Nucleic acid nano preparation with long-term stability as well as preparation method and application of nucleic acid nano preparation

Abstract

The invention discloses a nucleic acid nano preparation with long-term stability as well as a preparation method and application thereof. The nucleic acid nano preparation is prepared from a nucleic acid aqueous solution which is prepared by taking a polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic acid-glycolic acid) copolymer, cationic lipid, ionizable lipid, phospholipid, polyethylene glycol lipid and a cholesterol material as an organic phase. The preparation method is carried out in a micro-channel reactor, and then organic solvent evaporation, concentration and freeze-drying are carried out to obtain the product. The nucleic acid nano preparation disclosed by the invention can realize large-scale, continuous and intelligent preparation based on a micro-channel reactor, and has the advantages of long-term stability and high efficiency. The preparation method provided by the invention can be used for large-scale preparation and long-term storage, can be applied to treatment of various diseases including tumors, virus infection, hereditary metabolic diseases and the like, and has huge clinical application potential.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a nucleic acid nanoparticle preparation with long-term stability which is continuously prepared based on a microchannel reactor, and a preparation method and application thereof. Background Art

[0002] Nucleic acid drugs, as a therapeutic agent composed of DNA or RNA, treat a range of diseases, including genetic diseases, cancer and viral diseases, by modifying or regulating the expression of target genes. There are many types of such drugs, covering small interfering RNA (siRNA), messenger RNA (mRNA), antisense oligonucleotides (ASOs), microRNA (miRNA) mimics or inhibitors, and CRISPR-Cas9 systems. In recent years, nucleic acid drugs have received widespread attention from the scientific research community due to their huge application potential. In 2018, the U.S. Food and Drug Administration (FDA) approved the first small interfering RNA (siRNA)-based therapeutic drug Onpattro, which is used to treat a rare genetic neurological disease. Two years later, in 2020, vaccines based on messenger RNA (mRNA) technology, such as the COVID-19 vaccine developed by Pfizer-BioNTech and Moderna, their rapid development and emergency use authorization marked a major breakthrough in the field of nucleic acid drugs, and also highlighted the huge clinical application potential of nucleic acid drug technology.

[0003] However, the application of these drugs faces a series of challenges, including drug stability and delivery issues, potential immune responses, and how to ensure long-term safety and effectiveness. In order to solve these problems, researchers have developed a variety of nucleic acid delivery systems. The nanocarriers currently under research and clinical application for nucleic acid drugs can be mainly divided into the following categories: 1. Lipid nanoparticles (LNPs). LNPs are the most commonly used nucleic acid delivery system, especially in the delivery of mRNA vaccines. LNPs are composed of phospholipids, cholesterol, auxiliary lipids and polyethylene glycol (PEG), which can effectively protect nucleic acids from degradation by enzymes in the body and help them enter cells; 2. Polymer delivery systems, such as polyethyleneimine (PEI) and polylactic-co-glycolic acid (PLGA), can form stable complexes with nucleic acids, promote cellular endocytosis and endosomal escape through charge interactions, thereby improving the delivery efficiency of nucleic acids; 3. Coupled delivery carriers are used to improve the bioavailability and efficacy of drug molecules by chemically modifying them with materials, for example, coupling siRNA with polymers, peptides, antibodies, etc.

[0004] Although the application prospects of nucleic acid drugs are very bright, some challenges still need to be solved before they can be widely used clinically. On the one hand, how to effectively deliver nucleic acid drugs to target cells and tissues will affect the efficacy of nucleic acids. On the other hand, the biggest problem for current mRNA-LNP vaccines is long-term storage, which is affecting the use, transportation and application scope of vaccines. There is almost no data on the long-term storage stability of mRNA-LNP preparations in public information. Moderna and BioNTech / Pfizer's new crown vaccines need to be stored at -15 to -25°C and -60 to -90°C, respectively. mRNA-LNP needs to be stored frozen, which is a great challenge for the global transportation, storage and circulation of vaccines. This storage challenge comes from the complex interactions between multiple lipid components and the instability of mRNA, which is sensitive to oxygen, moisture, enzymes, pH and other factors. Therefore, there is an urgent need to develop mRNA vaccines with more stable properties and no need for frozen storage.

[0005] In the previous study, the inventor used the clinically approved pharmaceutical excipients polyethylene glycol-polylactic acid and cationic lipids to prepare nanocarriers encapsulating siRNA by double emulsification. Specifically: polyethylene glycol-polylactic acid and cationic lipids were dissolved in organic phase dichloromethane (250 μL), and an aqueous solution of mRNA (100 μL) was added, and the first emulsification was performed through an ultrasonic probe; then the aqueous phase (2.5 mL) was added, and the carrier was emulsified to obtain a nanoformulation encapsulating mRNA. The nanoformulation can efficiently encapsulate nucleic acid drugs and achieve in vivo nucleic acid drug delivery. Therefore, obtaining excellent stability to prevent nucleic acids from being degraded, so that they can be stored for a long time, and nanoformulations with biological functions such as good transfection efficiency need further exploration. Summary of the invention

[0006] Based on this, the purpose of the present invention is to provide a large-scale, continuous and intelligent preparation of nucleic acid nanoformulations with long-term stability based on a microchannel reactor, as well as a preparation method and application thereof.

[0007] The first aspect of the present invention is to provide a nucleic acid nanoformulation with long-term stability, which is prepared by using an aqueous solution of nucleic acid as the inner aqueous phase and materials containing polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic-co-glycolic acid) copolymer, lipids (ionizable lipids and / or cationic lipids), cholesterol, and phospholipids as the organic phase.

[0008] As a preferred method, a buffer containing nucleic acid is used as an aqueous phase, and a feed containing a polymer, a lipid, cholesterol, and a phospholipid is dissolved in an organic solvent as an organic phase;

[0009] The buffer is a citric acid buffer or a sodium citrate buffer,

[0010] The polymer is polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic acid-glycolic acid) copolymer, and the lipid is an ionizable lipid, or a mixture of an ionizable lipid and a cationic lipid;

[0011] The concentration of the nucleic acid in the aqueous phase is 50 μg / mL-100 μg / mL; in the organic phase, the concentration of the polymer is 0.05 mg / mL-22 mg / mL, the concentration of the lipid is 0.5 mg / mL-4 mg / mL, the concentration of the cholesterol is 1 mg / mL-2 mg / mL, and the concentration of the phospholipid is 0.1 mg / mL-1 mg / mL;

[0012] The mass percentage of the polymer in the organic phase feed is 6%-60%, further 12%-60%.

[0013] The nucleic acid may be siRNA (small interfering RNA) or mRNA (messenger RNA).

[0014] The second aspect of the present invention is to provide a method for large-scale preparation of the above-mentioned nucleic acid nanoparticles, which specifically includes building a large-scale production platform for nucleic acid nanopharmaceutical preparations using microchannel reactors, etc., to achieve continuous preparation and online quality control of nanopharmaceuticals, and introduce automation and intelligent control technologies to improve the production process of nanopharmaceutical preparations, forming a complete set of original key technology reserves from research to production.

[0015] A method for preparing the above-mentioned nucleic acid nanoparticle preparation comprises the following steps: (1) continuous preparation in a microchannel reactor: dissolving the polymer, lipid, cholesterol, and phospholipid materials in an organic solvent as an organic phase, and a citrate buffer or sodium citrate buffer of nucleic acid as an aqueous phase;

[0016] In the microchannel reactor, the aqueous phase and the organic phase are respectively passed through the channels into the reaction plate for mixed reaction to obtain a nucleic acid nanoparticle preparation solution;

[0017] (2) dialysis to remove organic solvents;

[0018] (3) concentrating to obtain a concentrated nucleic acid nanoparticle solution;

[0019] (4) freeze-drying the nucleic acid nanoparticle solution to obtain the nucleic acid nanoparticle preparation.

[0020] The third aspect of the present invention is to provide the use of the above-mentioned nucleic acid nanoparticle preparation in disease treatment.

[0021] The use of the above nucleic acid nanoparticle preparation in the preparation of drugs for preventing or treating tumors.

[0022] The use of the above nucleic acid nanoparticle preparation in the preparation of drugs for preventing or treating viral infections.

[0023] In the present invention, the inventors added cholesterol, phospholipids, and polymer lipid materials to the two components of polyethylene glycol-polylactic acid and lipids. This multi-component composition can significantly improve the transfection effect of nucleic acid drugs, which is equivalent to or even better than the efficacy of LNP. In addition, by adjusting the types and proportions of polymers, ionizable lipids, and phospholipids, this multi-component nucleic acid delivery system can be stored stably for a long time.

[0024] The nucleic acid nanoparticle preparation of the present invention is prepared from nucleic acid drugs such as siRNA and mRNA, polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic acid glycolic acid) copolymer, cationic lipid material, ionizable lipid material, cholesterol material, phospholipid material and polyethylene glycol lipid, has excellent stability, simple preparation method, high nucleic acid drug encapsulation rate and drug loading, can protect nucleic acid drugs from degradation, and provides a convertible and intelligent preparation technology for the fields of nucleic acid drug preparations such as siRNA and mRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the process flow chart for the preparation of nucleic acid nanoparticles; wherein A is the emulsification reaction in a microchannel reactor, B is the rotational multiplication of the organic solvent, C is the tangential flow concentration, and D is the freeze-dried freeze-dried preparation.

