 30;

[0186] the mole fraction of the PEGylated lipid is 0 to 10;

[0187] the mole fraction of the sterol is 9 to 70.

[0188] In some embodiments of the method of preparation, in the first lipid composition:

[0189] the mole fraction of the ionizable lipid is 30 to 60 (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or any integer or decimal within this range);

[0190] the mole fraction of the helper lipid is 10 to 20, e.g., 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, or any integer or decimal within this range);

[0191] the mole fraction of the PEGylated lipid is 1 to 2.5 (e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, or any integer or decimal within this range);

[0192] the mole fraction of the sterol is 25 to 60 (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or any integer or decimal within this range).

[0193] In some embodiments of the method of preparation, the molar ratio of the ionizable lipid:the helper lipid:the PEGylated lipid:the sterol is 40:10:2:48, 40:10:2.5:47.5, 40:15:1.5:43.5, 40:15:2:43, 40:20:1.5:38.5, 45:5:2:48, 45:15:1.5:38.5, 50:10:1.5:38.5, 50:10:2:38, 50:15:2.5:32.5, 50:15:1.5:33.5, 50:15:2:33, 50:20:1.5:28.5, or 30:10:2:58.

[0194] In some embodiments of the method of preparation, the delivery vehicle has an average particle size ranging from 40 to 200 nm; e.g., 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nm, or any integer or decimal within this range.

[0195] In some embodiments of the method of preparation, the delivery vehicle has a zeta potential ranging from +15 to +35 mV; e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 mV, or any integer or decimal within this range.

[0196] In some embodiments of the method of preparation, the nucleic acid is selected from the group consisting of siRNA, mRNA, sgRNA, DNA, or a combination thereof; preferably mRNA.

[0197] In some embodiments of the method of preparation, the mass ratio of the delivery vehicle and the nucleic acid to be delivered is 1 : 1 to 80: 1; preferably 10: 1 to 80: 1; such as 2.5: 1, 5: 1, 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, 55: 1, 60: 1, 65: 1, 70: 1, 75: 1, 80: 1.

[0198] In some embodiments of the method of preparation, the mass ratio of the delivery vehicle and the nucleic acid to be delivered is 15: 1 to 20: 1; such as 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, or any integer or decimal number within this range.

[0199] The present disclosure also provides the nucleic acid delivery system prepared by the aforementioned method, and its use in the preparation of a medicament.

[0200] The present disclosure also provides the nucleic acid delivery system prepared by the method of the present disclosure for the preparation of a nucleic acid cell transfection reagent.

[0201] The present disclosure also provides the nucleic acid delivery system prepared by the method of the present disclosure for the preparation of a nucleic acid protein replacement drug.

[0202] The present disclosure also provides the nucleic acid delivery system prepared by the method of the present disclosure for the preparation of a nucleic acid vaccine for treating a tumor or preventing the recurrence of a tumor.

[0203] As further exemplary embodiments, the present disclosure also provides a method of preparing a cargo delivery system. The difference compared to the aforementioned method of preparing a nucleic acid delivery system according to the present disclosure is only that the nucleic acid to be delivered is replaced by other cargo molecules (such as proteins, complexes of nucleic acids and proteins, mixtures of nucleic acids and proteins). The method of preparing a cargo delivery system according to the present disclosure is considered to have been fully described in terms of the steps, parameters, conditions it comprises.

[0204] Abbreviations used in the present disclosure:

[0205] EPC: 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, a cationic lipid;

[0206] DOTMA: 1,2-dioleoyloxy-3-methylammonium propane (chloride salt), a cationic lipid;

[0207] DOTAP: (2,3-dioleyloxypropyl)trimethylammonium chloride, a cationic lipid

[0208] DC-chol: 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol;

[0209] DOPE: 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine;

[0210] mPEG-DMG: methoxypolyethylene glycol dimyristyl glycerol;

[0211] PEI: polyethylenimine;

[0212] EE: encapsulation efficiency;

[0213] PDI: polydispersity index;

[0214] HEK293T: human embryonic kidney cells;

[0215] ALC-0315: ((4-hydroxybutyl)azabicyclohexyl)bis(hexane-6,1-diyl)bis(2- hexyldecanoate);

[0216] DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine;

[0217] ALC-0159: methoxypolyethylene glycol bistrimethylammonium;

[0218] SM102: heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl) amino)octanoate);

[0219] Lipid-5: 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)heptadecan-9-yl octanoate;

[0220] DODAP: 1,2-dioleoyl-3-dimethylammonium-propane;

[0221] DLin-KC2-DMA: 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-(1,3)-dioxolane;

[0222] Lipid-29: 8-(((8-((1-ethylnonyl)oxy)-8-oxooctyl)(3-((2-(methylamino)-3,4- dioxo-1-cyclobuten-1-yl)amino)propyl)amino)octanoic acid-1-octyl nonyl ester;

[0223] C12-200: 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2- hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol);

[0224] PEG-DSG: pegylated-distearoyl-rac-glycerol;

[0225] DOSPA: 2,3-dioleyloxy-N-(2-(spermidinecarboxamido)ethyl)-N,N-dimethyl-1- propane ammonium hydrochloride. BRIEF DESCRIPTION OF DRAWINGS

[0226] Figure 1: Comparison of preparation process of delivery vehicle.

[0227] Figure 2A-2B: Effect of pH and EPC on encapsulation efficiency.

[0228] Figure 3: Effect of different molar ratios of EPC on expression.

[0229] Figure 4A-4B: Effect of different preparation methods on encapsulation efficiency and expression.

[0230] Figure 5A-5B: 10% EPC can promote delivery vehicle to produce stronger expression and immune effect in DC2.4 cells.

[0231] Figure 6: 10% EPC can improve the expression of delivery vehicle comparable to LNP in the prior art (abscissa from left to right: blank, group 1 to group 9).

[0232] Figure 7: Effect of different buffers on encapsulation efficiency.

[0233] Figure 8: Effect of different mixing methods on encapsulation efficiency.