[0026] Figure 2 Schematic diagram of nucleic acid nanoparticle preparation

[0027] Figure 3 The figure shows the effect of ionizable lipids on the long-term stability of siRNA nucleic acid nanoformulations; A shows the effect of different MC3 / DOTAP ratios on NP / siRNA encapsulation efficiency, B shows the effect of different MC3 / DOTAP ratios on NP / siRNA particle size, C shows the effect of different MC3 / DOTAP ratios on NP / siRNA potential, and D shows the comparison of the knockdown effects of different MC3 / DOTAP ratios on NP / siRNA in CT26 cells, Lewis cells, Panc02 cells, and 4T1 cells.

[0028] Figure 4 Figure 3 shows the effect of ionizable lipids on the long-term stability of siRNA nucleic acid nanoformulations and their in vivo effects; A shows the effect of different MC3 / DOTAP ratios on the particle size stability of NP / siRNA, B shows the stability comparison of NP / siRNA and other nucleic acid delivery systems, C shows the transfection stability of NP / siRNA stored at 4°C, and D shows the transfection stability of NP / siRNA stored for a long time at different temperatures.

[0029] Figure 5It is the basic characterization of NP / mRNA nanoparticles of different formulations; wherein, A is the particle size of NP / mRNA nanoparticles of different formulations, B is the particle size of NP / mRNA nanoparticles of different formulations, and C is the in vivo fluorescence intensity expression of NP / mRNA nanoparticles of different formulations after intramuscular administration.

[0030] Figure 6 is the efficiency of mRNA delivery at the cellular level by NP / mRNA nanoformulations; AD is the efficiency of NP / Luci-mRNA transfection of DC2.4 cells, B16F10 cells, 293T cells and C2C12 cells with different formulations, respectively; EG is the effect of NP / Luci-mRNA transfection of DC2.4 cells, B16F10 cells and 293T cells with different formulations on cell activity, respectively; H is the relative fluorescence intensity expression of 293T cells transfected with NP / Luci-mRNA with different formulations after being placed at 4°C for 7 days.

[0031] Figure 7 To deliver different formulations of NP / E6E7-mRNA to treat mouse cervical cancer model; wherein, A is a schematic diagram of the treatment plan for NP / E6E7-mRNA to treat mouse cervical cancer model, B is the tumor growth curve of each group, and C is the weight change of each group.

[0032] Figure 8 To deliver different formulations of NP / LMP2A-mRNA to treat mouse nasopharyngeal carcinoma model; wherein, A is a schematic diagram of the treatment plan for NP / LMP2A-mRNA to treat mouse nasopharyngeal carcinoma model, B is the tumor growth curve of each group, and C is the survival curve of each group.

[0033] Fig. 9 Figure 2 shows the changes in immune indicators in mice after delivering NP / LMP2A-mRNA of different formulations to treat nasopharyngeal carcinoma models; A is the CD8 + Statistical analysis of T cells, B is IFN-γ in peripheral blood + CD8 + Statistical analysis of T cells, C is Ki67 in peripheral blood + CD8 + Statistical analysis of T cells, D is CD8 + Statistical analysis of T cells, E is IFN-γ in tumor tissue + CD8 + Statistical analysis of T cells, F is Ki67 in tumor tissue + CD8 + Statistical analysis of T cells, G is CD8 + Statistical analysis of T cells, H is IFN-γ in spleen tissue + CD8 +Statistical analysis of T cells, I is Ki67 in spleen tissue + CD8 + Statistical analysis of T cells.

[0034] Fig.10 The figure shows the basic characteristics of NP / mRNA nanoformulations with different formulations before and after the removal of organic solvents; A shows the change in particle size of NP / mRNA nanoformulations with different formulations before and after the removal of organic solvents; B shows the change in PDI of NP / mRNA nanoformulations with different formulations before and after the removal of organic solvents; C shows the change in Zeta potential of NP / mRNA nanoformulations with different formulations before and after the removal of organic solvents. D shows the change in efficiency of transfecting 293T cells of NP / Luci-mRNA with different formulations before and after the removal of organic solvents.

[0035] Fig.11 is the effect of polymer on the stability of mRNA nucleic acid nanoformulation; where A is different PEG 2K -PLGA 2K / DMG-PEG2000 NP / Luci-mRNA nucleic acid nanoformulation transfected 293T cells, B is different PEG 2K -PLGA 2K / DMG-PEG2000 NP / Luci-mRNA nucleic acid nanoformulation transfected DC2.4 cells, C is different PEG 2K -PLGA 2K / DMG-PEG2000 NP / Luci-mRNA nucleic acid nanoformulations were transfected into DC2.4 cells under different storage conditions, D is PEG with different molecular weights 2K -PLGA X NP / Luci-mRNA nucleic acid nanoformulation transfected 293T cells (compared with LNP / Luci-mRNA), E is PEG of different molecular weights 2K -PLGA X The relative fluorescence intensity of NP / Luci-mRNA nucleic acid nanoparticles after transfection of 293T cells, F is the relative fluorescence intensity of PEG with different molecular weight after being placed at 4°C 2K -PLGA X Relative fluorescence intensity expression of NP / Luci-mRNA nucleic acid nanoformulation after transfection of 293T cells.

[0036] Fig.12The figure shows the changes in the mRNA transfection efficiency of 293T cells with different phospholipid formulas NP / Luci-mRNA; A shows the mRNA transfection of 293T cells with different phospholipid formulas NP / Luci-mRNA, B shows the mRNA transfection of 293T cells with different phospholipid formulas NP / EGFP-mRNA under microscope observation, C shows the transfection of 293T cells with different phospholipid formulas NP / EGFP-mRNA by flow cytometry analysis, D shows the average fluorescence intensity of EFGP expression of cells after 293T cells were transfected with 293T cells with different phospholipid formulas NP / EGFP-mRNA, and E shows the relative fluorescence intensity expression of 293T cells after being transfected with NP / Luci-mRNA nucleic acid nanoformulations of different phospholipids at 4°C.

[0037] Fig.13 is the effect of different ionizable lipids on the transfection effect and stability of NP / mRNA; wherein, AE is the relative transfection efficiency of NP / Luci-mRNA encapsulated with different ionizable lipids compared with NP / Luci-mRNA encapsulated with SM-102, and F is the transfection stability of NP / Luci-mRNA encapsulated with different ionizable lipids stored at 4°C.

[0038] Fig.14 The freeze-dried morphology of nucleic acid nanoformulations and the characterization results before and after reconstitution; among them, A is the picture of freeze-dried nanoformulations with different lyophilization protectants and before and after dialysis, B is the comparison of the particle size of NP / mRNA before and after reconstitution under different freeze-drying conditions, C is the picture of freeze-dried nanoformulations of LNP and NP / mRNA before and after dialysis, and D is the comparison of the transfection effect of freeze-dried nanoformulations of LNP and NP / mRNA before and after dialysis after reconstitution.

[0039] Figure 15-Figure 23 is the structural formula of the constructed degradable and ionizable lipid. DETAILED DESCRIPTION

[0040] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0041] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0042] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0043] The present invention is the first to build a large-scale, continuous and intelligent preparation technology platform for nanomedicines, forming a production process that can accurately control the performance of nucleic acid nanomedicines, and can quickly, efficiently and batch produce nucleic acid preparations, establish a quality evaluation system for standardized preparations, thereby realizing the online preparation of nucleic acid drugs and developing a number of new nanocarrier technologies with independent intellectual property rights. The present invention has developed a nucleic acid nanoparticle that can be stored for a long time, creatively applying microchannel reactors to the continuous and intelligent preparation of nucleic acid nanoparticles, and has great clinical application potential.

[0044] In some embodiments of the present invention, a method for preparing nucleic acid nanoformulations based on a microchannel reactor is provided, and a large-scale, continuous and intelligent preparation technology for nucleic acid nanoformulations can be achieved through continuous sampling of a microchannel reactor pump. The specific technical process includes a microchannel reactor, rotary evaporation of an organic solvent, tangential flow concentration, and freeze drying of nucleic acid nanoformulations in a freeze dryer. The microchannel reactor is a microchannel reactor based on continuous flow.

[0045] Some embodiments of the present invention relate to a nucleic acid nanoformulation with long-term stability, which is prepared by an aqueous solution or buffer solution of nucleic acid as an aqueous phase, and an organic phase comprising polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic-co-glycolic acid) copolymer, lipids, cholesterol, and phospholipids dissolved in an organic solvent.

[0046] In some of the embodiments, the nucleic acid nanoformulation is prepared by mixing an aqueous solution of nucleic acid or a buffer solution as an aqueous phase, polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic-co-glycolic acid) copolymer, cationic lipids, ionizable lipids, cholesterol, phospholipids, and polyethylene glycol lipids dissolved in an organic solvent as an organic phase, and mixing them in a microchannel reactor.

[0047] The buffer is citric acid / sodium citrate buffer.

[0048] In some embodiments, the microchannel reactor-based reaction is first prepared by mixing an aqueous phase containing nucleic acid drugs with an organic phase containing ionizable lipids, cholesterol, polyethylene glycol lipids, and phospholipids in a reaction plate to obtain a nanoformulation solution loaded with nucleic acid.

[0049] Alternatively, the aqueous phase containing the nucleic acid drug and the organic phase containing ionizable lipids, polymers (polyethylene glycol-polylactic acid), cholesterol, polyethylene glycol lipids, and phospholipids are mixed in a reaction plate to obtain a nanoparticle preparation solution loaded with nucleic acid.

[0050] Alternatively, an aqueous phase containing nucleic acid drugs and an organic phase containing polymers (eg, polyethylene glycol-polylactic acid), cationic lipids, and ionizable lipids are mixed in a reaction plate to obtain a nanoparticle preparation solution loaded with nucleic acid.

[0051] In some embodiments, the nucleic acid is siRNA or mRNA.

[0052] In some of these embodiments, the siRNA or mRNA is a tumor therapeutic drug or an anti-viral infection therapeutic drug.

[0053] In some embodiments, the cationic lipid material can be dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyl-2,3-dioleyloxypropylammonium bromide (DOTMA), trimethyl-2,3-dioleyloxypropylammonium bromide (DOTAP), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), dimethyldioctadecylammonium bromide (DDAB), dimethyl-2-hydroxyethyl-2,3-dioleyloxypropylammonium bromide (DORI), dimethyl-2-hydroxyethyl-2,3-dioleyloxypropylammonium bromide (DORIE), dimethyl-3-hydroxypropyl-2,3-dioleyloxypropylammonium bromide (DORIE-HP), dimethyl-4-hydroxyethyl ... One of hydroxybutyl-2,3-dioleyloxypropylammonium (DORIE-HB), dimethyl-5-hydroxypentyl-2,3-dioleyloxypropylammonium bromide (DORIE-HPc), dimethyl-2-hydroxyethyl-2,3-dihexadecyloxypropylammonium bromide (DPRIE), dimethyl-2-hydroxyethyl-2,3-dioctadecyloxypropylammonium bromide (DSRIE), dimethyl-2-hydroxyethyl-2,3-ditetradecyloxypropylammonium bromide (DMRIE), N-(2-sperminecarbonyl)-N',N'-dioctadecylglycinamide (DOGS), 1,2-dioleoyl-3-succinyl-sn-glycerocholine ester (DOSC), 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol), lipid poly-L-lysine (LPLL), and stearylamine (SA).

[0054] More preferably, the cationic lipid in step (1) is one or more of DOTMA, DOTAP, DORI, DSRIE, DOGS, and DOSC, and most preferably is DOTAP.

[0055] In some preferred embodiments, the mass ratio of the cationic lipid to the ionizable lipid in the organic phase is 1:0.5-3, more preferably 1:1-2, and more preferably 1:1.

[0056] In some embodiments, the nucleic acid drug includes small interfering RNA (siCD47) targeting CD47, and targeting HPV mRNA, etc.

[0057] In some embodiments, the particle size of the nucleic acid nanoformulation after reconstitution ranges from 50 nm to 600 nm, preferably from 50 nm to 200 nm.

[0058] In some embodiments, the ionizable lipid is DLin-MC3-DMA, SM-102, ALC-0315, DLin-KC2-DMA, and can also be an ionizable lipid synthesized using the following method:

[0059]

[0060] like:

[0061] A2-2-B 1: A2-3-B 1: A3-2-B1: A3-3-B 1:

[0062] The synthesis of these ionizable lipids is as follows.

[0063] A2-2-B1: Using linker 2, tail chain B1 (100 mg, 0.14 mmol), head group A2 (20 mg, 0.14 mmol) and ethyl isocyanate (16 mg, 0.14 mmol) were dissolved in a mixture consisting of 120 μL dichloromethane (DCM) and 80 μL ethanol. The reaction was stirred at room temperature overnight. The mixture was purified by silica gel chromatography (10% methanol in dichloromethane, containing 0.1% NH4OH) to give brown oily lipid A2-2-B1 (12 mg, 0.013 mmol, 9%).

[0064] A2-3-B1: Using linker 3, tail chain B1 (100 mg, 0.14 mmol), head group A2 (20 mg, 0.14 mmol) and tert-butyl isocyanate (20 mg, 0.14 mmol) were dissolved in a mixture consisting of 120 μL dichloromethane (DCM) and 80 μL ethanol. The reaction was stirred at room temperature overnight. The mixture was purified by silica gel chromatography (10% methanol in dichloromethane, containing 0.1% NH4OH) to give brown oily lipid A2-3-B1 (10 mg, 0.01 mmol, 7%).

[0065] A3-2-B1: Using linker 2, tail chain B1 (100 mg, 0.14 mmol), head group A3 (22 mg, 0.14 mmol) and ethyl isocyanate (16 mg, 0.14 mmol) were dissolved in a mixture consisting of 120 μL dichloromethane (DCM) and 80 μL ethanol. The reaction was stirred at room temperature overnight. The mixture was purified by silica gel chromatography (10% methanol in dichloromethane, containing 0.1% NH4OH) to give lipid A3-2-B1 (15 mg, 0.02 mmol, 14%) as a brown oil.

[0066] A3-3-B1: Linker 3, tail chain B1 (100 mg, 0.14 mmol), head group A3 (22 mg, 0.14 mmol) and tert-butyl isocyanate (20 mg, 0.14 mol) were dissolved in a mixture consisting of 120 μL dichloromethane (DCM) and 80 μL ethanol. The reaction was stirred at room temperature overnight. The mixture was purified by silica gel chromatography (10% methanol in dichloromethane, containing 0.1% NH4OH) to give lipid A3-3-B1 (20 mg, 0.02 mmol, 14%) as a brown oil.

[0067] In some embodiments, the phospholipid is one of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dieucoylphosphatidylcholine (DEPC), dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylglycerol (DOPG), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidic acid (DSPA), dilauroylphosphatidylcholine (DLPC), myristoyl lysophosphatidylcholine (M-LysoPC), and palmitoyl lysophosphatidylcholine (P-LysoPC). More preferably, the phospholipid in step (1) is one or more of DSPC, DOPC, DPPC, DMPC, and DSPE, and most preferably DSPC.

[0068] In some of the embodiments, the polyethylene glycol lipid is one of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000), distearoyl-rac-glycerol-polyethylene glycol 2000 (DSG-PEG2000), and 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000).

[0069] In some of the embodiments, the organic solvent is a solution of chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide, ethanol, acetonitrile, tetrahydrofuran, acetone, etc., preferably ethanol and acetonitrile. In some preferred embodiments, the organic solvent is a mixed solution of ethanol and DMSO (55:45).

[0070] In some embodiments, the polymer is a synthetic polyethylene glycol-modified aliphatic and / or polyester aliphatic polyester.

[0071] In some embodiments, the polyethylene glycol-modified aliphatic polyester is at least one of polyethylene glycol-modified polylactide (PEG-PLA) and polyethylene glycol-modified poly(glycolide-co-lactide) (PEG-PLGA).

[0072] In some embodiments, the molecular weight of the aliphatic polyester is in the range of 1000 to 11000 Daltons.

[0073] In some embodiments, the molecular weight of the polyethylene glycol is in the range of 1000 to 10000 Daltons.

[0074] In some embodiments, the aliphatic polyester is poly(glycolide-co-lactide), and its LA / GA ratio ranges from 95 / 5 to 50 / 50, specifically 95 / 5, 85 / 15, 75 / 25, 50 / 50 or a range between any two specific ratios.

[0075] In some embodiments of the present invention, a method for preparing the nucleic acid nanoparticle preparation based on the above-mentioned microchannel reactor comprises the following steps:

[0076] (1) In a microchannel reactor, the aqueous phase and the organic phase are respectively introduced into a reaction plate through channels for mixed reaction to obtain a nucleic acid nanoparticle preparation solution;

[0077] (2) The solution of step (1) is transferred to a rotary evaporator, and the pressure is gradually increased in a water bath at about 37° C. to evaporate the organic solvent in the solution to obtain an initial nucleic acid nanoparticle preparation solution.

[0078] (3) Concentrating the nucleic acid nanoparticle preparation solution in step (2) by tangential flow to obtain a concentrated nucleic acid nanoparticle preparation solution.

[0079] (4) Adding a lyophilization protective agent with a final concentration of 10 to 20% (w / v) to the concentrated nucleic acid nanoparticle preparation solution of step (3), and performing vacuum low-temperature freeze-drying to obtain a nucleic acid nanoparticle preparation.

[0080] In some embodiments, the aqueous siRNA solution or mRNA solution in the aqueous phase of step (1) is dissolved in citric acid / sodium citrate buffer.

[0081] In some embodiments, the organic solvent in step (1) is at least one of chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide, ethanol, acetonitrile, tetrahydrofuran, and acetone, preferably a mixture of dimethyl sulfoxide and ethanol.

[0082] In some embodiments, the nucleic acid concentration in the aqueous nucleic acid solution is 50 μg / mL-200 μg / mL, preferably 50 μg / mL-100 μg / mL, and further preferably 80 μg / mL-100 μg / mL.

[0083] In some embodiments, in the organic phase, the concentration of the polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic acid-glycolic acid) copolymer is 0.05 mg / mL to 22 mg / mL, preferably 0.05 mg / mL to 10 mg / mL.

[0084] In some embodiments, in the organic phase, the cationic lipid concentration is 1 mg / mL to 2 mg / mL.

[0085] In some embodiments, in the organic phase, the concentration of the ionizable lipid is 0.5 mg / mL to 4 mg / mL, preferably 1 mg / mL to 4 mg / mL.

[0086] In some embodiments, in the organic phase, the cholesterol concentration is 1 mg / mL to 2 mg / mL, preferably 1.2 mg / mL to 1.6 mg / mL.

[0087] In some embodiments, in the organic phase, the phospholipid concentration is 0.1 mg / mL to 1 mg / mL, preferably 0.3 mg / mL to 0.8 mg / mL.

[0088] In some embodiments, in the organic phase, the polyethylene glycol lipid concentration is 0.25 mg / mL to 0.5 mg / mL, preferably 0.3 mg / mL to 0.4 mg / mL.

[0089] In some embodiments, the volume ratio of the aqueous phase to the organic phase is 1:1-5.

[0090] In some embodiments, the flow rate of the aqueous phase is 10 mL / min to 100 mL / min, more preferably 5 mL / min to 30 mL / min.

[0091] In some embodiments, the flow rate of the organic phase is 3 mL / min to 30 mL / min, more preferably 1.0 mL / min to 10 mL / min.