[0234] Figure 9: Concentration optimization results.

[0235] Figure 10: Optimization of the ratio of delivery vehicle and nucleic acid.

[0236] Figure 11: Encapsulation ability for nucleic acids of different lengths.

[0237] Figure 12: Changes in physicochemical properties of delivery system after being placed at 25°C for 2 months. Changes in (a) particle size, (b) PDI, (c) encapsulation efficiency, and (d) in vitro cell expression after different storage times.

[0238] Figure 13: Luciferase expression imaging detection.

[0239] Figure 14: In vivo distribution of delivery vehicle of the present disclosure.

[0240] Figure 15: Organ targeting of delivery vehicle of the present disclosure.

[0241] Figure 16: Growth curve of mouse tumor.

[0242] Figure 17: Number of nodules in the lungs of mice.

[0243] Figure 18: IFN-γ expression level in the spleen of mice. DETAILED DESCRIPTION

[0244] The present disclosure is described in detail by the following examples, which are for illustrative purposes only and are not intended to be limiting.

[0245] Example 1

[0246] Example 1A. Preparation of delivery vehicle

[0247] It is understood that, although a specific luciferase mRNA is used as an exemplary cargo in the examples, the prepared delivery vehicles are equally suitable for delivery of other mRNAs, or protein, protein nucleic acid mixtures or complexes, and are independent of the specific sequence of the nucleic acid / protein.

[0248] 1. Preparation of solutions:

[0249] (1) Lipid mixture preparation: Different molar ratios of lipid solutions were prepared according to Table 1A, with a total lipid concentration of 10 mg / ml.

[0250] (2) mRNA working solution preparation: Take 20 μΐ of luciferase mRNA stock solution (1 mg / ml), add 380 μΐ of 10 mM pH 4.0 citric acid buffer and mix to obtain a 50 μg / ml mRNA solution.

[0251] 2. Preparation of blank liposomes (also referred to herein as empty vehicles, delivery vehicles):

[0252] Using a microfluidic device (INano L+, ImaTech), blank liposomes were prepared by setting the parameters as follows:

[0253] Total volume: 2.30 ml,

[0254] Flow rate ratio: lipid phase / aqueous phase = 1 / 3,

[0255] Flow rate: 20 ml / min,

[0256] Immediately after sample preparation, a 7-fold dilution was performed using 10 mM pH 4.0 citric acid buffer.

[0257] The mass ratio of vehicle / mRNA in this example is 20:1. The exemplary nucleic acid in this example is luciferase mRNA, which has a length ranging from 1000 nt to 5000 nt (as an example 1922 nt, linear).

[0258] 3. Ultrafiltration:

[0259] The sample prepared above was transferred to a 100 kd ultrafiltration tube and ultrafiltration was performed on a low temperature centrifuge with the following settings: speed: 3000 rpm, temperature: 4°C, time: 30 min.

[0260] The sample was ultrafiltrated to about 1 ml, 5 ml of 10 mM pH 4.0 citric acid buffer was added, and ultrafiltration was performed again. The ultrafiltration was stopped again when the sample was about 1 ml.

[0261] 4. Preparation of mRNA-loaded delivery vehicle

[0262] Take 200 μl of the blank liposome solution in step 2 (in the production scenario, it is more advantageous to use the empty vehicle after step 3 of ultrafiltration) in a 2 ml EP tube, take 200 μl of luciferase mRNA working solution, and add it to the blank liposome solution while vortexing. After adding, vortex for about 5S, and store the prepared sample at 4°C.

[0263] Table 1A

[0264] The data show that the optimal molar ratio of Lipid-5: DOPE: mPEG-DMG: cholesterol is 40 / 15 / 1.5 / 43.5.

[0265] Example 1B. Preparation of LNP (control method)

[0266] 1. Preparation of blank liposomes

[0267] Using a microfluidic device (INano L+, I-Nano), the parameters were set as follows to prepare LNP:

[0268] Total volume: 1.6 ml,

[0269] Flow rate ratio: lipid phase: aqueous phase = 1:3,

[0270] Flow rate: 20 ml / min,

[0271] Immediately after sample preparation, dilute 7 times with 1xPBS for subsequent ultrafiltration.

[0272] 2. Ultrafiltration: The prepared sample was moved into a 100 kd ultrafiltration tube, and ultrafiltration was performed on a low-temperature centrifuge. The centrifuge was set as follows: speed: 3000 RPM, temperature: 4°C, time: 30 min.

[0273] The sample was ultrafiltrated to about 1 ml, 5 ml of 10 mM pH 4.0 citric acid buffer was added, and ultrafiltration was performed again. The ultrafiltration was stopped again when the sample was about 1 ml.

[0274] Ultrafiltration was performed again, and the ultrafiltration was stopped again when the sample was about 1 ml.

[0275] The method of Example 1A first prepares blank liposomes, and then vortexes with mRNA; Example 1B is a lipid solution that directly mixes with mRNA solution to prepare LNP, and the mRNA solution concentrations used by the two are different, Example 1A is 50 μg / ml, and Example 1B is 167 μg / ml.

[0276] Example 1C. Effect of buffer type and pH on encapsulation

[0277] 1. Take 200 μl of blank liposome solution in step 3 of Example 1A above into a 2 ml EP tube, and disperse in different pH value acetic acid buffer (AB), citric acid buffer (CB), phosphoric acid buffer (PB) or PBS (pH 7.2-7.4) solution; take 200 μl of luciferase mRNA working solution, disperse in different pH value AB, CB, PB solution (the concentration of nucleic acid is 50 μg / ml), vortex and add to the blank liposome solution, after adding, vortex for about 5S, and store after preparation at 4°C.

[0278] Among them, Lipid-5: DOPE: mPEG-DMG: Cholesterol = molar ratio (40 / 15 / 1.5 / 43.5) accounts for 90%, and cationic lipid EPC accounts for 10%.

[0279] The results show that the encapsulation effect in CB buffer and PB buffer is better than that in acetic acid buffer (Figure 7).