[0092] In the actual preparation process, the flow rate can be adjusted accordingly as needed, and is usually 1:8-12:46-55 (e.g., 1:10:50) according to the volume used, and can be adjusted accordingly to ensure sufficient and timely reaction.

[0093] In some of the embodiments, the microchannel reactor described in step (1) mainly includes: a control panel, two pumps (pump 1 and pump 2 from left to right), three pipelines (pipeline 1 and pipeline 2 corresponding to pump 1 and pump 2, respectively), a microchannel reaction plate and an output pipeline.

[0094] In some embodiments, the method steps of preparing the microchannel reactor by the microchannel reactor described in step (1) include: ① Turn on the microchannel switch, and control the switch with a constant temperature panel; connect the microchannel reactor pump to the ethanol solution, and flush the pump and the pipeline; use a syringe to remove the air from the two pumps; then connect the organic solvent (chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide, ethanol, acetonitrile, tetrahydrofuran, acetone) to pump 1, and flush the pump and the pipeline; connect pump 2 to the citric acid / sodium citrate buffer, and flush the pump and the pipeline. ② Set the flow rate of pump 1 to 0.1-10 mL / min, which is the aqueous phase (siRNA citric acid / sodium citrate buffer solution); set the flow rate of pump 2 to 1.0-40 mL / min, which is the organic phase (cationic lipids, ionizable lipids, polymers, phospholipids, cholesterol, polymer lipid organic solvent solution); the volume ratio of the two phases is adjusted according to the needs by the mobile phase speed. ③ Remove pipe 1 connected to pump 1, drain the organic solvent retained in the pipe, and use a syringe (with a special injection needle) to absorb the prepared siRNA solution; inject the siRNA solution into pipe 1, leaving a corresponding volume of air column at both ends of pipe 1, and then reconnect pipe 1 to the middle of pump 1 and the reaction plate, turn on pump 1 (connecting the organic solvent solution) to push the siRNA solution column to about 3 cm from the entrance of the reaction plate, and pause pump 1. ④ Connect pump 2 to the organic phase, turn on pump 2, and turn on pump 1 at the same time after about 80 seconds (using a timer). After about 1 minute, the aqueous phase and the organic phase begin to enter the reaction plate and begin to mix; this is the nucleic acid nanoparticle solution.

[0095] In some embodiments, the rotary evaporator in step (2) includes but is not limited to a parallel rotary evaporator, which removes the organic solvent to obtain an initial nucleic acid nanoformulation solution.

[0096] In some embodiments, the method for concentrating the initial nucleic acid nanoparticle preparation solution in step (3) includes: concentrating to a desired volume of 10 mL or more through a 100 kDa membrane. Other conventional methods can also be used as long as the nucleic acid nanoparticle preparation solution can be concentrated.

[0097] In some embodiments, freeze-drying the nucleic acid nanoformulation in the freeze dryer in step (4) includes adding a freeze-drying protective agent with a final concentration of, for example, 5-11 wt% to the concentrated nucleic acid nanoformulation solution, and freeze-drying by sublimation under vacuum conditions to obtain a nucleic acid nanoformulation that can be stored for a long time. Specifically, the freeze-drying protective agent can be sucrose, but is not limited thereto, and can also be glucose, trehalose, mannitol, etc.

[0098] The present invention is further described in detail below with reference to specific embodiments.

[0099] The sources of raw materials used in the following examples are:

[0100] Cationic lipid DOTAP: (2,3-dioleyloxypropyl)trimethylammonium chloride, purchased from Avanti PolarLipids;

[0101] Ionizable lipids: DLin-MC3-DMA, SM-102 (1-octylnonyl8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((decyloxy)hexyl)amino)octanoate)), ALC-0315, Dlin-KC2-DMA, purchased from Avanti Polar Lipids; in addition, ionizable lipids synthesized using the above method can also be: A2-2-B1, A2-3-B1, A3-2-B1, A3-3-B1.

[0102] Phospholipids: DSPC, DOPC, DEPC, DPPS, DOPG, DMPE, POPC, DSPE, POPC, DPPE, DMPC, DPPC, DSPA, DLPC, M-LysoPC, P-LysoPC were purchased from Shanghai Avituo Pharmaceutical Technology Co., Ltd.; high-purity cholesterol CHOL: purchased from Avanti Polar Lipids (AVT); molecular weight: 386.65

[0103] Polyethylene glycol lipids: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) was purchased from Avanti Polar Lipids;

[0104] Polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic acid glycolic acid) copolymer: the molecular weight of the polyethylene glycol segment ranges from 1000 to 10000 Daltons, and the molecular weight of the polylactic acid or polyethylene glycol-poly (lactic acid glycolic acid) polymer segment ranges from 1000 to 15000 Daltons, purchased from Guangzhou Kelan Biotechnology Co., Ltd.;

[0105] Lipofectamine TM RNAiMAX Transfection Reagent, catalog number 13778075, purchased from Thermo Company;

[0106] RNAKit, catalog number ER101-01, purchased from Quanshijin Company;

[0107] PrimeScriptTM RT reagent Kit, catalog number RR036A, purchased from TaKaRa;

[0108] FastStart Essential DNAGreen Master, product number 06924204001, purchased from Roche;

[0109] siRNA was purchased from Suzhou Beixin Biotechnology Co., Ltd., where the siCD47 sense chain sequence is: 5'-GGACUUGGCCUCAUUGUAATT-3' (SEQ ID NO: 1), and the antisense chain sequence is: 5'-UUACAAUGAGGCCAAGUCCTT-3' (SEQ ID NO: 2); mRNA was purchased from Hefei Afana Biotechnology Co., Ltd., where the Luciferase mRNA sequence is: SEQ ID NO: 3, and the EGFP mRNA sequence is: SEQ ID NO: 4.

[0110] Organic solvent solution was purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0111] Other reagents not specified were used directly.

[0112] Instrument models and companies used in the examples:

[0113] Microchannel reactor: Corning Lab reactor system, Corning Incorporated, USA;

[0114] Rotary evaporator: model RV10 auto control, IKA, Germany;

[0115] Minimate tangential flow ultrafiltration system and 300 kDa membrane: model OAPMP220, PALL, USA;

[0116] R&D freeze dryer: model LyoStar TM 3. SP Scientific, USA;

[0117] Nanoparticle size and Zeta potential instrument: model: Nano ZSE, Malvern, UK;

[0118] Flow cytometer: model: BD FACSCelesta, BD Company, USA;

[0119] qPCR instrument: Model Roche Light Cycler 96, Roche, USA.

[0120] The present invention is further described in detail below with reference to specific embodiments.

[0121] Example 1. Process flow of preparing nucleic acid nanoparticles

[0122] like Figure 1The microchannel reactor shown in A mainly comprises: a control panel, two pumps (from left to right, pump 1 and pump 2), two feed pipes (corresponding to pipe 1 and pipe 2 of pump 1 and pump 2, respectively), a microchannel reaction plate and an output pipe.

[0123] The steps of the method for mixing by microfluidics include: ① turning on the microfluidic switch; connecting the microfluidic pump to the ethanol solution to flush the pump and the pipeline; then connecting the organic solvent to pump 1 to flush the pump and the pipeline; connecting pump 2 to the sodium citrate buffer to flush the pump and the pipeline. ② Set pump 1 to the organic phase (the volume of the organic phase is 800 μL, polyethylene glycol-modified poly(glycolide-co-lactide) (PEG-PLGA) / polyethylene glycol-modified polylactic acid (PEG-PLA), cationic lipid DOTAP, ionizable lipid DLin-MC3-DMA, cholesterol CHOL, phospholipid distearoylphosphatidylcholine, and polyethylene glycol lipid DMG-PEG2000 are dissolved in an organic solvent ethanol-DMSO (55:45) mixed solution, with concentrations of 0.03 mg / mL~12 mg / mL, 0.4 mg / mL~4 mg / mL, 0.4 mg / mL~4 mg / mL, 0.16 mg / mL~3 mg / mL, 0.08 mg / mL~2 mg / mL, and 0.03 mg / mL~1 mg / mL, respectively). Set pump 2 to the aqueous phase (citric acid / sodium citrate buffer containing siRNA / mRNA, nucleic acid concentration of 0.083 mg / mL); the volume ratio of the two phases (1:3) is regulated by the flow rate (6 mL / min, 18 mL / min, respectively). ③ Turn on both pumps at the same time and collect the nucleic acid nanoparticles in a 15 mL centrifuge tube.

[0124] The collected nucleic acid nanoparticles are placed in a dialysis bag, such as Figure 1 As shown in B, the mixture was placed in a beaker containing PBS (pH 7.4), and the organic solvent was removed by rotary evaporation to obtain an initial nucleic acid nanoparticle preparation solution.

[0125] like Figure 1 As shown in C, the initial nucleic acid nanoformulation solution is passed through a 300 kDa envelope using tangential flow and concentrated to the desired volume by tangential flow. Further, a trehalose solution with a concentration of 50% is prepared, as Figure 1 As shown in D, a trehalose solution with a final concentration of 5% is added to the concentrated nucleic acid nanoparticle solution, and the solution is freeze-dried by sublimation under vacuum conditions to obtain a nucleic acid nanoparticle that can be stored for a long time. The prepared nanosystem loaded with siRNA is named NP / siRNA; the prepared nanosystem loaded with mRNA is named NP / mRNA. Figure 2 As shown, the nucleic acid nanoparticle preparation is a schematic structural diagram of lipid / polymer nucleic acid nanoparticles.