[0280] 2. In addition, a wider range of different pH values was investigated for their effect on encapsulation efficiency. The results show, for example, that No. 11, 12, 13, 16, 17, 18, 21, 22, 23 all have good encapsulation potential, showing that the type and pH (4 to 9 (preferably 4 to 7.4)) of the dispersion solution has a significant effect on the encapsulation efficiency (Table 1B).

[0281] Table 1B

[0282] 3. In addition, the advantages obtained by using the above-mentioned optimized buffer as a dispersion solution also apply to other types of lipid composition (Table 1C).

[0283] Table 1C

[0284] Example 2. Physicochemical data detection

[0285] The delivery vehicle prepared in Example 1A and the LNP prepared in Example 1B were tested as follows.

[0286] 1. Particle size, PDI detection:

[0287] Take 20 μl sample, dilute 50 times with ultrapure water, and detect on Malvern ZETA particle size R PRO.

[0288] 2. Encapsulation efficiency detection: detected using Ribogreen kit and microplate reader.

[0289] 3. Cell transfection:

[0290] Transfect LNP or delivery carrier samples in HEK293T or DC2.4 cells (1 x 10 4 cells / well, 96-well plate) at a dose of 50 ng / well. After the transfection reagent was incubated with the cells for 4 h, the culture medium containing mRNA / delivery carrier was discarded, fresh cell culture medium was added, and the cells were incubated in a 37°C CO2 incubator overnight. The expression amount of luciferase protein was detected at 24 h after transfection.

[0291] Example 3. pH and cationic lipid content enhance encapsulation efficiency and improve cell expression

[0292] To improve the low encapsulation efficiency problem in the prior art LPX preparation method, on the basis of the composition of ionizable lipid, auxiliary phospholipid, cholesterol, and PEG lipid four components, this embodiment uses the method of reducing the pH of the solution or adding additional cationic lipids to improve the encapsulation efficiency of mRNA.

[0293] 1. Lipid solution preparation: prepare the corresponding lipid solution according to Table 2, wherein the molar ratio of ionizable lipid / auxiliary lipid (DOPE) / cholesterol / mPEG-DMG lipid = 40 / 15 / 43.5 / 1.5, and 10% molar amount of EPC is additionally added to prepare a 10 mg / ml lipid solution.

[0294] 2. The ultrafiltration medium corresponds to pH 7.4 PBS and pH 4 CBS solution, respectively.

[0295] 3. Prepare the delivery carrier according to the method in Example 1A, and characterize its particle size, PDI, and encapsulation efficiency according to Example 2.

[0296] The results of FIG. 2A and FIG. 2B show that the method of reducing the pH of the solution or adding additional cationic lipids can improve the encapsulation efficiency of mRNA.

[0297] Table 2

[0298] Example 4. Adjusting the content of cationic lipids improves the problem of high proportion of cationic lipids

[0299] To improve the problem of high cationic lipid ratio in the LPX preparation method in the prior art, on the basis of the composition of ionizable lipid, auxiliary phospholipid, cholesterol and PEG lipid, 10 mM of pH 4 citric acid buffer and different molar amounts of cationic lipid are added to improve the problem of high cationic lipid ratio and improve the mRNA encapsulation rate.

[0300] 1. Lipid solution preparation: prepare the corresponding lipid solution according to Table 3, wherein the molar ratio of ionizable lipid / DOPE / cholesterol / mPEG-DMG lipid is 40 / 15 / 43.5 / 1.5, and different molar amounts of EPC (1 / EPC, 2.5 / EPC, 5 / EPC, 17.5 / EPC, 33 / EPC, 55 / EPC) and 10% molar amounts of DOTAP and DC-cholesterol are additionally added to prepare a 10 mg / ml lipid solution.

[0301] 2. Prepare the delivery carrier in the manner of Example 1A, and characterize the particle size, PDI and cell expression according to Example 2.

[0302] 3. Table 3 and Figure 3 show that the addition of cationic lipids improves the encapsulation rate but reduces cell expression, which may be due to the possible toxicity caused by the high proportion of cations. The addition of 10% EPC cationic lipid has good particle physicochemical properties and cell expression, and is better than other types of cationic lipids DOTAP and DC-cholesterol.

[0303] Table 3

[0304] Example 5. Effect of lipid / mRNA ratio on encapsulation rate

[0305] Due to the high lipid / mRNA ratio in the traditional RTU composition, on the basis of the composition of ionizable lipid, auxiliary phospholipid, cholesterol and PEG lipid, 10 mM of pH 4 citric acid buffer and 10% EPC are added to investigate the effect of different lipid / mRNA mass ratios on the mRNA encapsulation rate.

[0306] 1. Lipid solution preparation: prepare the corresponding lipid solution according to Table 4, wherein the molar ratio of ionizable lipid / DOPE / cholesterol / mPEG-DMG lipid is 40 / 15 / 43.5 / 1.5, and 10% EPC is additionally added to prepare 1 / 2 / 3 / 4 / 5 / 7.5 / 10 mg / ml lipid solutions.

[0307] 2. Prepare the delivery carrier in the manner of Example 1A, and characterize the particle size, PDI and encapsulation rate according to Example 2.

[0308] 3. The results show that the mass ratio of the entire blank carrier:mRNA can reach 75-80% when the ratio is 15:1 to 20:1, which has a good encapsulation effect.

[0309] Table 4

[0310] Example 6. The preparation method of microfluidic mixing further enhances the encapsulation rate

[0311] Because the particles prepared by manual mixing in the traditional way have poor repeatability, there are problems of uneven particle size and incomplete encapsulation, so the differences in the physicochemical properties of samples prepared by different preparation methods are investigated.

[0312] 1. Preparation of solution: Prepare different compositions according to Table 5, wherein the molar ratio of ionizable lipid / DOPE / cholesterol / mPEG-DMG lipid is 40 / 15 / 43.5 / 1.5, and 10% EPC is additionally added to prepare a 10 mg / ml lipid solution.

[0313] Table 5

[0314] 2. Preparation of delivery system (carrying nucleic acid):

[0315] (1) Microfluidic method: Use microfluidic equipment (INano L+, Maianna) to set parameters according to the following method to prepare the delivery system:

[0316] Total volume: 0.4 ml,

[0317] Flow rate ratio: blank liposome: luciferase mRNA working solution = 1:1,

[0318] Flow rate: 20 ml / min,

[0319] After preparation, store at 4°C.

[0320] (2) Vortex method (control method):

[0321] Take 200 μl of blank liposome solution in a 2 ml EP tube, take 200 μl of luciferase mRNA working solution, and add it to the blank liposome solution while vortexing. After adding, vortex for about 5S, and store at 4°C after preparation.

[0322] 3. Characterize the particle size, PDI and cell expression of the delivery carrier according to the method in Example 2.

[0323] 4. The results of FIGS. 4A and 4B show that the sample prepared by microfluidic method has smaller particle size, more uniform particles, and higher encapsulation rate, and the encapsulation rate is further improved to the level of 90%, and the cell expression is also improved, which shows the superiority of the new preparation process.

[0324] Example 7. Cationic lipid enhanced delivery vehicle for cell expression and immunogenicity in immune cells

[0325] The delivery vehicle with additional 10% EPC and without 10% EPC after optimization were evaluated in immune cells to test their feasibility as mRNA tumor vaccine.

[0326] Figures 5A and 5B results show that the delivery vehicle with additional 10% EPC has significantly better effect than the traditional vehicle, and is more suitable for mRNA tumor vaccine.

[0327] Example 8. Cationic lipid enhanced delivery vehicle has universality and can be applied to ionizable lipids and compositions widely studied in clinic

[0328] The lipid solution was prepared according to the molar ratio of Table 6, and the lipid concentration was 10 mg / ml. The sample preparation of delivery vehicle, LNP, particle characterization and cell verification were carried out according to Example 1A and Example IB.

[0329] Figure 6 results show that the method of adding cationic EPC has universality, can improve the encapsulation efficiency and expression effect of the traditional delivery vehicle preparation method, and the expression effect can be comparable or higher than that of the traditional LNP preparation method.

[0330] Table 6

[0331] The encapsulation efficiency of LNP prepared in Example IB is about 80-90%, and the encapsulation efficiency of the delivery vehicle of Example 1A is comparable; the preferred composition can even reach an encapsulation efficiency of 90% by microfluidic method, and the effect is comparable to LNP, and the encapsulation efficiency of the traditional vortex mixing method is slightly lower than that of LNP.

[0332] Example IB is a traditional positive control preparation method of LNP, which has high encapsulation efficiency, good transfection effect, but the production and preparation are complex. As can be seen from Figure 6, the delivery system prepared by the method of the present application Example 1A can significantly improve the low encapsulation efficiency and poor cell transfection of the traditional LPX, and the improved effect is comparable to the positive control LNP, but the production process is simplified (Figure 1).

[0333] Example 9. Other ways of introducing cationic lipids

[0334] The lipid solution was prepared according to the molar ratio of Table 7A, and the lipid concentration was 10 mg / ml. The delivery vehicle, particle characterization and cell verification were carried out according to Example 1A and Example IB.

[0335] Table 7A NA: value not applicable.

[0336] The results show that the mRNA encapsulation efficiency can be improved by the introduction of cationic lipids or cationic polymers with two components, three components, four components.

[0337] Example 10. Alternative Preparation Examples

[0338] The ionizable lipids / helper lipids / sterols / PEGylated lipids as defined previously, following the procedure of Example 1A, were used to prepare the following alternative delivery vehicles:

[0339] 1. Lipid-5 is replaced with: DOTMA, DOTAP, DDAB, DOSPA, DODAC, DODAP, DC-Chol, DMRIE, DMOBA, DLinDMA, DLenDMA, CLinDMA, DMORIE, DLDMA, DMDMA, DOGS, N4-Cholyl- spermine, DLin-KC2-DMA, DLin-MC3-DMA, ALC-0159, Lipid-29, C12-200, ALC-0315, or SM-102;

[0340] 2. Replace DOPE with: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-didodecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyloleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, or lysophosphatidylethanolamine (LPE);

[0341] 3. Replace mPEG-DMG with: ALC-0159, mPEG-DSPE, PEG-DSG, mPEG-DPPE, mPEG-DOPE, mPEG-DMPE, or DSPE-PEG;

[0342] 4. EPC is replaced by: DC-Cholesterol, DOTAP, DOTMA, DDAB, DOSPA, DTAB, TTAB, CTAB, DORI, DORIE, DPRIE, DSRIE, DMRIE or DOGS;

[0343] 5. Cholesterol is replaced by: Coprostanol, Sitostanol, Ergostanol, Campestanol, Brassicasterol, Stigmasterol, Brassicasterol, Tomatidine, Ursolic acid or α-tocopherol;

[0344] The delivery carriers obtained above are tested with reference to Example 2, and compared with conventional LPX, similar improvement in encapsulation rate can be observed, and the improved effect is equivalent to that of positive control LNP, but the production process is simplified.

[0345] Table 7B * No cationic lipid, only first lipid composition.

[0346] Table 7C * No cationic lipid, only first lipid composition.

[0347] Table 7D * Cationic lipid EPC is replaced by 5% or 10% DOTMA.

[0348] Table 7E Note: Lipid-5 / DSPC / PEG lipid / Cholesterol = 50:10:1.5:38.5. No cationic lipid, only first lipid composition.

[0349] Table 7F Note: Lipid-5 / DSPC / mPEG-DMG / sterol = 50:10:1.5:38.5; No cationic lipid, only first lipid composition.

[0350] Table 7G Note: ALC-0315 / co-lipid / PEG lipid / Cholesterol = 50:10:1.5:38.5; No cationic lipid, only first lipid composition.

[0351] Table 7H Note: No cationic lipid, only first lipid composition.