[0126] Example 2: Effects of ionizable lipids on basic characterization, long-term storage and transfection effects of nucleic acid nanoformulations

[0127] In order to investigate the effect of the ratio of lipid DLin-MC3-DMA and DOTAP and copolymer on the basic characterization and long-term storage of nanoparticles, the nanoparticles were prepared according to the method of Example 1. First, mPEG was fixed in the organic phase. 5K -PLGA 10K (mPEG block molecular weight of 5000, PLGA block molecular weight of 10000) and DOTAP feeding amount, according to the feeding mass ratio of DOTAP to 0:1, 1:2, 1:1, 2:1 or 3:1, the feeding amount of DLin-MC3-DMA was changed, mPEG 5K -PLGA 10K The mass ratio of siRNA to DOTAP was 25:1, and the mass ratio of siRNA to DOTAP was 10:1. The obtained nanoformulation was NP 0:1 / siRNA、NP 1:2 / siRNA、NP 1:1 / siRNA、NP 2:1 / siRNA and NP 3:1 / siRNA, the specific amount of each component is shown in Table 1.

[0128] Table 1. Formula of NP / siRAN used in this example

[0129]

[0130] The prepared nanoparticles were measured for particle size and potential using a dynamic light scattering (DLS) instrument. Figure 3 As shown in A and 3B, the higher the ratio of DLin-MC3-DMA, the larger the particle size and potential of the particles. At the same time, the nucleic acid encapsulation rate of the nanoparticles was detected and it was found that the ratio of DLin-MC3-DMA slightly affected the loading efficiency of siRNA ( Figure 3 C). NP / siCD47 with different lipid ratios and loaded with siRNA targeting CD47 were incubated with B16F10 cells, CT26 cells, 4T1 cells, Panc02 cells, and Lewis cells for 6 hours (siRNA concentration was 50nM). After 24 hours, RNA of the cells was extracted for real-time fluorescence quantitative PCR detection. The results showed that after NP / siRNA improved the quality of DLin-MC3-DMA, its siRNA transfection effect was greatly increased ( Figure 3 D).

[0131] We further measured the stability of NP / siRNA. We took the nucleic acid nanoparticles NP / siRNA prepared above and diluted it to 1 mg / mL with DEPC water. We stored it at 4°C. Figure 4 As shown in A, the NPs were taken out on the 0th day, the 5th day, the 10th day, the 20th day, and the 30th day, and the particle size was detected by dynamic light scattering. The results showed that during the observation time, the NP / siRNA particle size did not change significantly, and the particle size distribution did not fluctuate greatly, indicating that the nucleic acid nanoparticle aqueous solution of the present invention can maintain the stability of the hydration radius under long-term storage at 4°C. 1:1 The universal transfection reagent Lipofectamine RNAiMAX and lipid nanoparticles LNP (Onpattro formula) were stored at room temperature (~25°C), 4°C, -20°C (with 5% lyophilized protective agent sucrose), and -80°C (with 5% lyophilized protective agent sucrose). After 5 days, the NPs stored under different conditions were 1:1 / siRNA, Lipofectamine RNAiMAX, and LNP were incubated with B16F10 cells, and the knockdown of CD47 mRNA in B16F10 cells was detected by real-time fluorescence quantitative PCR reaction 24 hours later. Figure 4 As shown in B, NP / siRNA 1:1 The stability is the best (relative quantitative target mRNA expression, the lower the target mRNA expression, the better the effect). 1:1 The nanoparticles were stored at 4°C and incubated with B16F10 cells on the 1st, 7th and 15th day after particle preparation. The knockdown of CD47 mRNA in B16F10 cells was detected by real-time fluorescence quantitative PCR reaction 24 hours later. Figure 4 As shown in C, NP / siRNA 1:1 The transfection effect of the nanoparticles was slightly reduced, and the nanoparticles had good stability. 1:1 The NPs stored in different conditions were stored at 4°C, -20°C (5% lyophilization protective agent sucrose was added), and -80°C (5% lyophilization protective agent sucrose was added). 1:1 / siRNA was incubated with B16F10 cells, and 24 hours later, real-time fluorescence quantitative PCR was used to detect the knockdown of CD47 mRNA in B16F10 cells. Figure 4 As shown in D, when the mass ratio of DLin-MC3-DMA to DOTAP is 1:1, long-term low-temperature storage hardly changes the efficiency of the particles.

[0132] In order to investigate the difference between the efficacy of the nanoparticles of the present invention in delivering siRNA to treat diseases and the efficacy of lipid nanoparticles LNP (Onpattro formula) in treating diseases, this experiment used NP 1:1 / siRNA and LNP / siRNA were used to deliver siRNA targeting CD47 and PD-L1 for anti-tumor immunotherapy, and PBS was injected as a negative control group to investigate the inhibition of mouse melanoma. Five mice were selected from each group, and 2.5x10 5 B16F10 cells, wait until the tumor volume grows to 50mm 3 Drug administration began at (day 8). Each mouse in the treatment group was administered 0.5OD siRNA (i.e. 0.25OD siCD47 and 0.25OD siPD-L1) once every two days for a total of 6 times. 3 The experiment was terminated, the mice were sacrificed and their tumor tissues were taken for comparison. The tumor growth curves and pictures of each treatment group are shown in Figure 4 E and 4F, NP 1:1 / siRNA and LNP / siRNA have comparable therapeutic effects in vivo.

[0133] Example 3: Effect of polymer on mRNA transfection efficiency and stability of nanoparticles NP / mRNA in vivo and in vitro

[0134] In order to screen the most suitable polymer for preparing NP / mRNA, this study kept the feeding amount of SM-102, distearoylphosphatidylcholine, and cholesterol unchanged, and prepared NP / mRNA of various different formulations according to the preparation method of Example 1 by changing the type of polymer and its feeding amount. Through particle size analysis, we excluded those polymers that were not suitable for preparing NP / mRNA. We used mRNA (Luci-mRNA) expressing luciferase as a model drug, evaluated the expression efficiency of mRNA by intramuscular injection, and further screened out suitable polymer types and optimal feeding ratios. Then the appropriate NP / mRNA ratio was incubated with different cells to explore the changes in its in vitro transfection efficiency. Finally, a mouse tumor model was constructed to explore the in vivo therapeutic effects of different NP / mRNAs.

[0135] First, different polymers were used to prepare NP / mRNA with different formulations. The specific amounts of each component are shown in Table 2, and the particle size was analyzed by DLS. The experimental results showed that when the molecular weight of the hydrophobic block of the polymer was less than or equal to 2000Da, adjusting the polymer feed amount could not produce nano-scale particles ( Figure 5 AB), so this type of polymer was excluded from the subsequent optimization.

[0136] Table 2. Formula of NP / mRNA used in this example

[0137]

[0138]

[0139]

[0140] Note: The organic phase is 800 μL.

[0141] Then, this study used the method of bioluminescence imaging to prepare different NP / Luci-mRNA according to the formula in Table 2, and detected the Luci-mRNA expression efficiency 6 hours after injection into the mouse hind limb muscle. The specific operation was to inject 10 μg of NP / Luci-mRNA with different formulas into the left and right hind leg muscles of each mouse. 6 hours after the injection, the mice were anesthetized by intraperitoneal injection of 125 μL of sodium pentobarbital (1%), followed by injection of 200 μL of luciferase substrate luciferin (15 mg / mL) through the tail vein, and the in vivo imaging system (IVIS) was used to detect the expression of Luminescence fluorescence. In each experiment, we used LNP / Luci-mRNA as a positive control. The experimental results show that when the feeding amount of distearoylphosphatidylcholine, cholesterol and copolymer is fixed, when the polymer is selected as PEG 2K -PLGA 6K When the mass percentage of the feed amount is 60%, the expression efficiency of mRNA is the highest, which is about 40% of LNP / Luci-mRNA ( Figure 5 C).

[0142] In order to explore the effect of polymer on the efficiency of NP / mRNA delivery at the cell level, this experiment used PEG 2K -PLGA 4K PEG 2K -PLGA 6K PEG 2K -PLGA 8K Six kinds of NP / Luci-mRNA particles were prepared according to the preparation method of Example 1. During the preparation process, SM-102 (1340 μg), phospholipid-DSPC (300 μg), cholesterol (560 μg) and polymers of different lengths and mass ratios were added to the organic phase. The six kinds of particles were NP 6K 30％ / Luci-mRNA(PEG 2K -PLGA 6K The mass percentage is 30%, the feeding amount is 942μg), NP 6K 40％ / Luci-mRNA(PEG2K -PLGA 6K The mass percentage is 40%, the feeding amount is 1466μg), NP 6K 50％ / Luci-mRNA(PEG 2K -PLGA 6K The mass percentage is 50%, the feeding amount is 2200μg), NP 6K 60％ / Luci-mRNA(PEG 2K -PLGA 6K The mass percentage is 60%, the feeding amount is 3300μg), NP 4K 60％ / Luci-mRNA(PEG 2K -PLGA 4K The mass percentage is 60%, the feeding amount is 3300μg), NP 8K 60％ / Luci-mRNA(PEG 2K -PLGA 8K The mass percentage is 60%, the feeding amount is 3300μg), and the aqueous phase is Luci-mRNA (100μg). These 6 kinds of NP / Luci-mRNA and LNP / Luci-mRNA were used to transfect mouse melanoma B16F10 cells, human renal epithelial 293T cells, mouse dendritic DC2.4 cells and mouse myoblast C2C12 cells, wherein the lipid nanoparticles LNP / mRNA were prepared according to the preparation method of Example 1, and SM-102 (1340μg), phospholipid-DSPC (300μg), cholesterol (560μg) and DMG-PEG2000 (140μg) were added to the organic phase during the preparation process, and the aqueous phase was 100μg mRNA. The experimental results show that most NP / Luci-mRNA nanoparticles can deliver mRNA more effectively than LNP / Luci-mRNA. Different NP / Luci-mRNA nanoparticles have different transfection effects on different cell lines. For example, in B16F10 cells and DC2.4 cells, NP 6K30％ / Luci-mRNA transfection has the best effect ( Figure 6 A and 6B), while in 293T cells and C2C12 cells NP 4K60％ / Luci-mRNA transfection has the best effect ( Figure 6 C and 6D). In addition, NP / Luci-mRNA nanoparticles have excellent biocompatibility with B16F10 cells, 293T cells and DC2.4 cells ( Figure 6 E, 6F and 6G).