[0352] Comparative Example 1. Effect of different nucleic acid loading methods on encapsulation rate

[0353] Table 8

[0354] The results show that Method One and Method Three have better encapsulation rates, Method One and Method Two have smaller dispersion coefficients, and the cell expression of the three is close, and Method One and Method Three are preferred (Figure 8).

[0355] In production scenario, the encapsulation efficiency achieved by microfluidics is slightly higher than manual / vortex (as shown in the results in Example 6). However, in clinical application scenario, a more convenient operation mode is more desirable. The purpose of this comparative example is to demonstrate that, in the absence of microfluidic device loading nucleic acid drugs, by vortex mixing or Schlenk flask manual mixing only, the optimized lipid composition of the present disclosure can still meet the clinically available encapsulation effect.

[0356] Comparative Example 2. Effect of different concentrations of blank liposomes and nucleic acids on preparation

[0357] Table 9

[0358] High concentration mixing slightly increases particle size and improves cell expression, but has no significant effect on encapsulation efficiency and PDI. Nanoparticles can be prepared in the range of 0.01-1 mg / ml mRNA (Figure 9), preferably 0.2-1 mg / ml.

[0359] Comparative Example 3. Effect of different volume ratios on encapsulation

[0360] Table 10

[0361] The range of 20 / 1 to 1 / 20 can achieve good mixing and encapsulation effect (Figure 10).

[0362] Example 1. Encapsulation ability for nucleic acids of different sequence lengths

[0363] The blank liposome solution was mixed with GFP mRNA (here referring to 1000 nt mRNA), luciferase mRNA (here referring to 2000 nt mRNA) and nCoV mRNA (here referring to 4000 nt mRNA) respectively. And the physicochemical properties after mixing were investigated, the results showed that the blank liposome could well encapsulate RNA of different sequence lengths (Figure 11).

[0364] Herein, Lipid-5: DOPE: mPEG-DMG: Cholesterol = molar ratio (40 / 15 / 1.5 / 43.5) accounted for 90%, and cationic lipid EPC accounted for 10%.

[0365] Example 2. Evaluation of stability

[0366] The blank liposome (Lipid-5: DOPE: mPEG-DMG: cholesterol = molar ratio (40 / 15 / 1.5 / 43.5) 90%, cationic lipid EPC 10%) solution was placed at room temperature for different time, and the physicochemical properties after mixing with luciferase mRNA were characterized. The results showed that the blank liposome placed for 2 months still had good expression effect (Figure 12).

[0367] Effect Example 3. In vivo effect evaluation

[0368] Mouse live imaging:

[0369] 10 μg of luciferase mRNA + delivery vehicle (prepared in Example 1A; Lipid-5: DOPE: mPEG-DMG: cholesterol = molar ratio (40 / 15 / 1.5 / 43.5) 90%, cationic lipid EPC 10%) was taken; or

[0370] 10 μg of luciferase mRNA + control delivery vehicle (Lipid-5: DOPE: mPEG-DMG: cholesterol = molar ratio (40 / 15 / 1.5 / 43.5) 100%; prepared by the control method in Example 1B);

[0371] Balb / c (6-8 weeks) mice were injected intramuscularly, and 6 hours after administration, luciferin substrate was injected, and the luciferase expression imaging was detected by small animal live imaging instrument (Figure 13).

[0372] The delivery vehicle of the present disclosure had expression at the muscle injection site comparable to the control vehicle after intramuscular administration, but the expression in the liver was significantly lower than the control vehicle. It showed that the delivery vehicle of the present disclosure had better in vivo distribution characteristics as an mRNA tumor vaccine carrier, which could reduce the potential safety risk. Because excessive liver expression could cause potential systemic adverse reactions (Figure 14).

[0373] The delivery vehicle of the present disclosure was mainly distributed in the spleen and a small amount in the liver after intravenous administration; the control vehicle was mainly distributed in the liver and a small amount in the spleen, which showed that the delivery vehicle of the present disclosure had better spleen targeting (Figure 15).

[0374] Balb / c (6-8 weeks) mice were randomly divided into groups, and 2.5 x 10 5 CT26 cells, 3, 7, 10, and 14 days after tumor inoculation, 10 μg of mRNA + delivery vehicle of the present disclosure or mRNA + control vehicle was injected intramuscularly. During the treatment, the growth of tumor cells in mice was monitored (Figure 16). On the 16th day after tumor inoculation, the mice were dissected, and the number of nodules in the lungs of the mice was checked. At the same time, the specific immune response of the mice was detected (Figures 17-18).

[0375] The results show that the delivery vehicle of the present disclosure has a therapeutic effect on the personalized tumor vaccine comparable to the control vehicle.

[0376] Example 4. Encapsulation of different cargos

[0377] In addition to delivering nucleic acids as cargo molecules, other types of cargos can also be delivered. Different types of cargo molecules were encapsulated using the blank vehicle prepared in Example 1A (Lipid-5: DOPE: mPEG-DMG: Cholesterol = molar ratio (40 / 15 / 1.5 / 43.5) at 90%, cationic lipid EPC at 10%). Mass ratio of blank vehicle: cargo = 20:1.

[0378] Table 11

[0379] The results show that the blank vehicle prepared in this application can effectively encapsulate nucleic acids, proteins, or complexes of the two.

Claims

1. A delivery vehicle comprising or consisting of: • a first lipid composition; and • optionally, a second lipid composition consisting of a cationic lipid; wherein, the first lipid composition comprises or consists of: - an ionizable lipid, - a helper lipid, - a PEGylated lipid, and - a sterol; wherein, the ionizable lipid is selected from the group consisting of: ALC-0315, SM-102, Lipid-5, DODAP, DLin-KC2-DMA, Lipid-29, C12-200; the helper lipid is selected from the group consisting of: DMPC, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DUPC), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, palmitoyloleoyl phosphatidyl ethanolamine (POPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC); the PEGylated lipid is selected from the group consisting of: ALC-0159, mPEG-DMG, mPEG-DSPE, PEG-DSG, mPEG-DPPE, mPEG-DOPE, mPEG-DMPE; the sterol is selected from the group consisting of: cholesterol, campesterol, brassicasterol, sitosterol, coprostanol, ergosterol, stigmasterol, brassicacid sterol, tomatidine, ursolic acid, a-tocopherol; preferably, the cationic lipid is selected from the group consisting of: EPC, DC-Cholesterol, DOTAP, DOSPA, DOTMA; preferably EPC.

2. The delivery vehicle of claim 1, wherein, when the delivery vehicle comprises the second lipid composition: the first lipid composition represents from 45% to 99% w / w of the delivery vehicle; the second lipid composition represents from 1% to 55% w / w of the delivery vehicle; preferably, the first lipid composition represents 90% w / w of the delivery vehicle; the second lipid composition represents 10% w / w of the delivery vehicle.

3. The delivery vehicle according to claim 1, wherein in the first lipid composition: the mole fraction of the ionizable lipid is from 10 to 90, preferably from 30 to 600; the mole fraction of the helper lipid is from 0 to 30, preferably from 10 to 20; the mole fraction of the PEGylated lipid is from 0 to 10, preferably from 1 to 2.5; the mole fraction of the sterol is from 9 to 70; preferably from 25 to 60; More preferably, the molar ratio of said ionizable lipid: said helper lipid: said PEG lipid: said sterol is selected from: 40：10：2：48、 40：10：2.5：47.5、 40：15：1.5：43.5、 40：15：2：43、 40：20：1.5：38.5、 45：5：2：48、 45：15：1.5：38.5、 50：10：1.5：38.5、 50：10：2：38、 50：15：2.5：32.5 50：15：1.5：33.5、 50：15：2：33、 50：20：1.5：28.5、 30：10：2：58。 4. The delivery vehicle of claim 1 having a mean particle size ranging from: 40 to 1000 nm; preferably 40 to 200 nm; and / or a zeta potential ranging from: -10 to +60 mV; preferably +15 to +35 mV.