[0143] We further investigated the stability of NP / mRNA. We took the above six NP / Luci-mRNAs (i.e., NP 6K30％ / Luci-mRNA、NP 6K 40％ / Luci-mRNA、NP 6K 50％ / Luci-mRNA、NP 6K 60％ / Luci-mRNA、NP 4K30％ / Luci-mRNA、NP 8K30％ / Luci-mRNA) and LNP / Luci-mRNA were stored at room temperature (~25°C), 4°C, and -20°C (with 5% lyophilization protective agent sucrose added). Five days later, NP / Luci-mRNA and LNP / Luci-mRNA stored in different conditions were incubated with 293T cells, and the expression of cell luciferase was detected using an ELISA instrument after 24 hours. It can be found that the higher the polymer ratio in NP / mRNA, the better the stability of the nanoparticles ( Figure 6 H).

[0144] In order to investigate the efficacy of the nanoparticles of the present invention in delivering mRNA to treat diseases and the differences in the efficacy of NP / mRNA nanoparticles with different formulations in treating diseases, this experiment used the above-mentioned 6 kinds of NP / mRNA (i.e., NP 6K30％ / mRNA、NP 6K40％ / mRNA、NP 6K50％ / mRNA、NP 6K60％ / mRNA、NP 4K30％ / mRNA、NP 8K30％ / mRNA) and LNP / mRNA delivered mRNA encoding E6E7 antigen, E6E7-mRNA, to investigate the inhibition of HPV antigen-related tumors in mice. The lipid nanoparticles LNP / mRNA were prepared according to the preparation method of Example 1. During the preparation process, SM-102 (1340 μg), phospholipid-DSPC (300 μg), cholesterol (560 μg) and DMG-PEG2000 (140 μg) were added to the organic phase, and 100 μg mRNA was added to the aqueous phase. Figure 7 As shown in A, 5 mice were selected from each group. 1x106 TC-1 cells were implanted subcutaneously on the right side of the back of the mice 14 days before the first immunization. Nanoparticles loaded with E6E7-mRNA were injected intramuscularly starting from day 0. The dosage was 6.6 μg each time, and the drug was given once every three days. The tumor volume was measured and it was observed that the intramuscular injection of 6 different formulations of NP / E6E7-mRNA could effectively inhibit the growth of HPV antigen-related tumors in mice, and NP 6K40％ / E6E7-mRNA(PEG 2K -PLGA 6K The tumor inhibition effect of LNP / E6E7-mRNA ( Figure 7 B) Monitoring the body weight of mice after administration of the nanoformulation indicated that both NP / E6E7-mRNA had good biocompatibility ( Figure 7 C).

[0145] The above six NP / mRNAs (i.e. NP 6K30％ / mRNA、NP 6K40％ / mRNA、NP 6K50％ / mRNA、NP 6K60％ / mRNA、NP 4K30％ / mRNA、NP 8K30％ / mRNA) and LNP / mRNA delivered mRNA encoding LMP2A antigen to investigate the inhibition of melanoma in mice carrying the nasopharyngeal carcinoma antigen LMP2A. Figure 8 As shown in A, 5 mice were selected from each group and 5x10 6 B16F10 cells were injected into the right hind limb muscle of each mouse on the 4th, 8th, 13th and 18th days respectively. Each mouse was given 10 μg LMP2A-mRNA. Intramuscular injection of 6 different formulations of NP / LMP2A-mRNA can effectively inhibit the growth of EBV antigen-related tumors in mice, and NP 6K 50％ / LMP2A-mRNA and NP 6K 60％ The tumor inhibition effect of LMP2A-mRNA was slightly better than that of LNP / LMP2A-mRNA ( Figure 8 B and 8C). Three days after the fourth administration, the mice were sacrificed and their tumors, spleens and peripheral blood were collected for immune analysis using flow cytometry. Fig. 9 AI shows the CD8 + T cells are stimulated to varying degrees.

[0146] Example 4: Study on the effect of different formulations of NP / mRNA on nanoparticles before and after removal of organic solvents

[0147] In order to explore the effect of organic solvents in NP / mRNA on transfection efficiency and stability, this study kept the feed amounts of SM-102, distearoylphosphatidylcholine, and cholesterol unchanged, and changed the feed amount of polymer. NP / mRNA with different formulations was prepared according to the preparation method of Example 1. The specific proportions of each component are shown in Table 3. After preparing NP / mRNA with different formulations, dynamic light scattering was used to analyze the particle size of NP / mRNA. The experimental results showed that when PEG 2K -PLGA 6K When the feed amount of the organic phase is greater than 12% of the total feed amount of the organic phase components, nanoparticles with a particle size of less than 200 nm and good uniformity can be prepared ( Fig.10 AB), and the particle size and uniformity of the particles did not change much after the organic solvent was removed. By comparing the Zeta potential changes of each formula NP / mRNA before and after the organic solvent was removed, it can be found that the Zeta potential was positive before the organic solvent was removed, while the Zeta potential was negative after the organic solvent was removed ( Fig.10 C).

[0148] Table 3. Formula of NP / mRNA used in this example

[0149]

[0150] In order to explore the effect of the amount of copolymer on the efficiency of mRNA delivery at the cellular level before and after the removal of organic solvent from NP / mRNA, this experiment prepared 7 kinds of NP / Luci-mRNA particles according to the preparation method of Example 1, and the proportions of each particle component are shown in Table 2. These 7 kinds of NP / Luci-mRNA were used to transfect 293T cells. The experimental results showed that when PEG 2K -PLGA 6K When the feed amount of PEG accounts for 6%, 12%, and 22% of the total feed amount, the transfection efficiency of NP / Luci-mRNA after removing the organic solvent is close to or even better than that before removing the organic solvent. 2K -PLGA 6K When the feed amount accounts for more than 22% of the total feed amount, the transfection efficiency of NP / Luci-mRNA after removing the organic solvent is much worse than the transfection efficiency before removing the organic solvent ( Fig.10 D).

[0151] Example 5: Effect of Poly(ethylene glycol)-poly(lactic acid-co-glycolic acid) copolymer on the long-term storage stability of nucleic acid nanoformulations

[0152] In order to investigate the effect of the ratio between the copolymer of the present invention and other components such as ionizable lipids, cationic lipids, cholesterol, and the amount of polyethylene glycol polylactic acid glycolic acid copolymer on the efficiency of nanoparticle nucleic acid delivery and long-term storage stability. The preparation method of nucleic acid nanoformulation NP / mRNA is the same as that of Example 1. During the preparation process, a lipid SM-102, distearoyl phosphatidylcholine, cholesterol, and DMG-PEG2000 are added to the organic phase, and mPEG having a molar mass ratio of 0:1, 1:1, 2:1, 5:1 or 10:1 with DMG-PEG2000 is also added. 2K -PLGA 2K The obtained nanoformulation is NP 0:1 / mRNA、NP 1:1 / mRNA、NP 2:1 / mRNA、NP 5:1 / mRNA and NP 10:1 / mRNA; and the organic phase contains mPEG 2K -PLGA 2K Particles without DMG-PEG2000 (i.e. NP 10:0 The components of the nanoparticles are shown in Table 4.

[0153] Table 4. Formula of NP / mRNA used in this example

[0154]

[0155] Nanoparticles were prepared using Luciferase mRNA as the inner aqueous phase. 0:1 / mRNA、NP 1:1 / mRNA、NP 2:1 / mRNA、NP 5:1 / mRNA and NP 10:1 / mRNA was co-incubated with DC2.4 cells or 293T cells. After 24 hours, 150ug / mL luciferin potassium salt was added to the cells and incubated in the dark for 5 minutes. The fluorescence generation was then detected using a multifunctional microplate reader. Fig.11 As shown in A and 11B, mPEG 2K -PLGA 2K When the molar mass ratio of NP to DMG-PEG2000 was 2.0-5.0, NP / mRNA had a good transfection effect in both 293T cells and DC2.4 cells, and the transfection effect was improved compared with the traditional Onpattro formula.

[0156] NP 0:1 / mRNA、NP 1:1 / mRNA、NP 2:1 / mRNA、NP5:1 / mRNA and NP 10:1 / mRNA was divided into three portions and stored at 4°C, -20°C, and -20°C with 5% lyophilization protective agent sucrose for 7 days. After 7 days, the nanoparticles stored under different conditions and the re-prepared nanoparticles were incubated with DC2.4 cells for 24 hours, and then 150ug / mL fluorescein potassium salt was added to the cells and incubated in the dark for 5 minutes, and the fluorescence generation was detected using a multifunctional microplate reader. Fig.11 As shown in C, compared with Onpattro formulated LNP, mPEG 2K -PLGA 2K The addition of effectively improves the stability of the nanoparticles, so that the particles can have a better transfection effect whether they are stored at 4°C or -20°C.

[0157] We further want to explore the effect of the molecular weight of polylactic acid-co-glycolic acid copolymer on the transfection efficiency and stability of NP / mRNA. According to the method of Example 1, different polymers were used to prepare NP / mRNA of different formulations. The specific dosage of each component is shown in Table 5, and Luciferase mRNA was used as the inner aqueous phase to prepare nanoparticles. NP / Luci-mRNA of different formulations was co-incubated with 293T cells (Luciferase mRNA concentration was 1 μg / mL), and LNP was used as a positive control. After 24 hours, 150ug / mL of fluorescein potassium salt was added to the cells and incubated in dark for 5 minutes, and then the generation of fluorescence was detected using a multifunctional microplate reader. The results show that as the molecular weight of the polylactic acid-co-glycolic acid copolymer fragment increases, the NP / mRNA transfection efficiency decreases significantly ( Fig.11 D and 11E). NP / Luci-mRNA and LNP / Luci-mRNA with different formulations were stored at 4°C for one week and then co-incubated with 293T cells (Luciferase mRNA concentration was 1 μg / mL). After 24 hours, 150ug / mL fluorescein potassium salt was added to the cells and incubated in the dark for 5 minutes, and then the fluorescence generation was detected using a multifunctional microplate reader. The results showed that the molecular weight of the polylactic acid-co-glycolic acid copolymer fragment increased, and the stability of NP / mRNA was stronger. In general, the effect of NP / mRNA was better than that of LNP / mRNA ( Fig.11 F).

[0158] Table 5. Formula of NP / mRNA used in this example

[0159]

[0160]

[0161] We then investigated the difference in mRNA transfection efficiency between NP / mRNA and LNP / mRNA in vivo. 2K5:1 / mRNA and LNP / mRNA, where NP 2K5:1 The specific dosage of each component of the / mRNA organic phase is shown in Table 5. Luciferase mRNA was used as the inner aqueous phase to prepare nanoparticles. The specific operation was to inject 3μg NP / Luci-mRNA and LNP / Luci-mRNA into the left and right hind leg muscles of the mouse respectively. 6 hours after the injection, the mice were anesthetized by intraperitoneal injection of 125μL sodium pentobarbital (1%), followed by injection of 200μL luciferase substrate luciferin (15mg / mL) through the tail vein, and the expression of luminescence fluorescence was detected using an in vivo imaging system (IVIS). The results showed that even in the in vivo environment, NP 2K5:1 / mRNA mRNA transfection efficiency is still higher than LNP / mRNA ( Fig.11 G).

[0162] Example 6: Effect of phospholipid dosage and type on the delivery efficiency of NP / mRNA nanoparticles

[0163] In order to screen NP / mRNA with high transfection efficiency and explore the effect of phospholipid dosage and type on the delivery efficiency of NP / mRNA nanoparticles, this study prepared NP / mRNA encapsulated with Luciferase mRNA of different formulations by changing the dosage and type of phospholipids while keeping the dosage of SM-102, cholesterol and polyethylene glycol lipids unchanged according to the preparation method of Example 1. The specific proportions of each component are shown in Table 6.

[0164] Table 6. Formula of NP / mRNA used in this example

[0165]

[0166] After preparing different formulations of NP / mRNA, these three NP / Luci-mRNAs were used to transfect 293T cells. The experimental results showed that when distearoyl phosphatidylcholine and dioleoyl phosphatidylcholine were added to the phospholipid formulation of NP / mRNA, the transfection efficiency of NP / Luci-mRNA was optimal ( Fig.12A). Further, we compared the five-component NP / mRNA with the classic LNP / mRNA formula (SM-102 is 1340μg, cholesterol is 560μg, polyethylene glycol lipid is 140μg, and distearoylphosphatidylcholine is 300μg). LNP and NP3 / mRNA encapsulated with EGFP mRNA were prepared according to the preparation method of Example 1, and 293T cells were transfected with these two nanoparticles. The results showed that both fluorescence microscopy imaging ( Fig.12 B) or flow cytometry ( Fig.12 C, D), the transfection efficiency of the five-component NP3 / mRNA is up to 2 times that of LNP. The NP / Luci-mRNA prepared from LNP / Luci-mRNA and different phospholipids was stored at 4°C for one week and then co-incubated with 293T cells (Luciferase mRNA concentration was 1μg / mL). After 24 hours, 150ug / mL of fluorescein potassium salt was added to the cells and incubated in the dark for 5 minutes, and then the fluorescence generation was detected using a multifunctional microplate reader. The results showed that the screened ionizable lipid structure had no significant effect on the stability of NP / mRNA ( Fig.12 E).

[0167] Example 7: Study on the effect of ionizable lipids on the delivery efficiency of NP / mRNA nanoparticles

[0168] In order to explore the effect of ionizable lipids on the delivery efficiency and stability of NP / mRNA nanoparticles, this study maintained ionizable lipids (1340 μg), cholesterol (560 μg), distearoylphosphatidylcholine (300 μg), polyethylene glycol lipids (140 μg), mPEG 2K -PLGA 2K Under the condition that the feed amount of 114 μg is unchanged, by changing the type of ionizable lipid, NP / Luci-mRNA encapsulated with Luciferase mRNA of different formulations were prepared according to the preparation method of Example 1. The structure of the ionizable lipid is as follows: Figure 15-Figure 23 NPs with different formulations of NP / Luci-mRNA were co-incubated with 293T cells (Luciferase mRNA concentration was 1 μg / mL) to encapsulate NPs with ionizable lipid SM-102. SM-102 / Luci-mRNA was used as a positive control. After 24 hours, 150ug / mL luciferin potassium salt was added to the cells and incubated in the dark for 5 minutes, and then the fluorescence generation was detected using a multifunctional microplate reader. The results showed that NP / mRNA using ionizable lipids A2-1-B1, A3-1-B1, A2-2-B1, and A2-3-B1 showed the same fluorescence as NP SM-102 / Luci-mRNA similar transfection efficiency ( Fig.13 AE). NP / Luci-mRNA and LNP / Luci-mRNA prepared with different ionizable lipids were stored at 4°C for one week and then co-incubated with 293T cells (Luciferase mRNA concentration was 1μg / mL). After 24 hours, 150ug / mL fluorescein potassium salt was added to the cells and incubated in the dark for 5 minutes, and then the fluorescence generation was detected using a multifunctional microplate reader. The results showed that the screened ionizable lipid structure had no significant effect on the stability of NP / mRNA ( Fig.13 F).

[0169] Example 8 Basic characterization of nucleic acid nanoformulations with long-term stability and their lyophilized reconstitution

[0170] In order to explore the conditions of NP / mRNA freeze-drying process, 5% sucrose (Trehalose) was added to NP / mRNA before and after dialysis and classic formula LNP / mRNA as freeze-drying protective agent, and freeze-dried. The freeze-drying steps include: ① Check the air and nitrogen cylinder pressure to ensure that it is sufficient, and the nitrogen is at least greater than 3MPa. Open the air valve and adjust the pressure drop valve to above 0.8MPa. The pressure drop valve in the purification room is adjusted to 0.5MPa for air and 0.7MPa for nitrogen according to the instructions; ② Turn on the freeze dryer and place the sample in the sample tray with the rubber cover half-covered. Push the sample tray into the freeze-drying chamber, pull out the tray, close the freeze-drying chamber door and the condensation chamber door below; ③ Log in after the freeze-drying software automatically opens, adjust the freeze-drying program on the freeze dry page, and start freeze-drying. Observe the status bar to ensure good air tightness and that the ambient temperature is below 25°C; ④ During the freeze-drying process, when the PVG (vacuum degree including humidity) is less than 3 from the set vacuum degree, you can enter the temperature rise step (-28°C and later steps), and you can adjust the corresponding time and steps on the freeze dry page; ⑤ After the freeze-drying is completed, first backfill nitrogen on the semi auto page (cooperate with opening the backfill pipeline) to 500, raise the tray, press the cover, then close the backfill, release the vacuum, and open the hatch after it is completely released. Quickly remove the sample and press the cover.

[0171] The NP / mRNA nanoparticles were prepared according to the method of Example 1. During the preparation process, SM-102 (1340 μg), cholesterol (560 μg), distearoylphosphatidylcholine (300 μg), polyethylene glycol lipid (140 μg), mPEG 2K -PLGA 2K (114 μg), 100 μg mLuciferase was added to the aqueous phase. Half of the prepared NP / mRNA was dialyzed, and the other half was stored at 4°C. The NP / mRNA before and after dialyzation was then freeze-dried according to the above steps.

[0172] like Fig.14 As shown in A, the non-dialyzed NP / mRNA appears collapsed or viscous solid after freeze-drying, while the dialyzed NP / mRNA appears white, uniform in size and quality as a powder cake after freeze-drying.

[0173] Characterization of reconstituted nucleic acid nanoformulations: After lyophilizing the NP / mRNA in the above steps, 1 mL of DEPC-treated ultrapure water was added to the lyophilized mRNA nanoformulation (NP / mRNA) for reconstitution. The particle size of the nanoformulation was measured by dynamic light scattering (DLS). Figure 3 As shown in B, the particle sizes of the mRNA nanoformulation with sucrose protective agent added after dialysis and before and after reconstitution are approximately 133.6 nm and 171.3 nm, respectively.