5. The delivery vehicle of claim 1 selected from: A)： 1) 100% of a first lipid composition comprising: Lipid-5: DOPE: mPEG-DMG: Cholesterol in a molar ratio of 40: 15: 1.5: 43.5; 2) 100% of a first lipid composition comprising: ALC-0315: DSPC: ALC-0159: Cholesterol in a molar ratio of 50: 10: 1.5: 38.5; 3) 100% of a first lipid composition comprising: SM102: DSPC: mPEG-DMG: Cholesterol in a molar ratio of 50: 10: 1.5: 38.5; 4) 100% of a first lipid composition comprising: SM-102: DSPC: mPEG-DMG: Sitosterol in a molar ratio of 40: 10: 2: 48; 5) 100% of a first lipid composition comprising: SM-102: DSPC: mPEG-DMG: Sitosterol in a molar ratio of 45: 5: 2: 48; 6) 100% of a first lipid composition comprising: SM-102: DSPC: mPEG-DMG: Sitosterol in a molar ratio of 45: 15: 1.5: 38.5; 7) 100% of a first lipid composition comprising: SM-102: DSPC: mPEG-DMG: Sitosterol in a molar ratio of 50: 10: 1.5: 38.5; 8) 100% of a first lipid composition comprising: SM-102: DSPC: mPEG-DMG: Sitosterol in a molar ratio of 50: 15: 2: 33; 9) 100% of a first lipid composition comprising: SM-102: DOPE: mPEG-DMG: Sitosterol in a molar ratio of 50: 20: 1.5: 28.5; 10) 100% of a first lipid composition comprising: SM-102: DOPE: mPEG-DMG: Sitosterol in a molar ratio of 50: 15: 1.5: 33.5; 11) 100% of a first lipid composition comprising: SM-102: DSPC: mPEG-DMG: (Sitosterol + Cholesterol) in a molar ratio of 45: 15: 1.5: 38.5; 12) 100% of a first lipid composition comprising: Lipid-5: DSPC: mPEG-DPPE: Cholesterol in a molar ratio of 50: 10: 1.5: 38.5; 13) 100% first lipid composition comprising: Lipid-5: DSPC: mPEG-DMG: brassicasterol in a molar ratio of 50:10:1.5:38.5; 14) 100% first lipid composition comprising: ALC-0315: DSPC: ALC-0159: cholesterol in a molar ratio of 50:10:1.5:38.5; 15) 100% first lipid composition comprising: ALC-0315: DSPE: ALC-0159: cholesterol in a molar ratio of 50:10:1.5:38.5; 16) 100% first lipid composition comprising: ALC-0315: DMPC: ALC-0159: cholesterol in a molar ratio of 50:10:1.5:38.5; 17) 100% first lipid composition comprising: ALC-0315: DSPC: mPEG-DMG: cholesterol in a molar ratio of 50:10:1.5:38.5; 18) 100% first lipid composition comprising: ALC-0315: DSPE: mPEG-DMG: cholesterol in a molar ratio of 50:10:1.5:38.5; 19) 100% first lipid composition comprising: ALC-0315: DSPC: mPEG-DMG: cholesterol in a molar ratio of 50:10:1.5:38.5; 20) 100% first lipid composition comprising: ALC-0315: DSPC: mPEG-DMG: cholesterol in a molar ratio of 40:10:2.5:47.5; 21) 100% first lipid composition comprising: ALC-0315: DSPC: mPEG-DMG: cholesterol in a molar ratio of 40:15:2:43; 22) 100% first lipid composition comprising: ALC-0315: DSPC: mPEG-DMG: cholesterol in a molar ratio of 40:20:1.5:38.5; 23) 100% first lipid composition comprising: ALC-0315: DSPC: mPEG-DMG: cholesterol in a molar ratio of 50:10:2:38; 24) 100% first lipid composition comprising: ALC-0315: DSPC: mPEG-DMG: cholesterol in a molar ratio of 50:15:2.5:32.5; 25) 100% first lipid composition comprising: ALC-0315: DSPC: mPEG-DMG: cholesterol in a molar ratio of 30:10:2:58; B): 1) 90% first lipid composition, 10% cationic lipid; The first lipid composition comprises: Lipid-5: DOPE: mPEG-DMG: Cholesterol at a molar ratio of 40: 15: 1.5: 43.5; 2) 90% first lipid composition, 10% cationic lipid; The first lipid composition comprises: ALC-0315: DSPC: ALC-0159: Cholesterol at a molar ratio of 50: 10: 1.5: 38.5; 3) 90% first lipid composition, 10% cationic lipid; The first lipid composition comprises: SM102: DSPC: mPEG-DMG: Cholesterol at a molar ratio of 50: 10: 1.5: 38.5; 4) 90% first lipid composition, 10% cationic lipid; The first lipid composition comprises: SM-102: DSPC: mPEG-DMG: Sitosterol at a molar ratio of 40: 10: 2: 48; 5) 90% first lipid composition, 10% cationic lipid; The first lipid composition comprises: SM-102: DSPC: mPEG-DMG: Sitosterol at a molar ratio of 45: 5: 2: 48; 6) 90% first lipid composition, 10% cationic lipid; The first lipid composition comprises: SM-102: DSPC: mPEG-DMG: Sitosterol at a molar ratio of 45: 15: 1.5: 38.5; 7) 90% first lipid composition, 10% cationic lipid; The first lipid composition comprises: SM-102: DSPC: mPEG-DMG: Sitosterol at a molar ratio of 50: 10: 1.5: 38.5; 8) 90% first lipid composition, 10% cationic lipid; The first lipid composition comprises: SM-102: DSPC: mPEG-DMG: Sitosterol at a molar ratio of 50: 15: 2: 33; 9) 90% first lipid composition, 10% cationic lipid; The first lipid composition comprises: SM-102: DOPE: mPEG-DMG: Sitosterol at a molar ratio of 50: 20: 1.5: 28.5; 10) 90% first lipid composition, 10% cationic lipid; The first lipid composition comprises: SM-102: DOPE: mPEG-DMG: Sitosterol at a molar ratio of 50: 15: 1.5: 33.5; 11) 90% first lipid composition, 10% cationic lipid; The first lipid composition comprises: SM-102: DSPC: mPEG-DMG: (Sitosterol and Cholesterol) at a molar ratio of 45: 15: 1.5: 38.5; 12) 90% first lipid composition, 10% cationic lipid; The first lipid composition comprises: Lipid-5: DSPC: mPEG-DPPE: Cholesterol at a molar ratio of 50: 10: 1.5: 38.5; 13) 90% first lipid composition, 10% cationic lipid; The first lipid composition comprises: Lipid-5: DSPC: mPEG-DMG: Brassinosteroid at a molar ratio of 50: 10: 1.5: 38.5; 14) 90% first lipid composition, 10% cationic lipid; the first lipid composition comprises ALC-0315 : DSPC : ALC-0159 : cholesterol at a molar ratio of 50 : 10 : 1.5 : 38.5; 15) 90% first lipid composition, 10% cationic lipid; the first lipid composition comprises ALC-0315 : DSPE : ALC-0159 : cholesterol at a molar ratio of 50 : 10 : 1.5 : 38.5; 16) 90% first lipid composition, 10% cationic lipid; the first lipid composition comprises ALC-0315 : DMPC : ALC-0159 : cholesterol at a molar ratio of 50 : 10 : 1.5 : 38.5; 17) 90% first lipid composition, 10% cationic lipid; the first lipid composition comprises ALC-0315 : DSPC : mPEG-DMG : cholesterol at a molar ratio of 50 : 10 : 1.5 : 38.5; 18) 90% first lipid composition, 10% cationic lipid; the first lipid composition comprises ALC-0315 : DSPE : mPEG-DMG : cholesterol at a molar ratio of 50 : 10 : 1.5 : 38.5; 19) 90% first lipid composition, 10% cationic lipid; the first lipid composition comprises ALC-0315 : DSPC : mPEG-DMG : cholesterol at a molar ratio of 50 : 10 : 1.5 : 38.5; 20) 90% first lipid composition, 10% cationic lipid; the first lipid composition comprises ALC-0315 : DSPC : mPEG-DMG : cholesterol at a molar ratio of 40 : 10 : 2.5 : 47.5; 21) 90% first lipid composition, 10% cationic lipid; the first lipid composition comprises ALC-0315 : DSPC : mPEG-DMG : cholesterol at a molar ratio of 40 : 15 : 2 : 43; 22) 90% first lipid composition, 10% cationic lipid; the first lipid composition comprises ALC-0315 : DSPC : mPEG-DMG : cholesterol at a molar ratio of 40 : 20 : 1.5 : 38.5; 23) 90% first lipid composition, 10% cationic lipid; the first lipid composition comprises ALC-0315 : DSPC : mPEG-DMG : cholesterol at a molar ratio of 50 : 10 : 2 : 38; 24) 90% first lipid composition, 10% cationic lipid; the first lipid composition comprises ALC-0315 : DSPC : mPEG-DMG : cholesterol at a molar ratio of 50 : 15 : 2.5 : 32.5; 25) 90% first lipid composition, 10% cationic lipid; the first lipid composition comprises ALC-0315 : DSPC : mPEG-DMG : cholesterol at a molar ratio of 30 : 10 : 2 :

58.

6. Use of the delivery vector of any one of claims 1-5 in the manufacture of a medicament, wherein: the drug is selected from the group consisting of: nucleic acid cell transfection reagent, nucleic acid protein replacement drug, nucleic acid vaccine; Preferably, the nucleic acid vaccine is used for treating tumor or preventing recurrence of tumor; Preferably, the nucleic acid is selected from the group consisting of siRNA, mRNA, sgRNA, DNA, or a combination thereof; preferably mRNA.

7. Use of the delivery vehicle of any one of claims 1-5 for delivering nucleic acid to target cells; Preferably, the nucleic acid is selected from the group consisting of siRNA, mRNA, sgRNA, DNA, or a combination thereof; Preferably, the target cells are located in an organ selected from the group consisting of: liver, spleen; more preferably spleen; Preferably, the delivery vehicle is prepared into a dosage form selected from the group consisting of: intravenous administration, local administration, intratumoral administration, intramuscular administration, subcutaneous administration.

8. The use of claim 6 or 7, wherein: the mRNA is linear or circular; the mRNA is negatively charged; the mRNA has a length of 20 nt to 20000 nt.

9. The use of claim 6 or 7, wherein: the mass ratio of the delivery vehicle and the nucleic acid is 1:1 to 80:1; preferably 15:1 to 20:

1.

10. A nucleic acid delivery system, comprising: the delivery vehicle of any one of claims 1-5; and nucleic acid; wherein, the nucleic acid is selected from the group consisting of siRNA, mRNA, sgRNA, DNA, or a combination thereof; preferably mRNA; the mass ratio of the delivery vehicle and the nucleic acid is 1:1 to 80:1; preferably 15:1 to 20:

1.

11. A method of preparing a nucleic acid delivery system, comprising the steps of: 1) providing a first lipid composition and a second lipid composition to form a delivery vehicle, such that the total concentration of lipids in the delivery vehicle is 1 mg / ml to 80 mg / ml, preferably 10 mg / ml; contacting (preferably mixing) the delivery vehicle and a buffer with pH<9 (preferably pH<7, more preferably 10 mM pH 4.0 citric acid buffer) by microfluidics, preferably the volume ratio of the delivery vehicle and the buffer is 1:3; The parameters of the microfluidics include: flow rate 1 ml / min to 200 ml / min, temperature 4°C to 70°C; preferably, flow rate 12 ml / min to 20 ml / min, temperature 20°C to 30°C; 2) diluting the solution obtained in step 1) with a buffer with pH<9 (preferably pH<7, more preferably 10 mM pH 4.0 citric acid buffer) by 1 to 20 times, preferably 7 times, in volume; 3) subjecting the solution obtained in step 2) to ultrafiltration, the parameters of the ultrafiltration include: ultrafiltration rotation speed 0 rpm to 5000 rpm, centrifugation time 0 min to 180 min, centrifugation temperature 2°C to 30°C; preferably, ultrafiltration rotation speed 2000 rpm to 3000 rpm, centrifugation time 15 min to 30 min, centrifugation temperature 2°C to 8°C; 4) providing nucleic acid and the solution obtained in step 3) in a buffer, respectively, such that the concentration of the nucleic acid is 10 μg / ml to 1000 μg / ml, preferably 50 μg / ml; the pH of the buffer is independently 4 to 9 (preferably 4 to 7.4), respectively; the buffer is independently selected from the group consisting of: phosphate buffer, citrate buffer, acetate buffer, histidine buffer, histidine salt buffer, HEPES, Tris salt, preferably: phosphate buffer, citrate buffer; 5) contacting (preferably mixing) the delivery vehicle and the nucleic acid by microfluidics, the volume ratio of the delivery vehicle and the nucleic acid is 20:1 to 1:20; the parameters of the microfluidics include: flow rate 1 ml / min to 200 ml / min, flow rate ratio 0.1:1 to 1:0.1, volume ratio 1:10 to 10:1, temperature 4°C to 70°C; preferably, flow rate 12 ml / min to 20 ml / min, flow rate ratio 0.3:1 to 1:0.3, volume ratio 1:5 to 5:1, temperature 20°C to 30°C; 6) obtaining the nucleic acid delivery system; wherein, steps 1) to 3) as a whole and step 4) can be interchanged in order or performed in parallel; the first lipid composition and the second lipid composition are as defined in any one of claims 1-3; the nucleic acid is selected from the group consisting of siRNA, mRNA, sgRNA, DNA, or a combination thereof; preferably mRNA; the mass ratio of the delivery vehicle and the nucleic acid is 1:1 to 80:1; preferably 15:1 to 20:

1.

12. A method for preparing a nucleic acid delivery system, comprising steps of: steps 1) to 4) as defined in claim 11; 5) contacting the delivery vehicle and the nucleic acid by manual mixing and / or vortex mixing in a container, the volume ratio of the delivery vehicle and the nucleic acid is 20:1 to 1:20; 6) obtaining the nucleic acid delivery system; wherein steps 1) to 3) as a whole and step 4) can be interchanged in order or performed in parallel; the first lipid composition and the second lipid composition are as defined in any one of claims 1-3; the nucleic acid is selected from the group consisting of siRNA, mRNA, sgRNA, DNA, or a combination thereof; preferably mRNA; the mass ratio of the delivery vehicle and the nucleic acid is 1:1 to 80:1; preferably 15:1 to 20:

1.

13. A nucleic acid delivery system, which is obtained by the method of claim 11 or 12.

14. Use of the nucleic acid delivery system of claim 10 or 13 in the preparation of a medicament, wherein: the medicament is selected from the group consisting of: nucleic acid cell transfection reagent, nucleic acid protein replacement drug, nucleic acid vaccine; preferably, the nucleic acid vaccine is used for treating tumor or preventing recurrence of tumor.

15. Use of the delivery vehicle of any one of claims 1-5 for delivering cargo to target cells; the cargo is selected from the group consisting of: protein, complex of nucleic acid and protein, mixture of nucleic acid and protein; preferably, the target cells are located in an organ selected from the group consisting of: liver, spleen; more preferably spleen; preferably, the delivery vehicle is prepared into a dosage form for administration selected from the group consisting of: intravenous administration, local administration, intratumoral administration, muscle administration, subcutaneous administration.

16. The use of claim 15, wherein the mass ratio of the delivery vehicle and the cargo is 1:1 to 80:1; preferably 15:1 to 20:

1.

17. A cargo delivery system comprising: the delivery vehicle of any one of claims 1-5; and a cargo; wherein, the cargo is selected from the group consisting of: a protein, a nucleic acid, and a complex of a protein and a nucleic acid, and a mixture of a protein and a nucleic acid; the mass ratio of the delivery vehicle to the cargo is 1:1 to 80:1; preferably 15:1 to 20:1.

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