[0174] Further, we compared the freeze-drying effect of LNP / mRNA and NP / mRNA, and prepared NP / Luci-mRNA according to the above method. The LNP / mRNA nanoparticles were prepared according to the method of Example 1. During the preparation process, SM-102 (1340 μg), cholesterol (560 μg), distearoyl phosphatidylcholine (300 μg), polyethylene glycol lipid (140 μg), mPEG 2K -PLGA 2K (114μg), 100μg mLuciferase was added to the aqueous phase. Part of LNP / Luci-mRNA and NP / Luci-mRNA was dialyzed, and the other half was stored at 4°C. The nanoparticles before and after dialysis were then freeze-dried according to the above steps. It can also be found that if the nanoparticles are not dialyzed, the freeze-dried nanoparticles will not be in the form of a white powder with uniform size and quality ( Fig.14 C). After adding 1X PBS to re-dissolve the freeze-dried NP / mRNA and LNP / mRNA, they were co-incubated with 293T cells (Luciferase mRNA concentration was 1μg / mL). After 24 hours, 150ug / mL of luciferin potassium salt was added to the cells and incubated in the dark for 5 minutes. The fluorescence generation was then detected using a multifunctional microplate reader. The results showed that the freeze-dried effect of NP / mRNA was slightly better than that of LNP / mRNA ( Fig.14 D).

[0175] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A nucleic acid nanoparticle preparation, characterized in that: It is prepared by using a buffer containing nucleic acid as an aqueous phase and an organic phase containing polymers, lipids, cholesterol, and phospholipids dissolved in an organic solvent; The buffer is citric acid / sodium citrate buffer, The polymer is polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic acid-glycolic acid) copolymer, and the lipid is an ionizable lipid, or a mixture of an ionizable lipid and a cationic lipid; The concentration of the nucleic acid in the aqueous phase is 50 μg / mL-100 μg / mL; in the organic phase, the concentration of the polymer is 0.05 mg / mL-22 mg / mL, the concentration of the lipid is 0.5 mg / mL-4 mg / mL, the concentration of the cholesterol is 1 mg / mL-2 mg / mL, and the concentration of the phospholipid is 0.1 mg / mL-1 mg / mL; The mass percentage of the polymer in the organic phase feed is 12%-60%.

2. The nucleic acid nanoparticle preparation according to claim 1, characterized in that: The concentration of the nucleic acid in the inner aqueous phase is 80 μg / mL-100 μg / mL; in the organic phase, the concentration of the polymer is 0.05 mg / mL-10 mg / mL, the concentration of the lipid is 1 mg / mL-4 mg / mL, the concentration of the cholesterol is 1.2 mg / mL-1.6 mg / mL, and the concentration of the phospholipid is 0.3 mg / mL-0.8 mg / mL; and / or the mass percentage of the polymer in the organic phase feed is 30%-60%.

3. The nucleic acid nanoparticle preparation according to claim 1, characterized in that: The polymer is polyethylene glycol-modified polylactide or polyethylene glycol-modified poly(glycolide-co-lactide). Preferably, the ratio of LA / GA in the polylactide or poly(glycolide-co-lactide) is in the range of 95 / 5 to 50 / 50.

4. The nucleic acid nanoparticle preparation according to claim 3, characterized in that: The molecular weight of polyethylene glycol in the polymer is 1000 Dalton to 10000 Dalton, preferably 2000 Dalton to 6000 Dalton; and / or the molecular weight of polylactide or poly(glycolide-co-lactide) is 2000 Dalton to 10000 Dalton, preferably 2000 Dalton to 8000 Dalton.

5. The nucleic acid nanoparticle preparation according to claim 1, characterized in that: The phospholipid is at least one of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dieucoylphosphatidylcholine (DEPC), dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylglycerol (DOPG), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidic acid (DSPA), dilauroylphosphatidylcholine (DLPC), myristoyl lysophosphatidylcholine (M-LysoPC), and palmitoyl lysophosphatidylcholine (P-LysoPC). Preferably, the phospholipid is at least one of DSPC, DOPC, DPPC, DMPC, and DSPE.

6. The nucleic acid nanoparticle preparation according to claim 5, characterized in that: The phospholipids are distearoyl phosphatidylcholine and dioleoyl phosphatidylcholine, and the dosage ratio of the distearoyl phosphatidylcholine and dioleoyl phosphatidylcholine is 4:

3.

7. The nucleic acid nanoparticle preparation according to claim 1, characterized in that: The nucleic acid is siRNA or mRNA.

8. The nucleic acid nanoparticle preparation according to claim 1, characterized in that: The cationic lipid is selected from at least one of the following: the cationic lipid material can be dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyl-2,3-dioleyloxypropylammonium bromide (DOTMA), trimethyl-2,3-dioleyloxypropylammonium bromide (DOTAP), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), dimethyldioctadecylammonium bromide (DOTAP), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), dimethyldioctadecylammonium bromide (DOTAP), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), tri ...dioctadecylammonium bromide (DOTAP), trimethyltetradecyl DAB), dimethyl-2-hydroxyethyl-2,3-dioleyloxypropylammonium bromide (DORI), dimethyl-2-hydroxyethyl-2,3-dioleyloxypropylammonium bromide (DORIE), dimethyl-3-hydroxypropyl-2,3-dioleyloxypropylammonium bromide (DORIE-HP), dimethyl-4-hydroxybutyl-2,3-dioleyloxypropylammonium bromide (DORIE-HB), dimethyl-5-hydroxypentyl-2,3-dioleyloxypropylammonium bromide (DORIE-HPc), dimethyl-2-hydroxyethyl -2,3-dihexadecyloxypropylammonium (DPRIE), dimethyl-2-hydroxyethyl-2,3-dioctadecyloxypropylammonium bromide (DSRIE), dimethyl-2-hydroxyethyl-2,3-ditetradecyloxypropylammonium bromide (DMRIE), N-(2-sperminecarbonyl)-N',N'-dioctadecylglycinamide (DOGS), 1,2-dioleoyl-3-succinyl-sn-glycerocholine ester (DOSC), 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-C hol), lipid poly-L-lysine (LPLL), stearylamine (SA); preferably at least one selected from the following: trimethyl-2,3-dioleoyloxypropylammonium bromide, trimethyl-2,3-dioleoyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dioleoyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dioctadecyloxypropylammonium bromide, N-(2-spermine formyl)-N',N'-dioctadecylglycinamide, 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester.

9. The nucleic acid nanoparticle preparation with long-term stability according to claim 1, characterized in that: The ionizable lipid is selected from at least one of the following: DLin-MC3-DMA, 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((decyloxy)hexyl)amino)octanoate (SM-102), ALC-0315, DLin-KC2-DMA, and the following:

10. The nucleic acid nanoparticle preparation with long-term stability according to claim 1, characterized in that: The mass ratio of the cationic lipid to the ionizable lipid in the organic phase is 1:(0.5-3), preferably 1:(1-2).

11. The nucleic acid nanoparticle preparation according to claim 1, characterized in that: The organic solvent is selected from at least one of chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide, ethanol, acetonitrile, tetrahydrofuran and acetone, and is preferably a mixture of dimethyl sulfoxide and ethanol.

12. The nucleic acid nanoparticle preparation according to any one of claims 1 to 11, characterized in that: The material of the organic phase also includes polyethylene glycol lipids, which are one of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000), distearoyl-rac-glycerol-polyethylene glycol 2000 (DSG-PEG2000), and 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000).

13. The nucleic acid nanoparticle preparation according to claim 12, characterized in that: The molar mass ratio of the polyethylene glycol lipid to the polymer is 1:2.0-5.

0.

14. The nucleic acid nanoparticle preparation according to any one of claims 1 to 13, characterized in that: The particle size range of the nucleic acid nanoparticle preparation before and after re-dissolution is 50nm to 200nm.

15. A method for preparing the nucleic acid nanoparticle preparation according to any one of claims 1 to 14, characterized in that: The following steps are involved: (1) Continuous preparation in a microchannel reactor: the polymer, lipid, cholesterol, and phospholipid materials are dissolved in an organic solvent as an organic phase, and a citric acid / sodium citrate buffer of nucleic acid is an aqueous phase; In the microchannel reactor, the aqueous phase and the organic phase are respectively passed through the channels into the reaction plate for mixed reaction to obtain a nucleic acid nanoparticle preparation solution; (2) dialysis to remove organic solvents; (3) concentrating to obtain a concentrated nucleic acid nanoparticle solution; (4) freeze-drying the nucleic acid nanoparticle solution to obtain the nucleic acid nanoparticle preparation.

16. The preparation method according to claim 15, characterized in that: The volume ratio of the aqueous phase to the organic phase is 1:1-5, preferably 1:3; When polyethylene glycol lipid is added to the organic phase, the concentration of the polyethylene glycol lipid in the organic phase is 0.25-0.5 mg / mL.

17. The preparation method according to claim 15, characterized in that: A freeze-drying protective agent with a final concentration of 5% to 15% w / v is added to the concentrated nucleic acid nanoparticle solution, and / or the freeze-drying protective agent added during the freeze-drying is sucrose or glucose, trehalose, or mannitol.

18. The preparation method according to claim 15, characterized in that: The removal of the organic solvent is carried out by rotary evaporation; and / or the concentration is carried out by tangential flow concentration.

19. The preparation method according to any one of claims 15 to 18, characterized in that: The organic phase feedstock of the nucleic acid nanoparticle preparation is: the polymer, ionizable lipid, cholesterol, and phospholipid, wherein the phospholipid is selected from one or two of DSPC, DOPC, DPPC, DMPC, and DSPE; or The organic phase feedstock of the nucleic acid nanoparticle preparation is: the polymer, ionizable lipid, cholesterol, phospholipid and polyethylene glycol lipid, and the phospholipid is selected from one of DSPC, DOPC, DPPC, DMPC and DSPE; or The organic phase feedstock of the nucleic acid nanoparticle preparation comprises: the polymer, ionizable lipid and cationic lipid, cholesterol, phospholipid and polyethylene glycol lipid, and the phospholipid is selected from one of DSPC, DOPC, DPPC, DMPC and DSPE.

20. Use of the nucleic acid nanoformulation according to any one of claims 1 to 14 in the preparation of a drug for preventing or treating tumors or viral infections.

Images